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 .
Applicant’s preliminary amendment filed 4/15/2026 is acknowledged. Claims 1, 4, 7, 8, 10-12, 14, 20, 21, 27, 28, 30, 31, 34, 36, 39, 58, 64 and 67 have been amended. Claims 5-6, 9, 15-19, 23-25, 29, 32-33, 35, 37-38, 40-46, 48-57, 60-63, 65-66 and 68-69 have been canceled. Claims 1-4, 7-8, 10-14, 20-22, 26-28, 30-31, 34, 36, 39, 47, 58-59, 64 and 67 are pending and under examination.
Election/Restrictions
Applicant's election with traverse of Group I, claims 104, 7-8, 10-14, 20-22, 26-28, 30-31, 34, 36, 39 and 47 in the reply filed on 4/15/2026 is acknowledged.
The traversal is on the ground(s) that the invention of groups I and II are unified by special technical features that define a contribution over the art of record and that the Examiner has mischaracterized the teachings of both the present application and the cited reference. Applicant summarizes the Examiner’s arguments and states that the cited art prior art of Oliver is directed to mapping and sequencing biomolecules in which the target biomolecule (for example, a strand of DNA or RNA) is coated with protein, such as E. coli single-stranded binding protein, after hybridization with a probe. Applicant states that the technical rationale in Oliver is to improve the physical properties of the target biomolecule to facilitate uniform translocation and enhance signal-to-noise in nanopore or channel-based electrical detection. Id. The coating step in Oliver is thus directed to the analyte being measured, not to the probe or conjugate introduced for specific target recognition. Applicant states that in contrast, the present application unambiguously claims and describes the coating of the oligonucleotide component of a target-binding moiety-oligonucleotide conjugate, such as an antibody-oligonucleotide conjugate, with a nucleic acid binding protein. Applicant states that a purpose of this coating is to address a biological problem of non-specific binding of the oligonucleotide to cellular components, which would otherwise preclude effective targeting and quantification of intracellular molecules. Applicant additionally states that a purpose of this coating is to address a biological problem of non-specific binding of the oligonucleotide to cellular components, which would otherwise preclude effective targeting and quantification of intracellular molecules and further cites passages of the specification to support preparation of the composition. Applicant states that Oliver does not suggest, teach, or contemplate the coating of a probe oligonucleotide or a conjugate for example a probe or conjugate intended for intracellular protein quantification cells. Applicant states there is no disclosure or suggestion in Oliver of coating the oligonucleotide portion of a binding moiety-oligonucleotide conjugate prior to cellular introduction, nor of using such a coated composition to address the problem of non-specific oligonucleotide interactions within permeabilized cells for the purpose of barcoded intracellular protein quantification.
All of the amendments and arguments have been thoroughly reviewed and considered but are not found persuasive for the reasons that follows: The examiner acknowledges Applicant’s arguments but notes that Applicant’s arguments are not commensurate fully in scope with the claims. Specifically, lack of unity is based on the broadest first named invention which is directed to the oligonucleotide conjugated to a target binding moiety that specifically binds to a target cellular molecule as recited in the claim 58 of which the examiner asserts is taught by the cited prior art of Oliver. Arguments directed to the intended use of the first broadly named invention directed to the product, or directed to teachings in the specification are not found persuasive for establishing lack of unity. As indicated in the restriction requirement, Oliver establishes that unity of invention is lacking as the reference teaches the limitation of the broadest first named product, the oligonucleotide conjugated to a target-binding moiety that specifically binds to a target cellular molecule (see restriction requirement at 2/19/2026). Applicant’s arguments are not found persuasive and thus the requirement is still deemed proper and is therefore made FINAL. The claims 58-59, 64 and 67 directed to an oligonucleotide and a kit are withdrawn from consideration as being drawn to a non-elected invention.
Priority
This application is a 371 of PCT/US2022/013885 filed 01/26/2022 which claims benefit of 63/141,818 filed 01/26/2021.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 10/11/2023 is acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Drawings
The drawings were received on 7/21/2023 is acknowledged. These drawings are found acceptable by the examiner.
Claim Objections
Claim 14 is objected to because of the following informalities:
(a) Claim 14 comprise of the grammatical error in the line 5 at the recitation of “one or more of the cellular proteins are an intracellular protein”. It is suggested amending the limitation “are” in line 5 to ---is---. Appropriate correction is required.
Claim Rejections - 35 USC § 112
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 1-4, 7-8, 10-14, 20-22, 26-28, 30-31, 34 and 36 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.
(a) Claims 1-4, 7-8, 10-14, 20-22, 26-28, 30-31, 34, 36, 39 and 47 is indefinite at the recitation “target-binding-moiety barcode sequence contained in oligonucleotide components of conjugates” in the claim 1 and 27 because the specification does not provide a limitation definition of the limitation and it is unclear what is meant by the “… moiety barcode sequence contained in oligonucleotide components of conjugates”. The metes and bounds of the limitation in the context of the claims is unclear.
(b) Claim 11 is indefinite at the recitation of “preferentially” because it implies the feature is optional and thus it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
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 text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
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(s) 1-4, 7-8, 10-14, 20-22, 26-28, 30-31, 34, 36, 39, and 47 is/are rejected under 35 U.S.C. 103 as being unpatentable over Nolan et al {Nolan, used interchangeably herein} (US 20200208197, July 2, 2020) in view of Stoeckius et al {Stoeckius, used interchangeably herein} (CN 110475864, November 2019) and Oliver (20100243449, September 30, 2010).
Regarding Claims 1-4, 7-8, 10-14, 36, 39, and 47, Nolan discloses a method of quantifying the levels of a plurality of cellular proteins present in a cell (The present invention relates in part to the... quantification of individual target molecules in single cells, Para. [0005]; the target molecule is selected from the group consisting of a peptide, Para. [0011]), the method comprising incubating a cell with a population of binding moiety-oligonucleotide conjugates comprising a plurality of conjugates in which each conjugate comprises a target binding moiety that specifically binds to a cellular protein to be quantified conjugated to an oligonucleotide that comprises a target-binding-moiety barcode sequence, said target binding-moiety barcode sequence differing in sequence from target-binding-moiety barcode sequences contained in oligonucleotide components of conjugates that comprise different target binding moieties (providing: (i) a population of cells potentially comprising at least one target molecule, (ii) a first unique binding agent (UBA) specific for a first target molecule, Para. [0032]; a) a first target molecule, b) a first unique binding agent (UBA) specific for the first target molecule, c) a first linkable UBA-dependent epitope specific barcode (ESB) the target molecule is selected from the group consisting of a peptide, Para. [0011]; the UBA comprises an antibody, Para. [0010]): and quantifying the level of barcode sequences for each binding moiety associated with the cell, thereby quantifying the level of each of the cellular proteins bound to the target binding moiety (the APS comprises a nucleic acid. Nolan teaches wherein the cells are permeabilized ([0306]).
In some embodiments, the tag comprises multiple APSs, an ESB, and a UBA linked by ligation, Para. [0010]; one or more APSs may further comprise a random tag region allowing for subsequent normalization of the detected COBs (FIGS. 6-11) In some cases, the random region can function as a molecular counter to estimate the number of template molecules associated with each sequence variant, Para. [0122]; the quantity of the target molecule of the molecule is estimated using the counter tag, Para. [0022]). Nolan et al teach wherein the method comprises a step of performing an amplification reaction to amplify oligonucleotide sequences of conjugates bound to cellular proteins to obtain an amplification product (para. [0010] and [0100]). Nolan further discloses comprising incorporating a cellular identification sequence, a unique molecular identifier (UMI) sequence, and/or a sample identification sequence during amplification (Para. [0022]). Nolan further discloses wherein quantifying the level of barcode sequences comprise a quantitative amplification reaction (Examples of PCR techniques that can be used include, but are not limited to, quantitative PCR, Para. [0133]). Nolan teaches wherein the oligonucleotide comprises a detectable label (the ESB can be a detectable bead such as a bead with a unique spectral signature (e.g. a bead that has been internally dyed with red and infrared fluorophores), Para. [0096]) and further comprising detecting single from the label to identify the position of a target binding complex ([0214]).
In a similar embodiment to Nolan, Stoeckius et al teaches compositions comprising a construct, the construct comprising a via a linker to a polymer construct (i.e., oligonucleotide sequence) or conjugated ligand. target-specific ligand is designed as in the combination of biological sample. bar code for specific recognition ligand polymer construct, for example, oligonucleotide sequence, comprising amplifying the handle; an optional random molecular tag (RMT) or unique molecular identifier (UMI), hereinafter referred to as a "UMI" positioned adjacent to bar 5 'or 3' end, and an anchor, the anchor for hybridization with the capture sequence and for subsequently generating a double-stranded sequence, the capture sequence comprises sequence complementary to the anchor. In another aspect, the composition may further comprise one or more "additional" construct, different at any other construct the "additional" construct in the composition at least by at least one target, the ligand and the bar code and UMI (page 4, under “summary of the invention”).
Regarding Claims 20-22, 26-28, 30-31, 34, Nolan discloses a method of quantifying the levels of a plurality of cellular proteins present in a cell (, Para. [0005]); the target molecule is selected from the group consisting of a peptide, Para. [0011]), the method comprising (a) incubating a plurality of cells with a population of binding moiety oligonucleotide conjugates comprising a plurality of conjugates in which each conjugate comprises a target binding moiety that specifically binds to a cellular protein to be quantified conjugated to an oligonucleotide that comprises a target-binding-moiety barcode sequence, said target-binding-moiety barcode sequence differing in sequence from target-binding moiety barcode sequences contained in oligonucleotide components of conjugates that comprise different target binding moieties (providing: (i) a population of cells potentially comprising at least one target molecule, (ii) a first unique binding agent (UBA) specific for a first target molecule, Para. [0032]; a) a first target molecule, b) a first unique binding agent (UBA) specific for the first target molecule, c) a first linkable UBA-dependent epitope specific barcode (ESB) the target molecule is selected from the group consisting of a peptide, Para. [0011]; the UBA comprises an antibody, Para. [0010]); (b) distributing subpopulations of cells of the population into compartments (during the binding step multiple APSs are added to the tag in an ordered manner during successive rounds of split pool synthesis, Para. [0010]); (c) incorporating a cellular identification sequence during an amplification step performed on nucleic acids from the each of the subpopulations of cells of (b), wherein the cellular identification sequence for each subpopulation of (b) differs from the cellular identification sequence of other subpopulations of (b) distributed to other compartments (a cell origination barcode (COB) is generated with the APSs from the ordered set of APSs, Para. [0022]; UBA/ESB/COB complexes COB/ESB complexes and/or a combination thereof are amplified by polymerase chain reaction (PCR), Para. [0201]); (d) pooling the subpopulations to obtain a pooled population of cells ((c) splitting the population into two or more samples (e) pooling the two or more samples from step (c) into one sample, Para. [0032]); (e) distributing subpopulations of the pooled population of (d) into compartments ((f) splitting the sample from step ([e]) into two or more samples, Para. [0032]); (f) incorporating a cellular identification sequence during an amplification step performed on nucleic acids from each of the subpopulations of (e), wherein the cellular identification sequence for each subpopulation (e) differs from the cellular identification sequence of other subpopulations distributed to other compartments in step (e); and wherein steps (d)-(f) are optionally repeated ((g) adding one APS from the population of APSs per sample to the two or more samples from step ([f]), where a third complex is formed with the least one target molecule, the first UBA probe, the first ESB, the first APS, and the second APS, where the second common linker moiety from the second APS is bound to the third linker moiety from the first APS, and where the first APS and the second APS form a cell origination barcode (COB), Para. [0032]); and (g) quantifying the level of barcode sequences for each binding moiety associated with the cell in the amplified product, thereby quantifying the level of each of the cellular proteins bound to the target binding moiety (and (c) detecting the third complex or at least part of the third complex. In some embodiments, the methods further comprise repeating steps (e), through (g), Para. [0032]: the APS comprises a nucleic acid. In some embodiments, the tag comprises multiple APSs, an ESB, and a UBA linked by ligation, Para. [0010]; one or more APSs may further comprise a random tag region allowing for subsequent normalization of the detected COBs (FIGS. 6-11) In some cases, the random tag region can function as a molecular counter to estimate the number of template molecules associated with each sequence variant, Para. [0122]; the quantity of the target molecule of the molecule is estimated using the counter tag, Para. [0022]). Nolan teaches wherein the target molecule is an intracellular protein [0173].
Regarding Claims 20-22, 26- 29, Nolan discloses a method of quantifying the levels of a plurality of cellular proteins present in a cell (, Para. [0005]); the target molecule is selected from the group consisting of a peptide, Para. [0011]), the method comprising (a) incubating a plurality of cells with a population of binding moiety oligonucleotide conjugates comprising a plurality of conjugates in which each conjugate comprises a target binding moiety that specifically binds to a cellular protein to be quantified conjugated to an oligonucleotide that comprises a target-binding-moiety barcode sequence, said target-binding-moiety barcode sequence differing in sequence from target-binding moiety barcode sequences contained in oligonucleotide components of conjugates that comprise different target binding moieties (providing: (i) a population of cells potentially comprising at least one target molecule, (ii) a first unique binding agent (UBA) specific for a first target molecule, Para. [0032]; a) a first target molecule, b) a first unique binding agent (UBA) specific for the first target molecule, c) a first linkable UBA-dependent epitope specific barcode (ESB) the target molecule is selected from the group consisting of a peptide, Para. [0011]; the UBA comprises an antibody, Para. [0010]); (b) distributing subpopulations of cells of the population into compartments (during the binding step multiple APSs are added to the tag in an ordered manner during successive rounds of split pool synthesis, Para. [0010]); (c) incorporating a cellular identification sequence during an amplification step performed on nucleic acids from the each of the subpopulations of cells of (b), wherein the cellular identification sequence for each subpopulation of (b) differs from the cellular identification sequence of other subpopulations of (b) distributed to other compartments (a cell origination barcode (COB) is generated with the APSs from the ordered set of APSs, Para. [0022]; UBA/ESB/COB complexes COB/ESB complexes and/or a combination thereof are amplified by polymerase chain reaction (PCR), Para. [0201]); (d) pooling the subpopulations to obtain a pooled population of cells ((c) splitting the population into two or more samples (e) pooling the two or more samples from step (c) into one sample, Para. [0032]); (e) distributing subpopulations of the pooled population of (d) into compartments ((f) splitting the sample from step ([e]) into two or more samples, Para. [0032]); (f) incorporating a cellular identification sequence during an amplification step performed on nucleic acids from each of the subpopulations of (e), wherein the cellular identification sequence for each subpopulation (e) differs from the cellular identification sequence of other subpopulations distributed to other compartments in step (e); and wherein steps (d)-(f) are optionally repeated ((g) adding one APS from the population of APSs per sample to the two or more samples from step ([f]), where a third complex is formed with the least one target molecule, the first UBA probe, the first ESB, the first APS, and the second APS, where the second common linker moiety from the second APS is bound to the third linker moiety from the first APS, and where the first APS and the second APS form a cell origination barcode (COB), Para. [0032]); and (g) quantifying the level of barcode sequences for each binding moiety associated with the cell in the amplified product, thereby quantifying the level of each of the cellular proteins bound to the target binding moiety (and (c) detecting the third complex or at least part of the third complex. In some embodiments, the methods further comprise repeating steps (e), through (g), Para. [0032]: the APS comprises a nucleic acid. In some embodiments, the tag comprises multiple APSs, an ESB, and a UBA linked by ligation, Para. [0010]; one or more APSs may further comprise a random tag region allowing for subsequent normalization of the detected COBs (FIGS. 6-11) In some cases, the random tag region can function as a molecular counter to estimate the number of template molecules associated with each sequence variant, Para. [0122]; the quantity of the target molecule of the molecule is estimated using the counter tag, Para. [0022]).
Stoeckius teaches the composition and constructs may be used in high-throughput methods for detecting a desire target. The reference teaches by using any number of assays and method for detection, the compositions and kits can be used in different environments to detect different target or the totality of target. In one embodiment in the composition, one or more target method for detecting in a biological sample using the herein described. the method comprises the steps of contacting a biological sample with one or more of the above-described composition. In one embodiment, the sample is contacted with a composition comprising a first construct, the first construct has via a linker to a polymer construct (e.g., a construct oligonucleotide sequence) a first ligand is linked or conjugated. In one embodiment, the first ligand with the cell or on the cell surface of the first target (e.g., a cell surface epitope specifically binds). construct oligonucleotide sequence comprising: amplifying the handle; the specific identification code of the first ligand, an optional unique molecular identifier, which is located adjacent to the barcode 5 'or 3' end, and an anchor for hybridization with complementary sequence to produce double-stranded oligonucleotide sequence. In another embodiment, the biological sample and comprises substantially the same "first" construct of the composition, wherein each first construct substantially the same only in that no UMI in sequence or construct of optional UMI constructs of the difference with the reference "first". Thus, the biological sample with multiple ligands contact with respect to the same target cell surface epitope (page 33, section entitled “The method for using the composition”).
In the methods and compositions described herein, one or more UMI can construct oligonucleotide sequence with a single polymer construct/association. UMI can locate the bar code in the composition of 5 'or 3'. In another embodiment, as part of the method described, the UMI can inserted in polymer/construct oligonucleotide sequence. In one embodiment of the method as described herein, for RNA sequencing method according to which, during the method adding the UMI. However, not all RNA-seq method both using the UMI. in the embodiments of the single-cell droplets RNA sequencing described below, introducing another UMI during reverse transcription. each UMI is specific for its construct oligonucleotide sequence. Therefore, when the composition or method comprises a plurality of "first construct", each of the first construct different only in sequence of the UMI. each additional construct will also have its own UMI, the UMI is not present on the other construct repeating or not present in the ligand, the bar code on the target, and the anchor specific construct is different to each other. in various methods described herein with the use of similar, UMI can be associated with polymer (e.g., oligonucleotide or polynucleotide sequence) used in the specific assay format, or associated with polymer (e.g., oligonucleotide or polynucleotide) fixed on the substrate. each of the polymer for each UMI construct (e.g., an oligonucleotide or polynucleotide) is different from any other UMI for use in the composition or method (page 25). Stoeckius additionally teach wherein compartmentalization of single cells for analysis may be performed (page 30).
Neither Nolan nor Stoeckius teach wherein each of the plurality of conjugates comprises a nuclei acid binding protein bound to each oligonucleotide component.
Oliver teaches a non-specific nucleic acid binding moiety, such as T4 gene 32 protein (the probes have a tag, particularly when used in conjunction with a protein coating step, Para. [0025]; The coating step may include at least partially coating the detectable tag with one or more proteins, wherein the one or more proteins in the coating step may include one or more of T4 gene 32 protein, Para. [0044]).
It would have been obvious to one of ordinary skill in the art at the effective filing date of the claimed invention would have been motivated to have modified the method of Nolan in view of Stoeckius by using a non-specific nucleic acid binding protein such as T4 gp32 as taught by Oliver for the benefit of improving assay quality by preventing binding of undesired oligonucleotides within the sample that could mask the oligonucleotides from detection in subsequent steps.
Conclusion
No claims are allowed. Any inquiry concerning this communication or earlier communications from the examiner should be directed to CYNTHIA B WILDER whose telephone number is (571)272-0791. The examiner can normally be reached Flexible.
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/CYNTHIA B WILDER/ Primary Examiner, Art Unit 1681
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