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Application Status and Withdrawn Rejections
Applicant’s amendments filed May 6, 2026, amending claim 1 and adding new claims 2-10 is acknowledged. Claims 1-10 are pending and under examination.
The amendment to claim 1 overcomes the §103 rejection over Santos in view of Thomson and Enyeart since the rejection did not address newly added step (a) of amended claim 1. The Terminal Disclaimer (see below) overcomes the nonstatutory double patenting rejection.
The substitute Drawings are sufficient for overcoming the objection in the previous office action.
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.
Terminal Disclaimer
The terminal disclaimer filed on May 6, 2026 disclaiming the terminal portion of any patent granted on this application which would extend beyond the expiration date of US Patent 11,674,145 has been approved.
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 8 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 necessitated by amendment.
Claim 8 recites “the method of claim 6, where the protein that promotes sequence-specific gene transcription…” Claims 6 and 1 do not recite a protein that promotes sequence-specific gene transcription. As such, the recitation of “the protein that promotes…” lacks clear antecedent basis.
For the purpose of compact prosecution, claim 8 will be interpreted as depending from claim 7 which does recite a protein that promotes sequence-specific gene transcription.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 3, 5-6 and 9-10 are rejected under 35 U.S.C. 103 as being unpatentable over Santos (Santos et al, Nature Communications (2013), 4:2503, DOI: 10.1038/ncomms3503; of record) 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; of record) and Enyeart (Enyeart, Peter James, (2014) Studies in Bacterial Genome Engineering and Its Applications (Doctoral Dissertation); of record). This is a new rejection necessitate by amendment.
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 step (a), Santos teaches introducing a first plasmid encoding a lambda-RED recombinase coding sequence and a landing pad into a bacterial host cell (Fig 2a, step 1). Santos teaches the landing pad is integrated into the bacterial host chromosome upon expression of the lambda-RED recombinase gene from the plasmid (Fig 2a, step 1). Santos teaches the landing pad comprises a cat selectable marker (rectangle) flanked by LoxP and lox5171 sites (arrowheads) that are in turn flanked by sequences recognized by the lambda-RED recombinase (squares) (Fig 2a, step 1). 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 step (b), Santos teaches providing (ii) pALG3.4 (i.e., a second plasmid) comprising ALG3.4 (i.e., a gene of interest) and kan (i.e., a second selection marker) that are together 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 step (c), Santos teaches the bacterial cell comprising the landing pad is transformed with pALG3.4 (i.e., the second 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 (i.e., via RCME) (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 using a transposon for initial integration of the landing pad into the bacterial host genome such that the lox sites of the integrated landing pad flanked by inverted repeat sequences and the
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.
Regarding claim 3, Santos teaches the bacterial host cell is an E. coli cell (i.e., a proteobacteria). Enyeart demonstrates successful use of Mariner transposons in E. coli strains (Chapter 4).
Regarding claims 5-6, as indicated above for claim 1, the first and second lox sites on the landing pad are identical to the first and second lox sites, respectively, on the second plasmid. Additionally, the first lox site and the second lox sites are respectively loxP and lox5171 (i.e., heterospecific to each other).
Regarding claims 9-10, Santos teaches the gene of interest on the second plasmid flanked by the loxP and lox5171 is ALG3.4, a 34 kb fragment encoding the alginate metabolic pathway (i.e., a coding sequence for one or more enzymes) (page 3, ¶2).
Claims 2 and 4 are rejected under 35 U.S.C. 103 as being unpatentable over Santos (Santos et al, Nature Communications (2013), 4:2503, DOI: 10.1038/ncomms3503; of record), Thomson (Thomson and Blechl, "Recombinase technology for precise genome engineering." Advances in new technology for targeted modification of plant genomes (2015): 113-144; of record) and Enyeart (Enyeart, Peter James, (2014) Studies in Bacterial Genome Engineering and Its Applications (Doctoral Dissertation; of record) as applied to claims 1, 3, 5-6 and 9-10 above, and further in view of Santos2 (US 20120329115 A1) and Le Breton (Le Breton et al., Applied and Environmental Microbiology (2006), 72: 327-333). This is a new rejection necessitate by amendment.
The teachings of Santos, Thomson and Enyeart are recited above and applied as for claims 1, 3, 5-6 and 9-10. Santos, Thomson and Enyeart do not specifically teach the RAGE method or the function of Mariner transposons in bacterial cells other than E. coli.
Santos2 is a patent publication related to the Santos reference. Santos teaches each of the steps in claim 1, except wherein step (a) uses lambda-recombination instead of transposon-mediated landing pad integration and in step (c) Cre recombinase is expressed from a plasmid in trans instead of in cis. Santos2 also teaches the method of landing pad integration and RCME can be performed in other bacterial species including in Bacillus subtills (i.e., a non-E. coli host cell, a Firmicutes).
Le Breton teaches mariner transposase and transposon systems are functional in B. subtilis (Abstract). Le Breton teaches B. subtilis has served as a model for gram-positive bacterial and microbial differentiation (page 327, ¶1).
Regarding claims 2 and 4, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have adapted the variation of Santos’s RAGE integration method using transposon-based integration rendered obvious above to be used in B. subtilis. It would have amounted to the simple combination of known elements by known means to yield predictable results. The skilled artisan would have predicted such a method could be adapted for B. subtilis because Santos2 teaches that the RAGE method can be used to integrate metabolic pathways into B. subtilis, and Le Breton demonstrates that mariner transposon systems are functional in B. subtilis. The skilled artisan would have been motivated to adapt the system for B. subtilis because Santos2 suggests it and for the purpose of using the method for straight-forward gene integration into a model bacterial species.
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 but is not persuasive as it applies to the current rejection above that addresses the steps to produce the bacteria comprising the landing pad. Because the art recognizes different means to integrate a lox site landing pad into a bacterial genome, and it was obvious to use the means/steps taught in Enyeart to construct the initial landing pad bacterial strain for the reasons recited in the rejection above.
Allowable Subject Matter
Claim 7 is objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Claim 8 would be allowable if rewritten to overcome the rejection(s) under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), 2nd paragraph, set forth in this Office action. It is suggested that claim 8 depend from claim 7 once rewritten in independent form, which is otherwise allowable as indicated in the paragraph above.
The closest prior art is Santos in view of Thomson and Enyeart, whose teachings are recited in the §103 rejections above. Claim 7 requires that the landing pad comprise a coding sequence for “a protein that promotes sequence-specific gene transcription” outside the lox sites, but between the inverted repeats. Figures 8 and 11 illustrate such a landing pad with the coding sequence of T7 RNA Polymerase (T7 RNAP) included in the landing pad but outside the lox sites, such that the T7 RNAP coding sequence remains after recombinase-mediated cassette exchange (RMCE) to integrate the gene of interest. The figures also illustrate the gene of interest downstream of a T7 promoter. Most instances of using RMCE in bacteria use promoters that utilize RNAPs and transcription factors that are endogenous to the host bacteria or bacterial host strains in which the gene encoding the heterologous polymerase has previously been integrated into the host chromosome. For instance, both Santos and Chiang (Chiang et al., Process Biochemistry (2012) 47: 2246-2254) integrate recombinant genes (i.e., ALG3 operon and PHB operon) with its promoter, which is recognized by the endogenous E. coli transcriptional machinery such that an additional RNAP or TF is not required for transcription. Chiang also integrated heterologous coding sequences under the control of a lambda promoter that is also recognized by the endogenous RNA polymerase (Section 2.3, ¶1).
Another close prior art is de las Heras, which discloses a transposon-mediated method of integrating an operon under the control of the T7 promoter in the bacteria P. putida (de Las Heras and de Lorenzo. "Engineering whole-cell biosensors with no antibiotic markers for monitoring aromatic compounds in the environment." Microbial Metabolic Engineering: Methods and Protocols. New York, NY: Springer New York, 2011. 261-281). P. putida cannot recognize T7 promoters unless engineered to express T7 RNAP. De las Heras provides the T7 RNAP gene and the gene of interest under the control of the T7 promoter in separate transposon integration cassettes (Fig. 1). There is no teaching or suggestion by de las Heras to include the coding sequence for the T7 RNAP in the initial landing pad that will be used to integrate the gene of interest. Therefore, it was not obvious to modify the landing pad of Santos to include an RNAP or TF coding sequence that would be used to drive expression of the gene of interest after RMCE of the lox-flanking sequences.
Conclusion
Claim 7 is objected to. Claims 1-6 and 8-10 are rejected.
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 CATHERINE KONOPKA whose telephone number is (571)272-0330. The examiner can normally be reached Mon - Fri 7- 4.
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/CATHERINE KONOPKA/Primary Examiner, Art Unit 1635
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