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 . Applicant’s amendment filed 6/18/2026 is acknowledged. Claims 1, 5-6, 10-11, 13, 16, 19, 22 have been amended. Claims 2 and 23 have been canceled. Claims 24-36 have been added. Claims 1, 3-22, 24-36 are pending.
Any rejection not reiterated in this action has been withdrawn as being obviated by the amendment of the claims.
This action is made Final.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Previous Rejection
The objection to the sequence disclosure is withdrawn in view of Applicant’s submission of the sequence listing and CRF. The claim objection is withdrawn in view of Applicant’s amendment of the claims. The claim rejection under 35 USC 112 directed to the claims 1-22 are withdrawn in view of Applicant’s amendments of the claims. The claim rejection under 35 USC 102/103 as being anticipated/unpatentable over Vesey et al is withdrawn in view of Applicant’s amendment of the claims.
New Ground(s) of Rejections
THE NEW GROUND(S) OF REJECTIONS WERE NECESSITATED BY APPLICANT’S AMENDMENT OF THE CLAIMS:
Claim Rejections - 35 USC § 103
9. 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.
10. 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.
11. Claim(s) 1, 3-22 and 24-26 is/are rejected under 35 U.S.C. 103 as obvious over Vesey et al (WO 2023019290, February 2023, effective filing date August 2021, see entire document) in view of Zhang et al (US 20240043828, effective filing date October 2020).
Regarding claims 1, 3-22, and 24-26, Vasey teach a method for the detection of a target in a sample, the method comprising: (a) contacting the sample with: (i) a first target binding construct; (ii) a second target binding construct to immobilize or capture the target; (iii) a type V or type VI CRISPR/Cas effector protein; (iv) a trigger nucleic acid sequence; (v) a guide RNA comprising: a region that binds to the type V or type VI CRISPR/Cas effector protein, and a guide sequence that hybridizes with the trigger nucleic acid sequence, wherein hybridization between the guide sequence and the trigger nucleic acid sequence activates the nuclease activity of the CRISPR/Cas effector protein; and (vi) a labelled reporter construct, wherein said reporter construct is a nucleic acid that does not hybridize with the guide sequence of the guide RNA and is cleavable by the nuclease activity of the activated type V or type VI CRISPR/Cas effector protein; and
(b) measuring a detectable signal produced following cleavage of the labelled nucleic acid reporter by the type V or type VI CRISPR/Cas effector protein, thereby detecting the immobilized or captured target; wherein the type V or type VI CRISPR/Cas effector protein, the trigger nucleic acid sequence, the guide RNA, or the type V or type VI CRISPR/Cas effector protein in combination with the guide RNA optionally in further combination with the trigger nucleic acid, is conjugated to the first target binding construct to thereby co-locate the type V or type VI CRISPR/Cas effector protein and the target when present in the sample; and wherein the target is not a nucleic acid sequence.
According to a third aspect, the invention provides a method for the detection of a target in a sample, the method comprising:
(a) contacting the sample with: (i) a first target binding construct to thereby immobilize or capture the target; (ii) a second target binding construct; (iii) a third binding construct which binds to the second target binding construct; (iv) a type V or type VI CRISPR/Cas effector protein; (v) a trigger nucleic acid sequence; (vi) a guide RNA comprising: a region that binds to the type V or type VI CRISPR/Cas effector protein, and a guide sequence that hybridizes with the trigger nucleic acid sequence, wherein hybridization between the guide sequence and the trigger nucleic acid sequence activates the nuclease activity of the CRISPR/Cas effector protein; and (vii) a labelled reporter construct, wherein said reporter construct comprises a nucleic acid that does not hybridize with the guide sequence of the guide RNA and is cleavable by the nuclease activity of the activated type V or type VI CRISPR/Cas effector protein; and
(b) measuring a detectable signal produced following cleavage of the labelled nucleic acid reporter by the type V or type VI CRISPR/Cas effector protein, thereby detecting the immobilized or captured target; wherein the type V or type VI CRISPR/Cas effector protein, the trigger nucleic acid sequence, the guide RNA, or the type V or type VI CRISPR/Cas effector protein in combination with the guide RNA optionally in further combination with the trigger nucleic acid, is conjugated to the third binding construct to thereby co-locate the type V or type VI CRISPR/Cas effector protein and the target when present in the sample; and wherein the target is not a nucleic acid sequence ([011] at pages 3-5).
Vesey et al teach wherein the first and second target binding construct is immobilized on a substrate (claim 11).
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Vessey et al teach at paragraph Figure 1 shows schematics for the CRISPR/Cas12a-involved whole cell biosensing system for detection. A simple 3 step procedure has been applied to realize the Cryptosporidium whole cell detection, using a similar setup as a common ELISA: (A) Step 1: Adding sample for target Cryptosporidium cell capture; (B) Step 2: Adding pre-made anti- Cryptosporidium Abs-ssDNA conjugate to form the antibody-cell sandwich structure; (C) Step 3: Adding the prepared CRISPR/Cas12a reaction mixture to recognize the triggering ssDNA on the Ab-ssDNA conjugate for Cas12a RNP activation. (D) Finally, collateral cleavage of activated CRISPR/Cas12a RNPs cuts the fluorescent quenched reporters to generate amplified signal for detection.
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At [025], Figure 8 shows preparation of anti-Cryptosporidium Abs-ssDNA conjugate for CRISPR/Cas12a-based signal amplification.
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At para. [055], Figure 35 shows schematics of CRISPR-based Universal Immunoassay Signal Enhancer (CRUISE). platform. The key elements for CRUISE to bridge the CRISPR/Cas12a and immunoassays is the Abs-ssDNA conjugate, which has 3 components, including the selected antibody, streptavidin, and biotinylated triggering ssDNA. The prepared Abs-ssDNA conjugate can be used either as primary antibody or secondary antibody in different immunoassay schemes. For the primary antibody-based approach, (A) Abs-ssDNA can directly recognized the target analyte as the single antibody in the system or (B) as the detection antibody on a typical antibody-analyte sandwich structure; (C) for the secondary antibody-based approach, instead of targeting the analyte, the Abs-ssDNA targeting the Fc region of another antibody. Both these two CRUISE approaches share a similar three-step procedure: 1. Recognition of the designated target by the Abs-ssDNA; 2. Addition of CRISPR/Cas12a reaction mixture, which includes the CRISPR/Cas12a RNP able to recognize the triggering ssDNA and fluorescent quenched reporters; 3. Activation of CRISPR/Cas12a RNPs for random cutting the surrounding reporters to generate amplified fluorescent signal. This fluorescent signal intensity is detected to reveal the concentration of target analyte present in the sample.
Vesey et al teach that the first and second target binding constructs are attached to a solid support or substrate [0159], [0165]. An immobilized substrate may refer to any material that is suitable for, or may be modified to, the attachment of a polypeptide or polynucleotide. Possible substrates include, but are not limited to, glass and modified functionalized glass, plastic (including acrylics, polystyrene and copolymers of styrene with other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon etc.), polysaccharides, nylon or nitrocellulose, ceramics, resins, silica or silica-based materials (including silicon and modified silicon), carbon, metals, inorganic glass, plastics, fiber optic strands, and various other polymers [0159] and wherein the substrate in a vessel (see paragraph [0180] which teaches reagents for fluorometric, chemiluminescent or colorimetric detection, an apparatus or other component that can be use with the method, also including instructions for use and vials or other container for housing one or more of the aforementioned components).
Vesey additionally teach the following to support the inventive entity:
[0187] The plate based CRISPR/Cas biosensing assay was developed stepwise as below. The high binding plate was rinsed with PBS buffer to clean the wells. Then 100 μL of 10 μg/mL streptavidin solution was added in each well of the high binding plate to create a streptavidin-modified interface. After 1 hour incubation at room temperature (RT), the excess streptavidin molecules were washed away using 350 μL of a PBS buffer. Subsequently, 350 μL of Crypto incubation buffer was applied to each well for 1 hour at RT for the blocking of empty spots.5 μg/mL biotinylated Crypto antibody was applied to the well blocked plate to generate the biosensing interface. The Crypto sample was then added to the antibody modified plate for 1 hour incubation at RT, then the non-specifically attached Crypto were washed away by using the Crypto wash buffer. After capturing the Crypto, 5 μg/mL of antibody-ssDNA conjugate was applied for 1 hour at RT to form the sandwich structure, and the residual unbound conjugates was washed away using the Crypto wash buffer. Finally, 100 μL CRISPR/Cas solution was added into each well and incubated for 30 min before testing the fluorescence signal. The fluorescent signal was detected using ID3 several times at 30 mins intervals with excitation wavelength of 570 nm and emission wavelength of 615 nm. The characterization of Crypto using confocal microscopy.
[0188] In all confocal microscopy imaging, the same parameters were applied: intensity 3%, PMT 500 volts, 20 magnification, 630×630 μm2 field of view, offset 3%, 1.0 pinhole. The excitation wavelength for Tex-Crypto was 561 nm, and the emission range for Tex-Crypto was 570-670 nm. The excitation wavelength for FAM-antibody was 488 nm, and the emission range for FAM-antibody was 500-540 nm. Results 1.
Assay principle [0189] Figure 1 shows the basic schematics of the principle of the CRISPR/Cas12a-based whole cell level biosensing for Cryptosporidium microorganisms. After the sensing interface has been prepared on the surface of a high-binding 96-well plate coated with streptavidin and biotinylated anti-Cryptosporidium antibody, the Cryptosporidium oocysts in the sample are captured onto the sensing interface (Fig 1 A). Then, pre-made anti-Cryptosporidium antibodies and single strand DNA (Abs-ssDNA) conjugates are added to recognize and bind to the remaining free anti-Cryptosporidium antigens on the surface of the captured Cryptosporidium oocyst (Fig 1 B). These two steps realize a standard “sandwich” immunoassay scheme where the Cryptosporidium oocysts act as the “analyte”; this is possible because of the much larger size of Cryptosporidium oocyst compared to the antibodies. Afterwards, the CRISPR/Cas12a reaction mixture with the guide RNA integrated with the CRISPR/Cas12a RNP having a complementary sequence to the triggering ssDNA on the Ab-ssDNA conjugate is introduced (which is interpreted as the disruptable linker). This complementarity allows this RNP to be specifically activated by the recognition of the triggering ssDNA by the Ab-ssDNA conjugate (Fig 1 C). An abundant supply of quenched ssDNA-based fluorescent reporters is also provided (Fig 1 C). The activated CRISPR/Cas12a RNPs then indiscriminately and continuously cut the surrounding reporter molecules, owing to their collateral cleavage property upon RNP activation by the conjugate. This, in turn, unquenches the ssDNA-based fluorescent reporter and generates the amplified fluorescence signal required for analyte detection (Fig 1 D). In this way our sensing scheme is able to take advantage of the highly efficient collateral cleavage activity of the programmable Cas12a nuclease which is triggered by an antibody-cell sandwich immunoassay. See also paragraphs [0321] – [0348] which teaches pro-signal conjugates in anti-HRP ssDNA Abs conjugate. With regards to the newly added limitations, Vesey et al teach wherein the first components comprises an enzyme or enzyme component that is immobilized to a solid support, wherein the enzyme is peroxidase and the support is a bead (claims 7-8 at pages 8 and 9). Vesey teach with regards to the steps of releasing said first component, wherein upon releasing the first components flows to the second binding region wherein it couples and accumulates to a second component (see [0143] –[0144], [0149]-[0150]).
With regards to the pro-signaling molecule incorporated subsequent the step (b) of contacting the sample with a solid support, Vesey et al teach at paragraph [0326] wherein anti-HRP (horse radish peroxidase) ssDNA-Abs conjugate was added onto a high-binding 96 well plate and incubated at room temperature. Three washes of 1X PBS were performed, HRP labeled antibody was applied and incubated and then excel antibodies were removed by 1X PBS wash and TMB (3,3',5,5'- tetramethylbenzidine) (pro-signal agent) substrate for colorimetric signal generation was applied.
While Vesey teaches various aspects of the instant invention, Vesey does not teach wherein the enzyme in the first components comprise of a holoenzyme, apoenzyme, DNAzyme or ribozyme.
Regarding claims 1, 2-22, and 34-26, Zhang et al provides a general method for targeted gene modification, wherein the method comprises the use of CRISPR system (abstract [0004]). Zhang et al teach in the method the CAS protein or variants may be associated (e.g., fused) to one or more functional domains. The association can be by direct linkage of the Cas protein to the functional domain, or by the association with crRNA. The crRNA comprises an added or inserted sequence that can be associated with a functional domain of interest, including , for example an aptamer or nucleotide that binds to a nucleic acid binding adapter protein. The functional domain may be a functional heterologous domain [0135]. For example, the one or more heterologous functional domains may comprise one or more nuclear localization signal (NLS) domains. The one or more heterologous functional domains may comprise at least two or more NLS domains. Other examples of functional domains include beads, ribonucleases, spliceosomes, light inducible/controllable domain or chemically inducible/controllable domain [0137].
At paragraph Zhang et al teach at para. [0170], wherein the system may be responsive to one or more energy sources in order to produce a detectable signal. Finally, Zhang et al teach wherein the composition and systems may comprise one or more RNAse domains, including RNAse P [0231 and 0236], wherein the RNAse P comprises, ribozymes, holoenzymes which consisting of apoenzyme. Zhang teaches that these enzyme forms an active enzyme system by combination with a coenzyme and determines the specificity of the systems for a substrate [0236].
It would have been prima facie obvious to one of ordinary skill in the art at the time of the effective filing date of the claimed invention to have been motivated to have utilized in the analyte detection method of Vesey enzymes such as peroxidase as taught therein and ribozymes as taught by Zhang because both have been found to be effective in detecting signal strength and for the ability to detect specificity of a target detection system for a substrate. The ordinary artisan could expect a reasonable expectation of success in detecting a desired target in view the teachings of the cited prior art.
Conclusion
12. No claims are allowed. Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
13. Any inquiry concerning this communication or earlier communications from the examiner should be directed to CYNTHIA B WILDER whose telephone number is (571)272-0791. The examiner can normally be reached Flexible.
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/CYNTHIA B WILDER/Primary Examiner, Art Unit 1681
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