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 .
DETAILED ACTION
Pursuant to a preliminary amendment filed June 17, 2026, claims 28-36, 38-56, 84, 114 and 115 are currently pending in the instant application.
Response to Election/Restriction
Applicant's election of Group I with traverse claims 28-36, 38-47 and 54-56 directed to a method of decoding a nucleic acid barcode in situ in a sample; and Applicant’s election of Species (A) and (B) with traverse; and Species (C)-(H) with traverse as follows:
Species (A): wherein the nucleic acid barcode comprises only one target region (claim 29);
Species (B): wherein at least one of said encoding probes comprises a sequence complementary to the primary variable sequence (claim 30);
Species (C): wherein the linear probe comprises a binding region and an overhang region (claim 35);
Species (D): wherein the nucleic acid barcode is from a library of nucleic acid barcodes, the second part of each nucleic acid barcode comprises the same sequence (claim 40);
Species (E): wherein upon padlock probe hybridization, the 5' and 3' end regions of the padlock probe are immediately next to each other (claim 42);
Species (F): wherein when the 5' and 3' end regions of the padlock probe are hybridized to the first part of the nucleic acid barcode, the length of the gap is shorter than the length of the target region (claim 44);
Species (G): wherein the encoding probe comprises two or more of a plurality of unique readout regions (claim 49); and
Species (H): wherein each nucleic acid barcode comprises the same sequence (claim 53), in the reply filed June 17, 2026 is acknowledged.
Response to Arguments
Applicant’s arguments filed June 17, 2026 have been fully considered but they are not persuasive. Applicants essentially assert that: (a) Group II is dependent from the claims of Group I (Applicant Remarks, pg. 11, third full paragraph); and (b) each of claims 84, 114 and 115 depend from independent claim 28 and thereby incorporate by reference all of the limitations of claim 28 (Applicant Remarks, pg. 11, fourth full paragraph).
Regarding (a), amended claim 48 recites a claim depending from Group I, such that Applicant’s argument is moot.
Regarding (b), each of claims 84, 114 and 115 clearly recite distinct independent claims that appear to refer back to independent claim 28. Such claims do not incorporate by reference all of the limitations recited in independent claim 28. An independent claim is a stand-alone claim that contains all the limitations necessary to define an invention. For example, independent claim 28 recites a method of decoding a nucleic acid barcode in situ in a sample; independent claim 84 recites a method of performing an in situ genetic screen comprising three unique steps; independent claim 114 recites a method of performing an in situ genetic screen comprising six unique steps; and independent claim 115 recites a method of determining cellular positions in a single-cell sequencing. In contrast, a proper dependent claim such as claim 29 recites for example, “the method of claim 28, wherein the nucleic acid…target region,” such that the claim does not recite a completely different method from the method as recited in claim 28. Thus, the requirement for election/restriction is proper.
Claims 84, 114 and 115 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a non-elected invention, there being no allowable generic or linking claim.
Claims 31-33, 38, 39, 41, 43-45 and 48-53 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a non-elected species, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on June 17, 2026.
Please Note: in the reply filed June 17, 2026, Applicant elected for Species (D), wherein the nucleic acid barcode is from a library of nucleic acid barcodes, the second part of each nucleic acid barcode comprises the same sequence (claim 40). Instant claim 48 is withdrawn as being directed to a non-elected species; and claims 49-53 are withdrawn as depending from withdrawn claim 48.
The restriction requirement is still deemed proper and is therefore made FINAL.
The claims will be examined insofar as they read on the elected species.
Therefore, claims 28-30, 34-36, 40, 42, 46, 47 and 54-56 are under consideration to which the following grounds of rejection are applicable.
Interview Summary
Applicant contacted the Examiner to set up an interview, where such telephonic interview was conducted between the Examiner and Applicant’s representatives Hongfan Chen, Erika Csatary, Irina Vainberg on May 13, 2026, where the novelty of the invention, the confusion regarding the status of claim 48 (independent or dependent claim language); and potential amendments to the claims were discussed.
Priority
The present application filed September 14, 2023 is a 35 U.S.C. 371 national stage filing of International Application PCT/US22/20546, filed March 16, 2022, which claims the benefit of US Provisional Patent Application 63162257, filed March 17, 2021.
Information Disclosure Statement
The information disclosure statements (IDSs) submitted on September 14, 2023 and June 17, 2026 have been considered. Initialed copies of the IDSs accompany this Office Action.
Claim Objections/Rejections
Claim Interpretation: the term “the 5’ and 3’ end regions are immediately next to each other” as recited in claim 42 is interpreted to refer to the 5’ and 3’ regions of a single padlock probe that are bound to one another, hybridized to one another, and/or within any distance from one region to the other region.
Claim Objections
Claims 28-30, 34-36, 40, 42, 46, 47 and 54-56 are objected to because of the following informalities: Claims 28-30, 34-36, 40, 42, 46, 47 and 54-56 recite a mixture of pronouns including “the” and “said” within each claim, such that for consistency, a single pronoun reciting either “the” or “said” should be used.
Appropriate correction is required.
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 28-30, 34-36, 40, 42, 46, 47 and 54-56 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 applicant regards as the invention.
Claims 28 and 34 are indefinite for the recitation of the term “the target region” such as recited in claim 28, lines 4 and 5. There is insufficient antecedent basis for the term “the target region” in the claim because claim 28, line 3 recites the term “at least a target region”. The Examiner suggests that Applicant amend claim 28, line 3 to recite, for example, “amplifying a target region.”
Claim 28 is indefinite for the recitation of the term “contacting the sample” such as recited in claim 28, lines 6 and 11 because it is unclear whether the sample is contacted with a plurality of encoding probes; or whether the amplified nucleic acids obtained from (a) are contacted with a plurality of encoding probes and, thus, the metes and bounds of the claim cannot be determined.
Claims 28, 30 and 47 are indefinite for the recitation of the terms “said encoding probes” and/or “each encoding probe” such as recited in claim 28, lines 7-9 and 12. There is insufficient antecedent basis for the terms “said encoding probes” and/or “each encoding probe” in the claim because claim 28, line 6 recites the term “a plurality of encoding probes”. The Examiner suggests that Applicant amend the claim to recite, for example, “allow hybridization of the plurality of encoding probes.”
Claim 28 is indefinite for the recitation of the term “one or more of a plurality of unique readout regions” such as recited in claim 28, lines 9-10 because the term is unclear and confusing. It is unclear whether the encoding probe can comprise one unique readout region; whether it comprises one plurality of unique readout regions; whether it can comprise more than one plurality of unique readout regions; or whether the term refers to something else and, thus, the metes and bounds of the claim cannot be determined.
Claim 28 is indefinite for the recitation of the terms “a unique readout region” and “a different unique readout region” such as recited in claim 28, lines 13 and 18. There is insufficient antecedent basis for the terms “a unique readout region” and “a different unique readout region” in the claim because claim 28, lines 9-10 recites the term “one or more of a plurality of unique readout regions”.
Claim 28 is indefinite for the recitation of the terms “said labeled readout probes”; “the readout probes” and/or “one or more additional readout probes” such as recited in claim 28, lines 12-13, 15 and 17. There is insufficient antecedent basis for the terms “said labeled readout probes”; “the readout probes” and/or “one or more additional readout probes” in the claim because claim 28, line 11 recites the term “one or more labeled readout probes”. The Examiner suggests that Applicant amend the claim to recite, for example, “wherein each of said one or more labeled readout probes.”
Claim 28 is indefinite due to the use of use parentheses to comment on or qualify part of the sentences, for example, “probe(s)” and/or “labels(s)” in claim 28, lines 12, 14 and 15. It is unclear whether the limitations in parentheses are meant to be limitations in the claims or whether they are only suggestions, examples of a preferred embodiment, or synonym. Accordingly, the metes and bounds of the claim are not clear.
Claim 28 is indefinite for the recitation of the term “the label(s)” such as recited in claim 28, line 14. There is insufficient antecedent basis for the terms “the label(s)” in the claim.
Claim 28 is indefinite for the recitation of the term “optionally” such as recited in claim 28, lines 15 and 17 because the claim does not particularly point out and distinctly claim the subject matter which the applicant regards as his invention; and it is unclear whether the recited steps are carried out and, thus, the metes and bounds of the claim cannot be determined.
Claim 28 is indefinite for the recitation of the term “eliminating signal” such as recited in claim 28, line 15 because none of the steps prior to step (e) recite the generation of a signal, such that it is unclear what signal is eliminated from the label(s) and, thus, the metes and bounds of the claim cannot be determined.
Claim 28 is indefinite for the recitation of the terms “the presence” and “the identity” such as recited in claim 28, lines 19-20. There is insufficient antecedent basis for the terms “the presence” and “the identity” in the claim.
Claim 29 is indefinite for the recitation of the term “only one target region” such as recited in claim 29, lines 1-2 because claim 29 depends from instant claim 28, wherein claim 28, line 3 recites that the nucleic acid barcode comprises “at least a target region”, such that claim 29 does not recite that the nucleic acid barcode can comprise more than one target region or a plurality of target regions and, thus, the metes and bounds of the claim cannot be determined.
Claim 30 is indefinite for the recitation of the term “at least one of said encoding probes comprises a sequence…variable sequence” such as recited in claim 30, lines 1-2 because claim 30 depends from instant claim 28, wherein claim 28, lines 7-8 recites that the encoding probe comprises a sequence complementary to a sequence in said amplified nucleic acids, such that dependent claim 30 cannot recite that the at least one of said encoding probes comprises something different than what is recited in the independent claim and, thus, the metes and bounds of the claim cannot be determined. The Examiner suggests that Applicant amend the claim to recite, for example, “the at least one of said encoding probes further comprises a sequence…variable sequence.”
Claim 34 is indefinite for the recitation of the term “(a) comprises” such as recited in claim 34, line 1 because claim 34 depends from instant claim 28, wherein claim 28, lines 3-5 recites that (a) comprises amplifying at least a target region of a nucleic acid barcode, while dependent claim 34 recites amplifying circular padlock probes to generate amplified nucleic acids. Dependent claim 34 cannot recite that “(a) comprises” something different than what is recited in the independent claim and, thus, the metes and bounds of the claim cannot be determined. The Examiner suggests that Applicant amend claim 34 to recite (in part), for example, “wherein amplifying a nucleic acid barcode by rolling circle amplification comprises…”
Claim 34 is indefinite for the recitation of the terms “a pair of oligonucleotide probes”; “padlock probes”; “respective target sequences”; “a first part of the nucleic acid barcode”; “linear probe”; and “copies of the target region” such as recited in claim 34, lines 1-16 because claim 34 depends from instant claim 28, wherein claim 28 does not recite the presence of a pair of oligonucleotide probes; padlock probes; respective target sequences; a nucleic acid barcode comprising parts; linear probe; and copies of the target region and, thus, the metes and bounds of the claim cannot be determined.
Claim 34 is indefinite for the recitation of the term “the reverse complementary sequence” such as recited in claim 34, lines 10-11. There is insufficient antecedent basis for the terms “the reverse complementary sequence” in the claim.
Claim 34 is indefinite for the recitation of the term “in situ” such as recited in claim 34, line 15 because claim 34 depends from instant claim 28, wherein claim 28 does not recite the term “in situ” in the body of the claim, such that the preamble is not accorded any patentable weight because it merely recites the purpose of a process or the intended use of a structure, and where the body of the claim does not depend on the preamble for completeness (See; In re Hirao, 535 F.2d 67, 190 USPQ 15 (CCPA 1976) and Kropa v. Robie, 187 F.2d 150, 152, 88 USPQ 478, 481 (CCPA 1951)) and, thus, the metes and bounds of the claim cannot be determined.
Claim 35 is indefinite for the recitation of the term “the linear probe comprises a binding region and an overhang region” such as recited in claim 35, lines 1-2 because claim 35 depends from instant claims 28 and 34, wherein claim 34, line 12-13 recites that the linear probe comprises a region that is complementary to at least a region of the padlock probe, such that claim 35 cannot comprise something different than what is recited in claim 34 and, thus, the metes and bounds of the claim cannot be determined.
Claims 35 and 40 are indefinite for the recitation of the term “second part of the nucleic acid barcode” such as recited in claim 35, lines 2-3 because claim 35 depends from instant claims 28 and 34, wherein claims 28 and 34 do not recite that the nucleic acid barcode comprises a second part and, thus, the metes and bounds of the claim cannot be determined.
Claims 36 and 42 are indefinite for the recitation of the term “the 5’ and 3’ end regions” such as recited in claim 36, line 1. There is insufficient antecedent basis for the terms “the 5’ and 3’ end regions” in the claims because claim 34, lines 7-8 recites the term “a 5’ end region and a 3’ end region”.
Claim 54 is indefinite for the recitation of the term “the number” such as recited in claim 54, line 1. There is insufficient antecedent basis for the terms “the number” in the claim.
Claim 54 is indefinite for the recitation of the term “unique readout regions” such as recited in claim 54, lines 1-2 because claim 54 depends from claim 28, wherein claim 28, lines 9-10 and 18 recite the terms “one or more of a plurality of unique readout regions” and “a different unique readout region”.
Claims 55 and 56 are indefinite for the recitation of the term “the length” such as recited in claim 55, line 1. There is insufficient antecedent basis for the terms “the length” in the claim. Moreover, claims 55 and 56 depend from instant claim 28, wherein claim 28 does not recite a length of the variable sequence and, thus, the metes and bounds of the claim cannot be determined.
Claims 55 and 56 are indefinite for the recitation of the term “the variable sequence” such as recited in claim 55, line 1. There is insufficient antecedent basis for the term “the variable sequence” in the claim because claim 28, line 5 recites the term “a primary variable sequence”.
Claims 46 and 47 are indefinite insofar as they ultimately depend from instant claim 28.
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 29, 30, 34, 35, 40 and 54-56 are rejected under 35 U.S.C. 112(d) as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 29 recites (in part): “wherein the nucleic acid barcode comprises only one target region” such as recited in claim 29, lines 1-2 because claim 29 depends from instant claim 28, wherein claim 28, line 3 recites that the nucleic acid barcode comprises “at least a target region”, such that claim 29 does not recite that the nucleic acid barcode can comprise more than one target region or a plurality of target regions. Thus, claim 29 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 30 recites (in part): “wherein at least one of said encoding probes comprises a sequence complementary to the primary variable sequence” in lines 1-2 because claim 30 depends from instant claim 28, wherein claim 28, lines 7-8 recites that the encoding probe comprises a sequence complementary to a sequence in said amplified nucleic acids, such that dependent claim 30 cannot recite that the at least one of said encoding probes comprises something different than what is recited in the independent claim. Thus, claim 30 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 34 recites (in part): “wherein step (a) comprises the following steps…comprising copies of the target region” in lines 1-16 because claim 34 depends from instant claim 28, wherein claim 28, lines 3-5 recites that (a) comprises amplifying at least a target region of a nucleic acid barcode, while dependent claim 34 recites amplifying circular padlock probes to generate amplified nucleic acids. Dependent claim 34 cannot recite that “(a) comprises” something different than what is recited in the independent claim. Moreover, claim 28 does not recite the term “in situ” in the body of the claim, such that the preamble is not accorded any patentable weight. Additionally, claim 28 does not recite the presence of a pair of oligonucleotide probes; padlock probes; respective target sequences; a nucleic acid barcode comprising parts; linear probe; and copies of the target region. Thus, claim 34 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claims 35 and 40 recite (in part): “the second part of each nucleic acid barcode” in lines 1-3 because claims 35 and 40 depend from instant claims 28 and 34, wherein claims 28 and 34 do not recite that the nucleic acid barcode comprises parts including a second part. Thus, claims 35 and 40 are improper dependent claims for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 54 recites (in part): “wherein the number of unique readout regions is about 2 to 6000” lines 1-2 because claim 54 depends from instant claim 28, wherein claim 28 does not recite numbers of unique readout regions. Thus, claim 54 is an improper dependent claim for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claims 55 and 56 recite (in part): “the length of the variable sequence” line 1 because claims 55 and 56 depend from instant claim 28, wherein claim 28 does not recite a length of the variable sequence. Thus, claims 55 and 56 are improper dependent claims for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Applicant may cancel the claim, amend the claim to place the claim in proper dependent form, rewrite the claim in independent form, or present a sufficient showing that the dependent claim complies with the statutory requirements.
Claim Rejections - 35 USC § 102
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 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 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 28-30, 34-36, 40, 42, 46, 47 and 54-56 are rejected under 35 U.S.C. 102(a1)/102(a2) as being anticipated by Bava et al. (hereinafter “Bava”) (US Patent No. 12173360, issued December 24, 2024; filed February 19, 2021) as evidenced by Szemes et al. (hereinafter “Szemes”) (Nucleic Acids Research, 2005, 33(8), 1-13).
Regarding claim 28, Bava teaches an integrated assay of a biological sample comprising an in situ assay module and a spatial assay module, wherein the in situ assay comprises analyzing binding between nucleic acid probes and a first analyte at a spatial location of the biological sample; and providing conditions to allow spatially barcoded capture agents to capture a second analyte for analysis in the spatial assay module (interpreted as nucleic acid barcodes; and a sample, claim 28) (Abstract). Bava teaches that the product (of an analyte and/or a probe for the analyte) can be a hybridization product, a ligation product, an extension product (e.g., by a DNA or RNA polymerase), a replication product, a transcription/reverse transcription product, and/or an amplification product, such as a rolling circle amplification product (interpreted as amplifying; and rolling circle amplification, claim 28) (col 2, lines 52-57). Bava teaches that the binding interactions can be analyzed using microscopy, such as high resolution optical microscopy, to provide readouts of the presence and/or absence, distribution, location, amount, level, expression, or activity of the target analyte (e.g., nucleic acid molecules), wherein the in situ assay comprise in situ sequencing and/or in situ hybridization, such as sequential hybridization of probes to analyze about 20, about 50, about 100, about 200, about 500, about 1,000, about 2,000, about 5,000, or about 10,000 genes, e.g., mRNA transcripts from the genes in a tissue sample (interpreted as comprising readout probes; and primary variable sequence, claim 28) (col 13, lines 1-11 and 17). Bava teaches a dual readout, such as a microscopy readout and a sequencing readout (interpreted as comprising readout probes, claim 28) (col 13, lines 50-52). Bava teaches that agents, such as probes (e.g., templated ligation probes), labelling agents, analyte capture agents, and/or capture agents for the spatial assay module can be contacted with the sample prior to, together with, or after 207, 208, 209, and/or 210, wherein spatially resolved analyte information is obtained, such as by analyzing the capture probes and/or the captured analytes, in 211, such that steps of the exemplary workflow can be performed in any suitable order, wherein any one or more of steps 206, 207, 208 and 209 are performed prior to steps 210 and 211 (interpreted as amplifying a target region, then contacting the amplified product with encoding probes, claim 28a,b) (col 18, lines 63-67; and col 19, lines 1-6; and Figure 2). Figure 2A-B is shown below:
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Bava teaches that the method further comprises generating a rolling circle amplification (RCA) product in situ in the biological sample, the RCA product comprising a sequence of the first nucleic acid probe or complement thereof, the method can further comprise detecting a signal (e.g., fluorescent signal) associated with the RCA product at a spatial location of the biological sample on a first substrate (interpreted as amplifying; and rolling circle amplification (RCA), claim 28a) (col 8, lines 38-45). Bava teaches that Figure 3 shows a further exemplary workflow, wherein a sample comprises or is contacted with various nucleic acid molecules, including an optionally barcoded probe targeting Nucleic Acid 1; a barcoded circularizable probe such as a padlock probe targeting Nucleic Acid 2 and barcoded probes for ligation (e.g., RNA-templated ligation probes) which target the same or a different nucleic acid; a barcoded circularizable probe (e.g., padlock probe) targeting Nucleic Acid 3 and barcoded probes using a sequence of the circularizable probe (e.g., a barcode sequence of the circularizable probe) as a template for ligation; Nucleic Acid 4 which can be part of a labelling agent such as a reporter oligonucleotide (Nucleic Acid 4 conjugated to an antibody recognizing an analyte in the sample is shown as an example) or an endogenous molecule in the sample, such as an RNA (e.g., mRNA molecule), which undergoes reverse transcription to generate a cDNA in situ, such that the various nucleic acid molecules can be present in or contacted with the sample in any suitable combination and in any suitable temporal order (interpreted as contacting the sample with encoding probes to allow hybridization to amplified nucleic acids; comprising unique readout regions; and interpreting labelling agents as contacting the sample with labeled readout probes; and interpreting rRNA and antibodies to comprise primary variable sequences, claim 28b,c) (col 19, lines 10-28; and Figure 3). Figure 3 is shown below:
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Bava teaches that signals of the in situ assay module can be detected and optionally analyzed, for example, the barcoded circularizable probe (e.g., padlock probe) targeting Nucleic Acid 2 can be amplified using RCA, and the RCA product can be detected in situ using detection probes (e.g., including detectably labelled probes and/or intermediate probes) (interpreted as detecting labels of one or more labeled readout probes, claim 28d) (col 19, lines 40-46). Bava teaches that spatially-resolved analyte information is obtained, such as by analyzing the capture probes and/or the captured analytes, after removing captured molecules from the spatial array and collecting the captured molecules and/or products thereof for sequencing (interpreting sequencing as determining the identity of the nucleic acid barcodes, claim 28g) (col 19, lines 61-65). Bava teaches that the in situ assay comprises in situ sequencing of one or more barcode sequences of a probe that directly or indirectly binds an analyte including detecting signals indicating in situ hybridization of one or more detection oligos to one or more barcode sequences of a probe that directly or indirectly hybridizes to a nucleic acid moiety of an analyte, and a spatial and/or temporal pattern of the signals from the sequential hybridization is used to analyze the analyte (interpreting sequencing as determining the identity of the nucleic acid barcodes, claim 28g) (col 17, lines 10-19).
Regarding claims 29 and 30, Bava teaches that the method further comprises generating a spatially labeled polynucleotide comprising (i) a sequence of the nucleic acid label (corresponding to the binder and/or the protein target) or complement thereof and (ii) a sequence of the spatial barcode or complement thereof, such that the biological sample can be contacted with the binder for the protein analyte before, during, or after detecting the one or more probes at a spatial location of the sample including a binder for the protein analyte before, during, or after an in situ sequencing module performed on the sample for the first target which is a nucleic acid, wherein the protein analyte or a subunit or polypeptide sequence thereof can be encoded by a sequence of the nucleic acid analyte (interpreted as a nucleic acid barcode comprising one target region including in a protein/antibody, claims 29 and 30) (col 111, lines 35-50). Bava teaches that the capture probe can further comprise a universal domain which is 5' to the spatial barcode, wherein the universal domain comprises an amplification domain; and/or a cleavage domain for releasing the generated spatially labeled polynucleotide from the surface of the substrate (interpreting the capture probe as a nucleic acid barcode comprising one target region, claim 29) (col 7, lines 41-46). Bava teaches that the capture agent can capture the one or more nucleic acid probes hybridized to the first target nucleic acid (interpreting the capture probe as a nucleic acid barcode comprising one target region, claim 29) (col 8, lines 1-3).
Regarding claim 34, Bava teaches the one or more nucleic acid probes can comprise a primary probe that directly hybridizes to the first target nucleic acid or the complement or the amplification product thereof, wherein the primary probe can comprise a padlock probe, a circular probe, or a circularized probe comprising one or more barcode sequences which optionally correspond to a sequence of the first target nucleic acid; wherein the detecting step can comprise contacting the biological sample with one or more detectably labelled probes capable of directly or indirectly hybridizing to the primary probe (or a complement or amplification product thereof), optionally wherein the one or more detectably labelled probes hybridize to one or more barcode sequences of the primary probe (or a complement or amplification product thereof) (interpreted as a pair of probes including a linear probe and padlock probe complementary to nucleic acid barcodes, claim 34) (col 6, lines 33-36; 41-43; and 45-54). Bava teaches a method of analyzing a biological sample, comprising: (a) contacting the biological sample on a first substrate with one or more first nucleic acid probes that hybridize to a first target nucleic acid in the biological sample, wherein the first target nucleic acid is an RNA and the one or more first nucleic acid probes comprise one or more barcode sequences which correspond to a sequence of the first target nucleic acid; (b) contacting the biological sample with one or more detectably labeled probes that directly or indirectly bind to (i) the one or more barcode sequences in the one or more first nucleic acid probes or (ii) a complement of the one or more barcode sequences (interpreted as contacting a pair of probes to a nucleic acid barcode; and contacting the sample with a mixture of probes including a linear probe and/or padlock probes, claim 34); (c) using a plurality of capture agents joined directly or indirectly to a second substrate to directly or indirectly capture a ligation product from the biological sample, wherein the ligation product is generated from a templated ligation of a pair of probes hybridized to a: (i) a capture domain capable of capturing the ligation product, and (ii) a spatial barcode; and (d) generating, on the second substrate, a spatially labeled polynucleotide (cols 129-130, claim 1). Bava teaches that the one or more first nucleic acid probes comprise a primary probe that hybridizes to the first target nucleic acid, and wherein the primary probe is a circularizable probe, wherein comprising generating a rolling circle amplification (RCA) product of the circularizable probe (interpreting a padlock probe as a linear probe that is a circularizable probe; and amplifying, claim 34) (col 130, lines 49-55, claims 2, 3 and 6). Bava teaches that the pair of probes are contacted with the biological sample in (a) or after (b), wherein the pair of probes are linear probes (interpreted as contacting a pair of probes including a padlock probe and a linear probe, claim 34) (col 131, lines 8-14, claims 9 and 10). Bava teaches in Figure 3 (in part, as shown below), the circularization of a padlock probe (first panel), and a linear probe comprising a region that is complementary to at least a region of the padlock probe (second panel), wherein padlock probes are reverse complementary to a target region (interpreted as a pair of probes; circularization; and padlock probe reverse complementary to a target region, claim 34a1,a2) (Figure 3), wherein padlock probes comprise a 5’ end region and a 3’ end region that are brought into juxtaposition for circularization as [AltContent: oval]evidenced by Szemes (pg. 2, Figure 1).
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Bava teaches generating a rolling circle amplification (RCA) product in situ (interpreted as amplifying in situ to generate copies of a target region, claim 34a3) (col 8, lines 38-39).
Regarding claim 35, Bava teaches that the capture probe is immobilized on the feature of the array indirectly, e.g. via hybridization to a surface probe capable of binding the capture probe, the capture probe can further include an upstream sequence (5' to the sequence that hybridizes to the nucleic acid, e.g. RNA of the tissue sample) that is capable of hybridizing to 5' end of the surface probe (interpreted as a probe comprising a binding region, claim 35) (col 91, lines 14-20). Bava teaches that to generate probes using these oligonucleotides, a primer complementary to a portion of an oligonucleotide (e.g., a constant sequence shared by the oligonucleotides) can be used to hybridize with the oligonucleotide and extend (using the oligonucleotide as a template) to form a duplex and to create a 3' overhang (interpreted as comprising an overhang region, claim 35) (col 94, 56-61).
Regarding claim 36, Bava teaches with one or more first nucleic acid probes that hybridize to a first target nucleic acid in the biological sample, wherein the first target nucleic acid is an RNA and the one or more first nucleic acid probes comprise one or more barcode sequences which correspond to a sequence of the first target nucleic acid (interpreted as hybridizing to the first part of a nucleic acid barcode, claim 36) (col 129, claim 1, lines 43-50). Bava teaches in Figure 4B shows an in situ assay workflow, where an exemplary primary probe set binds to an RNA target, exemplary secondary probes each hybridizes to a barcode sequence of the primary probe or an amplification product (e.g., RCA product) thereof (interpreted as hybridizing to the first part of a nucleic acid barcode, claim 36) (col 10, lines 32-36; and Figure 4B).
Regarding claim 40, Bava teaches that the first strand cDNA can be amplified using PCR in 1006, wherein the forward and reverse primers flank the spatial barcode and target analyte regions of interest, generating a library associated with a particular spatial barcode (interpreted as a library of nucleic acid barcodes, claim 40) (col 52, lines 22-25). Bava teaches that for multiple capture probes that are attached to a common array feature, the one or more spatial barcode sequences of the multiple capture probes can include sequences that are the same for all capture probes coupled to the feature, and/or sequences that are different across all capture probes coupled to the feature (interpreted to include barcodes that comprise the same sequence, claim 40) (col 67, lines 25-30).
Regarding claim 42, Bava teaches that the padlock probe can be circularized using RNA-templated ligation, see, e.g., the first and third padlock probes in Figure 5, where an RNA-templated ligase can be used to close the circle of a linear DNA probe to circularize the padlock; See, e.g., PCT/EP2018/077161, which is incorporated herein by reference in its entirety (interpreted as using a ligase to circularize a padlock probe, claim 40) (col 41, lines 3-11).
Regarding claims 46 and 47, Bava teaches that a padlock probe or a probe set that comprises a padlock probe contains one or more barcodes one or more barcodes are indicative of a sequence in the target nucleic acid, such as a single nucleotide of interest (e.g., SNPs or point mutations), a dinucleotide sequence, a short sequence of about 5 nucleotides in length, or a sequence of any suitable length (interpreted as a padlock probe encoding a primary and secondary variable sequence, claims 46 and 47) (col 38, lines 65-67; and col 39, lines 1-6).
Regarding claim 54, Bava teaches that smFISH is applied to a multiplexed workflow, wherein consecutive/sequential hybridizations are used (e.g., as in seqFISH or scqFISH+) to impart a temporal barcode on target transcripts, wherein sequential rounds of fluorescence in situ hybridization can be accompanied by imaging and probe stripping, detecting individual transcripts (e.g., RNA transcripts) within a biological sample of interest (e.g., a tissue sample, a single cell, or extracted RNA) including where each round of hybridization comprises a pre-defined set of probes, such as between about 10 and about 50 probes such as 24 to 32 probes that target unique RNA transcripts (interpreted as >2 unique readout regions, claim 54) (col 35, lines 21-32).
Regarding claims 55 and 56, Bava teaches that a padlock probe or a probe set that comprises a padlock probe contains one or more barcodes one or more barcodes are indicative of a sequence in the target nucleic acid, such as a single nucleotide of interest (e.g., SNPs or point mutations), a dinucleotide sequence, a short sequence of about 5 nucleotides in length, or a sequence of any suitable length (interpreted as a variable sequence of 15-300 nucleotides, claims 55 and 56) (col 38, lines 65-67; and col 39, lines 1-6).
Bava does not specifically exemplify a padlock probe having a sequence complementary to the secondary variable sequence (claims 47, in part).
Bava meets all the limitations of the claims and, therefore, anticipates the claimed invention.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and
103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis 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.
Claims 28-30, 34-36, 40, 42, 46, 47 and 54-56 are rejected under 35 U.S.C. 103 as being unpatentable over Bava et al. (hereinafter “Bava”) (US Patent No. 12173360, issued December 24, 2024; filed February 19, 2021) in view of Deng et. al. (hereinafter “Deng”) (Chem, 2018, 4, 1373-1387; and Supplemental Information, 2018, 4, S1-S46) as evidenced by Szemes et al. (hereinafter “Szemes”) (Nucleic Acids Research, 2005, 33(8), 1-13).
The teachings of Hacohen as applied to claims 28-30, 34-36, 40, 42, 46, 47 and 54-56 are described supra.
Bava does not specifically exemplify padlock probes having a sequence complementary to the secondary variable sequence (claims 47, in part).
Regarding claim 47 (in part), Deng teaches that the expression and spatial profile of multiple RNA species at high precision in single cells is key information for understanding cellular behaviors and functions (Abstract, lines 1-2). Deng teaches that because the DNA sequence can store and encode abundant information, it is a superior candidate for molecular barcodes, such that the sequence programmability and well-understood hybridization thermodynamics could potentially enable encoding with the DNA sequence in a thermodynamic manner including using a method termed sequence-encoded amplicon (SeqEA), which is a DNA sequence-encoded fluorescence barcoding method based on the thermodynamic modulation of DNA hybridization, which achieves multiplex in situ RNA tagging, allowing for highly multiplexed imaging of RNAs in single cells with single-molecule and single-nucleotide resolution (pg. 1375, first full paragraph, lines 12-21). Deng teaches that the key design of SeqEA is the utilization of the programmable DNA hybridization process to encode RCA amplicons for multiplex RNA tagging (Figure 1), wherein SeqEA is performed with an encoded padlock probe composed of two modules: the recognition (R) and barcode (B1 and B2) modules, such that single-target RNAs hybridized with the R module are specifically recognized by the padlock probes, initiating RCA to produce a long DNA amplicon with hundreds of repeats of the padlock probe, such that the RCA amplicon forms a nano-clew that can be visualized by being hybridized with the fluorophore-labeled detection probes P1 and P2 (pg. 1375, last full paragraph, lines 1-8). Deng teaches that the fluorescence barcodes (amplicons with different fluorescence intensities) for tagging different target RNAs are simply constructed by tuning the sequence of barcode module B1 (Figure S1), where benefiting from its small size, the padlock probe can penetrate into fixed cells, enabling in situ recognition of single-molecule target RNA inside the cells, such that multiple RNA species can thus be visualized specifically with single-molecule resolution by in situ amplification and the sequence-encoding method (pg. 1375, last full paragraph, lines 11-17). Figure 1 and S1 are shown below:
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Fig. 1
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Fig. S1
Deng teaches in Figures 3(A) and 3(C), the hybridization reaction for sequence encoding; where the sequence of B1 (sequence marked in red is complementary with the P1 probe), the hybridization efficiency of B1-P1, and the histogram plots for the fluorescence intensity of amplicons 1–9 in the barcode B1 channel, wherein the fluorescence intensity of amplicons was the average fluorescence intensity of all pixels within the amplicons (interpreted as padlock probes having a sequence complementary to the secondary variable sequence, claim 47) (pg. 1378, Figure 3C). Deng teaches in Figure S2, where underlines in the barcode module B1 indicate the complementary sequence hybridized to detection probe P1 (interpreted as padlock probes having a sequence complementary to the secondary variable sequence, claim 47) (pg. 1378, Figure S2). Deng teaches in Table S3 that
underlines in the barcode modules B1 and B2 indicate complementary sequence hybridized to
the detection probes P1 and P2, respectively (interpreted as padlock probes having a sequence complementary to the secondary variable sequence, claim 47) (pg. S35, Table S3).
It is prima facie obvious to combine prior art elements according to known methods to yield predictable results; the court held that, "…a conclusion that a claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination would have yielded nothing more than predictable results to one of ordinary skill in the art. KSR International Co. v. Teleflex Inc., 550 U.S. ___, ___, 82 USPQ2d 1385, 1395 (2007); Sakraida v. AG Pro, Inc., 425 U.S. 273, 282, 189 USPQ 449, 453 (1976); Anderson’s-Black Rock, Inc. v. Pavement Salvage Co., 396 U.S. 57, 62-63, 163 USPQ 673, 675 (1969); Great Atlantic & P. Tea Co. v. Supermarket Equipment Corp., 340 U.S. 147, 152, 87 USPQ 303, 306 (1950)”. Therefore, in view of the benefits of
simultaneously visualizing individual mRNA species and spatial patterns associated with cancer-associated oncogene expression as exemplified by Deng, it would have been prima facie obvious for one of ordinary skill in the art at the time the invention was made to modify the method for the in situ hybridization and analysis of labeled encoding probes such as padlock probes to target nucleic acids in a biological sample including from a patient with cancer as disclosed by Bava to include the method of DNA-sequence-encoded fluorescent barcoding as taught by Deng with a reasonable expectation of success in encoding RCA amplicons for multiplex in situ RNA tagging; in providing a simple method of constructing and tagging different target RNAs by tuning the sequence of barcode module B1; and/or in allowing for highly multiplexed imaging of multiple RNA species within single cells including a cancer cell with single-molecule and single-nucleotide resolution including for the spatial detection of one or more analytes in a biological sample.
Thus, in view of the foregoing, the claimed invention, as a whole, would have been obvious to one of ordinary skill in the art at the time the invention was made. Therefore, the claims are properly
rejected under 35 USC §103(a) as obvious over the art.
Conclusion
Claims 28-30, 34-36, 40, 42, 46, 47 and 54-56 are rejected.
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/AMY M BUNKER/Primary Examiner, Art Unit 1684
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