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 .
Please note: The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure statement (e.g., see paragraphs [0026 and 0058]). 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
New IDSs were filed on 8/27/2025, 11/5/2025, 12/12/2025, and 2/20/2025. However, these IDSs do not contain all of the references noted in the specification paragraphs above. Accordingly, those references not included on an IDS are not considered.
Claim Interpretation
Claim 52 is directed to the method of claim 51 in which the “first region is adjacent to said second region when said first oligonucleotide and said second oligonucleotide are bound to said first portion and said second portion” of the target nucleic acid. The examiner is interpreting adjacent to mean that the target sequences (that share complementarity to either the first or second oligonucleotide) of the first and second oligonucleotides are anywhere between 15 to 0 (contiguous) nucleotides apart on the target nucleic acid as per the description provided in the specification in paragraph [0040].
Maintained Claim Rejections - 35 USC § 112a – New Matter
Claims 49-73 and 75 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. Amended claim 49 contains the limitation “wherein said tissue sample comprises a thickness of at least 50 micrometers (µm)”. There is no support in the disclosure for any thickness above 200µm, which the open-ended “at least” implies in this claim. For example, there is no support in the disclosure for tissue thicknesses of 1,000µm.
Response to Remarks
Applicant's arguments filed 2/20/2026 have been fully considered but they are not persuasive for the following reasons. Applicant traverses the 112a-Written Description rejection of claims 49-73 and 75 on pages 5-6 of Remarks. Applicant argues that claims 49-73 and 75 have support in the description and cite the recitations of “whole tissue” (paragraph [0103]), “entire adult mouse brain is roughly 400 cubic millimeters” (paragraph [0208]) which translates to roughly “7370 micrometers”, and “intact tissue includes brain tissue” (paragraph [0047]). While these recitations are present within the specification, support for the open-ended use of “at least” is not. With regard to range limitations, the MPEP states “the analysis must take into account which ranges one skilled in the art would consider inherently supported by the discussion in the original disclosure. In the decision in In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976), the ranges described in the original specification included a range of "25%- 60%" and specific examples of "36%" and "50%." A corresponding new claim limitation to "at least 35%" did not meet the description requirement because the phrase "at least" had no upper limit and caused the claim to read literally on embodiments outside the "25% to 60%" range, however a limitation to "between 35% and 60%" did meet the description requirement.” Even though the specification recites that an entire adult mouse brain is roughly 400 cubic millimeters, which would be 7370 micrometer sections, the specification does not disclose performing the methodology on an intact whole mouse brain. The specific example citing the size of a mouse brain discusses performing the methodology on “thick-section[s]”, or flat sections obtained from the whole tissue sample (paragraphs [0221] and [0229]). Furthermore, the “at least” provides no upper limit to the claim which now encompasses any embodiment even above a whole, intact mouse brain. For these reasons the rejection of claims 49-73 and 75 under 35 USC 112a-Written Description is maintained.
Maintained Claim Rejections - 35 USC § 103
Claims 49-62, 65-69, 71, and 73-75 are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (Science, July 27, 2018; cited on IDS from 7/15/2022) in view of Nuovo et al. (US 5538871 A, issued July 23, 1996).
Wang et al. teach a method of targeted 3D in situ transcriptomics within intact tissue samples (Abstract).
Specifically, Wang et al. teach in situ gene sequencing of a nucleic acid in a cell in a tissue sample comprising contacting said tissue sample with a plurality of oligonucleotides under conditions sufficient to allow for specific hybridization (claim 49(a); Structured Abstract). Wang et al. teach applying this methodology to thick tissue sections (claim 49(a); 150µm, Supplementary Materials page 6 – Sample preparation). Once hybridization to target nucleic acids is achieved, Wang et al. teach performing an amplification reaction by contacting said tissue sample with nucleotides to generate one or more amplicons from said plurality of oligonucleotides (claim 49(c); Structured Abstract and Figure 1). They then teach imaging said one or more amplicons to identify said nucleic acid (claim 49(d); Structure Abstract and Figure 1). Wang et al. teach heating said plurality of oligonucleotides prior to applying the oligonucleotide probes to the tissue sample (claim 65; “The probe mixture was heated at 90C for 2 to 5 min and then cooled down at r.t” Supplementary Materials page 3 – Library construction). Wang et al. teach that the target nucleic acid is a ribonucleic acid, more specifically a messenger ribonucleic acid (claims 68-69; Figure 1A).
As shown in the Figure below (Figure S1A from Wang et al. Supplementary Material), the method of Wang et al. employs first and second oligonucleotides, in which the first oligonucleotide comprises a first region that binds to a first portion of said nucleic acid, the first nucleotide binds to said second oligonucleotide, and said second oligonucleotide binds to said nucleic acid (claim 50; Figure S1A). Additionally, as exemplified by the figure below, the second oligonucleotide comprises a second region that binds to a second portion of said nucleic acid (claim 51), and the first region is adjacent to said second region when said first oligonucleotide and said second oligonucleotide are bound to said first portion and said second portion of nucleic acid, respectively (claim 52). The short arrows on the padlock probe and the corresponding complementary sequences on the hybridized DNA oligonucleotide represent the third region, fourth region, fifth region, and sixth region discussed in claims 53-54.
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Wang et al. go on to teach, subsequent to adding the plurality of oligonucleotides to the tissue sample, adding a ligase to said tissue sample to generate a closed nucleic acid circle using said second oligonucleotide and amplifying the target via rolling circle amplification (claims 55 and 58; Fig S1G, Supplementary Material – Library construction). The amplicons are “then copolymerized with acrylamide to embed within a hydrogel network…followed by clearance of unbound lipids and proteins (claims 56-57 and 59-60; Figure 1A, Figure 1A legend, and Figure S2).
Wang et al. teach including a barcode sequence within the second oligonucleotide, which, upon generation of amplicons via rolling circle amplification, generates one or more amplicons [that] comprise a barcode (claim 61) which is then “read-out through in situ sequencing” via imaging (claim 62; Figure 1 and Figure 1 legend). Wang et al. teach using an imaging system, specifically a confocal microscope, to perform in situ sequencing, which allows identification of the target nucleic acid through detection of a barcode sequence within the generated amplicons (claims 71 and 73; Figure 1A and Methods summary).
Regarding the tissue sample used, Wang et al. teach applying this methodology to both thin tissue sections and thick tissue sections (claim 74; 150um, Supplementary Materials page 6 – Sample preparation). In addition, Wang et al. teach that this method can be performed on a fresh-frozen tissue sample (claim 67; Figure S16A). Wang et al. teach adding a permeabilization agent to said tissue sample prior to hybridization of the oligonucleotides with the tissue sample (claim 75; “Brain slices were fixed with 4% PFA in PBS at r.t. for 10 min, permeabilized with -20oC methanol and then placed at -80oC for 15 min before hybridization.” Supplementary Materials page 3 – Sample preparation).
Wang et al. does not teach preincubating said tissue sample with a polymerase in an absence of nucleotides (claim 49(b)) or using a formalin-fixed paraffin embedded tissue sample. However, withholding reaction reagents to delay start of amplification is a strategy that is known in the art, as is using formalin-fixed paraffin embedded tissue samples for in situ amplification, as taught by Nuovo et al.
Nuovo et al. teaches a method of in situ PCR amplification in which “at least one reagent, preferably enzyme but quite possibly primers, dNTPs, or MgCl2, is omitted from the reaction mixture” at the start and subsequently added to the sample to control amplification start time (col 12, lines 9-18). Nuovo et al. also teach using formalin-fixed samples that are then embedded in paraffin and sliced for use in in situ PCR reactions (col 11, lines 37-55).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Wang et al. (which teaches contacting the tissue sample with an amplification mixture including both nucleotides and polymerase) to perform a pre-incubation of the tissue without nucleotides and potentially use a formalin-fixed paraffin embedded tissue sample (as taught by Nuovo et al.). One would be motivated to delay addition of nucleotides until a desirable reaction condition was achieved, as taught by Nuovo et al. In the case of Nuovo et al., the desirable reaction condition was temperature of the sample at which they wanted PCR to occur. However, Nuovo et al. acknowledge the unique challenge of performing amplification within fixed and permeabilized tissues, namely that “membranes must be permeabilized sufficiently to allow externally applied PCR reagents to reach the target nucleic acid” (column 5, lines 40-45). This highlights a need for allowing time for amplification reagents to reach target nucleic acids for amplification, namely a pre-incubation of a subset of amplification reagents until a desirable reaction condition (adequate diffusion into tissue) is reached. One would have a reasonable expectation of success given that addition of a single incubation step prior to addition of a nucleotides would not hinder the ability of the amplification reaction to proceed once all necessary components were present in the sample. One would be motivated to employ a formalin-fixed paraffin embedded tissue sample, as this is a commonly used method to preserve and work with tissue samples “in the art of clinical pathology” and allows one to section the sample into desirable thicknesses for downstream analyses according to need (col 11, lines 55-60).
Wang et al. in view of Nuovo et al. do not explicitly teach that that the pre-incubation of the thick tissue section with a polymerase in the absence of nucleotides is done “for a sufficient time to allow uniform diffusion of said polymerase throughout said tissue sample” (claim 49(b)). However, Wang et al. teaches developing their methodology to “overcome limitations in diffusional access” and teaches optimizing their methodology by testing the “specificity and penetration depth” of labelling components throughout 150µm thick tissues (Results - Adapting STARmap to thick tissue blocks for 3D analyses). Wang et al. additionally teaches that for thick tissue sections, the amplification reaction is carried out for 12 to 24 hours, rather than the 2 hours used for thin tissue sections (Supplementary Materials pages 4 and 6). Therefore, the necessity for adapting a methodology to account for uniform diffusion of reaction components throughout a tissue sample by optimizing time of incubation with said reaction components is taught by Wang et al. It would therefore be obvious, through routine optimization, to determine the sufficient amount of time to allow uniform diffusion of a polymerase throughout said tissue sample.
Claims 63-64 are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (Science, July 27, 2018; cited on IDS from 7/15/2022) in view of Nuovo et al. (US 5538871 A, issued July 23, 1996) as applied to claims 49-62, 65-69, 71, and 73-75 above, and further in view of Eid et al. (WO2007064905A2, 2007; cited on IDS from 11/19/2024).
Methods as taught by Wang et al. in view of Nuovo et al. are detailed above. Relevant to the instantly rejected claims, Wang et al. in view of Nuovo et al. teaches use of a buffer applied to tissue samples before imaging (Wang et al., Supplementary Materials page 4 – Imaging and sequencing).
Wang et al. in view of Nuovo et al. does not teach the inclusion of an antioxidant, specifically N-propyl gallate, in the buffer added prior to imaging. However, use of antioxidants or “anti-fade” reagents in buffers applied to samples before imaging was known in the art, as taught by Eid et al.
Eid et al. teach methods of limiting photodamage (or “photobleaching”) to samples requiring sustained imaging, such as for sequencing reactions (paragraph [0029]). Specifically, Eid et al. teach inclusion of “anti-fade” reagents in “illuminated reaction mixture” (reads on a buffer which is added prior to imaging). The specification of the instant application describes antioxidants added to buffers as “anti-fade buffers” (paragraph [0093]). One such anti-fade reagent named by Eid et al. is N-propyl gallate (paragraph [0045]).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Wang et al. in view of Nuovo et al. (which teaches applying an imaging buffer to a sample prior to imaging for in situ sequencing) to add an antioxidant such as n-propyl gallate to the imaging buffer, as taught by Eid et al. One would be motivated to include n-propyl gallate in the imaging buffer in order to reduce the amount of photobleaching that would occur during in situ sequencing, as taught by Eid et al. An antioxidant would enable this photosensitive reaction to proceed for a longer period of time and minimize damage to reagents used in the reaction, as taught by Eid et al. (paragraphs [0063-0064]). One would have a reasonable expectation of success given that Eid et al. performs sequencing reactions that include various reducing agents (antioxidants) successfully.
Claim 70 is rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (Science, July 27, 2018; cited on IDS from 7/15/2022) in view of Nuovo et al. (US 5538871 A, issued July 23, 1996) as applied to claims 49-62, 65-69, 71, and 73-75 above, and further in view of Bagasra (Nature Protocols, November 1, 2007).
Methods as taught by Wang et al. in view of Nuovo et al. are detailed above. Relevant to the instantly rejected claim, Wang et al. in view of Nuovo et al. teach detection and sequencing of mRNA in a tissue sample
Wang et al. in view of Nuovo et al. do not teach DNA as the target nucleic acid. However, detection of target DNA in a tissue sample through amplification was known in the art, as taught by Bagasra.
Bagasra teaches methods of amplifying both RNA and DNA targets within fixed tissue samples, the amplicons of which can then be detected by labelled probes (Abstract). Bagasra teaches that amplification of DNA in a tissue sample is very similar to amplification of RNA in a tissue sample and involves the use of a PCR reaction mixture as well as probes designed for sequence specific amplification.
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Wang et al. in view of Nuovo et al. (which teaches detection of amplicons of mRNA) to detect DNA amplicons as well, as taught by Bagasra. One would be motivated to detect DNA sequences through such a method given its potential application in diagnostics (such as detecting tumor genes, copy number variations, etc.) within tissue samples, as taught by Bagasra (Introduction). One would have a reasonable expectation of success given the similarity in how DNA would be amplified as compared to mRNA in the method of Wang et al., the DNA amplification reaction would also require hybridization of sequence specific probes and subsequent amplification and identification of barcoded amplicons through imaging.
Claim 72 is rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (Science, July 27, 2018; cited on IDS from 7/15/2022) in view of Nuovo et al. (US 5538871 A, issued July 23, 1996) as applied to claims 49-62, 65-69, 71, and 73-75 above, and further in view of Shah et al. (Development, August 1, 2016; cited on IDS from 7/15/2022).
Methods as taught by Wang et al. in view of Nuovo et al. are detailed above. Relevant to the instantly rejected claim, Wang et al. in view of Nuovo et al. teach using confocal microscopy as their imaging system.
Wang et al. in view of Nuovo et al. do not teach the use of a light sheet microscope in their imaging systems. However, use of light sheet microscopes for detection of nucleic acids in situ is known in the art, as taught by Shah et al.
Shah et al. teach a method of mRNA detection in situ in intact tissues within a hydrogel (Abstract). Detection of the mRNA is achieved through fluorescent amplification polymers (Fig 1A). Shah et al. teach using selective plane illumination microscopy (SPIM; reads on light sheet microscope according to the instant specification paragraph [0096]).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Wang et al. in view of Nuovo et al. (which teaches using confocal microscopy as the imaging system) to employ light sheet microscopy, as taught by Shah et al. One would be motivated to utilize this type of imaging system given that “SPIM…offers a fast alternative (~100 times faster than confocal microscopy) that rejects out-of-focus noise by illuminating and capturing images only from a thin selective plane” (Shah et al., pg 2865 col 2 para 3 – p 2866 col 1 para 1). One would have a reasonable expectation of success given that Shah et al. successfully applied this imaging method to whole mount tissues in which amplified target nucleic acids were embedded within a hydrogel.
Response to Remarks
Applicant’s arguments submitted on 2/20/2026 have been carefully considered but are not deemed persuasive for the following reasons.
Applicant has two arguments pertaining to the combination of Wang and Nuovo regarding the 35 USC 103 rejection of claim 49:
1) Applicant argues that “modifying Wang to include a pre-incubation step would obviate one of the stated advantages of Wang” (pg 6 of Remarks). Specifically, Applicant argues that adding a pre-incubation step would “increase the time of the workflow of Wang” (pg 6 of Remarks) and would “obviate the stated advantages of the Wang protocol as simplified and efficient” (pg 7 of Remarks). This argument is not persuasive. Nuovo teaches that withholding a reagent until a specific reaction condition has been achieved “increase[s] amplification specificity and sensitivity” (paragraph [0022]) offering an increase in “convenience” to achieve said sensitivity and specificity (paragraph [0020]), therefore motivating a skilled artisan to apply said teaching to the methodology of Wang, and supporting the stated advantage of Wang of “efficiency”. Moreover, the convenient solution of withholding reagents until a desired reaction condition has been achieved does not necessarily equate to obviating simplicity through the inclusion of an extra step if said extra step is recognized as being simple and provides a motivation for doing so (such as increasing sensitivity and specificity).
2) Applicant argues that “Nuovo teaches away from pre-incubating a tissue sample in the absence of nucleotides” (pg 6 of Remarks). Applicant specifically highlights the “preferred mode” of Nuovo in which an “enzyme” is withheld (pg 7 of Remarks). This argument is not persuasive. “‘A reference may be said to teach away when a person of ordinary skill, upon reading the reference, would be discouraged from following the path set out in the reference, or would be led in a direction divergent from the path that was taken by the applicant.’ . . . A reference does not teach away, however, if it merely expresses a general preference for an alternative invention but does not ‘criticize, discredit, or otherwise discourage’ investigation into the invention claimed.” DePuy Spine, Inc. v. Medtronic Sofamor Danek, Inc., 567 F.3d 1314, 1327 (Fed. Cir. 2009) (citing Ricoh Co., Ltd. v. Quanta Computer Inc., 550 F.3d 1325, 1332 (Fed. Cir. 2008) (quoting In re Kahn, 441 F.3d 977, 990 (Fed. Cir. 2006)) and In re Fulton, 391 F.3d 1195, 1201 (Fed. Cir. 2004)) (emphasis added). Furthermore, “the prior art’s mere disclosure of more than one alternative does not constitute a teaching away from any of these alternatives because such disclosure does not criticize, discredit, or otherwise discourage the solution claimed….” Fulton, 391 F.3d at 1200-01. Nuovo does not discredit withholding dNTPs at any point in the disclosure, but rather mentions a preferred embodiment of withholding enzyme.
Applicant argues that claims 50-62, 65-69, 71, and 73-75 are non-obvious for the reasons presented above as applied to claim 49. The response to these arguments are detailed above.
Applicant’s arguments are not deemed persuasive and the rejection of claims 49-62, 65-69, 71, and 73-75 under 35 USC 103 over Wang in view of Nuovo is maintained.
Regarding the rejections of claims 63-64 under 35 USC 103 over Wang in view of Nuovo and further in view of Eid, claim 70 under 35 USC 103 over Wang in view of Nuovo and further in view of Bagasra, and claim 72 under 35 USC 103 over Wang in view of Nuovo and further in view of Shah: Applicant’s arguments regarding these rejections are presented on pages 7-8 of Remarks of 2/20/2026. Each of these arguments depend on the arguments discussed above as they pertain to the initial rejection of claims 49-62, 65-69, 71, and 73-75 over Wang in view of Nuovo. These arguments have been addressed above, and for those same reasons these rejections are maintained.
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.
U.S. Patent No. 12,359,253
Claims 49-62, 65-75 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-4, 7-10, 14, 17, 21, 23, 25, and 28 of U.S. Patent No. 12,359,253 in view of Wang et al. (Science, July 27, 2018; cited on IDS from 7/15/2022) in view of Nuovo et al. (US 5538871 A, issued July 23, 1996).
Regarding claim 49: Claims 1, 17, and 28 of ‘253 teach a method of in situ sequencing of a nucleic acid in a tissue sample that comprises contacting a thick tissue sample with a plurality of oligonucleotides under conditions sufficient to allow for specific hybridize, performing amplification to generate amplicons, and imaging said one or more amplicons. These claims of ‘253 do not teach preincubating said tissue sample with a polymerase in an absence of nucleotides for a sufficient time to allow uniform diffusion of said polymerase through the sample. However, this methodology is known in the art, as taught by Wang et al. in view of Nuovo et al. and as detailed in the rejection of the instant claims above.
Regarding claims 61-62: ‘253 does not teach that the one or more amplicons comprise a barcode or detecting said barcode to identify the nucleic acid. However, including a barcode in amplicons to detect a target nucleic acid is known in the art, as taught by Wang et al.
Wang et al. teach including a barcode sequence within the second oligonucleotide, which, upon generation of amplicons via rolling circle amplification, generates one or more amplicons [that] comprise a barcode (claim 61) which is then “read-out through in situ sequencing” via imaging (claim 62; Figure 1 and Figure 1 legend).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of ‘253 to include a barcode in the second oligonucleotide to produce barcoded amplicons, as taught by Wang et al. One would be motivated to do so given the assertion by Wang et al. that this enables “multiplexed gene detection” (Results - Design and validation of STARmap principles). One would have a reasonable expectation of success given that Wang et al. demonstrates successful usage of barcoded amplicons for multiplexed gene detection using in situ sequencing in intact tissue samples.
Regarding claims 66-67: ‘253 does not teach that the tissue sample is a formalin-fixed paraffin embedded tissue sample or a fresh frozen tissue sample.
However, use of formalin-fixed paraffin embedded tissue samples and fresh=frozen tissue samples for sequencing target nucleic acids is known in the art, as taught by Wang et al. and Nuovo et al.
Wang et al. teach that the method of in situ sequencing can be performed on a fresh-frozen tissue sample (claim 67; Figure S16A). Nuovo et al. teach using formalin-fixed samples that are then embedded in paraffin and sliced for use in in situ PCR reactions (col 11, lines 37-55).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of ‘253 to use either type of tissue sample. One would be motivated to use fresh-frozen tissue samples given the assertion by Wang et al. that flash freezing of tissues allows “maximal preservation of native transcriptional signatures” (Results - Spatial cell typing in primary visual cortex with 160-gene STARmapping). One would be motivated to employ a formalin-fixed paraffin embedded tissue sample, as this is a commonly used method to preserve and work with tissue samples “in the art of clinical pathology” and allows one to section the sample into desirable thicknesses for downstream analyses according to need (Nuovo et al.: col 11, lines 55-60). One would have a reasonable expectation of success with either tissue type given the successful demonstration of amplification reactions within these types of fixed tissues by both groups.
Claims 63-64 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-4, 7-10, 14, 17, 21, 23, 25, and 28 of U.S. Patent No. 12,359,253 in view of Wang et al. (Science, July 27, 2018; cited on IDS from 7/15/2022) in view of Nuovo et al. (US 5538871 A, issued July 23, 1996) and further in view of Eid et al. (WO2007064905A2, 2007; cited on IDS from 11/19/2024) according to the citations and rationales provided above.
Response to Remarks
Applicant's arguments filed 2/20/2026 have been fully considered but they are not persuasive for the following reasons. Applicant argues on page 9 of Remarks that the rejection of claim 49 over ,253 in view of Wang and Nuovo “has not made clear why a skilled artisan would conclude that the invention of claim 49 would be an obvious variation of the ‘253 patent”. As detailed in the 103 rejection above, one would be motivated to delay addition of nucleotides until a desirable reaction condition was achieved, as taught by Nuovo et al. In the case of Nuovo et al., the desirable reaction condition was temperature of the sample at which they wanted PCR to occur. However, Nuovo et al. acknowledge the unique challenge of performing amplification within fixed and permeabilized tissues, namely that “membranes must be permeabilized sufficiently to allow externally applied PCR reagents to reach the target nucleic acid” (column 5, lines 40-45). This highlights a need for allowing time for amplification reagents to reach target nucleic acids for amplification, namely a pre-incubation of a subset of amplification reagents until a desirable reaction condition (adequate diffusion into tissue) is reached. One would have a reasonable expectation of success given that addition of a single incubation step prior to addition of a nucleotides would not hinder the ability of the amplification reaction to proceed once all necessary components were present in the sample.
The arguments against the double patenting rejection of claims 63 and 64 rely on the argument against the double patenting rejection of claim 49, which has been addressed above.
For these reasons the double patenting rejection of claims 49-75 are maintained.
Conclusion
No claims are allowed.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KAILEY E CASH whose telephone number is (571)272-0971. The examiner can normally be reached Monday-Friday 8:30am-6pm ET.
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/KAILEY ELIZABETH CASH/Examiner, Art Unit 1683
/ANNE M. GUSSOW/Supervisory Patent Examiner, Art Unit 1683
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