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 .
Election/Restrictions
Applicant’s election without traverse of Group II, claims 5-13 and the specie tumor DNA in the reply filed on 06/16/2026 is acknowledged.
Applicant is reminded that upon the cancelation of claims to a non-elected invention, the inventorship must be corrected in compliance with 37 CFR 1.48(a) if one or more of the currently named inventors is no longer an inventor of at least one claim remaining in the application. A request to correct inventorship under 37 CFR 1.48(a) must be accompanied by an application data sheet in accordance with 37 CFR 1.76 that identifies each inventor by his or her legal name and by the processing fee required under 37 CFR 1.17(i).
Claims Status
Claims 5-13,24-25,36-39 and 50-51 are pending and currently under examination.
Claims 30-35 are canceled in the claim set filed 06/16/2026.
Priority
This U.S. Patent application 18/523,606, filed on November 29, 2023 is a nonprovisional of and claims the benefit of U.S. Provisional Patent Application No. 63/428,694, entitled "FRAGMENTOMICS IN URINE AND PLASMA," filed on November 29, 2022. Accordingly, the priority date of the instant claim set is determined to be November 29, 2022.
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.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 5-13, 24-25, 36-39 and 50-51 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 applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 5 is indefinite over the limitation “clinically relevant DNA molecules”. It is unclear as to what are considered “clinically relevant DNA molecules”. The Specification teaches “clinically-relevant DNA, e.g., type(s) of transrenal and non-transrenal urinary cfDNA” (Para. 6) The recited “clinically relevant DNA molecules” encompasses any DNA molecule. Claims 6-13,24-25,36-39 and 50-51 depend on claim 5.
Claim 24 is indefinite over the limitation “transrenal DNA molecules”. It is unclear as to what are considered transrenal DNA molecules. The Specification teaches “transrenal DNA that exists before passing through a kidney, as opposed to non-transrenal DNA (such as from the kidney or bladder)” (Para. 90). The recited “transrenal DNA molecules” encompasses any DNA molecule not from the kidney or bladder.
Claim 50 is indefinite over the limitation “DNA stabilization agent”. It is unclear as to what is considered DNA stabilization agent. The specification teaches “the urine sample is processed using a DNA stabilization agent prior to obtaining the cell-free DNA molecules. Different DNA stabilization agents can be used, such as EDTA and Collipee stabilization agent. EDTA can inhibit the cleavage activity of the DNASE1 family by chelating magnesium and calcium, which are the essential ions required for DNASE1 digestion. The stabilizers can potentially stabilize the urinary DNA from degradation. The stabilizers could be but not limited to preservatives provided by Collipee company, diazolidinyl urea (DU), dimethylolurea, 2-bromo-2-nitropropane-1,3-diol, 5- hydroxymethoxymethyl-1-aza-3,7-dioxabicyclo (3.3.0) octane and 5-hydroxymethyl-1-aza-3,7- dioxabicyclo (3.3.0) octane and 5-hydroxypoly[methyleneoxy]methyl-1-aza-3.7-dioxabicyclo (3.3.0) octane, bicyclic oxazolidines (e.g. Nuosept95), DMDM hydantoin, imidazolidinyl urea (IDU), sodium hydroxymethylglycinate, hexamethylenetetramine chloroallyl chloride (Quaternium-15), biocides (such as Bioban, Preventol and Grotan), a water-soluble zinc salt, EDTA, other metal ion chelators such as N, N'-bis-(dithiocarboxy) piperazine (BDP),
diethyldithiocarbamate (DDTC), iminodisuccinic acid (IDS), polyaspartic acid, S,S- Ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycinediacetic acid (MGDA), etc.” (Para. 177). The recited “DNA stabilization agent” encompasses any agent that can stabilize DNA.
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 50 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 50 recites the limitation “wherein the urine sample is processed using a DNA stabilization agent prior to obtaining the plurality of cell-free DNA molecules from the urine sample” There is insufficient antecedent basis for this limitation in the claim. Claim 5 does to recite a limitation “processed” or “obtaining the plurality of cell-free DNA molecules from the urine sample”. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
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.
Claims 5-13, 24-25, 36-39 and 50-51 are rejected under 35 U.S.C. 103 as being unpatentable over Lo et al. (“Lo”; Patent App. Pub. US 20190341127 A1, Nov. 7, 2019).
Lo discloses “Various applications can use fragmentation patterns related of cell-free DNA, e.g., plasma DNA and serum DNA. For example, the end positions of DNA fragments can be used for various applications. The fragmentation patterns of short and long DNA molecules can be associated with different preferred DNA end positions, referred to as size-tagged preferred ends. In another example, the fragmentation patterns relating to tissue-specific open chromatin regions were analyzed. A classification of a proportional contribution of a particular tissue type can be determined in a mixture of cell-free DNA from different tissue types. Additionally, a property of a particular tissue type can be determined, e.g., whether a sequence imbalance exists in a particular region for a tissue type or whether a pathology exists for the tissue type.” (Abstract)
Regarding claim 5, Lo teaches a method wherein “Various embodiments are directed to applications (e.g., diagnostic applications) of the analysis of the fragmentation patterns related of cell-free DNA, …For example, the end positions of DNA fragments (molecules) can be used for various applications. Some embodiments can determine a classification of a proportional contribution of a particular tissue type in a mixture of cell-free DNA from different tissue types. For example, specific percentages, range of percentages, or whether the proportional contribution is above a specified percentage can be determined as a classification. In other embodiments, a property of a particular tissue type can be determined, e.g., whether a sequence imbalance exists in a particular region for a tissue type or whether a pathology exists for the tissue type” (Para. 4). Lo teaches a method comprising “analyzing a biological sample to determine a classification of a proportional contribution of the first tissue type in a mixture according to embodiments of the present disclosure. The biological sample includes a mixture of cell-free DNA molecules from a plurality of tissue types that includes a first tissue type.”(Para. 236). Lo teaches a method comprising “biological sample can be a bodily fluid, such as … urine” (Para. 62). Lo teaches a method comprising “fragmentation patterns relating to tissue-specific open chromatin regions were analyzed. A set of genomic positions relative to a center of a tissue-specific open chromatin region for a first tissue type can be used. In particular, knowledge of whether a DNA fragment has an upstream end or a downstream end at this set of genomic positions (e.g., relative to the center of an open chromatin region of a particular tissue type) can be used in a quantitative analysis” (Para. 8). Lo teaches a method comprising “we use short size and fragment end characteristics to enrich for the clinically relevant DNA molecules… Such enriched samples for clinically-relevant DNA (e.g., fetal, tumor, and transplant) can be used to detect sequence imbalance with higher accuracy” (Para 228). Thus, Lo suggests a method comprising analyzing a plurality of cell-free DNA molecules from the urine sample, wherein analyzing the plurality of cell-free DNA molecules includes: identifying, from the plurality of cell-free DNA molecules, a set of cell-free DNA molecules that are from open chromatin regions of one or more tissues associated with the clinically-relevant DNA molecules; and creating an enriched sample using the set of cell-free DNA molecules that are from the open chromatin regions of the one or more tissues, wherein the enriched sample has a higher concentration of clinically-relevant DNA compared to the urine sample.
Regarding claim 6, Lo teaches a method wherein “the separation value can be used to determine a proportional contribution of a first tissue type in a test mixture, e.g., by comparing to a similar measurement in a calibration sample for which the proportional contribution is known. In other embodiments, the separation value can be used as an indicator of a pathology in the first tissue type, e.g., when there is a statistically significant deviation from a reference value. Examples of such a pathology include an abnormally high fractional concentration of cell-free DNA from the first tissue type, is a rejection of a transplanted organ of the first tissue type, or cancer.” (Para. 9). Thus, Lo suggests a method further comprising determining a property associated with the clinically-relevant DNA molecules in the enriched sample, wherein the property associated with the clinically-relevant DNA molecules in the urine sample is (1) a fractional concentration of the clinically-relevant DNA molecules or (2) a level of pathology of a subject from whom the urine sample was obtained, the level of pathology associated with the clinically-relevant DNA molecules.
Regarding claim 7, Lo teaches a method comprising “we use short size and fragment end characteristics to enrich for the clinically relevant DNA molecules… Such enriched samples for clinically-relevant DNA (e.g., fetal, tumor, and transplant) can be used to detect sequence imbalance with higher accuracy” (Para 228). Lo also teaches a method wherein “short DNA fragments correspond to a range that has a minimum and/or a maximum that is less than a minimum and/or a maximum of a range for the long DNA fragments” (Para. 113). Furthermore, Lo teaches a method wherein “A threshold value may be a value above or below which a particular classification applies, e.g., a classification of a condition, such as whether a subject has a condition or a severity of the condition. A cutoff or threshold may be “a reference value” or derived from a reference value that is representative of a particular classification or discriminates between two or more classifications” (Para. 82).Thus, Lo suggests a method wherein creating the enriched sample further includes using the set of cell-free DNA molecules that are from the open chromatin regions of the one or more tissues and that have sizes that are less than a specified size threshold.
Regarding claim 8, Lo teaches a method wherein “The size of Window A and Window B can be adjusted to achieve the desired performance. The performance of difference window sizes can be obtained experimentally. The size of Window A can be set, for example but not limited to 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 15 bp, 20 bp, 25 bp and 30 bp. The size of Window B would be larger than that of Window A and can be set, for example but not limited to 20 bp, 25 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 100 bp, 120 bp, 140 bp, 160 bp, 180 bp and 200 bp” (Para. 141). Thus, Lo suggests a method wherein the specified size threshold is 40 base pairs, 50 base pairs, 60 base pairs, 70 base pairs, 80 base pairs, 90 base pairs, 100 base pairs, 110 base pairs, 120 base pairs, 130 base pairs, 140 base pairs, 150 base pairs, or 160 base pairs.
Regarding claim 9, Lo teaches a method wherein “the clinical potential of fragmentation patterns, especially in tracing the tissue-of-origin of cfDNA molecules. We first profiled the coverage and cfDNA fragment end signatures around known well-positioned nucleosome arrays and open chromatin regions. During the analysis, we separated the plasma DNA fragment ends into two groups where the orientation information was considered, namely ends on an upstream or downstream side ” (Para. 272). Thus, Lo suggests a method wherein creating the enriched sample further includes using the set of cell-free DNA molecules that are from the open chromatin regions of the one or more tissues and that have one or more ending sequences that correspond to a sequence end signature.
Regarding claim 10, Lo teaches a method wherein “hybridization capture of loci with high density of preferred ends could be performed on the cell-free DNA samples to enrich the sample with cell-free DNA molecules with such preferred ends following but not limited to detection by sequencing, microarray, or the PCR. Yet, alternatively, amplification based approaches could be used to specifically amplify and enrich for the cell-free DNA molecules with the preferred ends, e.g. inverse PCR, rolling circle amplification. The amplification products could be identified by sequencing, microarray, fluorescent probes, gel electrophoresis and other standard approaches known to those skilled in the art.” (Para.110, Para. 112, Para. 355). Thus, Lo suggests a method wherein identifying the set of cell- free DNA molecules or creating the enriched sample includes: subjecting the plurality of cell-free DNA molecules to probe molecules that have sequences from the open chromatin regions, thereby obtaining the set of cell-free DNA molecules.
Regarding claim 11, Lo teaches a method wherein “the positive amplification of a plasma DNA molecule with a preferred end detected via a fluorescent probe that binds to the middle bases of the amplicon. For instance, an end could be identified by the positive hybridization of a fluorescent probe that binds to some bases on a middle section of a plasma DNA molecule, where the fragment size known. In this way, one could determine the genomic identity or genomic coordinate of an end by working out how many bases are external to the fluorescent probe with known sequence and genomic identity.” (Para. 112). Thus, Lo suggests a method wherein creating the enriched sample includes: amplifying the set of cell-free DNA molecules using the one or more probe molecules.
Regarding claim 12, Lo teaches a method wherein “hybridization capture of loci with high density of preferred ends could be performed on the cell-free DNA samples to enrich the sample with cell-free DNA molecules with such preferred ends … and other standard approaches known to those skilled in the art.” (Para. 110). “discarding other cell-free DNA molecules of the plurality of cell-free DNA molecules” reads on other standard approaches known to those skilled in the art. Lo also teaches a method wherein “Since the tissue-specific open chromatin regions only accounted for a very small proportion of the human genome, through designing hybridization probes to capture these regions” (Para. 355). Furthermore. Lo teaches a method wherein “a sample holder can be a flow cell that includes probes” (Para. 395). Thus, Lo suggests a method wherein creating the set of cell-free DNA molecules includes: capturing the set of cell-free DNA molecules using the one or more probe molecules; and discarding other cell-free DNA molecules of the plurality of cell-free DNA molecules.
Regarding claim 13, Lo teaches a method wherein “a flow cell that includes probes” (Para.395). Thus, Lo suggests a method wherein one or more probe molecules are attached to a surface.
Regarding claim 24, Lo 2022 teaches a method wherein “contribution of a particular tissue type in a mixture of cell-free DNA from different tissue types.” (Para.4). Lo also teaches a method wherein “More recently, an approach has been applied to the detection of cfDNA derived from the brain (78), cells of the erythroid lineage (75), the heart (109), and the liver” (Para. 269). “cfDNA derived from the brain (78), cells of the erythroid lineage (75), the heart (109), and the liver” reads on transrenal DNA molecules. Thus, Lo suggests a method wherein the clinically-relevant DNA molecules are transrenal DNA molecules.
Regarding claim 25, Lo teaches a method wherein “We then analyzed … DNA fragmentation patterns in various tissue-specific open chromatin regions and further quantified the fragmentation patterns in various clinical scenarios to investigate the feasibility in inferring the tissue-of-origin of cfDNA, including predicting the tumor location in cancer patients” (Para. 272). Lo teaches a method wherein “The cell-free plasma DNA preferred ends associated with short DNA fragments can also reveal the tumor DNA fraction or fractional concentration” (Para.107). Thus, Lo suggests a method wherein the clinically-relevant DNA molecules include fetal DNA or tumor DNA.
Regarding claim 36, Lo teaches a method wherein “liver, lung, pancreatic, brain, colorectal, nasopharyngeal, ovarian, stomach, and blood cancers” (Para. 85). Lo also teaches a method wherein “besides T-cells and the liver, we mined tissue-specific open chromatin regions for 5 additional major human tissues (i.e., the placenta, lungs, ovary, breast and small intestines” (Para. 306). Thus, Lo suggests a method wherein the one or more tissues includes at least one of heart, lungs, colon, liver, or white blood cells.
Regarding claim 37, Lo teaches a method wherein “In some implementations, we used (but not limited to) 60 bp as the peak and 10 bp as the bin size for the quantification. Other example values for the peak offset are 40, 45, 50, 55, 65, 70, and 75 bp. Other example values for the window are 2, 3, 4, 5, 6, 7, 8, 9, 15, 20, 25, and 30 bp. One peak can be identified as a downstream peak, where more downstream ending positions are expected. Another peak can be identified as an upstream peak, where more upstream ending positions are expected. For each case, OCF values were calculated for the 7 tissue types investigated in this study using their tissue-specific open chromatin regions separately” (Para.315). OCF stands for Orientation-aware CfDNA Fragmentation. Lo also teaches a method wherein “we measured the differences of U and D end signals in 20 bp windows around the peaks… in the tissue-specific open chromatin regions as the OCF value for the corresponding tissue” (Para. 320). Thus, Lo suggests a method wherein the set of cell-free DNA molecules end at one or more positions in a window around the open chromatin regions of the one or more tissues.
Regarding claim 38, Lo teaches a method wherein “we used the publicly available DNase-seq (DNase I hypersensitive sites sequencing) data to mine the open chromatin regions” (Para. 393). Thus, Lo suggests a method wherein the open chromatin regions include Dnasel hypersensitivity sites.
Regarding claim 39, Lo teaches a method wherein “at least 10,000 or 50,000 or 100,000 or 500,000 or 1,000,000 or 5,000,000 cell-free DNA molecules” (Para. 242). Thus, Lo suggests a method wherein the set of cell-free DNA molecules include at least 5,000 cell-free DNA molecules.
Regarding claim 50, Lo teaches a method wherein “collected in EDTA-containing tubes and centrifuged” (Para. 381). Thus, Lo suggests a method wherein the urine sample is processed using a DNA stabilization agent prior to obtaining the plurality of cell-free DNA molecules from the urine sample.
Regarding claim 51, Lo teaches a method wherein “DNA molecule that is detected or determined by an analytical method, such as but not limited to massively parallel sequencing or next-generation sequencing, single molecule sequencing, double- or single-stranded DNA sequencing library preparation protocols” (Para.111) Lo also teaches a method wherein “sequence reads obtained from sequencing a sample from the subject” (Para. 86). Thus, Lo suggests a method wherein analyzing the plurality of cell-free DNA molecules includes receiving sequence reads obtained from a sequencing of the plurality of cell-free DNA molecules.
Therefore, the invention as recited in claims 5-13, 24-25,36-39 and 50-51 is prima facie obvious over the prior art Lo et al. It would have been obvious to provide a method of enriching a urine sample for clinically-relevant DNA molecules according to the limitations of the instant application claims 5-13, 24-25,36-39 and 50-51 based on Lo et al. 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. Thus, one of ordinary skill in the art would have had a reasonable expectation of success given the obviousness of the claim limitations in view of the teachings of Lo et al. (Patent App. Pub. No. US 20190341127 A1).
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Zhou et al. (2022). Next-Generation Sequencing-Based Analysis of Urine Cell-Free mtDNA Reveals Aberrant Fragmentation and Mutation Profile in Cancer Patients. Clinical chemistry, 68(4), 561–573. (Whole document- Claims 5-13, 24-25, 37-38 and 51)
Sun et al. (2019). Orientation-aware plasma cell-free DNA fragmentation analysis in open chromatin regions informs tissue of origin. Genome research, 29(3), 418–427. (Whole document- Claims 5-13, 24-25, 37-38 and 51)
Han, D. S., & Lo, Y. D. (2021). The nexus of cfDNA and nuclease biology. Trends in Genetics, 37(8), 758-770. (Whole document- Claims 5-13, 24-25, 37-38 and 51)
Murtaza et al. (“Murtaza”; Patent App. Pub. MURTAZA, Jan. 14, 2021). (Whole document- Claims 5-9)
Lo et al. (“Lo”; Patent App. Pub. WO 2021139716 A1, Sept. 15, 2021). (Whole document- Claims 5-13, 24-25, 36-39 and 50-51)
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
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