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, encompassing previously presented claims 1-16 and new claims 52-55 in the reply filed on 13 February 2026 is acknowledged.
Applicant’s election of "target transcripts capable of defining a cellular differentiation state" from the species of target transcripts and "paired-end NGS method" from the species of methods of obtaining a sequence from the fused amplicon in the reply filed on 13 February 2026 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Applicant is reminded that upon the cancelation of claims to a non-elected invention, the inventorship must be corrected in compliance with 37 CFR 1.48(a) if one or more of the currently named inventors is no longer an inventor of at least one claim remaining in the application. A request to correct inventorship under 37 CFR 1.48(a) must be accompanied by an application data sheet in accordance with 37 CFR 1.76 that identifies each inventor by his or her legal name and by the processing fee required under 37 CFR 1.17(i).
Information Disclosure Statement
The information disclosure statement (IDS) filed 21 August 2023, 5 December 2023, and 23 April 2025 are considered, initialed, and attached hereto.
Claim Status
Claims 1-16 and 52-55 are pending and under examination.
Claims 17-51 are canceled.
Specification
The use of the term Sigma-Aldrich, Lynx Therapeutics, 454 Life Sciences, Roche, Applied Biosystems, Complete Genomics, Fluka, Dupont, Krytox, Operon, Amersham, Sigma, Life Technologies, ArrayIt, Olympus, DNAsis, Hitachi, and Invitrogen, which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 15-16 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 15 contains the trademarks/trade names MiSeq, NextSeq, and HiSeq. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademarks/trade names are used to identify/describe various sequencing methods provided by Illumina, Inc. and, accordingly, the identification/description is indefinite.
Claim 15 recites the limitation "the one or more exogenous polynucleotides capable of interacting with a polynucleotide-guided protein or an expressed polynucleotide proxy therefor" in lines 12-14. There is insufficient antecedent basis for this limitation in the claim.
Claim 16 recites the limitation "the population of droplets or emulsions" in line 2, 11, and 19. There is insufficient antecedent basis for this limitation in the claim, since claim 2 on which claim 16 depends only recites a population of individually sequestered or discretely identifiable cells that is droplet-encapsulated or emulsion-encapsulated, which does not inherently include a population of droplets or emulsions as the population of individually sequestered or discretely identifiable cells could conceivably be encapsulated in a single droplet or emulsion.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-4, 13, 15-16, 52, and 54 are rejected under 35 U.S.C. 102(a)(1) and (a)(2) as being anticipated by Johnson et al. (U.S. Patent Application Publications Cite No 1 in IDS filed 23 April 2025)(US 2014/0057799, published 27 February 2014, effectively filed 16 December 2010), herein Johnson.
Regarding claim 1, Johnson teaches a method for identifying, within a population of individually sequestered or discretely identifiable cells, one or more target transcripts and one or more exogenous polynucleotides in an individual cell (“method for analyzing at least two nucleic acid sequences in a single cell contained within a population of at least 10,000 cells […] a first target nucleic acid […] a second target nucleic acid” [0010]; “In one aspect, the single cell is isolated in an emulsion microdroplet” [0013]; “In some embodiments, the first or second target nucleic acid sequence is an RNA sequence” [0015]; “The method includes introducing a unique barcode sequence comprising at least six nucleotides into each of the plurality of single cells” [0026] since the barcode is introduced into the cells, the barcode is an exogenous polynucleotide), the method comprising: (a) preparing or providing the population of individually sequestered or discretely identifiable cells, wherein a plurality of said population of individually sequestered or discretely identifiable cells comprises: an individually sequestered or discretely identifiable cell harboring the one or more exogenous polynucleotides or comprising a nucleic acid vector capable of expressing the one or more exogenous polynucleotides ([0010], [0013], [0015], and [0026] as above, the barcode being introduced into the cell teaches that the cell harbors the one or more exogenous polynucleotides); nucleic acid amplification reagents (“The primers are used with standard PCR conditions, for example, 1 mM Tris-HCl pH 8.3, 5 mM potassium chloride, 0.15 mM magnesium chloride, 0.2-2 µM primers, 200 µM dNTPs, and a thermostable DNA polymerase” [0108]; “In one embodiment, a set of nucleic acid probes (or primers) are used to amplify a first target nucleic acid sequence and a second target nucleic acid sequence to form a fusion complex” [0109]); and a plurality of oligonucleotides comprising: (i) a first pair of oligonucleotide primers for amplifying an exogenous polynucleotide in the individually sequestered or discretely identifiable cell (“For each of the plurality of single cells, the method provides at least one set of nucleic acid probes, the set comprising a first probe comprising a sequence that is complementary to a nucleic acid sequence that is located at the 5' end of the barcode sequence, a second probe comprising a sequence that is complementary to a nucleic acid sequence that is located at the 3' end of the barcode sequence and a second region of sequence that is complementary to a non-human, exogenous sequence” [0027]); and (ii) a second pair of oligonucleotide primers for amplifying a target transcript of the individually sequestered or discretely identifiable cell (“the set comprising […] a third probe comprising a sequence that comprises the non-human, exogenous sequence and a sequence that is complementary to a first subsequence of a second target nucleic acid sequence, and a fourth probe comprising a sequence that is complementary to a second subsequence of the second target nucleic acid sequence” [0027]), wherein the first pair of oligonucleotide primers includes a first primer having a first 5’-terminal region of sequence that is the same as or complementary to a second 5’-terminal region of sequence of a second primer of the second pair of oligonucleotide primers, wherein the first 5’-terminal region and the second 5’-terminal region are of sufficient length to allow for amplification-mediated joining of a first amplicon of the first pair of oligonucleotide primers and a second amplicon of the second pair of oligonucleotide primers into a fused amplicon (“hybridizing the exogenous sequence to its complement; amplifying the first target nucleic acid sequence, the second target nucleic acid sequence, and the exogenous sequence using the first and fourth probes; performing bulk sequencing of the fused complexes” [0028], teaching that the probes of [0027] recited above allow the fused amplicon to be made), wherein the individually sequestered or discretely identifiable cell is lysed to render lysed cell contents of the individually sequestered or discretely identifiable cell accessible in a manner that maintains sequestering or discrete identification of the lysed cell contents (“The method can also include lysing the cells prior to performing the amplification step” [0017]); (b) performing polymerase-mediated primer extension upon the lysed contents of the population under conditions suitable for generating fused amplicons comprising the first amplicon of the first pair of oligonucleotide primers and the second amplicon of the second pair of oligonucleotide primers by overlap extension, thereby generating the fused amplicons within the lysed cell contents (“The method continues by amplifying the first and second nucleic acid sequences independently, wherein the first target nucleic acid sequence is amplified using the first probe and the second probe, and wherein the second target nucleic acid sequence is amplified using the third probe and the fourth probe; hybridizing the exogenous sequence to its complement; amplifying the first target nucleic acid sequence, the second target nucleic acid sequence, and the exogenous sequence using the first and fourth probes; performing bulk sequencing of the fused complexes” [0028]; “FIG. 5A shows an example of single cell sequence linkage by intracellular overlap extension polymerase chain reaction” [0045]; FIG. 5A-6B showing the general process of a fused amplicon being generated from two target nucleic acids (g) and (h) by overlap extension PCR); (c) recovering the fused amplicons from the lysed cell contents of the population (“After thermocycling and PCR, the amplified material must be recovered from the emulsion” [0117]); and (d) obtaining sequence information from the fused amplicons using a sequencing method capable of obtaining sequences from both ends of individual fused amplicon sequences (“performing bulk sequencing of the fused complexes” [0028]; “The term "bulk sequencing" or "next generation sequencing" or "massively parallel sequencing" refers to any high throughput sequencing technology that parallelizes the DNA sequencing process. […] The terms "bulk sequencing," […] refer only to general methods […]. Any bulk sequencing method can be implemented in the invention, such as reversible terminator chemistry (e.g., Illumina), pyrosequencing using polony emulsion droplets (e.g., Roche), ion semiconductor sequencing (IonTorrent), single molecule sequencing (e.g., Pacific Biosciences), massively parallel signature sequencing, etc.” [0083]; “a next-generation sequencing machine (Illumina) to obtain >500 k paired-end 80 bp sequence” [0194] though this recitation is from a specific embodiment that did not include the creation of a fusion amplicon between an exogenous barcode and a transcript via overlap extension, it provides evidence that among the applicable bulk sequencing methods used in [0028] includes paired-end methods that obtain sequences from both ends) and identifying, as a pair, said sequences obtained from both ends of the same individual fused amplicon, thereby identifying, in the population of individually sequestered or discretely identifiable cells, the one or more target transcripts and the one or more exogenous polynucleotides within the individually sequestered or discretely identifiable cell (“identifying a single cell for each of the fused complexes based on the barcode sequence” [0028]).
Regarding claim 2, Johnson teaches the method of claim 1 (as discussed in the 35 U.S.C. 102 rejection of claim 1 above), wherein the population of individually sequestered or discretely identifiable cells: is droplet-encapsulated or emulsion-encapsulated (“the single cell is isolated in an emulsion microdroplet” [0013]); is present in a hydrogel, wherein the population of individually sequestered or discretely identifiable cells has been split and pool labeled; is present in a microfluidic chip (“In some embodiments, a microfluidic device is used to generate single cell emulsion droplets” [0092]); or is present in an array (“In some embodiments, the methods of the invention use single cells in reaction containers, rather than emulsion droplets. Examples of such reaction containers include 96 well plates” [0102], the single cells being distributed in multiple reaction chambers in the case of a 96 well plate is considered an array since each cell’s reaction takes place in a unique and discrete addressable location on the plate).
Regarding claim 3, Johnson teaches the method of claim 1 (as discussed in the 35 U.S.C. 102 rejection of claim 1 above), wherein the nucleic acid amplification reagents comprise reagents selected from the group consisting of PCR reagents, RPA reagents, RCA reagents, LAMP reagents, and other isothermal amplification reagents (“The primers are used with standard PCR conditions, for example, 1 mM Tris-HCl pH 8.3, 5 mM potassium chloride, 0.15 mM magnesium chloride, 0.2-2 µM primers, 200 µM dNTPs, and a thermostable DNA polymerase” [0108]; “In one embodiment, a set of nucleic acid probes (or primers) are used to amplify a first target nucleic acid sequence and a second target nucleic acid sequence to form a fusion complex” [0109]).
Regarding claim 4, Johnson teaches the method of claim 1 (as discussed in the 35 U.S.C. 102 rejection of claim 1 above), wherein the polymerase-mediated primer extension performed upon the lysed cell contents of the population under the conditions suitable for generating the fused amplicons comprising the first amplicon of the first pair of oligonucleotide primers and the second amplicon of the second pair of oligonucleotide primers by the overlap extension comprises performing one or more rounds of amplification selected from the group consisting of PCR, RPA, RCA, LAMP, and other isothermal amplification, upon the lysed cell contents of the population (“FIG. 5A shows an example of single cell sequence linkage by intracellular overlap extension polymerase chain reaction” [0045]).
Regarding claim 13, Johnson teaches the method of claim 1 (as discussed in the 35 U.S.C. 102 rejection of claim 1 above), wherein the population of individually sequestered or discretely identifiable cells is a population of individually sequestered or discretely identifiable mammalian cells (“A cell includes any kind of cell (prokaryotic or eukaryotic) from a living organism. Examples include, but are not limited to, mammalian mononuclear blood cells” [0076]).
Regarding claim 15, Johnson teaches the method of claim 1 (as discussed in the 35 U.S.C. 102 rejection of claim 1 above), wherein: the population of individually sequestered or discretely identifiable cells comprises a population of individually sequestered or discretely identifiable non-mammalian cells (“A cell includes any kind of cell (prokaryotic or eukaryotic) from a living organism. Examples include, but are not limited to, […] yeast cells, or bacterial cells” [0076]); the nucleic acid amplification reagents comprise a reverse transcriptase, a DNA polymerase and one or more primers selected from the group consisting of: poly-T-tailed oligonucleotide primers, primers for specific amplification of the one or more exogenous polynucleotides capable of interacting with a polynucleotide-guided protein or an expressed polynucleotide proxy therefor, and primers for amplification of a targeted transcript of interest (“Targeting and amplification of genetic loci in cells can be performed using PCR, LCR, padlock probes, RT-PCR, or multi-probe circularization. Any combination of these methods to target and amplify different loci can be used. For example, a combination amplification approach is used to amplify a genomic DNA locus and an RNA transcript. In one embodiment, a thermostable reverse transcriptase enzyme, such as ThermoScript RT (Lucigen) or GeneAmp Thermostable rTth (Life Technologies), is combined with a thermostable DNA polymerase, such as the Stoffel fragment or Taq DNA polymerase. Thermocycling can induce first strand cDNA synthesis from the RNA transcript target. Once cDNA from the RNA transcript is synthesized, overlap extension PCR is performed using the cDNA and the genomic DNA target sequences” [0157], this teaches using a reverse transcriptase and a DNA polymerase when one of the target nucleic acids is an RNA transcript; “a third probe comprising […] a sequence that is complementary to a first subsequence of a second target nucleic acid, and a fourth probe comprising a sequence that is complementary to a second subsequence of the second target nucleic acid sequence” [0010]; “In some embodiments, the […] second target nucleic acid sequence is an RNA sequence” [0015]); the first pair of oligonucleotide primers amplifies a gRNA sequence, an RNAi agent sequence, or a nucleic acid sequence that identifies expression of a plurality of gRNAs or RNAi agents; the one or more target transcripts: are capable of defining a cellular differentiation state; comprise one or more interferon stimulated gene transcripts (ISGS); comprise one or more target transcripts selected from the group consisting of IRF3, DNA JC13, STING1, TBK1, and TCF7; comprise a panel of transcripts for assessment of B-cell activation and differentiation status; or comprise a plurality of target transcripts, wherein individual droplets, hydrogel elements, microfluidic chip chambers, or array elements contain individual cells of the population of individually sequestered or discretely identifiable cells and comprise respective pairs of oligonucleotide primers for amplifying each target transcript of the plurality of target transcripts (“methods and systems for massively parallel genetic analysis of single cells in emulsion droplets or reaction containers. Genetic loci of interest are targeted in a single cell using specially-designed probes, and a fusion complex is formed by molecular linkage and amplification techniques. Multiple genetic loci can be targeted, and many sets of probes can be multiplexed by PCR into a single analysis, such that several loci or even the entire transcriptome or genome is analyzed. The invention is useful for analyzing genetic information in single cells in a high-throughput, parallel fashion for a large quantity of cells” [0073-0074]); the individually sequestered or discretely identifiable cell is lysed by heating, by contact with a Betaine solution, or by another chemical means (“The membranes of the resuspended cells are then disrupted using alkaline lysis buffer or proteinase K solutions” [0168]); the population of individually sequestered or discretely identifiable cells does not comprise microbeads (“In some embodiments, the barcode sequence is affixed to a bead or a solid surface” [0029] teaching embodiments not using a bead, such as using a solid surface or the embodiments that are not part of these “some embodiments”, see also [0147] for the addition of barcodes to cells without using beads); in step (c), recovering the fused amplicons comprises: breaking open a population of droplets or emulsions having an oil-water interface; and separating a fused amplicon-containing aqueous phase from an oil phase (“the amplified material must be recovered from the emulsion. In one embodiment, ether is used to break the emulsion, and then the ether is evaporated from the aqueous/ether layer to recover the amplified DNA in solution” [0117]); obtaining sequence from the fused amplicons comprises use of: a paired-end NGS method (“sequenced on a next-generation sequencing machine (Illumina) to obtain >500 k paired-end” [0194]); fused amplicon sequence data are obtained and then used to assemble a matrix of digital gene-expression measurements comprising counts of each expressed target transcript detected in each cell; paired transcript and exogenous polynucleotide sequences of fused amplicons are obtained for at least 10,000 individual cells (“The method also provides performing a bulk sequencing reaction to generate sequence information for at least 100,000 fused complexes from at least 10,000 cells within the population of cells, wherein the sequence information is sufficient to co-localize the first target nucleic acid sequence and the second target nucleic acid sequence to a single cell from the population of at least 10,000 cells” [0012]); gene perturbation effects of at least 1000 different exogenous polynucleotides are assessed in the population of individually sequestered or discretely identifiable cells; or the plurality of oligonucleotides further comprises a third pair of oligonucleotide primers for amplifying an exogenous polynucleotide or a second target transcript of the individually sequestered or discretely identifiable cells (“The method includes providing a second set of nucleic acid probes, the second set comprising a fifth probe comprising a sequence that is complementary to a third target nucleic acid subsequence, a sixth probe comprising a sequence that is complementary to a second subsequence of the third target nucleic acid sequence” [0020]).
Regarding claim 16, Johnson teaches the method of claim 2 (as discussed in the 35 U.S.C. 102 rejection of claim 2 above), wherein: the population of droplets or emulsions comprises water-in-oil emulsions (“any of the emulsions disclosed herein may be a water-in-oil (W/O) emulsion” [0080]); the population of droplets or emulsions comprises mean droplet or emulsion volumes of between about 10 pL and about 1.2 nL (“the average volume of droplets in an emulsion, may be […] less than about one nanoliter (or between about one nanoliter and one picoliter)” [0081]); and the population of droplets or emulsions comprises mean droplet or emulsion sizes of between about 20 microns and about 200 microns in diameter per individual droplet or emulsion (“A droplet may have a diameter (or an average diameter) of less than about 1000, 100, or 10 micrometers, or of about 1000 to 10 micrometers” [0081]).
Regarding claim 52, Johnson teaches the method of claim 1 (as discussed in the 35 U.S.C. 102 rejection of claim 1 above), and claim 52 differs from claim 1 in that:
(1) instead of a plurality of the population of individually sequestered or discretely identifiable cells comprising the cells, nucleic acid amplification reagents, and the plurality of oligonucleotides, in claim 52 the individual cells of the population of individually sequestered or discretely identifiable cells are contacted with nucleic acid amplification reagents and a plurality of oligonucleotides; Johnson teaches contacting the cell with nucleic acid amplification reagents and the plurality of oligonucleotides (“The method includes isolating the single cells with at least one set of nucleic acid probes; amplifying the first and second target nucleic acid sequences” [0011]; “The primers are used with standard PCR conditions, for example, 1 mM Tris-HCl pH 8.3, 5 mM potassium chloride, 0.15 mM magnesium chloride, 0.2-2 µM primers, 200 µM dNTPs, and a thermostable DNA polymerase” [0108]; “In one embodiment, a set of nucleic acid probes (or primers) are used to amplify a first target nucleic acid sequence and a second target nucleic acid sequence” [0109]);
(2) instead of limiting the oligonucleotides such that the first 5’-terminal region and the second 5’-terminal region are of sufficient length to allow for amplification mediated joining, in claim 52 the oligonucleotides are limited by the complementary 5’-terminal regions being “for generation of fused amplicons by overlap extension”; Johnson teaches generation of the fused amplicons by overlap extension (“FIG. 5A shows an example of single cell sequence linkage by intracellular overlap extension polymerase chain reaction” [0045]; FIGS. 5A-6B);
(3) claim 52 does not require that lysing be in a manner that maintains sequestering or discrete identification of the cell contents; as this limitation is strictly broader than the limitation in claim 1, Johnson’s teaching of claim 1 teaches this limitation;
(4) claim 52 does not require that obtaining sequence information from the fused amplicons use a sequencing method capable of obtaining sequences from both ends and the related identifying limitation; as this limitation is strictly broader than the limitation in claim 1, Johnson’s teaching of claim 1 teaches this limitation.
Regarding claim 54, Johnson teaches the method of claim 52 (as discussed in the 35 U.S.C. 102 rejection of claim 52 above), and claim 54 differs from claim 52 in that instead of contacting the individual cells with nucleic acid amplification reagents and the plurality of oligonucleotides, in claim 54 the individual cells are encapsulated with nucleic acid amplification reagents and the plurality of oligonucleotides; Johnson teaches encapsulation of the individual cells with nucleic acid amplification reagents and the plurality of oligonucleotides (“The method includes isolating the single cells with at least one set of nucleic acid probes; amplifying the first and second target nucleic acid sequences” [0011]; “In one aspect, the single cell is isolated in an emulsion microdroplet” [0013]; “FIG. 5A shows an example of single cell sequence linkage by intracellular overlap extension polymerase chain reaction, according to one embodiment of the invention […] The steps of FIG. 5 can be performed in a reaction container or an emulsion droplet” [0045]; FIGS. 5A-6B; “The primers are used with standard PCR conditions, for example, 1 mM Tris-HCl pH 8.3, 5 mM potassium chloride, 0.15 mM magnesium chloride, 0.2-2 µM primers, 200 µM dNTPs, and a thermostable DNA polymerase” [0108]; “In one embodiment, a set of nucleic acid probes (or primers) are used to amplify a first target nucleic acid sequence and a second target nucleic acid sequence” [0109]).
Therefore, Johnson anticipates claims 1-4, 13, 15-16, 52, and 54.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 5-12, 14, 53, and 55 are rejected under 35 U.S.C. 103 as being unpatentable over Johnson et al. (U.S. Patent Application Publications Cite No 1 in IDS filed 23 April 2025)(US 2014/0057799, published 27 February 2014, effectively filed 16 December 2010), herein Johnson, in view of Feldman et al. (Foreign Patent Documents Cite No 2 in IDS filed 21 August 2023)(WO 2019/222284, published 21 November 2019, effectively filed 14 May 2018), herein Feldman.
Regarding claim 5, Johnson teaches the method of claim 1 (as discussed in the 35 U.S.C. 102 rejection of claim 1 above). However, Johnson does not teach that the population of individually sequestered or discretely identifiable cells harbors of expresses a polynucleotide-guided protein capable of interacting with the one or more exogenous polynucleotides. This deficiency is made up for in the teachings of Feldman.
Regarding claim 5, Feldman teaches a method for identifying, within a population of individually sequestered or discretely identifiable cells, one or more target transcripts and one or more exogenous polynucleotides in an individual cell, wherein the population of individually sequestered or discretely identifiable cells harbors or express a polynucleotide-guided protein capable of interacting with the one or more exogenous polynucleotides (“a method for screening cells for presence of one or more genetic elements comprising: a) culturing a cell or cell population in one or more discrete volumes; b) introducing one or more polynucleotides into the cell or cell population, wherein each polynucleotide comprises nucleic acid sequences encoding a sequence defining one or more optical barcodes and the one or more genetic elements […]; c) incubating the cell or cell population to allow for expression of RNA transcripts comprising the one or more optical barcodes; d) detecting genomic, genetic, epigenetic, proteomic, and/or phenotypic differences caused by the one or more genetic elements in the cell or cell population; and e) detecting the optical barcode by an in situ sequencing method to identify the one or more genetic elements present in the cell or cell population” [0010]; “the polynucleotide sequence encoding one or more genetic elements encodes a CRISPR-Cas system […] the polynucleotide sequence encodes one or more guide sequences” [0011]; “In certain example embodiments, the polynucleotides or vectors encode a Cas nuclease, a short guide RNA (sgRNA) and the optical barcode” [0102]; the guide sequence/sgRNA is the exogenous polynucleotide, which interacts with the Cas nuclease, which is a polynucleotide-guided protein).
Regarding claim 6, Johnson teaches the method of claim 1 (as discussed in the 35 U.S.C. 102 rejection of claim 1 above), and Feldman teaches their method (as discussed in the 35 U.S.C. 103 rejection of claim 5 above) wherein the one or more exogenous polynucleotides is capable of interacting with a polynucleotide-guided protein (“the polynucleotides or vectors encode a Cas nuclease, a short guide RNA (sgRNA)” [0102], the sgRNA interacts with the Cas nuclease in a CRISPR-Cas system).
Regarding claim 7, Johnson teaches the method of claim 1 (as discussed in the 35 U.S.C. 102 rejection of claim 1 above), and Feldman teaches their method (as discussed in the 35 U.S.C. 103 rejection of claim 5 above) wherein the one or more exogenous polynucleotides comprise a nucleic acid sequence that identifies expression of one or more polynucleotides capable of interacting with a polynucleotide-guided protein (“In certain example embodiments, the polynucleotides or vectors encode a Cas nuclease, a short guide RNA (sgRNA) and the optical barcode” [0102]; “a unique optical barcode is assigned to each type of genetic perturbation (or each genetic perturbation is an optical barcode)” [0109], the unique assignment of the barcode to the genetic perturbation identifies expression of the sgRNA that interacts with the Cas nuclease).
Regarding claim 8, Johnson teaches the method of claim 1 (as discussed in the 35 U.S.C. 102 rejection of claim 1 above), and Feldman teaches their method (as discussed in the 35 U.S.C. 103 rejection of claim 5 above) wherein identifying, in the population of individually sequestered or discretely identifiable cells, the one or more target transcripts and the one or more exogenous polynucleotides identifies the one or more target transcripts and the one or more exogenous polynucleotides as co-expressed (“Because a unique optical barcode is assigned to each type of genetic perturbation (or each genetic perturbation is an optical barcode), read-out of the optical barcode allows the observed phenotypes described above to be correlated to a particular genotype” [0109] here the observed phenotype is the expression of RNA transcripts, which is correlated to a genotype having a specific genetic perturbation from expression of the introduced genetic element, which is the exogenous polynucleotide, thereby identifying the transcript and exogenous polynucleotide as co-expressed in the a cell).
Regarding claim 9, Johnson teaches the method of claim 1 (as discussed in the 35 U.S.C. 102 rejection of claim 1 above), and Feldman teaches their method (as discussed in the 35 U.S.C. 103 rejection of claim 5 above) wherein the population of individually sequestered or discretely identifiable cells comprises a nucleic acid vector or nucleic acid insert capable of expressing the one or more exogenous polynucleotides, and wherein the population of individually sequestered or discretely identifiable cells expresses the one or more exogenous polynucleotides (“A pooled library of transcriptional effectors for introducing one or more genetic perturbations is designed and cloned into a suitable vector” [0096]; “Expression of the polynucleotide or vector” [0098]).
Regarding claim 10, Johnson teaches the method of claim 1 (as discussed in the 35 U.S.C. 102 rejection of claim 1 above), and Feldman teaches their method (as discussed in the 35 U.S.C. 103 rejection of claim 5 above) wherein the one or more exogenous polynucleotides comprise a guide RNA (“the polynucleotides or vectors further encode a site specific nuclease capable of introducing the genetic perturbation into a target sequence within a cell or population of cells. Site specific nucleases include […] a CRISPR system comprising a Cas protein and sgRNA” [0101]).
Regarding claim 11, Johnson teaches the method of claim 1 (as discussed in the 35 U.S.C. 102 rejection of claim 1 above), and Feldman teaches their method (as discussed in the 35 U.S.C. 103 rejection of claim 5 above) further comprising comparing identities and levels of target transcripts and exogenous polynucleotides in the population of individually sequestered or discretely identifiable cells to identify exogenous polynucleotide-mediated gene perturbations in individual cells of the population of individually sequestered or discretely identifiable cells (“detecting genomic, genetic, epigenetic, proteomic, and/or phenotypic differences caused by the one or more genetic elements in the cell or cell population” [0010]; “Because a unique optical barcode is assigned to each type of genetic perturbation (or each genetic perturbation is an optical barcode), read-out of the optical barcode allows the observed phenotypes described above to be correlated to a particular genotype” [0109]).
Regarding claim 12, Johnson teaches the method of claim 1 (as discussed in the 35 U.S.C. 102 rejection of claim 1 above), and Feldman teaches their method (as discussed in the 35 U.S.C. 103 rejection of claim 5 above) wherein the population of individually sequestered or discretely identifiable cells is capable of acting as a cellular factor and wherein the population of individually sequestered or discretely identifiable cells comprises Chinese Hamster Ovary (CHO) cells and/or Human Embryonic Kidney (HEK) cells (“Applicants achieved comparably robust mapping of CRISPR sgRNAs to the frameshift reporter phenotype with the CROP-seq vector in […] HEK293 cells” [0320]).
Regarding claim 14, Johnson teaches the method of claim 1 (as discussed in the 35 U.S.C. 102 rejection of claim 1 above), and Feldman teaches their method (as discussed in the 35 U.S.C. 103 rejection of claim 5 above) wherein the population of individually sequestered or discretely identifiable cells is a population of primary cells (“The embodiments disclosed herein provide approaches based on in situ sequencing methods and are highly suited to screening in cultured and primary cells” [0094]; The above described constructs are introduced into a single cell or population of cells. The cells may be cultured cells, primary cells” [0104]).
Regarding claim 53, Johnson teaches the method of claim 52 (as discussed in the 35 U.S.C. 102 rejection of claim 52 above), and Feldman teaches their method (as discussed in the 35 U.S.C. 103 rejection of claim 5 above) wherein the one or more exogenous polynucleotides comprise at least 1000 different exogenous polynucleotides, and the method further comprises: assessing gene perturbation effects of the at least 1000 different exogenous polynucleotides based on the sequence information from the fused amplicons (“The method disclosed herein may analyze 10,000 perturbations replicated 1000 fold at the single-cell level, for a total of 10,000,000 single-cell assays in a screen” [0108], each perturbation correlating to a different exogenous polynucleotide).
Regarding claim 55, Johnson teaches the method of claim 54 (as discussed in the 35 U.S.C. 102 rejection of claim 54 above), and Feldman teaches their method (as discussed in the 35 U.S.C. 103 rejection of claim 5 above) wherein the population of individually sequestered or discretely identifiable cells harbors or expresses a polynucleotide-guided protein capable of interacting with the one or more exogenous polynucleotides (“the polynucleotide sequence encoding one or more genetic elements encodes a CRISPR-Cas system […] the polynucleotide sequence encodes one or more guide sequences” [0011]; “In certain example embodiments, the polynucleotides or vectors encode a Cas nuclease, a short guide RNA (sgRNA) and the optical barcode” [0102]; the guide sequence/sgRNA is the exogenous polynucleotide, which interacts with the Cas nuclease, which is a polynucleotide-guided protein).
It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to perform the simple substitution of the method of using overlap extension to fuse the amplicons of a transcript and an exogenous polynucleotide into a fused amplicon to allow for identification of their expression in the same cell, as taught by Johnson, for the barcoding of a transcript and barcoding of an exogenous polynucleotide to allow for identification of their expression in the same cell, as taught by Feldman (MPEP §2143 I. B.). One of ordinary skill in the art could have performed this substitution and would have found the results of this substitution predictable because both references are concerned with the analyzing transcripts at the level of an individual cell and merely use alternative methods of linking of the cell identity to transcripts and exogenous polynucleotides in the cell (physically linking into a fused amplicon in Johnson and linking via a shared unique barcode in Feldman) that could easily be substituted for one another by designing primers like those of Johnson that would permit the fusion of amplicons of the transcript and the exogenous genetic element of Feldman. Therefore, the invention as a whole of claims 5-12, 14, 53, and 55 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Conclusion
Claims 1-16 and 52-55 are rejected.
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/JEFFREY BELLAH/Examiner, Art Unit 1683
/ANNE M. GUSSOW/Supervisory Patent Examiner, Art Unit 1683
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