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 .
DETAILED ACTION
The Examiner of your application has changed. Please address future correspondence to Amy M. Bunker, AU1684.
Pursuant to a preliminary amendment filed January 1, 2026, claims 1-4, 6-11, 14-18 and 21-25 are currently pending in the instant application.
Response to Election/Restriction
Applicant's election with traverse of Group III, claims 16, 18 and 21-23, directed to an assay method; and the election of Species as follows:
Species (E): wherein in step (c) at least some of the compartments have two or more cells loaded therein, and cell surface protein expression profiles are determined (claim 21), in the reply filed January 30, 2026 is acknowledged.
Response to Traversals:
The traversal of is on the grounds that: (a) the Office incorrectly identifies claim 17 as independent (Group IV) because claim 17 depends from instant claim 16 (Group III) (Applicant Remarks, pg. 7, second full paragraph).
Regarding (a), Group III (claim 16) is directed to an assay method comprising steps (a)-(f); while Group IV (claim 17) is directed to a method, comprising carrying out the method of claim 16 under different conditions, and including unrelated components. It is noted that claims depending from Group III (claim 16) recite the phrase ”[T]he method of claim 16 wherein,” while claim 17 clearly recites language consistent with an independent claim. Instant claim 17 clearly recites independent process steps, which are compared to the steps recited in claim 16. Thus, the restriction is proper.
Claims 1-4, 6-11, 14, 15, 17, 24 and 25 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a non-elected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on January 30, 2026.
Claim 18 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a non-elected species, there being no allowable generic or linking claim.
Therefore, claims 16 and 21-23 are under consideration to which the following grounds of rejection are applicable.
Priority
The present application filed September 14, 2022 is a 371 PCT/US2021/023039, filed March 18, 2021, which claims the benefit of US Provisional Patent Application 62991529, filed March 18, 2020.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on September 18, 2025 have been considered. Initialed copies of the IDSs accompany this Office Action.
Claim Objections/Rejections
Specification Objection
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code (See; paragraphs [0012] and [0130]). See MPEP § 608.01.
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 16 and 21-23 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 applicant regards as the invention.
Claim 16 is indefinite due to the use of parentheticals such as “(i-a),” “(iii-a),” “(v-a),” “(vi-a),” “(vii-a),” “(viii-a),” and “i-a)” such as recited in claim 16, lines 8, 17, 18, 20 and 23. It is not clear whether the parenthetical is used to indicate a step, a limitation, a preferred embodiment, or synonym, etc. Accordingly, the metes and bounds of the claim are not clear.
Claim 16 is indefinite for the recitation of the term “an antibody barcode complement segment” such as recited in claim 16, line 16 because claim 16, lines 1-15 do not recite the presence of an “antibody barcode” such that it is unclear how a pool oligonucleotides can comprise “an antibody barcode complement segment” that identifies ‘binding specificity,’ when no antibody barcode is present, and no binding step is recited in claim 16. Moreover, it is unclear what component or specific binding partner the antibody binds, such that the ‘binding specificity’ of a sequence is identified and, thus, the metes and bounds of the claim cannot be determined.
Claim 16 is indefinite for the recitation of the term “a pool barcode complement segment” such as recited in claim 16, line 19 because claim 16, lines 1-18 do not recite the presence of a “pool barcode.” Moreover, it is unclear how or why a “pool oligonucleotide” comprises “a pool barcode complement segment” including when no pool barcode is present in each vessel and, thus, the metes and bounds of the claim cannot be determined.
Claim 16 is indefinite for the recitation of the term “the same pool barcode complement segments” such as recited in claim 16, lines 21-22. There is insufficient antecedent basis for the term “the same pool barcode complement segments” in the claim because claim 16, line 19 recites the term “a pool barcode complement segment.” The Examiner suggests that Applicant amend the claim to recite, for example, “wherein each pool barcode complement segment within each vessel has an identical sequence.”
Claims 16 and 21 are indefinite for the recitation of the term “at least some vessels” such as recited in claim 16, lines 23, 29 and 39 for failing to particularly point out and distinctly claim the subject matter which applicant regards as the invention. In the present instance claim 16, lines 23, 29 and 39 comprises several ranges including “at least”, “some”, and “at least some.” The limitation of “at least” starts at a specific number of vessels (or compartments, and/or sequence fragment structures), the limitation of “some” refers to an unspecified number, and/or the term “at least some” can refer to a portion of a total quantity, or the entire quantity of vessels (or compartments, and/or sequence fragment structures). Therefore the recitation of “at least some” is a recitation of multiple ranges making the claims indefinite. Examiner suggest that Applicant amend the claim to recite, for example, “some,” “at least 30%,” “some or all,” or “all” for clarity.
Claim 16 is indefinite for the recitation of the term “at least one staining construct is to a cell surface protein in i-a)” such as recited in claim 16, line 23 because the term appears to be missing a verb, such that it is completely unclear what the “at least one staining construct” is to a cell surface protein. For example, is unclear whether the at least one staining construct bound to a cell surface protein (and/or attached to, separate from, adjacent, near, associated with, cleaved from, etc.) and, thus, the metes and bounds of the claim cannot be determined.
Claim 16 is indefinite for the recitation of the term “optionally combining the contents of all or some of said plurality of vessels” such as recited in claim 16, line 24 because it is unclear whether the step of “combining” is carried out, or whether the step of combining is not carried out and, thus, the metes and bounds of the claim cannot be determined.
Claim 16 is indefinite for the recitation of the term “the contents” such as recited in claim 16, line 24. There is insufficient antecedent basis for the term “the contents” in the claim.
Claim 16 is indefinite for the recitation of the term “loading individual stained cells or combinations of individual stained cells” such as recited in claim 16, line 23 because the step is completely unclear given that claim 16 does not recite an origin or use of the stained cells with regard to the vessels or contents recited in 16(a) or 16(b). For example, claim 16 does not recite staining cells, the formation of stained cells, a process step of staining constructs bound to cells or cell surface proteins, whether stained cells are added to the vessels comprising the components (a), whether stained cells are added to the optionally combined contents, are added to a different, new compartment, or whether the term refers to something else and, thus, the metes and bounds of the claim cannot be determined.
Claim 16 is indefinite for the recitation of the term “the cell” such as recited in claim 16, line 28. There is insufficient antecedent basis for the term “the cell” in the claim because claim 16, line 3 recites the terms “a plurality of cells” and “each cell.”
Claim 16 is indefinite for the recitation of the term “one or more stained cells” such as recited in claim 16, line 29. There is insufficient antecedent basis for the term “one or more stained cells” in the claim because claim 16, lines 25 recites the terms “individual stained cells” and “combinations of stained cells.” Additionally, instant claim 16 does not recite staining cells, such that the origin of any stained cell is completely unclear and, thus, the metes and bounds of the claim cannot be determined.
Claim 16 is indefinite for the recitation of the term “each stained cell comprises one or more staining constructs bound to a cell surface protein of the cell” in claim 16, lines 27-28 because claim 16 does not recite any step of binding staining constructs (e.g., each comprising a pool oligonucleotide and handle-tagged antibodies) to cell-surface proteins, such that it is unclear if stained cells are added to the vessels or compartments already comprising the cells from the population of cells recited in 16(i-a) and, thus, the metes and bounds of the claim cannot be determined.
Claim 16 is indefinite for the recitation of the term “a plurality of droplet oligonucleotides” such as recited in claim 16, lines 29-33 because claim 16 does not recite the presence and/or the formation of droplets, such that the steps of the method relative to “droplet oligonucleotides” is completely unclear and, thus, the metes and bounds of the claim cannot be determined.
Claim 16 is indefinite for the recitation of the terms “droplet bar code” and “the same droplet barcode” such as recited in claim 16, lines 31-32 because claim 16 does not recite the presence and/or the formation of droplets, such that the steps of the method relative to a “droplet bar code” or “the same droplet barcode” is completely unclear and, thus, the metes and bounds of the claim cannot be determined.
Claim 16 is indefinite for the recitation of the terms “the droplet oligonucleotides” and “droplet oligonucleotides” such as recited in claim 16, line 32-33. There is insufficient antecedent basis for the terms “the droplet oligonucleotides” and “droplet oligonucleotides” in the claim because claim 16, lines 29-30 recites the term “a plurality of droplet oligonucleotides.”
Claim 16 is indefinite for the recitation of the term “in a compartment” such as recited in claim 16, line 32 because it is unclear whether the “compartment” refers to the same compartments as recited in claim 16, line 26, whether the “compartment” refers to some other container (e.g., vessels, droplets, oligonucleotides within droplets, etc.), and/or whether the term refers to some other compartment and, thus, the metes and bounds of the claim cannot be determined.
Claim 16 is indefinite for the recitation of the term “the same droplet barcode” such as recited in claim 16, line 32. There is insufficient antecedent basis for the term “the same droplet barcode” in the claim because claim 16, line 31 recites the term “a droplet barcode.”
Claim 16 is indefinite for the recitation of the term “producing sequence fragment structures corresponding to the capture constructs” such as recited in claim 16, line 36 because the location, purpose, structure, and/or origin of the sequence fragment structures, and/or how they relate the cells, cell surface proteins, compartments, capture segment, droplet oligonucleotides, barcodes, etc. is completely unclear given that claim 16 does not recite the presence of “capture constructs,” such that it is unclear how sequence fragment structures are produced such that the comprise a droplet barcode, a pool barcode, and an antibody barcode; and/or how they “correspond” to an unidentified and unrecited “capture construct” and/or what they have to do with determining a distribution of cell surface proteins and, thus, the metes and bounds of the claim cannot be determined.
Claim 16 is indefinite for the recitation of the term “the capture constructs” such as recited in claim 16, line 36. There is insufficient antecedent basis for the term “the capture constructs” in the claim.
Claim 16 is indefinite for the recitation of the terms “the plurality of sequence fragment structures” and “individual sequence fragment structures” such as recited in claim 16, lines 39-41. There is insufficient antecedent basis for the term terms “the plurality of sequence fragment structures” and “individual sequence fragment structures” in the claim because claim 16, line 36 recites the term “sequence fragment structures.”
Claim 16 is indefinite for the recitation of the term “the sequences” such as recited in claim 16, lines 39-40. There is insufficient antecedent basis for the term “the sequences” in the claim.
Claim 16 is indefinite for the recitation of the term “individual cells” such as recited in claim 16, lines 42-43 because it is unclear which cells are being referred to by the term “individual cells” given that claim 16 recites many different types of cells including “a plurality of cells,” “each cell,” “individual stained cells,” “combinations of stained cells,” “one or more stained cells,” etc.
Claim 21 is indefinite for the recitation of the term “step (c)” such as recited in claim 21, line 1. There is insufficient antecedent basis for the term “step (c)” in the claim because claim 16, line 25 recites the term “(c).”
Claim 21 is indefinite for the recitation of the terms “step (c),” “compartments containing cells comprise two or more cells loaded therein” and/or the determination of “cell surface protein expression profiles” in claim 21, lines 1-3 because claim 21 depends from instant claim 16, wherein claim 16 does not recite loading cells into compartment, and/or a “step (c),” such that the particular compartments and its contents are completely unclear and, thus, the metes and bounds of the claim cannot be determined.
Claim 22 is indefinite for the recitation of the term “at least 50% of the compartments containing cells comprise two or more cells” in claim 22, lines 1-2 because claim 21 depends from instant claim 16, wherein claim 16 recites that the compartments are loaded with stained cells, such that it is unclear whether the compartments comprise unstained cells, stained cells, and/or a mixture of stained and unstained cells and, thus, the metes and bounds of the claim cannot be determined.
Claim 23 is indefinite for the recitation of the term “are a compound barcode” in claim 23, line 2 because claim 23 depends from claim 16, wherein claim 16 does not recite the presence of a compound. Moreover, it is unclear whether the term “compound barcode” refers to a compound that is associated with the sequence fragment structure or capture constructs, whether the term refers to barcodes that have the same sequence, and/or whether the term refers to something else and, thus, the metes and bounds of the claim cannot be determined.
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.
Claims 21-23 are rejected under 35 U.S.C. 112(d) 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 21 recites (in part): “wherein in step ( c) at least some of the compartments have two or more cells loaded therein, and cell surface protein expression profiles of said two or more cells are determined” in lines 1-3 because claim 21 depends from instant claim 16, wherein claim 16 recites that compartments are loaded with stained cells (e.g., not unstained cells). Additionally, claim 16 does not recite the determination of cells surface protein expression profiles and/or “step (c)”. Thus, claim 21 is an improper dependent claim 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 22 recites (in part): “wherein at least 50% of the compartments containing cells comprise two or more cells” in lines 1-2 because claim 21 depends from instant claim 16, wherein claim 16 recites that compartments are loaded with stained cells (e.g., not unstained cells). Thus, claim 22 is an improper dependent claim 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 23 recites (in part): “wherein the pool barcode and antibody barcode are a compound barcode” in lines 1-2 because claim 23 depends from instant claim 16, wherein claim 16 does not recite the presence of a compound. Thus, claim 23 is an improper dependent claim 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.
Applicant may cancel the claim, amend the claim to place the claim in proper dependent form, rewrite the claim in independent form, or present a sufficient showing that the dependent claim complies with the statutory requirements.
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 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 may not be obtained though the invention is not identically disclosed or described as set forth in section 102 of this title, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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 16 and 21-23 are rejected under 35 U.S.C. 103 as being unpatentable over Engreitz et al. (hereinafter “Engreitz”) (US Patent No. 11549135, issued January 10, 2023; previously published March 19, 2020; effective filing date September 14, 2018) in view of Stoeckius et. al. (hereinafter “Stoeckius”) (Genome Biology, 2018, 19(224), 1-12).
Regarding claim 16 (in part), Engreitz teaches that compositions comprise an antibody-tagged oligonucleotide, including an origin specific barcode handle sequence, a first primer handle sequence, a second primer handle sequence, and a target binding region, wherein the composition can include an adapter sequence, a unique molecular identifier (UMI), and a poly-A sequence, such that methods for simultaneously measuring target oligonucleotides and protein in single cells generally involve delivering a mixture of the composition to a population of cells and encapsulating individual cells in an individual discrete volume comprising PCR primers on a bead, where the individual discrete volume can be suspended in a reverse transcription mixture and the nucleotide sequence of the origin specific barcode handle sequence can be detected, thereby assigning the target oligonucleotide and protein of interest to a specific individual discrete volume, while maintaining information about sample origin of the target oligonucleotide (interpreting discrete volumes as a plurality of vessels comprising cells, handle-tagged antibodies as staining constructs; a handle oligonucleotide attached to an antibody; barcode or UMI as identifying specificity of the antibi; and antibody specific for a cell surface protein, claim 16(i-a)-(iv-a)) (Abstract). Engreitz teaches methods for visualizing nucleic acids including direct fluorescence hybridization, wherein the oligonucleotide is single-stranded or double-stranded DNA, RNA, or a synthetic oligonucleotide; and wherein the synthetic oligonucleotide is a peptide nucleic acid; and the target binding region is sequence specific (interpreted as an antibody specific for a cell surface protein, claim 16(iii-a)) (col 3, lines 7-14). Engreitz teaches delivering one or more ligation dependent probes to the cells (interpreted as pool oligonucleotides), wherein the one or more ligation dependent probe comprise (i) sequences that bind in proximate locations on a target RNA, and (ii) the first primer handle sequence, the second primer handle sequence, or both; linking the bound proximity probes; amplifying the oligonucleotide of the composition and the linked ligation dependent probes using barcoded PCR primers, wherein the barcode is incorporated into each resulting amplicon, and quantifying target protein abundance and/or determining target protein localization based at least in part on sequencing of amplicons (interpreting the ligation-dependent probes as pool oligonucleotides comprising a handle complement segment; a capture complement segment; an antibody barcode complement segment; a pool barcode complement segment; loading stained cells, and sequencing, claim 16(viii-a), 16(c) and 16(e)) (col 3, lines 22-33). Engreitz teaches that
the invention provides a method for quantifying protein in individual molecule complexes comprising fixing a population of cells, lysing the cells, and encapsulating the resulting individual molecule complexes with any of the compositions described herein in an individual discrete volume (interpreted as combining the contents of all or some of the plurality of vessels, claim 16(b)) (col 5, lines 8-13). Engreitz teaches that the oligonucleotide tag can be used to encode identifying information about the binding specificity of a protein binding molecule and/or enable amplification and detection of said target protein (interpreted as a sequence that identifies the binding specificity of the antibody, claim 16(iv-a) and (vii-a)) (col 9, lines 27-30). Engreitz teaches that some examples of nucleic acids include the probes disclosed herein, wherein the oligonucleotide tag can range from approximately 30 to 300 nucleotides in length, and can comprise one or more of the following elements; an origin specific barcode, a first primer handle sequence, a second primer handle sequence, a target binding region, and an adapter, such that these elements can be directly adjacent to another or separated by a spacer sequence; and/or they can be ordered in any combination 5' to 3' (interpreted as segments position between v-a and vi-a, claim 16(viii-a)) (col 15, lines 14-23). Engreitz teaches that a "primer" as used herein refers to short nucleic acid molecules, which can be annealed to a complementary nucleic acid molecule by nucleic acid hybridization to form a hybrid between the primer and the nucleic acid strand (interpreting the handle oligonucleotide to be annealed to the handle complement segment, claim 16(v-a)) (col 17, lines 60-65). Engreitz teaches that in array-based methods, single-stranded DNA is annealed to beads and amplified via EmPCR (interpreting the handle oligonucleotide to be annealed to the handle complement segment, claim 16(v-a)) (col 26, lines 63-64). Engreitz teaches that the invention can comprise amplifying the target nucleic acid sequence including amplification by polymerase chain reaction (PCR), in which a sample is contacted with a pair of oligonucleotide primers under conditions that allow for the hybridization of the primers to a nucleic acid template in the sample, such that the primers are extended under suitable conditions, dissociated from the template, re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid, wherein this cycle can be repeated; and the product of amplification can be characterized by such techniques as electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing (interpreted as loading a PCR compartment; interpreting primers as droplet oligonucleotides comprising a capture segment; interpreting sequencing as producing sequence fragment structures and determining sequences of the droplet barcode, claims 16(d) and 16(e)) (col 28, lines 18-30). Engreitz teaches that the method can comprise admixing the population of cells with monomers of a polymerizable hydrogel and polymerizing the gel in an oil emulsion under conditions that allow covalent anchoring of nucleic acids and/or proteins to the hydrogel; and that an emulsion can be achieved by a variety of methods known in the art (see, for example, US 2006/0078888, of which paragraphs [0139]-[0143] are incorporated by reference herein) (interpreting hydrogels and emulsions as compartments and/or droplets, claim 16) (col 31, lines 8-17). Engreitz teaches that the hydrogel can comprise primer pairs comprising releasable linkers, wherein the primers are barcoded using combinatorial indexing, and wherein barcoded primer are incorporated into DNA or cDNA amplicons by the amplification step (interpreted as loading a PCR compartment; interpreting primers as droplet oligonucleotides comprising a capture segment and droplet barcodes; and interpreting forming amplicons as producing sequence fragment structures, claim 16(e)) (col 32, lines 4-8).
Regarding claims 21 and 22, Engreitz teaches delivering a mixture of any of the compositions described herein to a population of cells including encapsulating individual cells in an individual discrete volume, which can comprise PCR primers on a bead, such that the individual discrete volume can be suspended in a reverse transcription mixture and the nucleotide sequence of the origin specific barcode sequence can be detected, thereby assigning the target oligonucleotide and protein of interest to a specific individual discrete volume (interpreting a population of cells in a mixture, and discrete volumes suspended in RT mixture as compartments having two or more cells loaded therein, claims 21 and 22) (col 2, lines 3-12). Engreitz teaches that the method can comprise admixing the population of cells with monomers of a polymerizable hydrogel and polymerizing the gel in an oil emulsion under conditions that allow covalent anchoring of nucleic acids and/or proteins to the hydrogel, wherein individual cells are encapsulated in the individual discrete volume; and the emulsions can then be broken to release the individual discrete volume (interpreted as combining the contents of the plurality of vessels), where the oligonucleotide-tagged protein binding molecules can then be added, either before or after hydrogel polymerization, PCR can be performed in the individual discrete volumes, wherein cell-barcoded amplicons are generated, and the resulting amplicons can be sequenced (interpreting admixing a population of cells, and cells in an emulsion including within a PCR or sequencer as a compartment having two or more cells loaded therein, claims 21 and 22) (col 2, lines 41-53). Engreitz teaches the cells or population of cells can be obtained from a biological sample, wherein the biological sample can be obtained from a subject suffering from a disease (interpreting a biological sample and a subject as compartments comprising two or more cells, claims 21 and 22) (col 23, lines 64-66).
Regarding claim 23, Engreitz teaches that unique molecular identifiers (UMIs) can be used, for example, to normalize samples for variable amplification efficiency including featuring a solid or semisolid support (for example a hydrogel bead), to which nucleic acid barcodes such as a plurality of barcodes sharing the same sequence are attached, each of the barcodes can be further coupled to a unique molecular identifier, such that every barcode on the particular solid or semisolid support receives a distinct unique molecule identifier, wherein the unique molecular identifier can then be transferred to a target molecule with the associated barcode, such that the target molecule receives not only a nucleic acid barcode, but also an identifier unique among the identifiers originating from that solid or semisolid support (interpreting UMIs and/or barcodes that are the same as a compound barcode, claim 23) (col 17, lines 9-23).
Engreitz does not specifically exemplify determining distribution of cell surface proteins on individual cells (claim 16, in part).
Regarding claim 16 (in part), Stoeckius teaches cell hashing, where oligo-tagged antibodies against ubiquitously expressed surface proteins uniquely label cells from distinct samples, which can be subsequently pooled, such that by sequencing these tags alongside the cellular transcriptome, each cell can be assigned to its original sample, robustly identify cross-sample multiplets, and “super-load” commercial droplet-based systems for significant cost reduction, wherein a complementary genetic approach was used to validate the approach and demonstrate how hashing can generalize the benefits of single cell multiplexing to diverse samples and experimental designs (Abstract). Stoeckius teaches that recent developments have demonstrated how sample multiplexing can simultaneously overcome multiple challenges, such as the demuxlet algorithm, which enables the pooling of samples with distinct genotypes together into a single scRNA-seq experiment, such that here, the sample-specific genetic polymorphisms serve as a fingerprint for the sample of origin and therefore can be used to assign each cell to an individual after sequencing; and this workflow also enables the detection of multiplets originating from two individuals, reducing non-identifiable multiplets at a rate that is directly proportional to the number of multiplexed samples (pg. 1, col 2, last partial paragraph). Stoeckius teaches that CITE-seq was recently introduced, where oligonucleotide-tagged antibodies are used to convert the detection of cell surface proteins into a sequenceable readout alongside scRNA-seq, wherein a defined set of oligo-tagged antibodies against ubiquitous surface proteins could uniquely label different experimental samples, which enabled the pooling of these together and use the barcoded antibody signal as a fingerprint for reliable demultiplexing (pg. 2, col 1, first full paragraph, lines 1-9). Stoeckius teaches that by labeling and pooling eight human PBMC samples and running them simultaneously in a single droplet based scRNAseq run, cell hashtags allow for robust sample multiplexing, confident multiplet identification, and discrimination of low-quality cells from ambient RNA; and “super-loading” of commercial scRNA-seq platforms to substantially reduce costs, this strategy represents a generalizable approach for multiplet identification and multiplexing that can be tailored to any biological sample or experimental design (pg. 2, col 1, first full paragraph, lines 14-24). Stoeckius teaches in Figure 1, sample multiplexing using DNA-barcoded antibodies; (a) a schematic overview of sample multiplexing by cell hashing, wherein cells from different samples are incubated with DNA-barcoded antibodies recognizing ubiquitous cell surface proteins; and distinct barcodes (referred to as hashtag-oligos, HTO) on the antibodies allow pooling of multiple samples into one scRNA-seq experiment, such that after sequencing, cells can be assigned to their sample of origin based on HTO levels (“Methods” section); (b) representative scatter plot showing raw counts for HTO A and HTO B across all cell barcodes, where both axes are clipped at 99.9% quantiles to exclude visual outliers; (c) heatmap of scaled (z-scores) normalized HTO values based on our classifications, wherein multiplets express more than one HTO; and negative populations contain HEK293T and mouse NIH-3T3 cells that were spiked into the experiments as negative controls; (d) tSNE embedding of the HTO dataset, wherein cells are colored and labeled based on our classifications; eight singlet clusters and all 28 cross-sample doublet clusters are clearly present; (e) distribution of RNA UMIs per cell barcode in cells that were characterized as singlets (red), multiplets (violet) or negatives (grey); (f) transcriptome-based clustering of single-cell expression profiles reveals distinct immune cell populations interspersed across donors. B, B cells; T, T cells; NK, natural killer cells; mono, monocytes; DC, dendritic cells, wherein cells are colored based on their HTO classification (donor ID), as in (d) (interpreted as protein expression profiles; and interpreting (a), (d), (e) and (f) as determining the distribution of cell surface proteins on each cell, claim 16) (pg. 3, Figure 1). Stoeckius teaches that UMI distributions of each classification group indicated that only barcodes classified as doublets by both techniques exhibited a positive shift in transcriptomic complexity (Figure 2d) (pg. 4, col 2, first full paragraph, lines 7-11; and Figure 2d). Figure 1 is shown below:
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Stoeckius teaches that Figure 2(b) illustrates count distribution of the most highly expressed HTO for groups of concordant and discordant singlets, wherein both groups have identical classification strength based on cell hashing (pg. 5, Figure 2). Figures 2b and 2d are shown below:
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It is prima facie obvious to combine prior art elements according to known methods to yield predictable results; the court held that, "…a conclusion that a claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination would have yielded nothing more than predictable results to one of ordinary skill in the art. KSR International Co. v. Teleflex Inc., 550 U.S. ___, ___, 82 USPQ2d 1385, 1395 (2007); Sakraida v. AG Pro, Inc., 425 U.S. 273, 282, 189 USPQ 449, 453 (1976); Anderson’s-Black Rock, Inc. v. Pavement Salvage Co., 396 U.S. 57, 62-63, 163 USPQ 673, 675 (1969); Great Atlantic & P. Tea Co. v. Supermarket Equipment Corp., 340 U.S. 147, 152, 87 USPQ 303, 306 (1950)”. Therefore, in view of the benefits of uniquely labeling expressed cell surface proteins as exemplified by Stoeckius, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of simultaneously measuring cell proteins and/or target oligonucleotides in single cells via combinatorial indexing with oligonucleotide-tagged proteins comprising an antibody bound to an oligonucleotide handle, which can comprise one or more of a barcode, barcoded primer handle sequences, UMIs, cell-barcoded amplicons, and/or poly-A sequences within a gel or emulsion as disclosed by Engreitz to include the method of cell hashing following a combination of Cite-seq and scRNA-seq as taught by Stoeckius with a reasonable expectation of success in simultaneously uniquely labeling and analyzing ubiquitous cell surface proteins including surface protein expression within pooled cell samples from multiple subjects in a single droplet-based scRNA-seq run; in identifying low-quality cells; and/or in substantially reducing assay costs, while providing a generalizable approach that can be tailored to any biological sample or experimental design.
Thus, in view of the foregoing, the claimed invention, as a whole, would have been obvious to one of ordinary skill in the art at the time the invention was made. Therefore, the claims are properly
rejected under 35 USC §103(a) as obvious over the art.
Conclusion
Claims 16 and 21-23 are rejected.
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/AMY M BUNKER/Primary Examiner, Art Unit 1684