DETAILED CORRESPONDENCE
Status of the Application
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 February 19, 2026 has been entered.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 34-57 and 59-69 are pending in the application.
Applicant’s amendment to the claims, filed February 19, 2026, is acknowledged. This listing of the claims replaces all prior versions and listings of the claims.
Applicant’s remarks filed February 19, 2026 in response to the final rejection mailed November 20, 2025 and the advisory action mailed January 29, 2026 have been fully considered.
Claim 58 has been canceled and rejections previously applied to claim 58 are withdrawn.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Restriction/Election
In response to a requirement for restriction/election mailed May 14, 2025, applicant elected with traverse the invention of Group II, pending claims 46-52, 55, and 59-69, and species (E6), the at least one mutated gene is a gene involved in Lpp functionality and consists of the complete deletion of the ybiS gene, in the reply filed on July 11, 2025. The requirement was deemed proper and made FINAL in the non-final rejection mailed August 1, 2025.
Claims 34-45, 53, 54, 56, and 57 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claims 49, 51, 52, 55, 59, 62, 63, and 66-69 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to nonelected species, there being no allowable generic or linking claim.
Claims 46-48, 50, 60, 61, 64, and 65 are being examined on the merits with claim 50 being examined only to the extent the claim reads on the elected subject matter.
Claim Objections
The objection to claim 46 for reciting “a polypeptide having at least 75% amino acid sequence identity to SEQ ID NO: 2” is withdrawn in view of applicant’s amendment to claim 46 to delete the phrase at issue.
Claims 46, 60, and 61 are objected to because of the following informalities:
Claim 46 is objected to in the recitation of “envelop” in lines 4 and 6, which appears to be a misspelling of “envelope.”
Claim 46 is also objected to in the recitation of “the at least extra-genomic nucleic acid molecule” in the last line of step a) and in the interest of improving claim form, it is suggested that the term “one” be inserted between “least” and “extra-genomic.”
Claim 46 is also objected to in the recitation of “said amplified extra-genomic nucleic acid molecule or said at least one polypeptide” in step c) and in the interest of improving claim form, it is suggested that the noted phrase be amended to recite “said at least one amplified extra-genomic nucleic acid molecule or said at least one amplified polypeptide.”
Claim 60 is objected to in the recitation of “said amplified extra-genomic nucleic acid molecule” and in the interest of improving claim form, it is suggested that the noted phrase be amended to recite “said at least one amplified extra-genomic nucleic acid molecule.”
Claim 61 is objected to in the recitation of “said at least one polypeptide” and in the interest of improving claim form, it is suggested that the noted phrase be amended to recite “said at least one amplified polypeptide.”
Claim Rejections - 35 USC § 112(b)
Claims 46-48, 50, 60, 61, 64, and 65 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
Claim 46 (claims 47, 48, 50, 60, 61, 64, and 65 dependent therefrom) is indefinite in the recitation of “compared to a bacterium with unaltered envelop integrity” because it is unclear as to the reference bacterium that is considered to have “unaltered envelop integrity.” In the interest of advancing prosecution, applicant may consider amending the phrase “genetically modified E. coli bacteria comprising at least one mutated or deleted gene encoding a protein involved in the envelope integrity, said bacteria having an altered envelop integrity and being oversensitive to bacterial lysis as compared to a bacterium with unaltered envelop integrity, wherein the at least one mutated or deleted gene is ybiS” in claim 46 to recite “genetically modified E. coli bacteria comprising a mutated or deleted ybiS gene, said bacteria having an altered envelop integrity and being oversensitive to bacterial lysis as compared to an E. coli bacterium that has not been genetically modified to mutate or delete the ybiS gene.”
Claim 50 is confusing in the recitation of “The method according to claim 46, wherein the genetically modified E. coli bacteria comprises at least one mutated gene is a gene involved in Lpp functionality which consists of a mutation in the lpp gene…and/or the complete deletion of the ycfS gene and/or the complete deletion of the erfK gene,” which is inconsistent with the recitation of “wherein the at least one mutated or deleted gene is ybiS” in claim 46. In the interest of advancing prosecution, it is suggested that claim 50 be amended to recite “The method according to claim 46, wherein the genetically modified E. coli bacteria comprises a complete deletion of the ybiS gene.”
Claim Rejections - 35 USC § 102/103
The rejection of claims 46-48, 60, and 61 under 35 U.S.C. 102(a)(1) as anticipated by or, in the alternative, under 35 U.S.C. 103 as obvious over Chen et al. (Microbial Biotechnology 7:360-370, 2014; cited on the IDS filed on September 2, 2022; hereafter “Chen”) is withdrawn in view of applicant’s amendment to claim 46 to recite “wherein the at least one mutated or deleted gene is ybiS.” Chen does not teach a modified E. coli bacterium comprising a mutated or deleted ybiS gene.
Claim Rejections - 35 USC § 103
Claims 46-48, 50, 60, 61, 64, and 65 are rejected under 35 U.S.C. 103 as being unpatentable over Chen in view of Sanders (Microbiology 159:1842-1852, 2013; cited on Form PTO-892 mailed August 1, 2025; hereafter “Sanders”) and Yu et al. (Nucleic Acids Research 36:e84, 2008, 8 pages; cited on Form PTO-892 mailed August 1, 2025; hereafter “Yu”).
As amended, the claims are drawn to a method for the production and the purification of at least one extra-genomic nucleic acid molecule or at least one polypeptide comprising the steps of:
a) culturing genetically modified E. coli bacteria comprising at least one mutated gene encoding a protein involved in the envelope integrity, said bacteria having an altered envelop integrity and being oversensitive to bacterial lysis as compared to a bacterium with unaltered envelop integrity, wherein the at least one mutated or deleted gene is ybiS, said bacteria comprising at least one extra-genomic nucleic acid molecule or comprising a nucleic acid molecule encoding at least one polypeptide, so as to amplify the at least extra-genomic nucleic acid molecule or the at least one polypeptide;
b) lysing the bacteria obtained at step a) so as to obtain a lysis mixture; and,
c) purifying said amplified extra-genomic nucleic acid molecule or said at least one polypeptide from the lysis mixture obtained at step b).
Chen is related to the construction of leaky strains and extracellular production of exogenous proteins in recombinant Escherichia coli (see title). Chen teaches that while fermentation conditions have been explored to achieve extracellular production of recombinant proteins in E. coli, there is a disadvantage in that fermentation conditions vary greatly with different target proteins and to overcome the uncertainty of the fermentation conditions, the construction of leaky strains will become a main alternative to transport periplasmic-directed recombinant proteins into media (p. 361, column 1, middle). Chen teaches further studies to improve the extracellular production of the target proteins have become inevitable (p. 361, column 1, bottom) and teaches construction of leaky strains of E. coli by knocking out genes related to the biosynthesis of cell wall and membrane, especially of the outer membrane genes such as lpp encoding Braun’s lipoprotein (p. 361, column 1, middle). Chen teaches expression and analysis of recombinant proteins expressed in the leaky strains, including a determination of the secretory efficiency by comparing the intracellular and extracellular protein levels of the recombinant proteins (p. 365, column 1, top; p. 367, paragraph bridging columns 1-2).
Regarding claim 46, step a) and claims 50, 64, and 65, Chen teaches E. coli JM109 (DE3) is a popular host for the expression of recombinant proteins (p. 361, column 1, last paragraph). Chen teaches that in theory, the disruption of mrcA and mrcB genes encoding peptidoglycan synthetase or the disruption of pal gene encoding the peptidoglycan-associated outer membrane lipoprotein may cause the deficiencies in the structures of cell walls and outer membranes (p. 361, column 2, second full paragraph). Chen teaches E. coli JM109 (DE3) with single or double deletion of genes mrcA, mrcB, pal, and lpp (p. 361, column 2, third paragraph). Chen teaches the E. coli mutants were transformed with an expression vector for reteplase (rPA) (p. 364, column 1) and cultured in TB medium for expression of rPA in the cytoplasm (p. 365, column 1, top). Chen teaches the results suggested that double deletion of peptidoglycan synthetase genes and outer membrane genes may increase the outer membrane permeability enough for the leakage of periplasmic protein without affecting the growth of these strains significantly in complex media (p. 366, column 1, top).
Regarding claim 46, step b), Chen teaches preparing samples for analysis of recombinant proteins including a step of ultrasonication (p. 367, column 2, top). Chen’s step of ultrasonication is considered to be encompassed by step b) of claim 46 because one of ordinary skill in the art would have recognized that ultrasonication results in cell lysis.
Regarding claim 46, step c) and claims 60 and 61, Chen teaches centrifugation of the sonicate and collecting a supernatant fraction and a precipitate fraction (p. 367, column 2, top). Chen’s step of centrifuging the sonicate and collecting a supernatant fraction is considered to be encompassed by step c) of claim 46 and claim 61 because the cytoplasmic rPA within the sonicate is separated from the cellular debris by centrifugation and collecting of the supernatant fraction, which resulted in at least some level of purification of the cytoplasmic rPA as compared to the whole sonicate. Chen’s step of centrifuging the sonicate and collecting supernatant and precipitate fractions is also considered to be encompassed by claim 60 because the expression vector for rPA within the sonicate is separated by centrifugation into the supernatant fraction and/or precipitate fraction and the supernatant fraction and precipitate fraction are each collected, which resulted in at least some level of purification of the expression vector for rPA as compared to the whole sonicate.
Regarding claims 47 and 48, as stated above, Chen teaches the E. coli mutants harboring an expression vector (i.e., plasmid) for rPA were cultured in TB medium for expression of rPA in the cytoplasm (p. 365, column 1, top).
The difference between the claimed method and the method of Chen is that Chen does not teach or suggest a mutated or deleted ybiS gene.
Sanders teaches ErfK, YbiS, and Ycfs enzymes function by covalently attaching Braun’s lipoprotein to peptidoglycan (p. 1843, column 1, bottom) and are responsible for the attachment of Braun’s lipoprotein to the cell wall (p. 1848, column 1). Sanders teaches an E. coli mutant with deletion of ybiS, erfK, and ycfS genes (referred to as Δldt3), which leaked periplasmic proteins into a culture supernatant (p. 1842, abstract; p. 1846, column 1, bottom).
Yu teaches a method for gene deletion in E. coli (see whole document).
In view of Chen, Sanders, and Yu, it would have been obvious to one of ordinary skill in the art before the effective filing date to completely delete ybiS, erfK, and ycfS genes in an E. coli JM109 (DE3) host cell and determine the resulting secretory efficiency according to Chen. One would have been motivated to do this because Chen taught the inevitability of further studies to improve the extracellular production of target proteins and taught constructing leaky strains by knocking out genes related to the biosynthesis of cell wall and membrane will become a main alternative to transport periplasmic-directed recombinant proteins into media, and Sanders taught knocking out ybiS, erfK, and ycfS genes, which are related to the biosynthesis of cell wall, had the effect of leaking periplasmic proteins. Given that the sequences for each of ErfK, YbiS, and Ycfs enzymes are taught by Sanders (p. 1845, Figure 1), Yu taught a method for gene deletion in E. coli, and Chen taught a method for determining secretory efficiency of a leaky strain, one would have expected success to completely delete ybiS, erfK, and ycfS genes in a E. coli JM109 (DE3) host and determine its secretory efficiency of a recombinantly-expressed protein.
Regarding the limitation “said bacteria…being oversensitive to bacterial lysis” in step a) of claim 46, the phrase “oversensitive to bacterial lysis” is interpreted as meaning any level or amount of increased sensitivity to bacterial lysis as compared to a bacterium with unaltered envelop integrity. The combination of Chen, Sanders, and Yu does not explicitly teach an E. coli JM109 (DE3) host cell with deletion of ybiS, erfK, and ycfS genes is “oversensitive to bacterial lysis.” However, given that an E. coli JM109 (DE3) host cell with deletion of ybiS, erfK, and ycfS genes has a cell wall deficiency and leaked periplasmic proteins into a culture supernatant, it is presumed that an E. coli JM109 (DE3) host cell with deletion of ybiS, erfK, and ycfS genes has the property of being “oversensitive to bacterial lysis” as recited in step a) of claim 1.
In the interest of clarity, it noted that ybiS gene deletion is interpreted as being encompassed by a “mutated” ybiS gene in claim 46 and “at least a mutation of the ybiS gene” in claim 65.
Therefore, the invention of claims 46-48, 50, 60, 61, 64, and 65 would have been obvious to one of ordinary skill in the art before the effective filing date.
RESPONSE TO REMARKS: Applicant argues Sanders does not correlate YbiS enzyme function with oversensitivity to bacterial lysis and the technical effect of improving extra-genomic nucleic acids or amplified polypeptides following lysis.
Applicant’s arguments are not found persuasive. The cited prior art need not teach or suggest a correlation between YbiS enzyme function and oversensitivity to bacterial lysis and/or the technical effect of improving extra-genomic nucleic acids or amplified polypeptides following lysis to support obviousness. The reason or motivation to modify the reference may often suggest what the inventor has done, but for a different purpose or to solve a different problem. It is not necessary that the prior art suggest the combination to achieve the same advantage or result discovered by applicant (see MPEP 2144.IV).
Applicant argues Figure 5 shows that an E. coli MG1655 with deletion of the ybiS gene exhibits a more pronounced effect due to osmotic shock compared to wild-type E. coli MG1655 and mutants without deletion of ybiS and neither Chen nor Sanders teaches or suggests this superior, unexpected result.
Applicant’s arguments and allegation of unexpected results are not found persuasive. According to MPEP 716.02(e), applicant’s results must be compared with the closest prior art, which is Chen or Sanders. However, applicant’s results are not a comparison with the deletion mutants of Chen and/or Sanders. Also, according to MPEP 716.02(d), applicant’s results must be commensurate in scope with the claimed invention. In this case, applicant’s results are based on the effects of osmotic shock as a method of lysing bacteria, however, the claims do not recite “osmotic shock.” Rather, claim 46 step b) encompasses lysing bacteria by any lysis method and while nonobviousness of a broader claimed range can be supported by evidence based on unexpected results from testing a narrower range (MPEP 716.02(d).I), there is no evidence of record or line of reasoning to support the position that applicant’s results due to osmotic shock would be expected to extend to all methods for lysing bacteria.
For these reasons, it is the examiner’s position that the applicant’s results fail to rebut a prima facie case of obviousness and the claimed invention would have been obvious to one of ordinary skill in the art before the effective filing date.
Claim Rejections - 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 §§ 706.02(l)(1) - 706.02(l)(3) 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 USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The 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/process/file/efs/guidance/eTD-info-I.jsp.
Claims 46-48, 50, 60, 64, and 65 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 2, 5, and 8 of co-pending application no. 18/413,543 (reference application).
Regarding instant claims 46, 47, 50, 60, 64, and 65, claim 1 of the reference application recites a method for producing plasmid DNA, comprising the following steps (a) to (c):
(a) a step of preparing E. coli, which has a mutation in a gene region associated with maintaining outer membrane properties and has a desired plasmid;
(b) a step of culturing E. coli of (a); and
(c) a step of recovering a desired plasmid from bacterial cells after culture;
claim 2 of the reference application recites wherein step (c) is a step of recovering the desired plasmid by dissolving the outer membrane of the bacterial cell after culturing;
claim 5 of the reference application recites (in relevant part) wherein the gene region directly associated with maintaining physical and/or mechanical outer membrane properties is ybiS; and
claim 8 of the reference application recites wherein the mutation is a complete disruption.
Regarding instant claim 48, while claim 48 limits the “at least one polypeptide” in claim 46 to “at least one cytoplasmic polypeptide,” claim 48 does not limit the alternatives of “at least one extra-genomic nucleic acid molecule or at least one polypeptide” to the “at least one polypeptide.” As such, the alternative of “at least one extra-genomic nucleic acid molecule” is still encompassed by claim 48.
Therefore, claims 46-48, 50, 60, 64, and 65 of this application are unpatentable over claims 1, 2, 5, and 8 of the reference application. This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
RESPONSE TO REMARKS: Applicant requests that the provisional rejection be withdrawn in accordance with MPEP 804.I.B.1.(b).(i), however, this is not the only remaining rejection in the application.
Conclusion
Status of the claims:
Claims 34-57 and 59-69 are pending.
Claims 34-45, 49, 51-57, 59, 62, 63, and 66-69 are withdrawn from consideration.
Claims 46-48, 50, 60, 61, 64, and 65 are rejected.
No claim is in condition for allowance.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to DAVID J STEADMAN whose telephone number is (571)272-0942. The examiner can normally be reached Monday to Friday, 7:30 AM to 4:00 PM.
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/David Steadman/Primary Examiner, Art Unit 1656