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 .
Status of the Application
Claim(s) are 1-20 pending and under examination
The following Office Action is in response to Applicant's communication dated 05/02/2024.
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.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim(s) 16 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 applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 16 recites “ determining a functional score for each of the selected allosteric biosensors using confidence scoring”, but specification describe functional score and confidence scores as distinct measurements. Functional scores are determined from barcode RNA/DNA measurements, whereas confidence scores are separately calculated from sequencing counts variability among technical replicates, and variability among biological replicates. Specification also states confidence scores are used as filter for selecting biosensors for clonal validation [¶0121-0122]. Hence, it is unclear whether confident scores determine, modifies or merely accompany the functional scores. Since one of ordinary skill in the art cannot determine with reasonable certainty the scope of protection, the metes and bounds of the claim are unascertainable.
For purposes of examination, the functional score and confidence scores are interpreted according to the specification as distinct measurements.
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.
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.
Spoonamore et al. and Klein et al.
Claim(s) 1-5, 8-9, 11, 13-15, 17-20 is/are rejected under 35 U.S.C. 103 as being unpatentable over Spoonamore et al. (US20190204303A1, EFD: August 15th 2017) in view of Klein et al. (Nat Methods 17, 1083–1091 (2020)).
Regarding claim 1, Spoonamore discloses method for selecting biosensor comprising: 1) expression construct encodes allosteric protein variant under a first promoter 2) a functionally linked reporter containing a barcode identifying that variant and controlled by a second promoter (e.g. method for selecting engineered allosteric transcription factor sensors (TF sensors) that is responsive to target ligand. Spoonamore discloses making libraries of potential mutations to the TF sensors and screening the library for desired candidates [¶0055 and 0147]. The TF sensors and reporter gene may be on the same plasmid, which comprises one or more of these elements: the TF sensor gene; the regulatory sequences (e.g. promoter, ribosome binding site, transcriptional terminator, 5′-untranslated region, riboswitch, and/or degradation signal) controlling the expression of the TF sensor gene; the reporter gene; the regulatory sequences controlling the reporter gene (as above); the sensor DNA binding site; the origin of replication [¶0157]. The reporter gene comprises barcode sequence that is unique to each plasmid encoding a TF sensor gene library member [¶0083].)
ligand binding to expressed variant activate reporter system expression and absent of ligand inactivate reporter system expression (e.g. Screening sensor variants for ligand responsiveness includes detecting different reporter expression in presence/absent of ligand [¶0158]. Specifically, when a cell makes the TF sensor, it stops the reporter gene from turning on. The TF sensor only turns on the reporter gene when the target molecule binds to the sensor [¶0150])
mapping each variant to barcode sequence and assigning variant-barcode pairs (e.g. The reporter gene comprises barcode sequence that is unique to each plasmid encoding a TF sensor gene library member, such that the abundance of each sensor library member can be ascertained by sequencing the expressed barcode sequences, once each sensor and barcode combination has been determined [¶0083].)
grow cells transfected with the library in the presence of ligand (e.g. introducing nucleic acids encoding TF sensors and reporter system into host cells, contact cells with ligand/target molecules, and screen TF sensor libraries under induction conditions to identify variants exhibiting the desired ligand response behavior [¶0063-0065, ¶0158-0159]. )
select ligand-responsive TF sensor candidates (e.g. the reference teaches screening and recovering TF sensor variants having desired ligand responsive reporter characteristics, such as difference in reporter expression [¶0157-0158])
Spoonamore does not disclose plasmid comprises primer binding sites enabling next generation sequencing (NGS) of variant and reporter and the steps of isolate both RNA and plasmid DNA; use NGS to quantify each barcode in the total RNA; and determining fold enrichment for each variant in the total RNA, which is normalized using plasmid.
Klein discloses that in a pooled barcoded reporter assay, DNA and RNA corresponding to barcode constructs are extracted from the cells, separably sequenced, and reporter activity is determined from RNA counts normalized to corresponding DNA. Klein further teaches that resulting RNA/DNA normalized measurements provide a quantitative activity score for each library member [pages 1084-1086 and Method section]. The reference also uses construct specific PCR/NGS primer sequence, P5/P7 primers, and custom NexSeq primers to amplify and sequence candidate libraries [Method section].
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to apply Klein’s known RNA/DNA normalization method to Spoonamore’s barcode sensor library because Spoonamore discloses using barcode sequencing results to identify the TF sensors with desirable ligand interaction activity, but did not go into detail on data analysis aspect of sequencing. One of ordinary skill in the art would have been motivated to use Klein’s established framework for sequencing barcode containing RNA, corresponding DNA, and normalizing the RNA counts to DNA counts, hence, correcting differences in plasmid representation among library candidates and provide reliable quantitative measure of reporter activity for each TF sensor variant. Such modification represents applying a known quantitative analysis workflow for its established purpose in similar pooled barcoded reporter system. A skilled artisan would have had reasonable expectation of success since both references use expressed barcode sequences associate with respective library member to quantify transcriptional reporter activity by sequencing. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Regarding claims 2-4, Spoonamore discloses allosteric protein comprises a DNA binding domain, in the presence of the target ligand, either inactivates or activates expression of the reporter from the second promoter. Allosteric protein is an allosteric transcription factor (aTF), such as TetR, LacI, TtgR, MphR, AraC, or LysR (e.g. engineered allosteric DNA-binding sensors that binds to ligand and regulate transcription of reporter. For example, the reference discloses both repressor and activator configurations: for a repressor, the sensor binds DNA and block transcription until the target molecule/ligand binds the sensor. For an activator, target molecule binding enable recruitment of RNA polymerase and activate transcription [¶0081 and ¶0149-150]. Engineered TF sensor is an engineered version of TetR, LacI, TtgR, MphR, AraC, or LysR [¶0090-0092].)
Regarding claims 5, Spoonamore discloses the allosteric protein or allosteric domain associates, directly or indirectly, with a DNA binding domain that, in the presence of the target ligand, either inactivates or activates expression of the reporter from the second promoter. (e.g. protein sensor and/or switch may be an engineered version of a two-component or hybrid two-component system that directly bind both a ligand and DNA or work through a protein cascade. [¶0093]. )
Regarding claims 8, Although Klein does not disclose the exact barcode length 16 to 24 nucleotides, Klein discloses 15 nucleotides barcode [Method]. it would have been prima facie obvious to a person of ordinary skill in the art to increase number of nucleotide in barcode when working with larger population of variants to prevents stochastic overlap when sampling large pools of variants. This modification represents optimization of a result effective variable. The discovery of an optimum or workable range would have been within the ordinary skill in the art through routine experimentations. See MPEP § 2144.05 (II) "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955)
Regarding claims 9, Klein discloses each variant is associated with 10 to 100 barcodes (e.g. the median number of barcode counts per element/variant was >100 [page 1085])
Regarding claims 11, Klein discloses mapping comprises direct sequencing of the expression constructs (e.g. Klein’s construct library comprising barcode sequences, which are associated with candidate sequences. Klein further teaches amplifying and sequencing region comprising library sequence and associated barcode to link the barcode to their respective variant sequences. Hence establishing sequence-barcode associations prior to use of constructs in reporter assays. [page 1084])
Regarding claims 13, Klein discloses the reporter further comprises a coding sequence for a detectable marker protein operably linked to the second promoter. (e.g. reporter gene system (which is regulated by second promoter-see claim 1 rejection) comprises green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), Renilla Reniformis green fluorescent protein, GFPmut2, GFPuv4, yellow fluorescent protein (YFP), etc. [0175]).
Regarding claims 14, Klein discloses pooled barcode reporter assay using plasmid library comprising both experimental and positive/negative control constructs, specifically library consisting of 2,236 candidate along with 204 controls. The controls consist of sequences that previously demonstrated activity (100 positives) or lack thereof (100 negatives) [page 1084]. Klein further discloses transfecting plasmids into cells, extracting DNA and RNA, amplifying and sequencing the corresponding barcodes, and using the barcode counts to normalize activity score for each respective library element. Klein calculates activity from normalized RNA barcode count relative to DNA barcode count and determine activity as log2(RNA/DNA). Klein performs the same RNA/DNA activity calculation for the positive and negative controls and compare control activity distribution with experimental library members [pages 1084-1087].
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to use control sample’s average activity as normalization baseline for activity or fold enrichment of sensor variants because Klein explicitly use control constructs to establish reference activity distribution and calculate normalized RNA/DNA activity for both controls and experimental candidates. Hence, applying the control average would have been predictable quantitative use of Klein’s control measurements to account for background, experimental variation, and place experimental measurement relative to a common control to ensure consistency in quantitative data analysis.
Regarding claims 15, Spoonamore discloses validating the subpopulation selected allosteric biosensors by determining expression of the detectable marker protein in the presence and absence of the target ligand. (e.g. TtgR regulate promoter that drive GFP expression in response to naringenin ligand. FIG. 9 shows a ligand dose response of TtgR and TtgR variants to naringenin. FIG. 10 shows GFP distributions for the TtgR-GFP parent system and mutants 1, 2 and 3 from FIG. 9. Right shifting of curves indicates binding in the mutants.)
Regarding claims 17, Spoonamore discloses target ligand is a biological molecule, a drug (e.g. In some embodiments, the target molecule is an antibiotic [¶0118])
Regarding claims 18, Klein discloses barcoded reporter assay is a high throughput assay. (e.g. massively parallel reporter assay [Abstract])
Regarding claims 19 and 20, Spoonamore discloses device comprising a substrate, cell or chamber comprising the allosteric biosensor selected by the method of claim 1. wherein the device is suitable for use in high throughput screening, single cell analysis, online monitoring (e.g. In certain embodiments, the present invention provides sorting of cells, for either reporter negative (e.g. dark) or reporter positive (e.g. bright) cells, for their inability to either correctly repress the reporter gene system or remain inactive, depending on the logic of the gene reporter system, using, e.g., a reporter gene system and a fluorescence activated cell sorting (FACS) apparatus or microbial colony picker (e.g., QPix), or a microfluidics apparatus, or a bead-based apparatus [0084])
Spoonamore et al., Klein et al., and Raman et al.
Claim(s) 6 is/are rejected under 35 U.S.C. 103 as being unpatentable over Spoonamore et al. (US20190204303A1, EFD: August 15th 2017) in view of Klein et al. (Nat Methods 17, 1083–1091 (2020)) and Raman et al. (US20190062730A1, EFD: April 4th 2018).
Regarding claims 6, Spoonamore discloses OmpR family transcriptional regulators and lists KdpE, OmpR, PhoP, and ComA as transcriptional factors that may be engineered [¶0090-0101].
However, Spoonamore does not disclose the exact claimed allosteric protein pairs: PhoP/PhoQ, EnvZ/OmpR, KdpE/KdpD, and ComA/ComP.
Raman discloses DNA binding proteins are useful in inducible biosensor system and may comprise two component signaling proteins [0054] and expressly identifies PhoP/PhoQ, EnvZ/OmpR, KdpE/KdpD, and ComA/ComP as prokaryotic two component transcriptional systems [¶0056,Table 1].
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to use Raman’s known two component signaling systems in place of the two component or hybrid two-component system expressly suggested by Spoonamore with reasonable expectation of success because Raman identifies these systems as suitable two component transcriptional regulators for such inducible reporter system. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Spoonamore et al., Klein et al., and Nishikawa et al.
Claim(s) 7 is/are rejected under 35 U.S.C. 103 as being unpatentable over Spoonamore et al. (US20190204303A1, EFD: August 15th 2017) in view of Klein et al. (Nat Methods 17, 1083–1091 (2020)) and Nishikawa et al.(Nat Commun 12, 5562 (2021), disclosed in IDS).
Regarding claims 7, Spoonamore discloses sensors expression of the allosteric DNA binding sensor can be modulated, e.g. by mutating the allosteric DNA binding sensor promoter [¶0156]. However, Spoonamore does not disclose first promoter is a constitutively active promoter.
Nishikawa discloses allosteric sensor system, wherein the TtgR expression plasmid comprises constitutive promoter-RBS combination apFAB61-BBa_J61132 operably linked to TtgR gene [Library synthesis section]. Nishikawa further discloses screening libraries if engineered TtgR protein variants using ligand responsive reporter expression [pages 2-3].
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to employ Nishikawa’s constitutive promoter for expression of Spoonamore’s allosteric transcription factor sensors, including TtgR, in order to ensures a steady, baseline level of the regulator protein inside the cell, enable cell to detect the target binding activity quick and consistent. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Spoonamore et al., Klein et al., and Taylor et al.
Claim(s) 10 and 12 is/are rejected under 35 U.S.C. 103 as being unpatentable over Spoonamore et al. (US20190204303A1, EFD: August 15th 2017) in view of Klein et al. (Nat Methods 17, 1083–1091 (2020)) and Taylor et al. (Nat Methods 13, 177–183 (2016), disclosed in IDS).
Regarding claims 10, Spoonamore does not discloses the library comprises 5,000 to 20,000 allosteric protein variants or allosteric domain variants.
Taylor discloses engineered aTF proteins using computational protein design, single-residue saturation mutagenesis or random mutagenesis, then multiplex assembly and screening 6,800 allosteric sensor variants to engineer aTF response to inducer molecules [Abstract and page 178].
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to employ Taylor’s library of 6,800 aTF variants in Spoonamore’s sensor screening method to provide broad sampling of allosteric protein sequence space and facilitate identification of sensor variants having desired ligand specificity with reasonable expectation of success since Taylor able to identify new variants comparable in specificity and induction to wild-type [abstract]. As set forth in MPEP 2144.05, in the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists.
Regarding claims 12, Klein discloses 8 LacI variants nootkatone sensors [¶0033], 3 TetR variants apigenin sensors [¶0037], 5 AcuR variants chassis methylacrylate sensors [¶0052]. However, Klein’s sub populations do not reach the claimed 10 to 100 variants.
Taylor teaches screening allosteric LacI variants for ligand responsiveness and identifies a set of 31 sucralose-responsive variants for further engineering and characterization [page 181].
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to select a larger population of ligand responsive variants, such as 31 variants, for further engineering and evaluation to capture diverse specificity profiles, and lower the risk of losing allosteric function during downstream optimization. See MPEP § 2144.05 (II) "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) and KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Spoonamore et al., Klein et al., and Ashuach et al.
Claim(s) 10 and 12 is/are rejected under 35 U.S.C. 103 as being unpatentable over Spoonamore et al. (US20190204303A1, EFD: August 15th 2017) in view of Klein et al. (Nat Methods 17, 1083–1091 (2020)) and Ashuach et al. (Genome Biol. 2019;20(1):183).
Regarding claims 16, Spoonamore and Klein do not disclose selecting variant using confidence scoring.
Ashuach discloses MPRAnalyze, a statistical framework for analyzing MPRA count data. Ashuach calculate quantitative transcriptional activity for each library member from corresponding RNA and DNA barcode counts [pages 1-2]. Ashuach further teaches leveraging information from multiple barcodes with each sequence, providing multiple replicates within a single experiment and a single sequencing library to ensure robust analysis of MPRA data. The reference also teaches evaluate results using Wald statistical test to determine the significance of activity differences, allow measure of confidence with determined activity for each variant [pages 7-8].
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to apply Ashuach’s statistical confidence analysis to Spoonamore and Klein barcoded base activity measurements to account for variability and uncertainty in barcode sequencing measurements, allow reliable assessments of functional activity of each variant. One of ordinary skill in the art would have had reasonable expectation of success since Wald statistical test works best in large sample size which fit the pooled massively parallel reporter assays. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Conclusion
No claims are allowed
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Khai Quynh Tien Pham whose telephone number is (571)272-6998. The examiner can normally be reached M-T, 9-4 ET.
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/KHAI QUYNH TIEN PHAM/ Examiner, Art Unit 1684
/JEREMY C FLINDERS/ Primary Examiner, Art Unit 1684
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