DETAILED ACTION
Response to Amendment
Applicant’s response to the office action filed on January 7, 2026 has been entered. The claims pending in this application are claims 1, 2, and 4-20 wherein claims 9-18 and 20 have been withdrawn in the restriction requirement mailed on June 10, 2025. The objections and rejection not reiterated from the previous office action is hereby withdrawn in view of applicant’s amendment filed on January 7, 2026. Claims 1, 2, 4-8, and 19 will be examined.
Drawings
New Figures 2B, 3B, 4B, 4D to 4F, 6F, 11A to 11F, 13A to 13F, and 14A filed on January 7, 2026 have been accepted by the office.
Claim Objections
Claim 1 is objected to because of the following informalities: (1) “the target nucleic acid does not comprise a protospacer adjacent motif (PAM)” in a) should be “the target nucleic acid which does not comprise a protospacer adjacent motif (PAM); (2) “PAM” in b) should be “the PAM”; and (3) “an RNA guide nucleic acid” in c) should be “a guide RNA”.
Claim 8 is objected to because of the following informality: “the sensitivity of the method is between about 80% and 100% when the frequency of the target nucleic acid is between about 0.1% and 5% of the total nucleic acids in the sample” should be “the more than 0.001% of the total nucleic acids is about 0.1% to 5% of the total nucleic acids”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
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.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
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 of carrying out his invention.
Scope of Enablement
Note that this rejection is different from the rejection under 35 U.S.C. 112(a) mailed on October 7, 2025.
Claims 1, 2, 4-8, and 19 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for performing an amplification reaction in claim 1, does not reasonably provide enablement for selective amplification of a target nucleic acid in a sample using the methods recited in claims 1, 2, 4-8, and 19. 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.
Factors to be considered in determining whether a disclosure meets the enablement requirement of 35 USC 112, first paragraph, have been described by the court in In re Wands, 8 USPQ2d 1400 (CA FC 1988). Wands states at page 1404,
“Factors to be considered in determining whether a disclosure would require undue experimentation have been summarized by the board in Ex parte Forman. They include (1) the quantity of experimentation necessary, (2) the amount of direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims.”
The Nature of The Invention
The claims are drawn to a method of selective amplification of a target nucleic acid in a sample. The invention is a class of invention which the CAFC has characterized as “the unpredictable arts such as chemistry and biology.” Mycogen Plant Sci., Inc. v. Monsanto Co., 243 F.3d 1316, 1330 (Fed. Cir. 2001).
The Breadth of The Claims
Claims 1, 2, 4-8, and 19 encompass a method of selective amplification of a target nucleic acid in a sample, comprising: amplifying the target nucleic acid in an amplification reaction comprising: a) the target nucleic acid which does not comprise a protospacer adjacent motif (PAM); b) a non-target nucleic acid comprising the PAM; c) a guide RNA comprising a protospacer target sequence that forms a guide/non-target hybrid with the non-target nucleic acid; d) a Cas endonuclease having an affinity for the guide/non-target hybrid; and e) a polymerase; wherein the amplifying step is carried out with the polymerase for up to about 120 min, and wherein the target nucleic acid in the sample is present at a frequency of about 0.001% of total nucleic acids in the sample or more than 0.001% of the total nucleic acids in the sample.
Working Examples
The specification provides two working examples (see pages 8-14 of US 2023/0052289 A1, which is US publication of this instant case). However, the specification provides no working example for selective amplification of a target nucleic acid in a sample using the methods recited in claims 1, 2, 4-8 and 19.
The Amount of Direction or Guidance Provided and The State of The Prior Art
The specification provides two working examples (see pages 8-14 of US 2023/0052289 A1, which is US publication of this instant case). However, the specification provides no guidance for selective amplification of a target nucleic acid in a sample using the methods recited in claims 1, 2, 4-8, and 19. Furthermore, there is no experimental condition and/or experimental data in the specification to support the claimed invention. During the process of the prior art search, the examiner has not found any prior art which is related to selective amplification of a target nucleic acid in a sample using the methods recited in claims 1, 2, 4-8, and 19.
Level of Skill in The Art, The Unpredictability of The Art, and The Quantity of Experimentation Necessary
While the relative skill in the art is very high (the Ph.D. degree with laboratory experience), there is no predictability whether selective amplification of a target nucleic acid in a sample can be performed using the methods recited in claims 1, 2, 4-8, and 19.
Since the specification teaches that “FIG. 1A-1C: Programmable Enzyme-Assisted Selective Exponential Amplification (PASEA) enriches exponentially mutant alleles’ fraction. Directed by a single-guide RNA (sgRNA), Cas9 selectively cleaves WT alleles with protospacer adjacent motif (PAM) site while sparing oncogenic mutation lacking PAM. While both WT and mutant alleles amplify, the rate of amplification of mutant alleles far exceeds that of the WT. Nearly all the amplification products are mutant alleles” and “[F]IG. 4D is a diagram showing the principle of real-time PASEA. Directed by a single-stranded guided RNA (sgRNA), Cas9 selectively cleaves WT alleles with protospacer adjacent motif (PAM) site while sparing oncogenic mutation lacking PAM (FIG. 4E, SEQ ID NOs: 17, 18 and 20). The dotted frame illustrates WT and mutant allele sequences, the location of PAM site in the WT KRAS and its absence in KRAS G12. Cleavage takes place between the third nucleotide and the fourth nucleotide upstream from the PAM site. While PASEA amplifies both WT and mutant alleles, the rate of amplification of mutant alleles far exceeds that of the WT, resulting in a product dominated by mutant alleles” (see Figures 1A to 1C, 4D, and 4E, paragraphs [0021] and [0024] of US 2023/0052289 A1, which is US publication of this instant case), and a paper from the inventors teaches that “we conceived a new assay (Programmable Enzyme-Assisted Selective Exponential Amplification, PASEA) that combines the cleavage of wild type alleles with concurrent polymerase amplification. While PASEA increases the numbers of both wild type and mutant alleles, the numbers of mutant alleles increase at much greater rates, allowing PASEA to achieve an unprecedented level of selective enrichment of targeted alleles. By combining CRISPR-Cas9 based cleavage with recombinase polymerase amplification, we converted samples with 0.01% somatic mutant allele fractions (MAFs) to products with 70% MAFs in a single step within 20 min, enabling inexpensive, rapid genotyping with such as Sanger sequencers. Furthermore, PASEA’s extraordinary efficiency facilitates sensitive real-time detection of somatic mutant alleles at the point of care with custom designed Exo-RPA probes. Real-time PASEA’ performance was proved equivalent to clinical amplification refractory mutation system (ARMS)-PCR and NGS when testing over hundred cancer patients’ samples. This strategy has the potential to reduce the cost and time of cancer screening and genotyping, and to enable targeted therapies in resource-limited settings”, “we devised a new assay dubbed Programmable Enzyme-Assisted Selective Exponential Amplification (PASEA, Fig. 1) that concurrently amplifies both WT and mutant alleles in the presence of guided endonuclease that targets only the WT allele. Given time, the variant that exhibits a superior trait (the mutant allele being less susceptible to cleavage) will dominate. PASEA requires temperature-matched polymerase and endonuclease. Herein, we use CRISPR-Cas9 programmed to cleave WT alleles in combination with isothermal recombinase polymerase amplification (RPA). We converted samples with 0.01% somatic MAFs to products with 70% MAFs in a single step (single pot) within 20 min, enabling inexpensive, rapid genotyping with such as Sanger sequencers. Previously, we reported the broad outlines of our approach [20]. In this paper, we expound yet unpublished experimental data that demonstrates PASEA's capabilities and its suitability for resource poor settings. Furthermore, we used PASEA to test 108 clinical tissue samples and 10 blood samples from cancer patients and compared PASEA with NGS and amplification refractory mutation system (ARMS)-PCR” and “[P]ASEA relies on selective cleavage to obtain much greater amplification rates of mutant alleles than of WT alleles. The contrast between the PASEA and RPA [in the absence of Cas9 and sgRNA ribonucleoprotein (RNP)] amplification rates of WT genomic DNA is striking (Fig. 2a). Within 10 min, RPA produced about 109
amplicons while PASEA produced less than 105-four orders of magnitude less. When PASEA acts on a standard KRAS G12V (MAF 5%) sample (Fig. 2a), the numbers of both KRAS G12V and WT KRAS amplicons increase as time increases but the KRAS G12V amplifies at a much greater rate than WT KRAS. Due to selective amplification, the number of amplicons of WT
KRAS in the blend should be much less than the number of amplicons when PASEA is applied to pure WT KRAS (blue bars). After ∼3 min, the products of the standard sample (5% MAF) are dominated by the mutant allele (green bars)” (see pages 4126 and 4127, and Figures 1 and 2 from Chen et al., Chinese Chemical Letters, 33, 4126-4132, 2022), the specification and the paper from the inventors clearly indicate that, since Cas9 selectively cleaves WT alleles with a protospacer adjacent motif (PAM) site and does not cleave mutant alleles because the mutant alleles lack the PAM, nearly all of amplification products are mutant alleles after the PASEA and the scopes of claims 1, 2, 4-8, and 19 are much broader than the teachings of the specification since claim 1 does not require that Cas endonuclease is a Cas endonuclease which requires a PAM for target recognition such as Cas9. Since it is known that Type III CRISPR-Cas systems does not require a PAM for target recognition (see page 9791, right column, third paragraph from Artamonova et al., Nucleic Acids Research, 48, 9787-9803, 2020) and a typical type III CRISPR system encodes Cas10, a protein with two catalytic domains: the HD domain that cleaves ssDNA and the Palm domain that synthesizes cyclic oligoadenylates
(cOA) using ATP (page 28, third paragraph from Stella et al., Trends in Biochemical Sciences, 49, 28-37, 2024), and claim 1 does not require that a Cas endonuclease is a Cas endonuclease which requires a PAM for target recognition such as Cas9, if the Cas endonuclease is Cas10 or Cas10-Csm which does not require a PAM for target recognition, both the target nucleic acid and the non-target nucleic acid can be cleaved by the Cas endonuclease such as Cas10 or Cas10-Csm such that the target nucleic acid cannot be selectively amplified and it is unpredictable how selective amplification of a target nucleic acid in a sample can be performed using the methods recited in claims 1, 2, 4, 5, 7, 8 and 19. Furthermore, since claim 1 does not require amplifying the target nucleic acid in an amplification reaction using primers specifically amplifying the target nucleic acid, if primers used for amplifying the target nucleic acid can hybridize with other nucleic acids, the other nucleic acids also can be amplified and the target nucleic acid cannot be selectively amplified such that it is unpredictable how selective amplification of a target nucleic acid in a sample can be performed using the methods recited in claims 1, 2, 4-8, and 19. In addition, since it is known that Cas9 cleaving reaction is carried out at 37ºC (see page 2 of “In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (NEB #M0386)” from New England Biolabs) and claim 1 does not indicate that a polymerase is what kind of polymerase and the amplification reaction is what kind of amplification reaction, if the polymerase is Taq DNA polymerase and the amplification reaction is PCR which comprises denaturation, annealing and extension steps, during the process of the PCR, a Cas endonuclease is denatured such that some of non-target nucleic acid cannot be cleaved by the Cas endonuclease and may be amplified in the amplification reaction, and it is unpredictable how selective amplification of a target nucleic acid in a sample can be performed using the methods recited in claims 1, 2, 4-8, and 19.
Case law has established that “(t)o be enabling, the specification of a patent must teach those skilled in the art how to make and use the full scope of the claimed invention without ‘undue experimentation’.” In re Wright 990 F.2d 1557, 1561. In re Fisher, 427 F.2d 833, 839, 166 USPQ 18, 24 (CCPA 1970) it was determined that “[T]he scope of the claims must bear a reasonable correlation to the scope of enablement provided by the specification to persons of ordinary skill in the art”. The amount of guidance needed to enable the invention is related to the amount of knowledge in the art as well as the predictability in the art. Furthermore, the Court in Genentech Inc. v Novo Nordisk 42 USPQ2d 1001 held that “[I]t is the specification, not the knowledge of one skilled in the art that must supply the novel aspects of the invention in order to constitute adequate enablement”.
In view of above discussions, the skilled artisan will have no way to predict the experimental results. Accordingly, it is concluded that undue experimentation is required to make the invention as it is claimed. The undue experimentation at least includes to test whether selective amplification of a target nucleic acid in a sample can be performed using the methods recited in claims 1, 2, 4-8, and 19.
Conclusion
In the instant case, as discussed above, the level of unpredictability in the art is high, the specification provides one with no guidance that leads one to claimed methods. One of skill in the art cannot readily anticipate the effect of a change within the subject matter to which the claimed invention pertains. Thus given the broad claims in an art whose nature is identified as unpredictable, the unpredictability of that art, the large quantity of research required to define these unpredictable variables, the lack of guidance provided in the specification, the absence of any working example related to claimed invention and the no teaching in the prior art balanced only against the high skill level in the art, it is the position of the examiner that it would require undue experimentation for one of skill in the art to perform the method of the claim as broadly written.
Conclusion
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.
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Frank Lu, Ph. D., whose telephone number is (571)272-0746. The examiner can normally be reached Monday to Friday, 9 AM to 5 PM.
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/FRANK W LU/
Primary Examiner, Art Unit 1683
March 17, 2026