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 .
Claim Status
Claims 1, 15, 27, 32. 37, 39, 42, 45, 52 have been amended. Claims 6-12, 16-18, 21-26, 28-31, 33-34, 36, 38, 41, 43-44, and 47-51 were cancelled in an earlier amendment and claims 13 and 14 are cancelled in the most recent correspondence. Claims 1-5, 15, 19-20, 27, 32, 35, 37, 39-40, 42, 45-46 and 52 pending in the application. Claims 1 and 52 are independent claims.
Response to Arguments
Rejections Withdrawn
The rejection of claim 27 under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement is withdrawn following the applicants’ amendments.
The rejection of claims 27, 32, 37, 39, 42, 45 and 52 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or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention is withdrawn following the applicants’ amendments.
The rejection of claim 14 under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, 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of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends is withdrawn following the applicants’ amendments and cancellation of the claim.
The rejection of claims 1-5, 13-15, 19-20, 27, 35, 37, 39-40, and 42 under 35 U.S.C. 102(a)(1) as being anticipated by Cusanovich et al. (“Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing”, Science, Vol 348, Issue 6237, 22 May 2015), is withdrawn following the applicants’ amendments and cancellation of claims 13 and 14.
The rejection of claims 1-5, 13-15, 19-20, 27, 32, 35, 37, 39-40, 42, 45-46 and 52 are rejected under 35 U.S.C. 103 as being unpatentable over Cusanovich as applied to claims 1-5, 13-15, 19-20, 27, 35, 37, 39-40, and 42 above and incorporated here for reason supra, in further view of Frisen et al. (US 2014/0066318 A1, published Mar. 6, 2014, on IDS) is withdrawn following the applicants’ amendments and cancellation of claims 13 and 14.
New Rejections
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.
Claims 1-5, 15, 19-20, 27, 32, 35, 37, 39-40, 42, 45-46 and 52 are rejected under 35 U.S.C. 103 as being unpatentable over So et al. (US 2018/0245142 A1 published Aug. 30, 2018) in view of Cusanovich et al. (Science, 2015, of record).
So is in the field of nucleic acid detection and teaches “methods and compositions for spatial detection and analysis of nucleic acids in a tissue sample. The methods can enable the characterization of transcriptomes and/or genomic variation in tissues while preserving spatial information about the tissue” (see Abstract).
In regards to claims 1 and 52, So teaches methods for spatial detection and analysis of nucleic acids in tissue samples while preserving spatial information related to the origin of nucleic acids in the tissue (see Abstract, [0002] and throughout). So explains that nucleic acids originating from different regions of a tissues sample can be tagged with location-specific sequence information (“spatial address”) and subsequently sequenced to map nucleic acids to their regions of origin in the tissue (see [0088]-[0090]). So further discloses capture arrays comprising capture probes immobilized at spatially distinct capture sites where each capture probe includes a spatial address region and a capture region (see [003], [0007], [0141]-[0142], and throughout). The spatial address region corresponds to the position of the capture probe on the array and thereby correlates nucleic acids capture by the probe with a specific location in the tissue sample (see [0141]-[0142]). So further teaches using synthetic spatial tags incorporated into the capture probes on the array rather than a sequence derived from endogenous nucleic acids, the tag is not contiguous with endogenous DNA or RNA sequences within the cell (see So [0098]). So also teaches that a tissue section may be placed directly on the capture array surface such that nucleic acids from the tissue interact with the spatially encoded capture probes, thereby maintaining spatial orientation information of the nucleic acids relative to the tissue section (see Fig. 68, [0125], [0156]-[0157]). Thus So teaches that barcoding occurs while cells are located in an endogenous position within tissue sections as recited in claim 1.
So teaches “methods for barcoding (tagging) eukaryotic cell nuclei comprising: (a) transferring a plurality of oligonucleotides into the nuclei of a plurality of cells to obtain barcoded nuclei, wherein the cells are in an endogenous location within a tissue section.” So achieves this by utilizing an almost identical method to the one described in the current specification in relation to Fig. 1. See In re Best 562 F.2d 1252, 1254, 195 USPQ 430, 433 (CCPA 1977), In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990). Specifically, wherein a substrate with an array of oligonucleotides with spatial addresses adhered to the surface, adding transposase to the array to form transposome homodimers, placing a tissue section atop the oligonucleotide/transposome array, and tagmenting the DNA in the cell with transposome complex (see So Figs. 68-69 [0584]-[0608]). “(c) performing single-cell or nuclei analysis to identify the sequence of the barcode;” So teaches using sequencing by synthesis and sequencing by ligation workflows, which function as the basis for the single-cell analysis methods, and is used by So to identify individual cells within the tissue sample (see [0609]-[0627], [0630]-[0635]). Furthermore, So teaches the use of oligonucleotides that comprises a barcode region (address tag) and a target region (SBS primer region) (see Fig. 69, [0592]-[0593]).
Thus, So teaches “A method for barcoding eukaryotic cell nuclei comprising: (a) transferring a plurality of oligonucleotides into the nuclei of a plurality of cells to obtain barcoded nuclei, wherein the cells are in an endogenous location within a tissue section; (c) performing single-cell or nuclei analysis to identify the sequence of the barcode; wherein each oligonucleotide comprises a barcode region and a target region” of claim 1 and “wherein the barcode sequence is non-contiguous with endogenous DNA or RNA sequences and wherein the barcode corresponds to the endogenous location of a cell within a tissue section” of claim 52.
However, So does not explicitly disclose combining barcoded nuclei into a suspension wherein the nuclear envelope remains intact.
Cusanovich teaches transposase mediated barcoding of nuclei for high-throughput sequencing analysis, wherein oligonucleotides are introduced into nuclei using transposase complexes to index chromatin for sequencing analysis. Specifically, Cusanovich teaches that “we molecularly tag nuclei in 96 wells with barcoded transposase complexes. We then pool, dilute and redistribute 15 to 25 nuclei to each of 96 wells of a second plate using a cell sorter” prior to library construction and sequencing analysis (see pg. 910, 3rd col. 3rd para.). Cusanovich further teaches that permeabilized nuclei are exposed to transposases loaded with sequencing adapters, thereby introducing sequencing adapters into chromatin within nuclei prior to sequencing analysis (see pg. 910, 3rd col. 3rd para.). Thus Cusanovich teaches transferring oligonucleotides into nuclei to obtain barcoded nuclei, pooling the nuclei, and subsequently processing them for sequencing, which necessarily requires that nuclear envelopes remain intact during barcoding and pooling steps prior to lysis. Cusanovich further teaches analyzing barcoded DNA using single-cell ATAC-seq, reading on the single-cell analysis limitation of claim 1.
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to combine the spatial barcoding techniques of So with the nuclei-based transposase barcoding workflow of Cusanovich to enable spatially resolved single-nucleus sequencing, thereby preserving spatial chromatin information (e.g. chromatin accessibility) while enabling scalable sequencing analysis (e.g. human cell atlas with spatial data). Combining a familiar chromatin accessibility sequencing technique with a familiar spatial barcoding and sequencing technique to obtain the combined predictable results of spatial and chromatin accessibility data would have been obvious with a reasonable expectation of success.
To the extent applicant argues that So does not explicitly disclose transferring oligonucleotides into nuclei, such argument is not persuasive. So employs spatially encoded oligonucleotide capture probes designed to interact with nucleic acids originating from cells within tissue samples (see So Figs. 68-69, [0141]-[0142], [0584]-[0608]). The present application describes a substantially identical spatial array system utilizing transposase-mediated delivery of barcoded oligonucleotides into nuclei (see Fig. 1, Spec. [0171]-[0174]. Where the prior art and the claimed invention employ the same or substantially identical structure or process, the burden shifts to the applicant to demonstrate that the prior art does not necessarily possess the claimed property. See In re Best 562 F.2d 1252, 1254, 195 USPQ 430, 433 (CCPA 1977), In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990).
Accordingly, the combination of So and Cusanovich teaches or renders obvious transferring a plurality of oligonucleotides into the nuclei of a plurality of cells to obtain barcoded nuclei, wherein the cells are in an endogenous location within a tissue section; combining the barcoded nuclei in a suspension and wherein the nuclear envelope is intact in the suspension; and performing single-cell or nuclei analysis to identify the sequence of the barcode; wherein each oligonucleotide comprises a barcode region and a target region as recited in claim 1.
In regards to claim 2, both So and Cusanovich utilize transposase to introduce the spatial barcode into cell nuclei (see So Figs. 68-69, [0584]-[0608]; Cusanovich pg. 1, 1st col. para. 1, 3rd col. para. 2-3).
In regards to claim 3, both So and Cusanovich teach that the oligonucleotide comprises a transposome adaptor region (see So Fig. 69, [0592]-[0605]; Cusanovich pg. 910, 3rd col. 2nd para).
In regards to claims 4 and 5, both So and Cusanovich teaches implementing barcodes so that they correspond with a to a cellular characteristic. So teaches that the barcodes (spatial tags) correspond to the location of the cell in a tissue (see So Fig. 68-69, [0592]-[0608]), which Cusanovich teaches barcodes that correspond to cell type, and uses the barcodes to distinguish samples from each other utilizing different clonal populations of cells, such as Patski, HEK293T, GM12878, and HL-60 cells/nuclei. The HL-60 cell line is a well-established human leukemia (cancer) cell line (Fig. 2, pg. 912, 1st col., para. 1-2).
In regards to claim 15, both So and Cusanovich teach analysis methods that comprise sequencing the barcodes and/or cellular nucleic acids. (see So [0609]-[0627]; Cusanovich pg. 910, 3rd col. 1st para).
In regards to claims 19 and 20, Cusanovich teaches providing cells with 2 barcodes which correspond to a cellular characteristic (pg. 910, 3rd col., 1st para.).
In regards to claim 27, So teaches using methods to identify single nucleotide variants (see So [0124], [0584]-[0607]) while Cusanovich teaches single-cell analysis to identify DNA chromatin profiling (Title, Abstract, and throughout).
In regards to claim 32, So teaches using tissue sections that have previously been fixed or frozen (see [0125]-[0126]).
In regards to claim 35, So teaches using synthetic spatial tags incorporated into the capture probes on the array rather than a sequence derived from endogenous nucleic acids, the tag is not contiguous with endogenous DNA or RNA sequences within the cell (see So [0098]).
In regards to claim 37 and 39, So teaches methods wherein the transposome adaptor region comprises a transposase recognition sequence and/or wherein the transposome adaptor region comprises a complementary sequence capable of base-pairing with a transposome nucleic acid component (see Figs. 68-69, [0584]-[0608]).
In regards to claim 40, Cusanovich teaches a method in which the nucleic acids are fragmented and then a barcode is added (supplemental materials, pg. 2, 2nd para.)
In regards to claim 42, So teaches that the target region comprises a primer binding region (see Figs. 68-69, [0584]-[0608]).
In regards to claim 45 and 46, So teaches methods in which oligonucleotides are deposited on a substrate, a transposome is formed using the oligonucleotides and then a tissue sample with cells is deposited on top of the oligonucleotides and substrate (see So Fig. 68). So further teaches that the oligos on the substrate contain cleavage sites for their release, including photocleavable sites, (see So Fig. 69, [0163], [0196], [0584]-[0608]).
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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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/MATTHEW HAROLD RAYMONDA/Examiner, Art Unit 1684 /AARON A PRIEST/Primary Examiner, Art Unit 1681