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 .
Claims 1-18 are pending and currently under examination.
Response to Amendment
The Amendment filed 10/20/25 has been entered. Claims 1-18 are pending. Applicant’s amendments to the specification and claims 2-3 have overcome the objections and 112(b) rejections previously set forth in the Non-Final Office Action mailed 7/25/25.
Response to Arguments
Applicant’s arguments, see pages 7-9, filed 10/20/25, with respect to the rejections of claims 1-18 under 35 USC 103 have been fully considered and are found unpersuasive, and the 103 rejections documented in the Non-Final mailed 7/25/25 have been revised to address claim amendments filed 10/20/25 in this Final Office Action. More detailed responses to Applicant’s arguments are provided at the end of each maintained rejection.
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 1-18 remain/are rejected under 35 U.S.C. 103 as being unpatentable over Fan et al. (2015; WO 2015/031691 A1; FOR citation 2 in IDS filed on 3/10/23) in view of Chang et al. (2020; WO 2020/214642 A1; FOR citation N in PTO-892 filed 7/25/25) and de Rutte et al. (2020; NPL citation U in PTO-892 filed 7/25/25; "Massively parallel encapsulation of single cells with structured microparticles and secretion-based flow sorting". bioRxiv preprint doi: https://doi.org/10.1101/2020.03.09.984245; this version posted March 11, 2020).
This rejection is revised/updated in response to claim amendments filed 10/20/25.
(i) Fan et al. teaches “Massively parallel single cell analysis” (Title).
Relevant to claim 1, Fan et al. “FIG. 1 depicts an exemplary solid support conjugated with an exemplary oligonucleotide” (paragraph 0019), wherein the “solid support” is a bead. This teaching reads on claim 1 wherein the beads are linked to a plurality of clonal cell-barcoding oligonucleotides having a 3' capture sequence.
Further relevant to claim 1, Fan et al. Fig. 43 depicts the workflow of loading cells into an array with beads, reading on claim 1 contacting the beads containing the cells to an array of spots of array oligonucleotides linked to a solid planar surface.
Further relevant to claim 1, Fan et al. “EXAMPLE 5: Evaluating efficacy of split-pool synthesis to produce beads with clonal copies of one cell label combination” teaches "In this example, the efficacy of split-pool synthesis to produce beads with clonal copies of one cell label combination was evaluated" (paragraph 00513).
This teaching reads on claim 1 wherein the spots have clonal copies of array oligonucleotides.
Further relevant to claim 1, Fan et al. Fig. 1 teaches that the Molecular Label is "Different for different oligo on one bead". This teaching reads on claim 1 different spots have unique array oligonucleotides such that the sequence of the array oligonucleotides identifies the spot.
Further relevant to claim 1, Fan et al. Fig. 2B teaches that “The primer fragment may comprise a sequence that may hybridize to the linker label sequence of the oligonucleotide (e.g., the oligonucleotide coupled to the solid support)” (paragraph 00288).
This teaching reads on claim 1 wherein the array oligonucleotides comprise a 3' end sequence that is a reverse complement of the 3' capture sequence of the cell-barcoding oligonucleotides; and claim 1 wherein a portion of the cell-barcoding oligonucleotides anneal to the array oligonucleotides.
Further relevant to claim 1, Fan et al. Fig 8C reads on the spots have a size that only accommodates a single cell such that single cells reside on single spots.
Further relevant to claim 1, Fan et al. teaches that “As shown in FIG. 2C, a set of oligonucleotides was added to each well of a third plate. An oligonucleotide in a set of oligonucleotides comprises a linker, cell label, molecular label, and an oligodT. The linker and oligodT sequences are the same for each set of oligonucleotides. However, the cell label is different for each set of oligonucleotides. Thus, each well has a different cell label” (paragraph 00505).
This teaching reads on claim 1 wherein the sequence and location of the array oligonucleotides on the array is known; and claim 1 wherein the cell barcode on the cellular nucleic acid indicates the cell origin of the cellular nucleic acid and the array oligonucleotide indicates the location of the cell on the array.
Further relevant to claim 1, Fan et al. teaches "Detection of the labeled nucleic acids or any products thereof may comprise an emulsion or a droplet… The term emulsion, as used herein, can refer to a mixture of immiscible liquids (such as oil and water). Oil-phase and/or water-in-oil emulsions allow for the compartmentalization of reaction mixtures within aqueous droplets" (paragraph 00381).
This teaching reads on claim 1 encapsulating the beads containing cells into aqueous droplets in a water-in-oil emulsion.
Further relevant to claim 1, Fan et al. teaches "The molecular barcode, sample tag or molecular identifier label may comprise a target specific region. The target specific region may comprise a sequence that is complementary to the molecule. In some instances, the molecule is an mRNA molecule and the target specific region comprises an oligodT sequence that is complementary to the poly A tail of the mRNA molecule" (paragraph 00261).
This teaching reads on claim 1 tagging in the droplets at least one cellular nucleic acid from the cells with the cell-barcoding oligonucleotides.
Further relevant to claim 1, Fan et al. teaches "One aspect provided is a method, comprising contacting a sample with any solid support disclosed herein, hybridizing a target nucleic acid from the sample to an oligonucleotide of the plurality of oligonucleotides. In some embodiments, the method further comprises amplifying the target nucleic acid or complement thereof. In some embodiments, the method further comprises sequencing the target nucleic acid or complement thereof, wherein the sequencing comprises sequencing the molecular label of the oligonucleotide to which the target nucleic acid or complement thereof is bound" (paragraph 0010).
This teaching reads on claim 1 nucleotide sequencing tagged cellular nucleic acids from the cell and nucleotide sequencing cell-barcoding oligonucleotides linked to the array oligonucleotides.
Relevant to claim 2, Fan et al. teaches "Single cell nucleic acid libraries may also be produced by contacting the cells with an agent prior to lysing the cell. The agent may be an antigen, drug, cell, toxin, etc. Thus, specialized single cell nucleic libraries may be produced. Analysis of the nucleic acid libraries may be used to generate single cell drug expression profiles" (paragraph 00528).
This teaching reads on claim 2 after the providing and before the contacting, inducing the phenotype in the cells, thereby allowing for subsequent sorting of the beads for beads containing cells that generate a signal for sorting that is indicative of the phenotype, thereby forming a population of beads enriched for beads containing cells.
Further relevant to claim 2, Fan et al. Fig. 43A reads on the contacting comprises contacting the population of beads enriched for beads containing cells to the array of spots of array oligonucleotides linked to the solid planar surface.
Relevant to claim 3, Fan et al. teaches that "Determining the number of different labeled nucleic acids may comprise flow cytometry sorting of a sequence-specific label. Determining the number of different labeled nucleic acids may comprise detection of the labeled nucleic acids attached to the beads. Detection of the labeled nucleic acids attached to the beads may comprise fluorescence detection" (paragraph 00726).
This teaching reads on claim 3 wherein the signal for sorting that is indicative of the phenotype is a fluorescent signal and the sorting comprises sorting the cells with fluorescence-activated cell sorting (FACS).
Relevant to claims 4-6, Fan et al. teaches "In another example, an oligonucleotide is attached to the 5' end of an RNA molecule to produce a labeled-RNA molecule. Reverse transcription of the labeled-RNA molecule may occur by the addition of a reverse transcription primer. In some instances, the reverse transcription primer is an oligodT primer, random hexanucleotide primer, or a target-specific oligonucleotide primer. Generally, oligodT primers are 12-18 nucleotides in length and bind to the endogenous poly(A)+ tail at the 3' end of mammalian mRNA" (paragraph 00336).
This teaching reads on claim 4 wherein the tagging comprises annealing the 3' capture sequence of the cell-barcoding oligonucleotides to the at least one cellular nucleic acid.; claim 5 wherein the 3' capture sequence is a poly T sequence comprising at least five contiguous deoxythymidines; and claim 6 wherein the 3' capture sequence is a gene-specific capture sequence.
Relevant to claims 7-8, Fan et al. teaches "Conducting the one or more amplification reactions may comprise the use of one or more primers. The one or more primers may comprise one or more oligonucleotides. The one or more primers may anneal to the 3' end and/or 5' end of the plurality of labeled nucleic acids… The one or more primers may comprise at least one or more custom primers… The one or more custom primers may anneal to at least a portion of a molecular barcode. The one or more custom primers may anneal to the first sample tag, the second sample tag, the molecular identifier label, the nucleic acid or a product thereof… The one or more custom primers may anneal to two or more different labeled nucleic acids" (paragraph 00696).
This teaching reads on claim 7 wherein bridge oligonucleotides are present in the droplets and the tagging comprises annealing a first end of the bridge oligonucleotide to the cell-barcoding oligonucleotides and a second end of the bridge oligonucleotide to the cellular nucleic acid from the cell; and claim 8 wherein the beads are linked to at least a first set and a second set of clonal cell-barcoding oligonucleotides having a 3' capture sequence, wherein the first set and second set have different 3' capture sequences and wherein the 3' capture sequence of the first set anneals to the array oligonucleotides and (i) the 3' capture sequence of the second set anneals to the at least one cellular nucleic acid or (ii) the 3' capture sequence of the second set anneals to a first end of a bridge oligonucleotide and a second end of the bridge oligonucleotide anneals to the at least one cellular nucleic acid.
Relevant to claims 9-10, Fan et al. teaches "Contacting the plurality of nucleic acids with the plurality of sample tags may comprise ligating one or more sample tags to one or more nucleic acids… Contacting the plurality of nucleic acids with the plurality of sample tag may comprise performing one or more nucleic acid extension reactions" (paragraph 00715).
This teaching reads on claim 9 wherein the tagging comprises ligating the cell-barcoding oligonucleotides to the at least one cellular nucleic acid; and claim 10 wherein the tagging comprises primer extension wherein the cell-barcoding oligonucleotides are extended by a polymerase using the at least one cellular nucleic acid as a template.
Relevant to claims 11-12, Fan et al. teaches "In another example, an oligonucleotide is attached to the 5' end of an RNA molecule to produce a labeled-RNA molecule. Reverse transcription of the labeled-RNA molecule may occur by the addition of a reverse transcription primer" (paragraph 00336).
This teaching reads on claim 11 wherein the one or more cellular nucleic acids are RNA; and claim 12 wherein the tagging comprises reverse transcription.
Relevant to claim 13, Fan et al. teaches "In some embodiments, the target nucleic acid is DNA" (paragraph 00772)
This teaching reads on claim 13 wherein the one or more cellular nucleic acids are DNA.
Relevant to claims 14-15, Fan et al. teaches "In some embodiments, certain sequence types can be linked to a DNA or RNA profile. For example, T-cell receptor and/or B-cell receptor sequences can be linked to a transcription profile, microRNA profile, or genomic mutation profile of a sample, such as a single cell" (paragraph 00397).
This teaching reads on claim 14 wherein the cells are B cells and the at least one cellular nucleic acid from the cells encodes at least a portion of an antibody variable region; and claim 15 wherein the cells are T-cells and the at least one cellular nucleic acid from the cells encodes at least a variable portion of a T-cell receptor.
Relevant to claim 16, Fan et al. teaches "Determination of the sequence of a nucleic acid (e.g., amplified nucleic acid, labeled nucleic acid, cDNA copy of a labeled nucleic acid, etc.) may be performed using variety of sequencing methods including, but not limited to,… fluorescent in situ sequencing (FISSEQ) (paragraph 00366).
As FISSEQ requires in situ hybridization, this teaching reads on claim 16 wherein the assaying comprises… in situ hybridization to produce the signal.
Relevant to claim 17, Fan et al. teaches "Single cell nucleic acid libraries may also be produced by contacting the cells with an agent prior to lysing the cell. The agent may be an antigen, drug, cell, toxin, etc. Thus, specialized single cell nucleic libraries may be produced. Analysis of the nucleic acid libraries may be used to generate single cell drug expression profiles" (paragraph 00528).
This teaching reads on claim 17 wherein the assaying comprises adding target cells to the array and assaying the ability of the cells in the hollow hydrophilic beads to alter a phenotype of the target cells.
Relevant to claim 18, Fan et al. teaches "Detection of the labeled nucleic acids or any products thereof may comprise an emulsion or a droplet… The term emulsion, as used herein, can refer to a mixture of immiscible liquids (such as oil and water). Oil-phase and/or water-in-oil emulsions allow for the compartmentalization of reaction mixtures within aqueous droplets" (paragraph 00381).
This teaching reads on claim 18 wherein the beads containing the cells are encapsulated in a droplet before the assaying.
(ii) Fan et al. is silent to specifics regarding phenotypes and hollow beads relevant to claim 1. However, these limitations were known in the prior art and taught by Chang et al.
Chang et al. teaches “Methods of associating phenotypical data and single cell sequencing data” (Title).
Relevant to claim 1, Chang et al. teaches "There are provided, in some embodiments, methods of correlating phenotypical information and/or agent exposure with sequencing data in single-cell multi-omics workflows" (paragraph 0344).
This teaching reads on claim 1 A method of determining the phenotype and genotype of cells… assaying the cells on the spots to determine a phenotype and recording for the spots a signal indicative of the phenotype... and correlating the signal on the array to the nucleotide sequence of the tagged cellular nucleic acids.
Further relevant to claim 1, Chang et al. teaches “In some embodiments, the solid support is a bead. The bead can comprise one or more types of solid, porous, or hollow sphere, ball, bearing, cylinder, or other similar configuration which a nucleic acid can be immobilized (e.g., covalently or non-covalently)" (paragraph 0174); and "A bead can be hydrophilic" (paragraph 0183).
These teachings read on claim 1 providing a plurality of hollow hydrophilic beads.
Further relevant to claim 1, Chang et al. teaches "Analytes and/or reagents, such as oligonucleotide barcodes, for example, may be coupled/immobilized to the interior surface of a gel bead (e.g., the interior accessible via diffusion of an oligonucleotide barcode and/or materials used to generate an oligonucleotide barcode) and/or the outer surface of a gel bead or any other microcapsule described herein" (paragraph 0161).
Further relevant to claim 1, Chang et al. teaches “In some embodiments, coupling/immobilization of a reagent to a gel bead or any other microcapsule described herein may be reversible, such as, for example, via a labile moiety (e.g., via a chemical cross-linker, including chemical cross-linkers described herein). Upon application of a stimulus, the labile moiety may be cleaved and the immobilized reagent set free. In some embodiments, the labile moiety is a disulfide bond. For example, in the case where an oligonucleotide barcode is immobilized to a gel bead via a disulfide bond, exposure of the disulfide bond to a reducing agent can cleave the disulfide bond and free the oligonucleotide barcode from the bead” (paragraph 0161).
These teachings read on claim 1 releasing the oligonucleotides from the array such that released oligonucleotides from spots diffuse into beads residing on the spots.
(ii) Fan et al. and Chang et al. are silent to specifics regarding beads containing a cell relevant to claim 1. However, this limitation was known in the prior art and taught by de Rutte et al.
de Rutte et al. teaches “Massively parallel encapsulation of single cells with structured microparticles and secretion-based flow sorting” (Title).
Relevant to claim 1, de Rutte et al. Fig. 1 "(1) Particle Seeding and Cell Loading" and caption teach “(1) Prefabricated cavity-containing microparticles are seeded into a well plate and settle with their cavities remaining upright. Cells are seeded and adhere to the particle matrix via integrin binding sites (RGD).”
These teachings read on claim 1 the beads containing a cell.
Although Fan et al. does not explicitly teach the Chang et al. phenotypes and hollow beads or the de Rutte et al. beads containing a cell, it would have been prima facie obvious to the skilled artisan. Fan et al., Chang et al., and de Rutte et al. are analogous disclosures to the instant determination of phenotype and genotype.
The skilled artisan would have been motivated to combine the analogous art. Chang et al. teaches that their “novel and inventive methods of correlating phenotypical information with sequencing for single cells provided herein will open potential applications such as high-throughput drug screening for single cell multi-omics workflows” (paragraph 0426). Thus, the skilled artisan would have been motivated to include the Chang et al. phenotypic and bead methodologies within the methodology rendered obvious by Fan et al. in order to take advantage of the expanded “potential applications” within single cell multi-omics workflows.
Additionally, the skilled artisan would have been motivated to include the de Rutte et al. cell-containing beads within the methodology rendered obvious by Fan et al. because de Rutte et al. teaches "Moving beyond cells, the compartments formed can enable digital nucleic acid amplification assays and immunoassays, where the solid phase provides potential for barcoding and capturing of amplified assay signals. Given the ability to rapidly deploy our approach with established lab infrastructure we anticipate widespread applications of lab on a particle technology across a range of these single cell and single molecule assays in the near future" (last sentence of page 14 continued to page 15). Thus, the skilled artisan would be motivated by the rapid deployment of the “widespread applications” of the de Rutte et al. methodology.
The skilled artisan would have a reasonable expectation of success based on the disclosures of Fan et al. in view of Chang et al. and de Rutte et al. as discussed in the preceding paragraphs.
Applicant’s Arguments
Applicant argues that “The Office Action (p. 12) relies on Chang to teach ‘releasing the oligonucleotides from the array such that released oligonucleotides from spots diffuse into beads residing on the spots’ and apparently ‘an array of spots of array oligonucleotides linked to a solid planar surface.’ However, the section of Chang quoted in the office action for this aspect relates to linking barcodes to beads, not cleaving oligonucleotides and not cleaving from spots on an array as claimed. Indeed, it is not clear where in the art the office action considers ‘an array of spots of array oligonucleotides’ nor release of oligonucleotides as claimed occurs in the cited art. No other reference is cited as teaching this limitation. Thus, none of the cited art teaches or suggests: ‘contacting the beads containing the cells to an array of spots of array oligonucleotides linked to a solid planar surface’” (Remarks 10/20/25, last paragraph of page 8 continued to first paragraph of page 9).
Response to Applicant’s Arguments
The Examiner respectfully disagrees with the assertion that “none of the cited art teaches” the aforementioned limitations.
The Non-Final mailed 7/25/25 includes page 4 excerpt reproduced below:
Further relevant to claim 1, Fan et al. Fig. 43 depicts the workflow of loading cells into an array with beads, reading on claim 1 contacting the beads containing the cells to an array of spots of array oligonucleotides linked to a solid planar surface.
For clarity of record, Fig. 43 of Fan et al. (2015; WO 2015/031691 A1) is shown below.
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Absent a limiting definition for “contacting the beads containing the cells to an array of spots of array oligonucleotides linked to a solid planar surface”, the broadest reasonable interpretation would include Fan et al. Fig. 43 depiction of contacting the beads containing the cells to an array of spots of array oligonucleotides linked to a solid planar surface.
Applicant is reminded that the cited prior art reference must be read in its entirety, not
merely selective portions. As set forth in MPEP 2141.02:
Ascertaining the differences between the prior art and the claims at issue requires
interpreting the claim language, and considering both the invention and the prior art references
as a whole… A prior art reference must be considered in its entirety, i.e., as a whole,
including portions that would lead away from the claimed invention… However, ‘the prior art’s
mere disclosure of more than one alternative does not constitute a teaching away from any
of these alternatives because such disclosure does not criticize, discredit, or otherwise discourage
the solution claimed….’ In re Fulton, 391 F.3d 1195, 1201, 73 USPQ2d 1141, 1146 (Fed. Cir.
2004) (emphasis added)
Chang et al. does teach an embodiment of releasing the oligonucleotides from the array such that released oligonucleotides from spots diffuse into beads residing on the spots. Chang et al. teaches “Analytes and/or reagents, such as oligonucleotide barcodes, for example, may be coupled/immobilized to the interior surface of a gel bead (e.g., the interior accessible via diffusion of an oligonucleotide barcode and/or materials used to generate an oligonucleotide barcode) and/or the outer surface of a gel bead or any other microcapsule described herein… In some embodiments, coupling/immobilization of a reagent to a gel bead or any other microcapsule described herein may be reversible, such as, for example, via a labile moiety (e.g., via a chemical cross-linker, including chemical cross-linkers described herein). Upon application of a stimulus, the labile moiety may be cleaved and the immobilized reagent set free. In some embodiments, the labile moiety is a disulfide bond. For example, in the case where an oligonucleotide barcode is immobilized to a gel bead via a disulfide bond, exposure of the disulfide bond to a reducing agent can cleave the disulfide bond and free the oligonucleotide barcode from the bead” (paragraph 0161).
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/SARAH JANE KENNEDY/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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