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 .
Specification
The disclosure is objected to because page 28, line 7 of the specification contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 09/14/2023 and 03/15/2024 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Status of Claims
This office action is in response to Applicant's Response to Election / Restriction filed on March 10, 2026.
Claims 1-11, 13, 15, 18-20, 22-25 and 27 were previously pending and subject to restriction and election requirement. No claim amendments are made in the response filed on March 10, 2026.
Claims 1-11, 13, 15, 18-20, 22-25 and 27 are currently pending, with claims 10, 19-20, 23-25 and 27 withdrawn.
Claims 1-9, 11, 13, 15, 18 and 22 are under examination. This is the first action on the merits.
Election/Restrictions
Applicant’s election without traverse of Group I (claims 1-11, 13, 15, 18-20, 22-24) in the reply filed on March 10, 2026 is acknowledged 1.
Applicant’s election without traverse of the following species in the reply filed on March 10, 2026 is acknowledged:
Species of first and second oligonucleotide probes: E) the first and second oligonucleotide probes circularise on recognition of their specific target polynucleotide sequence (claim 8) 2;
Species of step (iii): J) wherein step (iii) comprises immobilizing the RCA-Products on a surface (claim 13) 3;
Species of surface: J1) a glass surface (claim 15) 4;
Species of processing RCA products: M) wherein the RCA-Products are labelled with a detectable moiety (claim 3) 5.
Claims 10, 19-20, 23-25 and 27 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention.
Examination on the merits commences on claims 1-9, 11, 13, 15, 18 and 22.
Priority
The priority date of the instant claims 1-9, 11, 13, 15, 18 and 22 is March 15, 2021, filling date of the UNITED KINGDOM Patent Application Number 2103543.1, to which the present application claims priority.
Claim Interpretation
In evaluating the patentability of the claims presented in this application, claim terms have been given their broadest reasonable interpretation (BRI) consistent with the specification, as understood by one of ordinary skill in the art, as outlined in MPEP§ 2111.
For the purpose of applying prior art, claim 1 recites "genetic-editing procedure," which is expressly defined in the specification as follows:
"By “genetic-editing procedure” we include any such procedure in which a target polynucleotide sequence is “edited” by addition and/or deletion and/or mutation of one or more nucleotide base in that molecule. " (page 6, lines 22-24).
Thus, as defined by the application's specification, the term “genetic-editing procedure” broadly encompasses any procedure that changes nucleotide base composition in a polynucleotide sequence, either in vitro or in vivo.
Claim Rejections - 35 USC § 112(b)
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.
Claim 3 is rejected under 35 U.S.C. 112(b), 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.
Regarding claim 3, it recites "preferably differentially-labelled such that RCA-Products from the correctly-edited polynucleotide sequence and/or the unedited polynucleotide sequence can be distinguished," which is indefinite because "preferably" suggests subjectivity and does not clearly delineate whether the feature is a requirement of the claim or merely an illustrative example. This uncertainly prevents determination of the scope with reasonable certainly.
Claim Rejections - 35 USC § 103
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.
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 1-9, 11, 13, 15, 18 and 22 are rejected under 35 U.S.C. 103 as being unpatentable over Germini (Germini et al. A Comparison of Techniques to Evaluate the Effectiveness of Genome Editing. Trends Biotechnol. 2018 Feb;36(2):147-159. doi: 10.1016/j.tibtech.2017.10.008. Epub 2017 Nov 17. PMID: 29157536; cited as Non-Patent Literature Document #4 in IDS filed 09/14/2023) , in view of Lizardi (Lizardi et al. , Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat Genet. 1998 Jul;19(3):225-32. doi: 10.1038/898. PMID: 9662393).
A) Germini provides an overview of main methods used to test for genome editing efficiency (entire document, Abstract for example).
Regarding claim 1, Germini teaches determining the efficiency of a genetic-editing procedure and notes that most techniques for evaluating genome editing effectiveness "search for genetic aberrations created in the proximity of the endonuclease cutting site, including point mutations, deletions, or insertions, which are the result of the DNA repair process. " (page 148, para 3). Thus, evaluating genome editing efficiency involves detecting mutations as evidence that editing has occurred.
All approaches described in Germini for assessing genome editing efficiency include an amplification step (see Table 1, PCR). Although Germini does not explicitly teach performing Rolling Circle Amplification in testing for genome editing efficiency, this approach is obvious in view of Lizardi.
Lizardi teaches methods for Mutation detection and single-molecule counting using isothermal rolling-circle amplification (entire document, see Abstract for example).
Regarding claim 1, Lizardi teaches:
providing a sample from a genetic-editing procedure (page 228, right-hand col, lines 1-4, genomic DNA samples comprising edits that simulate somatic mutations), the sample comprising one or more correctly-edited polynucleotide sequence and/or one or more unedited polynucleotide sequence (page 228, right-hand col, lines 4-5, wt and edited DNA comprising simulated mutations are mixed for RCA detection);
performing Rolling Circle Amplification, to generate RCA-Products from the one or more polynucleotide sequences in the sample (Fig. 6, 7; page 228, right-hand col, lines 7-8).
Regarding the limitation "determining the efficiency of the genetic-editing procedure based on the presence of the RCA-Products," Lizardi discloses measuring the ratio of the edited simulated mutant and wild type polynucleotides in a mixture using RCA (Page 228, right-hand col; Fig. 7). Accordingly, a person of ordinary skill in the art would have recognized that the RCA approach of Lizardi could be applied to evaluate genome editing efficiency by quantifying the relative amounts of edited (mutant) and unedited sequences in a sample. As noted in Germini, genome editing evaluation relies on detecting mutations, and Lizardi expressly teaches mutation detection using RCA.
Lizardi further teaches its RCA methods are rapid, technically simple and exhibit low variability (page 230, right-hand col, para 2, lines 1-6), which largely align with the characteristics identified in Germini for desirable genome editing evaluation methods:
“In general, researchers tend to limit tedious preliminary experiments to undertake the research program they have in mind as rapidly as reasonable. A good method to evaluate the efficiency of newly designed nucleases should combine simplicity in the experimental procedure with reliability of the method. In addition, a good method should save time and limit cost.” (Germini, page 148, para 2).
Therefore, It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to apply the RCA-based mutation detection method of Lizardi to the evaluation of genome editing efficiency as described in Germini. Both references involve amplification and detection of target polynucleotides and associated mutations. Because Germini teaches evaluation of genome editing efficiency relies on detecting mutations, and Lizardi provides a simple and rapid amplification method specifically suited for detecting and measuring mutant-to-wild-type ratios, a skilled artisan seeking a simple and rapid method for evaluating genome editing efficiency would have been motivated to select Lizardi's RCA method as a predictable and advantageous alternative to the amplification methods of Germini.
The person of ordinary skill would have had a reasonable expectation of success because genome editing introduces mutations into target polynucleotides, and methods capable of detecting such mutations, as taught by Lizardi, would predictably be suitable for evaluating genome editing outcomes.
B) Regarding claim 2, Lizardi teaches prior to performing Rolling Circle Amplification step (ii), the method comprises the step of generating circular single- stranded polynucleotide substrates from the polynucleotide sequences in the sample (Fig. 1), wherein the circular single-stranded polynucleotide substrates are specific for the correctly- edited polynucleotide sequence, and for the unedited polynucleotide sequence (Fig. 1, 2; page 225, right-hand col, para 2, 8-11).
Regarding claim 3, Lizardi teaches RCA-Products are labelled with a detectable moiety (Fig. 6 ).
Regarding claim 4, Lizardi teaches quantifying the RCA-Products generated in step (ii), and determining the amounts of the correctly- edited polynucleotide sequence and/or unedited polynucleotide sequence in the sample (Fig. 7).
Regarding claim 5, Lizardi teaches quantifying the total number of polynucleotide sequences in the sample (Fig. 5 and legends, “net DNA input for each amplification reaction consisted of 28 ng of genomic DNA, which corresponds to 17,000 copies of the gene locus”; page 231, lines 7-9).
Regarding claim 6, Lizardi teaches the circular single-stranded polynucleotide substrates are generated using a first oligonucleotide probe which specifically targets the correctly-edited polynucleotide sequence, and a second oligonucleotide probe which specifically targets the unedited polynucleotide sequence (Fig. 1, 2).
Regarding claim 7, Lizardi teaches gap- fill probe (Fig. 1, 2).
Regarding claim 8, Lizardi teaches the first and second oligonucleotide probes circularise on recognition of their specific target polynucleotide sequence (Fig. 1, 2).
Regarding claim 9, Lizardi teaches circularisation of the first and/or second oligonucleotide probes is mediated and/or improved by one or more Joining probe (Fig. 1, Fig. 2, 8- base gap probe).
Regarding claim 11, Lizardi teaches Rolling Circle Amplification of the circular single-stranded polynucleotide substrates is initiated by an amplification primer that is complementary to the circular singe-stranded polynucleotide substrate (Fig. 4)
Regarding claim 13, Lizardi teaches immobilizing the RCA-Products on a surface (Fig. 6).
Regarding claim 15, Lizardi teaches a glass surface (Fig. 6, legend, “A derivatized glass surface contains an oligonucleotide probe (P1) which is immobilized via a spacer (L), bound covalently to the glass.”).
Regarding claim 18, Lizardi teaches glass surface is modified to interact with an RCA-Product (Fig. 6, the glass surface is modified to be tethered to the RCA product).
Regarding claim 22, Lizardi teaches determining based on the relative amounts of the correctly- edited polynucleotide sequence and the unedited polynucleotide sequence in the sample (Fig. 7) .
Prior Art
Below are relevant prior art not used in rejection but pertinent to the claims or disclosure.
Applying RCA within the context of evaluating efficiency (e.g. specificity) of cellular genetic editing is known in the art:
See Urnov (WO2019204661A1- Methods for assessing specificity of cell engineering tools; Published 2019-10-24) see [0254]; [0259]; [0272] ;
See Lundin (Lundin et al. Spatiotemporal mapping of RNA editing in the developing mouse brain using in situ sequencing reveals regional and cell-type-specific regulation. BMC Biol. 2020 Jan 14;18(1):6. doi: 10.1186/s12915-019-0736-3. PMID: 31937309; PMCID: PMC6961268; cited as Non-Patent Literature Document #2 in IDS filed 09/14/2023) in Abstract, Figure 1.
Methods and/or benefits of detecting mutations via imaging of RCA products immobilized and/or additionally compacted on substrate are well known in the art:
See Zhong (Zhong et al. Visualization of oligonucleotide probes and point mutations in interphase nuclei and DNA fibers using rolling circle DNA amplification. Proc Natl Acad Sci U S A. 2001 Mar 27;98(7):3940-5. doi: 10.1073/pnas.061026198. PMID: 11274414; PMCID: PMC31158.) see Figure 1 and 3 ;
See Clausson (Clausson et al. Compaction of rolling circle amplification products increases signal integrity and signal-to-noise ratio. Sci Rep 5, 12317 (2015).) see Figure 1 ;
See Ciftci (Ciftci et al., Digital Rolling Circle Amplification-Based Detection of Ebola and Other Tropical Viruses. J Mol Diagn. 2020 Feb;22(2):272-283. doi: 10.1016/j.jmoldx.2019.10.014. Epub 2019 Dec 16. PMID: 31837428; cited as Non-Patent Literature Document#14 in IDS filed 03/15/2024) see Figure 1;
See Smolina (Smolina et al., High-density fluorescently labeled rolling-circle amplicons for DNA diagnostics. Anal Biochem. 2005 Dec 1;347(1):152-5. doi: 10.1016/j.ab.2005.06.002. Epub 2005 Jun 21. PMID: 16243289), see Fig.2 ;
See Kühnemund (Kühnemund et al., Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules. Nucleic Acids Res. 2017 May 5;45(8):e59. doi: 10.1093/nar/gkw1324. PMID: 28077562; PMCID: PMC5416848) see Figure 1 ;
See Hatch (Hatch et al., Rolling circle amplification of DNA immobilized on solid surfaces and its application to multiplex mutation detection. Genet Anal. 1999 Apr;15(2):35-40. doi: 10.1016/s1050-3862(98)00014-x. PMID: 10191983) , see Figure 2;
See Heo (Heo et al., A valveless rotary microfluidic device for multiplex point mutation identification based on ligation-rolling circle amplification. Biosens Bioelectron. 2016 Apr 15;78:140-146. doi: 10.1016/j.bios.2015.11.039. Epub 2015 Nov 14. PMID: 26606304), see Figs. 3-5.
Conclusion
No claims are allowed.
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/TIAN NMN YU/Examiner , Art Unit 1681 /AARON A PRIEST/Primary Examiner, Art Unit 1681
1 Claims 25 and 27 are withdrawn as being drawn to non-elected Groups II-III.
2 Claim 10 is withdrawn as being drawn to non-elected species F.
3 Claim 20 is withdrawn as being drawn to non-elected species K and L.
4 Claims 23-24 are withdrawn as being drawn to non-elected species J3.
5 Claim 19 is withdrawn as being drawn to non-elected species N.
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