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 .
Election/Restrictions
Applicant’s election without traverse of Invention I in the reply filed on 05 January 2026 is acknowledged. For clarity, the election of species restriction is not between Group A and Group B, the restriction requires the election of one enzyme from Group A and one enzyme for Group B. Applicant’s election of the species of Group A first DNA enzyme is a Cre recombinase and the species of Group B second DNA enzyme is a Dre recombinase in the reply filed on 05 January 2026 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claims 20-24 and 26 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species; and claim 36 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention.
Claims 1-19, 25, 27-35 are pending and being examined on the merits.
Priority
The current application is a 371 PCT of EP2021/058952 filed 04/06/2021. Acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. 119 (a)-(d). based on Application No. GB2005180.1, filed on 04/08/2020. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Information Disclosure Statement
The information disclosure statement filed 10/05/2022 and 05/21/2025 has been acknowledged.
Claim Objections
Claims 1, 3, 6, and 11 are objected to because of the following informalities:
Claim 1 is missing step iii.
Claim 3 refers to a missing step (step iii).
Claim 11 recites steps in part (A) that seem to be a continuation of the steps from claim 1. These steps are numerated by capital roman numerals. It would be remedial to numerate these steps with a lower-case roman numeral for consistency.
Claim 11 recites a method compositing (A)VII and (A)IX. Step A(VIII) is missing. It would be remedial to renumber the claims in numerical number.
Claim 11 recites “(A)II to (A)IX”; however the claim only recites (A)VII to (A)IX. It would be remedial to add (A)II to (A)VI to the claims in part A or refer the steps ii to vi of claim 1.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
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.
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 3-4, 6-7, 8-9, 11, and 13 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Regarding claim 3 and 25, the phrase "such as" renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Claim 6 recites “isolated in step vi” and depends from claims 3, 2, and 1. The cell isolated in step v. Step vi comprises excising a nucleic acid sequence flanked by the nucleic acid sequences E1 and E2 from the pre-defined genomic location and does not require a cell isolation. There is insufficient antecedent basis for this limitation in the claim.
Claim 8 recites “ the excised nucleic acid sequence” and depends from claim 1. The nucleic acid is excised in claim 2. There is insufficient antecedent basis for this limitation in the claim.
Claim 11 recites the method comprising A, B, C. It is unclear if the method comprises A, B, and C; or if the method comprises A, B, or C.
Claim 11 recites “isolated in step vii” and depends from claim 10 and claim 1. The cell isolated in step vii is in claim 6. There is insufficient antecedent basis for this limitation in the claim.
Claim 11 recites “the donor vector of step II)” and depends from claim 1 and 10. Claim 1 comprises of a ‘step ii’ not step II. Therefore, there is insufficient antecedent basis for this limitation in the claim.
Claim 11 recites “(A)II to (A)IX”. The claim set does not mention (A)II to (A)VII. There is insufficient antecedent basis for (A)II to (A)VII in the claim.
Those claims identified in the statement of rejection but not explicitly referenced in the rejection are also rejected for depending from a rejected claim but failing to remedy the indefiniteness therein.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 18-19 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 18 is dependent on claim 14 which requires that I1 and I2 to comprise two recombination sites. Since claim 18 requires I1 and I2 to comprise a single recombinase recognition site, the claim fails to include all the limitation of the claim upon which it depends. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
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.
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 1-4, 6, 8-14, 18-19, 25, 27-31, and 33-35 are rejected under 35 U.S.C. 103 as being unpatentable over Akbudak (Akbudak et al. Mol Biotechnol (2011) 49:82–89) in view of Obayashi (Obayashi et al. Journal of Bioscience and Bioengineering VOL. 113 No. 3, 381–388, 2012).
Regarding claim 1, 14, and 34-35, Akbudak teaches that Site-specific recombination systems, such as FLP–FRT and Cre–lox, carry out precise recombination reactions on their respective targets in plant cells (regarding claim 34) [abstract]. Akbudak molecular strategy for marker-free site-specific gene integration that consists of site-specific integration (SSI) of a transgene cassette followed by site-specific excision of the marker genes [pg. 84, see Molecular strategy]. Akbudak teaches a target construct, pAKT, that comprises a recognition site for a first DNA enzyme (lox76); a recognition site for a second DNA enzyme (FRT site); and a promotor nucleic acid sequence (UBI promoter) [pg. 84, see Molecular strategy; Fig. 1]. Akbudak teaches a donor vector comprising a nucleic acid sequence (lox75), a nucleic acid sequence of interest, GOI (GUS), a nucleic acid sequence comprising a recognition site for said second DNA enzyme (FRT); a nucleic acid sequence encoding a first selection marker (NPT) [pg. 84, see Molecular strategy; Fig. 1]. Akbudak teaches contacting the donor with the target construct in the presence of a first DNA enzyme (CRE) to enable recombination between the donor vector and the target construct [pg. 84, see Molecular strategy; Fig. 1]. Akbudak teaches selection and isolation of the cell lines of the transgenic clones by using the NPT selectable marker gene [pg. 84, col. 2, para 2].
Regarding claim 2, Akbudak teaches using a second enzyme (FLP recombinase, FLPe) to excise the nucleic acid sequences flanked by the FLP sites [84, col. 2, para 2 – pg. 85, col. 1, para 2].
Regarding claims 3-4, Akbudak teaches the excision of the nucleic acid sequence after selection and isolation [pg. 84, col. 2, para 2].
Regarding claim 6, Akbudak teaches the isolation of embryos derived from upon transient expression of FLPe [pg. 84, col. 2, para 2; Fig. 2].
Regarding claim 8, Akbudak teaches that the GOI is the b-glucuronidase (GUS) gene [pg. 84, col. 2, para 2].
Regarding claim 9 and 28, Akbudak teaches that the excision fragment lacks the GOI and comprised the NPT selection marker[Fig. 1].
Regarding claim 10, Akbudak teaches that the donor vector comprises a nucleic acid sequence comprising a recognition site for a first DNA enzyme (loxP), and a promotor nucleic acid sequence (UBI) [Fig. 1].
Regarding claims 18-19, Akbudak teaches the use of Lox76 and Lox75.
Regarding claim 25, Akbudak teaches FRT E1 and E2 sites [pg. 84, see Molecular strategy; Fig. 1].
Regarding claim 27, Akbudak teaches that the UBI promoter is functionally fused to the 5' -part of a split intron [Fig. 1d].
Regarding claims 30 and 31, Akbudak teaches that the Cre and FLP recombinases were provided in the form of a plasmid [pg. 84; see Plasmid Constructs].
Regarding claim 33, Akbudak teaches that the Cre enzyme is in the pAK7 target construct [pg. 84; see Plasmid Constructs].
Akbudak do not teach targeted integration into a genomic location of a eukaryotic cell.
Obayashi teaches using site-specific gene recombination systems, such as Cre/loxP, for genetic modification of cells and organisms [abstract]. Obayashi teaches an accumulative gene integration system (AGIS), in which target gene cassettes could be repeatedly integrated into a pre-determined site on a plasmid or cellular genome by recombinase-mediated cassette exchange (RMCE), using Cre and mutated loxPs [abstract; pg. 384, col. 2, last paragraph – pg. 385, col. 1, last paragraph]. Obayashi teaches that the integration site loxP1 can be introduced into the cell genome [pg. 384, col. 2, last paragraph – pg. 385, col. 1, para 1].
It would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to that the method of Akbudak could be used for cellular genome site-specific recombination upon the integration of loxP sites into the genome of the cells given the teachings of Obayashi. The combination of prior art elements according to known methods to yield predictable results supports can support a conclusion of obviousness. See MPEP 2143(I). One of ordinary skill in the art would have a reasonable expectation of success since both Akbudak and Obayashi teaches using site-specific gene recombination using Cre/loxP system.
Regarding claim 11 and 12, Akbudak do not teach performing a sequential targeted integration of n additional donor vectors into the pre-defined genomic location of the eukaryotic cell. Obayashi teaches accumulative gene integration efficiency using donor vectors comprising loxP sites, a GOI, and a selection marker; introducing the donor vector in the cell in the presence of the CRE enzyme to facilitate recombination, and selecting cells comprising the donor vector integrated in the genome [pg. 382, col. 2, para 7 – pg. 384, col. 1, para 3; pg. 384, col. 2, last paragraph – pg. 385, col. 1, para 5]. It would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to include an FRT nucleic acid sequence in the additional donors for the advantage of genome excision of the marker to facilitate marker-free SSI, and isolating the cells before and after excision as taught by Akbudak. The combination of prior art elements according to known methods to yield predictable results supports can support a conclusion of obviousness. See MPEP 2143(I). One of ordinary skill in the art would have a reasonable expectation of success since both Akbudak and Obayashi teaches using site-specific gene recombination using Cre/loxP system.
Regarding claim 13, the teachings of Akbudak are discussed above as applied to claims 10. Akbudak do not teach that the excised sequence comprises the expression cassette containing a gene encoding protein of interest. Akbudak teaches that the GOI remains integrated after the sequence has been excised. It would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to design the donor GOI in not flanked between the sites recognized by the second DNA enzyme. One of ordinary skill would be motivated to make this modification for the advantage of testing site specific recombination by assaying cells that do not contain the GOI, i.e. negative selection.
Regarding claim 29, Akbudak does not teach wherein said first selection marker is a fluorescent protein. Obayashi teaches selecting EGFP positive clones [pg. 385, col. 1, para 2-3; Figs. 1-3]. It would have been obvious to one ordinary skilled in the art before the effective filing date to substitute the NPT gene for EGFP as this would have been a simple substitution of one known selection marker for another known to be used in site-specific recombination. The combination of prior art elements according to known methods to yield predictable results supports can support a conclusion of obviousness. See MPEP 2143(I). One of ordinary skill in the art would have a reasonable expectation of success since both Akbudak and Obayashi teaches using site-specific gene recombination using Cre/loxP system.
Claims 5, 7, and 32 are rejected under 35 U.S.C. 103 as being unpatentable over Akbudak (Akbudak et al. Mol Biotechnol (2011) 49:82–89) in view of Obayashi (Obayashi et al. Journal of Bioscience and Bioengineering VOL. 113 No. 3, 381–388, 2012) as applied to claim 1-4, and further in view of Wang (Wang et al. Plant Cell Rep (2011) 30:267–285).
The teaching of Akbudak and Obayashi are discussed above as applied to claims 1-4 and similarly apply to claims 5, 7, and 32.
Akbudak and Obayashi do not teach where the donor vector comprises an expression cassette encoding a second selection marker and wherein the cell isolated in step v) additionally has been selected based on its non-expression of the second selection marker.
Wang reviews multiple uses of site-specific recombination and their application toward plant genomic engineering [abstract]. Wang teaches alternative strategies for the combined use of multiple site-specific recombinase systems for genome engineering to precisely insert transgenes into a pre-determined locus, and removal of unwanted selectable marker genes (i.e., antibiotic resistant genes) [abstract]. Wang teaches that the use of a positive/negative selectable marker cassette allow direct selection of plants that have undergone an excision event [pg. 276, col. 2, para 1; pg. 280, col. 1]. Wang teaches that negative selection prior to molecular characterization would save time and effort invested in screening for candidate lines [pg. 276, col. 2, para 1]. Wang teaches that scientist have demonstrated targeting efficiencies approaching 100%, from a preexisting chromosomal site, through the use of a negative selection marker exchange vector. They have reported that the technique is powerful enough to specifically integrate into a targeted location without the use of any selection and still obtain an integration frequency of 1% [pg. 278. col. 1]. Wang teaches that a positive/negative selectable marker scheme has been very effective for replacement strategies and makes the site-specific excision event completely dependent on precise integration [pg. 279, col. 2, para 1]. Wang teaches When the ‘EXCH’ vector is site-specifically inserted into the chromosomal ‘TAG’ cassette, the recognition sites required for excision will align stimulating the removal of the positive/negative selectable marker gene [pg. 279, col. 2, para 1]. Wang teaches that this technique is meant to eliminate undesirable random genomic integration, thereby reducing background and screening time [pg. 279, col. 2, para 1]. Wang teaches that the presence of the negative marker gene (e.g. codA) eliminates cells that experienced site-specific integration of the ‘EXCH’ cassette without subsequent excision, and those cells that had only random integration events [pg. 279, col. 2, para 1].
It would have been obvious to one ordinary skilled in the art before the effective filing date to modify the donor construct as taught and suggested by Akbudak and Obayashi to further comprise a second selection marker such as a negative selection antibiotic resistance genes/second DNA enzyme. One of ordinary skill would be motivated to make the modification for the advantage of eliminating cells that experienced site-specific integration without subsequent excision, and those cells that had only random integration events. The combination of prior art elements according to known methods to yield predictable results supports can support a conclusion of obviousness. See MPEP 2143(I). One of ordinary skill in the art would have a reasonable expectation of success since both Akbudak, Obayashi, and Wang teaches site-specific gene recombination using recombinase systems.
Claims 15-17 are rejected under 35 U.S.C. 103 as being unpatentable over -Akbudak (Akbudak et al. Mol Biotechnol (2011) 49:82–89) in view of Obayashi (Obayashi et al. Journal of Bioscience and Bioengineering VOL. 113 No. 3, 381–388, 2012) as applied to claim 1, and further in view of Suzuki (Suzuki et al. Nucleic Acids Research, 2011, Vol. 39, No. 8 e49).
The teaching of Akbudak and Obayashi are discussed above as applied to claims 1 and similarly apply to claims 15-17.
Akbudak and Obayashi do not teach where (a) I1 comprises two recombinase recognition site variants I1a and I1b; and (b) I2 comprises two recombinase recognition site variants I2a and I2b; and (c) I1a is capable of recombination with I2a and I1b is capable of recombination with I2b in the presence of said first DNA enzyme; wherein I1a is identical to I2a and I1b is identical to I2b; and wherein they are selected from loxP and the enzyme is Cre recombinase.
Suzuki teach eenome engineering of mouse ES cells using modified Cre/LoxP systems [pg. 10, col. 1, para 2]. Suzuki teach the use of two lox recombinase recognition site variants, loxP and lox2272 to knock in a Lin28 allele [pg. 10, col. 1, para 2; Fig. Fig. 3A].
It would have been obvious to one ordinary skilled in the art before the effective filing date to modify the method as taught and suggested by Akbudak and Obayashi to comprise two lox recombinase recognition site variants in both the genome and donor vector where each variant in the genome is identical to the variant in the vector. This modification would amount to an substitution one known method of site-specific recombination know to result in genome knock in for another. The combination of prior art elements according to known methods to yield predictable results supports can support a conclusion of obviousness. See MPEP 2143(I). One of ordinary skill in the art would have a reasonable expectation of success since both Akbudak, Obayashi, and Suzuki teaches site-specific gene recombination using Cre/Lox recombinase systems.
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 1-19, 25, 27-35 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-5 of copending Application No. 17995478 (reference application) in view of Akbudak (Akbudak et al. Mol Biotechnol (2011) 49:82–89), Obayashi (Obayashi et al. Journal of Bioscience and Bioengineering VOL. 113 No. 3, 381–388, 2012), Wang (Wang et al. Plant Cell Rep (2011) 30:267–285) and Suzuki (Suzuki et al. Nucleic Acids Research, 2011, Vol. 39, No. 8 e49)
Although the claims at issue are not identical, they are not patentably distinct from each other because the claim1 of the reference application anticipates the claim 1 of the current application. For additional limitations of the instant claims, see the additional teachings of the patented claims. To the extent that there are limitations that are not provided for by the patented claims, the teachings of Akbudak, Obayashi, Wang, Suzuki are discussed above. It would have been obvious to have modified the subject matter of the patented claims to arrive at the subject matter of the instant claims for substantially the same reasons as discussed above in view of the teachings of these references.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Conclusion
No claims allowed.
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/TIFFANY NICOLE GROOMS/Examiner, Art Unit 1637
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