DETAILED ACTION
Status of the Application
Claims 1-2, 5, 10, 13, 17, 19, 23, 30, 33-35, 39-40, 44-45, 47-48, 50, 52, 54-55, 58, 61-62, 64, 66, 68-69, 71, 73-74, 78, 80, 86-89 are pending.
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 of claims 61, 66, 86, and amendments to the specification as submitted in a communication filed on 5/8/2026 is acknowledged.
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 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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 filed on 5/8/2026 has been entered.
Applicant elected Group II, claims 61-62, 64, 66, 68-71, 73, 78, 80, 86, drawn in part to a system, the election of a dead Cas12 enzyme, Sau LF polymerase, and PcrA helicase, as indicated in a communication filed on 11/22/2023.
Claims 1, 2, 5, 10, 13, 17-19, 23, 30, 33-35, 39-40, 42, 44-45, 47-48, 50, 52, 54-55, 58, 64, 68, 74, 78 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention , there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 11/22/2023.
Claims 61-62, 66, 69, 71, 73, 80, 86-89 are at issue and will be examined to the extent they encompass the elected invention.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Rejections and/or objections not reiterated from previous office actions are hereby withdrawn.
Specification
The sequence listing submitted on 8/12/2024 remains objected to for the following reasons. The Examiner has been unable to locate support for the sequence of SEQ ID NO: 26. While it is agreed that paragraph [00179] of the specification as originally filed refers to the GenBank entries WP_003870487.1, WP_034654680.1, WP_095390358.1, and WP_055343022.1, and paragraph [00186] refers to a variant of GenBank entry WP_003870487.1 having the substitutions D403A/D404A, a variant of WP_034654680.1 having the substitutions D415A/D416A, a variant of WP_095390358.1 having the substitutions D407A/D408A, and a variant of WP_055343022.1 having mutations D402A/D403A, which correspond to SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29 as shown in the alignments provided in the final action of 2/11/2026, the specification is completely silent as to a GenBank entry that corresponds to SEQ ID NO: 26. There is no support for a protein having SEQ ID NO: 26 in view of the fact that there is absolutely no disclosure in the specification as originally filed for a protein having the exact same sequence. Please note that while paragraphs [00179] and [00186] of the specification as originally filed refer to a pcrA helicase from Bacillus sp. FJAT-27231, naturally occurring variants of a protein from the same organism are a common occurrence in nature. Therefore, the mere reference to a pcrA helicase from Bacillus sp. FJAT-27231, which encompasses a genus of proteins, does not inherently provide support for the specific sequence of SEQ ID NO: 26. Thus, one cannot reasonably conclude that the protein of SEQ ID NO: 26 was disclosed in the specification as originally filed. Therefore, there is no support for SEQ ID NO: 26 of the sequence listing. As such, the sequence listing submitted on 8/12/2024 introduces new matter into the disclosure. Applicant is required to cancel the new matter in the sequence listing in the reply to this Office action.
The amendment filed 5/8/2026 is objected to under 35 U.S.C. 132 (a) because it introduces new matter into the disclosure. 35 U.S.C. 132(a) states that no amendment shall introduce new matter into the disclosure of the invention. The added material which is not supported by the original disclosure is as follows. Paragraph [00186] of the amended specification refer to the pcrA helicase from Bacillus sp. FJAT-27231 with mutations D407A/D408A as having SEQ ID NO: 26. However, there is no support for SEQ ID NO: 26 in the specification as originally filed. Please note that there is no specific GenBank entry associated with the intended pcrA helicase from Bacillus sp. FJAT-27231 in the specification as originally filed, let alone a variant having mutations D407A/D408A. Applicant is required to cancel the new matter in the reply to this Office action.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 5/8/2026 is acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Claim Objections
Claim 62 is objected due to the recitation of “…Cas9 enzyme a dead Cas12a ..”. A comma should be introduced between the term “enzyme” and “a dead”. Appropriate correction is required.
Claim Rejections - 35 USC § 112(b) or Second Paragraph (pre-AIA )
Claims 61-62, 66, 69, 71, 73, 80, 86-89 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 pre-AIA the applicant regards as the invention.
Claim 61 (claims 62, 66, 69, 71, 73, 80, 86-89 dependent thereon) is indefinite in the recitation of “…a helicase selected from…PcrA helicase…and T7 Gp4 helicase, wherein the helicase comprises one or more alanine substitutions at two consecutive aspartate residues” for the following reasons. The term “at two consecutive aspartate residues” is unclear in the absence of which is the protein that comprises the consecutive aspartate residues and the location of those residues within the protein. In addition, the term is unclear in the absence of the sequence identifier of the reference protein that should be used to determine if the substitution required is present and the positions in that reference protein where those aspartate residues are. Please note that, for example, the term “PcrA helicase” encompasses a genus of helicases and it is unclear if any PcrA helicase has two consecutive aspartate residues. As known in the art, the term “PcrA helicase” is not solely limited to naturally occurring variants since man-made variants of a PcrA helicase could be also considered as PcrA helicases. Also, since only one substitution is required, it is unclear if the variant could have a deletion of one aspartate residue, namely replacing two aspartate residues with one alanine. For examination purposes, no patentable weight will be given to the term. Correction is required.
Claim 80 is indefinite in the recitation of “from about X to about Y” for the following reasons. The term “about” encompasses a range which includes values which are higher and lower than the recited reference value (i.e., 2.5%, 6.5%, 0.75 mM, 5 mM). The term “from X to Y”, implies a range where X and Y are the endpoints. Therefore, in the absence of a clear definition of what is encompassed by the term “about”, the term "from about X to about Y" is unclear and confusing because X and Y are endpoints which are undefined ranges. If the intended ranges are “from X to Y”, the claim should be amended accordingly. Correction is required.
When amending the claims, applicant is advised to carefully review all examined claims and make the necessary changes to ensure proper antecedent basis and dependency.
Claim Rejections - 35 USC § 112(a) or First Paragraph (pre-AIA )
Claim 86 was 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 due to the introduction of new matter. In view of the amendment of claim 86 removing a reference to SEQ ID NO: 26, this rejection is hereby withdrawn.
Claim Rejections - 35 USC § 103 (AIA )
Claims 61, 62, 66, 69, 80, 86-87 were rejected under 35 U.S.C. 103 as being unpatentable over Mandell (WO 2016/077350 published 5/19/2016; cited in the IDS) in view of Kong et al. (U.S. Publication No. 2004/0058378 published 3/25/2004; cited in the IDS), Korfhage (U.S. Publication No. 2013/0224799, published 8/29/2013; cited in the IDS), Ma et al. (eLife 7:e34186, page 1-21, published 4/17/2018), Yamamoto et al. (GenBank accession No. BAA00048.1), and GenBank accession No. WP_049660019.1 (7/24/2015). In view of the amendment of claim 86, which no longer requires a PcrA helicase that comprises SEQ ID NO: 26, this rejection is hereby withdrawn.
Claims 73 and 89 remain rejected under 35 U.S.C. 103 as being unpatentable over Mandell (WO 2016/077350 published 5/19/2016; cited in the IDS) in view of Kong et al. (U.S. Publication No. 2004/0058378 published 3/25/2004; cited in the IDS), Korfhage (U.S. Publication No. 2013/0224799, published 8/29/2013; cited in the IDS), and further in view of Deiman et al. (Molecular Biotechnology 20:163-179, 2002).
Claims 61, 62, 66, 69, 80, 87 remain rejected under 35 U.S.C. 103 as being unpatentable over Mandell (WO 2016/077350 published 5/19/2016; cited in the IDS) in view of Kong et al. (U.S. Publication No. 2004/0058378 published 3/25/2004; cited in the IDS) and Korfhage (U.S. Publication No. 2013/0224799, published 8/29/2013; cited in the IDS).
Claims 62 and 80 remain rejected under 35 U.S.C. 103 as being unpatentable over Mandell (WO 2016/077350 published 5/19/2016; cited in the IDS) in view of Kong et al. (U.S. Publication No. 2004/0058378 published 3/25/2004; cited in the IDS), Korfhage (U.S. Publication No. 2013/0224799, published 8/29/2013; cited in the IDS), and further in view of Begemann et al. (U.S. Publication No. 2017/0233756 published 8/17/2017).
Claims 71 and 88 remain rejected under 35 U.S.C. 103 as being unpatentable over Mandell (WO 2016/077350 published 5/19/2016; cited in the IDS) in view of Kong et al. (U.S. Publication No. 2004/0058378 published 3/25/2004; cited in the IDS), Korfhage (U.S. Publication No. 2013/0224799, published 8/29/2013; cited in the IDS), and further in view of Gootenberg et al. (Science 360:439-444, published online 2/15/2018; cited in the IDS).
These rejections have been discussed at length in the prior Office action. They are maintained for the reasons of record and those set forth below.
Applicant argues that claim 61 has been amended to require a helicase that comprises one or more alanine substitutions at two consecutive aspartate residues. Applicant submits that no cited reference teach or suggest this limitation. Applicant states that the specification demonstrates experimentally that alanine substitutions at two consecutive aspartate residues substantially increase helicase processivity and activity and that these substitutions were identified through screening. According to Applicant, Mandell et al., Kong et al. and Korfhage et al. do not teach helicase variants. According to Applicant, the teachings of Ma et al., Kong et al. and Mandell et al. do not establish that a helicase bearing alanine substitutes will function within an HAD amplification system, much less within a CRISPR R-loop initiated HAD system. Applicant refers to the experimental work provided in the specification in support of the argument that the claimed invention is nonobvious. Applicant is of the opinion that the cited references fail to teach an essential enabling claim limitation.
Applicant’s arguments have been fully considered but not deemed persuasive to overcome the instant rejections. The Examiner acknowledges the amendments made to claim 61. However, for the reasons extensively discussed above under Claim Rejections - 35 USC § 112(b) or Second Paragraph (pre-AIA ), no patentable weight has been given to the limitation “wherein the helicase comprises one or more alanine substitutions at two consecutive aspartate residues”. Therefore, these rejections are maintained for the reasons of record in view of the interpretation of claim 61.
Claims 61, 62, 66, 69, 80, 86-87 remain rejected under 35 U.S.C. 103 as being unpatentable over Mandell (WO 2016/077350 published 5/19/2016; cited in the IDS) in view of Kong et al. (U.S. Publication No. 2004/0058378 published 3/25/2004; cited in the IDS), Korfhage (U.S. Publication No. 2013/0224799, published 8/29/2013; cited in the IDS), Ma et al. (eLife 7:e34186, page 1-21, published 4/17/2018), Yamamoto et al. (GenBank accession No. BAA00048.1 6/15/2010), and GenBank accession No. WP_003870487.1 (5/7/2013).
Claims 61, 62, 66, 69, 80, 86-87 remain rejected under 35 U.S.C. 103 as being unpatentable over Mandell (WO 2016/077350 published 5/19/2016; cited in the IDS) in view of Kong et al. (U.S. Publication No. 2004/0058378 published 3/25/2004; cited in the IDS), Korfhage (U.S. Publication No. 2013/0224799, published 8/29/2013; cited in the IDS), Ma et al. (eLife 7:e34186, page 1-21, published 4/17/2018), Yamamoto et al. (GenBank accession No. BAA00048.1), and GenBank accession No. WP_034654680.1 (12/23/2014).
Claims 61, 62, 66, 69, 80, 86-87 remain rejected under 35 U.S.C. 103 as being unpatentable over Mandell (WO 2016/077350 published 5/19/2016; cited in the IDS) in view of Kong et al. (U.S. Publication No. 2004/0058378 published 3/25/2004; cited in the IDS), Korfhage (U.S. Publication No. 2013/0224799, published 8/29/2013; cited in the IDS), Ma et al. (eLife 7:e34186, page 1-21, published 4/17/2018), Yamamoto et al. (GenBank accession No. BAA00048.1), and GenBank accession No. WP_095390358.1 (8/30/2017).
Claims 61, 62, 66, 69, 80, 86-87 remain rejected under 35 U.S.C. 103 as being unpatentable over Mandell (WO 2016/077350 published 5/19/2016; cited in the IDS) in view of Kong et al. (U.S. Publication No. 2004/0058378 published 3/25/2004; cited in the IDS), Korfhage (U.S. Publication No. 2013/0224799, published 8/29/2013; cited in the IDS), Ma et al. (eLife 7:e34186, page 1-21, published 4/17/2018), Yamamoto et al. (GenBank accession No. BAA00048.1), and GenBank accession No. WP_055343022.1 (10/27/2015).
These rejections have been discussed at length in the prior Office action. They are maintained for the reasons of record and those set forth below.
Applicant argues that claim 61 has been amended to require a helicase that comprises one or more alanine substitutions at two consecutive aspartate residues. Applicant submits that no cited reference teach or suggest this limitation. Applicant states that the specification demonstrates experimentally that alanine substitutions at two consecutive aspartate residues substantially increase helicase processivity and activity and that these substitutions were identified through screening. According to Applicant, Mandell et al., Kong et al. and Korfhage et al. do not teach helicase variants. According to Applicant, the teachings of Ma et al., Kong et al. and Mandell et al. do not establish that a helicase bearing alanine substitutes will function within an HAD amplification system, much less within a CRISPR R-loop initiated HAD system. Applicant refers to the experimental work provided in the specification in support of the argument that the claimed invention is nonobvious. Applicant is of the opinion that the cited references fail to teach an essential enabling claim limitation.
Applicant’s arguments have been fully considered but not deemed persuasive to overcome the instant rejections. The Examiner acknowledges the amendment made to claim 61, the experimental work performed by Applicant, as well as the teachings of the cited prior art. However, the Examiner disagrees with Applicant’s contention that the claimed invention is nonobvious.
Claim 61 encompasses helicases that comprise SEQ ID NO: 25, 27, 28 or 29 as evidenced by claim 86. Therefore, even if the argument is made that the term “wherein the helicase comprises one or more alanine substitutions at two consecutive aspartate residues” is definite, which is not, the helicases of SEQ ID NO: 25, 27, 28 and 29 are helicases which are variants of known helicases where two consecutive aspartate residues were replaced with two alanine residues.
As previously indicated, GenBank accession No. WP_003870487.1 discloses a T. ethanolicus ATP-dependent DNA helicase PcrA that comprises all of SEQ ID NO: 25 except for two substitutions. See alignment previously provided. The polypeptide of SEQ ID NO: 25 comprises alanines at positions corresponding to positions 403 and 404 (aspartate residues) of the polypeptide of GenBank accession No. WP_003870487.1. An alignment of the E. coli UvrD protein disclosed by Yamamoto et al. and the helicase of GenBank accession No. WP_003870487.1 shows that positions 403 and 404 of the E. coli UvrD protein correspond to positions 403 and 404 of the protein of GenBank accession No. WP_003870487.1.
GenBank accession No WP_034654680.1 discloses a Bacillus megaterium ATP-dependent DNA helicase PcrA that comprises all of SEQ ID NO: 27 except for two substitutions. See alignment previously provided. The polypeptide of SEQ ID NO: 27 comprises alanines at positions corresponding to positions 415 and 416 (aspartate residues) of the polypeptide of GenBank accession No. WP_034654680.1 . An alignment of the E. coli UvrD protein disclosed by Yamamoto et al. and the helicase of GenBank accession No. WP_034654680.1 shows that positions 403 and 404 (aspartate residues) of the E. coli UvrD protein correspond to positions 415 and 416 of the protein of GenBank accession No. WP_034654680.1.
GenBank accession No WP_095390358.1 discloses a Bacillus simplex DNA helicase PcrA that comprises all of SEQ ID NO: 28 except for two substitutions. See alignment previously provided. The polypeptide of SEQ ID NO: 28 comprises alanines at positions corresponding to positions 407 and 408 (aspartate residues) of the polypeptide of GenBank accession No. WP_095390358.1. An alignment of the E. coli UvrD protein disclosed by Yamamoto et al. and the helicase of GenBank accession No. WP_095390358.1 shows that positions 403 and 404 (aspartate residues) of the E. coli UvrD protein correspond to positions 407 and 408 of the protein of GenBank accession No. WP_095390358.1.
GenBank accession No WP_055343022.1 discloses a P. sordellii ATP-dependent DNA helicase PcrA that comprises all of SEQ ID NO: 29 except for two substitutions. See alignment previously provided. The polypeptide of SEQ ID NO: 29 comprises alanines at positions corresponding to positions 402 and 403 (aspartate residues) of the polypeptide of GenBank accession No. WP_055343022.1. An alignment of the E. coli UvrD protein disclosed by Yamamoto et al. and the helicase of GenBank accession No. WP_055343022.1 shows that positions 403 and 404 (aspartate residues) of the E. coli UvrD protein correspond to positions 402 and 403 of the protein of GenBank accession No. WP_055343022.1.
With regard to the arguments that (i) Mandell et al., Kong et al. and Korfhage et al. do not teach helicase variants, (ii) the teachings of Ma et al., Kong et al. and Mandell et al. do not establish that a helicase bearing alanine substitutes will function within an HAD amplification system, much less within a CRISPR R-loop initiated HAD system, and (iii) the cited references fail to teach an essential enabling claim limitation, it is noted that (i) the instant rejections are obviousness rejections where it is the combined teachings of the references that render the claimed invention obvious, (ii) as set forth in MPEP § 2144 (I), the rationale to modify or combine the prior art does not have to be expressly stated in the prior art; the rationale may be expressly or impliedly contained in the prior art or it may be reasoned from knowledge generally available to one of ordinary skill in the art, established scientific principles, or legal precedent established by prior case law, and (iii) as set forth in MPEP § 2144(II), the strongest rationale for combining references is a recognition, expressly or impliedly in the prior art or drawn from a convincing line of reasoning based on established scientific principles or legal precedent, that some advantage or expected beneficial result would have been produced by their combination.
As previously indicated, Ma et al. teach that the E. coli UvrD double mutant D403A/D404A is a protein with hyper-helicase activity (page 4, first full paragraph; page 9, Molecular mechanism for the UvrD303 mutant). Ma et al. teach that these mutations (UvrD303) favor the closed conformation and will lead to better unwinding activity (page 9, last full paragraph). Ma et al. teach that UvrD belongs to the SF1 family, such as the PcrA and Rep helicases. Ma et al. teach that PcrA is highly homologous to UvrD and that the low-FRET “open” conformation of PcrA could be similar to the tilted conformation of UvrD (page 14, Functional insights for UvrD and its homologs).
The teachings of Mandell and Kong et al. as extensively discussed in prior Office actions render a system for helicase-dependent amplification that comprises two CRISPR/Cas complexes, each binding a different strand of a target nucleic acid, a PcrA helicase, a primer pair and a polymerase obvious. One of skill in the art would reasonably conclude that since Kong et al. teach that a PcrA helicase can be used for amplification, this reference clearly suggests that any PcrA helicase can be used for unwinding double stranded nucleic acids in an amplification method. Ma et al. teach making mutations to a helicase to increase its helicase (unwinding) activity, which is highly desirable in applications that require a helicase. While it is true that Ma et al. did not examine UvrD mutations for integration with CRISPR systems or isothermal amplification applications, the issue in the instant case is whether PcrA helicases having mutations that were shown to result in helicase hyperactivity would have been obvious to use in a system as claimed at the time of the invention. Please note that the enzymatic activity of a helicase is the same regardless of its intended use. The proteins of SEQ ID NO: 25, 27-29 are PcrA helicases that are variants of PcrA helicases known in the prior art, namely those disclosed by GenBank accession No. WP_003870487.1, GenBank accession No. WP_034654680.1, GenBank accession No. WP_095390358.1 and GenBank accession No. WP_055343022.1. These variants differ from those disclosed in the prior art only by two substitutions at two contiguous positions that substitute a D residue with an alanine, which are the same substitutions taught by Ma et al. in another helicase that is structurally similar to PcrA. As previously indicated, Ma et al. teach that the E. coli UvrD double mutant D403A/D404A is a protein with hyper-helicase activity. Ma et al. teach that these mutations (UvrD303) favor the closed conformation and will lead to better unwinding activity. Ma et al. teach that UvrD belongs to the SF1 family, such as the PcrA and Rep helicases, and that these helicases have a four-subdomain tertiary arrangement that includes two RecA-like domains (1A/2A) and a flexible domain (2B), believed to have a regulatory role in helicase activity and be a molecular switch. Ma et al. teach that the tilted state and related motions disclosed can possibly help connect structural information with function for other SF1 helicase. Ma et al. teach that PcrA is highly homologous to UvrD and that the low-FRET “open” conformation of PcrA could be similar to the tilted conformation of UvrD. Ma et al. teach that the D403A/D404A substitutions in the 2B domain stabilizes the UvrD closed conformation and that their analysis provides potential target residues to guide future experimental designs to obtain UvrD-like helicases with tunable unwinding activities.
It is reiterated herein that in view of the teachings of Ma et al. and the specific reference to the structural similarity between the E. coli UrvD helicase and PcrA helicases, including certain structural conformations, one of skill in the art would have been highly motivated to make the two substitutions that correspond to the substitutions D403A/D404A in the E. coli UrvD helicase of Ma et al. in the PcrA helicases of GenBank accession No. WP_003870487.1, GenBank accession No. WP_034654680.1, GenBank accession No. WP_095390358.1 and GenBank accession No. WP_055343022.1 to obtain variants having hyper-helicase activity to use in any application that uses a helicase, such as an amplification method, which is the subject matter of the claimed invention. As shown in the alignments previously provided, making the two substitutions that correspond to substitutions D403A/D404A in the E. coli UrvD helicase of Ma et al. to the PcrA helicases of GenBank accession No. WP_003870487.1, GenBank accession No. WP_034654680.1, GenBank accession No. WP_095390358.1 and GenBank accession No. WP_055343022.1 result in proteins that comprise SEQ ID NO: 25, 27-29, respectively.
While it is agreed that Ma et al. do not teach the helicases of SEQ ID NO: 25, 27-29, the prior art strongly suggests the substitutions of these consecutive D residues with alanine for increasing helicase activity in helicases such as PcrA, which is the activity that is required in the method of Mandell and Kong et al. As previously indicated, the helicase in the method of Mandell and Kong et al. is required for unwinding a double stranded nucleic acid. Therefore, contrary to Applicant’s assertions, the PcrA helicases of SEQ ID NO: 25, 27-29 are deemed obvious and functional equivalents of the PcrA helicases of the method of Mandell and Kong et al. As such, the claimed invention is deemed obvious over the cited prior art.
Conclusion
No claim is in condition for allowance.
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Any inquiry concerning this communication or earlier communications from the examiner should be directed to DELIA M RAMIREZ, Ph.D., whose telephone number is (571) 272-0938. The examiner can normally be reached on Monday-Friday from 8:30 AM to 5:00 PM. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Robert B. Mondesi, can be reached at (408) 918-7584. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
/DELIA M RAMIREZ/Primary Examiner, Art Unit 1652
DR
September 18, 2026
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