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 .
Election/Restrictions
Applicant’s election without traverse of Group I: claims 1-20, 23-26, and 29-36 in the reply filed December 18, 2025 is acknowledged.
Claim Status/Action Summary
This action is in response to the papers filed on June 9, 2026.
Claims 1-20, 23-26, 29-32, and 35 are pending in the present application. Claims 33-34, 36, and 38-39 were canceled in the response filed on June 9, 2026. Claims 1-20, 23-26, 29-32, and 35 are under examination.
Any objections and rejections not reiterated below are hereby withdrawn.
The objections to the specification have been withdrawn in view of the substitute specification filed on June 9, 2026.
The 102 rejection over Mellen et al. has been withdrawn in view of the amendments to the claims now incorporating the limitations of claim 33, which was not included in the 102 rejections of record, into independent claim 1.
Priority/Effective filing date
The present application, filed on August 4, 2023, claims benefit of U.S. Provisional Patent Application No. 63/395,722, filed on August 5, 2022. No other priority claims presently exist in the application. Therefore, the effective filing date is determined to be August 5, 2022.
Drawings
The drawings filed on August 4, 2023 are acceptable.
Claim Rejections - 35 USC § 112(b)- Indefiniteness
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.
Claims 1-20, 23-26, 29-32, and 35 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 the inventor or a joint inventor regards as the invention.
This is a new grounds of rejection necessitated by the amendments to the claims.
Claim 1 recites “a first detectable marker… is indicative of… a cell nucleus, and… using a measured intensity of the first detectable marker… to determine… [an] estimate of a number of cells…”. It is unclear whether this claim language is intended to limit the type of cells that may be detected/quantified by the claimed method because the recited relationship between a measured intensity of a cell nuclear marker (e.g. a nuclear protein such as a histone or DNA such as DAPI staining) does not correlate with cell number for all cell types. For example, a) multinucleate cells such as osteoclasts, skeletal and cardiac muscle cells, syncytiotrophoblast, b) anucleate cells such as mammalian erythrocytes, or c) populations of cells having diverse ploidy status (i.e. tumor cells having diverse aneuploidies or exhibiting micronuclei). It is unclear whether methods comprising determining an estimate of a number of cells based upon the measured intensity of a detectable nuclear marker in a capture region that may comprise multiple cells of diverse lineage or identity or cancer status is intended to encompass embodiments wherein the multiple cells comprise multinucleate, anucleate, or aneuploid cells.
Claim 35 recites “a measured intensity of [a] second detectable marker [that is indicative of the presence of a cell membrane]… to validate… the… estimate of the number of cells [based upon the abundance of a first marker that is indicative of the presence of a cell nucleus]”. It is unclear whether the claim requires: a) comparing a ratio of the intensities of the first and second detectable markers summed over each of the capture spots (e.g. a ratiometric pattern of total “nuclear” signal to total “cell membrane” signal for a given capture spot, b) a spatial pattern of the second detectable marker relative to the first detectable marker within each of the capture spots (e.g. “rings” of second marker signal surrounding first marker signal), or c) something else.
Claims 2-20, 23-26, 29-32, and 35 are rendered indefinite because they depend from, and thus include the indefinite limitations of claim 1.
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 8 recites the broad recitation “fluorescent dyes or stains”, and the claim also recites several species of “fluorescent dyes or stains” including: “trypan blue, Giemsa, Toluidine blue, acridine orange, Bismarck brown, carmine, Coomassie blue, cresyl violet, DAPI eosin, ethidium bromide, acid fuchsin, hematoxylin, methyl green, methylene blue, neutral red, Nile blue, Nile Red, propidium iodide, rhodamine, and safranin” which is the narrower statement of the limitation. The claim is considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims.
Response to arguments
The response asserts that the claimed methods are definite because “the examiner is not fully considering that detection step in context of the complete method of claim 1”, which requires both a “first spatial dataset…indicative of a cell nucleus…” and a “second spatial dataset comprising nucleic acid quantification data comprising, for each respective capture spot…”
This assertion has been thoroughly considered and is not persuasive.
As written, the claims are generic with respect to the types of tissue samples that are analyzed. As was previously described, multiple tissue types are well known in the art that comprise a heterogenous mixture of uninucleate cells and multinucleate cells. It is well known in the cell biology and genetics arts that euploid, uninucleate cells have a 1c, 2c, or 4c amount of DNA per nucleus depending on the mitotic and/or meiotic state of each cell in the heterogenous mixture, wherein c=the quantity of DNA in a haploid gamete of the organism from which the tissue sample is derived. The claimed methods recite measuring spatial datasets comprising a measured intensity over the entirety of each of the capture spots upon which the (generic) tissue sample is overlaid. Each capture spot encompasses a plurality of cells within the tissue, and the identity and characteristics of the cells within a tissue may be heterogenous such that a significant proportion of cells are not euploid and/or uninucleate (i.e. do not comprise an amount of DNA that is an integer multiple of the “c” value for the organism in question and/or comprise more than one nucleus per cell membrane). Because the claim as written encompasses embodiments wherein the estimate of cell number based upon the measured intensity of a first detectable marker that is indicative of a cell nucleus integrated over the entirety of a capture spot comprising a plurality of cells that may be significantly heterogenous does not correlate with the estimate of cell number based upon the measured nucleic acid quantification data in all encompassed embodiments, it is unclear whether the claim is meant to be limited to some specific subset of said embodiments or is intended to encompass embodiments wherein the two estimates do not correlate with the number of cells present in a capture spot.
For example: a single capture spot comprises a number of cells that are polynucleate (e.g. osteoclasts in a section of bone tissue) and a number of cells that are uninucleate (e.g. osteoblasts and osteocytes). Osteoclasts are known to comprise variable numbers of nuclei per cell (i.e. all osteoclasts do not have a fixed number of nuclei per cell), while osteoblasts and osteocytes are uninucleate.
Therefore, an estimate of cell number in a capture spot depending on a marker that is indicative of the presence of a nucleus would report a higher number of cells than are actually present in the capture spot, because the multinucleate cells are essentially counted “N” times, where N=the number of nuclei per cell, which is variable in a population of osteoclasts. An estimate of cell number based upon the quantity of DNA in a capture spot will similarly over-count multinucleate cells. In other examples (e.g. tissues comprising a heterogenous population of cancer cells comprising unstable genomes resulting in frequent and complex aneuploid karyotypes, micronuclei, and/or syncytia; virus-infected cells; etc.), the heterogenous genetics are more complicated than the example above and would similarly not provide accurate estimates of cell number per capture spot.
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.
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 1-20, 25-26, 29-32, and 35 are rejected under 35 U.S.C. 103 as being unpatentable over Mellen et al., US 2021/0097684 A1 (filed September 30, 2020 and published April 1, 2021) as evidenced by Lucas et al., “Altered subcellular localization of the NeuN/Rbfox3 RNA splicing factor in HIV-associated neurocognitive disorders (HAND)” Neuroscience Letters 558(2014) 97-102 in view of Kjaerulff et al., US 2012/0045757 A1 (published February 23, 2012).
This is rejection has been updated as necessitated by the amendments to the claims.
It is noted that Mellen et al. does not share any named inventors with the present application.
Regarding claim 1, Mellen et al. teach methods of analyzing tissue samples comprising: obtaining sets of images of the tissue sample overlayed on an array of capture spots, wherein a first dataset is acquired for (a) detectable marker(s) for each capture spot indexed by a spatial barcode corresponding to each capture spot and a second dataset is acquired comprising nucleic acid quantification data indexed by a spatial barcode to each capture spot, wherein the two datasets are co-registered on a display by the spatial barcodes (Mellen et al., Figure 20 and paragraph 0313).
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Mellen et al. further teach the plurality of capture probe spots comprises at least 1000 capture spots (Mellen et al., paragraph 0121).
Mellen et al. do not teach using a measured intensity of a detectable marker that is indicative of the presence of a nucleus to estimate a number of cells in each capture spot.
However, Mellen et al. do teach measuring an intensity of NeuN protein (Mellen et al., figure 20, see above).
Additionally, Lucas et al. demonstrate “the well-known anti-NeuN antibody… In use for more than 15 years, NeuN reactivity is found largely to be restricted to neuronal nuclei” (Lucas et al., page 99, column 2, paragraph 2). Therefore, as evidenced by Lucas et al., it is clear that NeuN is a detectable marker that is indicative of the presence of a nucleus.
However, Kjaerulff et al. teach various methods for automatic detection of fluorescence signals from a sample (Kjaerulff et al., paragraph 0028-0029) comprising staining nuclei with DAPI and viable cells with DACM (N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide) (a dye that reacts with thiols found in proteins in living cells (i.e. cells with intact membranes) (Kjaerulff et al., paragraph 0011 and 0015). Kjaerulff et al. further teach that automated methods comprising staining cell nuclei and viable cells having intact membranes provide for rapid detection and quantitation of live cells (Kjaerulff et al., paragraph 0146).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have combined the methods taught by Mellen et al., comprising automated brightfield and fluorescent imaging of tissues (including nuclear stains such as DAPI, NeuN antibodies or Hematoxylin and cytosolic stains such as Eosin and cell-membrane associated stains such as fluorescently-labeled anti-CD3 antibodies) associated with specific capture spots with the methods taught by Kjaerulff et al. comprising automatically detecting and counting intact cells (having nuclei stained with a DNA binding dye and intact membranes) on a substrate.
The ordinary artisan would have been motivated to count the number of cells per capture spot in the method taught by Mellen et al. by the cell-staining methods taught by Kjaerulff et al. because the ordinary artisan would have recognized the benefits of a) normalizing measured mRNA expression values to a concrete count of the number of cells from which the mRNA molecules are derived, b) excluding background counts from capture spots that do not contain a cell (i.e. due to diffusion of mRNA from neighboring spots or contamination), and/or c) determining which capture spots are representative of a single cell type (i.e. a cancer cell) within a mixed population of cells (i.e. a section of a tumor sample).
Regarding claim 2, Mellen et al. teach the datasets are spatially displayed in an interactive manner on a computer system (Mellen et al., paragraph 0329).
Regarding claim 3, Mellen et al. teach that users can perform computer-implemented actions such as zooming, panning, and adjusting opacity of all or a portion of the first and second datasets (Mellen et al., paragraphs 0285-0288).
Regarding claim 4, Mellen et al. teach the obtaining uses fluorescence microscopy (Mellen et al., paragraph 0102).
Regarding claim 5, Mellen et al. teach exposing the tissue sample to the set of detectable markers prior to the obtaining step (Mellen et al., paragraph 0103).
Regarding claims 6 and 7, Mellen et al. teach the detectable markers are fluorescently-labeled antibodies (Mellen et al., paragraph 0103).
Regarding claim 8, Mellen et al. teach detectable markers in the set of detectable markers includes: “Haemotoxylin and Eosin, a Periodic acid-Schiff reaction stain, a Masson’s trichome stain, an Alcian blue stain, a Van Gieson stain, a reticulin stain, an Azan stain, a Giemsa stain, a Toluidine blue stain, an isamin blue/eosin stain, a NissI and methylene blue stain, a Sudan black and/or osmium staining (Mellen et al., paragraphs 0018 and 0091-0102). Mellen et al. further teach the samples can be stained with “acridine orange, Bismarck brown, carmine, Coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetraoxide, propidium iodide, rhodamine, safranine, and/or an immunofluorescence stain” (Mellen et al., paragraph 0147).
Regarding claim 9, Mellen et al. teach each image in the set of images comprises at least 100,000 pixel values (Mellen et al., paragraph 0010).
Regarding claim 10, Mellen et al. teach that a spatial dataset comprises obtaining sequence reads from 5000 molecularly barcoded, spatially encoded capture probes in probe spots (i.e. 1000 or more capture probes per capture spot), wherein the capture probes associate with native mRNA (i.e. a nucleic acid from the tissue sample), wherein the capture probes incorporate a spatial barcode corresponding to the particular capture spot and a unique molecular index into a resulting cDNA sequencing library (Mellen et al., paragraph 0138 or 0309). Mellen et al. further teach grouping sequence reads by spatial barcodes and unique molecular indices, and assigning the sequence reads to a respective capture spot (Mellen et al., paragraph 0171). Furthermore, Mellen et al. teach collecting one hundred thousand or more (i.e. at least 10,000) sequence reads for a tissue sample (Mellen et al., paragraph 0135).
Regarding claim 11, Mellen et al. teach each capture probe comprises a poly-T sequence and a unique spatial barcode corresponding to a particular capture spot (Mellen et al., figure 19)
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Regarding claim 12, Mellen et al. teach that each capture probe in the respective capture spot includes the same spatial barcode (Mellen et al., figure 19).
Regarding claim 13, Mellen et al. teach that each capture probe in the respective capture spot includes the same spatial barcode AND a Unique Molecular Identifier (UMI) (i.e. each capture probe includes a different spatial barcode; a “unique spatial barcode”) (Mellen et al., paragraph 0081-0082).
Regarding claim 14, Mellen et al. teach the obtaining comprises high-throughput sequencing (Mellen et al., paragraph 0309).
Regarding claim 15, Mellen et al. teach that each capture probe in the respective capture spot includes the same spatial barcode (Mellen et al., figure 19).
Regarding claim 16, Mellen et al. teach that teach representation of a number of molecules is a count of a number of unique sequence reads having the same spatial barcode associated with a particular capture spot (Mellen et al., paragraph 0024-0026).
Regarding claim 17, Mellen et al. teach the plurality of loci (i.e. distinct transcripts; nucleic acids) comprises more than 1000 loci (Mellen et al., paragraph 0027 or paragraph 0175).
Regarding claim 18, Mellen et al. teach the plurality of nucleic acids comprises RNA or DNA (Mellen et al., paragraph 0313).
Regarding claim 19, Mellen et al. teach the plurality of nucleic acids comprises mRNA (Mellen et al., paragraph 0313).
Regarding claim 20, Mellen et al. teach the tissue sample is a sectioned tissue sample having a thickness of 10 µm (i.e. a depth of 30 microns or less) (Mellen et al., paragraph 0140).
Regarding claim 25, Mellen et al. teach the capture spots are circular (i.e. a closed-form shape) (Mellen et al., figure 19).
Regarding claim 26, Mellen et al. teach the capture spots have a diameter of 55 microns (Mellen et al., paragraph 0138) (i.e. between 3 and 90 microns).
Regarding claim 29, Mellen et al. teach each capture spot is at a different position in a two-dimensional array of capture spots on the substrate (Mellen et al., figure 19).
Regarding claim 30, Mellen et al. teach the capture probes (i.e. the capture spots) can be affixed to the substrate as a solid array (i.e. directly coupled), or through a bead (i.e. indirectly coupled) (Mellen et al., paragraph 0085).
Regarding claim 31, Mellen et al. teach a first detectable marker that is indicative of a particular cell type (i.e. having a wild type or variant receptor), and filtering from the display portions of the datasets not associated with the detectable marker (Mellen et al., figure 6 and paragraph 0303)
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Regarding claim 32, Mellen et al. teach a first detectable marker (immunohistochemistry fluorescence for CD3 protein expression) is used to filter those portions of the dataset not associated with CD3 protein expression (i.e. the first detectable marker that is a dye-labeled antibody to a protein expressed by a particular cell type) (Mellen et al., paragraph 0276).
Regarding claim 35, Kjaerulff et al. teach that DNA-binding dyes such as propidium iodide, ethidium bromide, and DAPI stain DNA in nuclei in intact cells as well as in dead or dying cells (Kjaerulff et al., paragraph 0009).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have combined the methods taught by Mellen et al. for quantitative, spatial assessment of mRNA expression from discrete cells or cell populations associated with a particular capture spot with the methods taught by Kjaerulff et al. for determining whether a particular cell or cells are viable (i.e. stained with a DNA-binding dye such as DAPI and a dye indicator of intact cell membranes such as DACM) (i.e. determining a count of cells per capture spot based on detection of nuclei AND an intact membrane).
Claims 23 and 24 are rejected under 35 U.S.C. 103 as being unpatentable over Mellen et al. as evidenced by Lucas et al. in view of Kjaerulff et al. as applied to claims 1-20, 25-26, 29-32, and 35 above, and further in view of Weisenfeld et al., US 2021/0150707 A1 (filed November 18, 2020 and published May 20, 2021).
This rejection has been updated as necessitated by the amendments to the claims.
Regarding claims 1-20, 25, 26, 29-32, and 36, the teachings of Mellen et al. are described in the rejection under 35 U.S.C. 103 in detail above.
Regarding claims 23 and 24, Mellen et al. teach capture spots have a diameter of 100 µm or less and are distributed with a center to center distance of 200 µm or less, and in particular examples, have a diameter of 55 µm and a center to center distance of 100 µm).
Mellen et al. as evidenced by Lucas et al. in view of Kjaerulff et al. do not teach capture spots having a diameter of 2 µm (i.e. contained within a 2 micron by 2 micron square or capture spots having a center to center distance of between 2 microns and 8 microns (i.e. having a diameter between 2 and 8 microns, assuming capture spots do not overlap).
However, Weisenfeld et al. teach similar methods of spatial analysis of tissue samples comprising preparing a tissue slice on an array of capture spots, imaging the tissue and/or array using fluorescent and/or bright field microscopy for multiple detectable markers, preparing spatially indexed, UMI-containing, next-generation sequencing libraries from nucleic acids released by the tissue onto the array of capture spots, sequencing the libraries, and overlaying the detectable marker signals and the sequencing data for each capture spot (Weisenfeld et al., figure 3A, 3B, and 12)
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Weisenfeld et al. further teach that the capture spots on the array can be a variety of sizes including, a diameter no larger than 2 microns (i.e. fitting within a 2 micron by 2 micron square, or having a center to center distance between 2 microns and 8 microns) (Weisenfeld et al., paragraph 0455).
Weisenfeld et al. further teach that resolution of data collected on such arrays of capture spots is dependent upon the diameter, and thus density, of the capture spots, wherein “low resolution arrays” have capture spots with 20 micron diameters or greater (i.e. Weisenfeld et al. describe the arrays taught by Mellen et al. as “low resolution”) and “high resolution arrays” have capture spots with a diameter of about 1 micron to about 10 microns. Weisenfeld additionally suggests “resolution of an array can be improved by constructing an array with smaller capture spots” (Weisenfeld et al., paragraphs 0473-0475).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the methods for analyzing tissue samples taught by Mellen et al. as evidenced by Lucas et al. in view of Kjaerulff et al. with the teachings and suggestions of Weisenfeld et al. to increase the resolution of the capture array by decreasing the diameter of each individual capture spot to between 1 and 10 microns, wherein smaller diameters correspond to higher expected resolution of analytes (i.e. mRNA expression) on a given biological sample. Therefore, the ordinary artisan would have been motivated to select the smallest practicable diameter capture spots in order to predictably attain the highest possible resolution spatial gene expression data by the methods taught by Mellen et al. as evidenced by Lucas et al. in view of Kjaerulff et al. and Weisenfeld et al. (i.e. 1 micron diameter capture spots).
Claims 1-20, 25, 26, 29-32, and 36 are rejected under 35 U.S.C. 103 as being unpatentable over Mellen et al., US 2021/0097684 A1 (filed September 30, 2020 and published April 1, 2021) in view of Kjaerulff et al., US 2012/0045757 A1 (published February 23, 2012).
This rejection has been updated as necessitated by the amendments to the claims.
Regarding claim 1, Mellen et al. teach methods of analyzing tissue samples comprising: obtaining sets of images of the tissue sample overlayed on an array of capture spots, wherein a first dataset is acquired for (a) detectable marker(s) for each capture spot indexed by a spatial barcode corresponding to each capture spot and a second dataset is acquired comprising nucleic acid quantification data indexed by a spatial barcode to each capture spot, wherein the two datasets are co-registered on a display by the spatial barcodes (Mellen et al., Figure 20 and paragraph 0313).
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Mellen et al. further teach the plurality of capture probe spots comprises at least 1000 capture spots (Mellen et al., paragraph 0121).
Mellen et al. do not teach using a measured intensity of a detectable marker that is indicative of the presence of a nucleus to estimate a number of cells in each capture spot.
However, Mellen et al. do teach measuring an intensity of NeuN protein (Mellen et al., figure 20, see above).
However, Kjaerulff et al. teach various methods for automatic detection of fluorescence signals from a sample (Kjaerulff et al., paragraph 0028-0029) comprising staining nuclei with DAPI and viable cells with DACM (N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide) (a dye that reacts with thiols found in proteins in living cells (i.e. cells with intact membranes) (Kjaerulff et al., paragraph 0011 and 0015). Kjaerulff et al. further teach that automated methods comprising staining cell nuclei and viable cells having intact membranes provide for rapid detection and quantitation of live cells (Kjaerulff et al., paragraph 0146).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have combined the methods taught by Mellen et al., comprising automated brightfield and fluorescent imaging of tissues (including nuclear stains such as DAPI, NeuN antibodies or Hematoxylin and cytosolic stains such as Eosin and cell-membrane associated stains such as fluorescently-labeled anti-CD3 antibodies) associated with specific capture spots with the methods taught by Kjaerulff et al. comprising automatically detecting and counting intact cells (having nuclei stained with a DNA binding dye and intact membranes) on a substrate.
The ordinary artisan would have been motivated to count the number of cells per capture spot in the method taught by Mellen et al. by the cell-staining methods taught by Kjaerulff et al. because the ordinary artisan would have recognized the benefits of a) normalizing measured mRNA expression values to a concrete count of the number of cells from which the mRNA molecules are derived, b) excluding background counts from capture spots that do not contain a cell (i.e. due to diffusion of mRNA from neighboring spots or contamination), and/or c) determining which capture spots are representative of a single cell type (i.e. a cancer cell) within a mixed population of cells (i.e. a section of a tumor sample).
Regarding claim 2, Mellen et al. teach the datasets are spatially displayed in an interactive manner on a computer system (Mellen et al., paragraph 0329).
Regarding claim 3, Mellen et al. teach that users can perform computer-implemented actions such as zooming, panning, and adjusting opacity of all or a portion of the first and second datasets (Mellen et al., paragraphs 0285-0288).
Regarding claim 4, Mellen et al. teach the obtaining uses fluorescence microscopy (Mellen et al., paragraph 0102).
Regarding claim 5, Mellen et al. teach exposing the tissue sample to the set of detectable markers prior to the obtaining step (Mellen et al., paragraph 0103).
Regarding claims 6 and 7, Mellen et al. teach the detectable markers are fluorescently-labeled antibodies (Mellen et al., paragraph 0103).
Regarding claim 8, Mellen et al. teach detectable markers in the set of detectable markers includes: “Haemotoxylin and Eosin, a Periodic acid-Schiff reaction stain, a Masson’s trichome stain, an Alcian blue stain, a Van Gieson stain, a reticulin stain, an Azan stain, a Giemsa stain, a Toluidine blue stain, an isamin blue/eosin stain, a NissI and methylene blue stain, a Sudan black and/or osmium staining (Mellen et al., paragraphs 0018 and 0091-0102). Mellen et al. further teach the samples can be stained with “acridine orange, Bismarck brown, carmine, Coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetraoxide, propidium iodide, rhodamine, safranine, and/or an immunofluorescence stain” (Mellen et al., paragraph 0147).
Regarding claim 9, Mellen et al. teach each image in the set of images comprises at least 100,000 pixel values (Mellen et al., paragraph 0010).
Regarding claim 10, Mellen et al. teach that a spatial dataset comprises obtaining sequence reads from 5000 molecularly barcoded, spatially encoded capture probes in probe spots (i.e. 1000 or more capture probes per capture spot), wherein the capture probes associate with native mRNA (i.e. a nucleic acid from the tissue sample), wherein the capture probes incorporate a spatial barcode corresponding to the particular capture spot and a unique molecular index into a resulting cDNA sequencing library (Mellen et al., paragraph 0138 or 0309). Mellen et al. further teach grouping sequence reads by spatial barcodes and unique molecular indices, and assigning the sequence reads to a respective capture spot (Mellen et al., paragraph 0171). Furthermore, Mellen et al. teach collecting one hundred thousand or more (i.e. at least 10,000) sequence reads for a tissue sample (Mellen et al., paragraph 0135).
Regarding claim 11, Mellen et al. teach each capture probe comprises a poly-T sequence and a unique spatial barcode corresponding to a particular capture spot (Mellen et al., figure 19)
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Regarding claim 12, Mellen et al. teach that each capture probe in the respective capture spot includes the same spatial barcode (Mellen et al., figure 19).
Regarding claim 13, Mellen et al. teach that each capture probe in the respective capture spot includes the same spatial barcode AND a Unique Molecular Identifier (UMI) (i.e. each capture probe includes a different spatial barcode; a “unique spatial barcode”) (Mellen et al., paragraph 0081-0082).
Regarding claim 14, Mellen et al. teach the obtaining comprises high-throughput sequencing (Mellen et al., paragraph 0309).
Regarding claim 15, Mellen et al. teach that each capture probe in the respective capture spot includes the same spatial barcode (Mellen et al., figure 19).
Regarding claim 16, Mellen et al. teach that teach representation of a number of molecules is a count of a number of unique sequence reads having the same spatial barcode associated with a particular capture spot (Mellen et al., paragraph 0024-0026).
Regarding claim 17, Mellen et al. teach the plurality of loci (i.e. distinct transcripts; nucleic acids) comprises more than 1000 loci (Mellen et al., paragraph 0027 or paragraph 0175).
Regarding claim 18, Mellen et al. teach the plurality of nucleic acids comprises RNA or DNA (Mellen et al., paragraph 0313).
Regarding claim 19, Mellen et al. teach the plurality of nucleic acids comprises mRNA (Mellen et al., paragraph 0313).
Regarding claim 20, Mellen et al. teach the tissue sample is a sectioned tissue sample having a thickness of 10 µm (i.e. a depth of 30 microns or less) (Mellen et al., paragraph 0140).
Regarding claim 25, Mellen et al. teach the capture spots are circular (i.e. a closed-form shape) (Mellen et al., figure 19).
Regarding claim 26, Mellen et al. teach the capture spots have a diameter of 55 microns (Mellen et al., paragraph 0138) (i.e. between 3 and 90 microns).
Regarding claim 29, Mellen et al. teach each capture spot is at a different position in a two-dimensional array of capture spots on the substrate (Mellen et al., figure 19).
Regarding claim 30, Mellen et al. teach the capture probes (i.e. the capture spots) can be affixed to the substrate as a solid array (i.e. directly coupled), or through a bead (i.e. indirectly coupled) (Mellen et al., paragraph 0085).
Regarding claim 31, Mellen et al. teach a first detectable marker that is indicative of a particular cell type (i.e. having a wild type or variant receptor), and filtering from the display portions of the datasets not associated with the detectable marker (Mellen et al., figure 6 and paragraph 0303)
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Regarding claim 32, Mellen et al. teach a first detectable marker (immunohistochemistry fluorescence for CD3 protein expression) is used to filter those portions of the dataset not associated with CD3 protein expression (i.e. the first detectable marker that is a dye-labeled antibody to a protein expressed by a particular cell type) (Mellen et al., paragraph 0276).
Regarding claim 35, Kjaerulff et al. teach that DNA-binding dyes such as propidium iodide, ethidium bromide, and DAPI stain DNA in nuclei in intact cells as well as in dead or dying cells (Kjaerulff et al., paragraph 0009).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have combined the methods taught by Mellen et al. for quantitative, spatial assessment of mRNA expression from discrete cells or cell populations associated with a particular capture spot with the methods taught by Kjaerulff et al. for determining whether a particular cell or cells are viable (i.e. stained with a DNA-binding dye such as DAPI and a dye indicator of intact cell membranes such as DACM) (i.e. determining a count of cells per capture spot based on detection of nuclei AND an intact membrane).
Regarding claim 35, Kjaerulff et al. teach that DNA-binding dyes such as propidium iodide, ethidium bromide, and DAPI stain DNA in nuclei in intact cells as well as in dead or dying cells (Kjaerulff et al., paragraph 0009).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have combined the methods taught by Mellen et al. for quantitative, spatial assessment of mRNA expression from discrete cells or cell populations associated with a particular capture spot with the methods taught by Kjaerulff et al. for determining whether a particular cell or cells are viable (i.e. stained with a DNA-binding dye such as DAPI and a dye indicator of intact cell membranes such as DACM) (i.e. determining a count of cells per capture spot based on detection of nuclei AND an intact membrane).
Claims 23 and 24 are rejected under 35 U.S.C. 103 as being unpatentable over Mellen et al. in view of Kjaerulff et al. as applied to claims 1-20, 25-26, 29-32, and 35 above, and further in view of Weisenfeld et al., US 2021/0150707 A1 (filed November 18, 2020 and published May 20, 2021).
This rejection has been updated as necessitated by the amendments to the claims.
Regarding claims 1-20, 25, 26, 29-32, and 36, the teachings of Mellen et al. are described in the rejection under 35 U.S.C. 103 in detail above.
Regarding claims 23 and 24, Mellen et al. teach capture spots have a diameter of 100 µm or less and are distributed with a center to center distance of 200 µm or less, and in particular examples, have a diameter of 55 µm and a center to center distance of 100 µm).
Mellen et al. in view of Kjaerulff et al. do not teach capture spots having a diameter of 2 µm (i.e. contained within a 2 micron by 2 micron square or capture spots having a center to center distance of between 2 microns and 8 microns (i.e. having a diameter between 2 and 8 microns, assuming capture spots do not overlap).
However, Weisenfeld et al. teach similar methods of spatial analysis of tissue samples comprising preparing a tissue slice on an array of capture spots, imaging the tissue and/or array using fluorescent and/or bright field microscopy for multiple detectable markers, preparing spatially indexed, UMI-containing, next-generation sequencing libraries from nucleic acids released by the tissue onto the array of capture spots, sequencing the libraries, and overlaying the detectable marker signals and the sequencing data for each capture spot (Weisenfeld et al., figure 3A, 3B, and 12)
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Weisenfeld et al. further teach that the capture spots on the array can be a variety of sizes including, a diameter no larger than 2 microns (i.e. fitting within a 2 micron by 2 micron square, or having a center to center distance between 2 microns and 8 microns) (Weisenfeld et al., paragraph 0455).
Weisenfeld et al. further teach that resolution of data collected on such arrays of capture spots is dependent upon the diameter, and thus density, of the capture spots, wherein “low resolution arrays” have capture spots with 20 micron diameters or greater (i.e. Weisenfeld et al. describe the arrays taught by Mellen et al. as “low resolution”) and “high resolution arrays” have capture spots with a diameter of about 1 micron to about 10 microns. Weisenfeld additionally suggests “resolution of an array can be improved by constructing an array with smaller capture spots” (Weisenfeld et al., paragraphs 0473-0475).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the methods for analyzing tissue samples taught by Mellen et al. in view of Kjaerulff et al. with the teachings and suggestions of Weisenfeld et al. to increase the resolution of the capture array by decreasing the diameter of each individual capture spot to between 1 and 10 microns, wherein smaller diameters correspond to higher expected resolution of analytes (i.e. mRNA expression) on a given biological sample. Therefore, the ordinary artisan would have been motivated to select the smallest practicable diameter capture spots in order to predictably attain the highest possible resolution spatial gene expression data by the methods taught by Mellen et al. in view of Kjaerulff et al. and Weisenfeld et al. (i.e. 1 micron diameter capture spots).
Response to arguments
The response asserts “applicant has amended 1 to incorporate the limitations of non-rejected claim 33. Applicant notes that the remaining claims depend from claim 1… In light of the amendment to claim 1… requests withdrawal of the rejections under 35 U.S.C. 103.
This assertion has been thoroughly reviewed and is not persuasive.
Claim 33 is/was previously rejected as unpatentable under 35 U.S.C. 103 over Mellen et al. in view of Kjaerulff et al. in the office action dated March 10, 2026 (see page 19, item # 16).
The rejection over Mellen et al. in view of Kjaerulff et al. does not appear to have been addressed in the response.
These arguments fail to comply with 37 CFR 1.111(b) because they amount to a general allegation that the claims define a patentable invention without specifically pointing out how the language of the claims patentably distinguishes them from the references.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claim 1 is rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 8, and 15 of U.S. Patent No. 11,514,575 B2, (herein referred to as ‘575) in view of Kjaerulff et al., US 2012/0045757 A1 (published February 23, 2012).
This rejection has been updated as necessitated by the amendments to the claims.
Claim 1 of ‘575 recites a method comprising steps using a computer system to: for each capture spot in an array of capture spots, obtaining one or more spatial projections of a biological sample, obtaining one or more two-dimensional images of a tissue section from the biological sample overlaid on a substrate having the array of capture probes, obtaining a corresponding set of discrete attribute values obtained from two-dimensional spatial sequencing of the tissue section, wherein each attribute value is for a different loci (i.e. mRNA molecule counts in each capture spot); obtaining cluster assignments of each capture spot based on the two-dimensional images and the attribute values, and displaying pixel values of all or a portion of the two-dimensional images overlaid with each capture spot (‘575, claim 1). Claim 8 of ‘575, which depends on claim 1, further recites the two-dimensional images comprise bright-field images and immunohistochemistry images taken at different wavelengths (i.e. emission images of detectable immunohistochemistry markers). Finally, claim 15 of ‘575, which depends on claim 1, further recites that each of the “loci” for which “attribute values” are collected, recited by claim 1, are each a respective gene, and that each attribute value is a count of unique molecular indices that also map to a respective gene (i.e. the attribute values assigned to each capture spot are representations of numbers of molecules of a nucleic acid originating from the tissue sample).
The claims of ‘575 do not recite using a measured intensity of a detectable marker that is indicative of the presence of a nucleus to estimate a number of cells in each capture spot.
However, Kjaerulff et al. teach various methods for automatic detection of fluorescence signals from a sample (Kjaerulff et al., paragraph 0028-0029) comprising staining nuclei with DAPI and viable cells with DACM (N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide) (a dye that reacts with thiols found in proteins in living cells (i.e. cells with intact membranes) (Kjaerulff et al., paragraph 0011 and 0015). Kjaerulff et al. further teach that automated methods comprising staining cell nuclei and viable cells having intact membranes provide for rapid detection and quantitation of live cells (Kjaerulff et al., paragraph 0146).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have combined the methods claimed by ‘575, comprising automated brightfield and fluorescent imaging of tissues associated with specific capture spots with the methods taught by Kjaerulff et al. comprising automatically detecting and counting intact cells (having nuclei stained with a DNA binding dye and intact membranes) on a substrate.
The ordinary artisan would have been motivated to count the number of cells per capture spot in the method recited by the claims of ‘575 by the cell-staining methods taught by Kjaerulff et al. because the ordinary artisan would have recognized the benefits of a) normalizing measured mRNA expression values to a concrete count of the number of cells from which the mRNA molecules are derived, b) excluding background counts from capture spots that do not contain a cell (i.e. due to diffusion of mRNA from neighboring spots or contamination), and/or c) determining which capture spots are representative of a single cell type (i.e. a cancer cell) within a mixed population of cells (i.e. a section of a tumor sample).
Claim 1 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 and 64 of copending Application No. 16/951,864 (herein referred to as ‘864) in view of Kjaerulff et al., US 2012/0045757 A1 (published February 23, 2012).
This rejection has been updated as necessitated by the amendments to the claims.
Claim 1 of ‘864 recites steps comprising: placing a sample on a substrate comprising fiducial markers and at least 1000 capture spots, obtaining one or more images of the sample, obtaining sequence reads from the capture spots wherein each capture probe a capture spot comprises a unique spatial barcode associated with a particular location on the substrate, and providing a composite representation of the one or more images aligned to the capture spots and a representation of the sequence reads at each position within the one or more images that maps to a capture spot (i.e. spatially displaying the two datasets co-registered by the plurality of spatial barcodes) (‘864, claim 1).
Claim 64 depends from claim 1 and further claims that the one or more images comprises brightfield or fluorescence images.
The claims of ‘864 do not recite using a measured intensity of a detectable marker that is indicative of the presence of a nucleus to estimate a number of cells in each capture spot.
However, Kjaerulff et al. teach various methods for automatic detection of fluorescence signals from a sample (Kjaerulff et al., paragraph 0028-0029) comprising staining nuclei with DAPI and viable cells with DACM (N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide) (a dye that reacts with thiols found in proteins in living cells (i.e. cells with intact membranes) (Kjaerulff et al., paragraph 0011 and 0015). Kjaerulff et al. further teach that automated methods comprising staining cell nuclei and viable cells having intact membranes provide for rapid detection and quantitation of live cells (Kjaerulff et al., paragraph 0146).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have combined the methods claimed by ‘864, comprising automated brightfield and fluorescent imaging of tissues associated with specific capture spots with the methods taught by Kjaerulff et al. comprising automatically detecting and counting intact cells (having nuclei stained with a DNA binding dye and intact membranes) on a substrate.
The ordinary artisan would have been motivated to count the number of cells per capture spot in the method recited by the claims of ‘864 by the cell-staining methods taught by Kjaerulff et al. because the ordinary artisan would have recognized the benefits of a) normalizing measured mRNA expression values to a concrete count of the number of cells from which the mRNA molecules are derived, b) excluding background counts from capture spots that do not contain a cell (i.e. due to diffusion of mRNA from neighboring spots or contamination), and/or c) determining which capture spots are representative of a single cell type (i.e. a cancer cell) within a mixed population of cells (i.e. a section of a tumor sample).
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Response to arguments
The response asserts:
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This assertion has been thoroughly reviewed and is not persuasive.
These arguments fail to comply with 37 CFR 1.111(b) because they amount to a general allegation that the claims define a patentable invention without specifically pointing out how the language of the claims patentably distinguishes them from the references.
Conclusion
No claim is allowed.
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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/Z.M.T./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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