DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
2. 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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 06 Nov 2025 has been entered.
Status of Claims
3. This action is written in response to applicant’s response received 06 Nov 2025. Claims 1-3, 6-14, and 17-19 are currently pending and examined herein.
4. Any rejection or objection not reiterated herein has been overcome by amendment. Applicant’s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
Priority
5. The instant application claims foreign priority to Japanese Patent Application No.2014-
043348 (filed March 5, 2014), which was filed in the Japanese language. On June 29, 2018,
Applicants filed an English translation of Japanese Patent Application No. 2014-043 348
accompanied by a sworn declaration by Takeshi S. Komatani attesting to the accuracy of the
translation. The translation filed June 29, 2018 of Japanese Patent Application No. 2014 -043348
indicates that the priority document describes the fusion between a Cas9 nickase and a deaminase
([0054]-[0058]), wherein protein binding domains such as SH3 domain and a binding partner thereof may be used ([0024]), wherein a plurality of sequence recognizing modules are used to simultaneously target different target nucleotide sequences in proximity ([0046]), and the different types of cells recited by claim 7 ([0024]), use of an inducible promoter ([0027]), and wherein the Cas9 nickase has the D10A or H840A mutation ([0056]). Accordingly, the effective filing dates of instant claims 1, 2, 6, 7, 9, 11-14, and 17-19 is March 5, 2014.
However, support for the recitations “wherein the different target nucleotide sequences are present in different genes” as recited in instant claim 3, "A method of modifying one or more nucleotides of a site of a double stranded DNA in any targeted alleles on homologous chromosomes in a polyploid cell" as recited by instant claim 8, or "wherein the target nucleotide sequence in the double stranded DNA is present in a gene essential for survival of the cell" as recited in instant claim 10 could not be found in the English translation of Japanese Patent Application No.2014-043348. Therefore, instant claims 3, 8, and 10 are not afforded the effective filing date of March 5, 2014.
The instant application also claims foreign priority to Japanese Paten t Application No.2014-201859 (filed on September 30, 2014), which was filed in the Japanese language. On June 29, 2018, Applicants filed an English translation of Japanese Patent Application No. 2014 -201859 accompanied by a sworn declaration by Takeshi S. Komatani attesting to the accuracy of the translation. The translation filed June 29, 2018 of Japanese Patent Application No. 2014-201859 describes "in one preferable embodiment of the present invention, two or more kinds of nucleic acid sequence-recognizing modules that specifically bind to different target nucleotide sequences (which may be present in one gene of interest, or two or more different genes of interest" ([0049]) and "wherein the cell is a polyploid cell, and a site in any targeted allele on a homologous chromosome is modified" ([17]), and wherein the target gene is an "essential gene" for survival of the cell [0068]. Accordingly, the effective filing date of instant claims 3, 8, and 10 is September 30, 2014.
Declaration Filed under 37 CFR 1.132
6. The Declaration of Keiji Nishida under 37 CFR 1.132 filed Oct 23, 2023 has been considered but it is insufficient to overcome the rejection of claims 1-3, 6-14, and 17-19 based upon 35 U.S.C. 103 as set forth in this Office action because the Affidavit recites CRISPR complexes with Cas9 nickase and does not recite SH3 domain and ligand at all.
Claim Rejections - 35 USC § 103
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 CPR 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.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-3, 6-7, 9-14, and 17-19 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (US 8697359 B1; Filling Date: Oct 15, 2013) in view of Liu (US 2015/0166980; PRO: 61/915,386 filed on Dec 12, 2013) in view of Dickson (WO 2007139573 A1; Published Date: Dec 6, 2007), and further in view of Dueber (Synthetic protein scaffolds provide modular control over metabolic flux, Nat. Biotech. 2009, 27 (8), 753-759).
Regarding claim 1, Zhang teaches a method of modifying expression of a polynucleotide in a eukaryotic cell comprising a CRISPR enzyme complex with a guide sequence hybridized to a target sequence within said polynucleotide (i.e. nucleic acid-sequence recognizing molecule that specifically binds to a target nucleotide sequence in a targeted site of the double stranded DNA) (column 26, lines 45-49). Zhang teaches wherein the CRISPR enzyme directs cleavage of one strand of the double stranded DNA at the location of a target sequence (i.e. a Cas9 nickase) (Fig 21A-D; column 18, lines 33-35), and “the availability of a nickase can significantly reduce the likelihood of off-target modifications” (column 49, lines 56-58).
Zhang does not teach a Cas9 complex coupled with a nucleic acid-base converting enzyme that converts a target nucleotide to another nucleotide by introducing one or more mutations consisting of a substitution, a deletion, and an insertion.
Liu teaches a fusion protein comprising a nuclease-inactive Cas9 domain and a deaminase domain (i.e. nucleic acid-base converting enzyme) to convert one or more nucleotides in the targeted site to a different nucleotide without introducing a double strand break ([009]). Liu teaches the mutation consists of a substitution mutation, specifically a T>C or A>G point mutation to correct a point mutation in the targeted sequence associated with a disease.
It would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to modify Zhang’s Cas9 nickase by incorporating the deaminase domain of Liu, specifically by substituting the nuclease-inactive Cas9 domain of Liu with the nickase Cas9 of Zhang because it would have merely amounted to a simple substitution of prior art elements according to known methods to yield predictable results. One would have been motivated to have done so for the advantage of reducing off-target modifications with the nickase variant while maintaining the nucleotide-converting functionality of the deaminase. One of ordinary skill in the art would had a reasonable expectation of success because both Zhang and Liu teaches methods for targeted DNA editing using CRISPR Cas9 complexes.
Neither Zhang or Liu teaches ligation with SH3 domain and ligand.
However, Dickson teaches the fundamental binding structures of SH3 domains (section 5.5.1, lines 5-10). SH3 domain and ligand’s structural modularity and feasibility of fusion to heterologous proteins have been evidenced by Dueber, who refers to building synthetic protein scaffolds by fusing SH3 ligands to hydroxy-methylglutaryl-CoA synthanse (HMGS) and SH3 domains to hydroxy-methylglutaryl-CoA reductase (HMGR) (Fig. 2A; pg. 735, left-column, first paragraph). HMGS and HMGR interacted in an SH3-dependent manner as “no significant interaction was observed between HMGS and HMGR without engineered SH3 interactions” but “an observable amount of HMGR was pulled down when a single ligand was tethered to HMGS”. Further, Dueber teaches that mevalonate titers increased by 2-10fold in vivo via HMGR and HMGS co-recruitment by SH3 domain/ligand (Fig 2c, pg. 755 left-column, second paragraph). Dueber’s teachings confirmed that SH3 domain/ligand maintains its functional properties in vitro and in vivo, and this pair can be used as a modular linker to recruit or colocalize protein scaffolds in close proximity with each other.
It would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to have used SH3 domain/ligand linkage, as taught by Dickson and evidenced by Dueber, with the Cas9 nickase taught by Zhang and deaminase taught by Liu because SH3 domain/ligand are known modular linkers with independent folding, tight affinity, and successful use in previous synthetic biology applications including engineered fusion proteins. One of ordinary skill in the art would have had a reasonable expectation of success in doing so because it would have been a substitution of a known element into the dCas9-deaminase complex taught by Liu, and an addition into the known method of modifying expression of polynucleotides using CRISPR-Cas9 complexes taught by Zhang.
Regarding claim 2, Zhang teaches CRISPR system can mediate multiplexed editing, wherein two or more CRISPR complexes that each comprise a nucleic sequence-recognizing module that specifically binds to a different target nucleotide sequence (column 49, lines 60-67).
Regarding claim 3, Zhang teaches wherein the different target nucleotide sequences are present in different genes, EMX1 and PVALB, (column 49, lines 60-67).
Regarding claim 6, Zhang teaches introducing “DNA molecules encoding one more gene product” comprising components of CRISPR complex (column 2, lines 30-62).
Regarding claim 7, Zhang teaches wherein the cell is a eukaryotic cell (column 3, line 38), microbial cell, yeast cell, insect yell or mammalian cell (column 14, lines 25-28).
Regarding claim 9, Zhang teaches wherein the expression one or more elements of the CRISPR system is driven by a regulatory element (Column 15, lines 55-57), and expression of CRISPR system elements were under bicistronic expression vectors in eukaryotic cells (Fig. 8A-B).
Regarding claim 10, Liu teaches using CRISPR dCas9 systems with deaminase to correct point mutations in disease-associated genes, including PI3KCA ([0009]) whose function is essential for survival of the cell. Thus, it would be obvious to one of ordinary skill in the art before the effective filling date of the invention to further apply this teaching of Liu, with the teachings discussed in claim 1, to edit a target nucleotide sequence in genes essential for cell viability. One of ordinary skill in the art would have had a reasonable expectation of success following the method taught by Liu.
Regarding claim 11, the obviousness of ligating a Cas9 nickase (i.e. nucleic acid sequence-recognizing module) and a deaminase (nucleic acid base-converting enzyme) with SH3 domain and SH3 ligand linkers to perform on-target functional editing is discussed above as applied to claim 1.
Regarding claim 12, Zhang teaches “DNA molecules encoding…one or more vectors comprising…b) a second regulatory operably linked to a Type-II Cas9 protein” (Column 2, lines 30-42). Liu also teaches “a nucleic acid construct that comprises a sequence encoding a nuclease-inactive Cas9 sequence… a sequence encoding a nucleic-acid-editing enzyme…and, optionally, a sequence encoding a linker positioned between the Cas9 encoding sequence and the cloning site” for the nucleic-acid-editing enzyme ([0011]). The obviousness of fusing a Cas9 nickase (i.e. nucleic acid sequence-recognizing module) and a deaminase (nucleic acid base-converting enzyme) with SH3 domain and SH3 ligand linkers to perform on-target functional editing is discussed above as applied to claim 1. As evidenced by teachings of Dueber, these two fusion proteins would yield predictable results “wherein when expressed, the first fusion protein and the second fusion protein link with each other to form a complex that is capable of converting a target nucleotide to another nucleotide by catalyzing a reaction for converting a substituent on a purine or pyrimidine ring on a DNA base to another group or atom, without cleaving the other strand of the double-stranded DNA strand in the targeted site” as required by claim 12.
Regarding claim 13, Zhang teaches wherein “an aspartate-to-alanine Substitution (D10A) in the RuvC I catalytic domain of Cas9 from S.pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand)” (column 18, lines 44-47).
Regarding claim 14, Zhang teaches wherein “other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A” (column 18, lines 47-49).
Regarding claim 17, Zhang and Liu’s teachings is discussed as applied to claim 12 and the obviousness of fusing a Cas9 nickase (i.e. nucleic acid sequence-recognizing module) and a deaminase (nucleic acid base-converting enzyme) with SH3 domain and SH3 ligand linkers to perform on-target functional editing is discussed above as applied to claim 1.
Regarding claim 18 and 19, Zhang’s teachings is discussed as applied to claims 13 and 14.
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang (US 8697359 B1; Filling Date: Oct 15, 2013) in view of Liu (US 2015/0166980; PRO: 61/915,386 filed on Dec 12, 2013) in view of Dickson (WO 2007139573 A1; Published Date: Dec 6, 2007), in view of Dueber (Synthetic protein scaffolds provide modular control over metabolic flux, Nat. Biotech. 2009, 27 (8), 753-759) and further in view of Kim (WO 2014/065596 A1; Published Date: May 1, 2014; Filing Date: Oct 23, 2013)
The teachings of Zhang, Liu, Dickson, and Dueber are discussed above.
However, Zhang, Liu, Dickson, and Dueber do not teach modifying a targeted site in a double stranded DNA in each of two or more targeted alleles on homologous chromosomes in a polyploid cell.
Regarding claim 8, Kim teaches using CRISPR Cas9 systems to introduce modifications at a targeted site on alleles in a diploid cell. Fig 22 shows a conceptual diagram of a diploid cell containing different biallelic mutations (C) and identical biallelic mutations (D). It is noted that the biology definition for polyploid is cells containing more than two sets of chromosomes found in a diploid cell, which indicates that diploid cells are not polyploid cells. However, the specification defines a polyploid cell as “diploid, triploid, tetraploid and the like” ([0035]) and Example 8 (pg. 40) discloses “simultaneous editing of Ade1 and Can1 genes were performed in budding yeast YPH501 strain as a diploid strain”. Thus, the diploid cell and teachings of Kim meet the requirement of the claim. Further, the introduction of mutations (either different or identical biallelic mutations) into homologous alleles inherently satisfies the limitation of each of two or more targeted alleles on homologous chromosomes.
It would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to have applied the method of claim 1 to modify targeted sites in in a polyploid cell because one would have the ability to introduce point mutation modifications on multiple alleles simultaneously. Given the substantial amount of guidance provided by Kim in targeting multiple alleles and detecting successful editing, one of ordinary skill in the art would have had performed the method of claim 8 with a reasonable expectation of success.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-2, 6,7,9, 13-14, and 18-19 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-6 of U.S. Patent No. 10,655,123 B2 (referred to as Nishida 1) in view of Dueber (Synthetic protein scaffolds provide modular control over metabolic flux, Nat. Biotech. 2009, 27 (8), 753-759). Although the claims at issue are not identical, they are not patentably distinct from each other.
Regarding claims 1-2, 6,7,9, 11-14, and 17-19, Nishida 1 teaches a method of modifying a nucleic acid base in a particular region of a genome, without cleaving double-stranded DNA. Nishida 1 further teaches a contacting step performed using a CRISPR-Cas9 nickase protein, the same current claimed cell types, an expression vector comprising the CRISPR-Cas9 system, Cas9 nickase’s mutations and cleavage activity, and nucleotide modifications.
Nishida 1 does not teach the use of SH3 domain and SH3 ligand.
Dueber teaches that SH3 domain/ligand maintains its functional properties in vitro and in vivo, and this pair can be used as a modular linker to recruit or colocalize protein scaffolds in close proximity with each other (Fig 2c, pg. 755 left-column, second paragraph).
Thus, it would be obvious to one of ordinary skill in the art before the effective filling date of the invention to have modified the CRISPR-Cas9 system of Nishida 1 with SH3 domain/ligand linkage because SH3 domain/ligand are known modular linkers with independent folding, tight affinity, and successful use in previous synthetic biology applications including engineered fusion proteins. One of ordinary skill in the art would have had a reasonable expectation of success in doing so because it would have been a substitution of linkers
Regarding claims 11, 12, and 17, Nishida 1 does not explicitly teach a nucleic acid-modifying enzyme complex, a nucleic acid or a nucleic acid combination encoding fusion proteins for the nucleic acid-modifying enzyme complex. However, these products recited in claims 11, 12, and 17 represent the essential components for practicing the method of Nishida 1, and do not recite any structural or functional features that patentably distinguish them from the CRISPR-Cas9 complexes inherently used in the method of Nishida 1. Any differences between the claimed products and the elements required by Nishida 1 are obvious variants.
Claim 8 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-6 of U.S. Patent No. 10,655123 B2 (referred to as Nishida 1) in view of Kim (WO2014/065596 A1; Published Date: May 1, 2014). Although the claims at issue are not identical, they are not patentably distinct from each other.
The teachings of Nishida 1 are discussed above.
Nishida 1 does not teach modifying a targeted site in double stranded genomic DNA in each of two or more targeted alleles on homologous chromosomes in a polyploid cell.
Kim teaches a method of using CRISPR Cas9 systems to introduce modifications at a targeted site by error-prone non-homologous end-joining repairs on alleles in a diploid cell. The specification defines a polyploid cell as “diploid, triploid, tetraploid and the like” ([0035]). Thus, it would be obvious to one of ordinary skill in the art before the effective filling date of the invention to have applied the method of Nishida 1 to modify targeted sites in two or more alleles on homologous chromosomes in a polyploid cell because one would have the ability to introduce point mutation modifications on multiple alleles simultaneously. It would be predictable to one skilled in the art to combine the teachings as Kim teaches targeting multiple alleles using CRISPR-Cas9 systems and detecting successful editing.
Claim 11 is rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-4 of U.S. Patent No. US11718846B2 (referred to as Nishida 2). Although the claims at issue are not identical, they are not patentably distinct from each other because they claim a nucleic acid-modifying enzyme complex that specifically binds to a target nucleotide sequence in a double stranded DNA, wherein the complex comprises a nucleic acid base converting enzyme fused to SH3 ligand and a nucleic acid sequence-recognizing module CRISPR-Cas9 protein fused to SH3 domain.
Response to Arguments
Applicant’s arguments have been fully considered but are not persuasive. The prior rejection over Dickson (using SH3 domain/ligand in the context of in vitro ribosome-display library selection) is maintained, as Dickson continues to teach the fundamental properties of SH3 domain/ligand binding pairs including their binding affinity, structural modularity, and feasibility of fusing to heterologous proteins. However, in view of Applicant’s Remarks (received on Nov 06, 2025) regarding the following:
i) “the conditions of Dickson are not analogous to those of the present claims”, “an in vitro ribosomal-display environment fundamentally differs from the intracellular genome-editing context”
ii) the rationale “overlooks the complex structural and enzymatic constraints inherent in Cas9-based fusion constructs, the predictability in using SH3 domains in genome-editing and enzyme-fusion systems”
and iii) “there is no teaching or suggestion in Dickson, Zhang, or Liu to employ SH3 domain/ligand modules to couple distinct protein complexes for functional editing”
Examiner additionally cites Dueber, which explicitly teaches using SH3 domain/ligan to couple HMGS and HMGR for purpose fusion protein co-recruitment in vitro and intracellularly. It is noted that Dueber was referenced by Applicant’s own non-patent literature (Nishida, Science 353, 6305 (2016)) for SH3 domain/ligand. Nishida stated (pg. 1, middle column) “…PmCDA1 from sea lamprey by either a fusion protein or attaching a SH3 (Src 3 homology) domain (in text citation Dueber)”. This confirms the concept of using SH3 domain/ligand to bring intracellular proteins together in close proximity was known by Applicant before the effective filling date. Thus, Dueber’s teachings undermine Applicant’s arguments set above and directly corresponds to the claimed mechanism.
The prior rejection is maintained for additional reasons set forth below:
i) Considering all teachings of the prior art, SH3 domain/ligand is taught in the reference as a high affinity binding pair that brings the fused protein partners into close proximity. The claimed invention uses this functional property for the same purpose, applying SH3 domain/ligand affinity interactions to bring Cas9 nickase and nucleobase-converting enzyme together (i.e. the first fusion protein and the second fusion protein link with each other to form a complex (claims 12 and 17)). Whether the prior art used SH3 domain/ligan for in vitro selection, engineered metabolic pathways, or genome editing is not structurally different. A person of ordinary skill in the art would have recognized the same intrinsic binding property could be exploited in other contexts with induced proximity of fusion proteins. If the prior art as a whole teaches some suggestion to combine the elements, it is not required that these elements be combined for the same motivation as the Applicant’s stated purpose of genome-editing systems (see MPEP 2141.02). The fact that prior art teaches SH3 domain/ligand bring different fused proteins together, in contexts different than genome editing, does not diminish its relevance. The disclosure of a reference is not limited to the specific invention disclosed but extends to all disclosed subject matter. In addition, the prior art must be considered for what it teaches, and it is not limited to the context of its specific examples (i.e. in vitro selection and engineering metabolic pathways). One of ordinary skill in the art need not see the identical problem addressed in a prior reference to be motivated to apply its teachings.
ii) Further, claims must be distinguished over the prior art based on structural limitations, not merely on a new use of an existing structure. In the instant case, Applicant argues that SH3 domain/ligand is distinguished over prior art based on its application on genome editing. The claims recite the same structural features as disclosed in the prior art, wherein protein A is fused to SH3 domain, protein B is fused to SH3 ligand, and protein A and B are colocalized upon the high affinity binding interactions of SH3 domain and its ligand. Applicant claimed SH3 domain/ligan usage in CRISPR complexes for genome editing is not a structural feature, and this distinct usage or intended use does not enable claimed invention to be patentably distinct.
iii) Applicant argues “the use of the SH3 domain-SH3L binding pair to link the dCas9 protein and the nucleic acid base-converting enzyme achieves an unexpectedly enhanced mutation frequency relative to the level of mutation frequency of a standard fusion protein configuration in which the dCas9 and the enzyme domains are linked by an in-frame peptide linkage”. This argument has been considered, but is not persuasive because unexpected results must be commensurate in scope with the claims (see MPEP 716.02 (d)). All the constructs shown in Fig 1B of Nishida comprised a nuclease-inactive Cas9 (dCas9), which functions significantly different than Cas9 nickase, as evidenced in Fig 2A where the mutation frequency between dCas9 and nCas9(H840A) differs by approximately 100-fold. Unexpected results for dCas9 do not establish unexpected results for Cas9 nickase unless applicant proves why the results would extend across the claimed scope. Although Applicant did not include Fig 2A in their Remarks, it is noted that the constructs in Fig 2A comprised of a Cas9 nickase and it is not clear whether nCas9(D10A)-PmCDA and nCas9(H840A)-PmCDA contained SH3 domain/ligand or conventional linker as such critical structural features were not disclosed. Thus, the evidence for unexpected results provided by the Applicant is insufficient to overcome the case of prima facie obviousness.
vi) Applicant also states an on-target mutation frequency with approximately 100-fold enhancement was achieved as compared with conventional fusions (Nishida, Figure 1B). Applicant relies on a 100-fold enhancement observed in construct 4 (PmCDA1-SHL attached to dCas9-SH3-gRNA via SH3 and SHL interaction) and construct 5 (PmCDA1 fused to dCas9 with short linker). However, this comparison is not proper under MPEP 716.02 (c) as it does not include the results of construct 3 (PmCDA1 and dCas9-gRNA without direct attachment) and construct 4. The appropriate comparison is between construct 3 (no linker) and construct 4 (SH3 domain/ligand), which differs only in the presence of the claimed modular linkage. Nishida Fig 1B shows only less than 10-fold enhancement, with some datapoint overlap. Such modest enhancement is consistent with Dueber’s teachings where HMGR tagged with one SH3 domain resulted in approximately 4-fold increase compared to untagged HMGR (Fig 2C; pg. 755, left-column). A person of ordinary skill in the art would have reasonably expected an improvement of the same order of magnitude when employing this known SH3 domain/ligand linkage. Applicant has not shown “the present application provides expectedly superior results not taught in the prior art” (Remarks, last paragraph of pg. 10) or the “SH3 domain-SH3L modular configuration responsible for the enhanced on-target editing efficiency” (Remarks, bottom of pg. 11).
v) Fig 2 shows that nCas9(D10A)-PmCDA achieved nearly 100-fold enhancement of on-target mutation frequency as compared with dCas9-PmCDA, indicating that the key contribution to activity enhancement arises from the differences between Cas9 variants (nickase as compared to nuclease-inactive) which is consistent with Zhang’s teaching that nickase can significantly reduce the likelihood of off-target modifications (column 49, lines 56-59). This data further confirms that SH3 domain/ligand was not responsible for the significant enhanced on-target editing efficiency.
Applicant’s response is not sufficient to rebut the prima facie case of obviousness, and the combination of known elements yielded expected results, strengthening the prima facie case of obviousness. Accordingly, Applicant’s arguments haven been considered but are not persuasive, and the rejection under 35 U.S.C. 103 is maintained.
Applicant’s remarks regarding the prior rejection of claim 8 based on Cost (WO2013/169802A1), which discloses the use of zinc-finger nucleases for simultaneous editing of multiple alleles, have been fully considered and is withdrawn. However, claim 8 remains unpatentable in view of Kim (WO 2014/065596 A1; Published Date: May 1, 2014), as set forth in the current Office Action (see obviousness discussion above under 35 U.S.C. 103 rejection). As further support of the state of the art, Wang (Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew, Nat. Biotech. 2014, 32 (9), 947-951; DOI: 10.1038/nbt.2969; Published online: July 20, 2014) is cited as relevant art demonstrating using CRISPR systems for simultaneous editing of multiple alleles in polyploid genome (not diploid) was also known prior to the priority date of the claim (Sept 30, 2014) (see discussion below in Conclusion).
Applicant’s remarks regarding the prior rejection of claim 10 based on Doudna (US 2014/0068797 A1) have been considered and is withdrawn. However, claim 10 remains unpatentable in view of Liu (US 2015/0166980; PRO: 61/915,386 filed on Dec 12, 2013) as set forth in the current Office Action (see obviousness discussion above under 35 U.S.C. 103 rejection).
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant’s disclosure. Wang (Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew, Nat. Biotech. 2014, 32 (9), 947-951; DOI: 10.1038/nbt.2969; Published online: July 20, 2014) teaches simultaneously introduce mutations in three homoeoalleles in hexaploidy bread wheat by using zinc-finger nucleases (Abstract). Although Wang states “we have yet to cover CRISPR-Cas9 lines mutated in all three TaMLO alleles”, Wang also teaches using CRISPR-Cas9 system to mutate a single TaMLO allele and identifying four independent mutants, each carrying different mutations in the TaMLO-A1 allele (Fig. 1d, e; pg. 950, left-column, second paragraph). Thus, Wang demonstrates the state of the art for multi-allelic genome editing in polyploid cells, and teaches known strategies a person ordinary skill in the art would use to expand CRISPR-Cas9 editing to more than two alleles. One also would have had a reasonable expectation of success by following the method taught by Wang.
No claims are allowable.
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QIWEN SU-TOBON
Examiner
Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636