DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 3/2/2026 has been entered.
Status of Claims
Receipt of Arguments/Remarks filed on 3/2/2026 is acknowledged. New claim 20 was added. Claims 1-9 and 11-20 are pending. Claims 11-19 are withdrawn as being directed to a non-elected invention.
Information Disclosure Statement
The information disclosure statements (IDS) filed on 12/8/2025, 1/16/2026, 4/7/2026, 8/19/2026, 9/15/2026, and 9/24/2026 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 3 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 3 recites that the genetic modification (1), increasing expression of a kpsS gene as compared to expression of the kpsS gene of an unmodified strain, is achieved by modifying the expression control region of the kpsS gene or by increasing the copy number of kpsS. Claim 1, upon which claim 3 depends, recites that the expression of kpsC is not increased compared to the WT strain. kpsS and kpsC are in the same operon and thus have the same expression control region (see instant specification pp. 14-15 para. 73).
As kpsS and kpsC are on the same operon and have the same expression control region, it is unclear how a modification to the expression control region would result in increased expression of kpsS but not kpsC. The instant specification further states that “an effect of particularly improving production of heparosan is obtained by increasing expression of only the kpsS gene among them. Accordingly, as the genetic modification that increases expression of the kpsS gene, a genetic modification for increasing the copy number of the kpsS gene is particularly preferable” (instant specification p. 15 para. 73). This statement in the specification appears to indicate that if only the kpsS expression is increased, the modification for increasing the copy number would be used rather than a modification to the expression control region.
Therefore, claim 3 is indefinite as it is unclear how only kpsS expression, and not kpsC expression, would be increased if the only modification is modifying the expression control region.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1, 3, 6-7, and 20 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Bronner et al., Journal of bacteriology. 1993 Sep;175(18):5984-92.; and as evidenced by Pazzani et al., Journal of bacteriology. 1993 Sep;175(18):5978-83, and NCBI GenBank X74567.1.
Regarding claim 1, Bronner teaches the production of capsular K5 polysaccharide (or heparosan, see instant specification p. 1 para. 2-5 and Bronner p. 5984 para. 1 showing K5 polysaccharide structure) by Escherichia coli (Bronner p. 5984 para. 1). Bronner teaches E. coli strain LE392(pGB118::1), which expresses all of the region 1 E. coli K5 capsule gene cluster except for kpsS (Bronner Table 2). LE392(pGB118::1) is capable of producing capsular polysaccaharide, or heparosan (Bronner p. 5988 para. 1; Fig. 3). Bronner teaches that the LE392(pGB118::1) strain is modified with a genetic modification to increase expression of the kpsS gene, by expressing plasmid pHC4 which has a kpsS insert, yielding strain LE392(pGB118::1/pHc4) (Bronner p. 5988 para. 2; Tables 1 and 2). The modification to increase kpsS expression in the LE392(pGB118::1) strain results in increased capsule (heparosan) production, i.e. fully encapsulated bacteria (Bronner p. 5988 para. 2; Fig. 3 showing amount of capsule in a fully encapsulated bacteria compared to pGB118::1 strain). Strain LE392(pGB118::1/pHc4) is not modified to increase expression of kpsC gene compared to LE392(pGB118::1) (Bronner Tables 1 and 2). Bronner teaches culturing the modified strain LE392(pGB118::1/pHc4) in a medium to produce heparosan in the medium (Bronner p. 5985 “Bacteria and cultivation”; p. 5986 “Isolation of the K5 polysaccharide”).
Regarding claim 3, the modification to increase expression of kpsS is increasing the copy number of the kpsS gene, as the original strain LE392(pGB118::1) does not express kpsS, so adding a plasmid which contains kpsS increases the copy number compared to the unmodified strain (Bronner Tables 1 and 2; p. 5987 first full para.).
Regarding claim 6, the bacterium is Escherichia coli (Bronner p. 5984 para. 3; p. 5985 “Bacteria and cultivation”).
Regarding claim 7, Bronner teaches that the region 1 sequence, comprising kpsS, is set forth in reference 34, Pazzani et al. (Bronner p. 5984 para. 3). The sequence of Pazzani has GenBank accession number X74567 (see Pazzani p. 5979 “Nucleotide sequence accession number”). SEQ ID NO: 33 is 99.9% identical to the kpsS gene of GenBank accession X74567 (see NCBI X74567.1 reference, sequence alignment in OA appendix). Regarding the limitation “having a property of increasing a heparosan-producing ability of the bacterium of the genus Escherichia having the heparosan producing ability when an expression level is increased in the bacterium”, this is a functional limitation directed to a property of the kpsS gene with a sequence according to SEQ ID NO: 33. As Bronner teaches a gene with the same structure, 99.9% sequence identity, this gene must have the same functional properties, absent any unclaimed essential features. Further, as discussed above, Bronner teaches that increasing expression of kpsS leads to increased capsular polysaccharide production.
Regarding claim 20, the bacterium which is modified with a genetic modification to increase kpsS production is LE392(pGB118::1) (Bronner p. 5988 para. 2; Tables 1 and 2). This strain originally has a capsule (heparosan) producing ability (Bronner p. 5988 para. 1; Fig. 3).
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 2, 4, and 8 are rejected under 35 U.S.C. 103 as being unpatentable over Bronner et al. as applied to claims 1, 3, and 6-7 above, in view of Zhang et al., Metabolic engineering. 2012 Sep 1;14(5):521-7 and as evidenced by UC San Diego “Escherichia coli Strains and NIH Guidelines”.
Bronner teaches the method of claim 1 as discussed above. Bronner does not teach that the bacterium of the genus Escherichia further has at least one of the following genetic modifications (2) and (3) as recited in claim 2.
Regarding claim 2, Zhang teaches a method for producing the capsular polysaccharide heparosan by culturing metabolically engineered Escherichia coli that has the ability to produce heparosan (Zhang “Abstract”). Zhang teaches that E. coli K5 is a strain that is used for heparosan production but it is a pathogenic bacterium potentially causing urinary tract infections and it would be advantageous to develop an alternative source of heparosan that avoids the potential crisis (Zhang p. 522 para. 2). Zhang teaches the overexpression of genes kfiA, kfiB, kfiC, and kfiD, which are part of the metabolic synthesis pathway for heparosan in E. coli K5 (Zhang Fig. 1; p. 522 para. 2-3).
Regarding claim 4, Zhang teaches increasing the copy number of kfiA, kfiB, kfiC, and kfiD by introducing these genes into an E. coli strain using medium and high copy plasmids, including pETDuet-1 and pRSFDuet-1 (Zhang pg. 523 Section 2.2).
Regarding claim 8, Zhang teaches an E. coli strain expressing kfiA, kfiB, kfiC, and kfiD derived from E. coli strain K5 (Zhang pg. 523 Section 2.1). The kfiA gene from E. coli K5 is 100% identical to instant SEQ ID NO: 34 (see sequence alignment in OA appendix). Similarly, kfiB, kfiC, and kfiD from E. coli K5 are 100% identical to SEQ ID NOs: 35, 36, and 37 (see sequence alignment in OA appendix). Regarding the limitation “having a property of increasing a heparosan-producing ability of the bacterium of the genus Escherichia having the heparosan producing ability when an expression level is increased in the bacterium”, this is a functional limitation directed to a property of the genes with a sequence according to SEQ ID NO: 34-37. As Zhang teaches genes with this same structure, they must be capable of performing the same function.
It would have been obvious for a skilled artisan to modify the strain used in the method of Bronner and incorporate additional genetic modifications to overexpress kfiA, kfiB, kfiC, and kfiD in addition to kpsS. Both Bronner and Zhang teach culturing E. coli strains to produce heparosan, or capsular polysaccharide. Zhang teaches that the expression of kfiA, kfiB, kfiC, and kfiD is beneficial for heparosan production, as these genes are part of the metabolic synthesis pathway for heparosan in E. coli K5. It would have been obvious that a strain of E. coli which has been modified to increase expression of kpsS as taught by Bronner could be additionally modified with genes in the heparosan biosynthetic pathway.
A person of ordinary skill in the art would have been motivated to make these modifications to the strain of Bronner because Zhang shows that expressing these genes in E. coli increases the production of heparosan (Zhang Fig. 2). Zhang additionally teaches that E. coli K5, which is the E. coli strain that is typically used for heparosan production, is pathogenic and potentially causes urinary tract infection (Zhang p. 526 Section 4 para. 1). Thus, exogenous expression of these genes in other E. coli strains which are not pathogenic is advantageous in that it allows for heparosan production strains which are safer (Zhang p. 526 Section 4 para. 6). Therefore, a skilled artisan would have been motivated to modify the strain as taught by Bronner, LE392(pGB118::1/pHc4), which produces heparosan as discussed above. E. coli LE392 is a non-pathogenic, K-12 derived laboratory strain (see UC San Diego ref). Therefore, a skilled artisan would have been motivated to modify this non-pathogenic, heparosan-producing strain with additional genetic modifications as taught by Zhang that are beneficial for heparosan production, and culture this strain in a method of producing heparosan.
A skilled artisan would have had a reasonable expectation of success in making this modification because Zhang teaches that the kfi genes can be exogenously expressed in E. coli and result in increased heparosan production. A skilled artisan could therefore expect success in exogenously expressing these genes in a different E. coli strain as taught by Bronner, used in a method of producing heparosan (capsular polysaccharide).
Claims 5 and 9 are rejected under 35 U.S.C. 103 as being unpatentable over Bronner in view of Zhang as applied to claims 2, 4, and 8 above, and further in view of Kalamorz et al., Molecular microbiology; 65(6):1518-33, and as evidenced by NCBI Reference Sequence: NC_000913.3:3347115-3347969 and UC San Diego “Escherichia coli Strains and NIH Guidelines”.
Bronner and Zhang teach the method of claim 2 as set forth above. These references do not teach a deletion of the yhbJ gene as recited in instant claim 5 having the sequence according to SEQ ID NO: 38 as recited in instant claim 9.
Regarding claim 5, Kalamorz teaches that E. coli with yhbJ deleted overproduces glucosamine-6-phosphate (GlcN-6-P) synthase (GlmS), which catalyzes the conversion of fructose-6-phosphate to GlcN-6-P (Kalamorz pg. 1518 “Introduction”).
It would have been obvious to a skilled artisan, before the effective filing date, to combine the teachings of Bronner and Zhang with the teachings of Kalamorz, utilizing an E. coli strain with yhbJ deleted for the production of heparosan. Bronner and Zhang teach methods of cultivating genetically modified E. coli strains for heparosan biosynthesis. It would have been obvious to a skilled artisan that an additional genetic modification, deletion of the yhbJ gene, could be made in the strain used in the method of Bronner and Zhang.
A person of ordinary skill in the art would have been motivated to incorporate a yhbJ deletion in the strain used in the method of Bronner and Zhang because Kalamorz teaches that deletion of yhbJ results in GlmS overproduction. Zhang teaches that the metabolic synthesis pathway for heparosan involves conversion of fructose-6-phosphate to GlcN-6-P, which would be catalyzed by GlmS as taught by Kalamorz (Zhang Fig. 1). Therefore, a yhbJ deletion strain would be considered advantageous for producing heparosan, as GlmS overproduction would allow for conversion of fructose-6-phosphate to GlcN-6-P, which is a precursor for heparosan biosynthesis.
A skilled artisan would have a reasonable expectation of success in making this combination to achieve the predictable outcome of increased heparosan production in a strain with yhbJ deleted, given the role of this gene in negatively regulating GlmS, which catalyzes the production of a heparosan precursor, and the successful use of a genetically modified E. coli strain in a method of heparosan production as taught by Bronner and Zhang.
Regarding claim 9, SEQ ID NO: 38 is 99% identical to the yhbJ gene of E. coli K-12 (see NCBI ref, sequence alignment in OA appendix). The strain taught by Bronner is LE392, which is a K-12 derivative (see UCSD ref). Therefore, the yhbJ gene in this strain would have a sequence that is 99% identical to instant SEQ ID NO: 38. As discussed above, it would have been obvious to make this deletion in a strain as taught by Bronner and Zhang, used in a method of heparosan production. Regarding the limitation “having a property of increasing a heparosan-producing ability of the bacterium of the genus Escherichia having the heparosan producing ability when an expression level is decreased in the bacterium”, this is a functional limitation directed to a property of the yhbJ gene with a sequence according to SEQ ID NO: 38. As the yhbJ gene in the strain taught by Bronner is 99% identical to SEQ ID NO: 38, it must be capable of performing the same function.
Response to Arguments
The rejection of claims 1-4 and 6-8 under 35 U.S.C. § 103 over Zhang and Willis; and the rejection of claims 5 and 9 under 35 U.S.C. § 103 over Zhang, Willis, and Kalamorz has been withdrawn. However, upon further consideration, new grounds of rejection of claims 1-9 and 20 are made as set forth above. Given these new grounds of rejection, the arguments presented regarding claims rejected under 35 U.S.C. § 103 over Zhang in view of Willis and/or Kalamorz are moot. However, responses to pertinent arguments are set forth below.
Applicant argues that the invention as claimed is not obvious in view of unexpected beneficial results, specifically that the amount of heparosan accumulated in the culture medium of an Escherichia bacterium is significantly increased by increasing expression of only kpsS, and not kpsC. Applicant argues that even assuming a person of ordinary skill in the art would have been motivated to culture a strain having overexpressed kpsS and not kpsC, there would not have been a reasonable expectation that the heparosan production would be increased because kpsS and kpsC have the same function.
In response to this argument, it is noted that the method of claim 1 is presently rejected under 35 U.S.C. § 102 over Bronner. Bronner teaches culturing a strain with a modification to increase kpsS and not kpsC, and teaches that heparosan, or capsular polysaccharide, is produced as discussed above. Therefore, a method as claimed is anticipated by Bronner, and must have the same results as the instant method, absent any unclaimed essential features.
Further, it is noted that the instant claims require culturing any Escherichia strain that can produce heparosan, has a modification to increased kpsS expression, and does not have a modification to increase kpsC expression. The claims do not require a specific amount of heparosan to be produced, specific media or culture conditions, or any specific bacterial parent strains. Therefore, as discussed above, the teachings of Bronner anticipate the method of claims 1, 3, 6-7, and 20.
Conclusion
Claims 1-9 and 20 are rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to EMILY F EIX whose telephone number is (571)270-0808. The examiner can normally be reached M-F 8am-5pm ET.
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/EMILY F EIX/Examiner, Art Unit 1653
/JENNIFER M.H. TICHY/Primary Examiner, Art Unit 1653
