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
Claims 1-20 are rejected.
Priority
The instant application claims the benefit of priority to provisional application 63/239,381 filed on 31 August 2021. As such, the effective filing date of claims 1-20 is 31 August 2021.
Information Disclosure Statement
No IDS has been filed herein.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the drawings are not identified by sequence identifiers in accordance with 37 CFR 1.821(d). Sequence identifiers for nucleotide and/or amino acid sequences must appear either in the drawings or in the Brief Description of the Drawings.
Required response – Applicant must provide:
Replacement and annotated drawings in accordance with 37 CFR 1.121(d) inserting the required sequence identifiers;
AND/OR
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers into the Brief Description of the Drawings, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Drawings
The drawings are objected to because Figure 7A includes enumerated nucleotide sequences without providing sequence identifiers (see above). Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Specification
The disclosure is objected to because of the following informalities:
Page 14, [0045] refers to Fig. 6e, Fig. 6f, Fig. 6g, and Fig. 6h but these figures are missing from the application.
Page 15, [0045] refers to “Fig. 6e” but this figure is missing from the application.
Appropriate correction is required.
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.
Claim 5-7 and 17 are rejected under 35 U.S.C. 112(b), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor, or a joint inventor, regards as the invention.
Claim 5 recites “identifying one or more replacement LBMs within the protein sequence (S), wherein the one or more replacement LBMs are assigned to replace one or more LBMs in the hybrid repressor.” Both the specification and Claim 1 define a “protein sequence (S) of a hybrid repressor” as “hybrid protein sequence comprises a plurality of DNA-binding modules (DBMs) and a plurality of ligand-binding modules (LBMs).” The specification also states that “in an aspect, …a hybrid repressor comprising an N-terminal domain that interacts with promoters (DNA-binding) and a C-terminal domain that senses molecular signals (ligand-binding).” These differing definitions create ambiguity as to the protein sequence in question and the selection of replacement LBMs for a sequence comprised of a plurality of LBMs. . Claim 6 is also rejected due to its dependency on 5.
Claim 7 recites “identifying a plurality of replacement LBMs within the protein sequence (S), wherein the plurality of replacement LBMs are assigned to replace one or more LBMs in the hybrid repressor to identify a plurality of mutation protein sequences.” Both the specification and Claim 1 define a “protein sequence (S) of a hybrid repressor” as “hybrid protein sequence comprises a plurality of DNA-binding modules (DBMs) and a plurality of ligand-binding modules (LBMs).” The specification also states that “in an aspect, …a hybrid repressor comprising an N-terminal domain that interacts with promoters (DNA-binding) and a C-terminal domain that senses molecular signals (ligand-binding).” Neither the claims nor the specification define the phrase “plurality of mutation protein sequences.” These issues regarding the terminology create ambiguity as to the protein sequences referenced in the claim.
Claim 7 further recites “determining a plurality of mutation compatibility scores”. Neither the claims nor the specification define the phrase “mutation compatibility score”. As a result, it is unclear what is meant by “determining a plurality of mutation compatibility scores for the plurality of mutation protein sequences.”
Claim 17 refers to “The method of step 13” and does not properly refer back to a claim. As a result, the scope of the claim is unclear. This rejection can be overcome by removing the word “step” and replacing with the word “claim”.
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Claim 1, and claims 2-10 due to their dependency on claim 1, are rejected under 35 U.S.C. 112(a), first paragraph, as failing to comply with the written description requirement. The claims contain 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 applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. Claim 1 recites “receiving a protein sequence (S) of a hybrid repressor, wherein the hybrid protein sequence comprises a plurality of DNA-binding modules (DBMs) and a plurality of ligand- binding modules (LBMs).” Additionally, claim 9 recites “wherein the compatibility score is further based on residue proximity between the plurality of LBMs.” However, the specification recites “the allosterically regulated transcription repressor is a hybrid repressor comprising an N-terminal domain that interacts with promoters (DNA-binding) and a C-terminal domain that senses molecular signals (ligand-binding).” [0021] (emphasis added) The specification fails to describe how one skilled in the art would receive or analyze a hybrid repressor with a plurality of DBMs and a plurality of LBMs given that the specification defines a repressor as containing a DBM and an LBM.
Claim 1, and claims 2-10 due to their dependency on claim 1, are rejected under 35 U.S.C. 112(a) because the specification, while being enabling for “… the compatibility refers to the extent to which the hybrid repressor comprising a DBM and LBM is able to carry out its intended function (e.g., transcriptional repression) when compared to a reference repressor”[0022], does not reasonably provide enablement for determining compatibility for protein sequence that “comprises a plurality of DNA-binding modules (DBMs) and a plurality of ligand- binding modules (LBMs)”. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
MPEP § 2164.01(a) explains how enablement for the claimed invention can be analyzed:
In order to determine compliance with the enablement requirement of 35 U.S.C. 112(a), the Federal Circuit developed a framework of factors in In re Wands, 858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988), referred to as the Wands factors to assess whether any necessary experimentation required by the specification is “reasonable” or is “undue.” . . . These factors include, but are not limited to:
(A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
The Wands factors are analyzed with respect to the claimed invention in turn below.
The claim is broad and refers to “determining an original compatibility score C(S), where the compatibility score C is a function of the protein sequence (S).” There is no explanation of how to determine said compatibility score.
The nature of the invention is a method of scoring protein sequences containing hybrid repressors and predicting the performance of those repressors based on the compatibility score.
The state of the art includes computational methods to determine the compatibility of hybrid repressors. Dimas et al. (NAR, August 2019) teaches a method to “predict the compatibility of a hybrid repressor with DNA-binding and ligand-binding modules from different LacI family members.” (Page 8923, col. 21, lines 56-58) Dimas et al (NAR, August 2019) also teaches “Allosterically regulated transcriptional regulators…comprise an N-terminal domain that interacts with promoters (DNA binding) and a C-terminal domain that senses molecular signals (ligand-binding).” (Page 8914, col. 3, lines 3-7) .
While one skilled in the art would be familiar with methods to rate a hybrid repressor with one DBM and one LBM, there is no method for evaluating sequences with a plurality of DBMs and a plurality of LBMs as described in the claims.
The level of predictability in the art is high as it is unknown which combinations of DBMs and LBMs would be most compatible. As indicated in the Specification, “one major challenge in creating hybrid repressors is that some DBMs and LBMs are incompatible.” [0022]
The specification provides no guidance as to how to determine the compatibility of a plurality of DBMS and a plurality of LBMs in a given protein sequence.
No working examples are provided in the specification for the scoring method of claim 1.
With no guidance from the specification, it would require an undue amount of experimentation to determine how to use the method of claim 1.
In view of the Wands factors discussed above, the disclosure of the instant application does not reasonably enable a person having ordinary skill in the art to use the full scope of the claimed invention.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1-20 are rejected under 35 U.S.C. 101 because the claimed inventions are directed to abstract ideas without significantly more.
Step 2A, Prong 1
In accordance with MPEP § 2106, claims found to recite statutory subject matter (Step
1: YES) are then analyzed to determine if the claims recite any concepts that equate to an abstract idea, law of nature or natural phenomenon (Step 2A, Prong 1). In the instant application, the claims recite the following limitations that equate to an abstract idea:
Claim 1 and dependent claims 2-10 recite determining an original compatibility score C(S), where the compatibility score C is a function of the protein sequence (S); and predicting, based on the compatibility score C, a performance of the hybrid repressor.
Claim 3 and dependent claim 4 recite evaluating the performance of the hybrid repressor.
Claim 5 and dependent claim 6 recite determining a second compatibility score for the protein sequence comprising one or more replacement LBMs.
Claim 7 recites determining a plurality of mutation compatibility scores for the plurality of mutation protein sequences.
Claim 6 recites identifying a second hybrid repressor characterized by a second compatibility score that is from about 5 fold to about 500 fold greater than the original compatibility score.
Claim 7 recites determining that one or more second compatibility scores of the plurality of compatibility scores are improved relative to the compatibility score by from about 5 fold to about 500 fold; and identifying a second protein sequence for a second hybrid repressor using one or more mutation protein sequences of the plurality of mutation protein sequences having the one or more second compatibility scores, wherein the second hybrid repressor demonstrates a greater functionality than the hybrid repressor.
Claims 8, 14, and 15 recite the compatibility score C(S) is based on inter-modular coevolutionary coupling strength parameters.
Claim 9 recites the compatibility score is further based on residue proximity between the plurality of LBMs.
Claim 10 recites determining a structure-based score SF(S), where the structure-based score SF is a function of the coevolutionary strength between residues, where predicting the performance of the hybrid repressor is further based on the structure-based score SF.
Claim 11 and dependent claims 12-20 recite determining an original compatibility score C(S), where the original compatibility score C is a function of the protein sequence (S).
Claim 11 recites computationally mutating at least one amino acid residue in the LBM of the repressor to generate a hybrid repressor with a mutated LBM.
Claim 12 recites wherein at least two amino acid residues in the hybrid repressor with a mutated LBM are mutated.
Claim 13 recites wherein at least three amino acid residues in the hybrid repressor with a mutated LBM are mutated.
Claims 16 and 17 recite step (c) [claim 11] is carried out a plurality of times to generate a plurality of hybrid repressors with a mutated LBM.
Claim 18 recites evaluating the performance of the repressor to obtain a base activity.
Claim 19 recites comprising evaluating the performance of the hybrid repressor with a mutated LBM to obtain a second activity.
Claim 20 recites identifying hybrid repressors with a mutated LBM having a second activity that is improved by from about 5 fold to about 500 fold when compared to the base activity.
The limitations for determining compatibility scores, predicting performances, evaluating performances, determining structure-based scores, or modifying are evaluations or judgements that can be made by making mental observations of the protein sequences, which fall under the "mental processes" grouping of abstract ideas. As such, claims 1-20 recite abstract ideas (Step 2A, Prong 1: YES).
Step 2A, Prong 2
Claims found to recite a judicial exception under Step 2A, Prong 1 are then further
analyzed to determine if the claims as a whole integrate the recited judicial exception into a
practical application or not (Step 2A, Prong 2). This judicial exception is not integrated into a
practical application because the claims do not recite an additional element that reflects an
improvement to technology or applies or uses the recited judicial exception in some other
meaningful way. Rather, the instant claims recite additional elements that amount to mere
instructions to implement the abstract idea or insignificant extra-solution activity. Specifically, the claims recite the following additional element:
Claim 4 recites the performance of the hybrid repressor is evaluated using one or more transcriptional assays.
The limitations for evaluating performance describe mental processes with additional elements. This judicial exception is not integrated into a practical application because these additional elements do not add any meaningful limitations. The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception because they only describe additional ways to evaluate performance. As such, these limitations equate to mere instructions to implement the abstract ideas. The above recited additional element does not provide a practical application of the recited judicial exception. As such, claims 1-20 are directed to an abstract idea (Step 2A, Prong 2: NO).
Step 2B
Claims found to be directed to a judicial exception are then further evaluated to determine if the claims recite an inventive concept that provides significantly more than the judicial exception itself (Step 2B). The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception because the claims recite additional elements that equate to mere instructions to apply the recited exception in a well-understood, routine and conventional activity.
As discussed above, there are no additional limitations to indicate that the claimed
methods require anything other than basic experimental techniques in order to carry out the
recited abstract ideas in the claims. Claims that amount to nothing more than an instruction to
apply the abstract idea do not render an abstract idea eligible.
Furthermore, the additional elements recited in the claims amount to well-understood,
routine and conventional activity, as evidenced by Zaslaver et al (Nature Methods, Volume 3, Number 8, August 2006, pages 623-628). Zaslaver et al. discloses regarding transcriptional assays that an “approach is to measure the transcriptional activity of promoters by means of reporter genes. Classic studies used the lacZ reporter, and in the last decade GFP has become widely used.” (Page 623, column 1, lines 30-32) As such, the combination of additional elements recited in the claims is well-understood, routine and conventional.
The additional elements do not comprise an inventive concept when considered individually or as an ordered combination that transforms the claimed judicial exception into a
patent-eligible application of the judicial exception. Therefore, the claims do not amount to
significantly more than the judicial exception itself (Step 2B: No). As such, claims 1-20
are not patent eligible.
Claim Interpretation
For the purposes of applying prior art:
Claim 1 is interpreted to as including a set of protein sequences that each contain a hybrid repressor wherein each repressor comprises a DBM and an LBM. This set of protein sequences includes many hybrid repressors made from a plurality of DBMs and LBMs, but each repressor contains only one DBM and one LBM.
Claim 5 is interpreted as replacing the LBM in a given hybrid repressor with another LBM from the plurality of available LBMs.
Claim 7 is interpreted as replacing the LBM in a given hybrid repressor with an LBM that has a mutation.
Claim Rejections - 35 USC § 102
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 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.
Claims 1-6, 8, and 9 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Dimas et al. (Nucleic Acids Research, 24 April 2019, pages 5449-5463) (Hereafter referred to as Dimas.)
Regarding claim 1, Dimas teaches to “…develop a module-swapping strategy to construct modular repressors: by fusing a DRM and an ESM from two different repressors, the resulting hybrid repressors possess the corresponding properties of DNA recognition and allosteric response from their respective native modules…(and) used two DRMs and three ESMs to construct a set of six engineered repressors…” (Page 5450, column 3, lines 12-19). In addition, Dimas teaches “…a methodology to predict the compatibility of hybrid repressors with non-native DRMs and ESMs. The compatibility score used in this study evaluates the possibility of two modules from different origins to integrate and work as a functional protein that preserves properties of homodimerization, ligand and promoter binding, and allosteric transition.” (Page 5451, column 5, lines 8-14)
Regarding claim 2, Dimas teaches a “Computational strategy for the prediction of hybrid repressor performance. Direct Coupling Analysis was used to infer the parameters of the global joint probability distribution estimated for the LacI homologs. First, these parameters, eij and hi, were used to compute direct information values for residue pairs to discern the most important residue-residue pairs between the ESM and the DRM domains. Then, a compatibility score C(S) of a specific repressor sequence was calculated using eij, only for those residue position pairs with the highest DI values.” (Page 5450, column 4, Figure 1 caption)
Regarding claim 3, Dimas teaches “…to develop a compatibility prediction model, which assigns compatibility score(s), C(S), to estimate compatibility between a DRM and an ESM…” (Page 5454, column 11, lines 57-60) and “…we validated this predictive model with experimental results from hybrid repressors.” (Page 5454, column 12, lines 3-4)
Regarding claim 4, Dimas teaches they “…assessed the ability of hybrid repressors in gene expression regulation and allosteric response, by using a transcriptional reporter assay.” (Page 5455, Figure 2(A) caption, lines 2-3)
Regarding claim 5, Dimas teaches “By combining DRMs and ESMs from different LacI family members, we can mix and match DNA recognition and allosteric response properties originated from different repressors.” (Page 5450, column 2, Figure 1 caption) Additionally, Dimas teaches “performing quantitative comparisons of the compatibility scores from repressors with the same DRM or ESM.” (page 5459, column 20, lines 51-52). Regarding claim 5, see also Figure 2 in Dimas. (Page 5455)
Regarding claim 6, Dimas teaches “Our model computes a compatibility score for particular DRM-ESM combinations, which serves as a metric to accurately predict whether the resulting repressor is capable of providing an induction dynamic range above a fixed threshold (20-fold induction; Figure 3).” (Page 5459, column 20, lines 44-48; See also Figure 3, page 5456, column 16)
Regarding claim 8, Dimas teaches “a computational approach based on coevolutionary information to model module compatibility” and “a coevolutionary modeling approach…to infer compatibility between DRMs and ESMs among LacI homologs. Coevolution among residues in a protein family plays an important role in stability and function of proteins.” (Page 5450, column 3, lines 31-33 and 42-46)
Regarding claim 9, Dimas teaches “In these two equations, i and j represent ith and jth residues in the LacI homolog alignment sequence. Ai and Aj refer to one of the 20 possible amino acids (or a gap in the sequence alignment) located at position i and j. In this distribution model, pairwise couplings, eij (Ai, Aj), provide information on the coevolutionary strength between residue position i and j in the case where the amino acids in these two positions are Ai and Aj, respectively.” (Supplementary Information, Supplementary Methods, “Identification of important coevolving DRM-ESM residue pairs”, paragraph 2)
Claim Rejections - 35 USC § 103
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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.
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Dimas (Nucleic Acids Research, 24 April 2019, pages 5449-5463) in view of Richards et al (ACS Synthetic Biology, 6 September 2016, pages 6-12). (Hereafter referred to as Richards)
Dimas teaches the methods of claim 1, as rejected above under 35 U.S.C. 102(a)(1). (NAR, 2019: Page 5450, column 3, lines 12-19; Page 5451, column 5, lines 8-14)
Dimas does not teach replacing one or more LBMs with a mutated LBM and does not teach assessing the function of hybrid repressor with a mutated LBM.
Richards teaches creating a series of engineered LacI repressor variants with mutated effector-binding sites. See Figure 2, page 8, for a visual representation of the locations of the mutations in the N-subdomain and C-subdomain of the repressor. These two subdomains are both part of what Richards refers to as the “effector-binding domain.” The effector-binding domain is synonymous with the “ligand binding module” referenced in this application. Richards also teaches evaluating and comparing the function of the mutated repressors. (Figure 3, page 9; Figure 4, page 10)
It would have been prima facie obvious to one of ordinary skill in the art at the effective filing date of the invention to substitute one known element for another and the results of the substitution would have been predictable. Specifically, Dimas teaches that repressors comprising a DBM and an LBM are modular. Further, Dimas teaches that it is possible to create a variety of hybrid repressors by selecting from a plurality of DBMs and LBMs. Richards teaches that it is possible to create a variety of functional mutated LBMs. Additionally, one of ordinary skill in the art would have had a reasonable expectation of success in creating hybrid repressors with mutated LBMs. Dimas explains that their “methodology can help navigate favorable mutations, reforming the repressors with specific signal-response linkages to exhibit improved compatibility between ESM and DRM while maintaining the functionality of each domain.” (Page 5460, column 22, lines 8-11) Therefore, one of ordinary skill in the art would have recognized that the repressor module swapping method could be used with mutant LBMs to study the impact on hybrid repressor function. The invention is therefore prima facie obvious.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Dimas (Nucleic Acids Research, 24 April 2019, pages 5449-5463) in view of Morcos et al (PNAS, 6 December 2011, pages E1293-E1301). (Hereafter referred to as Morcos)
Dimas teaches the methods of claim 1, as rejected above under 35 U.S.C. 102(a)(1). (NAR, 2019: Page 5450, column 3, lines 12-19; Page 5451, column 5, lines 8-14) In addition, Dimas teaches scoring protein sequences compatibility based inter-modular coevolutionary coupling strength parameters. (Page 5450, column 3, lines 31-33 and 42-46)
Dimas does not teach “determining a structure-based score SF(S), where the structure-based score SF is a function of the coevolutionary strength between residues, where predicting the performance of the hybrid repressor is further based on the structure-based score SF.”
Morcos teaches using Direct Coupling Analysis to evaluate ligand-mediated couplings based on coevolution. The method by Morcos includes quantifying strength between residue pairs and using this information to predict spatial contact. (Page E1294, column 3, lines 44-50; column 4, lines 39-41) Morcos further teaches equations to determine the strength of these coupling interactions. (Page 1300, equation 11-13) Morcos teaches that these residue pair interactions have can assist in determination of complex structure prediction. (Page E1298, column 11, lines 23-25)
It would have been prima facie obvious to one of ordinary skill in the art at the effective filing date of the invention to have applied the method of Morcos to further evaluate the performance of the hybrid repressor of Dimas. Dimas explains that “[t]his methodology might be limited by the total number of available sequences and the requirement of having structured independent-folding units that interact.” (Page 5460, column 22, lines 22-24) Therefore, one of ordinary skill in the art would have been motivated to utilize a methodology to evaluate coevolutionary strength between residues to determine structure of the hybrid repressors. The invention is therefore prima facie obvious.
Claims 11-20 are rejected under 35 U.S.C. 103 as being unpatentable over Dimas (Nucleic Acids Research, 24 April 2019, pages 5449-5463) in view of Garruss et al (PNAS, 29 June 2021, pages 1-10). (Hereafter referred to as Garruss)
Dimas teaches “Development of a computational model to predict compatibility among DRMs and ESMs from LacI homologs.” (Page 5450, column 4, Figure 1) Dimas further teaches “organizing the extensive set of LacI homologous sequences and a reweighting scheme for sequences with larger than 80% similarity, we used a mathematical strategy to identify DRM-ESM residue pairs that are strongly coupled.” (Supplementary Information, Supplementary Methods, page 2, paragraph 1) Dimas further teaches determining a compatibility score based on inter-modular coevolutionary strength parameters (Page 5450, column 3, lines 31-33 and 42-46) and evaluating the performance of a hybrid repressor. (Page 5455, Figure 2(A) caption, lines 2-3)
Dimas does not teach computationally mutating one or more amino acids in the LBM of the repressor, determining compatibility scores for the hybrid repressors with mutated LBMs, or evaluating the performance of hybrid repressors with mutated LBMs.
Garruss teaches utilizing computational models to analyze the effects of mutations in the LacI repressor, including mutations in the C-terminal domain (LBM). (Figure 2, page 4). Garruss further teaches evaluating one, two, and three computational mutations in the LacI repressor. (Page 9, Table 1) Garruss further acknowledges the significance of evolutionary conservation and coevolutionary coupling when evaluating LacI repressor mutations. (Page 3, column 4, lines 36-52; Page 5, column 6, lines 3-5)
It would have been prima facie obvious to one of ordinary skill in the art at the effective filing date of the invention to have applied the method of Garruss to computationally mutate the LacI LBM and evaluate the performance of the mutated hybrid repressors. Dimas explains that the method studying LacI hybrid repressors can also be used to “help navigate favorable mutations, reforming the repressors with specific signal-response link ages to exhibit improved compatibility between ESM and DRM while maintaining the functionality of each domain.”(Page 5460, column 22, lines 8-11) Therefore, one of ordinary skill in the art would have been motivated to computationally mutate amino acids in LacI homologous LBMs and to use evaluate the performance of hybrid repressors with mutated LacI homologous LBMs based on coevolutionary coupling strength parameters. One skilled in the art would have been motivated to computationally mutate at least one amino acid residue (claim 11), at least two amino acid residues (claim 12, at least three amino acid residues (claim 13), or to mutate amino acid residues a plurality of times (claims 16 and 17) Further, one skilled in the art would have recognized the benefit of combining the methods of Garruss and Dimas to determine the compatibility scores of hybrid LacI repressors with mutated LBMs utilizing inter-modular coevolutionary coupling strength parameters (claims 11, 14, and 15). Additionally, one skilled in the art would have recognized the benefit of combining the methods of Garruss and Dimas to evaluate and compare the performance of the hybrid repressors containing mutated LacI homologous LBMs (claims 18, 19, and 20) The invention is therefore prima facie obvious.
Conclusion
The prior art made of record and not relied upon is considered pertinent to the applicant’s disclosure. Dimas et al (NAR, August 2019) teaches using the modular swapping strategy to create hybrid TetR repressors. Cocco et al (Reports on Progress in Physics, January 2018) teaches methods for calculating coevolutionary pair interactions in proteins. Meinhardt et al (NAR, September 2012) teaches creating chimeric LacI repressors. Schmidl et al (Nature Chemistry Biology, July 2019) teaches DNA binding domain swapping in transcription factors. Masso et al (Bioscience Research and Applications, 2008) teaches using computational mutagenesis of LacI repressors.
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/S.L.G./Examiner, Art Unit 1687
/Karlheinz R. Skowronek/Supervisory Patent Examiner, Art Unit 1687