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 IDS received on August 9, 2024 is proper and is being considered by the Examiner.
Drawings
The drawings received on August 9, 2024 are acceptable.
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 1 is indefinite for reciting the phrase, “incubating the contents of a reaction vessel at a first isothermal temperature for a first period of time, the reaction vessel comprising” because it is unclear whether the phrase is intending to mean that all of the contents recited after the phrase, “the reaction vessel comprising” are exposed to that first isothermal temperature; or at least some of the contents of reagents that are in the reaction vessel are exposed to the first isothermal temperature. The latter interpretation is made. This is because dependent claim 5 appears to recite that some of the reagents of the reaction vessel are treated prior to their exposure to the “first isothermal temperature”.
Claims 2-19 are indefinite by way of their dependency on claim 1.
Claims 7 and 20 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 7 is indefinite for reciting the phrase, “the second period of time”. There is an insufficient antecedent basis for the phrase. For the purpose of prosecution, the claim has been construed to depend from claim 5.
Claim 10 is indefinite for reciting the phrase, “crRNA comprises a polynucleotide sequence complementary to a conserved sequence of Lba cas12a, a variable sequence of Lba cas12a, or both” because the term, “complementary” when it pertains to polynucleotide, it infers a base complementarity. Because Cas12a is a protein made up of amino acids, there can be no complementarity between a polynucleotide sequence and an amino acid sequence of Cas12a protein. For the purpose of prosecution, the phrase has been construed to mean what is well-known in the art of CRISPR assay where RNA molecules are bound to a scaffold region (or pocket) of a Cas protein.
Claim 11 is indefinite for using the trademarked product, “SplintR” ligase because a product identified its trademark does not define the good but its source. Because the product can change over a period of time while maintaining its tradename (for example, Coke®), usage of such term renders the metes and bounds indeterminable and indefinite.
Claim Rejections - 35 USC § 102
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.
Claim 20 is rejected under 35 U.S.C. 102(a)(1) as being anticipated by Tian et al. (Chem. Comm., 2020, vol. 56, pages 13445-13448; IDS ref).
For the instant rejection, the term, “one-pot” does not distinguish the presently claimed system as one-pot is based on the intended usages of the reagent recited in the claims.
Tian et al. teach a set of isothermal (the amplification is performed via RCA, which is a well-known isothermal amplification reaction) detection components comprising:
a Cas12a enzyme (“CRISPR-Cas-12a-based strategy is developed”, Abstract, also “Cas-TCA strategy … crRNA is generated by target-responsive amplification and further processed by Cas12a, without the involvement of any pre-synthesis and purification steps”, page 13446, 2nd column);
a ligase (“SplintR ligase”, page 13446, 2nd column, 2nd paragraph);
a polymerase (“T7 polymerase”, page 13446, 2nd column, 2nd paragraph); and
a reporter DNA capable of producing a CRISPR-generated detectable signal (see Fig. 1, reporter).
Therefore, Tian et al. anticipate the invention as claimed.
Claim 20 is rejected under 35 U.S.C. 102(a)(1) as being anticipated by Zhang et al. (Microchemical Journal, 2020, vol. 158, pages 1-7).
Zhang et al. teach a set of isothermal (the amplification is performed via RCA, which is a well-known isothermal amplification reaction) detection components comprising:
a Cas12a enzyme (“we propose a novel CRISPR-Cas12a enhanced RCA method for miRNA detection”, page 2, 1st column);
a ligase;
a polymerase (see T4 ligase and Phi29 DNA polymerase, page 6, 1st column, section 4.1); and
a reporter DNA capable of producing a CRISPR-generated detectable signal (“reporters, whose two terminals are labeled with a fluorophore (Cy3) and a corresponding quenching group (BHQ1)”, page 2, 1st column, 2nd paragraph).
Therefore, Zhang et al. anticipate the invention as claimed.
Claim Rejections - 35 USC § 103
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.
Claims 1-19 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (Microchemical Journal, 2020, vol. 158, pages 1-7) in view of Cao et al. (Analytical Chimica Acta, published on-line August 12, 2021, vol. 1182, pages 1-8).
With regard to claim 1, Zhang et al. teach a method of detecting a target polynucleotide in a sample comprising the below depicted steps (reproduced from Scheme 1):
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As seen, Zhang et al. teach providing: a) a sample comprising one or more microRNAs (“we propose a novel CRISPR-Cas12a enhanced RCA method for miRNA detection”, page 2, 1st column); b) a padlock probe comprising a ligation zone and a detection zone (see above “Padlock” that anneals to the target miRNA, and results in a ligation zone, also “padlock complementary repeat sequences”, that is recognized by CRISPR-12a, thus “detection zone”, “P’ sequence”, see page 2), said detection zone being recognized by a Cas12a associated RNA (sgRNA, herein), with the 5’ end of said padlock being phosphorylated (evidenced by 5’ and 3’ ends of the circularized template being ligated with a ligase); c) an RNP comprising a Cas12a CRISPR-associated (Cas) enzyme and sgRNA, wherein the sgRNA is capable of hybridizing to the detection zone of the padlock probe (see above); d) a ligase and e) polymerase (see T4 ligase and Phi29 DNA polymerase, page 6, 1st column, section 4.1); and a reporter DNA capable of producing a CRISPR-Cas generated detectable signal (see above, also “reporters, whose two terminals are labeled with a fluorophore (Cy3) and a corresponding quenching group (BHQ1)”, page 2, 1st column, 2nd paragraph).
The artisans teach the inclusion of all of the above reagents into a single vessel (see page 6, section 4.4).
With regard to claim 2, Zhang et al. teach the combination of the sample miRNA, the padlock probe, ligase, polymerase and reporter DNA in a single in a single reaction prior to their incubation in a first temperature (see section 4.4, after the addition of all of the reagents, the mixture is incubated for 30 minutes1).
With regard to claim 4, the first period of time is about 10 min to about 3 hours (see page 6, section 4.4, “[a]fter incubation at room temperature for 2 hours … reporter probes were added .. incubated for 30 min”).
With regard to claims 5-7, the sample of miRNA and padlock probe are incubated before RNP and the rest of the reagents are added (“2 mL miRNA, 2 mL padlock probe and 6 mL DEPC water were mixed and slowly cooled to room temperature after heating at 95oC for 5 min”, page 6, section 4.4).
With regard to claims 8 and 9, the polynucleotide sequence of the detection zone is a repeat sequence recognize by sgRNA, and not a PAM sequence recognized by the Cas enzyme, said Cas enzyme being Lba Cas12a (see page 6, section 4.1).
With regard to claims 11 and 12, the ligase is T4 ligase and DNA polymerase is Phi29 (see above).
With regard to claims 13 and 14, the reporter DNA comprises a polynucleotide, a detectable molecule and a quencher, wherein the detectable molecule is Cy3 (see above).
With regard to claim 15, the reaction vessel contains at least dNTPs, BSA, etc. (see page 6, section 4.4).
Zhang et al. do not teach that the RNA molecule which serves to provide binding to the RCA region and mediate the collateral cleavage of the reporter molecules are crRNA (claim 1, in-part) or that the cRNA comprises a polynucleotide sequence that binds to the a scaffold (or pocket) region conserved in Lba cas 12a (claim 102).
Zhang et al. do not explicitly teach that the isothermal temperature is about 16 to about 48oC (claim 3).
While Zhang et al. teach that their method is for the detection of miRNA of interest, said miRNA being correlated with breast cancer (see page 3, 2nd column, bottom paragraph), the artisans do not explicitly teach the application of all different types of miRNAs for other types of diseases, such as PDAC (claim 16).
Zhang et al. teach a particular assay condition for their assay and therefore, do not explicitly teach the concentration of RNP complex (claim 17) or the incubation condition of the Cas12a and crRNA (claims 18 and 19).
Cao et al. teach a method of detecting a target nucleic acid via generating an RCA product with a padlock probe, with a Cas12a enzyme complexed with crRNA (see Fig. 1).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Zhang et al. with the teachings of Cao et al., thereby arriving at the invention as claimed for the following reasons.
The motivation to combine the teachings of Zhang et al. with the teachings of Cao et al. is based on the alternative means of targeting the Cas12a enzyme complex to a target region found on an RCA product that yields the same predictable outcome, as discussed by the Supreme Court in KSR.
In KSR, the Supreme Court particularly emphasized “the need for caution in granting a patent based on the combination of elements found in the prior art,” Id. at 415, 82 USPQ2d at 1395, and discussed circumstances in which a patent might be determined to be obvious. Importantly, the Supreme Court reaffirmed principles based on its precedent that “[t]he combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results.” Id. at 415-16, 82 USPQ2d at 1395. The Supreme Court stated that there are “[t]hree cases decided after Graham [that] illustrate this doctrine.” Id. at 416, 82 USPQ2d at 1395. (1) “In United States v. Adams, . . . [t]he Court recognized that when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable result.”
As discussed above, Zhang et al. already teach that short sequences, such as miRNAs can be detected via the means of generating an RCA product via a padlock probe, wherein in the presence of the target miRNA, padlock probe is circularized and amplified via RCA, said RCA product comprising multiple repeat regions which are recognized by a Cas12a-complex:
“Many traditional miRNA detection methods, such as real-time quantitative polymerase chain reaction (qRT-PCR) … are reported to achieve sensitive miRNAs detection in solutions or in cell lysates … RCA assay has been attracted more attention due to its stable signal output and easy-to-operate characteristics … RCA can convert short-length RNA in to single-stranded DNA (ssDNA), which is much more stable in a complicated environment …” (page 1, 2nd column)
While Zhang et al. employed an sgRNA for this purpose, Cao et al. teach that crRNA complexed with the same Cas12a enzyme can also be employed for targeting RCA products, both of circumstance of which, result in the collateral cleavage of FRET labeled nucleic acid reporters.
Therefore, one of ordinary skill in the art would have had a reasonable expectation that the application of Zhang et al.’s method with crRNA/Cas12a complex would have resulted in the same outcome.
With regard to the application of Zhang et al.’s teachings for detecting miRNA biomarkers that correlate with cancers of other origins, such as PDAC, doing so would have been an obvious application of a diagnostics assay, as one of ordinary skill in the art would have been capable of designing a padlock probe specific to any desired miRNA targets, and when circularized upon binding, the detection process would have been the same regardless of the target.
Lastly, with regard to the actual assay conditions, such as the concentrations of the RNP complex, the incubation temperatures and time, such are well-established assay parameters that are result-effective, all of which are desired to be optimized through routine experimentation. Such a process and discovery are well-within the ordinarily skilled artisan and therefore, deemed obvious.
Therefore, the invention as claimed is deemed prima facie obvious over the cited references.
Conclusion
No claims are allowed.
Inquiries
Any inquiry concerning this communication or earlier communications from the Examiner should be directed to Young J. Kim whose telephone number is (571) 272-0785. The Examiner can best be reached from 7:30 a.m. to 4:00 p.m (M-F). The Examiner can also be reached via e-mail to Young.Kim@uspto.gov. However, the office cannot guarantee security through the e-mail system nor should official papers be transmitted through this route.
If attempts to reach the Examiner by telephone are unsuccessful, the Examiner's supervisor, Gary Benzion, can be reached at (571) 272-0782.
Papers related to this application may be submitted to Art Unit 1681 by facsimile transmission. The faxing of such papers must conform with the notice published in the Official Gazette, 1156 OG 61 (November 16, 1993) and 1157 OG 94 (December 28, 1993) (see 37 CFR 1.6(d)). NOTE: If applicant does submit a paper by FAX, the original copy should be retained by applicant or applicant’s representative. NO DUPLICATE COPIES SHOULD BE SUBMITTED, so as to avoid the processing of duplicate papers in the Office. All official documents must be sent to the Official Tech Center Fax number: (571) 273-8300. Any inquiry of a general nature or relating to the status of this application should be directed to the Group receptionist whose telephone number is (571) 272-1600.
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/YOUNG J KIM/Primary Examiner
Art Unit 1637 September 22, 2026
/YJK/
1 The Office notes that the term, “first temperature” does not contain any weight because the order of such temperature application appears to have no real meaning as applicants dependent claim introduced a second temperature that is applied to a mixture prior to the application of a first temperature.
2 See above 112b interpretation.
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