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 06/30/2026. Claims 78-89 and 92-93 are currently pending. Claims 90-91 have been cancelled. Claims 85-89 are withdrawn from prosecution as being drawn to non-elected subject matter. Accordingly, claims 78-84 and 90-91 are examined herein. The restriction requirement mailed 03/19/2025 is still deemed proper. Applicant elected the invention of Group I and the species of glucose-6-phosphate-1-dehydrogenase, cas3, an alcohol and lon with traverse in the reply filed 05/19/2025. As noted in the office action of 03/18/2026, the species election between deleted genes sspB and cas3 is withdrawn in light of the teachings of Brockman. This reference clearly teaches a microorganism in which the naturally occurring sspB gene has been deleted. The election among the other recited species of deleted genes is maintained.
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.
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 06/30/2026 has been entered.
Terminal Disclaimer
The terminal disclaimer filed on 06/30/2026 disclaiming the terminal portion of any patent granted on this application which would extend beyond the expiration date of U.S. Patent Numbers 11,142,761 and 11,268,111 has been reviewed and is accepted. The terminal disclaimer has been recorded. The rejections of the claims for non-statutory double-patenting, as set forth in the office action of 03/18/2026, are withdrawn in light of the terminal disclaimer.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
Claim 78 is rejected under 35 U.S.C. 103 as being unpatentable over Soma (Soma et al. Metabolic flux redirection from a central metabolic pathway toward a synthetic pathway using a metabolic toggle switch. Metabolic Engineering, vol. 23, 1 May 2014, pages 175-184; of record, cited on an IDS) in view of Nakashima (Nakashima et al. 2014. A Vector Library for Silencing Central Carbon Metabolism Genes with Antisense RNAs in Escherichia coli . Appl Environ Microbiol 80.) and Zhou (Zhou et al. Lycopene production in recombinant strains of Escherichia coli is improved by knockout of the central carbon metabolism gene coding for glucose-6-phosphate dehydrogenase. Biotechnol Lett (2013) 35:2137–2145).
Soma teaches a genetically modified microorganism (E. coli) comprising: a production pathway comprising at least one enzyme for the production of a product (a metabolic toggle switch in E. coli applied to isopropanol production), and at least one gene silencing synthetic metabolic valve comprising controlled transcriptional gene silencing of a gene encoding citrate synthase (gltA) (Abstract).
Soma notes that conditional gene silencing of genes responsible for bacterial growth and/or cell maintenance is advantageous over deletion of those genes because, “Deletion of these genes would increase the titer and yield of the desired product per cell, but decrease the growth rate and/or final cell density, and perhaps resulting in cell death.” (p. 175), but that, “An alternate approach is to keep the expression of these genes high until an adequate cell mass is achieved, then turn these genes off.” (Id.).
Soma further teaches wherein under conditions of depleting a limiting nutrient (minimal medium), a stationary phase or non-dividing cellular state is induced, and the valve is activated in that phase (p. 178, § 3.1, Fig. 3B showing the switch toggled during a non dividing phase).
Soma does not teach wherein transcriptional gene silencing comprises expression of small guide RNAs specific for the gene.
The broadest reasonable interpretation of “transcriptional gene silencing” encompasses gene silencing which targets transcripts for translational inhibition or degradation, such as antisense and miRNA/siRNA/shRNA technology, as well as systems which inhibit transcription at the pre-transcriptional stage, such as CRISPRi systems. The broadest reasonable interpretation of “guide RNA” encompasses both the guide strands of double-stranded RNA silencing duplexes as well as CRISPR/Cas-associated guide RNAs.
Nakashima teaches a vector library for silencing central carbon metabolism genes with antisense RNAs (transcriptional silencing) in E. coli (Title).
Nakashima further teaches that the vectors, “inducibly express antisense RNAs” with a high silencing efficacy (Abstract).
Nakashima further teaches a vector for silencing central carbon metabolism genes gltA (citrate synthase, non-elected species) and zwf (glucose-6-phosphate-dehydrogenase, elected species) (Table 1).
While Nakashima teaches targeting elected species zwf, Nakshima does not provide an explicit rationale to do so.
Zhou teaches that knockout of zwf results in improved lycopene production in E. coli (Title).
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 modified the conditional silencing of central carbon metabolism genes to improve production of a product, as taught by Soma, to instead use the inducible (conditional) transcriptional antisense silencing approach, as taught by Nakashima. The ordinary artisan would have been motivated to do so, and would have had a reasonable expectation of success, based on the combination of Soma’s teachings that conditional deactivation of genes responsible for bacterial growth and/or cell maintenance during non-growth phases was advantageous for optimizing both cell growth and product yield, along with Nakashima’s teachings that inducible (conditional) antisense silencing of central carbon metabolism genes (i.e., genes responsible for bacterial growth and/or cell maintenance) had high efficacy and could be performed on both gltA and zwf.
Claims 79 and 80 are rejected under 35 U.S.C. 103 as being unpatentable over Soma, Nakashima and Zhou as applied to claim 78, further in view of Qi (Cell. 2013 Feb 28;152(5):1173–1183; of record).
Soma, Nakashima and Zhou render obvious the invention of claim 78, from which the instantly rejected claims depend, as described above.
Soma, Nakashima and Zhou do not teach wherein transcriptional gene silencing further comprises expression of a gene encoding a dCas9 protein, or wherein a single plasmid expresses small guide RNAs for two or more genes.
Qi et al. teach the CRISPR interference (CRISPRi) system, based on a catalytically dead Cas9, which can efficiently repress expression of targeted genes in E. coli (§SUMMARY). Qi et al. further teach that, “the silencing effects of dCas9-sgRNA can be induced and reversed” (p. 4 §CRISPRi Gene Knockdown Is Inducible and Reversible). Therefore, Qi et al. teach controlled transcriptional gene silencing wherein the silencing comprises expression of small guide RNAs specific for the gene.
Qi further teaches that the CRISPRi system may target multiple genes at a time using plasmids encoding more than one guide RNA (Figure 4).
Qi further provides a teaching, suggestion or motivation to use the CRISPRi system, especially in microbes, by noting that, “CRISPRi could provide an RNAi-like method for gene perturbation in microbes” and that it is, “relatively simple”, “does not rely on the presence of complex host factors”, “is flexible and highly designable”, and “can efficiently silence genes in bacteria” with “no detectable off-target effects”.
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 modified the valve as taught by Soma, Nakashima and Zhou to use CRISPRi conditional transcriptional silencing instead of the antisense technology taught by Nakashima. The ordinary artisan would have been motivated to do so, and would have had a reasonable expectation of success, based on Qi’s teachings that the CRISPRi system offered several advantages over other technologies, such as flexibility, designability, simplicity, and no detectable off-target effects.
Claims 81-84 and 92-93 are rejected under 35 U.S.C. 103 as being unpatentable over Soma, Nakashima and Zhou as applied to claim 78, further in view of Brockman (of record).
Soma, Nakashima and Zhou render obvious the valve of claim 78, from which the instantly rejected claims depend, as described above.
Regarding claim 81, Soma, Nakashima and Zhou do not teach wherein the valve further comprises conditional expression of a proteolysis chaperone protein.
Brockman teaches a metabolic valve comprising controlled proteolysis (degradation) of an enzyme (Abstract) through conditional expression of a proteolysis chaperone protein (SspB) through aTc-inducible expression of SspB from a plasmid(p. 2, § 3.2).
Regarding claim 82, Brockman teaches wherein the protein is sspB (see above).
Regarding claim 83, Brockman teaches that the naturally occurring sspB gene is disrupted (“To make the default state “ON”…SspB was knocked out at its native locus”; p. 7 § 3.2).
Regarding the above claims and claims 84, 92, and 93, while Brockman does not teach a valve which combines controlled transcriptional silencing of gltA or zwf and controlled proteolysis of gltA or zwf.
However, Nakashima teaches, “The weakest point of antisense silencing is that complete removal of gene function is impossible.”
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 modified the valve comprising controlled transcriptional silencing of gltA or zwf, as taught by Soma, Nakashima and Zhou, to further comprise controlled proteolysis of the proteins encoded by those genes, as taught by Brockman and suggested by Nakashima. The ordinary artisan would have been motivated to do so based on Nakashima’s teachings that the conditional antisense silencing approach did not completely remove gene function. Based on common sense and sound scientific reasoning, the ordinary artisan would have recognized that adding the controlled proteolysis system, as taught by Brockman, would have addressed the problem raised by Nakashima by eliminating any residual gene function.
Brockman teaches conditional expression of the chaperone protein, i.e., expression of SspB from an inducible promoter (see above).
Response to Arguments
Applicant's arguments filed 06/30/2026 have been fully 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.
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.
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Claims 78-80 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-2, 31-33, 35 and 37 of copending Application No. 19/249,511 (filed 06/25/2025) in view of Qi.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
The instant claims compare to the copending claims as follows:
Instant claims
Copending claims
78. A genetically modified E.coli or S. cerevisiae microorganism comprising a production pathway comprising at least one enzyme for the production of a product and at least one gene silencing synthetic metabolic valve comprising controlled transcriptional gene silencing of a gene encoding an enzyme that is one of: enoyl-ACP reductase, citrate synthase, soluble transhydrogenase, glucose-6-phosphate-l-dehydrogenase, lipoamide dehydrogenase; wherein transcriptional gene silencing comprises expression of small guide RNAs specific for the gene;wherein, under conditions of depleting of a limiting nutrient from a growth medium in which the genetically modified microorganism is growing, a stationary phase or non-dividing cellular state is induced;wherein the synthetic metabolic valve of the microorganism is activated in the stationary phase or non-dividing cellular state.
37. The cell of any one of claims 1-36, wherein said cell is an E. coli cell
1. A cell for generating a product, wherein said cell comprises: a heterologous polynucleotide for controlled reduction of expression of an enzyme of a metabolic pathway wherein said controlled reduction of expression of said enzyme induces a stationary phase of said cell
2. The cell of claim 1 or 2, wherein said enzyme is selected from the group consisting of enoyl-ACP/CoA reductase, glucose-6-phosphate dehydrogenase, lipoamide dehydrogenase, citrate synthase, soluble transhydrogenase
31. The cell of claim 1, wherein said heterologous polynucleotide comprises a silencing polynucleotide, and said silencing polynucleotide comprises a guide RNA (gRNA) comprising a gRNA sequence that recognizes a promoter of a gene encoding said enzyme.
35. The cell of any one of claims 1-34, wherein expression of said heterologous polynucleotide is regulated by phosphate availability in said cell.
79. The genetically modified microorganism of claim 78, wherein transcriptional gene silencing further comprises expression of a gene encoding a dCas9 protein.
80. The genetically modified microorganism of claim 78, wherein a single plasmid expresses small guide RNAs for two or more genes.
32. The cell of claim 31, wherein said heterologous polynucleotide encodes a CRISPR enzyme, wherein said CRISPR enzyme specifically binds to said promoter sequence when bound to said gRNA
33. The cell of claim 32, wherein said CRISPR enzyme is catalytically inactive.
Although the claims at issue are not identical, they are not patentably distinct from each other because the copending claims as a whole are drawn to an E. coli cell comprising a production pathway (polynucleotide) comprising the same species of enzymes and a controlled metabolic pathway in which expression of the enzyme is reduced, and the expression of the enzyme is reduced using guide RNAs and a catalytically inactive Cas enzyme (i.e., dCas9). The controlled reduction of the copending claims, which induces a stationary phase, is induced be a limiting nutrient, phosphate, and this process is interpreted as a specific process encompassed by the generic activation of the metabolic valve (i.e., a heterologous polynucleotide for controlled reduction of expression) in a stationary phase induced by depletion of a limiting nutrient, as recited in the instant claims. Further, it would have been obvious to modify the heterologous polynucleotide for controlled reduction of expression into a CRISPRi conditional silencing system, using multiple encoded guide RNAs, for the same reasons described in the rejections of the claims under 35 USC 103, above.
Claims 78-84 and 92-93 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-2, 31-33, 35 and 37 of copending Application No. 19/268,525 (filed 07/14/2025) in view of Qi and Brockman.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
The instant claims compare to the copending claims as follows:
Instant claims
Copending claims
78. A genetically modified E.coli or S. cerevisiae microorganism comprising a production pathway comprising at least one enzyme for the production of a product and at least one gene silencing synthetic metabolic valve comprising controlled transcriptional gene silencing of a gene encoding an enzyme that is one of: enoyl-ACP reductase, citrate synthase, soluble transhydrogenase, glucose-6-phosphate-l-dehydrogenase, lipoamide dehydrogenase; wherein transcriptional gene silencing comprises expression of small guide RNAs specific for the gene;wherein, under conditions of depleting of a limiting nutrient from a growth medium in which the genetically modified microorganism is growing, a stationary phase or non-dividing cellular state is induced;wherein the synthetic metabolic valve of the microorganism is activated in the stationary phase or non-dividing cellular state.
79. The genetically modified microorganism of claim 78, wherein transcriptional gene silencing further comprises expression of a gene encoding a dCas9 protein.
80. The genetically modified microorganism of claim 78, wherein a single plasmid expresses small guide RNAs for two or more genes.
81. The genetically modified microorganism of claim 78, wherein the synthetic metabolic valve directed to controlled transcriptional gene silencing further comprises conditional expression of a proteolysis chaperone protein.
82. The genetically modified microorganism of claim 81, wherein the chaperone protein is a sspB protein.
83. The genetically modified microorganism of claim 78 wherein the genetically modified microorganism is characterized by disruption or deletion of a gene naturally occurring in the genetically modified microorganism, the naturally occurring gene one of a gene encoding lactate dehydrogenase, phosphate acetyltransferase, pyruvate oxidase, pyruvate- formate lyase, the methylglyoxal synthase, acetate kinase, alcohol dehydrogenase, ATP- dependent Lon protease, outer membrane protease, arcA transcriptional dual regulator, iclR transcriptional regulator, cas3, sspB or combinations thereof.
84. The genetically modified microorganism of claim 81 further comprising a synthetic metabolic valve comprising controlled proteolysis of an enzyme that is one of: enoyl- ACP reductase, citrate synthase, soluble transhydrogenase, glucose-6-phosphate- 1-dehydrogenase, lipoamide dehydrogenase, or combinations thereof.
92. A genetically modified E.coli or S. cerevisiae.microorganism comprising: a production gene silencing pathway comprising at least one enzyme for the production of a product, andat least one synthetic metabolic valve comprising controlled proteolysis of an enzyme that is one of enoyl-ACP reductase, citrate synthase, soluble transhydrogenase, glucose-6-phosphate- 1-dehydrogenase, lipoamide dehydrogenase, or combinations thereof;wherein, under conditions of depleting of a limiting nutrient from a growth medium in which the genetically modified microorganism is growing, a stationary phase or non-dividing cellular state is induced;wherein the synthetic metabolic valve of the microorganism is activated in the stationary phase or non-dividing cellular state.
93. The genetically modified microorganism of claim 92, further comprising a synthetic metabolic valve directed to controlled transcriptional gene silencing and this valve comprises conditional expression of a proteolysis chaperone protein, the chaperone protein required for controlled proteolysis.
18. A genetically modified microorganism comprising:(i) a production pathway for a product…(iii) a synthetic metabolic valve for dynamic and selective regulation of enoyl-ACP reductase, the synthetic metabolic valve comprising: gene expression-silencing of a enoyl-ACP reductase gene, selective enzymatic degradation of a enoyl-ACP reductase protein, or a combination thereof;(iv) a synthetic metabolic valve for dynamic and selective regulation of citrate synthase, the synthetic metabolic valve comprising: gene expression-silencing of a citrate synthase gene, selective enzymatic degradation of a citrate synthase protein, or a combination thereof; and (v) a synthetic metabolic valve for dynamic and selective regulation of glucose-6- phosphate dehydrogenase, the synthetic metabolic valve comprising: gene expression-silencing of a glucose-6-phosphate dehydrogenase gene, selective enzymatic degradation of a glucose-6- phosphate dehydrogenase protein or a combination thereof…wherein the synthetic metabolic valves are selectively activated to silence…a enoyl-ACP reductase gene, citrate synthase gene, glucose-6-phosphate dehydrogenase gene and/or degrade…a enoyl-ACP reductase protein, citrate synthase protein, glucose-6- phosphate dehydrogenase protein in response to depletion of phosphate as a limiting nutrient in the growth media in which the genetically modified microorganism is growing.
19. The genetically modified microorganism of claim 8, wherein the genetically modified microorganism is an Escherichia or Streptomyces microorganism.
20. The genetically modified microorganism of claim 8, wherein the genetically modified microorganism is an E. coli microorganism.
Although the claims at issue are not identical, they are not patentably distinct from each other because the copending claims are drawn to an E. coli cell comprising a metabolic valve for regulating the same enzymes as those instantly claimed, using a combination of dynamic and selective (i.e., conditional/controlled) gene silencing and proteolytic degradation, and it would have been obvious to use the CRISPRi system of Qi and the SspB-based proteolytic degradation system of Brockman to achieve such regulation, as described in the rejections of the claims under 35 USC 103 above.
Conclusion
No claims are allowed at this time.
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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