The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
This office action is in response to Applicants’ amendments/remarks received April 30, 2026.
Rejections and/or objections not reiterated from previous office actions are hereby withdrawn.
Claims 2, 4 are canceled. Claims 1, 3, 5-11 are under consideration.
Priority: This application is a 371 of PCT/KR2021/018486, filed December 7, 2021, which claims benefit of foreign application KR 10-2020-0169583, filed December 7, 2020. A copy of the foreign priority document has been received in the instant application and is not in the English language.
Objections and Rejections
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1, 3, 5, 7-8 are rejected under 35 U.S.C. 103 as being unpatentable over Noh et al. (2017 Cell Systems 5: 418-426; previously cited) view of Yoo et al. (2013 Nature Protocols 8(9): 1694-1707). Instant claim 1 appears to be a product-by-process claim. “[E]ven though product-by-process claims are limited by and defined by the process, determination of patentability is based on the product itself.” MPEP 2113. For prior art purposes, instant claim 1 has been interpreted as directed to a recombinant microorganism obtained by introducing a sequence encoding a 17-21 nt synthetic sRNA that inhibits eno expression into the recombinant microorganism. The additional limitations of the recombinant microorganism “having enhanced ability to produce spider silk protein, etc.” is an intended use of the recombinant microorganism and has not been accorded patentable weight. MPEP 2111.02.
Noh et al. disclose transforming an E. coli cell with plasmids constructed to generate a synthetic small RNA (sRNA) to knockdown a target gene, where the target gene is the eno gene (at least p. 420-421, also Fig. 2). Noh et al. disclose the strategy used is a fine-tuned gene expression strategy through varying the expression levels of synthetic sRNAs (at least p. 418-419). Noh et al. disclose that an alternate strategy for fine-tuning gene expression using synthetic sRNA is to modulate the target mRNA-binding ability of synthetic sRNA, which can be achieved by changing the sequence of the target-binding region within synthetic sRNA (at least p. 418-419). Noh et al. disclose this strategy has been successfully demonstrated to be effective, citing Yoo et al. 2013 (p. 419).
Yoo et al. disclose that it should be noted that synthetic sRNAs with longer binding sequences have greater chances of binding to off-target mRNAs (p. 1699). Yoo et al. disclose that for reliable repression, it is recommended that synthetic sRNAs have a target-binding sequence of 20-30 nt in length and a binding energy of -30 to 40 kcal mol-1 (p. 1699).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of the prior art and arrive at the claimed recombinant microorganism comprising a sequence encoding a 20 nt synthetic sRNA that inhibits the eno gene (instant claim 1). The motivation to do so is given by the prior art. Noh et al. disclose a recombinant microorganism obtained by generating synthetic sRNA to knockdown a target gene, where the target gene is the eno gene. It is disclosed an alternate strategy for fine-tuning gene expression is by the sequence of the target-binding region within synthetic sRNA (Noh et al.; Yoo et al.). Yoo et al. disclose that for reliable repression, it is recommended that synthetic sRNAs have a target-binding sequence of 20-30 nt in length. Therefore, one of ordinary skill would have reasonable motivation to incorporate a sequence encoding a 20 nt synthetic sRNA as suggested in Yoo et al. for the synthetic sRNA to knockdown or suppress the eno gene in the microorganism of Noh et al. because the prior art discloses that it is also an effective strategy that successfully suppresses a target gene. One of ordinary skill would have a reasonable expectation of success because the prior art discloses that synthetic sRNAs having a target-binding sequence of 20 nt in length are reliable and effective for fine-tuning gene expression.
Regarding instant claim 8, Noh et al. disclose the microorganism having a knockdown eno gene is an E. coli cell (at least p. 420-421, also Fig. 2).
Regarding instant claim 7, Noh et al. disclose synthetic sRNAs are composed of two modules: an mRNA-binding module and an Hfq-binding module (p. 419), the three best being MicC, MicF, and SgrS (p. 419). Yoo et al. also disclose synthetic sRNA is composed of two function parts: one is for target-mRNA binding and the other is for recruiting Hfq proteins (p. 1694), being MicC (p. 1696). Therefore, Noh et al. and Yoo et al. can be deemed to disclose that the synthetic sRNA for repressing the eno gene comprises the recited Hfq binding site derived from any one of MicC, MicF, and SgrS, and a region forming a complementary bond with an mRNA of the eno gene.
Regarding instant claim 3, Noh et al. disclose various target genes for knockdown, including the eno gene and ackA gene (at least p. 420). It would have been obvious to one of ordinary skill in the art to further include a synthetic sRNA to repress an additional gene ackA in the E. coli cell having inhibited expression of the eno gene noted above in Noh et al. in view of Yoo et al. The motivation to do so is given by the prior art, which disclose a synthetic sRNA-based knockdown strategy for production of a target substance of interest in E. coli. One of ordinary skill would have a reasonable expectation of success because the prior art Noh et al. disclose E. coli having inhibited eno expression and E. coli having inhibited ackA expression both successfully produced the target substance of interest. Therefore, the combination of the two inhibited genes would be obvious because it would have the same purpose for enhancing production of the target substance of interest production.
Regarding instant claims 3, 5, Noh et al. also disclose synthetic sRNA to knockdown the target gene pck (p. 420-421, also Fig. 2). It would have been obvious to one of ordinary skill in the art to further include a synthetic sRNA to repress an additional gene pck in the E. coli cell having inhibited expression of the eno gene noted above in Noh et al. in view of Yoo et al. The motivation to do so is given by the prior art, which disclose a synthetic sRNA-based knockdown strategy for production of a target substance of interest in E. coli. One of ordinary skill would have a reasonable expectation of success because the prior art Noh et al. disclose E. coli having inhibited eno expression and E. coli having inhibited pck expression both successfully produced the target substance of interest. Therefore, the combination of the two inhibited genes would be obvious because it would have the same purpose for enhancing production of the target substance of interest production.
Claims 1, 3, 5, 6, 7-8, 9-11 are rejected under 35 U.S.C. 103 as being unpatentable over Noh et al. (2017 Cell Systems 5: 418-426; previously cited) view of Yoo et al. (2013 Nature Protocols 8(9): 1694-1707) and Xia et al. (2010 PNAS 107(32): 14059-14063; IDS 06.10.23, previously cited). The teachings of Noh et al. and Yoo et al. over at least instant claims 1, 3, 5, 7-8 are noted above.
As noted above, Noh et al. in view of Yoo et al. reasonably disclose introducing a sequence encoding a 20 nt synthetic sRNA that inhibits a target gene, the gene being the eno gene, for production of a target substance. Noh et al. do not teach a target substance of interest being silk protein.
Xia et al. disclose spider dragline silk is a remarkably strong fiber that makes it attractive for numerous applications (p. 14059). Xia et al. disclose that native-sized recombinant spider silk protein expressed and produced in a metabolically engineered E. coli results in a strong fiber (p. 14059-14060). Xia et al. disclose the recombinant spider silk protein expression construct comprises silk protein monomer (p. 14060, Fig. 1), where the monomer sequence has 100% sequence identity with the amino acid sequence of instant SEQ ID NO: 1.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to incorporate the recombinant spider silk expression construct of Xia et al. for the target substance of interest into the engineered E. coli having a knocked down eno gene of Noh et al. in view of Yo et al., or alternatively, incorporate the engineered E. coli having a knocked down eno gene of Noh et al. in view of Yoo et al. for the metabolically engineered E. coli utilized in Xia et al. for expression of recombinant spider silk protein, to thereby arrive at the claimed microorganism having inhibited eno expression by a sequence encoding a 20 nt synthetic sRNA that inhibits the eno gene and a gene encoding a recombinant silk protein having the amino acid sequence of instant SEQ ID NO: 1 (instant claims 6, 9). The motivation to do so is given by the prior art, which disclose metabolically engineered E. coli cells can be transformed to express products of interest, including spider silk proteins. One of ordinary skill would have a reasonable expectation of success E. coli is a well-known host cell and can be adapted for expression of known recombinant proteins.
Regarding instant claims 10-11, Xia et al. disclose a process for culturing the metabolically engineered E. coli to produce recombinant spider silk protein and recovering the recombinant spider silk protein (p. 14060), where the recovered spider silk protein is precipitated with a salt (p. 14061). Therefore, it would be obvious that a metabolically engineered E. coli having a knocked down eno gene of Noh et al. in view of Yoo et al., and utilized in Xia et al. for expression of recombinant spider silk protein can be cultured to produce recombinant spider silk protein and recovering the recombinant spider silk protein by a salt precipitation step. One of ordinary skill would have a reasonable expectation of success because metabolically engineered E. coli are still host cells capable of being adapted for expressing known recombinant proteins.
Reply: In view of Applicants’ amendments/remarks, the previous 102 and 103 rejections have been withdrawn. However, the claims remain unpatentable under new 103 rejections for the reasons noted above and herein.
Applicants assert that in Noh et al., the eno and ackA genes were used solely for the purpose of producing putrescine – there is no description whatsoever regarding silk protein production.
Applicants’ remarks are not persuasive. As noted above, instant claim 1 has been interpreted as directed to a recombinant microorganism obtained by introducing a sequence encoding a 17-21 nt synthetic sRNA that inhibits eno expression into the recombinant microorganism. The additional limitations of the recombinant microorganism “having enhanced ability to produce spider silk protein, etc.” is an intended use of the recombinant microorganism and has not been accorded patentable weight. MPEP 2111.02. There is no requirement that the recombinant microorganism in instant claim 1 is actually producing silk protein. The recombinant microorganism in instant claim 1 just has the limitations of having a sequence encoding a 17-21 nt synthetic sRNA that inhibits eno expression.
Therefore, it is not necessary that Noh et al. disclose that the recombinant microorganism having an inhibited eno gene by a synthetic sRNA based strategy has to produce silk protein.
Applicants assert that Noh et al. at p. 430 disclose that when driven by a strong promoter, the expression of eight sRNAs (anti-poxB, -eno, -ackA, -pck, -glnA, -sucA, -argF, and -gltX sRNAs) reduced putrescine titer compared with the base strain. Applicants assert that thus, it is taught by Noh et al. that when the eno gene is suppressed, putrescine production actually decreases.
Applicants’ remarks are not persuasive. MPEP 2123 notes that “[t]he prior art’s mere disclosure of more than one alternative does not constitute a teaching away from any of these alternatives because such disclosure does not criticize, discredit, or otherwise discourage the solution claimed….” In re Fulton, 391 F.3d 1195, 1201, 73 USPQ2d 1141, 1146 (Fed. Cir. 2004). In this instance, Noh et al. disclose that alternatively, under a different strength promoter, the synthetic sRNAs for suppressing eno and ackA increased production of the target substance (at least p. 421, Fig. 2). Therefore, Noh et al. still disclose that suppressing eno and ackA efficiently produces a target substance (putrescine). Therefore, Noh et al. do not teach away from inhibiting the eno gene in a recombinant microorganism for production of a target substance.
Applicants assert that further, Noh et al. contain no mention or suggestion of the precise length of the sRNA that is required to suppress eno gene expression, much less any mention or suggestion of the synthetic sRNAs ranging from 17 to 21 nt in length that Applicants have demonstrated to be highly effective for suppressing eno expression (example 3, Fig. 4 of the instant specification).
Applicants’ remarks are not persuasive. Noh et al. is cited with new 103 reference Yoo et al., which disclose that for reliable repression, it is recommended that synthetic sRNAs have a target-binding sequence of 20-30 nt in length (p. 1699). Therefore, Applicants’ remarks that a synthetic sRNA having for instance, 20 nt in length, is highly effective for suppressing eno expression are not persuasive because the prior art discloses the same length recited for reliable repression by a synthetic sRNA.
Applicants assert that the present invention relates to a microorganism whose silk production capacity is enhanced by suppression of the eno gene, unlike Xia et al., and thus has a different mechanism of action (Xia uses tRNA modification).
Applicants’ remarks are not persuasive. Xia et al. disclose expressing recombinant spider silk protein in a metabolically engineered E. coil (p. 14060). Xia et al. disclose the spider silk protein is predominantly rich in glycine (p. 14059). Xia et al. disclose utilizing tRNAGly to increase the glycine pool (p. 14060). It is depicted in Fig. 1E of Xia et al. that glycine is synthesized from serine and downstream from 3-phosphoglycerate in the pathway (p. 14060). Noh et al. also disclose a metabolic pathway for E. coli where suppression of the eno gene is downstream of 3-phosphoglycerate (p. 421 Fig. 2B). Therefore, one of ordinary skill would have a reasonable expectation of success that an engineered E. coli cell having a knocked down eno gene can be utilized in the efficient or enhanced production of recombinant spider silk protein because the engineered E. coli cell still comprises the pathway genes/proteins for producing glycine and does not need to divert to a subsequent pathway since the eno gene is knocked down.
For at least these reasons, the 103 rejections are maintained.
No claim is allowed.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/Marsha Tsay/Primary Examiner, Art Unit 1656
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