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 .
Continued Examination Under 37 CFR 1.114
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 September 24, 2025 has been entered.
Application Status
Applicant’s amendments filed September 24, 2025, amending claim 1 is acknowledged. Claim 1 is pending and under examination.
The amendment to claim 1 overcomes the §103 rejection over Santos in view of Thomson. Santos uses lambda-RED recombineering to integrate the initial Lox-site landing pad into the bacterial genome, and as such, the lox sites are not flanked by inverted repeats.
The substitute Drawings were partially sufficient for overcoming the objections. However, the number text in FIGs 19C, 19D and 21C is still not legible. See objection below.
Any other 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.
Drawings
The drawings are objected to because the numbers and letters of FIGS. 19C, 19D and 21C are not sufficient to provide satisfactory reproduction characteristics. 37 CFR 1.84(l) states that “all drawings must be made by a process which will give them satisfactory reproduction characteristics. Every line, number, and letter must be durable, clean, black (except for color drawings), sufficiently dense and dark, and uniformly thick and well-defined.” In the instant case, the number values in each of the graphs in the above figures is very small, light grey, of poor resolution, and otherwise not sufficiently dense and dark to permit satisfactory reproduction characteristics. Appropriate correction is required.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Rejections - 35 USC § 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.
Claim 1 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention. This is a new rejection necessitate by amendment.
Claim 1 recites (a)(i) a first lox site and a second lox site of a landing pad that is in a bacterial host cell. Claim 1 also recites (a)(ii) a first lox site and a second lox site flanking a gene of interest that is in a plasmid. Claim 1 then recites “wherein the first and second lox sites are both together flanked by a first inverted repeat and a second inverted repeat” in lines 9-10. It is not clear if “the first and second lox sites” are referring to the lox sites in the bacterial cell, the lox sites in the plasmid or both. As such, the wherein clause in lines 9-10 lacks clear antecedent basis. Based on the figures, the inverted repeats (IR) can be flanking lox sites in a plasmid or flanking lox sites when integrated in the genome of bacteria (see e.g., Fig 3B). Thus, the Specification does not provide clarity about which set of lox sites the IRs are to flank.
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.
Claim 1 is rejected under 35 U.S.C. 103 as being unpatentable over Santos (Santos et al, Nature Communications (2013), 4:2503, DOI: 10.1038/ncomms3503; cited #8 on IDS filed 5/4/2023) in view of Thomson (Thomson and Blechl, "Recombinase technology for precise genome engineering." Advances in new technology for targeted modification of plant genomes (2015): 113-144) and Enyeart (Enyeart, Peter James, (2014) Studies in Bacterial Genome Engineering and Its Applications (Doctoral Dissertation)). This is a new rejection necessitate by amendment.
Claim 1 is indefinite for the reason described above in paragraph 11. For the purposes of compact prosecution, “the first and second low sites [that] are both together flanked by a first inverted repeat and a second inverted repeat” are interpreted to the be the lox sites that are already present in the bacterial host cell.
Santos teaches Recombinase-assisted genome engineering (RAGE) is a strategy that facilitates stable instalment (i.e., a method for integrating a gene of interest into a bacterial host genome) by recombinase-mediated cassette exchange (RCME) (Fig 2, legend). Santos teaches RAGE results in integration of ALG3.4 (i.e., a gene of interest) into the chromosome of a bacterial cell (Fig 2a, step 2). Regarding (a) (i), Santos teaches the method starts by producing (i.e., providing) a bacterial cell that comprises the cat gene (i.e., a first selectable marker) flanked by loxP and lox5171 (i.e., first and second lox sites) (Fig. 2a, Step1). Santos teaches the lox site landing pad was integrated into the genome using lambda-RED recombination, which utilized homologous sequences between insert and target for integration (page 8, ¶12). Regarding (a) (ii), Santos teaches providing (ii) pALG3.4 (i.e., a plasmid) comprising ALG3.4 (i.e., a gene of interest) and kan (i.e., a second selection marker) tougher flanked on either side by a loxP site and a lox5171 site (i.e., the first and second lox sites of the plasmid are different) (Fig. 2a, Step2). Regarding (b), Santos teaches the bacterial cell comprising the landing pad is transformed with pALG3.4 (i.e., the first plasmid is introduced into the bacterial cell) and an additional plasmid, pJW168, which comprises the coding sequence for Cre recombinase (Fig. 2a, Step2). Santos teaches that Cre expression is induced by IPTG (Fig 2a, legend). Santos teaches that upon Cre expression, ALG3.4 is integrated into the chromosome by recombination between the loxP sites on the plasmid and landing pad, and between the lox5171 site on the plasmid and landing pad (Fig 2a, step 2). Santos also teaches that chromosomal positioning can influence gene expression (page 5, ¶3). Santos teaches testing loci-dependent transcriptional variants by integrating the ALG3.4 pathway at 5 different positions, which affected growth rate of the bacteria (page 5, ¶3; Fig 3).
Santos does not teach the Cre Recombinase coding sequence is provided on the same plasmid as the gene of interest or integrated in the bacterial host genome. Santos does not teach the lox sites of the integrated landing pad flanked by inverted repeat sequences.
Thomson reviews recombinase technology for precise genome engineering (Title, Abstract). Thomson teaches that during Recombinase-Mediated Cassette Exchange (RMCE) technology combines the integration and excision reactions into a strategy to replace transgenes in situ (page 126, ¶3). Thomson teaches that during RMCE, a selectable marker gene flanked by inverted recognition sites the genome of an organism is excised and replaced by a gene of interest encoded by a plasmid and flanked by the matching inverted heterologous recognition sites using a recombinase such as Cre (Fig 7.4, legend). Thomson teaches that during RMCE the Recombinase can be provided in cis, encoded on the plasmid comprising the gene of interest and outside the recognition sites (page 126, ¶3).
Enyeart teaches a selectable marker flanked by two lox sites, which are then flanked by a first and second inverted repeat sequence (IR) (Fig 4.1). Enyeart teaches integrating the IR-lox-Marker-lox-IR into the E. coli genome using a mariner-type transposase (Fig 4.1; pages 103-104). Enyeart teaches using mariner-type transposons allows library creation since the transposon cassette is integrated randomly (Table 1.4 and 104-105). Enyeart teaches the mariner type transposons have an exceptionally broad host range, have very little insertion bias, do not rely on host factors for transposition, and are very efficient at transposition (page 13, ¶1).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have modified Santo’s RAGE method by 1) integrating the lox site landing pad using transposon-based integration which results in flanking inverted repeats, and 2) placing the Cre recombinase coding sequence on the pALG3.4 plasmid that also contains the gene of interest. It would have amounted to 1) substituting one known method for integrating a sequence of interest for another known method, and 2) the simple rearrangement of parts in the Santo’s RAGE genetic engineering machinery.
Regarding the use of transposon-based insertion of the landing pad, the skilled artisan would have predicted the Santo’s landing pad could be modified by including IRs and inserted via mariner transposition because Enyeart teaches that the mariner transposon has a wide host range, including in many bacteria and E. coli, and is very efficient. The skilled artisan would have been motivated use random transposition in order to make a library of cells in which the landing pad was integrated in order to test the best genomic location for expression of the alginate operon. Santos teaches that integration site can affect growth rate and transgene expression. By integrating the landing pad randomly, thereby creating a library of integration sites, the skilled artisan could screen for the optimal site of ALG3.4 pathway integration in a single step.
Regarding the placement of the Cre coding sequence, the skilled artisan would have expected that the Cre recombinase could be included in the pALG3.4 plasmid because 1) Santos demonstrates that the Cre coding sequence can be cloned and introduced into bacteria on a plasmid, and 2) Thompson teaches that the Cre coding sequence can be provided on the same plasmid as the cassette that will be integrated into the genome upon Cre expression. MPEP 2144.04.VI.C explains that rearrangement of prior art elements is prima facie obvious when the placement of parts – in this case Cre Recombinase gene on a different plasmid – does not change the operation of the device. Thompson teaches that the Cre coding sequence can be placed in either trans or cis for RMCE to function, and, as such, the prior art makes clear that the placement of Cre does not change how the RMCE machinery functions.
Response to Arguments
Applicant argues that the added limitation overcomes the previous obviousness rejection because neither Santos or Thomson teach the lox sites flanked by inverted repeats (Remarks, page 6). This argument has been fully considered and is persuasive. The previous §103 rejection over Santos in view of Thomson has been withdrawn. However, the art recognizes different means to integrate a lox site landing pad into a bacterial genome, and it was obvious to use the means taught in Enyeart for the reasons recited in the rejection above.
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.
Claim 1 is rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-10 of U.S. Patent No. 11674145. This is a maintained rejection. Although the claims at issue are not identical, they are not patentably distinct from each other because:
Patented claim 1 recites “A method for integrating a gene of interest into a chromosome of a bacterial host cell, the method comprising: (a) providing a bacterial donor cell comprising a donor plasmid, the donor plasmid comprising a transposase coding sequence and a donor sequence, wherein the donor sequence is flanked by inverted repeats recognized by the transposase wherein the donor sequence comprises a first selectable marker coding sequence and a first and a second lox site, and wherein the first selectable marker coding sequence is flanked by the first and the second lox sites; (b) introducing the donor plasmid into a bacterial host cell . . . wherein upon introduction of the donor plasmid into the bacterial host cell . . . the donor sequence is integrated into a chromosome of the bacterial host cell.” Components (a) and (b) of patented claim 1 anticipate step (a) (i) of instant claim 1. Patented claim 1 also recites “(c) providing a second plasmid comprising the gene of interest and a second selectable marker coding sequence, wherein the gene of interest and the second selectable marker are both flanked by a first lox site of the second plasmid and a second lox site of the second plasmid, wherein the first and the second lox sites of the second plasmid are different. Component (c) of patented claim 1 anticipates step (a) (ii) of instant claim 1. Patented claim 1 recites “(d) introducing the second plasmid into the bacterial host cell, wherein the donor sequence comprises a Cre recombinase coding sequence, and wherein upon introduction of the second plasmid into the bacterial host cell, Cre recombinase is expressed from the Cre recombinase coding sequence, and the gene of interest is integrated into the chromosome of the bacterial host cell by the Cre recombinase through recombinase-mediated cassette exchange (RMCE) between the first lox site of the second plasmid and the first lox site of the donor sequence, and between the second lox site of the second plasmid and the second lox site of the donor sequence.” Because instant claim 1 allows the Cre Recombinase to already be present in the host cell or originally comprised on the first plasmid, Component (d) of patented claim 1 anticipates step (b) of instant claim 1.
Response to Arguments
Applicant states that a Terminal Disclaimer will be filed upon determination of an allowable claim (Remarks, page 6, last ¶). Because a terminal disclaimer has not been filed, the nonstatutory double patenting rejection is maintained.
Conclusion
No claims are allowable.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CATHERINE KONOPKA whose telephone number is (571)272-0330. The examiner can normally be reached Mon - Fri 7- 4.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Ram Shukla can be reached at (571)272-0735. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/CATHERINE KONOPKA/Examiner, Art Unit 1635