DETAILED ACTION
CONTINUED EXAMINATION UNDER 37 CFR 1.114 AFTER FINAL REJECTION
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after the final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant’s submission of RCE and the amendments filed on July 13, 2026 have been entered. The claims pending in this application are claims 1, 2, 4-6, and 8-20 wherein claims 9-18 and 20 have been withdrawn in the restriction requirement mailed on June 10, 2025. The objections not reiterated from the previous office action are hereby withdrawn in view of applicant’s amendment filed on May 19, 2026. Claims 1, 2, 4-6, 8, and 19 will be examined.
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.
New Matter
Claims 1, 2, 4, 5, 8, and 19 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
A limitation “a Cas endonuclease requiring the PAM site for cleavage and having an affinity for the guide/non-target hybrid” is added to independent claim 1. Although paragraph [0013] pf the specification describes that “FIG. 4A, FIG. 4B, and FIG. 4C: Recombinase polymerase amplification (RPA) probe enables real-time detection. (FIG. 4A) Schematics of the Exo-RPA probe. (FIG. 4B) KRAS gene (SEQ ID NO: 16) with the locations of the primers, sgRNA protospacer (). and Exo-RPA probe indicated. (FIG. 4C) Real-time RPA monitoring of
serially diluted, wild-type alleles in the absence of RNP. Probe concentration: 240 nM. FIG.
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. Exo-probe indicates the number of
amplicons and enables quantification in real time (FIG. 4F)” and Cas12a from bacterial species Francisella novicida, Acidaminococcus or Lachnospiraceae, SaCas9 from bacterial species Staphylococcus aureus, CjCas9 from bacterial species Campylobacter jejuni, SpCas9 from bacterial species Streptococcus pyogenes, and NmCas9 from bacterial species Neisseria meningitidis are Cas endonucleases requiring the PAM site for cleavage and having an affinity for the guide/non-target hybrid (see claim 6), nowhere in the specification describes such limitation recited in claim 1 since nowhere in the specification has a phrase “a Cas endonuclease requiring the PAM site for cleavage and having an affinity for the guide/non-target hybrid” and describes all Cas endonucleases requiring the PAM site for cleavage and having an affinity for the guide/non-target hybrid such as Cas 12 b, which is another Cas endonuclease requiring the PAM site for cleavage and having an affinity for the guide/non-target hybrid (see page 1 of “CRISPR-associated endonuclease Cas12b”).
MPEP 2163.06 notes “If new matter is added to the claims, the examiner should reject the claims under 35 U.S.C. 112, first paragraph - written description requirement. In re Rasmussen, 650 F.2d 1212, 211 USPQ 323 (CCPA 1981).” MPEP 2163.02 teaches that “Whenever the issue arises, the fundamental factual inquiry is whether a claim defines an invention that is clearly conveyed to those skilled in the art at the time the application was filed...If a claim is amended to include subject matter, limitations, or terminology not present in the application as filed, involving a departure from, addition to, or deletion from the disclosure of the application as filed, the examiner should conclude that the claimed subject matter is not described in that application.” MPEP 2163.06 further notes “When an amendment is filed in reply to an objection or rejection based on 35 U.S.C. 112, first paragraph, a study of the entire application is often necessary to determine whether or not “new matter” is involved. Applicant should therefore specifically point out the support for any amendments made to the disclosure” (emphasis added).
Response to Arguments
In page 6, fifth paragraph bridging to page 7, third paragraph of applicant’s remarks, applicant argues that “[M]ore specifically, Applicant's disclosure provides a detailed description of the presently- claimed methods, and in particular states that the methods are realized by programmable endonucleases. Application at paragraphs [0069], [00126], and [00127]. Although the examples in the application provide embodiments of the claimed methods that utilize the programmable endonuclease Cas9, these embodiments do not limit the scope of Applicant's technology to the foregoing example programmable endonuclease, as Applicant’s disclosure explains that other programmable endonucleases can also be used in the methods. For example, the application states that ‘programmable endonucleases remove the dominant and interfering (background) wild-type sequences to facilitate detection of scarce mutant alleles.’ Application at paragraph [00126]. The application also states that the method ‘concurrently amplifies both wild type and mutant alleles while selectively cleaving the former. Given time, the variant that exhibits a superior trait (the mutant allele) will dominate.’ Application at paragraph [00127]. The selective cleaving is achieved through programmable endonucleases requiring programmable endonucleases that cleave the non-target nucleic acid comprising protospacer adjacent motif (PAM), but not the target nucleic acid lacking PAM. This is demonstrated at least in Applicant's FIG. 4E and described in paragraph [0013] of the application. Therefore, the application has sufficient description of programmable endonucleases requiring PAM site for cleaving the nucleic acids. Accordingly, the claims reciting ‘a Cas endonuclease requiring the PAM site for cleavage and having an affinity for the guide/non-target hybrid’ is fully supported by the application”.
The above arguments have been fully considered but they are not persuasive toward the withdrawal of the rejection because nowhere in the specification describes such limitation recited in claim 1 since nowhere in the specification has a phrase “a Cas endonuclease requiring the PAM site for cleavage and having an affinity for the guide/non-target hybrid” and describes all Cas endonucleases requiring the PAM site for cleavage and having an affinity for the guide/non-target hybrid such as Cas 12 b, which is another Cas endonuclease requiring the PAM site for cleavage and having an affinity for the guide/non-target hybrid (see “CRISPR-associated endonuclease Cas12b”).
Scope of Enablement
Note that some issues from the rejection under 35 U.S.C. 112(a) mailed on March 19, 2026 have been withdrawn in view of the amendment filed on July 13, 2026.
Claims 1, 2, 4-6, 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, 4-6, 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, 4-6, 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 requiring the PAM site for cleavage and having an affinity for the guide/non-target hybrid; and e) a polymerase having an optimal operating temperature substantially similar to an optimal operating temperature of the endonuclease; 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 a mutant form of the non-target nucleic acid and 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, 4-6, 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, 4-6, 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, 4-6, 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, 4-6, 8, and 19.
Since the specification teaches that “[I]nspired by Darwin theory of the survival of the fittest, described is a new assay dubbed Programmable Enzyme-Assisted Selective Exponential Amplification (PASEA) that concurrently amplifies both wild type and mutant alleles while selectively cleaving the former. Given time, the variant that exhibits a superior trait (the mutant allele) will dominate. PASEA exponentially increases the copy number of mutant alleles to nearly 100% of the amplicons. PASEA must be implemented with temperature-matched polymerase and endonuclease. Herein, CRISPR-Cas9 programmed to cleave wild type alleles is used in combination with isothermal recombinase polymerase amplification (RPA). With specifically designed primers, PASEA amplifies both RNA and DNA, providing an unprecedented enrichment, increasing the fraction of ultra rare somatic mutant alleles within 20 min to a level detectable with inexpensive sequencers such as Sanger in a single step. Furthermore, with the aid of a custom-designed Exo-RPA probe, PASEA identifies the presence of mutant alleles in real time, suggesting the possibility of mutant allele detection at the point-of-care (POC) in a closed tube or in a microfluidic chip. The real time PASEA performed on par with next-generation sequencing (NGS) and amplification refractory mutation system (ARMS)-PCR when testing 108 clinical tissue samples and 10 blood samples from cancer patients” and “[M]AF enrichment is essential for timely, sensitive detection of clinically critical, rare mutant alleles. Such an enrichment can be accomplished by hybridizing nucleic acids of interest to nucleic acid probes in the absence or presence of nucleic-acid guided endonucleases lacking catalytic activity (e.g., dCas9) (30, 31); by preferential enzymatic amplification of mutant alleles with specifically designed primers and DNA blocker (1, 2, 5); by suppression of the amplification of wild type alleles with capping nucleic acids (3); and by selective depletion of wild type nucleic acids with programmable endonuclease such as CRISPR Cas 9 (6, 19) and Argnoautes (7), wherein unwanted background sequences are selectively removed from the sample. These various methods can be used independently or in combination. The targeted nucleic acids can then be detected either directly or by sequencing” (see paragraphs [0159] and [0209] 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 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 a mutant of a non-target nucleic acid is amplified with specifically designed primers and the scopes of claims 1, 2, 4-6, 8, and 19 are much broader than the teachings of the specification since claim 1 does not require that the amplifying step is carried out in the presence of specifically designed primers. Without the primers in claim 1, it is unpredictable how selective amplification of the target nucleic acid in the sample can be performed using the methods recited in claims 1, 2, 4-6, 8, and 19. Furthermore, since claim 1 does not require amplifying the target nucleic acid in an amplification reaction using specifically designed primers, if primers used for amplifying the target nucleic acid can hybridize with other nucleic acids in the sample, the other nucleic acids in the sample also can be amplified and the target nucleic acid cannot be selectively amplified such that it is unpredictable how selective amplification of the target nucleic acid in the sample can be performed using the methods recited in claims 1, 2, 4-6, 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, 4-6, 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.
Response to Arguments
In page 7, fourth paragraph bridging to page 9, second paragraph of applicant’s remarks, applicant argues that “[T]he Song Declaration details that the application explains the clamed method with words, examples, graphs, figures, and tables such that those of ordinary skill in the art would be able to practice the claimed methods without undue experimentation. Song Declaration at ¶ ¶ 8-17. For example, although the office suggests that the claimed method would be unpredictable without primers specific to the target nucleic acid,¹ Applicant's disclosure explains that the selective amplification in the claimed method is achieved through the specific cleavage of the non-target nucleic acid, and not by the primers” and “[S]ong Declaration at ¶ 9, which declaration also explains that a person of ordinary skill in the art would be familiar with primer design. Song Declaration at ¶ 14”.
The above arguments have been fully considered but they are not persuasive toward the withdrawal of the rejection. Although the declaration of Jinzhao Song under 35 CFR 1.132 filed on July 13, 2026 indicated that “[I]n my view, a POSITA would also be familiar with primer design, as this is a routine task in the field. Also, the subject application provides substantial guidance related to the specificity of the primers. See for example, paragraph [00137] and Table 1, describing the location of the primers for both the wild type non-target nucleic acid and for the mutant KRAS DNA target nucleic acid, both utilizing the same RPA primer set” and “designing specific primers to detect wild type and mutant regions of interest is routine in the art” (see pages 6 and 7) and the specification and the paper from the inventors clearly indicate that a mutant of a non-target nucleic acid is amplified with specifically designed primers (see paragraphs [0159] and [0209] of US 2023/0052289 A1, which is US publication of this instant case, pages 4126 and 4127, and Figures 1 and 2 from Chen et al., Chinese Chemical Letters, 33, 4126-4132, 2022), there are no primers in claim 1. Without the primers in claim 1, it is unpredictable how selective amplification of the target nucleic acid in the sample can be performed using the methods recited in claims 1, 2, 4-6, 8, and 19. Furthermore, since claim 1 does not require amplifying the target nucleic acid in an amplification reaction using specifically designed primers, if primers used for amplifying the target nucleic acid can hybridize with other nucleic acids in the sample, the other nucleic acids in the sample also can be amplified and the target nucleic acid cannot be selectively amplified such that it is unpredictable how selective amplification of the target nucleic acid in the sample can be performed using the methods recited in claims 1, 2, 4-6, 8, and 19.
Conclusion
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
August 22, 2026
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