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 06/14/2024. Claims 1-20 are currently pending and are examined herein.
Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
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.
Claims 12-17 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 12, the term “in the vicinity thereof” is a relative term which renders the claim indefinite. The term is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. It is unclear how close an amino acid residue must be to be considered “in the vicinity” of an amino acid residue at the site of binding/at the protein’s active site.
Amending the claim to delete the term would obviate this rejection.
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.
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:
Determining the scope and contents of the prior art.
Ascertaining the differences between the prior art and the claims at issue.
Resolving the level of ordinary skill in the pertinent art.
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.
U.S. PGPUB 2016/0340665 - Falb
Claims 1-9, 11 and 19-20 are rejected under 35 U.S.C. 103 as being unpatentable over US PGPUB US 2016/0340665 A1 to Falb (hereinafter ‘Falb’) in view of Xenopoulos (Xenopoulos et al. Production and purification of plasmid DNA vaccines: is there scope for further innovation? Expert Rev. Vaccines 13(12), 1537–1551 (2014).).
Regarding claims 1 and 19-20, Falb teaches methods for preparing E. coli which has a mutation in a gene region associated with maintaining outer membrane properties, which includes causing a mutation and culturing the bacteria:
[0304] In certain embodiments, the genetically engineered bacteria are E. coli
[0199] One way to secrete properly folded proteins in gram-negative bacteria—particularly those requiring disulphide bonds—is to target the periplasm in a bacterium with a destabilized outer membrane…Destabilizing the bacterial outer membrane to induce leakiness can be accomplished by deleting or mutagenizing genes responsible for tethering the outer membrane to the rigid peptidoglycan skeleton, including for example, lpp, ompC, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl. Lpp is the most abundant polypeptide in the bacterial cell existing at ˜500,000 copies per cell and functions as the primary ‘staple’ of the bacterial cell wall to the peptidoglycan. Silhavy, T. J., Kahne, D. & Walker, S. The bacterial cell envelope. Cold Spring Harb Perspect Biol 2, a000414 (2010)…Moreover, a combination of these mutations may synergistically enhance the leaky phenotype of the cell without major sacrifices in cell viability. Thus, in some embodiments, the engineered bacteria have one or more deleted or mutated membrane genes. In some embodiments, the engineered bacteria have a deleted or mutated lpp gene. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from ompA, ompA, and ompF genes. In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from tolA, tolB, and pal genes…In some embodiments, the engineered bacteria have one or more deleted or mutated gene(s), selected from lpp, ompA, ompA, ompF, tolA, tolB, pal, degS, degP, and nlpl genes.
Falb teaches methods of producing proteins in the engineered bacteria (see above). Falb does not teach the method for producing plasmid DNA. However, Falb does teach that the engineered E. coli have destabilized membranes, which the ordinary artisan would have predicted would make the bacteria more amenable to lysis (i.e., cell disruption to release cytoplasmic contents).
Xenopoulos teaches that, “The demand for plasmid DNA (pDNA) has vastly increased over the past decade…The challenge has always been poor productivity and delivery of pDNA. Plasmid DNA-based vaccines have traditionally required milligram scale of GMP-grade product for vaccination due to the relatively low efficacy and duration of gene expression. However, efforts to increase pDNA vaccine effectiveness are evolving in genetic manipulations of bacterial host” (Abstract). Xenopoulos further teaches that cell lysis is a key step of pDNA production, stating, “The objective of cell disruption is to release pDNA and remove solids”. However, while alkaline lysis is “the most common approach”, “there are challenges with alkaline lysis methods, especially on a large scale”. Xenopoulos notes that, “A completely different method for cell lysis involves the use of newly developed autolytic E. coli strains”.
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 method of preparing genetically engineered E. coli for protein secretion, as taught by Falb, for isolation of plasmid DNA instead, as taught by Xenopoulos. The ordinary artisan would have been motivated by Falb’s teachings that mutations of genes encoding proteins that tether the outer membrane to the peptidoglycan skeleton of gram negative bacteria lead to a destabilized membrane, combined with Xenopoulos’s teachings that there was an art-recognized need for further methods of cell lysis for plasmid purification, and that genetic engineering of autolytic E. coli strains – in other words, strains which are easier to lyse, such as those taught by Falb – was one such solution.
Regarding claim 2, Xenopoulos teaches dissolving the outer membrane after culturing (see above).
Regarding claims 3 and 4, Falb teaches that the gene regions are directly associated with maintaining physical and/or mechanical outer membrane properties (“tethers the outer membrane to the peptidoglycan skeleton”; see above).
Regarding claims 5-7, Falb teaches wherein the gene region/mutated amino acid sequence is at least one selected from lpp, ompC, ompA, ompF, tolA, tolB, and pal (see above).
Regarding claims 8-9, Falb teaches wherein the mutation is a complete disruption (deletion) or partial mutation (mutation; see above).
Regarding claim 11, Falb teaches wherein the mutation is a partial mutation of a structural protein (see above).
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Falb and Xenopoulos, as applied to claims 1-9, 11 and 19-20, further in view of U.S. PGPUB 20210301297 A1 to Dassler (cited on an IDS, hereinafter ‘Dassler’).
Falb and Xenopoulos render obvious the method of claim 9, from which the instantly rejected claim depends, as described above.
Falb and Xenopoulos do not teach wherein the mutation is a partial mutation of a signal peptide, specifically.
Dassler teaches a mutant strain of E. coli in which the N-terminal signal peptide of the lpp protein has a partial mutation, i.e., a mutation at position 14 (Abstract and below):
[0038] Preferably, the bacterial strain is characterized in that the N-terminal signal peptide contains, instead of glycine, some other proteinogenic amino acid at amino acid position 14 and is identical to the signal peptide of the wild-type Lpp protein at all other amino acid positions.
[0039] More preferably, the bacterial strain is characterized in that the proteinogenic amino acid at position 14 of the N-terminal signal peptide is aspartic acid. In this case, an aspartic acid residue is present at position 14 instead of the glycine residue (G14D exchange) and all other amino acid positions are identical to the signal peptide of the wild-type Lpp protein.
Dassler is analogous to Falb, in that it teaches mutant “leaky” strains of bacteria with reduced outer membrane stability:
[0006] Suitable for the extracellular production of a target protein are, for example, so-called “leaky” strains of E. coli, which discharge proteins situated in the periplasm into the medium to an increased extent owing to the absence of or the change in certain structural elements of the cell envelope.
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 method for producing plasmids as taught by Falb and Xenopoulos to include the specific lpp mutation taught by Dassler. Falb and Xenopoulos render obvious methods for producing plasmids entailing destabilization of the outer membrane structure by deletion or generic partial mutation of proteins such as lpp. Dassler, which is analogous art to Falb, provides a specific deletion of specific regions of lpp suitable to predictably achieve the outcome of outer membrane destabilization.
Claims 12-14 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Falb and Xenopoulos, as applied to claims 1-9, 11 and 19-20, further in view of U.S. PGPUB 2021/0269843 A1 to Koch (cited on an IDS, hereinafter ‘Koch’).
Falb and Xenopoulos render obvious the method of claim 11, wherein various outer membrane proteins are deleted or mutated, from which the instantly rejected claims depend, as described above.
Falb and Xenopoulos do not teach specific partial mutations, such as an amino acid substitution or disruption at the site of binding or contacting an outer membrane or peptidoglycan layer.
Koch teaches preparation of a mutant E. coli strain in which the N-terminal region of pal (the region responsible for anchoring to the outer cell membrane) is deleted, i.e., pal has a partial mutation, resulting in membrane destabilization (Abstract, paras [0016]-[0017]).
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 method as taught by Falb and Xenopoulos to comprise a step of causing the N-terminal deletion of pal. Falb and Xenopoulos render obvious methods for producing plasmids entailing destabilization of the outer membrane structure by deletion or generic partial mutation of proteins such as ompA, tolB, pal, lpp, etc. Koch, which is analogous art to Falb in that it concerns causing mutations which induce membrane destabilization of gram negative bacteria to improve protein secretion, provides a specific deletion of specific regions of pal suitable to predictably achieve the outcome of outer membrane destabilization.
Regarding claims 13-14, Koch teaches wherein the structural protein is pal, which has a mutation in an N-terminal region. (see above).
Regarding claim 18, Koch teaches wherein the E. coli is derived from K-12 (para [0018]).
Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Falb, Xenopoulos and Koch, as applied to claims 12-14 and 18, further in view of U.S. PGPUB 2021/0301297 A1 to Dassler (cited on an IDS, hereinafter ‘Dassler’).
Falb, Xenopoulos and Koch render obvious the method of claims 12-14, from which claim 15 depends, as described above.
Falb, Xenopoulos and Koch do not teach wherein the partially mutated structural protein is lpp, which has a mutation in a C-terminal region.
However, Falb does teach that, “a combination of these mutations may synergistically enhance the leaky phenotype of the cell without major sacrifices in cell viability” (see above).
Analogously to Koch and Falb, Dassler teaches a mutant E. coli for protein secretion (Abstract). Dassler further teaches a mutant form of lpp in which the C-terminal lysine present in the wild-type lpp is mutated (para [0031]) to prevent post-translational modification needed to ensure the full function of the lpp protein, namely the connection of the outer membrane to the peptidoglycan layer (para [0022]).
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 method as taught by Falb, Xenopoulos and Koch to further comprise a step of causing the C-terminal mutation of lpp, as taught by Dassler. Falb and Xenopoulos render obvious methods for producing plasmids entailing destabilization of the outer membrane structure by deletion or generic partial mutation of proteins such as ompA, tolB, pal, lpp, etc. Koch, which is analogous art to Falb in that it concerns causing mutations which induce membrane destabilization of gram negative bacteria to improve protein secretion, provides a specific deletion of specific regions of pal suitable to predictably achieve the outcome of outer membrane destabilization. Dassler teaches a further destabilizing mutation. Finally, Falb suggests causing multiple mutations to genes such as lpp and pal to synergistically enhance membrane destabilization, which would have motivated the ordinary artisan to combine these mutations.
Claim 16 is rejected under 35 U.S.C. 103 as being unpatentable over Falb, Xenopoulos, Koch and Dassler, as applied to claim 15, further in view of Choi (Choi et al. Distinct Roles of Outer Membrane Porins in Antibiotic Resistance and Membrane Integrity in Escherichia coli. Front. Microbiol., 29 April 2019.).
Falb, Xenopoulos, Koch and Dassler render obvious the method of claims 12-14, from which claim 15 depends, as described above. Falb, Xenopoulos, Koch and Dassler also teach that ompA may be deleted or mutated to generate “leaky” mutants for protein and/or plasmid production (see above).
Falb, Xenopoulos, Koch and Dassler do not teach wherein the partially mutated structural protein is specifically ompA with a mutation in a C-terminal region.
Choi teaches E. coli mutants with partially mutated ompA proteins with decreased membrane integrity:
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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 method as taught by Falb, Xenopoulos, Koch and Dassler to further comprise a C-terminal deletion of ompA, as taught by Choi. Falb and Xenopoulos render obvious methods for producing plasmids entailing destabilization of the outer membrane structure by deletion or generic partial mutation of proteins such as ompA, tolB, pal, lpp, etc. Koch and Dassler, which are both analogous to Falb as described above, provide various mutations which destabilize the outer membrane. Choi provides an additional destabilizing partial mutation by deleting the C terminal of ompA. Finally, as previously described, Falb suggests causing multiple mutations to genes such as ompa, lpp and pal to synergistically enhance membrane destabilization, which would have motivated the ordinary artisan to combine these mutations.
Claim 17 is rejected under 35 U.S.C. 103 as being unpatentable over Falb, Xenopoulos and Koch, as applied to claims 12-14 and 18 above, further in view of Sanders (Sanders et al. Phenotypic analysis of Eschericia coli mutants lacking L,D-transpeptidases. Microbiology (2013), 159, 1842-1852.; of record, cited on an IDS).
Falb, Xenopoulos and Dassler render obvious the method of claim 12, from which claim 17 depends, as described above. Falb, Xenopoulos and Koch also teach that various genes may be deleted or mutated to generate “leaky” mutants for protein and/or plasmid production (see above).
Falb, Xenopoulos and Koch do not teach wherein the structural protein is ybiS, ycfS or erfK. However, Koch does teach partial mutations to the pal gene, which codes for a structural protein.
Sanders teaches E. coli mutants with deletions of ybiS, ycfS and erfK, which leads to a loss of outer membrane stability (Abstract):
Escherichia coli has five genes encoding l,d-transpeptidases (Ldt) with varied functions. Three of these enzymes (YbiS, ErfK, YcfS) have been shown to cross-link Braun’s lipoprotein to the peptidoglycan (PG)…We report that a triple deletion mutant lacking ybiS, erfK and ycfS is hypersusceptible to the metal-chelating agent EDTA, leaks periplasmic proteins and is resistant to the toxic effect of d-methionine….These data demonstrate that loss of the E. coli Ldt enzymes involved with coupling the PG to Braun's lipoprotein resulted in the loss of outer membrane stability
Sanders further notes that the catalytic sites and the catalytic residue for all three genes were known:
These Ldts belong to the YkuD superfamily (alternatively the ErfK/YcfS/YnhG family), based on a highly conserved set of amino acids that constitute the catalytic domain, characterized by an active site cysteine residue, which differs from classical PBPs, which generally rely on a serine residue for catalysis (p. 1843).
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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 method as taught by Falb, Xenopoulos and Koch to further comprise a partial mutation of the conserved active site cysteines of any of ybiS, ycfS or erfK, as taught by Sanders. Falb and Xenopoulos render obvious methods for producing plasmids entailing destabilization of the outer membrane structure by deletion or generic partial mutation of proteins involved in outer membrane stability. Koch teaches that partial mutations, as opposed to full deletions, of proteins involved in membrane stability (such as pal) destabilize the outer membrane. Sanders teaches an alternative or additional mutation by teaching that deletion of ybiS, ycfS and erfK leads to outer membrane destabilization, and that all of those proteins comprise active site residues which may be deleted to achieve the same effect (i.e., abrogation of the enzymes’ activity).
U.S. PGPUB 2024/0052393- Yamagiwa
Claims 1-11 and 18-20 are rejected under 35 U.S.C. 103 as being unpatentable over U.S. PGPUB 20240052393 to Yamagiwa in view of Xenopoulos (Xenopoulos et al. Production and purification of plasmid DNA vaccines: is there scope for further innovation? Expert Rev. Vaccines 13(12), 1537–1551 (2014).).
Note: the applied reference has a common applicant and inventors with the instant application. However, it includes at least one inventor not named in the present application (Yamagiwa). Based upon the earlier effectively filed date of the reference (3/22/21), it constitutes prior art under 35 U.S.C. 102(a)(2). This rejection under 35 U.S.C. 102(a)(2) might be overcome by: (1) a showing under 37 CFR 1.130(a) that the subject matter disclosed in the reference was obtained directly or indirectly from the inventor or a joint inventor of this application and is thus not prior art in accordance with 35 U.S.C. 102(b)(2)(A); (2) a showing under 37 CFR 1.130(b) of a prior public disclosure under 35 U.S.C. 102(b)(2)(B) if the same invention is not being claimed; or (3) a statement pursuant to 35 U.S.C. 102(b)(2)(C) establishing that, not later than the effective filing date of the claimed invention, the subject matter disclosed in the reference and the claimed invention were either owned by the same person or subject to an obligation of assignment to the same person or subject to a joint research agreement.
Note: It is noted that the instant application claims foreign priority to foreign application JP2021-117545, filed 07/16/2021. However, Applicant cannot rely upon the certified copy of the foreign priority application to overcome this rejection because a translation of said application has not been made of record in accordance with 37 CFR 1.55. When an English language translation of a non-English language foreign application is required, the translation must be that of the certified copy (of the foreign application as filed) submitted together with a statement that the translation of the certified copy is accurate. See MPEP §§ 215 and 216.
Regarding claims 1, 19 and 20 Yamagiwa teaches a method of preparing an E. coli strain which has a mutation in a gene associated with maintenance of the outer membrane structure (Abstract). Yamagiwa also teaches culturing the E. coli and isolating a protein after culturing:
[0004] When a protein is to be produced using E. coli, in general, a protein is produced in a cell. Accordingly, it is necessary to separate a culture medium into a microbial cell and a culture supernatant therein after fermentation culture and then to carry out periplasmic extraction, cell disruption, recovery of an inclusion body, or the like.
Yamagiwa does not teach the method used for plasmid production. The prior art and the instantly claimed invention differ in the use to which the mutant E. coli is put: protein production versus plasmid production.
However, Yamagiwa does teach that the mutations result in a higher recovery efficiency after osmotic shock:
[0132] As a result of comparison between the wild-type strain and the E. coli strain having modification in the gene associated with outer membrane formation, the recovery efficiency from the E. coli strains having modification in the gene associated with outer membrane formation by the cold osmotic shock method was found to be improved with the use of strains lacking ompA, tolA, mepS, nlpI, arcA, cyoA, dppA, ecnB, mrcA, mrcB, oppA, and slyB.
Yamagiwa also teaches that the method of extracting the protein is based on any one of the techniques: osmotic shock, treatment with a chelate agent, treatment with a cell-wall degrading enzyme, treatment with a chaotropic, heating, treatment with a surfactant, and freeze-thawing (claim 10).
Xenopoulos teaches that, “The demand for plasmid DNA (pDNA) has vastly increased over the past decade…The challenge has always been poor productivity and delivery of pDNA. Plasmid DNA-based vaccines have traditionally required milligram scale of GMP-grade product for vaccination due to the relatively low efficacy and duration of gene expression. However, efforts to increase pDNA vaccine effectiveness are evolving in genetic manipulations of bacterial host” (Abstract). Xenopoulos further teaches that cell lysis is a key step of pDNA production, stating, “The objective of cell disruption is to release pDNA and remove solids”. However, while alkaline lysis is “the most common approach”, “there are challenges with alkaline lysis methods, especially on a large scale”. Xenopoulos notes that, “A completely different method for cell lysis involves the use of newly developed autolytic E. coli strains”.
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 method of preparing genetically engineered E. coli for protein secretion, as taught by Yamagiwa, for isolation of plasmid DNA instead, as taught by Xenopoulos. The ordinary artisan would have been motivated by Yamagiwa’s teachings that certain mutations of certain genes involved in maintenance of the outer membrane improved recovery efficiency of the molecule of interest after osmotic shock, combined with Xenopoulos’s teachings that there was an art-recognized need for further methods of cell lysis for plasmid purification, and that genetic engineering of autolytic E. coli strains – in other words, strains which are easier to lyse, such as those taught by Yamagiwa – was one such solution.
Regarding claim 2, Yamagiwa teaches wherein step (c) is a step of recovering the desired plasmid by dissolving the outer membrane of the bacterial cell after culturing (see above).
Regarding claims 3-7, Yamagiwa teaches wherein the gene region is directly associated with maintaining membrane properties and is at least one of the lpp, ompA, tolA, mepS, nlpI, arcA, bamD, cyoA, dppA, ecnB, mrcA, mrcB, oppA, or slyB gene:
[0039] The term “gene associated with outer membrane formation” used herein refers to a gene of E. coli associated with maintenance of the outer membrane structure. More specifically, the term “gene associated with outer membrane formation” refers to an E. coli gene associated with expression of a protein associated with maintenance of the outer membrane structure of E. coli; i.e., a nucleic acid (DNA or RNA) of E. coli. A gene associated with outer membrane formation is not particularly limited, provided that such gene is associated with maintenance of the outer membrane structure. For example, such gene is the pal, lpp, ompA, tolA, mepS, nlpI, arcA, bamD, cyoA, dppA, ecnB, mrcA, mrcB, oppA, or slyB gene
2. The method according to claim 1, wherein the gene associated with maintenance of the outer membrane structure is at least one gene selected from the group consisting of pal, lpp, ompA, tolA, mepS, nlpI, arcA, bamD, cyoA, dppA, ecnB, mrcA, mrcB, oppA, and slyB.
3. The method according to claim 1, wherein two or more genes of the E. coli selected from the group consisting of pal, lpp, ompA, tolA, mepS, nlpI, arcA, bamD, cyoA, dppA, ecnB, mrcA, mrcB, oppA, and slyB are modified.
Regarding claims 8-11, Yamagiwa teaches wherein the modifications are complete or partial disruptions of a signal peptide or structural gene, which is interpreted as encompassing genes encoding structural proteins (Yamagiwa claims 5-8):
5. The method according to claim 1, wherein the modification is complete deletion.
6. The method according to claim 1, wherein the modification is partial mutation.
7. The method according to claim 6, wherein the modification is partial mutation of a signal sequence.
8. The method according to claim 6, wherein the modification is partial mutation of a structural gene.
Regarding claim 18, Yamagiwa teaches that the E. coli is derived from the K12 strain:
9. The method according to claim 1 , wherein the E. coli is derived from the B strain or the K12 strain.
Claims 12-14 are rejected under 35 U.S.C. 103 as being unpatentable over Yamagiwa and Xenopoulos, as applied to claims 1-11 and 19-20 above, further in view of U.S. PGPUB 2021/0269843 A1 to Koch (cited on an IDS, hereinafter ‘Koch’).
Yamagiwa and Xenopoulos render obvious the method of claim 11, wherein various outer membrane proteins are deleted or mutated, from which the instantly rejected claims depend, as described above.
Yamagiwa and Xenopoulos do not teach specific partial mutations, such as an amino acid substitution or disruption specifically at the site of binding or contacting an outer membrane or peptidoglycan layer.
However, Yamagiwa teaches that the proteins associated with outer membrane maintenance may be modified in various ways, including partial deletions of the coding region or the introduction of missense mutations:
[0059] When a part of the coding region of the amino acid sequence of the protein encoded by the target gene is to be deleted as the partial disruption, any region, such as the N-terminal region, the internal region, the C-terminal region, or other regions may be deleted. The reading frames of the upstream and downstream sequences of the region to be deleted may be inconsistent. At least a part of the coding region and/or the expression control sequence of the amino acid sequence of the target gene in the genomic DNA may be deleted, such as a region consisting of a number of nucleotides that is, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the total number of the nucleotides constituting the coding region and/or the expression control sequence. Alternatively, 100% of the region can be deleted (complete deletion). For example, a genetically-modified E. coli strain lacking at least a region from the start codon to the stop codon of the target gene of genomic DNA may be used.
[0061] Other examples of modification of the target gene of genomic DNA in the E. coli strain include introduction of missense mutation, introduction of a stop codon (nonsense mutation), and introduction of frameshift mutation via addition or deletion of 1 or 2 nucleotides into or from the amino acid sequence coding region of the gene in genomic DNA.
Koch teaches preparation of a mutant E. coli strain in which the N-terminal region of pal (the region responsible for anchoring to the outer cell membrane) is deleted, i.e., pal has a partial mutation, resulting in membrane destabilization (Abstract, paras [0016]-[0017]).
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 method as taught by Yamagiwa and Xenopoulos to comprise a step of causing the N-terminal deletion of pal. Yamagiwa and Xenopoulos render obvious methods for producing plasmids entailing destabilization of the outer membrane structure by deletion or generic partial mutation of proteins such as ompA, tolB, pal, lpp, etc. Yamagiwa further suggests partial disruptions of the target genes. Koch, which is analogous art to Yamagiwa in that it concerns causing mutations which induce membrane destabilization of gram negative bacteria to improve protein secretion, provides a specific deletion of specific regions of pal suitable to predictably achieve the outcome of outer membrane destabilization.
Regarding claims 13-14, Koch teaches wherein the structural protein is pal, which has a mutation in an N-terminal region. (see above).
Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Yamagiwa, Xenopoulos and Koch, as applied to claims 12-14, further in view of U.S. PGPUB 2021/0301297 A1 to Dassler (cited on an IDS, hereinafter ‘Dassler’).
Yamagiwa, Xenopoulos and Koch render obvious the method of claims 12-14, from which claim 15 depends, as described above.
Yamagiwa, Xenopoulos and Koch do not teach wherein the partially mutated structural protein is lpp, which has a mutation in a C-terminal region.
Analogously to Koch and Yamagiwa, Dassler teaches a mutant E. coli for protein secretion (Abstract). Dassler further teaches a mutant form of lpp in which the C-terminal lysine present in the wild-type lpp is mutated (para [0031]) to prevent post-translational modification needed to ensure the full function of the lpp protein, namely the connection of the outer membrane to the peptidoglycan layer (para [0022]).
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 method as taught by Yamagiwa, Xenopoulos and Koch to further comprise a step of causing the C-terminal mutation of lpp, as taught by Dassler. Yamagiwa, Xenopoulos and Koch render obvious methods for producing plasmids entailing destabilization of the outer membrane structure by deletion or generic partial mutation of proteins such as ompA, tolB, pal, lpp, etc. Koch, which is analogous art to Yamagiwa in that it concerns causing mutations which induce membrane destabilization of gram negative bacteria to improve protein secretion, provides a specific deletion of specific regions of pal suitable to predictably achieve the outcome of outer membrane destabilization. Dassler teaches a further destabilizing mutation which would predictably have achieved the same effect.
Claim 16 is rejected under 35 U.S.C. 103 as being unpatentable over Yamagiwa, Xenopoulos, Koch and Dassler, as applied to claim 15, further in view of Choi (Choi et al. Distinct Roles of Outer Membrane Porins in Antibiotic Resistance and Membrane Integrity in Escherichia coli. Front. Microbiol., 29 April 2019.).
Yamagiwa, Xenopoulos, Koch and Dassler render obvious the method of claims 12-14, from which claim 15 depends, as described above.
Yamagiwa, Xenopoulos, Koch and Dassler do not teach wherein the partially mutated structural protein is specifically ompA with a mutation in a C-terminal region.
Choi teaches E. coli mutants with partially mutated ompA proteins with decreased membrane integrity:
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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 method as taught by Yamagiwa, Xenopoulos, Koch and Dassler to further comprise a C-terminal deletion of ompA, as taught by Choi. Yamagiwa and Xenopoulos render obvious methods for producing plasmids entailing destabilization of the outer membrane structure by deletion or generic partial mutation of proteins such as ompA, tolB, pal, lpp, etc. Koch and Dassler, which are both analogous to Falb as described above, provide various mutations which destabilize the outer membrane. Choi provides an additional destabilizing partial mutation by deleting the C terminal of ompA, which would predictably have led to the same effect of membrane destabilization.
Claim 17 is rejected under 35 U.S.C. 103 as being unpatentable over Yamagiwa, Xenopoulos and Koch as applied to claims 12-14 above, further in view of Sanders (Sanders et al. Phenotypic analysis of Eschericia coli mutants lacking L,D-transpeptidases. Microbiology (2013), 159, 1842-1852.; of record, cited on an IDS).
Yamagiwa, Xenopoulos and Koch render obvious the method of claim 12, from which claim 17 depends, as described above.
Yamagiwa, Xenopoulos and Koch do not teach wherein the structural protein is ybiS, ycfS or erfK. However, Koch does teach partial mutations to the pal gene, which codes for a structural protein.
Sanders teaches E. coli mutants with deletions of ybiS, ycfS and erfK, which leads to a loss of outer membrane stability (Abstract):
Escherichia coli has five genes encoding l,d-transpeptidases (Ldt) with varied functions. Three of these enzymes (YbiS, ErfK, YcfS) have been shown to cross-link Braun’s lipoprotein to the peptidoglycan (PG)…We report that a triple deletion mutant lacking ybiS, erfK and ycfS is hypersusceptible to the metal-chelating agent EDTA, leaks periplasmic proteins and is resistant to the toxic effect of d-methionine….These data demonstrate that loss of the E. coli Ldt enzymes involved with coupling the PG to Braun's lipoprotein resulted in the loss of outer membrane stability
Sanders further notes that the catalytic sites and the catalytic residue for all three genes were known:
These Ldts belong to the YkuD superfamily (alternatively the ErfK/YcfS/YnhG family), based on a highly conserved set of amino acids that constitute the catalytic domain, characterized by an active site cysteine residue, which differs from classical PBPs, which generally rely on a serine residue for catalysis (p. 1843).
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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 method as taught by Yamagiwa, Xenopoulos and Koch to further comprise a partial mutation of the conserved active site cysteines of any of ybiS, ycfS or erfK, as taught by Sanders. Yamagiwa amd Xenopoulos render obvious methods for producing plasmids entailing destabilization of the outer membrane structure by deletion or generic partial mutation of proteins involved in outer membrane stability. Koch teaches that partial mutations, as opposed to full deletions, of proteins involved in membrane stability (such as pal) destabilize the outer membrane. Sanders teaches an alternative or additional mutation by teaching that deletion of ybiS, ycfS and erfK leads to outer membrane destabilization, and that all of those proteins comprise active site residues which may be deleted to achieve the same effect (i.e., abrogation of the enzymes’ activity).
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 1-10 and 18-20 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 3-9 and 16 of copending Application No. 18/466,984 in view of Falb and Xenopoulos.
Regarding instant claims 1, 19 and 20, although the claims at issue are not identical because while copending claim 1 recites methods of producing proteins which comprise causing a mutation in a gene region associated with maintaining outer membrane properties of E. coli, culturing the bacterium, and recovering the proteins instead of recovering a plasmid, they are not patentably distinct from each other because Falb and Xenopoulos render obvious methods with those strains and steps which are used to produce and recover plasmids instead of proteins, as already described in the rejections under 35 U.S.C. 103 above.
Regarding instant claims 3-7, copending claims 3-4 recite that the genes are selected from pal, ompA, tolA and lpp. Instant claim 5 evidences that these genes are inherently directly associated with maintaining outer membrane properties. Therefore, the recitation of those specific genes anticipates the limitations of 3-5 as well as the limitations of 5-7 which recite those same genes.
Regarding instant claims 8-9, copending claims 5-6 recite wherein the modification is either a complete or partial deletion.
Regarding instant claim 10, copending claim 7 recites wherein the modification is partial mutation of a signal sequence (i.e., signal peptide), and copending claim 16 recites various species of sequences encoding signal peptides, which anticipate the generic signal peptide instantly claimed.
Regarding instant claim 18, copending claim 9 recites wherein the E. coli is derived from B or K12.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 1-20 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 46-52, 55, 59-60, 62, 65-69 of copending Application No. 17/792,011.
Regarding instant claims 1-2 and 19-20, copending claims 46 and 47 recite methods of producing plasmids which involve culturing E. coli strains with mutations in at least one gene involved in envelope (i.e., outer membrane) integrity, followed by recovering the plasmid via lysis (i.e., dissolving the outer membrane), while copending claim 48 merely specifies what is encoded by the plasmid.
Regarding instant claims 3-17, copending claims 49-52, 55, 59-60, 62, 65-69 merely recite various species of mutations in species of genes which are generically recited in the instant claims.
Copending claims 34-45 and 54 recite a bacterium encoding various specific mutations in various specific genes recited in the instant claims, as described above for the copending process claims. As the copending process claims make clear by reciting limitations in which structurally similar strains are used, it would have been obvious to use said mutant E. coli in a process of plasmid purification.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Conclusion
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/AMANDA M ZAHORIK/Examiner, Art Unit 1636
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