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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 1/30/2026 has been entered.
Status of Claims
Receipt of Arguments/Remarks filed on 1/30/2026 is acknowledged. Claims 1 and 5-9 are pending. Claims 1, 6-7, and 9 were amended. Claim 4 was cancelled. Claims 5-9 are withdrawn as being directed to a non-elected invention.
Withdrawn Rejections
The rejections on the grounds of nonstatutory double patenting are withdrawn.
Priority
The certified copy of the foreign priority document is not in English. Should applicant desire to obtain the benefit of foreign priority under 35 U.S.C. 119(a)-(d) prior to declaration of an interference, a certified English translation of the foreign application must be submitted in reply to this action. 37 CFR 41.154(b) and 41.202(e).
Failure to provide a certified translation may result in no benefit being accorded for the non-English application. The effective filing date of the claims is considered to be 4/18/2022.
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.
Claim 1 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al., Systems Microbiology and Biomanufacturing; 1:291-301 (published 2/20/2021) in view of Jennewein et al., EP 2927316A1 (published 7/10/2015), and Zhu et al., Journal of Agricultural and Food Chemistry; 69(12):3702-11 (published 3/23/2021); and as evidenced by Millipore Sigma “Express yourself Competent cells, vectors, media and antibiotics for recombinant protein production” and Aryal “Characteristic of Genetic Code”.
Regarding claim 1, Zhang teaches metabolic engineering of Escherichia coli for the production of Lacto‑N‑neotetraose (LNnT) (Zhang Abstract). Zhang teaches an E. coli K12 strain that expresses lgtA and lgtB from Neisseria meningitidis using a pCDFDuet plasmid (Zhang p. 295 “Construction of the biosynthesis pathway of LNnT”). Zhang teaches optimizing the yield of LNnT by trying different Duet plasmids, including pETDuet and pRSFDuet (Zhang p. 296 “Increase LNnT yield by optimizing the expression of lgtA and lgtB”). Zhang teaches that the strain has lacZ knocked out and lacY overexpressed (Zhang p. 296 para. 3). Zhang further teaches strains with pgm or galE overexpressed, which are used to produce LNnT (Zhang p. 297 “Improved the synthesis of LNnT by the fine‑tuning of the gene expression”; Table 1).
Thus, Zhang teaches a genetically engineered bacterium comprising a pCDFDuet plasmid that expresses lgtA and lgtB from Neisseria meningitidis sequentially, overexpresses lacY and pgm or galE from E. coli K12, and has lacZ knocked out. As the strain used by Zhang is E. coli K12, it is considered that lacY, pgm, galE, and lacZ would have sequences which are identical to SEQ ID NOs: 28, 24, 25, and 23, respectively, as these sequences are from E. coli K12 (see instant specification para. 7).
Zhang does not teach expression of galETK in addition to pgm, lacY, lgtA and lgtB in the same strain; that lacY is expressed on a pETDuet-1 plasmid; a pRSFDuet-1 plasmid expressing pgm and galE, galT, and galK with sequences according to SEQ ID NOs: 25-27 sequentially, or that the sequences of lgtA and lgtB are SEQ ID NO: 1 and 2 respectively.
Regarding the expression of galETK in addition to pgm, lacY, lgtA and lgtB, Jennewein teaches E. coli strains developed for the production of human milk oligosaccharides (HMOs) such as lacto-N-neotetraose (LNnT) with a deletion of lacZ and integrations of the gale operon galETKM, as well as lacY and lgtA, referred to as E. coli BL21 (DE3) 724 (Jennewein pg. 17 Table 4). Jennewein additionally teaches batch fermentation of E. coli BL21 (DE3) 724 harboring plasmids pCDF-galE and plNTmalE-lgtB (Jennewein pg. 18 para. 132). Jennewein teaches that phosphoglucomutase (pgm) is an enzyme in the LNnT synthesis pathway (Jennewein Fig. 1 C (6); pg. 13 para 102). Jennewein teaches that galE, galT, galK, and lacY, are all from E. coli K-12 (Jennewein pg. 14 para. 108; pg. 16 para. 124-129). It is expected that since the genes taught by Jennewein are derived from K-12, they would have sequences identical to SEQ ID NOs: 25-28, which are derived from E. coli K-12.
It would have been obvious to a skilled artisan, before the effective filing date, to modify the strain of Zhang and overexpress the genes in the gal operon, galE, galT, and galK, in addition to lacY, pgm, lgtA, and lgtB. Both Zhang and Jennewein are directed to modified bacterial strains for LNnT production. All of these genes are known to be involved in the LNnT biosynthesis pathway as taught by both Zhang and Jennewein. Zhang teaches strains expressing galE, lacY, lgtA, and lgtB (Zhang Table 1). It would have been obvious that other genes in the same operon, galT and galK, could be expressed with galE, and that all of these could be expressed in a strain that also overexpresses lacY, pgm, lgtA, and lgtB. A skilled artisan would have been motivated to do so because metabolic engineering of E. coli is a known strategy for optimizing the production of human milk oligosaccharides such as LNnT. As taught by both Zhang and Jennewein, different combinations of genes in the LNnT pathway when expressed or deleted in E. coli result in different yields of LNnT and a skilled artisan would have been motivated to express all of these genes in the same microorganism, with a reasonable expectation of success, as all of the genes are part of the LNnT metabolic pathway and have been overexpressed in strains which are capable of producing LNnT.
Regarding the lgtA and lgtB sequences, Jennewein teaches that lgtA and lgtB are from Neisseria meningitidis strain MC58 (Jennewein p. 16 Ex. 2). lgtA from N. meningitidis MC58 is 80% identical to SEQ ID NO: 1 (see sequence alignment in OA appendix). lgtB from N. meningitidis MC58 is 98.91% identical to SEQ ID NO: 2 (see sequence alignment in OA appendix).
Regarding the lgtA sequence, Zhu teaches a genetically engineered bacterial strain for production of the HMO Lacto-N-triose II with lgtA overexpressed (Zhu pg. 3702 “Introduction”). Zhu teaches that the sequence of the lgtA gene is derived from N. meningitidis (Zhu p. 3703 “Bacterial Strains, Media, and Culture Conditions”; “Plasmid Construction”). The original lgtA sequence, with accession number U25839.1, is 80% identical to instant SEQ ID NO: 1 (see sequence alignment in OA appendix). Zhu teaches that the lgtA sequence is codon optimized for expression in E. coli and the codon optimized sequence as listed in Table S3 is 100% identical to instant SEQ ID NO: 1 (Zhu p. 3703 “Plasmid Construction”; supplemental contents Table S3; see sequence alignment in OA appendix). Zhu further teaches that lgtA can be introduced to produce LNT II as well as the derivative, LNnT (Zhu pg. 3702 para. 3).
It would have been obvious for a skilled artisan to overexpress an lgtA gene with a sequence according to Zhu in a genetically engineered strain as taught by Zhang and Jennewein. All of these references teach lgtA from N. meningitidis expressed in E. coli for production of LNnT. Jennewein teaches a sequence of lgtA from N. meningitidis which is 80% identical to instant SEQ ID NO: 1. Jennewein additionally teaches that the sequence is codon optimized for expression in E. coli (Jennewein p. 16 lines 36-40). The sequence of lgtA taught by Zhu, which is 100% identical to instant SEQ ID NO: 1, is codon optimized for expression in E. coli (Zhu p. 3703 “Plasmid Construction”). It would have been obvious for a skilled artisan to codon optimize the lgtA gene for E. coli expression, as this is a common technique taught by both Jennewein and Zhu for recombinant gene expression. Further, as the codon-optimized N. meningitidis lgtA gene for expression in E. coli has a sequence identical to SEQ ID NO: 1, it would have been obvious for a skilled artisan to use such a sequence in a recombinant E. coli strain.
Regarding the lgtB sequence, the difference between SEQ ID NO: 2 and the lgtB sequence from Neisseria meningitides MC58 as taught by Jennewein is 9 nucleotides (see sequence alignment in OA appendix). However, it is known in the art that the genetic code is degenerate, meaning that the same amino acid is encoded by more than one nucleotide codon triplet (see Aryal reference p. 3). The sequence as taught by Jennewein differs in the following codons: AAC vs AAT, GTT vs GTC, ATC vs ATT, AGC vs AGT, TTA vs CTT, GCT vs GCG, TCC vs TCG, and CCT vs CCA (see sequence alignment in OA appendix). All of these alternative codons encode for the same amino acid (see Aryal ref. p. 2). Therefore, the sequence according to SEQ ID NO: 2 and the sequence taught by Jennewein encode for identical proteins. Further, Zhu and Jennewein teach that codon optimization for exogenous gene expression in bacteria is a common technique, as discussed above, and Jennewein teaches that the lgtB gene is codon optimized for E. coli expression (Jennewein p. 16 lines 45-50). Thus, it would have been obvious to codon optimize the lgtB gene as taught by Jennewein and arrive at a sequence with 100% identity to instant SEQ ID NO: 2.
Regarding the choice of plasmids, it would have been obvious to express the genes using pETDuet, pRSFDuet, and pCDFDuet plasmids in the combinations as claimed. Zhang teaches lgtA and lgtB expressed sequentially on pCDFDuet (Zhang Table 1). Zhang additionally teaches trying different plasmids for lgtA and lgtB expression, including pETDuet and pRSFDuet, to determine the optimal plasmid for expression of a particular gene (Zhang p. 296 “Increase LNnT yield by optimizing the expression of lgtA and lgtB”). Further, Zhu teaches use of pRSFDuet-1, pETDuet-1, and pCDFDuet-1 plasmids in combination to express various genes for production of human milk oligosaccharides, and teaches that optimization of the multigene expression strength is a common approach to the combinatorial fine-tuning of pathways for target metabolite synthesis (Zhu p. 3705 “Fine-Tuning of LNT II Synthesis via the Optimization of the Gene Expression Strength”). Further, it is known in the art that Duet plasmids are specifically designed to express two target proteins in E. coli, and that Duet plasmids are designed with compatible replicons and drug resistance genes for maintenance of up to four Duet plasmids in a single cell (see Millipore Sigma reference, p. 30).
As these Duet vectors are designed to be compatible with one another for the purpose of expressing up to 8 proteins in the same cell, it would have been obvious to utilize pETDuet and pRSFDuet in combination with the pCDFDuet to express lacY, pgm, and the operon galETK. Based on the teachings of Zhang and Zhu, it would have been obvious for a skilled artisan to try different combinations of the genes on each plasmid and optimize gene expression by choosing the appropriate expression vector for each gene. Given the finite number of known options for expressing lacY, galETK, and pgm on up to four compatible Duet plasmids, as well as the teaching of Zhang and Zhu that plasmid choice is something that would be routinely optimized by a skilled artisan, it is considered obvious to try various combinations of genes expressed on each of the plasmids and arrive at the claimed plasmid configuration.
Response to Arguments
The declaration under 37 CFR 1.132 filed 1/30/2026 is insufficient to overcome the rejection of claim 1 based upon 35 U.S.C. § 103 as set forth in the last Office action because:
Table 1 of the Declaration provides newly generated data as set forth in Table 1 to demonstrate the effect of different plasmid combinations on the yield of lacto-N-neotetraose.
However, it is not clear from this data that the plasmid combinations are the only factor contributing to differences in lacto-N-neotetraose production. For example, it is not clear what growth conditions were used, or if there are any other differences between the strains or how they were cultivated that would result in different LNnT production. As discussed on p. 13 and as shown in Table 4 of the instant specification, changing the carbon source during fermentation can impact the yield of LNnT even when the strain is the same. Thus, the data presented is not sufficient to indicate that only modifying the plasmid combination would have a significant and unexpected impact on LNnT production.
Further, as discussed in the above rejection, it is considered that choice of plasmids, including the Duet plasmids as claimed, is something that is commonly and routinely optimized by a skilled artisan during experimentation. The claimed Duet plasmids are created for the specific purpose of being used together in a single strain to express multiple genes, and optimization of which genes are expressed on a particular plasmid is considered routine and within the knowledge of a skilled artisan. As there are a finite number of combinations when using compatible Duet vectors to express the claimed genes, it is considered obvious to try different plasmid combinations to arrive at the claimed combination.
Regarding the differences in the lgtA and lgtB sequences taught by the prior art, the declaration states that the inventors examined various homologous genes with similar functions but significant performance differences, and the selection strategy is based on a series of comprehensive judgments such as product structure, metabolic flux analysis, expression system matching, enzyme activity experiments. The declaration states that especially for low-similarity sequences such as lgtA, whether it can be expressed in the target strain after replacement, whether it has activity, and whether it affects the product structure, all require experimental verification, which is highly uncertain and very time consuming, and it cannot be concluded that the present invention lacks patentability simply based on the conclusory statement that there must exist some kind-of motivation to exchange these genes.
In response to these arguments, the genes in the cited references are all lgtA or lgtB genes from Neisseria meningitidis. These genes are known to encode for proteins which function as a β-1,3-acetylglucosamine transferase or β-1,4-galactosyltransferase, respectively. Thus, it is not considered in this situation that the selection strategy would require product structure, metabolic flux analysis, expression system matching, or enzyme activity experiments, as the function of each of the gene products are established, and they are all derived from the same microorganism.
Further, as discussed above, the difference between lgtA as taught by Jennewein, derived from Neisseria meningitidis MC58, and lgtA with a sequence according to SEQ ID NO: 1 as taught by Zhu, is codon-optimization, which is a commonly used and well-established technique for expression of exogenous genes in different microorganisms. Further, the degeneracy of the genetic code is well-known as discussed above. Thus, it is considered that it would have been obvious to utilize genes having the claimed sequences in a recombinant strain based on the teachings of the prior art, as these sequences can be derived from common techniques such as codon optimization and encode the same proteins, with a reasonable expectation of success. It is the position of the examiner that based on the prior art teachings discussed above, it would not be highly uncertain and time consuming to arrive at a recombinant strain expressing the genes as claimed.
Regarding the expression of pgm, Zhang is newly relied upon to teach a strain that expresses pgm, as well as lgtA, lgtB, and lacY, and is capable of producing LNnT. Therefore, it is considered that a strain expressing pgm in combination with the other claimed genes is obvious in view of the prior art, and the arguments regarding Jennewein and Parschat are moot in view of newly applied references.
For the above reasons, the declaration under 37 CFR 1.132 is not sufficient to overcome the rejection of claim 1 under 35 U.S.C. § 103.
Applicant’s arguments with respect to claim 1 have been 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. However, responses to pertinent arguments regarding rejections under 35 U.S.C. § 103 are set forth below, and relevant arguments have additionally been addressed with respect to the declaration under 37 CFR 1.132 as discussed above.
Applicant argues that the claimed invention encompasses a three-plasmid modular expression system that is based on an in-depth analysis and system optimization. Applicant argues that different from the legacy single-plasmid or double-plasmid designs described in the cited art, there is no report in the literature that combines expression of the above six genes in three Duet vectors in this modular way. It is the position of the inventors that this new strategy is not a technical path that can be directly or in any way reasonably derived from any combination of the cited and existing public documents and it is not selectable from a finite set of possible solutions that could reasonably yield success.
Regarding the choice of plasmids, as discussed above, the Duet plasmid system is established for the purpose of expressing multiple genes in a single strain and designed for use in combination. It is considered that the strategy used in the instant invention can be derived from the prior art teachings and would be selectable from a finite set of possible solutions. As there are a finite number of Duet plasmids, i.e. four, designed for use together (see Millipore Sigma reference) and a finite number of genes involved in LNnT biosynthesis, it is considered that it would have been obvious to try various combinations of genes and plasmids to arrive at the claimed combination, particularly in light of teachings that plasmid choice and combinations are routinely optimized in the process of developing recombinant bacterial strains for HMO production.
Conclusion
Claim 1 is rejected. No claims are allowed.
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
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