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 .
Application Status
This action is written in response to applicant’s correspondence received 04/27/2026. Claims 1-5, 7-8, and 16-20 are currently pending and are examined herein. Claims 6 and 9-15 have been cancelled. Claims 1-3, 8 and 16 are currently amended.
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
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Applicant’s submission, received on 04/27/2026, of a translation of the foreign priority document and statement that the translation is accurate is acknowledged. The translation of said application has been made of record in accordance with 37 CFR 1.55. Accordingly, the foreign priority claim has been perfected, and the claims benefit from the 12/23/2020 filing date of the foreign priority document. U.S. PGPUB 2020/0399659 to Onishi, which shares a common inventor with the instant application, is excepted as prior art under 102(b)(1)(A) because it was published after the foreign priority application, on 12/24/2020.
Additionally, Applicant’s clear statement in the remarks of 04/27/2026 (pp. 11-12) that the claimed invention and Onishi were owned by, or subject to an obligation of assignment to, the same person no later than the effective filing date of the claimed invention, is acknowledged. Accordingly, U.S. PGPUB 2020/0399659 to Onishi is excepted as prior under 102(b)(2)(C).
As Onishi does not qualify as prior art under 102(a)(1) or 102(a)(2), the rejections of claims 1-2. 4-7, 9-10, 12-15 and 17-20 over Onishi in view of Wacker, as set forth on pages 8-12 of the office action of 01/28/2026, as well as the rejections of claims 3, 8, 11, and 16 over Onishi and Wacker further in view of Mizutani as set forth on pages 12-14 (Id.), are hereby withdrawn.
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:
Determining the scope and contents of the prior art.
Ascertaining the differences between the prior art and the claims at issue.
Resolving the level of ordinary skill in the pertinent art.
Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1-5, 7-8, and 16-20 are rejected under 35 U.S.C. 103 as being unpatentable over PGPUB 2017/0121691 A1 to Wacker (hereinafter ‘Wacker’; of record, cited in a previous office action) in view of Mizutani (High-throughput plasmid construction using homologous recombination in yeast: its mechanisms and application to protein production for X-ray crystallography. Bioscience, Biotechnology, and Biochemistry, 2015, 79(1), 1–10.; of record, cited in a previous office action) and Chandran (Chandran et al. TREC-IN: gene knock-in genetic tool for genomes cloned in yeast. BMC Genomics 2014, 15:1180.).
Regarding claim 1:
Wacker teaches a method for producing a transformant which comprises introducing into a host cell a plasmid for transformation comprising a pair of homologous recombination sequences capable of causing homologous recombination, a counter selection marker, and a target-specific endonuclease:
[0006] In one aspect, provided herein are methods for inserting contiguous sequences of DNA, including large, contiguous sequences of DNA, into host cell genomes.
[0015] …wherein the donor plasmid comprises: (i) from 5′ to 3′: (1) the recognition sequence of the restriction endonuclease; (2) a first homology region of at least 0.5 kilobases (kb), (3) a heterologous insert DNA of at least 8 kb; and (4) a second homology region of at least 0.5 kb; and (ii) a counterselection marker.
Wacker teaches that the counter selection marker is used to repress growth of host cells that comprise the non-integrated plasmid (i.e., the host that retains the helper plasmid and where the donor DNA has not been excised; para [0091]).
Wacker teaches that when the endonuclease is expressed, it cleaves the endonuclease target sequences to excise, from the plasmid, the gene of interest and its associated homologous recombination sequences, which allows the gene of interest to be inserted into the genome via homologous recombination.
Wacker does not teach that the gene of interest is added to the host cell as linear nucleic acid molecules and that those molecules are incorporated into the helper plasmid in the host cell.
Wacker also does not teach the newly added limitation that the endonuclease gene is on the same plasmid as the gene of interest. Instead, Wacker teaches that the endonuclease is expressed from a second “helper” plasmid:
[0037] FIG. 1. Schematic map of the donor plasmid pDOC-C and relevant elements. ampR: DNA cassette encoding the gene conferring beta lactam resistance; sacB: expression cassette conferring sensitivity to sucrose; oriT: origin of replication; MCS: multiple cloning site; SceI: homing endonuclease restriction site for mobilizing the DNA from the donor plasmid; DNA insert: DNA stretch replacing the acceptor cell DNA between HR1 and HR2; HR1, HR2: homology regions 1 and 2, these are the DNA regions present in the host cell at which crossing over and homologous recombination occurs; selection cassette: selectable marker gene to screen for integrated clones, this cassette can be flanked by site specific homologous recombination sites which allow removal of the cassette; DNA of Interest: foreign DNA remaining in the target strain in place of the target strain DNA after selection cassette removal.
[0038] FIG. 2. Scheme of integration procedure. Different steps of the procedure are labeled by numbers in brackets. The target cell (rectangle) containing helper plasmid, donor plasmid (circles labeled pTKRED and pDOC) and chromosome (scribble) are grown (1) and induced with IPTG and arabinose (2) to induce expression of the homologous recombinase lambda red and the homing endonuclease SceI (scissors). The latter cuts the pDOC donor plasmid and thereby mobilizes the insert DNA resulting in the linearized insert DNA inside the cell (3). The linearized insert DNA is the optimal substrate for the lambda red recombinase which facilitates crossing over and homologous recombination at the homologous recombination sites HR1 and 2 (bold black bars). The enzymatic recombination is indicated by crossed over, thin black lines (4). The resulting strain contains the insert DNA in place of the DNA formerly present between HR1 and 2. .
Wacker does not teach the introduction of multiple linear nucleic acid fragments for plasmid construction in the host cell.
Mizutani teaches high-throughput plasmid construction in yeast (Title). Mizutani notes that, “Homologous recombination in S. cerevisiae can connect multiple fragments” (p. 1 right), and that, “plasmid construction can be done by homologous recombination in S. cerevisiae itself” (p. 4). The method encompasses, “Transformation of S. cerevisiae using three DNA fragments, an E. coli plasmid (cut at multiple cloning sites), a target gene (with homologous sequence of the multiple cloning site at both ends), and a helper plasmid (cut by two restriction enzymes),” and “enables construction of an expression plasmid by homologous recombination in yeast cells” (p. 5). Fig. 3B shows this approach, in which nucleic acid fragments are joined to each other and inserted into the plasmid via homologous recombination, using two sets of homologous recombination sequences. Lastly, Mizutani notes that this method, “This method has various advantages over the traditional method, including flexibility in the position of DNA insertion and ease of manipulation.” (Abstract). In summary, Mizutani teaches administering multiple linear DNA fragments comprising the gene of interest to the host cell, along with a plasmid comprising the other elements, and that the gene of interest is inserted into the plasmid in the host cell itself.
Chandran teaches a plasmid for transformation wherein the plasmid comprises restriction endonuclease target sites, homologous sequences for homologous recombination into the host genome, counterselection markers, a gene of interest, and a sequence encoding the necessary restriction endonuclease all on the same plasmid, as shown in Figure 1:
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In summary, Chandran shows that both the gene of interest and the endonuclease can be combined on a single plasmid, in cis, in an analogous SceI-based, counterselective, knock-in system, this system “enabling seamless gene knock-in with high efficiency” (Abstract).
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have combined the method for transformation comprising the helper plasmid with counter selection, as taught by Wacker, with the method of combining those elements onto a single plasmid, as taught by Chandran. The ordinary artisan would have had a reasonable expectation of success based on Chandran’s teachings that a single-plasmid construct in a closely analogous system enabled seamless gene knock-in with high efficiency. The ordinary artisan would further have been motivated to construct the plasmid in the host cell, as taught by Mizutani, based on Mizutani’s teaching that the method had various advantages over the traditional method, including flexibility in the position of DNA insertion and ease of manipulation.
Regarding claim 2, it is not entirely clear what is meant by “a pair of endonuclease target sequences provided on the other side of a site into which the linear nucleic acid fragment for genome-introduction is incorporated via the homologous recombination sequences”. The claimed structure is interpreted as requiring a pair of homologous recombination sequences around the gene insertion site that allow homologous recombination between the plasmid and the linear donor DNA, and that the endonuclease target sequences flank the homologous sequences. Wacker teaches those elements, as discussed above and shown in FIG. 1, which shows two SceI sites flanking a multiple cloning site (MCS) in the plasmid and then shows corresponding homology regions (HR1 and HR2) in both the donor nucleic acid and the plasmid. Claim 3 is interpreted as describing the linear DNA, which has the corresponding pair of homologous sequences flanking the donor DNA sequence and endonuclease target sequences which allow the donor DNA to be excised from the linear DNA and inserted into the plasmid, which is also shown in FIG. 1.
Regarding claims 4 and 5, Wacker and Chandran both teach the SceI-site specific SceI homing endonuclease is the endonuclease used, and teach that it is operably linked to a promoter, i.e., expressible (see above).
Regarding claim 7, Wacker teaches that the promoter is inducible:
[0126] The ability to control helper plasmid function is important to reduce recombination activity to a limited time during cell growth, as unwanted side reactions may occur if continuous recombination is promoted. Thus, inducible promoters and inducers may be utilized to ensure that certain components of the helper plasmids are expressed only when desired. Exemplary inducible promoters include, without limitation, the araBAD promoter system (inducible by the presence of arabinose) and the tac promoter (inducible by the presence of IPTG). Table 7 provides a further list of inducible components that can be used in accordance with the methods described herein.
Regarding claim 8, the claim is interpreted as describing a system wherein the multiple linear nucleic acid fragments comprise homologous regions that allow end to end homologous recombination with one another during plasmid construction. To that effect, Mizutani’s method teaches that multiple linear donor DNA molecules may be used and may have homology regions at their 5’ and 3’ ends, depending on their intended position in the plasmid, allowing multiple fragments to be joined (see Fig. 3B).
Regarding claims 16-20, these claims are merely drawn to the helper plasmid with the same structural features described in the method claims, and are rendered obvious by the above references for the reasons already described.
Response to Arguments
Applicant’s arguments with respect to claim(s) 1-20 have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument. The amendment to claim 1 which recites that the helper plasmid comprises an endonuclease gene was not previously present. The new references are being used in view of the amendment to claim 1, because the addition of the endonuclease in the helper plasmid alters the interpretation of the claim such that the endonuclease must be present on the helper plasmid prior to incorporation of the donor sequences, which was not previously required by the claim.
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 1-5, 7-8 and 16-20 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-8 of U.S. Patent No. 12,338,454 B2 in view of Wacker (cited above) and Mizutani (cited above).
Regarding claims 1-5, 7, and 17-20, patented claims 1-3 and 5-7 recite methods of producing a transformed yeast host comprising introducing one or more linear nucleic acid fragments and a helper plasmid comprising a pair of homologous recombination sequences for incorporation of a gene of interest into a genome, wherein the homologous recombination are sequences flanked by a pair of endonuclease target sequences, and the plasmid further comprises a homing endonuclease that specifically cleaves the endonuclease target sequences. While the patented claims do not recite that the plasmid has a counter selection marker that is used to induce death in cells comprising the plasmid, that limitation is rendered obvious by Wacker, as discussed above.
Regarding claims 8 and 16, patented claims 4 and 8 recite the linear nucleic acid fragments with the same homologous recombination sequences at their 5’ and 3’ termini, which permit homologous recombination between the fragments. These limitations are taught by Mizutani for the same reasons already discussed above.
Claims 1-5, 7-8 and 16-20 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 and 3-9 of copending Application No. 18/258,909 in view of Wacker and Mizutani (cited above).
Regarding claims 1-2, 4-5 and 17-21, copending claims 7-10 recite methods of producing a transformant comprising introducing the plasmid of copending claim 6. This plasmid comprises the same homologous sequences, endonuclease sites, counter selection marker, and homing endonuclease gene with an inducible promoter as recited in the instant claims.
The copending and instant claims differ in that the copending claims do not recite a plurality of linear nucleic acid molecules comprising the endonuclease sites and homologous sequences as recited in instant claims 1, 3, and 8 . However, those elements are rendered obvious by Mizutani, as already discussed above.
This is a provisional nonstatutory double patenting rejection.
Claims 1-5, 7-8 and 16-20 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 and 3-9 of copending Application No. 18/255,151 in view of Wacker and Mizutani (cited above).
Regarding claims 1-2, 4-5 and 17-21, copending claims 7-10 recite methods of producing a transformant comprising introducing the plasmid of copending claim 6. This plasmid comprises the same homologous sequences, endonuclease sites, counter selection marker, and homing endonuclease gene with an inducible promoter as recited in the instant claims.
The copending and instant claims differ in that the copending claims do not recite a plurality of linear nucleic acid molecules comprising the endonuclease sites and homologous sequences as recited in instant claims 1, 3, and 8 . However, those elements are rendered obvious by Mizutani, as already discussed above.
This is a provisional nonstatutory double patenting rejection.
Response to Arguments
Applicant's arguments filed 04/27/2026 have been fully considered but they are not persuasive for the following reasons:
Applicant argues that the patented and pending cases are all allowable over Mizutani because the patented and copending applications fail to teach the functional limitations recited in the amended claims (i.e., cleavage of the endonuclease target sequences with the encoded endonuclease, excising the gene of interest together with its flanking homology regions from the plasmid, and inserting the gene of interest into the genome by homologous recombination). Respectfully, this is not persuasive because, as described above, the relevant structures of the plasmid used are the same as those instantly claimed, and are thus inherently capable of the same functions.
Conclusion
No claim is allowed at this time.
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 AMANDA M ZAHORIK whose telephone number is (703)756-1433. The examiner can normally be reached M-F 8:00-16:00 EST.
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/AMANDA M ZAHORIK/Examiner, Art Unit 1636
/BRIAN WHITEMAN/Primary Examiner, Art Unit 1636
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