DETAILED ACTION
The amendment filed on 10/15/2025 has been entered. No new matter was added.
Claims 23 and 24 were amended in the claim set filed on 10/15/2025.
Applicant’s election without traverse of Group I (Claims 1-22 and 24-25) in the reply filed on 06/06/2025 is acknowledged. Claims 23 and 26-27 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to nonelected Group II, claims 23 and 26-27, drawn to a kit, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 06/06/2025. Applicant asks that the Examiner kindly rejoin and consider the remaining species when appropriate.
Claims 1-22, 25 and 27 are canceled.
Claims 28-40 are added.
Claims 24 and 28-40 filed are currently under examination.
Response to the Arguments
Objections to the claims in the previously mailed non-final have been withdrawn in light of Applicant’s claim amendments.
Applicant’s arguments (pg. 6) regarding previous rejections of claims 1-22 under 35 U.S.C. 112(b) and claims 3 and 5-6 under 35 U.S.C. 112(d) have been fully considered and are persuasive. The 35 U.S.C. 112(b) and U.S.C. 112(d) rejections documented in the previously mailed non-final have been withdrawn in light of applicant’s claim cancellations.
Applicant’s arguments (pg. 6-7) regarding previous rejections of claims 1-22 under 35 U.S.C. 103 have been fully considered and are persuasive. The 35 U.S.C. 103 rejections with regard to claims 1-22 documented in the previously mailed non-final have been withdrawn in light of applicant’s claim cancellations.
Applicant’s arguments regarding previous rejections of claims 24-25 under 35 U.S.C. 103 have been fully considered and are persuasive. The 35 U.S.C. 103 rejections documented in the previously mailed non-final have been withdrawn in light of applicant’s claim amendments and arguments on Pg. 7-8. As necessitated by amendment, new grounds of rejection for claims 24 and 28-40 are made, as documented below, under the 35 U.S.C. 103 rejections in this office action on Pg. 3-11.
The rejections for claims 24 and 28-40 are documented below in this Final Office Action are necessitated by claim amendments filed on 10/15/2025.
Priority
This application claims priority to U.S. Provisional Patent Application No. 63/177,165, filed 04/20/2021. The priority date of claim set filed on 10/15/2025 is determined to be 4/20/2021.
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 24 and 28-40 remain/are rejected under 35 U.S.C. 103 as being unpatentable over Kvastad et al. (“Kvastad”; (2021). The spatial RNA integrity number assay for in situ evaluation of transcriptome quality. Communications biology, 4(1), 57. Published online: January 08, 2021) in view of Bao et al. (“Bao”; (2009). Fluorescent probes for live-cell RNA detection. Annual review of biomedical engineering, 11, 25–47.).
This maintained rejection has been revised in response to claim amendments filed on 10/15/2025.
Kvastad discloses that the RNA integrity number (RIN) is a frequently used quality metric to assess the completeness of rRNA, as a proxy for the corresponding mRNA in a tissue. Current methods operate at bulk resolution and provide a single average estimate for the whole sample. Spatial transcriptomics technologies have emerged and shown their value by placing gene expression into a tissue context, resulting in transcriptional information from all tissue regions. Thus, the ability to estimate RNA quality in situ has become of utmost importance to overcome the limitation with a bulk rRNA measurement. Here we show a new tool, the spatial RNA integrity number (sRIN) assay, to assess the rRNA completeness in a tissue wide manner at cellular resolution. We demonstrate the use of sRIN to identify spatial variation in tissue quality prior to more comprehensive spatial transcriptomics workflows. (Abstract)
Regarding claim 24, Kvastad teaches a method comprising “a novel method capable of measuring a spatial RNA integrity number (sRIN) in situ from a single tissue section” (Pg. 2, Introduction, Para. 2). Thus, Kvastad teaches a method of determining RNA integrity of a biological sample.
Regarding claim 24 step (a), Kvastad teaches a method “placing single cell layer tissue sections on a glass slide with pre-printed DNA oligonucleotides complementary to the 18S rRNA”, each sRIN slide can contain up to 16 fully coated capture areas (7.5 × 7.5 mm). “pre-printed DNA oligonucleotides” and “capture areas” are interpreted as oligonucleotide with capture domains used to probe the sample analyte. Thus, Kvastad teaches a method comprising: (a) contacting the biological sample with a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises a capture domain.
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Regarding claim 24 step (b), Kvastad teaches a method wherein “we designed an assay based on placing single cell layer tissue sections on a glass slide with pre-printed DNA oligonucleotides complementary to the 18S rRNA, …The tissue section is non-covalently fixed, permeabilized, stained with hematoxylin and eosin (HE) for imaging, and incubated overnight to allow the synthesis of transcripts complementary-to-18S-rRNA (c18RNA).” (Pg. 2, The sRIN assay, Para. 1; Figure 1a -see below). Kvastad teaches a method wherein “rRNA molecules are captured from the tissue section onto a uniformly covered rRNA capture slide (Pg. 3, Discussion, Para. 2). Thus, Kvastad teaches a method comprising: (b) hybridizing a RNA analyte from the biological sample to the capture probe.
Regarding claim 24 step (c), Kvastad teaches a method wherein “synthesis of transcripts complementary-to-18S-rRNA (c18RNA)” (Pg. 2, The sRIN assay, Para. 1) and “rRNA molecules are captured from the tissue section onto a uniformly covered rRNA capture slide, copied by reverse transcription” (Pg. 3, Discussion, Para. 2). Thus, Kvastad teaches a method comprising: (c) extending a 3' end of the capture probe using the RNA analyte as a template to generate an extended capture probe.
Regarding claim 24 step (d), Kvastad teaches a method comprising “rRNA molecules are captured from the tissue section onto a uniformly covered rRNA capture slide, copied by reverse transcription fluorescently labeled oligonucleotide probes are sequentially and rapidly hybridized at multiple sites on the c18RNA (Fig. 1b)” (Pg. 2, The sRIN assay, Para. 1; Figure 1b- see above). Kvastad teaches a method comprising “we performed several rounds of hybridization using a mouse olfactory bulb tissue sample, alternating between a surface probe and P1 conjugated with Cy5 and Cy3” (Pg. 2, The sRIN assay, Col.2 Para. 3). Kvastad teaches a method comprising labeled oligonucleotide probes in Supplementary Table 1. (Supp. Table 1, Probes and primers). Thus, Kvastad teaches a method comprising: (d) hybridizing a first labeled oligonucleotide probe and a second labeled oligonucleotide probe to the extended capture probe.
Regarding claim 24 step (e), Kvastad teaches a method “After scanning each image and recording its fluorescence signal (measured in fluorescence units, FU), the probe was stripped off and another probe was hybridized. The results demonstrate reproducible sequential probe hybridizations (Supplementary Fig. 2)” (Pg. 2, The sRIN assay, Col 2., Para. 3). Thus, Kvastad teaches a method comprising measuring a first intensity of the first label and a second intensity of the second label of the plurality of labeled oligonucleotides probe.
Regarding claim 24 step (f-g), Kvastad teaches a method wherein “Control probes were designed to include complementary sequences of three detection probes at a time with a 20 bases spacer sequence between each probe (Supplementary Table 1, Integrated DNA Technologies (IDT)). For all probes used during sequential hybridization to generate sRIN heat maps, a single fluorophore (Cy3) was selected to increase consistency and minimize variability in fluorescent signal between probe positions within the synthesized c18RNA” (Pg. 5, Design of primers and probes, Para.1). The control probes are interpreted as being used for comparison and having known information such as lengths and intensity. Kvastad teaches a method comprising “The fraction of pixels with a positive signal was calculated for each probe by dividing the number of pixels with a positive Cy3 signal by the number of pixels with a positive Cy3 signal for probe 1 (P1)” (Pg. 7, Comparison between 18S+ and 18S− total RNA with sRIN assay, Para. 1). Kvastad teaches a method comprising “the P1 probe was used for normalization of P2-P4. Bars represent the mean fraction of normalized Probe 1 pixels covered on the capture area” (Supplementary Figure 6.). Thus, Kvastad teaches a method comprising correlating the ratio to one or more references, wherein each of the one or more reference comprises a labeled oligonucleotide having a known length, thereby detecting a length of the nucleic acid analyte and determining the RNA integrity of the biological sample; measuring a first FRET efficiency of the first and second labeled oligonucleotide probes; comparing the first FRET efficiency to a second FRET efficiency of one or more reference analytes having a known length; and correlating a ratio of the first and second FRET efficiencies to the known length.
Kvastad does not explicitly teach all of the limitations of steps (d-g). Specifically, “wherein the first labeled oligonucleotide probe is capable of transferring energy to the second labeled oligonucleotide probe by Forster resonance energy transfer (FRET); determining the energy transferred to the second labeled oligonucleotide probe; measuring FRET efficiency.
Bao discloses commonly used techniques for analyzing gene expression, such as polymerase chain reaction (PCR), microarrays, and in situ hybridization, have proven invaluable in understanding RNA processing and regulation. However, these techniques rely on the use of lysed and/or fixed cells and are therefore limited in their ability to provide important spatial-temporal information. This has led to the development of numerous techniques for imaging RNA in living cells, some of which have already provided important insight into the dynamic role RNA plays in dictating cell behavior. Here we review the fluorescent probes that have allowed for RNA imaging in living cells and discuss their utility and limitations. Common challenges faced by fluorescent probes, such as probe design, delivery, and target accessibility, are also discussed. It is expected that continued advancements in live cell imaging of RNA will open new and exciting opportunities in a wide range of biological and medical applications. (Abstract)
Regarding claim 24, Bao teaches a method wherein “As shown schematically in Figure 1b, linear FRET probes utilize two linear oligonucleotides that are fluorescently labeled at their 5′ and 3′ end with donor and acceptor fluorophores, respectively, forming a FRET pair … These probes are designed to hybridize to adjacent regions on a nucleic acid target such that the two fluorophores are brought into close proximity only when both probes are hybridized to the same RNA target. Excitation of the donor fluorophore leads to sensitized emission of acceptor fluorescence, producing a FRET signal indicative of target detection. Because a FRET signal is only generated when both probes hybridize to adjacent sequences on target RNA, this method provides a novel way to differentiate between target recognition and background fluorescence from unbound probes. This results in a significant improvement in signal-to-background ratio (i.e., fluorescent signal in the presence of target-to-fluorescent signal in the absence of target) compared with fluorescently labeled linear probes” (Pg. 29, Linear FRET Probes, Para.1; Figure 1b shown below). Detection of hybridized probes to one or more regions on the target RNA is interpreted as intuitively suggesting the length based on regions that the probes are
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designed to hybridize and detection of the probes.
Thus, Kvastad and Bao teach a method comprising steps (d-g) of determining RNA integrity of a biological sample, the method wherein the first labeled oligonucleotide probe is capable of transferring energy to the second labeled oligonucleotide probe by Forster resonance energy transfer (FRET) a dipole-dipole coupling mechanism; (e) determining the energy transferred to the second labeled oligonucleotide probe; (f) measuring a first FRET efficiency of the first and second labeled oligonucleotide probes; (g) comparing the first FRET efficiency to a second FRET efficiency of one or more reference analytes having a known length; and (h) correlating a ratio of the first and second FRET efficiencies to the known length, thereby detecting a length of the RNA analyte and determining the RNA integrity of the biological sample.
Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of determining spatial RNA quality in situ as taught by Kvastad to incorporate the method of wherein the first labeled oligonucleotide probe is capable of transferring energy to the second labeled oligonucleotide probe by a dipole-dipole coupling mechanism; determining the energy transferred to the second labeled oligonucleotide probe as taught by Bao and provide a method to detect the length of the RNA analyte using FRET efficiency thereby determining the RNA integrity of the biological sample. Doing so would allow for enhanced characterization of RNA quality using distinct fluorescence resonance energy transfer (FRET) measurements to determine the hybridization of the labeled probes along the length of the extended capture or complement thereof.
The teachings of Kvastad and Bao are documented above in the rejection of claim 24 under 35 U.S.C. 103 above. Claim 28-29, 31 and 38-40 depends on claim 24. Claim 30 depends on claim 29, which depends on claim 24. Claims 35-37 depend on claim 34. Claims 33 and 34 depend on claim 32, which depends on claim 31, which depends on claim 24.
Claim interpretations: Regarding claim 32, “if the RNA integrity is determined to be high” of the instant claim is interpreted as optional. Claims 33-37 depend on claim 32 and thus are considered optional as well.
Regarding claim 28, Kvastad teaches a method wherein “By comparing this sRIN heat map (Fig. 1d) to images of the tissue section acquired after staining with HE (Fig. 1e), one can determine the distribution of spatial RNA integrity at cellular resolution” (Pg. 2, The sRIN assay, Col. 2, Para. 1). Thus, Kvastad teaches a method further comprising generating an image of the extended capture probe and using the image of the extended capture probe to generate a spatial nucleic acid integrity number for a location on the substrate.
Regarding claims 29 and 30, Kvastad teaches a method wherein “to retrieve the spatial RNA information… complementary to the 18S rRNA, … allow the synthesis of transcripts complementary-to-18S-rRNA (c18RNA)” (Pg. 2, The sRIN assay, Para. 1). Thus, Kvastad teaches a method wherein the RNA analyte is a ribosomal RNA (rRNA) analyte or a transfer RNA (tRNA) analyte; and wherein the rRNA is 18S rRNA, 28S rRNA, or a combination thereof.
Regarding claims 32 and 33, Kvastad teaches a method wherein “by analyzing the ST results from the serial tissue sections located both before and after the section used in the sRIN analysis, we found that the spatial RNA quality distribution observed in the sRIN analysis was representative of that in the wider 3D sample space” (Pg. 3, Applied sRIN assay to a less well-preserved clinical sample, Para. 1; Figure 4 and Supplementary Figure 9). “if the nucleic acid integrity is determined to be high” of the instant claim is interpreted as optional. Thus, Kvastad teaches a method wherein if the RNA integrity is determined to be high, the method further comprises determining abundance and/or location of a plurality of analytes in a related biological sample, wherein the related biological sample is a serial tissue section from the biological sample and further comprising imaging the serial tissue section.
Regarding claim 38, Kvastad teaches a method wherein “Control probes were designed to include complementary sequences of three detection probes at a time with a 20 bases spacer sequence between each probe (Supplementary Table 1, Integrated DNA Technologies (IDT)). For all probes used during sequential hybridization to generate sRIN heat maps, a single fluorophore (Cy3) was selected to increase consistency and minimize variability in fluorescent signal between probe positions within the synthesized c18RNA” (Pg. 5, Design of primers and probes, Para.1). The control probes are interpreted as being used for comparison and having known information such as lengths and intensity. Kvastad teaches a method wherein “A sample’s RNA integrity number (RIN) is often defined based on the relative abundance of full length transcripts. High-quality samples consist predominantly of full-length transcripts while low-quality samples contain mostly short, fragmented transcripts” (Pg. 2, Introduction, Para.1). spatial distribution of RNA integrity is estimated from that of the 18S rRNA (Fig. 1c), which is visualized as a heat map covering the entire tissue section. By comparing this sRIN heat map (Fig. 1d) to images of the tissue section acquired after staining with HE (Fig. 1e), one can determine the distribution of spatial RNA integrity at cellular resolution” (Pg. 2, The sRIN assay., Para.1). Thus, Kvastad suggests a method wherein an increase in the length of the RNA analyte compared to the known length correlates to an increase of the RNA integrity of the biological sample.
Regarding claim 39, Kvastad teaches a method wherein “Fresh frozen tissues” (Pg. 5, Cell cultivation, frozen tissue sections, and RNA extraction, Para. 1). Thus, Kvastad teaches a method wherein the biological sample is a tissue sample, wherein the tissue sample is a fresh frozen tissue sample or a fixed tissue sample.
Regarding claim 40, Kvastad teaches a method comprises “subarrays with spatially barcoded printed oligonucleotides with a 3-end designed to capture poly-A tailed transcripts (Pg. 6, ST in brief, Para. 1). Thus, Kvastad teaches a method wherein the capture domain comprises a sequence complementary to the RNA analyte, wherein the RNA analyte hybridizes to the capture probe via the capture domain.
Response to Arguments
Applicant' s arguments filed on 10/15/2025 (Pg. 7-8) with respect to claims 24 and 28-40 have been considered but do not apply to the new grounds of rejections. To clarify some instances argued in the response filed 10/15/2025 that remain relevant to the new grounds of 103 rejection documented in this Final Office Action, examiner’s responses to each relevant argument made by Applicant are provided below:
Applicant’s argument: “However, Bao likewise does not disclose a method of determining RNA integrity of a biological sample using FRET as claimed at least because Bao fails to teach comparing a first FRET efficiency to a second FRET efficiency of one or more reference analytes having a known length, and correlating a ratio of both the first and second FRET efficiencies to the known length.” (Pg. 7)
Response: Applicant's arguments filed 10/15/2025 have been fully considered but do not apply to the new grounds of rejections. As necessitated by amendment, see the new grounds of rejection towards claim 24 under 35 U.S.C. 103. Furthermore, in response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Applicant’s argument: “Bao does not provide a reason to modify Kvastad to arrive at the claimed subject matter reciting use of FRET to determine RNA integrity. Assuming, arguendo, that one of ordinary skill in the art were to combine Kvastad and Bao, as proposed by the Office, this would not yield a method with all the claimed limitations. At least for these reason, amended claim 24 is not obvious over the cited art.” (Pg. 7-8)
Response: In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of determining spatial RNA quality in situ as taught by Kvastad to incorporate the method of wherein the first labeled oligonucleotide probe is capable of transferring energy to the second labeled oligonucleotide probe by a dipole-dipole coupling mechanism; determining the energy transferred to the second labeled oligonucleotide probe as taught by Bao and provide a method to detect the length of the RNA analyte using FRET efficiency thereby determining the RNA integrity of the biological sample. Doing so would allow for enhanced characterization of RNA quality using distinct fluorescence resonance energy transfer (FRET) measurements to determine the hybridization of the labeled probes along the length of the extended capture or complement thereof.
Conclusion of Response to Arguments
In view of the amendments, new grounds of rejections and responses to arguments are documented in this Final Office Action. No claims are in condition for allowance.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KENDRA R VANN-OJUEKAIYE whose telephone number is (571)270-7529. The examiner can normally be reached M-F 9:00 AM- 5:00 PM.
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/KENDRA R VANN-OJUEKAIYE/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682