DETAILED ACTION
Applicant’s response, filed 02 Sept. 2025 has been fully considered. The following rejections and/or objections are either reiterated or newly applied. They constitute the complete set presently being applied to the instant application.
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 .
Continued Examination Under 37 CFR 1.114
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 02 Sept. 2025 has been entered.
Status of Claims
Claims 18, 23-158, 163-167, and 169 are cancelled.
Claims 1-17, 19-22, 159-162, 168, and 170-171 are pending.
Claims 1, 17, 20-22, 159-162, 168, and 170-171 are rejected.
Claims 2-16 and 19 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Priority
The effective filing date of the claimed invention is 07 Feb. 2022.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 29 April 2025 and 18 Aug. 2025 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements were considered by the examiner.
Claim Rejections - 35 USC § 103
The rejection of claims 2, 4, and 19 under 35 U.S.C. 103 as being unpatentable over Brunner (2009) in view of Mair (2021) and Wu (2011) in the Office action mailed 02 May 2025 has been withdrawn in view of claim amendments received 02 Sept. 2025.
The rejection of claim 18 under 35 U.S.C. 103 as being unpatentable over Brunner (2009) in view of Mair (2021) and Wu (2011) in the Office action mailed 02 May 2025 has been withdrawn in view of the cancellation of this claim received 02 Sept. 2025.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 17, 20-22, 159-160, 162, 168, and 170-171 are rejected under 35 U.S.C. 103 as being unpatentable over Brunner (2009) in view of Mair (2021) and Wu (2011). Any newly recited portion is necessitated by claim amendment.
Cited References:
Brunner et al., Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetal liver, 2009, Genome Research, 19, pg. 1044-1056 and suppl. (previously cited);
Mair et al., Cell-free DNA technologies for the analysis of brain cancer, British Journal of Cancer, 2021, pg. 371-378 (previously cited); and
Wu et al., Statistical Quantification of Methylation Levels by Next-Generation Sequencing, 2011, PLoS ONE, 6(6), pg. 1-12; (previously cited).
Regarding claim 1 Brunner discloses a method for measuring methylation levels at CpG sites in a genome using DNA molecules (Abstract; Figure 1) comprising the following steps:
Brunner discloses analyzing a plurality of DNA molecules from a biological sample of a subject (pg. 1053, col. 1, para. 3; Suppl. Table 1) by sequencing the DNA molecules (Figure 1), wherein the analyzing includes sequencing DNA molecules that are only digestion with MSpI (Figure 1A; pg. 1053, col. 2, para. 2), which digests DNA regardless of methylation status (i.e. without treatment using a process that differentially modifies nor recognizes DNA molecules depending on their methylation status) (pg. 1045, col. 1, para. 3). Brunner further discloses performing next-generation sequencing of the DNA molecules (Suppl. pg. 3, para. 2-4).
Brunner discloses the analyzing includes each of the reads of DNA molecules to a reference genome to identify a position of each DNA molecule in the genome (pg. 1053, col. 2, para. 3; Figure 1).
Brunner discloses determining a first amount of DNA molecules that align to the genome and include a CGG on their 5’ ends for each treatment (i.e. an amount of DNA molecules ending at a CG, or CpG site, which is within -1 to + 1 of a window around the CpG site with C at the 0 position) (pg. 1053, col. 2, para. 3; Suppl. pg. 3, para. 4 to pg. 4, para. 1, e.g. usable reads that are aligned plus end with CGG are used; Suppl. Table 2).
Brunner discloses determining if a digestion site is assayable for methylation using the amount of DNA molecules treated with MSpI ending with the CGG (pg. 1045, col. 1, para. 3; pg. 1053, col. 2, para. 4; Figure 3).
Regarding claim 1 and dependent claims 20-21, Brunner does not disclose the following limitations:
Regarding claim 1¸ Brunner does not disclose the DNA is cell-free DNA.
However, Mair discloses cell-free DNA technologies for analyzing cancer (Abstract), and discloses various bisulfite-free methods for analyzing the epigenome of cfDNA, including using methylation-restriction enzymes to cleave unmethylated regions followed by PCR and sequencing, citing the method of Brunner above (i.e. reference [78]) (pg. 373, col. 2, para. 4 and pg. 378, col. 1, para. 4 to col. 2, para. 1). Mair further discloses that bisulfite conversion based methods for epigenome analysis present limitations for cfDNA analysis because it necessitates harsh conditions of pH and temperature (pg. 378, col. 1, para. 2).
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 have modified the method of Brunner to have analyzed cell-free DNA in order to use a bisulfite-free method that avoids the harsh conditions of pH and temperature, as shown by Mair (pg. 378, col.1 , para. 2). This modification would have had a reasonable expectation of success given Mair discloses the method of Brunner as an option for cfDNA epigenome analysis using bisulfite-free methods (pg. 378, col. 1, para. 4 to col. 2, para. 1).
Further regarding claim 1:Brunner does not explicitly disclose using the determined first amount in the determination of the classification for methylation. Instead, Brunner uses the first amount to determine whether the site is assayable, and then uses a count of DNA molecules processed by HpaII in determining a binary classification of whether methylation is present (Figure 1; pg. 1053, col. 2, para. 4).
Brunner further does not disclose the first amount is normalized using a ratio of the first amount to one or more other amounts of the plurality of cell-free DNA molecules.
Regarding claims 20-21, Brunner does not disclose the normalization uses a number of the plurality of cell-free DNA molecules covering the CpG site or uses an average or median depth of the plurality of cell-free DNA molecules in a region including the CpG site.
However, these limitations were obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, as shown by Wu.
Regarding claims 1 and 20, Wu discloses a method for statistical quantification of methylation levels by next-generation sequencing (Abstract), and discloses that the method of Brunner only estimates methylation using HpaII tag counts and it would be reasonable to combine the tag count information of MspI and HpaII to contribute to a proportion estimate in methylation quantification (pg. 2, col. 1, para. 2; pg. 3, col. 2, para. 3-4). Wu further discloses that using the MsPI tag count information, proportional estimates between 0 and 1 for methyl-seq data can be estimated using 1-∑ yij/ ∑ xi (pg. 3, col. 2, para. 4 to pg. 4, col. 1, para. 3; pg. 4, col. 2, para. 1-2; Figure 1-2), and that this proportional estimate uses the sum of counts for MspI across each site K in the region (pg. 4, col. 1, para. 2 and col. 1, para. 1). Wu further discloses adjusting the sequencing depth bias for the MspI and HpaII libraries, by using genome-wide CGG tags aligned to MspI sites (i.e. a number of the cell-free DNA molecules covering the CpG site), and defining in a ratio of the MspI library to each HpaII library to specify the sequencing depth bias dj, and therefore, the proportional estimate becomes max(1-dj ∑ yij/ ∑ xi, 0) (pg. 5, col. 1, para. 3, section 2.5). Given dj is the ratio of CGG tags in the form MspI/HpaII, the above formula is equivalent to ∑ yij / (HpaII CGG tags) / (∑ xi/ MspI CGG tags). Thus, the first amount (xi) is normalized to other amounts of the cell-free DNA molecules (e.g. the genome-wide CGG tags aligned to MspI sites in the MspI library).
Regarding claim 21, Wu further discloses that sequencing performed is generally deeper on MspI libraries than on HpaII libraries, and the bias is different between regions, and further remarks that because the bias information for region is lacking, a sequencing depth of genome-wide CGG tags aligned to MspI sites were used. Therefore, while Wu does not explicitly normalize the first amount to CGG tags within a region (i.e. a region including the CpG site) due to missing information, Wu suggests using CGG tags in a given region (i.e. also a number of the cell-free DNA molecules covering the CpG site) to account for bias differences between regions. A sequencing depth indicates the average number of reads covering a specific genome position by definition, as understood by one of ordinary skill in the art and also discussed in Applicant’s own specification as published at para. [0154]). Wu does not expressly state if a total number of CGG tags across the genome for each of the HpaII and MspI are included in the ratio for the sequencing depth bias dj, or if the depth of CGG tags across the genome (i.e. an average count) are used in the sequencing depth bias dj. However, using the total number of tags in a particular region or across the genome is equivalent to using the average number of tags in a region or across the genome, given the tag counts of CGG in MspI and HpaII are combined in a ratio. In other words, [CountMSPI/ (region length)] / [CountHpaII/ (region length)] = CountMSP1 / CountHpaII, given the region lengths (or genome length) are equal.
It is noted that while Wu also uses a number of DNA molecules treated with HpaII to determine the methylation, the claims use the transitional phrase “comprising”, which is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. Therefore, the claims are open to analyzing a second plurality of cell-free DNA molecules from the subject using an enzyme that differentially modifies DNA based on its methylation status.
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 have modified the method of Brunner in view of Mair, to have further used a normalized first amount of DNA molecules in the MspI library using a sequencing depth of reads of the MspI library aligned to MspI sites in a region, to determine the classification of methylation, as shown by Wu, discussed above. One of ordinary skill in the art would have been motivated to combine the methods of Brunner in view of Mair with Wu, in order to provide a continuous proportion estimate of methylation, rather than a binary classification of methylation, as disclosed by Wu (pg. 2, col. 1, para. 2; pg. 3, col. 1, para. 3-4), and further to account for sequencing depth bias differences in regions across the genome, as disclosed by Wu (pg. 5, col. 1, para. 2).This modification would have had a reasonable expectation of success given Wu applies the MspI tag counts of the method of Brunner to determine the methylation classification, and thus the method of Wu is applicable to the method of Brunner.
Regarding claim 17, Brunner further discloses the classification for the methylation classifies CpG sites as either mostly methylated at >80% (i.e. methylation density of at least 70%) or mostly unmethylated at less than 20% methylation (i.e. methylation density below 30% or less) (pg. 1053, col. 1, para. 1, e.g. methyl-seq distinguishes between >80% methylated sites and <20% methylated sites, which corresponds to hyper- and hypo-methylated as claimed).
Regarding claim 22¸ Brunner further discloses determining a classification for CpG sites in a region comprising 18 CpG sites each within 35-75 (base pairs of each other i.e. a different CpG site within 600 nucleotides downstream of the CpG site) (pg. 1053, col. 2, para. 4).
Regarding claim 159, Brunner further discloses sequencing the plurality of DNA molecules using an Illumina Sequencing platform (pg. 1045, col. 2, para. 2).
Regarding claim 160, Brunner further discloses analyzing the DNA molecules includes PCR (pg. 1053, col. 2, para. 2).
Regarding claim 162, Brunner further discloses analyzing at least 10,000 DNA molecules (Suppl. Table 2).
Regarding claim 168, Brunner further discloses determining the genomic position of each DNA molecule includes aligning the reads to the reference genome using software, which necessarily requires a computer system (pg. 1053, col. 2, para. 3). Furthermore, broadly providing an automatic or mechanical means to replace a manual activity which accomplished the same result is not sufficient to distinguish over the prior art. See MPEP 2144.04 III.
Regarding claim 170¸ Brunner further discloses the subject is a human (Suppl. Table 1; pg. 1050, col.2 , para. 2, c) and the reference genome is a human reference genome (pg. 1053, col. 2, para. 3, e.g. UCSC hg18).
Regarding claim 171, Brunner further discloses outputting the methylation classification on a browser of a computer (Figure 3).
Therefore, the invention is prima facie obvious.
Claim 161 is rejected under 35 U.S.C. 103 as being unpatentable over Brunner in view of Mair and Wu, as applied to claim 160 above, and further in view of Valencia (2014). This rejection is previously cited.
Cited reference: Valencia et al., Analytical Performance Assessment of Two Clinical Exome Pipelines: The CCHMC Experience, 2014, Journal of Molecular Diagnostics¸ pg. 699-789 (previously recited).
Regarding claim 161, Brunner in view of Mair and Wu disclose the limitations of claim 160, as applied above.
Further regarding claim 161, Brunner in view of Mair and Wu, as applied to claim 160 above, do not disclose the PCR targets sequences in a repeat region.
However, Valencia discloses a method for characterizing FMR1, a repeat region (pg. 708, col. 1, para. 2, Introduction), which comprises PCR and targeted sequencing around the repeat region using primers flanking the repeat region (pg. 708, col. 1, para. 2, Methods and Results, e.g. methylation sequencing using primer pairs upstream and downstream of CGG repeat region). Valencia further discloses characterizing the repeat region are important to testing paradigms for FMR1 expansion risk assessments (pg. 708, col. 1, para. 2, Introduction).
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 have modified the method of Brunner in view of Mair and Wu, to have used PCR that targets sequences in a repeat region, as shown by Valencia (pg. 708, col. 1, para. 2). One of ordinary skill in the art would have been motivated to combine the methods of Brunner in view of Mair and Wu with the method of Valencia in order to aid testing paradigms for FMR1 expansion risk assessment, as shown by Mair (pg. 708, col. 1, para. 2, Introduction). This modification would have had a reasonable expectation of success given Valencia discloses using Methyl-Seq (pg. 708, col. 1, para. 2, Results), as performed in Brunner.
Therefore, the invention is prima facie obvious.
Response to Arguments
Applicant's arguments filed 02 Sept. 2025 regarding 35 U.S.C. 103 have been fully considered but they are not persuasive as they pertain to the claims rejected above under 35 U.S.C. 103.
Applicant remarks that an average is a ratio of a number of sites, and not a ratio to another amount of cell-free DNA molecules, and thus the cited references do not teach “the first amount is normalized using a ratio of the first amount to one or more other amounts of the plurality of cell-free DNA molecules” and the rejections of claims 1 and 20-21 are flawed (Applicant’s remarks at pg. 8, para. 4 to pg. 9, para. 1-2 and 5-6).
This argument is not persuasive because the “average” is no longer relied upon to teach the claimed normalization step, and the arguments do not take into account the newly cited portions of Wu discussed in the above rejection. As discussed in the above rejection, Wu discloses normalizing the first amount using genome-wide CGG tags aligned to MspI sites from the MspI library and defining in a ratio of tags in the MspI library to the HpaII library to specify the sequencing depth bias dj, and therefore, the proportional estimate becomes max(1-dj ∑ yij/ ∑ xi, 0) (pg. 5, col. 1, para. 3, section 2.5). Given dj is the ratio of CGG tags in the form MspI/HpaII, the above formula is equivalent to ∑ yij / (HpaII CGG tags) / (∑ xi/ MspI CGG tags). Thus, the first amount (xi) is normalized to other amounts of the cell-free DNA molecules (e.g. the genome-wide CGG tags aligned to MspI sites in the MspI library). Specifically regarding claim 21, Wu does normalize the first amount using a sequencing depth of CGG tag counts in a region, which shows using an average depth of the cell-free DNA molecules in a region including the CpG site, as applied as the above rejection.
Allowable Subject Matter
Claims 2-16 and 19 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
The following is a statement of reasons for the indication of allowable subject matter:
Claims 1-2, 4, 17, 19-22, 159-162, 168, and 170-171 are patent eligible for the reasons discussed in the Office action mailed 02 May 2025.
Claims 3 and 5-16 are allowed for the reasons discussed in the Office action mailed 02 May 2025.
Claims 2, 4, and 19 are free of the prior art for the following reasons:
Claim 2, and dependent claim 4, recite “determining a second amount of cell-free DNA molecules ending at a second position within the window around the CpG site, the second position being different than the first position, wherein the ratio is of the first amount and the second amount”. Brunner in view of Wu make obvious using a first amount of cell-free DNA molecules ending at a first position within a window around the CpG site and then normalizing the first amount using a read depth of reads covering a CpG site to normalize the first amount by a sequencing depth bias (e.g. the CGG Tags aligned to MspI sites in Wu at pg. 5, col. 1, para. 2). However, Wu discloses the sequencing depth bias uses CGG tags aligned to MspI sites across the genome or in a specific region. Wu does not disclose the ratio is the first amount normalized to CGG tags specifically aligned to one particular position (i.e. the second position in the window), as required by claim 2.
Claim 19 recites “the normalization uses the number of the plurality of cell-free DNA molecules ending within a region including the CpG site”. Wu does not disclose the normalization uses the total number of cell-free DNA molecules in a particular region (i.e. the number…ending within a region), and instead only uses an amount of cell-free DNA molecules including the CGG tag within a region.
Conclusion
No claims are allowed.
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/KAITLYN L MINCHELLA/Primary Examiner, Art Unit 1685