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 .
Claim Status
Applicant’s amendment filed 12/9/2025 has been entered. Claims 1-2 and 6-9 are cancelled. Claims 3-5 and 10-13 are pending and currently under examination.
Independent claim 3 has been amended, including incorporating limitations “wherein the nuclease-assisted gene targeting system is CRISPR/Cas9” of cancelled claims 6-7 and “wherein the reduction of SIRT-1 gene expression is caused by a knockout of the SIRT-1 gene”. Claim 10 has been amended to depend from claim 11 and claim 12 has been amended to depend from claim 3.
Claims 1 and 2 (now cancelled) are withdrawn from prosecution as being drawn to non-elected subject matter. The restriction requirement mailed 3/10/2025 is still deemed proper. Applicant's elected Group II, claims 3-13 without traverse in the reply filed 7/24/2025.
Any rejection or objection not reiterated herein has been overcome by amendment. Applicant’s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
Specification - withdrawn
Objection to the specification for the use of trade names and marks is withdrawn in view of Applicant’s amendment to the specification filed 12/9/2025.
Claim Objections - withdrawn
Objection to claim 3 is withdrawn in view of Applicant’s amendment to remove the clerical error and consistently use the term “expression”.
Claim Rejections - 35 USC § 112 - withdrawn
Rejection of claim 7 under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement is withdrawn in view of Applicant’s cancellation of claim 7.
Rejection of claims 4-13 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or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention is withdrawn in view of Applicant’s cancellation of claims 6-9 and amendments to claims 3, 5, and 10-12.
Claim Rejections - 35 USC § 103 – new necessitated by amendment
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.
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.
Claims 3-5 and 10-13 are rejected under 35 U.S.C. 103 as being unpatentable over Fischer et al. (published 2015; of record) in view of Bernier et al. (published 4/5/2011; of record).
Fischer’s disclosure is directed to a review of the use of Chinese hamster ovary (CHO) cells as a host system for industrial manufacturing of recombinant protein therapeutics (entire document). Fischer teaches advances in bioprocess, media and vector optimizations, and advances in host cell engineering technologies comprising introduction, knock-out or post-transcriptional silencing of engineering genes that have paved the way for remarkable achievements in CHO cell line development (abstract). Fischer further teaches that analysis of cellular pathways and mechanisms is important to find novel target molecules to establish superior production cell factories (entire document). Fischer teaches that a major issue of eukaryotic expression systems is the continuous acidification of culture medium due to accumulation of lactate which is generated by the conversion of pyruvate by the lactate dehydrogenase (LDH) enzyme and the resulting lactate-mediated drop in the pH leads to an inhibition in cell growth (pp.1887-1888, Section 3.3.3). Fischer further teaches that targeted gene knockdown or knockout can lead to a decrease in lactate production and multi-fold increase in protein production in cells (Tables 2 and 3).
Regarding claim 3, Fischer teaches methods for producing recombinant mammalian (CHO) cells with improved productivity and reduced lactate production by targeting endogenous genes involved in various cellular pathways for knockout (pp.1887-1888; Fig. 1; Table 3). Fischer teaches that cell engineering approaches to improve the performance of CHO manufacturing cell lines classically comprised either overexpression of beneficial genes or repression of disadvantageous gene products by genomic knock-out or siRNA-mediated knockdown (Introduction). Fischer teaches targeting gene knockout by CRISPR/Cas9 nuclease-assisted targeting systems (p. 1882, Section 3.2; p. 1886, Section 3.2.3; and Fig. 1). Fischer further teaches the advantages of precise genome editing in CHO cells using CRISPR/Cas9 to create genetic knockouts appear to be easier to establish, less time-consuming and much more cost effective compared to the more sophisticated and expensive alternatives and that it has the potential to revolutionize current cell line optimization strategies to enable rational design of mammalian cell factories (p. 1886, Section 3.2.3). Fischer further teaches that reducing LDHA activity by targeting the gene for knock-down and erasing LDHA activity by LDH antisense mRNA expression reduced lactate levels in CHO cells (pp. 1887, Section 3.3.3). Fischer teaches selecting mammalian cells where the expression of the target gene has been reduced, whether by knockdown or knockout (p. 1880, Section 3.1; 1882, Section 3.2; and 1889, Section 3.4.4).
However, Fischer does not specifically teach targeting endogenous SIRT-1 gene.
Bernier’s disclosure is directed to the role of SIRT1 deacetylase, as a regulator of lactate and ATP production via STAT3 transcriptional activity (entire document). Bernier teaches Sirt1 gene knockout in murine embryonic fibroblasts (MEFs) (abstract).
Regarding claim 3, Bernier teaches a lower level of lactate production in Sirt1-KO cells (Fig. 5A; p. 19274, right column, para 5 through p. 19275, left column para 2). Bernier teaches that SIRT1 ectopically expressed in the Sirt1-KO cells results in an increase in lactate production compared to Sirt1-KO cells with a control plasmid (Fig. 5C). Bernier further teaches that Sirt1-KO cells increased STAT3 protein levels significantly compared to WT cells (Fig. 1). Bernier further teaches that siRNA knockdown of STAT3 increased lactate production (p. 19275, left column para 2; Fig. 5).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of producing knockout recombinant mammalian cells with improved recombinant productivity and reduced lactate production, as described in Fischer, by knocking out the endogenous SIRT-1 gene, as described by Bernier. Fischer teaches that reducing expression of certain endogenous target genes can reduce lactate production (Table 3) and that targeted gene knockout can lead to an increase in protein production in cells (Table 2). Bernier teaches that sirt1-/- knockout cells have reduced lactate production. One of ordinary skill would have recognized that targeted gene knockout in CHO cells with reduced lactate levels results in higher production of exogenous proteins and that sirt1-/- knockout cells exhibit reduced lactate levels and would have been motivated to produce recombinant cells targeting sirt1 for knockout to increase protein production. It would have been further obvious to reduce target gene expression with CRISPR/Cas9 because Fischer teaches advantages of using CRISPR/Cas9. Fischer specifically teaches that precise genome editing in CHO cells using CRISPR/Cas9 to create genetic knockouts appear to be easier to establish, less time-consuming and much more cost effective compared to the more sophisticated and expensive alternatives (p. 1886, Section 3.2.3). Accordingly, there would have been a reasonable expectation of success for using CRISPR/Cas9 to reduce SIRT-1 gene expression by knockout of the SIRT-1 gene. One would have had further had a reasonable expectation of success because Fischer and Bernier both teach improving cell culture by reducing lactate production and increasing protein production in mammalian cells. Thus, the claimed invention as a whole is prima facie obvious.
Regarding claim 4, Bernier teaches that the Sirt-1 gene knockout is homozygous in MEF cells (see entire document).
Regarding claim 5, Fischer teaches that in recombinant mammalian cells, the productivity of the recombinant mammalian cell with targeted knockout/knockdown is at least 10% increased product compared to WT (Tables 2 and 3). Thus, there would have been a reasonable expectation of success that the method made obvious by Fischer and Bernier would result in at least 10% increased productivity compared to a SIRT-1 parent mammalian cell line. Furthermore, the limitation is a ‘wherein’ clause that does not add any additional structural limitations. The method made obvious by Fischer and Bernier would inherently result in the claimed limitation because it made obvious the claimed method steps and materials recited in the instant claim.
Regarding claims 10-11, Fischer teaches the versality of CHO cells to allow combinatorial targeted FUT8 knockout and simultaneous integration of an antibody expression cassette (Table 2). Fischer teaches CHO-GS cell factories with the endogenous glutamine synthesis gene knocked out that can be transfected with an expression vector encoding a transgene (p. 1882, Section 3.2). Fischer further teaches targeted gene knockdown in mAb-producing CHO cells after introduction of the exogenous mAb (p. 1888, Section 3.3.4 and p. 1890, Section 3.4.5-3.4.6).
Regarding claims 12-13, Fischer teaches that the mammalian cell is a targeted integration CHO host cell (Table 2; and p. 1880, Section 3).
Therefore the invention as a whole would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Response to Arguments
Applicant's arguments filed 12/9/2025 have been fully considered but they are not persuasive. Applicant argues that Fischer fails to teach or suggest a method for producing a recombinant mammalian cell with a SIRT-1 gene knockout that has increased productivity and reduced lactate production.
In response to applicant's argument that Fischer fails to teach or suggest a method for producing a recombinant mammalian cell with a SIRT-1 gene knockout that has increased productivity and reduced lactate production, the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981). In this case, Fischer teaches improvements in CHO cell engineering as recombinant protein production by gene knock-down and gene knock-out of endogenous genes (entire document). Fischer teaches knockdown and knockouts of target genes resulting in increased recombinant productivity, reduced lactate production, or both (see 103 discussion above). Bernier teaches reduced lactate production in Sirt1-KO cells (abstract). One of ordinary skill would have recognized that targeted gene knockout in CHO cells with reduced lactate levels results in higher production of exogenous proteins and that sirt1-/- knockout cells exhibit reduced lactate levels and would have been motivated to produce recombinant cells targeting sirt1 for knockout to increase protein production.
Applicant argues that the Office Action states that Table 1 of Fischer “teaches that targeted gene knockdown or knockout can lead to a decrease in lactate production and multi-fold increase in protein production in cells," (Office Action, page 11), but Table 1 relates to "Targeted genes and pathways for CHO cell engineering using gene introduction”. Applicant further argues that Table 1 mostly mentions studies demonstrating product yields as a result of improved media utilization.
The Office agrees. The Office apologizes for the clerical error of citing Table 1 instead of Tables 2 (targeted gene knock-outs) and 3 (targeted gene knock-down approaches). The argument regarding the studies within Table 1 are moot in view of the admission of clerical error.
Applicant argues that Fischer fails to teach or even suggest that a single gene knockout, let alone knocking out a sirtuin protein such as SIRT-1, in CHO cells can lead to increased recombinant productivity and reduced lactate production. Applicant further argues that Fischer teaches that knocking down LDHA reduced lactate concentrations, but an accompanying increase in recombinant protein productivity was only seen when both LDHA and PDHK were knocked down (Table 3 and page 1888, top of left column). Applicant argues that a knockdown of 2 genes was required to the see the effect of increased productivity and reduced lactate production that the currently claimed invention achieved by the single gene knockout of SIRT-1. Applicant further argues that Fischer notes a "recent study revealed that a complete knockout of LDH is lethal in CHO cells even though PDHK-1, -2 and -3 expression is concomitantly down-regulated (Yip et al., 2014), a fact which always has to be taken into account for complete gene knockout strategies to promote particular cell phenotypes in host cell engineering," (page 1888, left column) and that given this difference, one of skill in the art would not have been able to predict the effect of a single gene knockout of SIRT-1 on both based on the recombinant protein productivity and lactate production based on the gene knockdown experiments summarized in Table 3 of Fischer. Applicant further argues that they have unexpectedly shown that for recombinant cells, knocking out SIRT-1 alone with CRISPR/Cas9 was sufficient to reduce lactate production and increase productivity (see pages 43-46 and Figs. 1-8 of the as-filed specification). As noted in the as-filed specification, the "results obtained by a knockout of the SIRT-1 gene are surprising as the knockout of other genes likewise potentially influencing productivity was without positive effect. Applicant further argues that 20 combinational knockout of different genes did not perform better than a single SIRT-1 knockout in a cell line producing a molecule comprising an antigen binding domain targeting Fibroblast Activation Protein (FAP) and a trimer of 4-1BB ligands (CD137L), named FAP-4-1BBL," (see page 43, lines 18-23 of the as-filed specification). Applicant concludes that as such, Fischer fails to teach that a single gene knockout in a recombinant protein- producing mammalian cell can lead to increased recombinant productivity and reduced lactate production, and in fact warns that complete gene knockouts (such as those achieved by the currently claimed invention), may lead to cell lethality.
The Office disagrees. In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., a single gene knockout in a recombinant protein-producing mammalian cell leading to increased recombinant productivity and reduced lactate production) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). Claim 3, as currently amended, does not limit the method for producing a recombinant mammalian cell to targeting SIRT-1 alone because the claim recites “the method comprises the following steps” which encompasses the SIRT-1 gene knockout and additional knockouts, knockdowns, and/or insertions.
Applicant’s arguments regarding unexpected results are moot in light of the amended claims not being limited to a single knockout cell line.
Applicant’s arguments regarding cell lethality in light of Fischer’s teachings that a complete knockout of LDH is lethal in CHO cells are not persuasive because one example of lethality would not have discouraged one of ordinary skill of the art from recognizing that Bernier successfully produced Sirt1-KO cells in a mammalian cell line without lethality.
Applicant argues that Bernier fails to remedy the deficiencies of Fischer, as Bernier also fails to teach or suggest a method for producing a recombinant mammalian cell with a SIRT-1 gene knockout that has increased productivity and reduced lactate production and that Bernier fails to teach or suggest that a knockout of SIRT-1 alone would be sufficient to also increase productivity of a recombinant protein along with reducing lactate production.
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). The Office is relying on Fischer to teach targeted gene knockout in CHO cells with reduced lactate levels resulting in higher production of exogenous proteins and on Bernier to teach that sirt1-/- knockout cells exhibit reduced lactate levels, such that it would have been obvious to one of ordinary skill that reduced lactate levels can lead to increased protein production. One would have been motivated to produce recombinant cells targeting sirt1 for knockout to increase recombinant protein production. Therefore the invention as a whole would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KHALEDA B HASAN whose telephone number is (571)272-0239. The examiner can normally be reached IFP, Monday - Friday 7:30am-5pm.
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/KHALEDA B HASAN/Examiner, Art Unit 1636
/BRIAN WHITEMAN/Primary Examiner, Art Unit 1636