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 .
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.
Claim(s) 1, 2, 4, 5, 6, 10, 12, 13, 14, and 23 is/are rejected under 35 U.S.C. 103 as being unpatentable over Iyer et al. (US 2020/0277663 A1) in view of Bruno.
The reference teaches a method for labeling nucleic acids in a cell of a cell culture or tissue, the method comprising contacting the cell culture or tissue with a first label for labelling nucleic acids and applying an electrical field to the cell, thereby increasing a permeability of a cell membrane of the cell, thereby allowing the first label to be introduced to the cell.
Paragraph 1119 teaches that in molecules having a plurality of barcodes are migrated from a substrate to cells of a biological sample. The reference teaches that the biological sample includes tissues and cell culture samples (¶34). The reference teaches that physical force is used to facilitate introduction of a nucleic acid having a barcode into a cell present in a biological sample, i.e. tissue. Physical force can be electroporation (¶ 1120, ¶ 1142). Thus, the reference clearly teaches using electroporation to introduce barcode molecules into cells.
With regard to claim 4, the label is a nucleic acid barcode.
With regard to claim 12, the barcoded nucleic acid was inherently in fluid that is contacted with the cell culture or tissue.
With regard to claim 13, the reference teaches extracting nucleic acid molecules from the cell and sequencing them (¶14-16, for example).
With regard to claim 14, the reference teaches obtaining the transcriptome of the cell culture or tissue based on the sequencing (¶14-16, for example).
Iyer et al. does not teach increasing permeability of a cell membrane of a first cell to introduce the first label into the first cell by applying an electric field to the first cell via an electrode of a multielectrode array.
Iyer et al. teaches employing electrodes in spatial analysis of samples to effect active migration. In figure 21 the reference shows steps of tissue fixation, and then cleavage of a probe from the array. The capture probe migrates into the sample. The reference teaches that the capture probe can diffuse towards the cells by active migration (¶1216), and the reference teaches that active migration includes via an electrophoretic transfer system (¶327). The reference teaches that an electrophoretic field can be applied to analytes to facilitate migration of analytes toward a capture probe (¶902). The reference teaches that negatively charged nucleic acid analytes can be pulled toward a positively charged anode, and that the skilled practitioner has the knowledge and experience to arrange the electrophoretic transfer system to facilitate capture of particular targets (i.e. movement of particular analytes) (¶908). Iyer teaches electrophoretic capture performed on a spatially-addressable microelectrode array (MEA), and that the density of the surface can be up to 500,000 electrodes per 1 mm2 (¶0907).
Additionally, Iyer teaches that the fixed tissue samples can include brain tissue, which inherently includes neurons (¶117 and ¶230).
Furthermore, Iyer teaches that the probes which comprise barcodes can be associated with spatial barcode sequences common to the probes in other regions (¶407), or that a particular point on an array can be coded with two spatial barcodes, where each spatial barcode identifies a particular defined region within the array, and an array point possessing both spatial barcodes identifies the sub-region where two defined regions overlap (¶418). A single barcode that is applied to more than one region on an array with a density that is less than or equal to cellular density would include inserting the same barcode into more than one cell, in which case a first barcode would enter a first and second cell. See instant claim 23. Alternatively, if the density of the array were such that more than one cell was present over a single electrode, then the activation of the electrode would result in a barcode entering more than one cell.
Iyer does not specifically teach a multielectrode array wherein the density of electrodes is equal to or higher than a density of cells in the cell culture or tissue, nor does Iyer teach an embodiment which directly combines the microelectrode array, electroporation, a neuron, and methods wherein the barcoded molecule enters the cell.
However, it would have been prima facie obvious to one having ordinary skill in the at before the invention was made to have contacted the cell culture or tissue with a multielectrode array, as taught by Iyer in order to provide a mechanism to facilitate entry of desired barcoded molecules into the cells or tissue. As Iyer teaches multielectrode arrays having a density of up to up to 500,000 electrodes per 1 mm2 (¶ 0907), it would have been obvious to have employed such a high-resolution MEA in order to facilitate high density delivery of molecules to cells embedded in tissues or cell cultures. Such a high density would have inherently been at least one electrode per cell in a brain tissue sample.
Furthermore, it would have been obvious to apply the methods of Iyer that include electroporation of barcoded molecules in to cells to neurons or brain tissue comprising neurons, as this is a specific embodiment of sample taught by Iyer, and the sample could have been used with the predictable outcome of spatial analysis of the sample.
With regard to claim 9, Iyer does not teach recording the time at which the first label is introduced into a cell. However, the examiner takes Official notice that before the invention was made it was is standard laboratory procedure to record the at which an experiment was run in a laboratory notebook or log. Therefore, it would have been obvious to one having ordinary skill in the art to have implemented standard laboratory procedure and recorded the time at which the assay which included adding the barcoded labels to the cell of Iyer was carried out. One would have been motivated to do this for careful and complete record keeping.
Bruno discloses providing a high-density microfluidic microelectrode array capable of electroporation and obtaining electrophysiological data in neuron cell culture (Electrophysiological Recordings and Electroporation; p. 4).
The reference further teaches increasing permeability of a cell membrane of a first cell of a plurality of cells to introduce a caffeine by applying an electric field to the first cell via an electrode of the multielectrode array that is located under the first cell (p. 4, 2nd column). The reference teaches that the MEA enables localized delivery of cargo, ensuring high spatial localization.
The reference teaches that the system is capable of delivery of nucleic acids and other molecules to examine gene expression on a chip (page 9, right hand column). The reference teaches that the electrode array provides a functional approach that combines high precision cell response monitoring with simultaneous molecule delivery.
It would have been obvious to have modified the method taught by Iyer so as to have employed the multielectrode array to induce cell permeability and allow the entry of the barcoded molecules into a cell or cells. One would have been motivated to do so by the teachings of Bruno which exemplify delivering cargos directly into cells and demonstrate using cell electroporation to permeabilize cells. It would have been prima facie obvious to have modified the method taught by Iyer so as to have included employing a microelectrode array system that was capable of both molecule delivery via electroporation and obtaining physiological data. This would have enabled the skilled artisan to obtain both gene expression and other physiological data about cells of interest by using the gene expression analysis technique taught by Iyer along with the physiological data collection technique taught by Bruno for an expanded analysis of cells, such as neurons in culture or tissue samples.
Claim(s) 7, 8, 21, and 22 is/are rejected under 35 U.S.C. 103 as being unpatentable over Iyer et al. in view of Bruno as applied to claims above, and further in view of Zhaung et al. (US 11098303).
The teachings of Iyer et al. in view of Bruno are given previously in this Office action are fully incorporated here.
Furthermore, Iyer teaches combining barcoding of the cells with in-situ sequencing (Example 3).
Iyer in view of Bruno does not teach the washing steps required in claims 7, 8, and 22.
Zhuang teaches a method for imaging or determining nucleic acids within cells, for example, determining the transcriptome of a cell. (Col. 1, lines 53-57). The reference teaches that the sample containing cells includes a cell culture or a biological tissue (Col. 13, lins 10-15). The reference teaches that primary probes that encode codewords (i.e. nucleic acid barcodes) are introduced into a cell and then hybridized with secondary labeled probes to label the cell and the primary probes (Col 8, lines 1-10; lines 15-18). The reference teaches that the hybridizing of primary and secondary probes can be repeated and probes may be removed between application of different rounds of probes (Col. 12, lines 3-5). The reference teaches repeating this process for at least three rounds (Col. 12, lines 17-18). The reference teaches that after each round of hybridization the labels used to visualize the probes may be removed by stringent washing (Col. 36, line 5). The reference teaches that each round of FISH includes at 30 minutes of hybridization (Col. 41, line 26), and so, two probes are introduced and then read thirty minutes apart, and the state of the cell with regard to hybridization is read at least 30 minutes apart. Finally, the reference teaches that the nucleic acid probes may be introduced into the cell by electroporation (Col. 14, lines 50-53).
Therefore, it would have been obvious to have additionally included steps of introducing codeword labels into cells in sequence in order to carry out sequencing by hybridization of the cells taught by Iyer et al. One would have been motivated by the direct teaching of Iyer that the barcoded cells should subsequently be exposed to sequencing by hybridization, and by the teaching of Zhuang et al. to do so, particularly also teaching introduction of the probes by electroporation.
Claim(s) 11 is/are rejected under 35 U.S.C. 103 as being unpatentable over Iyer et al. in view of Bruno as applied to claims above, and further in view of Dunne et al. (US 20170136458).
The teachings of Iyer et al. in view of Bruno are given previously in this Office action are fully incorporated here.
Iyer does not teach a method wherein applying electrical fields to cell of the cell culture or tissue via electrodes of the multielectrode array is repeated for each column and each row of the array, thereby introducing into cells of the cell culture or tissue that are proximate to electrodes of the same column or row is introduced a same label and different labels are introduced to different rows or columns.
Dunne et al. teaches labelling nucleic acids in a multiwell array by introducing a row-specific barcode to each row and a column specific barcode to each column as a way to label the cell with the precise position of the cell in the addressable array (¶ 33 and 34, for example).
It would have been prima facie obvious to one having ordinary skill in the art to have modified the methods taught by Iyer so as to have introduced column and row specific barcodes to the cells on the surfaces taught by Iyer et al. One would have been motivated to do so by the teachings of Dunne et al. who provide a precise way to label cells with their location on an array. One would have been motivated to introduce the barcode pairs (column, row) either simultaneously or sequentially because these are the only two choices and the order of steps would not affect the outcome of labeling the cells with a precise location. Therefore, based on the teachings of the prior art, the claimed invention would have been prima facie obvious before the effective filing date.
Response to Remarks
Any rejection not reiterated was overcome by amendment.
The rejections have been updated to address the amended claims. The remarks are addressed insofar as they are relevant to the new rejections.
Applicant argues that Iyer does not teach using electrodes for "increasing permeability," which is an electroporative effect. This is addressed in the newly set forth rejection by the teachings of Bruno which teaches using electrodes on a multielectrode array for an electroporative effect.
Applicant argues that Iyer's mention of electroporation to label cells is mentioned using a substrate with an array of immobilized barcoded capture probes, See Iyer Figure 12 and para 6. Applicant argues this is distinct form claim 1 which does not require an array with different probes. However, the instant method does not exclude a method which employs such an array. Further, Applicant argues that there is no teaching of suggestion in Iyer that individual cells could be selectively electroporated with a label that is in contact with all of the cells. The claims do not require this feature. The comprising language of the claims encompasses that more than one cell is labeled at the same time.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Juliet Switzer whose telephone number is (571)272-0753. The examiner can normally be reached Monday to Thursday, 8:00 AM-3:30 PM.
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Juliet Switzer
Primary Examiner
Art Unit 1682
/JULIET C SWITZER/Primary Examiner, Art Unit 1682
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