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 .
Claims 1-12, 15-17, 19-22, and 28 are pending. Claim 28 is withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Claims 13-14, 18, 23-27, and 29-41 are cancelled. Claims 1, 9, 17, and 19 are amended. Claims 1-12, 15-17, and 19-22 are currently under examination.
Response to Amendment
The Amendment filed 3/9/26 has been entered. Claims 1-12, 15-17, 19-22, and 28 are pending. Applicant’s amendments to claims 1, 9, 17, and 19 have overcome the objections and 112(b) rejections previously set forth in the Non-Final Office Action.
Response to Arguments
Applicant’s arguments, see pages 6-8 and page 8, filed 3/9/26, with respect to the rejections of claims 1-12, 15-17, and 19-22 under 35 USC 103 and claims 1-7, 15-17, and 22-25 under nonstatutory double patenting have been fully considered are found unpersuasive, and the 103 and nonstatutory double patenting rejections documented in the Non-Final mailed 9/8/25 have been revised to address claim amendments filed 3/9/26 in this Final Office Action. More detailed responses to Applicant’s arguments are provided at the end of each maintained rejection.
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.
Claims 1-12, 15-17, and 19-22 remain/are rejected under 35 U.S.C. 103 as being unpatentable over Credle et al. (2017; NPL citation 1 in IDS filed 6/30/22; “Multiplexed Analysis of Fixed Tissue RNA using Ligation in Situ Hybridization”. Nucleic Acids Research, Vol. 45, No. 14. Doi: https://doi.org/10.1093/nar/gkx471) in view of Frei et al. (2016; NPL citation U in PTO-892 filed 9/8/25; "Highly multiplexed simultaneous detection of RNAs and proteins in single cells"; Nat Methods 13, 269-275; https://doi.org/10.1038/nmeth.3742).
This rejection is revised/updated in response to claim amendments filed 3/9/26.
(i) Credle et al. teaches limitations relevant to claims 1-5, 9-10, 12, 15, 17, and 19-22.
Relevant to claim 1 a) – d), Credle et al. Figure 1 and associated caption teach "Workflow of the LISH [ligation in situ hybridization] assay. Step 1. Hybridization of pairs of chimeric 3'-diribonucleotide-containing and 5'-phosphorylated DNA probes on formalin fixed RNA within a tissue section. Step 2. Adjacently annealed probe pairs are then ligated in situ with Rnl2. Step 3. RNase H treatment (i) releases RNA-templated ligation products into solution for downstream analysis and (ii) destroys unwanted DNA-templated ligation products. Step 4. Amplification of ligation products by multiplex PCR (using universal ‘outside’ primers, ‘OF’ and ‘OR’)."
Relevant to claims 2-3, Credle et al. teaches "Tissues were fixed in formalin for at least 48 h prior to dehydration and paraffin wax embedding" (page 2, paragraph 1 of MATERIALS and METHODS section "Tissues and sections").
Relevant to claims 4-5, Credle et al. Figure 1 and associated caption teach "Workflow of the LISH [ligation in situ hybridization] assay. Step 1. Hybridization of pairs of chimeric 3'-diribonucleotide-containing and 5'-phosphorylated DNA probes on formalin fixed RNA within a tissue section. Step 2. Adjacently annealed probe pairs are then ligated in situ with Rnl2. Step 3. RNase H treatment (i) releases RNA-templated ligation products into solution for downstream analysis and (ii) destroys unwanted DNA-templated ligation products. Step 4. Amplification of ligation products by multiplex PCR (using universal ‘outside’ primers, ‘OF’ and ‘OR’)."
Relevant to claim 9, Credle et al. Figure 1 and associated caption teach “Step 2. Adjacently annealed probe pairs are then ligated in situ with Rnl2".
Relevant to claim 10, Credle et al. teaches that their LISH products were sequenced using Illumina platform (page 3, MATERIALS AND METHODS section "Illumina sequencing").
Relevant to claim 12, Credle et al. Abstract teaches "High-throughput DNA sequencing modalities, including single molecule sequencing, can be used to analyze ligation products from complex panels of LISH probes (‘LISH-seq’), which can be amplified efficiently and with negligible bias."
Relevant to claim 15, Credle et al. teaches that their LISH products were sequenced using Illumina platform (page 3, MATERIALS AND METHODS section "Illumina sequencing"). Additionally, Credle et al. Fig. 1 shows that the LISH products contain the ligated sequence formed by the probes.
Relevant to claim 17, Credle et al. Fig. 1 shows that the two multi-partite probes have universal adapter regions, or detection probes.
Relevant to claim 19, Credle et al. Fig. 1 shows that the two multi-partite probes have a spacer sequence between the universal adapter regions, or detection probes.
Relevant to claims 20-21, Credle et al. Fig. 3 caption teaches "The LISH assay is non-destructive and compatible with common histological stains", with Fig. 3B depicting imaging results and the targets within the samples.
Relevant to claim 22, Credle et al. Fig. 3 caption teaches "The LISH assay is non-destructive and compatible with common histological stains. (A) LISH-qPCR was performed twice on the same FFPE sections using two non-overlapping probe pools (Panel-A or Panel-B) in opposite order. Panel-A was measured after LISH-1 on section AB and after LISH-2 on section BA. Panel-B was measured after LISH-1 on section BA and after LISH-2 on section AB. (B) Hematoxylin and Eosin (H&E), Cresyl Violet (CV), Periodic Acid Schiff (PAS), Calcofluor White (CW) and -CD3 Immunohistochemical Staining (IHC) were used prior to performing LISH. qPCR analysis of RPS19 probe ligation product was used to measure loss of signal due to staining, by comparison with an unstained section. Scale bar (100 m for H&E, CV, PAS and IHC) and (50 m for CW). Error bars denote +/− s.d. of the mean."
(ii) Credle et al. is silent to specifics regarding rolling circle amplification relevant to claims 1, 6-8, 10-11, and 16. However, these limitations were known in the prior art and taught by Frei et al.
Relevant to claim 1 e) – h), Frei et al. teaches "PLAYR [proximity ligation assay for RNA] uses the concept of proximity ligation [citations] to detect individual transcripts in single cells, as shown schematically in Fig. 1a, and is compatible with immunostaining. Pairs of DNA oligonucleotide probes (probe pairs) are designed to hybridize to two adjacent regions of target transcripts in fixed and permeabilized cells. Each probe in a pair is composed of two regions with distinct function. The role of the first region is to selectively hybridize to its cognate target RNA sequence. The second region, separated from the first by a short spacer, acts as template for the binding and circularization of two additional oligonucleotides (termed backbone and insert). When hybridized to an adjacent probe pair, the backbone and insert oligonucleotides form a single-stranded DNA circle that can be ligated. The ligated, closed circle is then amplified through rolling circle amplification by phi29 polymerase initiated by the 3′ OH of one member of the probe pair. As phi29 continues to polymerize, it creates a linear molecule that contains hundreds of concatenated complementary copies of the original circle [citation]. Then, using a labeled oligonucleotide that is complementary to the insert region of the amplicon, one can detect any given probe pair through binding to the amplified product" (page 269, paragraph 1 of RESULTS section "Overview of PLAYR technology and probe design").
Relevant to claim 6, Frei et al. Fig. 1a shows that the backbone and insert contact the probes (step 3 - equivalent to step (e)) before ligation (step 4 - equivalent to step (d)).
Relevant to claim 7, Frei et al. Fig. 1a shows that the ligating steps are performed simultaneously (step 4 - equivalent to steps (d) and (f)).
Relevant to claim 8, Frei et al. Fig. 1a shows that the backbone and insert contact the probes (step 3 - equivalent to step (e)) before simultaneous ligation (step 4 - equivalent to steps (f) and (d)).
Relevant to claim 10, Frei et al. teaches "The ligated, closed circle is then amplified through rolling circle amplification by phi29 polymerase initiated by the 3′ OH of one member of the probe pair. As phi29 continues to polymerize, it creates a linear molecule that contains hundreds of concatenated complementary copies of the original circle [citation]. Then, using a labeled oligonucleotide that is complementary to the insert region of the amplicon, one can detect any given probe pair through binding to the amplified product" (page 269, paragraph 1 of RESULTS section "Overview of PLAYR technology and probe design").
Relevant to claim 11, Frei et al. teaches "PLAYR can be multiplexed via the use of designed oligonucleotides with different insert regions that act as cognate barcodes for given transcripts [citation]. Different insert sequences are designed to have the same melting temperatures and base compositions and form RCA products with similar efficiency (Supplementary Fig. 1). To ensure that RCA products uniquely barcode a particular transcript, the insert sequences do not have common substrings longer than 4 bases" (page 270, column 1, paragraph 1).
Relevant to claim 16, Frei et al. teaches "The ligated, closed circle is then amplified through rolling circle amplification by phi29 polymerase initiated by the 3′ OH of one member of the probe pair. As phi29 continues to polymerize, it creates a linear molecule that contains hundreds of concatenated complementary copies of the original circle [citation]. Then, using a labeled oligonucleotide that is complementary to the insert region of the amplicon, one can detect any given probe pair through binding to the amplified product" (page 269, paragraph 1 of RESULTS section "Overview of PLAYR technology and probe design").
(iii) Although Credle et al. did not explicitly teach the Frei et al. rolling circle amplification, it would have been prima facie obvious to the skilled artisan. Credle et al. and Frei et al. are analogous disclosures within the instant probe hybridization and ligation detection methodology.
The skilled artisan would be motivated to combine the analogous art. Credle et al. teaches “the ability to efficiently template the ligation of DNA probes directly on fixed RNA in situ may facilitate emerging technologies such as … PLAYR [Frei et al. citation]... For these reasons, LISH is a promising new methodology for biomedical researchers and pathologists” (page 8, column 2, paragraph 1). Credle et al. provides the motivation to include the Frei et al. rolling circle amplification within the multi-partite probe methodology. The skilled artisan would have a reasonable expectation of success based on the disclosures of Credle et al. in view of Frei et al.
Applicant’s Arguments and Response to Applicant’s Arguments
Applicant argues that “Credle et al., requires the ligation of adjacently annealed probes in situ and is silent with respect to a circularized and locked target nucleic acid. Frei et al., requires additional probes which become circularized, i.e., it is the probes which are circularized and not the entire target. Therefore, the combination of Credle et al., and Frei et al., would result in circularized probes attached to the target nucleic acid and not the target nucleic acid” (Remarks 3/9/26, page 7, paragraphs 2-3).
Applicant further argues “The combination of cited references fail to teach, disclose or otherwise suggest a circularized and locked target nucleic acid… Applicant has amended claim 1 to recite that the target nucleic acid is circularized and locked” (Remarks 3/9/26, page 8, paragraph 2).
Amended claim 1 requires “f)… forming a circularized probe that is hybridized to the target RNA, wherein circularization locks the circularized probe into place around the target mRNA, g) amplifying the circularized probe…” As written, the probe is circularized, not the target nucleic acid as argued above. Additionally, the combination of “Credle et al., and Frei et al., would result in circularized probes attached to the target nucleic acid”, thus reading on the instant circularized probe “locked” into place around the target mRNA via hybridization/base-pair complementarity.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-7, 15-17, and 22-25 remain/are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 33 and 42-54 of copending Application No. 18/209,992 (reference application). Although the claims at issue are not identical, they are not patentably distinct from each other because they are coextensive in scope.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
This rejection is revised/updated in response to claim amendments filed 3/9/26.
The instant claims are drawn to A method for detecting an immobilized target ribonucleic acid (RNA) comprising the steps of: a) contacting a biological sample comprising the target RNA in a reaction mixture with at least one probe set comprising (i) a first multi-partite probe comprising a 5' phosphorylated donor probe and a first bridge probe, wherein the 5' phosphorylated donor probe specifically hybridizes to the target RNA; and (ii) a second multi-partite probe comprising a 3' acceptor probe and a second bridge probe, wherein the 3' acceptor probe specifically hybridizes to the target RNA adjacent to the 5' donor probe and the second bridge probe is 5' phosphorylated; b) incubating the reaction mixture of step (a) under conditions that permit hybridization of the at least one probe set to the target RNA present in the biological sample; c) washing away unbound probe sets; d) ligating the 5' phosphorylated donor probe and the 3' acceptor probe of adjacent probe pairs, wherein the 5’ phosphorylated donor probe and the 3’ acceptor probe are hybridized to the target RNA; e) contacting the reaction mixture with at least one bridge primer that specifically hybridizes to the first bridge probe and the second bridge probe, wherein the first bridge probe and the second bridge probe anneal to the bridge primer adjacent to each other; f) ligating the first bridge probe of the 5’ phosphorylated donor probe and the second bridge probe of the 3’ acceptor probe thereby forming a circularized probe that is hybridized to the target RNA, wherein circularization locks the circularized probe into place around the target mRNA, g) amplifying the circularized probe by rolling circle amplification; and h) detecting the target RNA.
The copending claims are drawn to “A method of analyzing RNA from a subject, comprising: obtaining a fixed tissue sample from the subject; applying one or more multi-partite probes to the fixed tissue sample, wherein each of the one or more multi-partite probes includes at least two sub-probes; annealing at least one of the one or more applied multi-partite probes to a target nucleic acid within the fixed tissue sample; ligating the at least two sub-probes associated with the at least one annealed multi-partite probe to create a target nucleic acid proxy within the fixed tissue sample; and stamping the ligated probes onto a replica surface coated with a plurality of immobilized PCR primers to spatially amplify and detect the target nucleic acid proxy, thereby analyzing the subject’s RNA.”
The copending claims are required to perform the instant methods. Thus, they are obvious variants. Dependent claims are rejected as they are coextensive in scope.
Applicant’s Arguments and Response to Applicant’s Arguments
Applicant argues “The cited application claims do not recite a circularized and locked target nucleic acid” (Remarks 3/9/26, page 8, last paragraph).
Amended claim 1 requires “f)… forming a circularized probe that is hybridized to the target RNA, wherein circularization locks the circularized probe into place around the target mRNA, g) amplifying the circularized probe…” As written, the probe is circularized, not the target nucleic acid as argued above. Additionally, the combination of “Credle et al., and Frei et al., would result in circularized probes attached to the target nucleic acid”, thus reading on the instant circularized probe “locked” into place around the target mRNA via hybridization/base-pair complementarity.
Conclusion
THIS ACTION IS MADE FINAL Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/SARAH JANE KENNEDY/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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