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 .
Information Disclosure Statement
The information disclosure statements (IDSs) submitted on January 8th, 2025 is acknowledged. The submissions are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements have been considered by the examiner.
Claim Status
Claims 4, 6-7, 9-11, 15, 17-18, 25, 27-28, 31-21, and 34 have been amended. Claims 5, 8, 20-24, 26, 29-30, 33, and 35 have been cancelled. Claims 1-4, 6-7, 9-19, 25, 27-28, 31-32, and 34 are pending, under examination, and discussed in this office action.
Claim Objections
Claims 1 and 18 are objected to because of the following informalities:
Claim 1 recites “contacting the sample to a solution comprising”. This is grammatically incorrect. “Contacting the sample to a solution comprising” should instead read “contacting the sample with a solution comprising”.
Claim 18 recites “influenza BSARS”. This is grammatically incorrect. “influenza BSARS” should instead be read “influenza B, SARS”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
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 14 and 18 are 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 14 recites the limitation “hydrogen ion”. There is insufficient antecedent basis for this limitation in the claim. Claim 14 depends from claim 12, which gives examples of metal ions as the target analyte. Hydrogen ion is not a metal ion, and therefore claim 14 introduces a new and unsupported target analyte category without antecedent basis in the claim from which it depends. It is unclear whether the claim is intended to depend from a different independent or dependent claim.
Claim 18 recites the limitation “the virus is”. There is insufficient antecedent basis for this limitation in the claim. Claim 18 depends from claim 15, which recites “a viral nucleic acid” as the target analyte not a virus. The claim therefor introduces “the virus” without prior introduction of a virus. It is unclear whether the claim is intended to depend from a different independent or dependent claim.
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.
Claims 1-4, 6-7, 9-11, 15-17, 25, 31-32, and 34 are rejected under 35 U.S.C. 103 as being unpatentable over Baughman et al. (US 20200032324 A1; published on January 30th, 2020; cited on the IDS filed January 8th, 2024) in view of Doudna et al. (US 20190241954 A1; published on August 8th, 2019; cited on the IDS filed filed January 8th, 2024).
Regarding instant claim 1, Baughman teaches a method of detecting a target nucleic acid sequence in a biological sample. The method comprises combining a biological sample with a composition including a Cas protein programmed with a guide RNA having a spacer sequence complementary to the target sequence, incubating the sample mixture with an indicator device to create detection mixture, and applying a separating force to the detection mixture followed by detecting a signal from an untethered reporter molecule (claim 17; [0087]).
Baughman teaches that the indicator device used in this method comprises a reporter molecule (detectable marker) attached at one end of a tether molecule, which includes at least one indicator of nucleic acid sequence (oligonucleotide) and a solid support (surface) attached at the other end of the tether (claims 1; 17 [0042]). Therefore, the reporter molecule is immobilized on the solid support via tether.
Baughman teaches that the reporter molecule may be an enzyme, including horseradish peroxidase (claim 4; [0055]).
Baughman teaches a guide oligonucleotide that hybridizes to the target analyte, in the form of a guide RNA associated with the Cas protein and having a spacer sequence complementary to the target sequence (claim 13, [0087]).
Baughman teaches a Cas13 protein that cleaves the tether after the guide RNA hybridizes to the target, releasing the reporter molecule into solution (claim 7; [0040; 0093]).
Baughman teaches that this contacting occurs in a vessel. Baughman discloses that the detection device may be a microfuge tube or a muti-well plate [0097; 0018].
Baughman teaches removing the solution comprising the released reporter molecule is untethered, a separating force is applied to separate untethered reporter molecules from the solid support, and the untethered reporter molecules are collected in the detection area (claim 17; 23; [0096]).
Baughman teaches measuring a signal produced by the released detectable marker in the solution removed from the vessel. Baughman discloses that detecting the untethered reporter molecule in the detection area confirms the presence of the target sequence (claim 17; [0099; 0100]).
Baughman does not directly teach a cas12 protein the context of the claimed surface immobilized reporter detection method. Baughman’s methods are more directed towards Cas13 based detection. Baughman also doesn’t teach the guide oligonucleotide hybridizing to a nucleic acid sequence partially complementary to an aptamer.
Doudna, in the same field of endeavor, teaches that Cas12 family proteins exhibit trans-cleavage activity against a reporter ssDNA upon target recognition by the guide RNA, enabling sensitive detection of target DNA in a sample. Doudna teaches contacting a sample with a Cas12 effector protein, a guide RNA comprising a region that binds the Cas12 effector protein and a guide sequence that hybridizes with the target DNA, and a reporter that is cleaved by the activated Cas12 proteins to produce a detectable signal (claim 46; 57; [0006].
It would have been obvious to one of ordinary sill in the art, before the effective filing date of the claimed invention, to have modified the detection method of Baughman, in view of Doudna, with the Cas12 protein in place of, or combination with, the Cas13 protein of Baughman, using the Cas12 trans-cleavage detection approach taught by Doudna. Both Baughman and Doudna are in the same field of endeavor (e.g. CRISPR based nucleic acid diagnostics) and both disclose CRISPR proteins whole collateral cleavage activity against reporter molecule is activated by target recognition, generating a detectable signal. One of ordinary skill in the art would have been motivated to make the modification because Doudna demonstrates that Cas12 proteins exhibit the same fundamental collateral cleavage behavior [0359; 0386] as the Cas13 proteins of Baughman, while additionally targeting DNA rather than RNA. Therefore, expanding the detection platform of Baughman to DNA targets.
Regarding instant claim 2, Baughman, in view of Doudna, teaches the method of claim 1. Baughman further teaches that the molecule may include a plurality of tethered molecules attached to the solid support, and that a plurality of reporter molecules may be attached to a single tether (claim 6; [0079; 0055]), this corresponds to a reporter comprising two or more oligonucleotides conjugated to the detectable marker.
Regarding instant claim 3, Baughman, in view of Doudna, teaches the method of claim 1. Baughman further teaches embodiments in which a single reporter molecule is attached at one end of a single tether molecule (claim 1; 9; [0042]), this corresponds to reporter consisting of one oligonucleotide conjugated to one detectable marker.
Regarding instant claim 4, Doudna, in view of Baughman, teaches the method of claim 1. Claim 4 recites that the Cas12 protein comprises a sequence as set out in SEQ ID NO: 1, corresponds to the Lachnospiraceae bacterium ND2006 Cas12a protein (LbaCas12a).
Doudna teaches LbaCas12a, the protein of SEQ ID: 1 of the instant application throughout Examples 1-4 [0348-415].
It would have been obvious of one of ordinary skill in the art, before the effective filing date of the claimed invention, to use the specific LbCas12a protein of Doudna in the surface immobilized reporter detection method of Baughman. Doudna provides extensive characterization of LbCas12a trans-cleavage kinetics, demonstrating highly robust catalytic efficiency and validation on clinical samples [0363]. This establishes a clear motivation and reasonable expectation of success for selecting this specific protein for use in the Baughman detection. The LbaCas12a sequence of SEQ ID NO:1 was well characterized in the art prior the effective filing date of the instant application (see MPEP 2141)).
Regarding instant claim 6, Baughman, in view of Doudna, teaches the method of claim 1. Baughman further teaches that the signal is greater when the target analyte is present in the sample than when it is absent. Baughman discloses that a detected signal greater than a background value, which is determined from a sample lacking the target sequence, indicating the presence of the target (claim 17; [100]).
Regarding instant claim 7, Baughman, in view of Doudna, teaches the method of claim 1. Baughman further teaches that the signal produced by the released reporter molecule is greater when the target analyte is present in the sample than when it is absent [0100]. Doudna further supports this limitation by disclosing that LbaCas12a produces a robust fluorescent signal only in the presence of the cognate target, with no signal observed in target-absent conditions [0388; 0365]. Doudna further teaches that signal detected in the absence of the quencher moiety corresponding to the cleaved, target-present state of the reporter is at least 2 fold, at least 5 fold, at least 7 fold, at least 10 fold, or at least 20 fold greater than the signal detected in the presence of the quencher moiety [0131].
It would have been obvious to one of ordinary sill in the art, before the effective filing date of the claimed invention, to achieve the specific fold enhancement ranges by combining the Baughman surface immobilized reporter format of with the Cas12a trans-cleavage detection of Doudna.
Regarding instant claim 9, Baughman, in view of Doudna, teaches the method of claim 1. Baughman teaches that the guide oligonucleotide is RNA. Baughman discloses that the Cas protein includes a guide RNA sequence, and that the guide RNA may include natural or syntheitic nucleic acids including modified nucleic acids such as locked nucleic acids (LNA) or ssDNA (claim 13; [0030]). Doudna likewise teaches that the guide RNA may include DNA bases in addition to RNA bases [0162]
Regarding instant claim 10, Baughman, in view of Doudna, teaches the method of claim 1. Baughman teaches that the indicator nucleic acid sequence of the tether (oligonucleotides of the reporter) may be single-stranded RNA, double-stranded DNA, or combinations thereof [0053; claim 11; 12].
Regarding instant claim 11, Baughman, in view of Doudna, teaches the method of claim1. Baughman teaches that the target nucleic acid sequence may be derived from a fungus, bacterium, virus, protozoa, or mammalian cell (claim 19; [0036).
Regarding instant claim 15, Baughman, in view of Doudna, teaches the method of claim 1. Baughman teaches that the target sequence may be derived from a virus, and specifically lists DNA and RNA viruses as target sources (claim 19; [0065; 0070])
Regarding instant claim 16, as discussed above, Baughman, in view of Doudna, teaches the method of claim 1. Baughman teaches that the viruses targeted may be selected from both DNA and RNA viruses [0065].
Regarding instant claim 17, Baughman, in view of Doudna, teaches the method of claim 15. Doudna teaches detection of viral nucleic acids from Picornaviridae (polio virus) [0099; 0107]. Baughman likewise discloses Picornaviridae as a viral target family [0065].
Regarding instant claim 25, Baughman, in view of Doudna, teaches the method of claim 15. Baughman teaches that the disclosed device and method are useful for genetic screening, cancer screening, mutational analysis, microRNA analysis, and mRNA analysis [0036]. Doudna further supports the cancer-related nucleic acid limitation by disclosing SNP detection, cancer screening, detection of bacterial infection, detection of antibiotic resistance, and detection of viral infection as application of the Cas12 based detector [0109].
Regarding instant claim 31, Baughman, in view of Doudna, teaches the method of claim 1. Baughman teaches that the reporter molecule (detectable marker) may be an enzyme or catalyst. Baughman discloses a list of reporter enzymes including NanoLuc, horseradish peroxidase, alkaline phosphatase, and beta galactosidase [0055; 0044; 0048].
Regarding instant claim 32 Baughman, in view of Doudna, teaches the method of claim 1. Baughman teaches that the solid support may be a bead, fiber, particle, or filter (claim 6; 21; [0059; 0042]. Baughman further discloses embodiments in which the solid support is a tube or forms part of a multiwell plate assay [0097].
Regarding instant claim 34, Baughman, in view of Doudna, teaches the method of claim 1. Baughman teaches that the vessel in which the assay is conducted may be a tube or a multiwell plate [0097].
Claims 12-13 and 27-28 are rejected under 35 U.S.C. 103 as being unpatentable over Baughman et al. (US 20200032324 A1; published on January 30th, 2020; cited on the IDS filed January 8th, 2024) in view of Doudna et al. (US 20190241954 A1; published on August 8th, 2019), as applied to claims 1-3 above, and further view of Xiong et al. (“Functional DNA Regulated CRISPR-Cas12a Sensors for Point-of Care Diagnostics of Non-Nucleic Acid Targets” J Am Chem Soc. 2020 January 08; 142(1): 207–213. doi:10.1021/jacs.9b09211).
Regarding instant claim 12 and 13 Baughman and Doudna in view of Xiong teach detection of non-nucleic acid target using a Cas12a aptamer-based detection platform. Xiong teaches a Cas12a based sensor regulated by functional DNA molecules such as aptamers and DNAzymes that are selective for small organic molecule and metal ion detections (p. 1, para 1). Xiong discloses a DNAzyme-regulated CRISPR-Cas12a sensor for Sodium ion (Na+) detection and states that the platform is generalizable to a wide range of metal ion targets (p. 3, para 2; p. 7, para 2; p. 15; see attached supplement document for Fig. S11 and S12).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to apply the metal ion detection platforms of Xiong, which uses the same Cas12a trans cleavage reporter mechanism as Baughman and Doudna, to the specific metal ions recited in claim 12 and 13, including mercury, copper, silver, zinc, gold, and manganese, as Xiong explicitly teaches that the platform is applicable to any metal ion for which a suitable functional DNA can be obtained.
Regarding instant claim 28, Baughman and Doudna in view of Xiong, as applied to claim 11. Xiong teaches that an aptamer-regulated Cas12a sensor in which ATP binding aptamer locks a DNA activator, preventing activation of Cas12a trans-cleavage. Upon ATP binding, the aptamer undergoes a conformational change releasing the DNA activator, which activates Cas12a to cleave an ssNA-FQ reporter, generating a fluorescent signal. (p. 4, para 2; page 1, para 1; p. 12).
Claims 18 and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Doudna et al. (US 20190241954 A1; published on August 8th, 2019; cited on the IDS filed filed January 8th, 2024) in view of Baughman et al. (US 20200032324 A1; published on January 30th, 2020; cited on the IDS filed January 8th, 2024), as applied to claim 15 above, and further view of Broughton et al. (“CRISPR–Cas12-based detection of SARS-CoV-2” Nat Biotechnol 38, 870–874 (2020). https://doi.org/10.1038/s41587-020-0513-4.
Regarding instant claim 18, Doudna and Baughman, as applied to claim 15 above, teach that detecting viral nucleic acids using a CRISPR based detection platform. Baughman teaches that the Cas13a based platform may be applied to Orthomyxoviridae viruses [0065]. Influenza A (H1N1) virus belongs to the family of Orthomyxoviridae. Doudna further discloses “influenza virus” as a target pathogen [0107].
Broughton further teaches the detection of SARS-COV-2 and related Coronaviridae as an additional limitation of claim 18. Specifically, Cas12 E gene gRNA was able to detect three SARS-like coronavirus strains (p. 870, right column, para 2-3; Fig. 1), and that clinical samples from patients infected with seasonal coronaviruses HCoV-229E and HCoV-NL63 were specifically tested in the validation of the study (Fig. 2e). Broughton further identifies H1N1 and SARS as historical coronavirus/influenza epidemics motivating the development of rapid CRISPR based detection platforms, directly linking these viral families as targets for CRISPR based detection (p. 870, left column, para 1-2).
It would have been obvious to one of ordinary sill in the art, before the effective filing date of the claimed invention, to combine the viral detection platforms of Baughman and Doudna with the SARS-CoV-2 and coronavirus detection of Broughton, as all three references use the same CRISPR trans-cleavage detection mechanism and Broughton identifies the same viral families, providing both motivation and a reasonable expectation of success for applying the combined detection platform to the specific viruses (see MPEP 2141(III)).
Regarding instant claim 19, Doudna and Baughman in view of Baughman, as applied to claim 18 above, teach detecting SARS-CoV-2. Broughton teaches a Cas12a-based DECTECR assay specifically designed and validated for SARS-CoV-2 detection using LbCas12a with guide RNAs targeting the E (envelope) and N (nucleoprotein) genes of SARS-CoV-2 (p. 870, right column, para 2; p. 875, left column).
Claim 27 is rejected under 35 U.S.C. 103 as being unpatentable over Baughman et al. (US 20200032324 A1; published on January 30th, 2020; cited on the IDS filed January 8th, 2024) in view of Doudna et al. (US 20190241954 A1; published on August 8th, 2019) and further view of Xiong et al. (“Functional DNA Regulated CRISPR-Cas12a Sensors for Point-of Care Diagnostics of Non-Nucleic Acid Targets” J Am Chem Soc. 2020 January 08; 142(1): 207–213. doi:10.1021/jacs.9b09211), as applied to claim 11 above, and even further view of Hessels et al. (US 20160362746 A1; published on December 15th, 2016).
Regarding instant claim 27, Baughman and Doudna in view of Xiong, as applied to claim 11 teach a CRISPR based detection platform for target analytes using an oligonucleotide conjugated to a detectable marker. Xiong teaches proteins as a target class detectable using the fDN regulated Cas12a platform is applicable to protein targets, identifying proteins as a target class accessible via aptamer based functional DNA regulation (p. 1, para 1; p. 8, para 1). Hessels further teaches detection of PSA (Prostate-specific antigen) using oligonucleotides that hybridize to PSA mRNA as the target analyte (claim 40; 45). Hessels discloses contacting a biological sample with oligonucleotide that hybridize to PSA mRNA, wherein the oligonucleotides may be labeled with a detectable marker including an enzymatic label, enabling quantitative detection of PSA in the sample (claim 40-41; 45).
It would have been obvious to one of ordinary sill in the art, before the effective filing date of the claimed invention, to apply the aptamer-regulated Cas12a detection platform of Baughman, Doudna, and Xiong to detect PSA, as Hessels shows that PSA is a clinically important target detectable via oligonucleotide hybridization and labeled marker, just like the methods of claim 1 of the instant application and Xiong teaches proteins as targets accessible to the fDNA regulated Cas12a platform. A POSITA would have been motivated to combine these references because Hessels establishes the clinical need and feasibility of PSA detection via oligonucleotide based methods, Xiong provices the CRISPR-Cas12a aptamer mechanism for converting protein binding into a signal, and Baughman and Doudna provide the surface immobilized reporter detection format. All of them are using the same fundamental principle of oligonucleotide hybridization to a target analyte triggering a detectable signal, with a reasonable expectation of success (see MPEP 2141(III)).
Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over Baughman et al. (US 20200032324 A1; published on January 30th, 2020; cited on the IDS filed January 8th, 2024) in view of Doudna et al. (US 20190241954 A1; published on August 8th, 2019) and further view of Xiong et al. (“Functional DNA Regulated CRISPR-Cas12a Sensors for Point-of Care Diagnostics of Non-Nucleic Acid Targets” J Am Chem Soc. 2020 January 08; 142(1): 207–213. doi:10.1021/jacs.9b09211), as applied to claim 12-13 above, and even further view of Xu et al. (US 20160362746 A1; “Optimization of CMOS-ISFET-Based Biomolecular Sensing: Analysis and Demonstration in DNA Detection” IEEE Transactions on Electron Devices, vol. 63, no. 8, pp. 3249-3256, Aug. 2016, doi: 10.1109/TED.2016.2582845).
Regarding instant claim 14, Baughman and Doudna in view of Xiong, as applied to claims 12-13 above, teach detecting metal ion target analytes using a DNAzyme regulated CRISPR-Cas12 detection platform. Xiong’s DNAzyme-regulated Cas12a sensor for Na+ demonstrates the applicability of the platform to metal ions detected.
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to apply this platform to hydrogen ion detection using pH-sensitive DNAzymes or aptamers. The use of hydrogen ion concentration as a target analyte in biosensing contexts, including in conjunction with nucleic acid detection platforms was well established in the art as Xu, in the same field of endeavor teaches that hydrogen ion concentration, measured as pH is a known and clinically relevant target analyte in electrochemical biosensing platforms and specifically establishes that ISFET based sensors were originally developed for pH sensing and have been applied to detection of various biomolecules including DNA (p. 3249, right column, para 1). Xu therefore establishes hydrogen ion as a target analyte in a biosensing contexts and motivates its detection alongside nucleic acid targets (see MPEP 2141(III)).
Conclusion
All claims are rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Nura Choudhury whose telephone number is (571)272-6148. The examiner can normally be reached M-F, 9-5 ET.
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/NURA M. CHOUDHURY/Examiner, Art Unit 1683
/ANNE M. GUSSOW/Supervisory Patent Examiner, Art Unit 1683
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