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 .
Response to Amendment
The amendment filed February 20th, 2026 is acknowledged. Regarding the Office Action mailed September 8th, 2025:
The double patenting rejection is withdrawn in view of the abandonment of copending Application No. 17766636.
Maintained, modified, or new rejections are set forth below, as necessitated by the amendments. Responses to arguments, if necessary, follow their respective rejection sections.
Claim Summary
Claims 1 and 10 have been amended. Claims 2-4 and 9 have been canceled. Claims 1, 5-8, and 10-20 are pending. Claims 1, 5-8, and 10-20 are under examination and discussed in this Office action.
Claim Rejections - 35 USC § 112(b) - New - Necessitated by Amendment
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 5 and 6 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.
In the most recent amendment, claim 4 was cancelled. Claim 5 as written depends from claim 4 and therefore does not currently depend from any claim. This makes the claim indefinite. Given that previously claim 4 depended from claim 1, claim 5 will be interpreted to depend from claim 1. This will also serve to rectify dependency for claim 6, which depends from claim 5 and suffers the same issue of lack of proper dependency as claim 5.
Claim Rejections - 35 USC § 103 – Modified – Necessitated by Amendment
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.
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.
Claim(s) 1, 5-8, and 11-20 are rejected under 35 U.S.C. 103 as being unpatentable over Pellegrino et al. (High-throughput single-cell DNA sequencing of acute myeloid leukemia tumors with droplet microfluidics, Genome Research, August 2018, 28, 1345-1352; previously cited), in view of Shahi et al. (Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding, Scientific Reports, March 2017, 7, 1-12; previously cited), and Samuels (US 20120220494 A1).
Regarding instant claim 1, Pellegrino teaches a method for analyzing a plurality of cells, the method comprising: for one or more cells of the plurality of cells: encapsulating the cell in an emulsion comprising reagents (Page 1345, column 2, paragraph 2), the cell comprising at least one DNA molecule (Page 1345, column 2, paragraph 2); lysing the cell within the emulsion to generate a cell lysate comprising the at least one DNA molecule (Page 1345, column 2, paragraph 2; Figure 1); encapsulating the cell lysate comprising the at least one DNA molecule with a reaction mixture in a second emulsion (Page 1345, column 2, paragraph 2; Figure 1); performing a nucleic acid amplification reaction within the second emulsion using the reaction mixture to generate amplicons, the amplicons comprising: a first amplicon derived from one of the at least one DNA molecule (Page 1345, column 2, paragraph 2; Figure 1); sequencing the first amplicon (Page 1345, column 2, paragraph 2; Figure 1); determining one or more mutations of the cell using at least the sequenced first amplicon, wherein the one or more mutations comprise a single nucleotide variant (SNV) (Page 1347, column 2, paragraph 1); and distinguishing the one or more cells of the plurality of cells using the determined SNVs to identify a subpopulation of cells in the plurality of cells identifying a subpopulation of cells in the plurality of cells, the subpopulation of cells characterized by the one or more mutations (Page 1347, column 2, paragraph 1; Figure 3D and 3E).
Pellegrino does not teach wherein the cell also comprises at least one analyte-bound antibody conjugated oligonucleotide; lysing the cell within the emulsion to generate a cell lysate also comprising the oligonucleotide; encapsulating the cell lysate also containing the oligonucleotide with a reaction mixture in a second emulsion; performing a nucleic acid amplification reaction within the second emulsion using the reaction mixture to generate amplicons, the amplicons comprising: a second amplicon derived from the oligonucleotide; sequencing the second amplicon; determining one or more mutations of the cell using at least the sequenced first amplicon, wherein the one or more mutations also comprise a copy number variation (CNV); determining a presence or absence of an analyte using at least the second amplicon; and distinguishing the one or more cells of the plurality of cells using the determined CNVs and presence or absence of the analyte to identify a subpopulation of cells in the plurality of cells.
Shahi, in the same field of endeavor, teaches wherein the cell comprises at least one analyte-bound antibody conjugated oligonucleotide (Page 4, paragraph 3); lysing the cell within the emulsion to generate a cell lysate comprising the oligonucleotide (Page 4, paragraph 7); encapsulating the cell lysate comprising the oligonucleotide with a reaction mixture in a second emulsion (Page 5, paragraph 2); performing a nucleic acid amplification reaction within the second emulsion using the reaction mixture to generate amplicons, the amplicons comprising: a second amplicon derived from the oligonucleotide (Page 5, paragraph 3); sequencing the second amplicon (Page 6, paragraph 2); determining a presence or absence of an analyte using at least the second amplicon (Page 8, paragraph 2); and distinguishing the one or more cells of the plurality of cells using the determined presence or absence of the analyte to identify a subpopulation of cells in the plurality of cells (Page 8, paragraph 4; Figure 6A).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method of Pellegrino with the additional detection of an analyte of Shahi. Since both Pellegrino and Shahi are in the same field of endeavor (e.g. single cell analysis), one of ordinary skill in the art would combine the two teachings with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to make this modification because this can allow for simultaneous characterization of the genome and proteome. This is valuable in tracking the flow of information through cells, particularly in the progression of cancer (Shahi, Page 9, paragraph 6).
Neither Pellegrino, nor Shahi, teach determining one or more mutations of the cell using at least the sequenced first amplicon, wherein the one or more mutations also comprise a copy number variation (CNV); or distinguishing the one or more cells of the plurality of cells using the determined CNVs to identify a subpopulation of cells in the plurality of cells.
Samuels, in the same field of endeavor, teaches on using single-cell emulsion with multiple droplets, amplification of specific targets, and sequencing, equivalent to both Pellegrino and Shahi (Pages 20-21, paragraphs [0271]-[0278]; Figure 18). Samuels also teaches that if a locus has a copy number variation within the same sample target, this would be seen as variation in the number of sequenced reads that have the same barcode and sequence (Page 19, paragraph [0265]). This is cited in reference to haplotype determination. However, similar methods are used for both haplotype determinization and single-cell genomics (Page 20, paragraph [0271]), and it would be obvious that, given the sequencing performed for the single-cell genomics (Page 6, paragraph [0286]), these sequenced reads can also be used to determine a copy number variation in a target. Finally, Samuels teaches that the invention can identify one or more mutations (i.e. copy number variation) in subpopulations of cells in a sample, obviating that mutations can be used to identify a subpopulation of cells (Page 6, paragraph [0122]; Page 22, paragraph [0290]).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method of Pellegrino and Shahi with the method of Samuels. Since both Pellegrino, Shahi, and Samuels are in the same field of endeavor (e.g. single cell analysis), one of ordinary skill in the art would combine the two teachings with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to make this modification because Samuels teaches that “one or more mutations are identified in subpopulations of cancer cells in a sample using the labeling methods of the invention. The ability to identify multiple mutations existing in one cell better informs research and physicians on the possibility of drug resistance or reoccurrence of disease, and also inform treatment.” (Page 6, paragraph [0122]). This, in combination with the motivation from Shahi for looking at both the genome and proteome, provides motivation for distinguishing one or more cells using SNVs, CNVs, and the presence or absence of analytes.
Regarding instant claim 5, Pellegrino, in view of Shahi and Samuels, teaches the method of claim 4. Pellegrino further teaches wherein the SNV and/or CNV is identified in a gene relevant in acute myeloid leukemia (Page 1347, column 2, paragraph 1).
Regarding instant claim 6, Pellegrino, in view of Shahi and Samuels, teaches the method of claim 5. Pellegrino further teaches wherein the SNV and/or CNV is identified in any of DNMT3A (Page 1347, column 2, paragraph 1).
Regarding instant claim 7, Pellegrino, in view of Shahi and Samuels, teaches the method of claim 1. Shahi further teaches wherein determining presence or absence of the analyte comprises determining an expression level of the analyte, the analyte bound by the antibody conjugated to the oligonucleotide (Page 8, paragraph 4; Figure 6A).
Regarding instant claim 8, Pellegrino, in view of Shahi and Samuels, teaches the method of claim 7. Shahi further teaches wherein the analyte is any of CD19 or CD3 (Page 8, paragraph 4).
Regarding instant claim 11, Pellegrino, in view of Shahi and Samuels, teaches the method of claim 1. Shahi further teaches comprising: prior to encapsulating the cell in the emulsion, exposing the cell to a plurality of antibody-conjugated oligonucleotides; and washing the cell to remove excess antibody conjugated oligonucleotides (Page 10, Cell staining and in droplet cell lysis).
Regarding instant claim 12, Pellegrino, in view of Shahi and Samuels, teaches the method of claim 11. Shahi further teaches wherein the oligonucleotides conjugated to the plurality of antibodies comprise a PCR handle, a tag sequence, and a capture sequence (Figure 2A).
Regarding instant claim 13, Pellegrino, in view of Shahi and Samuels, teaches the method of claim 1. Pellegrino further teaches wherein the plurality of cells comprise cancer cells (Page 1346, column 2, paragraph 1).
Regarding instant claim 14, Pellegrino, in view of Shahi and Samuels, teaches the method of claim 13. Pellegrino further teaches wherein the cancer cells are any of acute myeloid leukemia (Page 1346, column 2, paragraph 1).
Regarding instant claim 15, Pellegrino, in view of Shahi and Samuels, teaches the method of claim 1. Pellegrino teaches the method further comprising encapsulating a first barcode and a second barcode in the second emulsion along with the at least one DNA molecule, the oligonucleotide, and the reaction mixture (Page 1345, column 2, paragraph 2; Figure 1).
Regarding instant claim 16, Pellegrino, in view of Shahi and Samuels, teaches the method of claim 15. Pellegrino further teaches wherein the first amplicon comprises the first barcode (Page 1346, column 1, paragraph 2).
Regarding instant claim 17, Pellegrino, in view of Shahi and Samuels, teaches the method of claim 15. Pellegrino further teaches wherein the second amplicon comprises the second barcode (Page 1346, column 1, paragraph 2). The obviousness analysis for claim 1 regarding the second amplicon also applies here. Additionally, it would be obvious that the second amplicon would comprise the second barcode given the teachings of Pellegrino regarding oligonucleotides containing both barcodes and gene specific primer sequences for amplifying specific targets (Page 1346, column 1, paragraph 2). This would necessarily include the barcode in amplicons produced using the oligonucleotides that also contain primer sequences. Oligonucleotides of Pellegrino as described above could have specific primer sequences designed for an analyte-bound antibody conjugated oligonucleotide as claimed given that primer design is very well known in the art. Furthermore, Shahi teaches that analyte-bound antibody conjugated oligonucleotides are amplified to link cell barcodes with the conjugated oligonucleotide sequence, necessarily resulting in an amplicon with a barcode (Page 2, paragraph 4; Figure 1D). This would amount to combining prior art elements according to known methods to yield predictable results (see MPEP 2141(III)).
Regarding instant claim 18, Pellegrino, in view of Shahi and Samuels, teaches the method of claim 15. Pellegrino further teaches wherein the first barcode and second barcode share a same barcode sequence (Page 1347, column 1 and 2: “…where two or more cells are assigned to the same barcode, and show that cells can be discreetly identified with the barcoding workflow.”; Figure S3).
Regarding instant claim 19, Pellegrino, in view of Shahi and Samuels, teaches the method of claim 15. Pellegrino further teaches wherein the first barcode and second barcode share different barcode sequences (Page 1345, column 2, paragraph 2; Figure 1).
Regarding instant claim 20, Pellegrino, in view of Shahi and Samuels, teaches the method of claim 15. Pellegrino further teaches wherein the first barcode and second barcode are releasably attached to a bead in the second emulsion (Page 1346, column 1, paragraph 2).
Response to Arguments
Applicant's arguments filed February 20th, 2026 have been fully considered but they are not persuasive.
The Applicant first acknowledges the Examiner’s previous rejection (Page 6 of the Remarks filed February 20th, 2026). The Applicant then notes the amendment to claim 1 which recites “distinguishing the one or more cells of the plurality of cells using the determined SNVs, CNVs, and presence of absence of the analyte to identify a subpopulation of cells in the plurality of cells” (Page 6 of the Remarks filed February 20th, 2026). The Applicant argues that the cited references fail to render obvious this amended language (Page 6 of the Remarks filed February 20th, 2026). The Applicant argues that, at best, Pellegrino teaches on identifying using SNVs, and not all three options presented in the claim amendment (Page 7 of the Remarks filed February 20th, 2026). The Applicant argues that neither Shahi nor Fu cure the deficiencies of Pellegrino as neither teach on all three options being used to distinguish subpopulations of cells (Page 7 of the Remarks filed February 20th, 2026). The Applicant then argues that there would be no motivation to combine Pellegrino, Shahi, and Fu given that combining aspects of Pellegrino and Shahi would be problematic for the functioning of the method of Fu (Pages 7-8 of the Remarks filed February 20th, 2026). The Applicant further states that the claimed method is substantially and surprisingly more effective at identifying distinct cellular subpopulations compared to detection of SNVs, CNVs, or proteins alone, reiterating that none of Pellegrino, Shahi, or Fu teach or suggest the amended claim 1 (Pages 8-9 of the Remarks filed February 20th, 2026).
In response to these arguments, it is noted that the 103 rejections have been modified to use the reference of Samuels to teach aspects related to the CNVs and also combining detecting subpopulations of cells with multiple mutations. This is considered sufficient to cure the deficiencies of Pellegrino and Shahi. Any arguments related to the Fu reference are moot given it is no longer relied upon to teach the invention as claimed. Therefore, given the combined teachings of Pellegrino, Shahi, and Samuels, it is found that Pellegrino, Shahi, and Samuels in combination adequately teach all aspects of the invention as currently claimed. The arguments are not found persuasive.
Conclusion
All claims stand rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Allison E Schloop whose telephone number is (703)756-4597. The examiner can normally be reached Monday-Friday 8:30-5 ET.
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/ALLISON E SCHLOOP/Examiner, Art Unit 1683
/ANNE M. GUSSOW/Supervisory Patent Examiner, Art Unit 1683
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