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 .
The response on 12/10/2025 has been received and entered. Claims 1, 3, 5, 10-12, 14-15, 17, 28-29, 31, 37, 41, and 45 are pending, all of which have been examined on the merits.
Claim Objections
Claims 5 and 10 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Status of Prior Rejections/Response to Arguments
RE: Deficiency in Nucleotide and/or Amino Acid Sequence Disclosures
Applicants submitted replacement drawings including FIG. 10 properly labeled with SEQ ID NOs. The objection is withdrawn.
RE: Objection to Specification
Acknowledgement is made that a substitute specification was filed on August 23, 2023 which does not include shaded tables. The objection is withdrawn.
RE: Objection to claims 17 and 22
Applicants amendments to claim 17 overcome the objection. The objection over claim 17 is withdrawn.
Claim 22 has been cancelled, rendering the objection moot.
RE: Rejection of claims 12 and 29 under 35 U.S.C. 112(b)
Claims 12 and 29 have been amended to remove the phrase “SPCas9 FIF1”. Amendments to the claims overcome the rejection of record. The rejection is withdrawn.
RE: Rejection of claims 1-3, 5, 10-12, 15, 17, 22, 37, and 45 under 35 U.S.C. 102 over Maselko et al (bioRxiv, April 2020).
Applicants amended claim 1 to require crossing a first insect comprising a first nucleic acid sequence encoding a CRISPR-associated (Cas) endonuclease to a second insect comprising two or more sgRNA sequences targeting a sequence proximal to a transcription start site of a developmental gene to produce progeny insects; and genetically crossing a female progeny comprising a mutation in the sgRNA target sequence with a male insect comprising a second nucleic acid sequence encoding a nuclease-defiance endonuclease fused to a transcriptional activator to produce an insect with a reproductive barrier.
Applicants assert Maselko et al does not disclose the method as claimed and rather discloses a complex genetic crossing scheme to produce mutant flies that are viable as homozygotes without any apparent phenotype.
In response, the argument has been fully considered but is not found persuasive. The broadest reasonable interpretation of the term “progeny” includes any decedents of the original cross. Maselko et al teaches a method of producing D. melanogaster with engineered genetic incompatibilities (EGI) which reads on method of engineering a reproductive barrier in an insect. Supplementary Fig. 2 of Maselko illustrates a cross between a male expressing a Cas9 and a female homozygous for sgRNA Hh which reads on claim 1(a) (See Fig. S2a cross 1). Cross 11 of Fig. S2a further illustrates a female descendent of the original cross comprising a ΔHh allele (reads on a mutation in the sgRNA target sequence) with a male expressing dCas9::VPR (reads on a nuclease deficient endonuclease fused to a transcriptional activator). The flies in cross 11 are crossed to produce Hh EGI flies. Thus, Maselko teaches the limitations of claim 1. The rejection has been modified accordingly.
Applicants can overcome the rejection by specifying all the alleles present in each cross or specifying the number of crosses required to produce the insects with reproductive barrier.
Applicants further modified claims 5 and 10 requiring the sgRNA target sequence be operably linked to a ubiquitous promoter. Amendments to claims 5 and 10 overcome the rejection of record.
The rejection over claims 1, 3, 11, 12, 15, 17, 37, and 45 is maintained.
The rejection over claims 5 and 10 is withdrawn.
Claims 2 and 22 have been cancelled rendering the rejection moot.
RE: Rejection of claims 1-3, 5, 10-12, 15, 17, 22, 37, 38, and 45 under 35 U.S.C. 103 over Maselko et al (bioRxiv, April 2020).
Applicants traverse the rejection of record on the grounds that Maselko does not teach the limitations of claim 1 as amended.
In response, the argument is not convincing. While the method of Maselko comprises different intermediate steps (i.e. crossing to balancers), the method of Maselko teaches the claimed steps (See response to 102 rejection above and detailed rejection below).
Applicants amendments to claims 5 and 10 overcome the rejection of record. Additionally, it would not have been obvious to operably link a ubiquitous promoter to the sgRNA target sequence of Maselko because the sgRNA target sequence of Maselko is a promoter region for an endogenous developmental gene important for patterning (e.g. hh, hid, pyr, upd1, upd2, upd3, wg, vn). Given that developmental genes of Maselko are necessary for patterning, their expression should be restricted. Thus it would not be obvious to modify the endogenous gene or add an additional copy of the gene comprising a promoter that is operably linked to a ubiquitous promoter because it could disrupt the expression pattern resulting in lethality or developmental abnormalities.
Claims 2, 22, and 38 have been cancelled rendering their rejection moot.
Given that claims 1, 3, 11, 12, 15, 17, 37, and 45 are anticipated by Maselko, the 103 rejection is withdrawn.
RE: Rejection of claims 1-3, 5, 10-12, 14, 15, 17, 22, 37, 38, and 45 under 35 U.S.C. 103 over Maselko et al (bioRxiv, April 2020) in view of Guilinger et al (Nat Biotechnol, 2014).
Applicants traverse the rejection of record on the grounds that Maselko and Guilinger do not teach the limitations of claim 1 as amended.
In response, the argument is not convincing. While the method of Maselko comprises different intermediate steps (i.e. crossing to balancers), the method of Maselko teaches the claimed steps (See response to 102 rejection above and detailed rejection below).
The rejection over claims 1, 3, 11, 12, 14, 15, 17, 37, and 45 is maintained.
Applicants amendments to claims 5 and 10 overcome the rejection of record. The rejection over claims 5 and 10 is withdrawn.
Claims 2, 22, and 38 have been cancelled rendering their rejection moot.
RE: Rejection of claims 1-3, 5, 10-12, 15, 17, 22, 27-29, 31, 37, 38, and 45 under 35 U.S.C. 103 over Maselko et al (bioRxiv, April 2020) as evidenced by Flybase and in view of Basset et al (Cell Reports 2013).
Applicants traverse the rejection of record on the grounds that Maselko, Flybase, and Basset do not teach the limitations of claim 1 as amended.
In response, the argument is not convincing. While the method of Maselko comprises different intermediate steps (i.e. crossing to balancers), the method of Maselko teaches the claimed steps (See response to 102 rejection above and detailed rejection below).
The rejection over claims 1, 3, 11, 12, 15, 17, 28, 29, 37, and 45 is maintained.
Applicants amendments to claims 5 and 10 overcome the rejection of record. The rejection over claims 5 and 10 is withdrawn.
Claims 2, 22, 27, and 38 have been cancelled rendering their rejection moot.
RE: Rejection of claims 1-3, 5, 10-12, 15, 17, 22, 27-29, 31, 34, 37, 38, and 45 under 35 U.S.C. 103 over Maselko et al (bioRxiv, April 2020) as evidenced by Flybase and in view of Basset et al (Cell Reports 2013) and Gilbert et al (Cell, 2013).
Applicants traverse the rejection of record on the grounds that Maselko, Flybase, Basset, and Gilbert do not teach the limitations of claim 1 as amended.
In response, the argument is not convincing. While the method of Maselko comprises different intermediate steps (i.e. crossing to balancers), the method of Maselko teaches the claimed steps (See response to 102 rejection above and detailed rejection below).
Applicants amendments to claims 5 and 10 overcome the rejection of record. The rejection over claims 5 and 10 is withdrawn.
Claims 2, 22, 27, 34, and 38 have been cancelled rendering their rejection moot.
Given that claims 1, 3, 11, 12, 15, 17, 28, 29, 37, and 45 are rendered obvious by Maselko, Flybase and Basset, their rejection is withdrawn.
RE: Rejection of claims 1-3, 5, 10-12, 14, 15, 17, 22, 37, 38, and 45 under 35 U.S.C. 103 over Maselko et al (bioRxiv, April 2020) in view of Carvalho et al (PLOS NTDs, 2015).
Applicants traverse the rejection of record on the grounds that Maselko and Carvalho do not teach the limitations of claim 1 as amended.
In response, the argument is not convincing. While the method of Maselko comprises different intermediate steps (i.e. crossing to balancers), the method of Maselko teaches the claimed steps (See response to 102 rejection above and detailed rejection below).
The rejection over claims 1, 3, 11, 12, 15, 17, 37, 41, and 45 is maintained.
Applicants amendments to claims 5 and 10 overcome the rejection of record. The rejection over claims 5 and 10 is withdrawn.
Claims 2, 22, and 38 have been cancelled rendering their rejection moot.
New/Maintained Rejections
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 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, 3, 11, 12, 15, 17, 31, 37, and 45 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Maselko et al (bioRxiv, April 2020).
Maselko et al teaches a method of engineered genetic incompatibilities (EGI) which produces D. melanogaster (flies) engineered to be mutually incompatible with wild-type flies or other EGI strains of flies (See abstract). The method comprises crossing flies homozygous for S. Pyogenes dCas9-VPR (or dxCas9-VPR) to flies homozygous for sgRNAs targeting several genes important for development including hh, hid, pyr, upd1, upd2, upd3, wg, vn (See lns 89-90 and Fig. 1). In the progeny of the EGI cross, dCas9-VPR drives overexpression of the selected gene, resulting in lethality (See lns 80-82). All transgenic flies were generated via ΦC31 mediated integration targeted to attP landing sites (See lns 274-275). Maselko et al further teaches producing flies harboring silent mutations in the gene promoter, which can be used to produce viable offspring (See lns 110-119). The silent mutations are made by crossing flies expressing a sgRNA or flies directly injected with sgRNA to flies expressing germline active Cas9 (See lns 112-113). The progeny comprising the resulting mutations were crossed to balancers. The method of Maselko et al required 19 crosses (See Fig. S2). Resulting flies were further crossed to the flies expressing dCas9 and produced viable offspring (See Fig.S2). Maselko et al further teaches an alternative method in which embryos from homozygous promoter mutant strains are injected with a single plasmid containing expression constructs for both the dCas9-VPR and the sgRNA resulting in a more direct approach requiring fewer crosses (See lns 126-129 and Fig. 3b).
Regarding claim 1: Maselko et al teaches a method of EGI in D. melanogaster which reads on method of engineering a reproductive barrier in an insect. The method comprises breeding (reads on genetically crossing) a fly expressing homozygous sgRNA (reads on a second insect comprising two sgRNA sequences) targeting a gene promoter (reads on a sgRNA target sequence is proximal to a transcription start site) to a fly expressing Cas9 (reads on a first insect comprising a first nucleic acid sequence encoding a Cas endonuclease) (See cross 1 in Fig. S2a). The exemplified target gene in Fig. S2 is Hh which reads on a developmental gene. Cross 11 of Fig. S2a further illustrates a cross between a female descendent comprising a ΔHh allele (reads on crossing a female progeny insect comprising a mutation in the sgRNA target sequence) and a male expressing dCas9::VPR (reads on a nuclease deficient endonuclease fused to a transcriptional activator). The flies in cross 11 are crossed to produce Hh EGI flies which reads on the flies are crossed to produce an insect with a reproductive barrier.
Regarding claim 3: Following the discussion of claim 1 above, Maselko et al discloses all transgenic flies in the study were generated via ΦC31 mediated integration targeted to attP landing sites which reads on step (a) comprises integrating the first nucleic acid sequence into the genome of the first insect. Additionally, a male fly expressing dCas9::VPR is used in the EGI method of Maselko thus Maselko teaches step (b) comprises integrating the second nucleic acid sequence into the genome of the male insect.
Regarding claims 11, 12, 15, and 17: Following the discussion of claim 1 above, Maselko et al discloses crosses using a fly expressing S. pyogenes dCas9-VPR or dxCas9-VPR which reads on the nuclease deficient endonuclease is fused to an effector domain comprising VPR.
Regarding claim 31: Following the discussion of claim 1 above, Maselko discloses producing flies with silent mutations in the sgRNA-binding sequence to prevent lethal over- or ectopic-expression in EGI strains which reads on mutations that prevent the sgRNA from targeting the sgRNA target sequence and the mutation does not alter expression of the developmental gene product.
Regarding claim 37: Following the discussion of claim 1 above, Maselko et al discloses flies expressing a sgRNA targeting several genes including hh, hid, and wg which reads on the gene product comprises at least one of eve, hid, hh or wg.
Regarding claim 45: Following the discussion of claim 1 above, Maselko discloses crossing a fly expressing a mutation in the promoter region of a developmental gene with a fly expressing dCas9-VPR to produce EGI flies (reads on a genetically modified insect produced by the method of 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.
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 1, 2, 11, 12, 14, 15, 17, 31, 37, and 45 are rejected under 35 U.S.C. 103 as being unpatentable over Maselko et al (bioRxiv, April 2020) in view of Guilinger et al (Nat Biotechnol, 2014).
The teachings of Maselko et al are set forth above.
Maselko et al anticipates claims 1, 3, 11, 12, 15, 17, 31, 37, and 45.
Regarding claim 14: Following the discussion of claim 1 above, Maselko et al discloses a fly expressing dCas9-VPR which reads on a nuclease deficient endonuclease comprising a Cas9.
Maselko et al does not disclose the Cas fusion comprising Cas9 fused with Fok1.
Guilinger et al teaches fusing FokI nuclease to Cas9 or dCas9 improves specificity and reduces off-target effects (See abstract and first paragraph of discussion section).
Given that Maselko et al discloses using a fly expressing dCas9-VPR and Guilinger et al teaches fusing FokI to dCas9 improves specificity, it would have been prima facie obvious to modify the dCas9-VPR of Maselko et al by fusing a FokI to the dCas9. One would have been motivated to modify the dCas9 of Maselko et al because Guilinger teaches fusing a FokI to a dCas9 improves specificity and reduces off-target effects. There is a reasonable expectation of success because Guilinger teaches FokI can be fused to dCas9.
Claims 1, 3, 11, 12, 15, 17, 28, 29, 31, 37, and 45 are rejected under 35 U.S.C. 103 as being unpatentable over Maselko et al (bioRxiv, April 2020) as evidenced by Flybase and in view of Basset et al (Cell Reports 2013).
The teachings of Maselko et al are set forth above.
Maselko et al anticipates claims 1, 3,11, 12, 15, 17, 31, 37, and 45.
Regarding claims 28 and 29: Following the discussion of claim 1 above, Maselko et al teaches a method of producing EGI flies by crossing flies expressing dCas9-VPR to flies expressing a sgRNA. Maselko et al further teaches producing flies with silent mutations (reads on a mutation in the genomic sequence that does not alter expression of the gene product) in the promoter region of the targeted genes. Maselko produces the mutant flies by crossing flies expressing an active S. pyogenes Cas9 (See Flybase page on Dmel\M{nanos-Cas9.P}ZH-2A) (reads on an endonuclease that cleaves the genomic sequence) to flies expressing sgRNA thereby producing offspring with the silent mutation in the target gene’s promoter. The method of Maselko requires several generations of breeding to produce the stable line. Flies expressing the silent mutation and an sgRNA were then crossed to flies expressing dCas9 resulting in viable offspring.
Maselko et al does not teach step (a) comprises introducing an endonuclease that cleaves the genomic sequence into the first insect.
Bassett et al teaches drosophila embryos can be edited by injecting the embryos with Cas9 mRNA, which is expressed transiently, and a sgRNA (See pg. 220, Sec. Introduction, last paragraph).
Given that both Maselko et al and Basset et al teach methods of editing drosophila using Cas9 and a sgRNA and Basset et al teaches Cas9 and sgRNA can be directly injected into the same fly embryo in order to directly edit the embryo, it would have been prima facie obvious to modify the method of Maselko et by injecting Cas9 mRNA into the fly expressing the sgRNA resulting in editing of said fly. One would have been motivated to modify the method of Maselko et al by injecting Cas9 mRNA into the sgRNA expressing fly because the modification would result in flies being directly edited whereas the current method of Maselko requires several generations of breeding. Therefore, the modified method would reduce the number of crosses required to achieve the desired mutant flies. There is a reasonable expectation of success because Basset et al teaches injecting Cas9 mRNA into fly embryos results in editing.
Claims 1, 3, 11, 12, 15, 17, 31, 37, and 45 are rejected under 35 U.S.C. 103 as being unpatentable over Maselko et al (bioRxiv, April 2020) in view of Carvalho et al (PLOS NTDs, 2015).
The teachings of Maselko et al are set forth above.
Maselko et al anticipates claims 1, 3, 11, 12, 15, 17, 31, 37, and 45.
Regarding claim 41: Following the discussion of claim 1 above, Maselko et al teaches a method of producing EGI flies (reads on insects) according to the method of claim 1.
Maselko et al does not teach the insects are mosquitos from the genera listed in claim 41.
Carvalho et al teaches dengue is primarily spread by aedes aegypti (mosquitos) and that genetic engineering can be used to control mosquito populations by introducing transgenes which result in embryo lethality (Introduction, first and third paragraphs).
Given that Maselko teaches a method of producing flies which when bred with wild type-flies resulting in non-viable embryos and Carvalho et al teaches mosquito populations can be controlled by introducing transgenes into mosquitos that lead to embryo lethality, it would have been prima facie obvious to modify the method of Maselko et al to produce EGI Aedes Aegypti mosquitos rather than EGI flies. One would have been motivated to modify the method of Maselko et al to produce EGI A. Aegypti mosquitos because Carvalho et al teaches dengue is primarily spread by A. Aegypti. There is a reasonable expectation of success because Carvalho et al teaches transgenic mosquitos can be used to control mosquito populations and Maselko et al teaches a method of producing transgenic insects.
Allowable Subject Matter
The following is a statement of reasons for the indication of allowable subject matter:
Claims 5 and 10 have been modified to require “the sgRNA target sequence is operably linked to a ubiquitous promoter”. Per claim 1, the sgRNA target sequence is proximal to a transcription start site of a developmental gene. Given that developmental genes in insects are expressed in specific patterns and disruption of said patterns can result in morphological abnormalities and lethality, it would not have been obvious to modify a sequence proximal to a transcription start site of a developmental gene (i.e. the sgRNA target sequence) by linking the sequence to a ubiquitous promoter, because the modification could result in altered patterning of the developmental gene.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to MARISOL A O'NEILL whose telephone number is (571)272-2490. The examiner can normally be reached Monday - Friday 7:30 - 5:00 EST.
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/MARISOL ANN O'NEILL/Examiner, Art Unit 1633
/ALLISON M FOX/Primary Examiner, Art Unit 1633