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 .
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 07/09/2024 is 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 August 24, 2026. No claims amendment are made in the response filed on 08/24/2026.
Claims 1-10 are currently pending and under examination. This is the first action on the merits.
Claim Objections
Claims 1 and 9 are objected to because of the following informalities:
In claim 1, part (d), lines 3, it should read "thereby forming a captured nucleic acid."
In claim 9, line 2, "5-methylcytosin" should read "5-methylcytosine."
Priority
The priority date of the instant claims 1-10 is 12/13/2021, filling date of the US provisional application NO. 63/288,805.
Election/Restrictions
Applicant’s election without traverse of the following species in the reply filed on August 24, 2026 is acknowledged:
Species of epigenetic sequencing workflow: A) A workflow for sequencing DNA with a 5hmC epigenetic marker as shown in FIG. 1.
Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
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 a method of "determining the epigenomic state of a single cell."
The specification defines the term "epigenome" as follows:
“Epigenetics” or epigenome” or “epigenetic markers” are terms that describe the heritable changes in gene expression patterns that are independent of primary DNA sequence changes and affect the outcome of a locus or chromosome without altering the underlying DNA sequence. As used herein, the epigenetic marker can be a genomic DNA modification marker, a histone modification marker, a DNA-transcription factor interaction marker, a DNA accessibility marker, a chromatin conformation marker, and a mRNA-nucleosome interaction marker. As described herein, exemplary epigenetic markers include, but are not limited to, 5-methylcytosin, 5-hydroxymethylcytosine, 5-formylcytosine, 5-carboxylcytosine, and 3-methylcytosin. (specification, page 7, para 1)
Accordingly, under BRI and in light of the specification, "determining the epigenomic state of a single cell" is interpreted as encompassing process that analyzes an epigenetic marker (e.g., DNA methylation, chromatin accessibility) of the single cell.
For the purpose of applying prior art, claim 1 recites binding functions, including
"a functional group capable of binding to a capture reagent";
"a functional group capable of binding to the functional group of the functionalized hydrogel"; and
"capture reagent is also capable of binding to a nucleic acid."
The term "binding" is not expressly defined in the application's disclosure.
Under BRI and in light of the specification (e.g., p. 15, step 8), "binding" is interpreted as encompassing both covalent binding (e.g., tetrazine covalently link to NZ-agarose) and non-covalent binding (e.g., Streptavidin bind with biotin on the DNA).
For the purpose of applying prior art, claim 1 recites a "capture reagent," which is a term not expressly defined in the application's disclosure.
Accordingly, under BRI and within the context of the claim, "capture reagent" is interpreted as any element capable of binding to a nucleic acid.
For the purpose of applying prior art, claim 1 recites "particles."
The application's disclosure does not provide express definition for the term "particle."
Under BRI and in light of the specification (e.g., p. 15, step 3; p. 7, lines 28-29), "particle" is interpreted to encompass both solid particles and aqueous droplets.
For the purpose of applying prior art, claim 1 recites "universal adaptor," which is a term not expressly defined in the application's disclosure.
Thus, because the application's disclosure does not define the term "universal adaptor" with any structural features that distinguishes it from adaptors known in the art, the term "universal adaptor" is interpreted under BRI and in light of the specification as encompassing any adaptor.
The commonly understood definition for "adaptor" is a short, synthesized, single-stranded or double-stranded oligonucleotide that can be attached to the ends of other DNA or RNA molecules 1.
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.
Claims 1-10 are 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.
A) Regarding claim 1, it recites "wherein said capture reagent is also capable of binding to a nucleic acid" in part (b), lines 3-4.
This limitation is indefinite because it is unclear which element "said capture reagent" refers to, whether it refers to "a functionalized capture reagent," introduced in part (b), line 1; or whether it refers to "a capture reagent," introduced in part (a), line 2.
Claims 2-10 are rejected for depending from claim 1 and not remedying the indefiniteness.
B) Claim 2 recites the hydrogel comprises a "hybridize hydrogel material."
This limitation is indefinite because it is unclear what "hybridize hydrogel material" encompasses.
The term "hybridize hydrogel material" is not a commonly understood term, and the application's disclosure does not provide a definition or clear description for this term. Although the specification includes the same phrase as the claim (p.2, line 9), it does not explain what the term means or what feature it requires.
It is unclear whether the claim and the specification intended to recite a "hybrid hydrogel material" instead of a "hybridize hydrogel material." Therefore, the metes and bounds of the term "hybridize hydrogel material" cannot be determined with reasonable certainty.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-4, 6 and 10 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Belhocine (US20190383804A1- Methods and systems for characterizing multiple analytes from individual cells or cell populations; published on 2019-12-19).
Belhocine teaches single-cell sequencing methods using “cell beads,” which are single cells encapsulated in hydrogel particles for multi-omics analysis ([0209]; [0628]; FIG. 49).
Belhocine teaches using cell beads to capture and sequence RNA and DNA from the same cell, thereby enabling the analysis of both transcriptional information (e.g., gene expression, RNA velocity) and genomic information (e.g., mutations, methylation status, chromatin accessibility) from the same cell.”). ([0663]). A skilled artisan would readily understand that both methylation status and chromatin accessibility are considered epigenomic states.
Regarding claim 1, Belhocine teaches a method of determining the epigenomic state of a single cell ([0736] “Characterization, Analysis, and Detection of DNA Methylation”; “the determination of methylated and/or hydroxymethylated DNA residues is performed with the aid of a cell bead and the cell bead processing methods for, e.g., gDNA, mRNA, and protein described herein”; [0663] “cell beads may be used to capture and process RNA and DNA from a single cell, thereby enabling the analysis of both transcriptional information (e.g., gene expression, RNA velocity) and genomic information (e.g., mutations, methylation status, chromatin accessibility) from the same cell.”) comprising the steps of:
(a) preparing a functionalized hydrogel ([0209] “the biological particle may be encased inside of or comprise a gel or matrix (e.g., polymer matrix) to form a “cell bead.” A cell bead can contain biological particles (e.g., a cell) or macromolecular constituents (e.g., RNA, DNA, proteins, etc.) of biological particles. A cell bead may include a single cell or multiple cells, or a derivative of the single cell or multiple cells. For example after lysing and washing the cells, inhibitory components from cell lysates can be washed away and the macromolecular constituents can be bound as cell beads” ;[0628] “polymer molecules or gel precursors are crosslinked to form a cell bead matrix or hydrogel. ”; see also [0634]; [0637]; FIG. 49, “shows a flowchart that depicts an example method 4900 of producing droplets containing a cell bead (e.g., a cell bead comprising multiple different components of a cell) and a gel bead comprising barcode sequences and generating sequence reads to identify and characterize at least two different types of macromolecular components (e.g., RNA and gDNA) from a cell.” ),
wherein said functionalized hydrogel is chemically modified with a functional group capable of binding to a capture reagent ([0628]lines 17-19, Polymer molecule comprise a first crosslink precursor comprising a first click chemistry moiety );
(b) preparing a functionalized capture reagent ([0628] lines 10-15, functionalized nucleic acid molecule comprises a functional group (e.g., a click chemistry moiety such as 13818 or 13820) to facilitate attachment to the cell bead matrix. ; see also [0634]),
wherein said functionalized capture reagent is chemically modified with a functional group capable of binding to the functional group of the functionalized hydrogel of (a) ([0628] lines 10-15), and
wherein said capture reagent is also capable of binding to a nucleic acid ([0637] functionalized nucleic acid molecules comprises a sequence configured to hybridize to a nucleic acid molecule; [0645] “in embodiments where cell beads comprise functional nucleic acid molecules comprising a poly-T sequence, cellular mRNA may be hybridized to the nucleic acid molecules”);
(c) encapsulating single cells into particles comprising a cell lysis buffer and the functionalized hydrogel of (a) under conditions that allow cell lysis ([0645] cell beads 4611 can be subjected conditions suitable to lyse or permeabilize biological particles (e.g., cells or nuclei) in the cell beads 4613, thereby releasing or otherwise allowing access to one or more cellular constituents (e.g., nucleic acids, such as mRNA and gDNA, proteins, etc.); [0469] lines 7-15; lysing single cell with lysis buffers; see also [0679] ; [0691]; FIG. 49);
(d) preparing a nucleic acid from the encapsulated single cells of (c) under conditions that allow binding of the capture reagent to (i) the nucleic acid, and (ii) the functional group of the functionalized hydrogel, thereby forming a captured nucleic ([0645] “ cell beads 4611 can be subjected conditions suitable to lyse or permeabilize biological particles (e.g., cells or nuclei) in the cell beads 4613, thereby releasing or otherwise allowing access to one or more cellular constituents (e.g., nucleic acids, such as mRNA and gDNA, proteins, etc.)) … where cell beads comprise functional nucleic acid molecules comprising a poly-T sequence, cellular mRNA may be hybridized to the nucleic acid molecules ”; [0634] “the first crosslink precursor 13906 and the second crosslink precursor 13907 are configured to form a crosslink 13909 thereby linking the nucleic acid molecule 13902 with the polymer or gel precursor”; [0628] lines 10-15);
(e) preparing the captured nucleic acid of (d) for sequencing, wherein said preparing comprises universal adaptor ligation and barcoding ([0641] “biological particle of the cell bead (e.g., mRNA, cDNA, gDNA, etc,) may be barcoded as described elsewhere herein.”; [0649] release captured nucleic acid in cell beads so that “nucleic acid barcode molecules can interact with the released cellular components (e.g., cellular nucleic acids) to generate barcoded nucleic acid molecules for nucleic acid sequencing as described elsewhere herein”; see also [0651]; [0652] The barcoded molecules is subjected to additional reactions to add other sequences (functional sequences for sequencing such as flow-cell adaptor sequences, sequencing primer binding sites, etc.) to the constructs; [0179] An adaptor can be coupled to a polynucleotide sequence by any approach, including ligation); and
(f) sequencing the captured nucleic acid and thereby determining the epigenomic state of a single cell ([0652]; [0663] lines 20-25 “cell beads may be used to capture and process RNA and DNA from a single cell, thereby enabling the analysis of both transcriptional information (e.g., gene expression, RNA velocity) and genomic information (e.g., mutations, methylation status, chromatin accessibility) from the same cell.”; see also [0664]).
Regarding claim 2, Belhocine teaches hydrogel comprises agarose or alginate ([0205] lines 8-17).
Regarding claim 3, Belhocine teaches hydrogel polymer comprising functional group azide (N3) ([0628]lines 17-19,” Polymer molecule comprise a first crosslink precursor comprising a first click chemistry moiety” ; [0207] line 20; [0488] “reaction of the first and second reactive moieties may comprise a chemical ligation reaction such as a copper-catalyzed 5′ azide to 3′ alkyne “click” chemistry reaction ”).
Regarding claim 4, Belhocine teaches capture reagent comprising functional group alkyne (([0628] lines 10-15, functionalized nucleic acid molecule comprises a functional group (e.g., a click chemistry moiety such as 13818 or 13820) to facilitate attachment to the cell bead matrix ; [0488] “reaction of the first and second reactive moieties may comprise a chemical ligation reaction such as a copper-catalyzed 5′ azide to 3′ alkyne “click” chemistry reaction ”).
Regarding claim 6, Belhocine teaches the nucleic acid is genomic DNA ([0637] functionalized nucleic acid molecules may comprise a sequence configured to hybridize to a nucleic acid molecule; [0687] “generating sequence reads to identify and characterize at least two different types of macromolecular components (e.g., RNA and gDNA) from a cell.”; [0689] random primers, primers specific for given DNA loci,).
Regarding claim 10, Belhocine teaches wherein the conditions of (d) that allow binding of the capture reagent to (i) the nucleic acid, and (ii) the functional group of the functionalized hydrogel comprises cross-linking the capture reagent to the hydrogel ([0634] “the first crosslink precursor 13906 and the second crosslink precursor 13907 are configured to form a crosslink 13909 thereby linking the nucleic acid molecule 13902 with the polymer or gel precursor”).
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.
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 5 and 7-9 are rejected under 35 U.S.C. 103 as being unpatentable over Belhocine (US20190383804A1- Methods and systems for characterizing multiple analytes from individual cells or cell populations; published on 2019-12-19), in view of Qiong (Qiong et al., Sulfinate Based Selective Labeling of 5 Hydroxymethylcytosine: Application to Biotin Pull Down Assay. Bioconjugate Chem. 21 February 2018; 29 (2): 245–249. doi.org/10.1021/acs.bioconjchem.7b00826); as evidenced by
Farka ( Farka et al., Surface design of photon-upconversion nanoparticles for high-contrast immunocytochemistry. Nanoscale 2020; 12 (15): 8303–8313. doi.org/10.1039/c9nr10568a) .
Belhocine teaches sequencing methods using “cell beads,” which are single cells encapsulated in hydrogel particles for multi-omics analysis ([0209]; [0628]; FIG. 49).
Belhocine teaches using cell beads to capture and sequence RNA and DNA from the same single cell, thereby enabling the analysis of both transcriptional information and genomic information, such as methylation status, from the same cell. ([0663]).
Belhocine teaches processing DNA to obtain methylation information, such as detecting 5hmC ([0736] lines 37-40).
Regarding claim 5, Belhocine teaches that its capture reagent for capturing a nucleic acid target is a functionalized nucleic acid comprising a functional group (e.g., a click chemistry moiety) ([0628] lines 10-15) and a sequence configured to hybridize to a target nucleic acid, such as an oligo-dT sequence for RNA, or a random primer for gDNA ([0391] lines 1-12; [0637] lines 4-10; [0688]-[0689]). Although Belhocine does not explicitly teach the capture reagent being streptavidin, this feature would have been obvious in view of Qiong.
Qiong teaches an efficient labeling method for conjugating biotin onto 5hmC sites in DNA, which enables enrichment of 5hmC-containing DNA using streptavidin (Abstract; p. 248, right-hand col., para 1). Qiong teaches that its labeling method is based on a modification of bisulfite conversion ꟷ in the presence of sulfinate derivative of biotin (BioS), bisulfite conversion of 5hmC yields a biotin-labeled, sulfinate conjugated 5-hmC adducts (Figure 1).
Qiong further teaches advantages of its method for labeling and enrichment of 5hmC, including a simple workflow with a one-step reaction for efficient conjugation, a mild biocompatible aqueous protocol, good yield with high selectivity, and ease of recovery without harsh conditions. Qiong also highlights application of the method in cellular 5-hmC quantification and epigenetic sequencing:
“In conclusion, we have developed a novel chemical method to label hmC with sulfinate reagents. Model reaction using ethanesulfinate achieved specific conversion of hmdC nucleoside as well as hmC in single- and double-stranded DNA with yields up to 80%. The labeling method affords an efficient conjugation of biotin onto hmC sites in DNA via biotin-sulfinate probes in one step reaction with decent yield. Further application to biotin/streptavidin pull-down assay showed remarkable potentials of the present methods in the enrichment assays for hmC containing DNA. Moreover cleavage of DNA from biotin labels was achieved via a unique step of alkylamine incubation without harsh conditions. The mild biocompatible aqueous protocol, with good labeling yield, high selectivity for hmC and unique potentials for DNA recovery are the desirable attributes of this chemical method. Hence, the innovative method developed here provides a novel strategy for chemical labeling of hmC as nucleosides, oligonucleotides, and duplexes, which could have wide applications in hmC involved biological studies, therapeutics and biotechnology, such as cellular 5-hmC quantification and epigenetic sequencing.” (p. 248, right-hand col., para 1).
Accordingly, a person of ordinary skill in the art before the effective filing date of the claimed invention would have found it prima facie obvious to modify Belhocine’s single-cell multi-omics method to label 5hmC in the cellular gDNA with biotin for streptavidin capture, as taught by Qiong. This would enrich 5hmC-containning DNA for cellular 5hmC quantification and epigenetic sequencing.
The skilled artisan would have been motivated to make this modification because Belhocine already teaches detecting 5hmC, and Qiong teaches an improved method of 5hmC-specific labeling and enrichment, useful for 5hmC quantification and sequencing. Qiong further teaches advantages including simplicity in workflow, good yield, recovery without harsh conditions.
There would have been a reasonable expectation of success because the references disclose technically compatible teachings. Belhocine teaches compatibility with bisulfite conversion reagents (e.g., [0689] line 13), and Qiong’s labeling method is a modification of bisulfite conversion. Qiong’s mild, aqueous, and biocompatible protocol conditions would have been compatible with Belhocine’s droplet-based method. Further, using streptavidin as a capture reagent in a hydrogel polymer would have been within the level of ordinary skill in the art because functionalized streptavidin for click chemistry was commercially available at the time of the effective filing date, as evidenced by Farka (p. 8305, left-hand col, lines 7-8, “streptavidin azide (7 Bioscience, Germany; 1 mg mL−1)”).
Regarding claims 7-9, as discussed above for claim 5, the combined teachings of Belhocine and Qiong teach streptavidin as capture reagent, which is capable of binding to biotin-labeled 5- hydroxymethylcytosine.
Prior Art
Below are relevant prior art not used in rejection but pertinent to the claims or disclosure.
The following prior art also teach cell-encapsulation in hydrogel particles for nucleic acid analysis:
US20200277672A1- Single Cell Genomic Sequencing Using Hydrogel Based Droplets ;
Lan et al. Single-cell genome sequencing at ultra-high-throughput with microfluidic droplet barcoding. Nat Biotechnol. 2017 Jul;35(7):640-646. doi: 10.1038/nbt.3880. Epub 2017 May 29. PMID: 28553940; PMCID: PMC5531050;
Zhu et al. ; Hydrogel Droplet Microfluidics for High-Throughput Single Molecule/Cell Analysis. Acc. Chem. Res. 17 January 2017; 50 (1): 22–31. doi.org/10.1021/acs.accounts.6b00370;
Kamperman et al. Single-Cell Microgels: Technology, Challenges, and Applications. Trends Biotechnol. 2018 Aug;36(8):850-865. doi: 10.1016/j.tibtech.2018.03.001. Epub 2018 Apr 12. PMID: 29656795: see Figure 5 for example;
US20190127789A1 - Immobilization-based systems and methods for genetic analysis and other applications; see abstract; claim 1 for examples;
US20190376118A1- Methods and systems for characterizing nucleic acid molecules; cited in IDS filed 07/09/2024.
The following prior art also teach capturing epigenetic DNA modification marker in streptavidin hydrogel beads:
US20180298431A1 - Selective oxidation of 5-methylcytosine by tet-family proteins; see [0423].
Conclusion
Claims 1 and 9 are objected to; claims 1-10 are rejected. No claims are allowed.
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/TIAN NMN YU/Examiner , Art Unit 1681
1 See Wikipedia (Adapter - Wikipedia; Archived Oct 09, 2021 on WaybackMachine):
" An adapter or adaptor, or a linker in genetic engineering is a short, chemically synthesized, single-stranded or double-stranded oligonucleotide that can be ligated to the ends of other DNA or RNA molecules."
See also US20120058468A1- Adaptors for nucleic acid constructs in transmembrane sequencing; abstract for example.
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