DETAILED ACTION
Status of the Application
Claims 1 and 7 are pending.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicant’s amendment of claim 1 as submitted in a communication filed on 3/6/2026 is acknowledged.
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 3/6/2026 has been entered.
Claim 1 was amended in a communication filed on 11/12/2024 to require the inducible regulation of two proteases, wherein one of the proteases is ftsH, and the second one is selected from the group consisting of lon, ClpXP, ClpAP and hslUV. As previously indicated, while this embodiment was not present in the claims as restricted, in the interest of advancing prosecution, the Examiner will continue to use the combination of proteases previously elected for further examination on the merits. In view of Applicant’s election of the combination of proteases ftsH, hsIUV and lon as submitted in the communication of 9/25/2023, the claims will be examined to the extent they encompass this elected combination. Claims 1 and 7 are at issue and will be examined to the extent they encompass the elected invention.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Rejections and/or objections not reiterated from previous office actions are hereby withdrawn.
Claim Objections
Claim 1 is objected to due to the recitation of “one or more gRNA under…wherein said guide RNAs..”. To enhance clarity and to be grammatically correct, the term should be amended to recite “one or more gRNAs under…wherein said guide RNAs..”. Appropriate correction is required.
Claim 1 is objected to due to the recitation of “…protease selected from ftsH, lon…and/or hslUV”. To enhance clarity and to be consistent with commonly used claim language, the term “and/or” should be replaced with “and”. Correction is required.
Claim Rejections - 35 USC § 112(b) or Second Paragraph (pre-AIA )
Claims 1 and 7 remain 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 pre-AIA the applicant regards as the invention. New grounds of rejection are necessitated by amendment.
Claim 1 (claim 7 dependent thereon) is indefinite in the recitation of “wherein (i) the endogenous cas 3 gene…is deleted, and/or (ii) introduce a C-terminal degron tag to endogenous E. coli ftsH,….and/or hsIUV genes and wherein the microorganism is further modified to express a chaperone…, or (iii) a combination of (i) and (ii)” for the following reasons. The term “wherein ..(ii) introduce a C-terminal degron tag…” is completely unclear and confusing as one cannot determine how the term further limits the claim. Furthermore, while a degron tag is a protein, it is unclear if the claim requires the introduction of a protein (i.e., tag) to a protease gene (polynucleotide). In view of the indefiniteness of the limitation “(ii)”, the term “a combination of (i) and (ii)” is also unclear.
For examination purposes, it will be assumed that the genetically modified E. coli microorganism requires inducible regulation of at least two proteases of the genetically modified microorganism, wherein the two proteases are ftsH and one or more of HsIUV and lon, wherein the genetically modified E. coli microorganism (i) has a deletion of the endogenous cas3 gene, and comprises a plasmid expressing one or more gRNAs under the control of a low-phosphate inducible promoter, wherein said guide RNAs form a complex with the mutated CRISPR cascade complex of E. coli lacking the protein encoded by the cas3 gene to bind to the promoters of the endogenous E. coli ftsH, lon, ClpXP, ClpAP and/or hslUV genes, and/or (ii) has a nucleic acid encoding a C-terminal degron tag introduced into the endogenous E. coli ftsH, lon, ClpXP, ClpAP and/or hslUV genes, and has been modified to express a chaperone that binds to the C-terminal degron tag, wherein said chaperone expression is under the control of a low-phosphate inducible promoter, and wherein the C-terminal degron tag promotes proteolytic degradation of the protease ftsH, lon, ClpXP, ClpAP and/or hslUV, upon expression of the chaperone. Correction is required.
When amending the claims, applicant is advised to carefully review all examined claims and make the necessary changes to ensure proper antecedent basis and dependency.
Claim Rejections - 35 USC § 112(a) or First Paragraph (pre-AIA )
Claims 1 and 7 remain rejected 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. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claims 1 and 7 remain rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a genetically engineered E. coli cell that expresses a recombinant protein by using an inducible expression system, wherein said E. coli cell has been further genetically modified to (i) delete the endogenous cas3 gene, and introduce a plasmid that expresses guide RNAs under the control of a low-phosphate inducible promoter, wherein said guide RNAs form a complex with the mutated CRISPR cascade complex of E. coli lacking the protein encoded by the cas3 gene to bind to promoters of endogenous E. coli protease genes, and /or (ii) introduce a C-terminal degron tag to endogenous E. coli proteases and a plasmid that expresses a chaperone that binds to the C-terminal degron tag, wherein said chaperone expression is under the control of a low-phosphate inducible promoter, does not reasonably provide enablement for a genetically engineered microorganism that expresses a recombinant protein by using an inducible expression system, wherein said genetically engineered microorganism has been modified by any means to inducibly reduce the level of an endogenous E. coli protease. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
These rejections have been discussed at length in the prior Office action. They are maintained for the reasons of record and those set forth below.
Applicant argues that the claims are not open to any means of inducibly reducing the level of any protease but requires the control of a low-phosphate inducible promoter, guide RNAs to form a complex with the mutated CRISPR cascade complex, and a C-terminal degron tag to endogenous E. coli proteases and a plasmid that expresses a chaperone that binds to the C-terminal degron tag. Applicant states that these rejections doe not apply to the instantly amended claims.
Applicant’s arguments have been fully considered but not deemed persuasive to overcome the instant rejection. The Examiner acknowledges the amendments made to the claims. However, the Examiner disagrees with Applicant’s contention that the entire scope of the claims is adequately described and enabled. Assuming that the limitation (ii) is clear and definite, which is not for the reasons set forth in Claim Rejections - 35 USC § 112(b) or Second Paragraph (pre-AIA ), the limitations of (i) and (ii) apply to any one of the proteases ftsH, lon, ClpXP, ClpAP or hsIUV. The deletion of the endogenous cas3 gene or the addition of a C-terminal degron tag are not required for the ftsH protease or the combination of two proteases recited in the preamble. Please note that the list of proteases in limitations “(i)” and “(ii)” are in the alternative and there is no statement indicating that the inducible regulation of the at least two proteases is obtained by the methods of “(i)” or “(ii)”. As such, the inducible regulation of ftsH and any of lon, ClpXP, ClpAP or hslUV is not necessarily limited by deleting the cas3 gene or the use of a C-terminal degron tag. For example, one could have a genetically modified E. coli microorganism that meets the limitation “(i)” applied to the endogenous proteases ClpAP and ClpXP and has inducible regulation of ftsH and lon by any means. Therefore, the claims still require placing any nucleic acid that encodes any RNA or protein that can reduce the level of the recited proteases under the control of an inducible promoter. As such, the amended claims are not adequately described or enabled for the reasons extensively discussed in prior Office actions. Briefly, the claims still require placing any nucleic acid that encodes any RNA or protein that can reduce the level of the recited proteases under the control of an inducible promoter. Such protein is not limited to a protease that can degrade the target protease but it encompasses unknown proteins that can interact with the target protease and reduce its catalytic activity. In the case of gene silencing, this encompasses not only RNAs that can block the expression of the target proteases, but also RNAs that can block the expression of any endogenous inducer/enhancer of the expression of the target proteases. The entire genus of nucleic acids encoding RNAs or proteins that when expressed can reduce the level of the recited proteases has not been adequately described or enabled. Therefore, contrary to Applicant’s assertions, the claims as amended do not meet the written description and enablement requirements.
Claim Rejections - 35 USC § 103 (AIA )
Claims 1 and 7 remain rejected under 35 U.S.C. 103 as being unpatentable over Menacho-Melgar et al. (Biotechnology and Bioengineering 117:2715-2727, published 5/22/2020) in view of Gottesman (U.S. Publication No. 2019/0055588 published 2/21/2019) and Li et al. (bioRxiv doi:10.1101/2020.07.26. 219949, pages 1-23, published 7/26/2020).
This rejection has been discussed at length in prior Office actions. It is maintained for the reasons of record and those set forth below.
Applicant argues that Menacho-Melgar and Li are silent as to the simultaneous inducible expression or over expression of a heterologous protein while at the same time reducing the two distinct proteases ftsH and one of lon, ClXP, ClpAp and hsIUV. Applicant submits that the invention provides surprising and unexpected results that cannot be determined by the combination of Menacho-Melgar, Gottesman and Li. Applicant states that expression levels were measured and that the disclosed synergy provides a 100-fold improvement. Applicant submits that the generic teaching of Menacho-Melgar regarding the effectiveness of synthetic metabolic valves for gene and protein regulation does not imply that any gene can be controlled by using the synthetic metabolic valve of Menacho-Melgar and expect routine results. Applicant states that Gottesman relies on host cells deficient in one or more proteases by chromosomal deletion and refers to BL21(DE3). Applicant states that the specification teaches that BL21(DE3) is a lon deficient strain and that there is room to further reduce lon protease activity to improve the expression of heterologous proteins. Applicant states that while there is still Lon activity when using BL21(DE3), the present invention has overcome that disadvantage with the optimized synthetic metabolic valves of the instant invention. Applicant states that nothing in the teachings of Menacho-Melgar, Gottesman or Li would have provided the ordinary artisan a reasonable expectation of the improved performance of silencing the lon protease in contrast with chromosomal deletion of the gene.
Applicant’s arguments have been fully considered but not deemed persuasive to overcome the instant rejection. The Examiner acknowledges the amendments made to the claims, the teachings of the prior art and the specification, as well as the results obtained by Applicant when using a synthetic metabolic valve to control protease activity. However, the Examiner disagrees with Applicant’s contention that the claimed invention is not obvious over the cited prior art. Claims 1 and 7 as interpreted are directed in part to an E. coli cell that has been genetically engineered to produce a heterologous protein upon induction, wherein said E. coli cell has inducible regulation of two endogenous proteases, wherein one of the proteases is ftsH and the other is hslUV, wherein the levels of the endogenous proteases are reduced upon induction, wherein this reduction is achieved through proteolysis and/or gene silencing, and wherein said E. coli cell has a deletion in a gene encoding the lon protease. See Claim Rejections - 35 USC § 112(b) or Second Paragraph (pre-AIA ) for claim interpretation.
With regard to the argument that Menacho-Melgar and Li are silent as to the simultaneous inducible expression or over expression of a heterologous protein while at the same time reducing the two distinct proteases ftsH and one of lon, ClXP, ClpAp and hsIUV, it is noted that the instant rejection is not based solely on the teachings of Menacho-Melgar et al. and Li et al. Instead, the instant rejection is based on the combination of references by Menacho-Melgar et al., Gottesman and Li et al. as well as the knowledge of the art at the time of the invention.
It is reiterated herein that a person of ordinary skill in the art is motivated to further genetically modify the E. coli cells of Menacho-Melgar et al. to reduce the levels of endogenous proteases, such as the endogenous ftsH, lon and hsIUV proteases because (i) Gottesman teaches the reduction or elimination of endogenous proteases, such as ftsH, lon, ompT and hsIUV, to increase the yield and/or stability of recombinant proteins produced in E. coli , and (ii) Gottesman and Menacho-Melgar et al. teach the use of E. coli cells that already have a genetic modification so that they are deficient in lon activity (BL21(DE3)) and how even those cells that are deficient in lon activity could be improved to further reduce protease activity.
As previously discussed, a person of ordinary skill in the art is motivated to use the low-phosphate inducible gene silencing system and low-phosphate inducible proteolysis system of Li et al. and replace the target enzymes glucose-6-phosphate dehydrogenase and citrate synthase of Li et al. with endogenous proteases, such as ftsH, and hslUV, for the benefit of (i) delaying the inactivation of proteases required for normal cell growth until the stationary phase, which is the phase when the protein of interest in being produced by the E. coli cells of Menacho-Melgar et al., and when the reduction/inactivation of proteolytic activity is needed, and (ii) starting the reduction/inactivation of proteases at the same time as the synthesis of the protein of interest is induced since the trigger for the reduction/inactivation of proteases and the synthesis of the protein of interest is the same, namely the depletion of phosphate in the culture medium. A person of ordinary skill in the art is motivated to combine the low-phosphate inducible gene silencing system, the low-phosphate inducible proteolysis system and a chromosomal deletion of a gene encoding a protease because some of the target proteases might be essential for cell growth while other target proteases might not have a significant impact on cell growth, thus requiring different control strategies. Since chromosomal deletion requires less gene manipulations compared to the introduction of the low-phosphate inducible systems of Li et al., one of skill in the art would use the low-phosphate inducible systems of Li et al. to reduce the levels of those proteases that are essential for growth and introduce a chromosomal deletion in the genes of those proteases that do not have a major impact on cell growth.
With regard to the argument that the generic teaching of Menacho-Melgar et al. regarding the effectiveness of synthetic metabolic valves for gene and protein regulation does not imply that any gene can be controlled by using the synthetic metabolic valve of Menacho-Melgar and expect routine results, it is noted that the Examiner is not relying on the teachings of Menacho-Melgar et al. for a synthetic metabolic valve. Instead, the Examiner in prior Office actions has previously stated that it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further genetically modify the E. coli cells of Menacho-Melgar et al. (i) to introduce an inducible gene silencing system and an inducible proteolysis system such as that of Li et al. to reduce the level of endogenous proteases, such as the endogenous ftsH protease and the hslUV protease, or (ii) to introduce an inducible gene silencing system and an inducible proteolysis system such as that of Li et al. to reduce the level of endogenous proteases, such as the endogenous ftsH protease and the hslUV protease, and additionally introduce a deletion in an endogenous gene encoding other endogenous proteases, such as the endogenous lon or ompT gene. Therefore, as previously stated in prior Office actions, the Examiner is relying on the teachings of Li et al. to show that an inducible gene silencing system and an inducible proteolysis system to reduce the levels of an undesirable protein using a low phosphate induction system (metabolic valves) were known in the prior art at the time of the invention.
With regard to the argument that while there is still Lon activity when using BL21(DE3), the present invention has overcome that disadvantage with the optimized synthetic metabolic valves of the instant invention and that nothing in the teachings of Menacho-Melgar, Gottesman or Li would have provided the ordinary artisan a reasonable expectation of the improved performance of silencing the lon protease in contrast with chromosomal deletion of the gene, it is noted that one of the embodiments encompassed by claims 1 and 7 is the deletion of the lon protease gene as set forth in claim 7. Therefore, it does not appear that the metabolic valves the instant invention would be required to reduce/eliminate lon activity in the case of the deletion of the lon protease. In addition, while it is agreed that none of the cited references would have predicted the improvement in protein levels, the issue in the instant case is whether one of skill in the art would have a reasonable expectation of success in replacing the target enzymes of Li et al. with the proteases recited and observing some improvement in the reduction of proteolytic activity of any of the recited proteases, particularly in view of the results shown by Li et al. when proteolysis and gene silencing were combined (Figure 1f-1g). The results shown by Li et al. clearly indicate a drastic reduction in enzymatic activity when gene silencing and proteolysis, both controlled by a low phosphate inducible system, are combined.
It is reiterated herein that at the time of the invention, there was a reasonable of expectation of success in making the genetic manipulations of Li et al. to the E. coli cells of Menacho-Melgar et al. wherein the target enzymes of Li et al. are replaced with endogenous E. coli proteases because the molecular biology techniques required were well known in the art as evidenced by Li et al., and E. coli protease genes such as ftsH, lon, ompT and hslUV genes were well known in the art as evidenced by Gottesman. While one could argue that there was no absolute certainty that replacing the target enzymes of Li et al. with the endogenous E. coli proteases recited would result in the reduction of the protease activity of the endogenous E. coli proteases recited, it is noted that all that is required in the obviousness analysis is a reasonable expectation of success in observing some reduction in the protease activity. There is no evidence in the prior art teaching or suggesting that (i) gRNAs that could target the E. coli protease genes for gene silencing cannot be made when the nucleotide sequences of the E. coli protease genes are known, (ii) C-terminal DAS+4 peptide tags attached to the desired proteases to make them a target of the ClpXP protease in E. coli could not be added, (iii) ClpXP protease cannot degrade the recited E. coli proteases, or (iv) the low phosphate inducible promoter used by Li et al. is not functional when operably linked to a nucleic acid encoding a gRNA that targets the E. coli protease genes. Therefore, in the absence of evidence to the contrary, one of skill in the art would have concluded that there was a reasonable expectation of success in making the necessary genetic replacements in the systems of Li et al. and observe some reduction in proteolytic activity in the E. coli cell of Menacho-Melgar et al., particularly in view of the results shown when gene silencing and proteolysis are combined. Thus, contrary to Applicant’s assertions, for the reasons of record and those set forth above, one would conclude that the claimed invention is obvious over the teachings of Menacho-Melgar et al., Li et al. and Gottesman.
Conclusion
No claim is in condition for allowance.
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Any inquiry concerning this communication or earlier communications from the examiner should be directed to DELIA M RAMIREZ, Ph.D., whose telephone number is (571) 272-0938. The examiner can normally be reached on Monday-Friday from 8:30 AM to 5:00 PM. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Robert B. Mondesi, can be reached at (408) 918-7584. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
/DELIA M RAMIREZ/Primary Examiner, Art Unit 1652
DR
April 17, 2026
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