DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
2. Claims 1-14 are pending and have been examined herein.
Objection to Drawings / Objection to the Specification
3. The drawings are objected to under 37 CFR 1.83(a). The specification describes the figures, for example Figures 2, and 4-6, in terms of particular colors. For instance, in describing Figure 6, the specification (para [0015]; paragraph numbering herein is with respect to the published application) states “Only cells adjacent or in direct contact with the printed area show red fluorescence.” Para [0013] recites “colors indicating clusters” and para [0014] recites “colors indicating expected clustering.” At para [0105], in describing Figure 2, it is stated that “As shown in FIG. 2 , cells in the hashed nanowells (blue circles) show strong red fluorescence” However, the figures have been filed in black and white. Thus, the description of the figures in the specification is not consistent with the drawing and the specification is thereby also objected to.
If the drawings are intended to be in black and white, the specification should be amended to delete the reference to the recited colors. Alternatively, corrected drawing sheets in compliance with 37 CFR 1.121(d) are required. Note that color drawings are only accepted on rare occasions when they are the only practical medium by which to disclose the subject matter to be patented. See MPEP 608.02(VIII).
Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
If color photographs or color drawings are submitted, it is noted that color photographs and color drawings are not accepted unless a petition filed under 37 CFR 1.84(a)(2) is granted.
A petition for color drawings must be accompanied by the appropriate fee set forth in 37 CFR 1.17(h), one set of color drawings or color photographs, as appropriate, if submitted via EFS-Web or three sets of color drawings or color photographs, as appropriate, if not submitted via EFS-Web, and, unless already present, an amendment to include the following language as the first paragraph of the brief description of the drawings section of the specification:
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Color photographs will be accepted if the conditions for accepting color drawings and black and white photographs have been satisfied. See 37 CFR 1.84(b)(2).
Appropriate correction is required.
Claim Rejections - 35 USC § 103
4. 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 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.
Claim(s) 1-14 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ramachandran Iyer et al (WO 2020/176788, published 03 September 2020).
Ramachandran Iyer (p. 3, lines 10-20) teaches:
“methods for identifying a location of a biological analyte in a biological sample, the method comprising: (a) providing a biological sample comprising live cells; (b) contacting the biological sample with a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises (i) a spatial barcode and (ii) a capture domain that binds specifically to a biological analyte in the biological sample; and (c) determining (i) all or a part of the sequence corresponding to the biological analyte specifically bound to the capture domain, or a complement thereof, and (ii) all or a part of the sequence of the spatial barcode or a complement thereof, and using the determined sequences of (i) and (ii) to identify the location of the biological analyte in the biological sample.”
In the method of Ramachandran Iyer, the capture probe comprising the spatial barcode constitutes a “labelling substrate.”
Ramachandran Iyer further teaches:
“In some embodiments, a live cell of the plurality of live cells is distributed onto the substrate” (p. 4, lines 8-10). It is also stated that “The sample can be dissociated into non-aggregated cells (e.g., single cells) and analyzed by the single cell / droplet methods described herein” (p. 107, lines 3-5). At p. 370, lines 12-15 and p. 372, lines 4-7, it is disclosed that the cells may be from a cell culture.
Thus, regarding step (a) of claim 1, the method of Ramachandran Iyer is one in which single live cells are distributed on a substrate. The substrates on which the cells are distributed are considered to constitute a “cell culture substrate.”
Regarding steps (b) and (c) of claim 1, Ramachandran Iyer states (p. 298, lines 9-20):
“a cell-tagging agent comprises a cell-penetrating agent (described below). In some embodiments, a cell-penetrating agent transports the cell-tagging agent into the cells of a biological sample. When a cell-tagging agent comprises a barcode (e.g., a nucleic acid that includes a spatial barcode), the barcode also penetrates into the cell. In some embodiments, a plurality of cell-tagging agents are cleaved (e.g., photocleaved) from an array via a cleavage domain, thus freeing the cell-tagging agents from the array and allowing at least one capture probe of the plurality to penetrate a cell. The cell-tagging agent can then interact with an intracellular biological analyte via the capture domain. In some embodiments, the plurality of capture probes is migrated from the array into cells of the biological sample via cell-penetrating agents. In some embodiments, migrating a plurality of capture probes from the array to cells of the biological sample includes applying a force (e.g., mechanical, centrifugal, or electrophorectic) to the biological sample.”
Accordingly, Ramachandran Iyer teaches preparing a labelling substrate / capture probe, wherein labelling substrate / capture probe comprises oligonucleotides comprising spatial barcodes and labelling the single cells with labelling substrates / capture probes.
Regarding step (e) of claim 1, Ramachandran Iyer teaches sequencing the single cells labeled with the labeling substrate / capture probes.
For example, Ramachandran Iyer (p. 272, lines 1-9) states:
“After analytes from the sample have hybridized or otherwise been associated with capture probes, analyte capture agents, or other barcoded oligonucleotide sequences according to any of the methods described above in connection with the general spatial cell- based analytical methodology, the barcoded constructs that result from hybridization/association are analyzed via sequencing to identify the analytes.”
Ramachandran Iyer does not specifically teach step (d) of claim 1 wherein, prior to sequencing, a suspension is prepared comprising the labeled single cells.
However, Ramachandran Iyer does teach:
“For analytes that have been barcoded via partitioning, barcoded nucleic acid molecules or derivatives thereof (e.g., barcoded nucleic acid molecules to which one or more functional sequences have been added, or from which one or more features have been removed) can be pooled and processed together for subsequent analysis such as sequencing on high throughput sequencers” (p. 275, lines 10-14).
“For analytes that have been barcoded via partitioning with beads, as described above, individual analytes (e.g., cells, or cellular contents following lysis of cells) can be extracted from the partitions by breaking the partitions, and then analyzed by sequencing to identify the analytes” (p. 331, lines 9-12).
Ramachandran Iyer teaches that the cells can be attached to beads which are suspected in solution in individual wells (e.g., p. 175, lines 22-30), e.g., “The beads can also be suspended in a solution and deposited on a surface (e.g., a membrane, a tissue section, or a substrate (e.g., a microscope slide).” See also p. 149, lines 25-34 and p. 249, lines 18-20.
Methods are also disclosed therein wherein a microfluidic device is used to partition individual cells into discrete droplets in discrete partitions (e.g., p. 248, lines 22-30; p. 249, lines 7-24).
Additionally, Ramachandran Iyer teaches that single cells portioned in a microcapillary array can be collected following being labeled with a barcoded capture probe and processed individually for sequencing or pooled for sequence analysis (p. 291, line 20 to p. 202, line 8).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have included a step in the method of Ramachandran Iyer of preparing a solution comprising the capture probe labelled cells prior to performing sequencing. One would have been motivated to have done so because Ramachandran Iyer teaches that the cells can be in solution / in suspension after being labeled with the capture probe so as to facilitate further analysis of the cells, particularly by sequencing the nucleic acids present in the single cells.
Regarding claim 2-4, Ramachandran Iyer teaches that the single cells are present in a series of wells, which thereby compartmentalize the single cells, including nano-wells (e.g., Fig. 29B; p. 12, lines 17-19; p. 202, lines 10-11;p. 247 line 31 to p. 248, line 4; p. 112, line 20 to p. 113, line 30).
Regarding claims 5 and 6, Ramachandran Iyer teaches that the cells may have regions that show morphological features that may indicate the presence of a disease or the development of a “disease phenotype” and that morphological and histological features can be identified by staining and imaging a cell (p. 245, line 12 to p. 246 line 7; p. 404, lines 20-30). It is stated that the staining and imaging are performed prior to contacting the sample (cell) with a spatial array (comprising the capture probe / labeling substrate; see, e.g., p. 146, lines 8-13). Accordingly, Ramachandran Iyer teaches methods which further comprise determining a phenotypic or morphologic measurement of the single cells after preparing the cell culture substrate and before labeling the single cells with the capture probe / labeling substrate.
Regarding claim 7, Ramachandran Iyer teaches that the capture probe / labelling substrate comprises a lipid-modified / conjugated oligonucleotide (e.g., p. 306, line 11 to p. 307, line 19):
“In some embodiments, a cell-tagging agent can attach to a surface of a cell through a combination of lipophilic and covalent attachment. For example, a cell-tagging agent can include an oligonucleotide attached to a lipid to target the oligonucleotide to a cell membrane, and an amine group that can be covalently linked to a cell surface protein(s) via 15 any number of chemistries described herein. In these embodiments, the lipid can increase the surface concentration of the oligonucleotide and can promote the covalent reaction.
As used herein, an "anchor oligonucleotide" and/or "co-anchor oligonucleotide" can include a lipid-conjugated oligonucleotide, wherein the lipid is capable of becoming
embedded within a cell membrane. In some embodiments, the lipid capable of becoming embedded within a cell membrane includes but is not limited to, sterol lipids such as cholesterol, tocopherol, steryl, palmitate, lignoceric acid, and derivatives thereof. In some embodiments, the sterol lipid of the anchor oligonucleotide and/or co-anchor oligonucleotide can be attached to either the 5' or 3' end of the oligonucleotide portion. In some embodiments, the anchor oligonucleotide and/or the co-anchor oligonucleotide can integrate 25 into the cell membrane of a cell in a biological sample (e.g., the sterol lipid of the anchor oligonucleotide and/or co-anchor oligonucleotide)” (p. 306, lines 11-26). “
Regarding claim 8, Ramachandran Iyer teaches that the capture probe / labelling substrate may be embedded within a gel scaffold (e.g., Fig. 20B and p. 12, lines 17-19).
Regarding claim 9, Ramachandran Iyer teaches that the capture probe / labelling substrate is attached to (i.e., deposited) onto a surface (e.g., p. 92, lines 3-12; p. 124, lines 1-4 and lines 20-24).
Regarding claim 10, Ramachandran Iyer teaches that the surface is an array, a slide, or patterned hydrogels (e.g., p. 4, lines 14-18; p. 112, lines 20-33; p. 202, lines 10-24).
Regarding claim 11, as discussed above, in the method of Ramachandran Iyer , the labelling step is carried out under conditions that allow capture probes / labelling substrates to contact the single cells (e.g., p. 298, lines 9-20).
. Regarding claim 12, Ramachandran Iyer teaches that the method comprises cleaving the capture probe / labeling substrate to release the capture probe / labeling substrate from the surface (e.g., p. 307, lines 21-34).
Regarding claim 13, modification of the method of Ramachandran Iyer as discussed above would have comprised disassociating the single cells from a support on which they were attached and collecting the labeled single cells. One would have been motivated to have included these steps in the method of Ramachandran Iyer so as to have accomplished the objectives set forth therein of facilitating the sequencing and further analysis of the single cells, either individually or as pools of single cells.
Regarding claim 14, Ramachandran Iyer (p. 17, lines 28-33) teaches “spatial analysis methodologies can be used to detect the differences in analyte levels (e.g., gene and/or protein expression) within different cells in histological slide samples, the data from which can be reassembled to generate a three-dimensional map of analyte levels (e.g., gene and/or protein expression) of a tissue sample obtained from a mammal, e.g., with a degree of spatial resolution (e.g., single cell resolution). Ramachandran Iyer also teaches methods wherein a sample that has been histologically evaluated by a pathologist is subsequently analyzed for gene expression levels correlated with cancer (p. 368, line 3 to p. 369, line 12). Thus, the teachings of Ramachandran Iyer suggest including a step of correlating the phenotypic or morphologic measurement with the sequencing information.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CARLA J MYERS whose telephone number is (571)272-0747. The examiner can normally be reached M-Th 6:30-5:00 EST.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Wu-Cheng Winston Shen can be reached on 571-272-3157. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/CARLA J MYERS/Primary Examiner, Art Unit 1682
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