DETAILED ACTION
Election/Restrictions
Applicant’s election without traverse of Group I, claims 1 and 3-18, drawn to a method for profiling genetic and epigenetic characteristics of a cell-free DNA (cfDNA) sample from a subject. in the reply filed on 12/16/2025 is acknowledged.
Claims 2, 20, 23-24 and 33 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected Groups II-V, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 12/16/2025.
Claims Status
Claims 1-18, 20, 23-24 and 33 are pending.
Claims 2, 20, 23-24 and 33 are withdrawn.
Claims 1 and 3-18 are currently under examination
Priority
This application is a 371 of PCT/IL2021/051382 filed November 18, 2021, which claims priority to Israeli Patent Application No. 278856, filed on November 19, 2020.The priority date of the instant claim set is determined to be November 19, 2020.
Specification
The abstract of the disclosure does not commence on a separate sheet in accordance with 37 CFR 1.52(b)(4) and 1.72(b). A new abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claim 18 is rejected under 35 U.S.C. 101 because the claimed invention is directed towards the abstract idea/Mental step related to data comparison and routine and conventional steps digesting, preparing a library, sequencing, and determining sequencing data methylation, without significantly more. The claim(s) recite(s) abstract ideas and routine and conventional methods. This judicial exception is not integrated into a practical application because no additional elements integrate the judicial exceptions into a practical application. The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception because no additional elements are considered significantly more than the judicial exceptions.
Claim analysis
The instant claim 18 is directed towards A method according to claim 1, further comprising: comparing the genetic and epigenetic profile of the cfDNA sample to one or more reference genetic and epigenetic profile selected from a cancer profile and a non-cancer profile, to detect cancer- associated genetic and epigenetic changes in the cfDNA sample.
The comparing the genetic and epigenetic profile of the cfDNA sample to one or more reference genetic and epigenetic profile selected from a cancer profile and a non-cancer profile is an abstract idea.
The method of claim 1 steps a-d, which claim 18 depends on recites the following steps (a) … digestion with at least one methylation-sensitive restriction endonuclease … (b) preparing a sequencing library … (c) sequencing the sequencing library …; and (d) determining from the sequencing data a methylation value … which are considered to be an active step requiring the analysis of a sample. The active step is routine and conventional as demonstrated by the 35 USC § 103 rejections stated below.
According to the 2019 Patent Eligibility Guidance an initial two step analysis is required for determining statutory eligibility.
Step 1. Is the claim directed to a process, machine, manufacture, or composition of matter? In the instant case, the Step 1 requirement is satisfied as the claims are directed towards a process.
Step 2A Prong one. Does the claim recite a law of nature, a natural phenomenon or an abstract idea? Yes, abstract idea.
With regard to claim 18, the claim recites “A method according to claim 1, further comprising: comparing the genetic and epigenetic profile of the cfDNA sample to one or more reference genetic and epigenetic profile selected from a cancer profile and a non-cancer profile, to detect cancer- associated genetic and epigenetic changes in the cfDNA sample.” The comparing the genetic and epigenetic profile of the cfDNA sample to one or more reference genetic and epigenetic profile selected from a cancer profile and a non-cancer profile is an abstract idea. The method of claim 1 steps a-d, which claim 18 depends on, are considered routine and conventional.
Step 2A prong two. Does the claim recite additional elements that integrate the judicial exception into a practical application? No, there are no additional steps that integrate the claims into a practical application.
Step 2B. Does the claim recite additional elements that are significantly more than the judicial exceptions? No, there are no additional elements that are significantly more than the judicial exceptions.
Regarding claim 18, the claim requires the routine and conventional active steps as recited in claim 1 steps a-d, which claim 18 depends on, similar to that of Makarov et al. (“Makarov”; Patent App. Pub. US 20180030527 A1, Feb. 1, 2018,) as demonstrated by the 35 USC § 103 rejections stated below.
Makarov discloses “The present invention regards a variety of methods and compositions for obtaining epigenetic information, such as DNA methylation patterns, through the preparation, amplification and analysis of Methylome libraries. In particular, the method employs preparation of a DNA molecule by digesting the DNA molecule with at least one methylation-sensitive restriction enzyme; incorporating a nucleic acid molecule into at least some of the digested DNA molecules by either (1) incorporating at least one primer from a plurality of primers that have a 5′ constant sequence and a 3′ variable sequence, wherein the primers are substantially non-self-complementary and substantially non-complementary to other primers in the plurality; or (2) incorporating an oligonucleotide having an inverted repeat and a loop under conditions wherein the oligonucleotide becomes blunt-end ligated to one strand of the digested DNA molecule, followed by polymerization from a 3′ hydroxyl group present in a nick in the oligonucleotide-linked molecule; and amplifying one or more of the DNA molecules” (Abstract).Thus, the claim does not provide additional steps which are significantly more.
Claim Rejections - 35 USC § 103
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.
Claim 1, 3-9 and 12-18 are rejected under 35 U.S.C. 103 as being unpatentable over Makarov et al. (“Makarov”; Patent App. Pub. US 20180030527 A1, Feb. 1, 2018).
Interpretations: Regarding claim 1, “optionally at least one additional genetic or epigenetic characteristic of the cell-free DNA sample selected from DNA mutation, copy number variation and nucleosome positioning, wherein an amount of cell-free DNA comprising 3000 haploid equivalents is sufficient for the method, wherein the cell-free DNA sample is not subjected to amplification prior to library preparation, and wherein determining the methylation value and the at least one additional genetic or epigenetic characteristic of the cell-free DNA sample is carried out based on the same sequencing data” Is interpreted as optional.
Makarov discloses “The present invention regards a variety of methods and compositions for obtaining epigenetic information, such as DNA methylation patterns, through the preparation, amplification and analysis of Methylome libraries. In particular, the method employs preparation of a DNA molecule by digesting the DNA molecule with at least one methylation-sensitive restriction enzyme; incorporating a nucleic acid molecule into at least some of the digested DNA molecules by either (1) incorporating at least one primer from a plurality of primers that have a 5′ constant sequence and a 3′ variable sequence, wherein the primers are substantially non-self-complementary and substantially non-complementary to other primers in the plurality; or (2) incorporating an oligonucleotide having an inverted repeat and a loop under conditions wherein the oligonucleotide becomes blunt-end ligated to one strand of the digested DNA molecule, followed by polymerization from a 3′ hydroxyl group present in a nick in the oligonucleotide-linked molecule; and amplifying one or more of the DNA molecules” (Abstract).
Regarding claim 1 steps (a-b), Makarov teaches a method comprising “The DNA molecules of the present invention for which the methods are employed such that a differential characteristic, for example, methylation, is determined may be of any kind… in particular the DNA molecules for the methods herein may be referred to as substantially fragmented and/or cell-free DNA” (Para. 53). Makarov teaches a method comprising “preparing plurality of fragments using restriction enzymes that differentiate between methylated and non-methylated regions, incorporating a known sequence at the end of said DNA fragments,” (Para. 51). Makarov teaches a method comprising “fragmented nucleic acid” (Para. 150). Thus, Makarov suggests a method comprising step (a) subjecting the cell-free DNA sample to digestion with at least one methylation-sensitive restriction endonuclease, to obtain restriction endonuclease-treated DNA in which methylated sites are intact and unmethylated sites are cut and (b) preparing a sequencing library from the restriction endonuclease-treated DNA while preserving the sequence information at the ends of the DNA molecules, wherein preparing the sequencing library comprises ligating sequencing adapters to DNA molecules in the restriction endonuclease-treated DNA, wherein each adapter is capable of ligation to both the digested and undigested DNA molecules.
Regarding claim 1 step (c), Makarov teaches a method comprising “the library that is generated, … is analyzed such that the one or more characteristics of the original DNA molecule may be identified. For example, the analysis may be of any kind sufficient to gain information, although in specific embodiments it comprises at least sequencing…” (Para.74). Makarov teaches a method comprising “easy to automate and apply in a high-throughput setting for disease diagnostics” (Para. 100). Thus, Makarov suggests a method comprising step (c) sequencing the sequencing library by a high-throughput sequencing method to provide sequencing data.
Regarding claim 1 step (d), Makarov teaches a method comprising “The present invention relates to novel methods and compositions for determining and analyzing methylation of a DNA molecule by preparing plurality of fragments” (Para. 51). Thus, Makarov suggests a method comprising step (d) determining from the sequencing data a methylation value for at least one restriction locus.
Regarding claim 1, Makarov teaches a method comprising “The reaction mixture preferably comprises about 0.5 to about 100 ng of thermally or mechanically fragmented DNA” (Para. 254). 3000 haploid equivalents is interpreted as 10 ng of DNA. 10 ng of DNA reads on about 0.5 to about 100 ng of DNA. Thus, Makarov suggests a method comprising
Regarding claims 3 and 5-6, Makarov teaches a method wherein “The reaction mixture preferably comprises about 0.5 to about 100 ng of thermally or mechanically fragmented DNA” (Para. 254). 6000 haploid equivalents is interpreted as 20 ng of DNA. “about 0.5 to about 100 ng of DNA” reads on 20 ng of DNA and 10-100 ng. Furthermore, the MPEP states "Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." (MPEP 2144.05). Thus, Makarov suggests a method wherein an amount of cell-free DNA comprising 6,000 haploid equivalents is sufficient for the method; wherein the amount of cell-free DNA is between 10-200 ng. and wherein the amount of cell-free DNA is between 20-100 ng.
Regarding claim 4, Makarov teaches a method wherein “Clinical samples representing … DNA from blood cells, serum, plasma, or other body fluids… can be used as a source for methylation analysis.” (Para. 266). Makarov further teaches a method wherein “Blood collected from healthy donors or from prostate cancer patients was aliquoted into 6 ml Vacutainer SST Serum Separation tubes” (Para. 582). “Blood collected … was aliquoted into 6 ml …tubes” reads on amount of blood obtained within an optimizable range of the claimed 9-10 ml. Thus, Makarov suggests a method wherein the cell-free DNA is plasma cell- free DNA, and wherein the amount of the cell-free DNA is an amount obtained from 9-10 ml of blood.
Regarding claim 7, Makarov teaches a method wherein “genomic DNA is digested with a methylation-sensitive restriction endonuclease” (Para. 285). Makarov teaches a method wherein “Generally, a library is prepared in at least 4 steps: first, randomly fragmenting the DNA into pieces… second, repairing the 3′ ends of the fragmented pieces and generating blunt, double stranded ends; third, attaching universal adaptor sequences to the 5′ ends of the fragmented pieces; and fourth, filling in of the resulting 5′ adaptor extensions. In an alternative embodiment, the first step comprises obtaining DNA molecules defined as fragments of larger molecules, such as may be obtained from a tissue (for example, blood, urine, feces, and so forth), a fixed sample, and the like, and may comprise substantially fragmented DNA” (Para. 286). Makarov also teaches a method wherein “A skilled artisan recognizes that random fragmentation can be achieved by at least three exemplary means: mechanical fragmentation, chemical fragmentation, and/or enzymatic fragmentation.” (Para. 287). “methylation-sensitive restriction endonuclease” and “enzymatic fragmentation” reads on methylation- sensitive restriction endonuclease produces non-blunt ends or blunt ends. Thus, Makarov suggests a method wherein the at least one methylation- sensitive restriction endonuclease produces non-blunt ends, and the method further comprises subjecting the restriction endonuclease-treated DNA to end repair prior to the ligation of sequencing adapters, to obtain DNA molecules with blunt ends.
Regarding claim 8, Makarov teaches a method comprising “the library that is generated, … is analyzed such that the one or more characteristics of the original DNA molecule may be identified. For example, the analysis may be of any kind sufficient to gain information, although in specific embodiments it comprises at least sequencing…” (Para.74). Makarov teaches a method comprising “easy to automate and apply in a high-throughput setting for disease diagnostics” (Para. 100). Thus, Makarov suggests a method wherein the high-throughput sequencing is whole genome high-throughput sequencing and wherein the high-throughput sequencing is target-specific high-throughput sequencing.
Regarding claim 12, Makarov teaches a method wherein “methylation status of one or more specific sequences is obtained by analyzing at least part of one or more DNA molecules… a nucleic acid molecule, such as genomic DNA, is digested with a restriction enzyme that cleaves DNA based on methylated CpG, such as McrBC, or it is digested with one or more, such as a mixture of several restriction enzymes unable to cleave sites having a methylated CpG” (Para. 54). Makarov teaches a method wherein “part or all of a particular group of 11 methylation-sensitive restriction endonucleases, specifically, Aci I, Bst UI, Hha I, HinP1, Hpa II, Hpy 99I, Ava I, Bce AI, Bsa HI, Bsi E1, and Hga I, that have 4-5 base pair recognition sites with at least one CpG dinucleotide, and that have the characteristic of being unable to digest recognition sites having a methylated CpG, may be used to selectively cleave unmethylated CpG regions within DNA… The spatial distribution of recognition sites for these particular nucleases in the human genome closely follows the distribution of the CpG dinucleotides, with their density being very high in the CpG-rich regions (CpG islands).” (Para. 55). Thus, Makarov suggests a method wherein the at least one restriction locus is a plurality of restriction loci.
Regarding claim 13, Makarov teaches a method wherein “cleavage with multiple methylation-sensitive restriction enzymes to occur simultaneously” (Para. 202). Thus, Makarov teaches a method wherein the at least one methylation- sensitive restriction endonuclease is a plurality of methylation-sensitive restriction endonucleases, and wherein the digestion with the plurality of methylation-sensitive restriction endonucleases is a simultaneous digestion.
Regarding claim 14-16, Makarov teaches a method wherein “In some embodiments, part or all of a particular group of 11 methylation-sensitive restriction endonucleases, specifically, AciI, … HinP1” (Para. 55). Thus, Makarov suggests a method wherein the plurality of methylation-sensitive restriction endonucleases comprises HinPlI; wherein the plurality of methylation-sensitive restriction endonucleases comprises AciI, and wherein the digestion is carried out using HinPlI and AciI.
Regarding claim 17, Makarov teaches a method wherein Genomic DNA that has not been digested by the methylation-sensitive enzyme mix may serve as positive control during library preparation and analysis. Makarov teaches a method wherein “The second aliquot is incubated in parallel but without restriction enzymes (“uncut” control).” (Para. 363). Thus, Makarov suggests a method wherein the step of subjecting the cell-free DNA sample to digestion with at least one methylation-sensitive restriction endonuclease further comprises determining digestion efficacy, and proceeding to preparing a sequencing library if the digestion efficacy is above a predefined threshold.
Regarding claim 18, Makarov teaches a method wherein “Methylome libraries synthesized from the serum DNA of cancer patients as compared to healthy donor controls. As expected, the level of methylation in serum DNA from cancer patients was much lower than in tumor tissue or cancer cell lines, since cancer DNA in circulation represents only a relatively small fraction of the total cell-free DNA. The method disclosed here is very sensitive to reliably detect methylation in body fluids and can be applied as a diagnostic tool for early detection, prognosis, or monitoring of the progression of cancer disease.” (Para.). Thus, Makarov suggests a method further comprising: comparing the genetic and epigenetic profile of the cfDNA sample to one or more reference genetic and epigenetic profile selected from a cancer profile and a non-cancer profile, to detect cancer- associated genetic and epigenetic changes in the cfDNA sample.
Therefore, the invention as recited in claims 1, 3-9 and 12-18 is prima facie obvious over the prior art Makarov et al. One of ordinary skill in the art would have had a reasonable expectation of success given the lack of novelty. It would have been obvious to provide a method for profiling genetic and epigenetic characteristics of a cell-free DNA (cfDNA) sample from a subject according to the limitations of the instant application claims 1, 3-9 and 12-18 based on Makarov et al (Patent App. Pub. US 20180030527 A1, Feb. 1, 2018).
Claims 10-11 are rejected under 35 U.S.C. 103 as being unpatentable over Makarov et al. (“Makarov”; Patent App. Pub. US 20180030527 A1, Feb. 1, 2018) as applied to claim 1 above, and further in view of Shih et al. (“Shih”; Patent App. Pub. US 20200283840 A1, Sept. 10, 2020)
The teachings of Makarov are documented above in the rejection of claims 1, 3-9 and 12-18 under 35 U.S.C. 103. Claim 11 depends on claim 10, which depends on claim 1.
Regarding claims 10-11, Makarov further teaches “Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention.” (Para. 756). Thus, the ordinary skilled artisan would be motivated to modify the methods of Makarov to incorporate further details of determining a methylation value for at least one restriction locus.
Makarov does not explicitly teach the limitations of claims 10 and 11.
Shih discloses “The invention relates to methods of utilizing epigenetic information to separate one type of DNA from a mixture of multiple DNAs. The applications of the methods of the invention include, for example, the detection of chromosomal abnormality (e.g., aneuploidy, cancer cells), identification of genome abnormality, direct detection of DNA with abnormal copy number and development of indicators for the above-mentioned detection and identification.” (Abstract)
Regarding claim 10-11, Shih teaches a method wherein “The pooled DNA that contains methylated and unmethylated test DNA was first treated with PvuI, which digests unmethylated DNA. The enzyme-treated DNA was then used for generating an NGS library using standard protocols. After PCR amplification, the DNA library was then sequenced using NGS. Reads were separated into methylated and unmethylated by the attached barcodes and were mapped to the reference using Bowtie 2. Reads of undigested DNA (568 bp, full length) would contain PvuI sites and reads of digested DNA (353 bp and 215 bp) would map to the same location without PvuI sites in the alignments. For the methylated DNA library, we yielded 6,208 fragment from NGS, in which 6135 were full length and 73 were digested fragments… our results confirm that Method 4 can distinguish methylated and unmethylated DNA from a mix of DNAs.” (Para. 162). “PvuI” reads on a methylation sensitive enzyme. “568 bp, full length” reads on at least 50bps. “6135 were full length” reads on methylation value. A sequence reads may be aligned against a reference genome and restriction loci, namely, restriction sites within the genome, are selected and analyzed. Thus, Makarov and Shih suggest a method wherein determining a methylation value for at least one restriction locus comprises:(i) selecting at least one restriction locus and determining the number of sequence reads covering a predefined genomic region of at least 50 bps in length that contains said restriction locus; and (ii) calculating a methylation value for the at least one restriction locus based on the read count determined in step (i) and a reference read count and wherein step (i) comprises determining the number of sequence reads covering a predefined genomic region of at least 100 bps in length that contains said restriction locus.
Makarov and Shih are both considered to be analogous to the claimed invention because they are in the same field of methods of preparing nucleic acids for sequencing. Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the methods of profiling genetic and epigenetic characteristics of a cell-free DNA (cfDNA) sample from a subject as taught by Makarov to incorporate the method of determining the number of sequence reads covering a predefined genomic region of at least 50 bps in length that contains at least one restriction locus and calculating a methylation value for the at least one restriction locus based on the read count as taught by Shih and provide a method for determining a methylation value for at least one restriction locus. These claim elements were known in the art and one of skill in the art could have combined these elements by known methods with no change in their respective functions, and the combination would have yielded the predictable outcome according to the limitations of claims 10 and 11. Doing so would allow for the ability to distinguish between methylated and unmethylated DNA using NGS and to screen for genomic variations and abnormalities to detect disease.
Conclusion
No claims are in condition for allowance.
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/KENDRA R VANN-OJUEKAIYE/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682