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.
Claim Status
Claims 1, 3, and 51, 53-56, 58-60, and 62-67 are pending.
Claims 1 and 51 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 8/11/2025.
Claims 3, 53-56, 58-60, and 62-67 are being examined on the merits.
Specification
Applicant’s amendments to the Specification to include the proper notation for trade names and marks is acknowledged. The notification of trade names and marks without proper notation as presented in the Office Action of 9/8/2025 was not a formal objection to the specification (as evidenced by box 10 not being checked on PTO-326). Therefore, there is no objection to withdraw.
Claim Objections
The objection to claims 64-65 is withdrawn in light of Applicant’s amendments to the claims.
Withdrawn Claim Rejections
35 USC § 112b - Indefiniteness
The rejection of claims 52 and 61 under 35 U.S.C. 112(b) is withdrawn in light of Applicant’s cancellation of the claims.
The rejection of claims 56-58, 62, and 66 under 35 U.S.C. 112(b) as laid out in the Office Action of 9/8/2025 is withdrawn in light of Applicant’s amendments to the claims.
35 USC § 102
The rejection of claims 3, 52-53, 55-60, and 63-67 under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Frisen I (Frisen et al., WO 2018091676 A1; cited on IDS of 1/19/2023) is withdrawn in light of Applicant’s amendments to the claims and cancellation of claim 52.
35 USC § 103
The rejection of claim 54 under 35 U.S.C. 103 as being unpatentable over Frisen I (Frisen et al., WO 2018091676 A1; cited on IDS of 1/19/2023) in view of Zhu (Zhu et al., Bio Techniques, 2001; cited on IDS of 1/19/2023) is withdrawn in light of Applicant’s amendments to the claims.
The rejection of claim 61 under 35 U.S.C. 103 as being unpatentable over Frisen I (Frisen et al., WO 2018091676 A1; cited on IDS of 1/19/2023) in view of Lutz (Lutz et al., Nano Research, 2018) and over Frisen I (Frisen et al., WO 2018091676 A1; cited on IDS of 1/19/2023) in view of Schroeder (Schroeder et al., US 20030211483 A1) is withdrawn in light of Applicant’s cancellation of the claim.
The rejection of claim 62 under 35 U.S.C. 103 as being unpatentable over Frisen I (Frisen et al., WO 2018091676 A1; cited on IDS of 1/19/2023) in view of Frisen II (Frisen et al., WO 2014060483 A1; cited on IDS of 4/3/2023) is withdrawn in light of Applicant’s amendments to the claims.
New Claim Rejections - 35 USC § 112b - Indefiniteness
Claims 3, 53-56, 58-60, and 62-67 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 3 recites the limitation “wherein the TSO blocking moiety prohibits the rcTSO from interacting with the TSO”. It is unclear how this is possible, given that the rcTSO can only be generated via the reverse transcription of the TSO (and thus necessitates an interaction between the two). For the purposes of examination, it is being interpreted that the TSO blocking moiety prohibits the rcTSO from interacting with the TSO primer. This is in line with the embodiments presented in the specification on page 4, lines 25-28 and page 36, lines 16-20. However, further clarification is required.
Additionally, no structure is provided for the TSO blocking moiety. The specification states that “in some embodiments” the TSO blocking moiety is a nucleotide sequence, however this would mean that the TSO blocking moiety could be anything that prevents interaction between the rcTSO and the TSO primer (small molecule inhibitor, caged nucleobases, etc.). Therefore, the scope of this claim is indefinite and clarification is required.
Claims 53-56, 58-60, and 62-67 depend from claim 3, inherit this deficiency, and are rejected on the same basis.
Claims 54 and 55 recite the limitation "the TSO". There is insufficient antecedent basis for this limitation in the claim. These claims depend from claim 3, which defines "a plurality of template switching oligonucleotides (TSOs)". Switching from plural "TSOs" to singular "the TSO" in these dependent claims makes it unclear whether these additional limitations apply to a single type of TSO among a plurality of different TSOs or if there is only one type of TSO and a plurality of copies of this one type of TSO. Further clarification is required.
Response to Remarks
Applicant’s amendments to the claims submitted on 12/8/2025 resolved most issues presented in the previous Office Action (as described in the Withdrawn Claim Rejections above). However, additional 112b’s have been made above to address indefiniteness in the newly amended claim 3, and indefiniteness that was not addressed in the Response to Remarks regarding claims 54-55.
New Claim Rejections - 35 USC § 103
Claims 3, 53, 55-56, 58-60, and 63-67 are rejected under 35 U.S.C. 103 as being unpatentable over Frisen I (Frisen et al., WO 2018091676 A1; cited on IDS of 1/19/2023) in view of Liu (Liu et al., Accounts of Biochemical Research 2013).
Claim 3: Frisen I teaches a method of processing an analyte in a biological sample (Abstract). Frisen I’s method comprises hybridizing an analyte from the biological sample to a capture probe that comprises a capture domain and a spatial barcode (claim 3(a); “positional domain”, pg 4, ln 10-14 and pg 33, ln 23-24). Frisen I teaches contacting the analyte with an aqueous reaction mixture that comprises a polymerase enzyme capable of extending the capture probes using the target nucleic acid analyte (pg 10, ln 30-34). The polymerase capable of doing so is a reverse transcriptase (claim 3(b)(ii)) and for reverse transcription Frisen I teaches using a “suitable buffer” and “dNTPs” (claim 3(b)(i) and 3(b)(iii); pg 68, ln 5). Reverse transcription of the target analyte is referred to as first strand synthesis. Frisen teaches including template switching oligonucleotides for performing second strand synthesis (pg 60, ln 21-35 and pg 61, ln 1-11). Frisen I teaches that first strand synthesis (reverse transcription) and second strand synthesis can occur in one reaction (claim 3(c); “second strand synthesis may occur contemporaneously with the first strand synthesis or may be performed immediately following the first strand synthesis reaction”, pg 16, ln 11-14). Frisen I teaches that reverse transcription generates an extended capture probe that comprise a complementary sequence of a TSO (rcTSO) in the plurality of TSOs and that TSO primers will bind to said rcTSO to generate the second strand (“the oligonucleotide probe that hybridises to the cDNA overhang contains an amplification domain sequence, the complement of which is incorporated into the synthesised first strand cDNA product. Primers containing the amplification domain sequence, which will hybridise to the complementary amplification domain sequence incorporated into the cDNA first strand, can be added to the reaction mix to prime second strand synthesis”, pg 60, ln 29-35).
Frisen I does not teach that a TSO blocking moiety is included in the reaction, or that this blocking moiety prohibits the rcTSO from interacting with the TSO primer (see claim interpretation and 112b rejection of claim 3 above). However, use of blocking moieties to selectively control the start of an amplification reaction is known in the art, as taught by Liu.
Liu teaches the use of photolabile moieties in the backbone of oligonucleotides that allows for the selective cleavage of oligonucleotides and their subsequent “activation” (Introduction, paragraph 3 and Scheme 1B). Liu teaches a specific example in which an oligonucleotide is prevented from interacting with its target until light-mediated cleavage allows for hybridization between the oligonucleotide and its target (Scheme 1B). Liu also teaches an example in which photo-caged nucleobases are included within an oligonucleotide strand, which allows for activation (hybridization between the strand and its target) upon application of light (Scheme 1D).
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 Frisen I with that of Liu to include a moiety for the selective activation of an oligonucleotide. One would be motivated to do so given the teaching by Liu that one can noninvasively and selectively control the activation of synthesis by providing these moieties as a blocker to prevent association between a primer and its target (Scheme 3). Liu teaches that this can reduce nonspecific amplification (2.2 Light-Activated and Light-Deactivated Polymerase Chain Reaction). One would have a reasonable expectation of success given that Liu specifically teaches that this is non-invasive, and thus would not require separation of steps such that first and second strand synthesis could be performed in the same reaction, as taught by Frisen I.
Claim 53: Frisen I teaches using a reverse transcriptase that has terminal transferase activity and template switching ability (Superscript I-III or M-MLV, pg 16, ln 22-23).
Claim 55: Frisen I teaches hybridizing the TSO to the terminal nucleotides added by the reverse transcriptase. This places the TSO directly adjacent to the 5’ end of the analyte (pg 60, ln 25-29). Given that the specification does not provide a specific example for “coupling” the examiner is interpreting coupling to mean that the TSO is directly adjacent to the 5’ end of the analyte. Frisen I teaches that upon further extension of the first strand (“cDNA molecule”), a complement of the oligonucleotide probe (TSO) is incorporated into the synthesized first strand cDNA (pg 60, ln 29-31). This generates an extended capture probe that is complementary to the analyte and the TSO.
Claim 56: Frisen I teaches hybridizing the TSO primer to the rcTSO and extending the TSO primer using the extended capture probe as a template to generate a second strand that is complementary to all or a portion of the analyte and all or a portion of the capture probe (pg 60, ln 29-35).
Claim 58: Frisen I teaches that the second strand synthesis occurs immediately after reverse transcription, meaning that the TSO primer would necessarily have to bind to the rcTSO of the extended capture probe immediately after the reverse transcription for second strand synthesis to occur (pg 16, ln 11-14).
Claim 59: Frisen I teaches that the second strand synthesis is performed immediately after the reverse transcription (pg 16, ln 11-14).
Claim 60: Frisen I teaches that the second strand synthesis is performed contemporaneously or immediately after the reverse transcription (pg 16, ln 11-14) and that all components for the reaction can be added in a single reaction mix (pg 72, ln 26-27), therefore indicating that there is no wash step between reverse transcription and second strand synthesis.
Claim 63: Frisen I teaches permeabilizing the biological sample (pg 50, ln 5-9).
Claim 64: Frisen I teaches that the capture probe furthers comprises a cleavage domain and a functional domain such as an amplification domain (pg 27, ln 8-12).
Claim 65: Frisen I teaches that the biological sample can be “fresh, frozen, fixed, or unfixed tissues” and that the fixation may be “formalin-fixation and paraffin-embedding (FFPE)” (pg 47 ln 12-17 and ln 33-34).
Claim 66: Frisen I teaches imaging the biological specimen after using a staining technique known in the art (pg 56, ln 1-8) and particularly teaches imaging using fluorescence microscopy, therefore indicating that the tissue was stained using immunofluorescence. Frisen teaches that imaging the biological sample can be accomplished before the nucleic acid is processed (pg 53, ln 31-34).
Claim 67: Frisen I teaches that the analyte is an RNA molecule (pg 7, ln 1-2 and pg 15, ln 28-32).
Claim 54 is rejected under 35 U.S.C. 103 as being unpatentable over Frisen I (Frisen et al., WO 2018091676 A1; cited on IDS of 1/19/2023) in view of Liu (Liu et al., Accounts of Biochemical Research 2013) as applied to claims 3, 53, 55-56, 58-60, and 63-67 above, and further in view of Zhu (Zhu et al., Bio Techniques, 2001; cited on IDS of 1/19/2023).
The teachings of Frisen I in view of Liu regarding the method of claim 3, from which claim 54 depends, are detailed in the rejection above. Briefly, and relevant to the instantly rejected claim, Frisen I in view of Liu teaches a method of processing an analyte in a biological sample in which first strand and second strand synthesis take place in the same reaction and wherein second strand synthesis is accomplished through the use of template switch oligonucleotides. Frisen I teaches that the template switch oligonucleotide (TSO) comprises a sequence that hybridizes to the capture probe (pg 60, ln 27-29).
Frisen I in view of Liu does not teach that the TSOs are 10 to 50 nucleotides in length or that they are comprised of DNA. However, use of TSOs that are 10 to 50 nucleotides in length that are comprises of DNA is known in the art, as taught by Zhu.
Zhu teaches performing second strand synthesis following reverse transcription of an RNA template using TSOs that are 39 nucleotides long and are composed of DNA (Materials and Methods - Oligonucleotides for cDNA Synthesis and Figure 1).
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 Frisen I in view of Liu with that of Zhu. One would be motivated to use the method as taught by Zhu given that Zhu teaches successful template switching with oligonucleotides to perform second strand synthesis with an oligonucleotide that effectively hybridizes and acts as a template at 39 nt in length. One would have a reasonable expectation of successful given the demonstration by Zhu that this methodology can be performed using a target such as polyA RNA and given the fact that Frisen I directly references use of the SMART™ technology that Zhu describes (pg 60, ln 21-23).
Claim 62 is rejected under 35 U.S.C. 103 as being unpatentable over Frisen I (Frisen et al., WO 2018091676 A1; cited on IDS of 1/19/2023) in view of Liu (Liu et al., Accounts of Biochemical Research 2013) as applied to claims 3, 53, 55-56, 58-60, and 63-67 above, and further in view of Frisen II (Frisen et al., WO 2014060483 A1; cited on IDS of 4/3/2023).
The teachings of Frisen I in view of Liu regarding the method of claims 3 and 56, from which claim 62 depends, are detailed in the rejection above. Briefly, and relevant to the instantly rejected claim, Frisen I in view of Liu teaches a method of processing an analyte in a biological sample in which first strand and second strand synthesis take place in the same reaction and wherein second strand synthesis is accomplished through the use of template switch oligonucleotides.
Frisen I in view of Liu does not teach releasing the second strand using physical denaturation or chemical denaturation. However, use of physical denaturation to release the second strand after second strand synthesis is known in the art, as taught by Frisen II.
Frisen II teaches a method of spatial detection of RNA in a tissue sample using capture probes that hybridize to the RNA targets (pg 11). Frisen II teaches first strand synthesis via reverse transcription (pg 11, ln 16-17) and second strand synthesis via template switching (pg 60, ln 1-4 and ln 23-27). Frisen II then teaches releasing the second strand cDNA molecule from the surface of the substrate (pg 11, ln 29-34). Frisen II teaches that the release of the second strand molecule is achieved by “physical means” such as “denaturing” (pg 64, ln 8-14).
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 Frisen I in view of Liu with that of Frisen II. One would be motivated to apply the “releasing” step from Frisen II given the assertion by Frisen II that this allows for sequencing analysis of the second strand to determine the positional information encoded thereon (pg 62, ln 33-35 and pg 63, ln 1-2). One would have a reasonable expectation of success given that Frisen II asserts that methods of releasing the second strand through physical means such as denaturation are “well known in the art” (pg 64, ln 12-14).
Response to Remarks
New rejections have been made above given Applicant’s amendment to claim 3 to include the limitations of what was claim 61 in the previous Office Action. In regards to Applicant’s arguments against the 103 rejection of inclusion of a blocking moiety, Applicant’s arguments have been fully and carefully considered and are deemed persuasive. For these reasons the rejection of the limitation of claim 61 over Lutz and Schroeder are withdrawn and new grounds of rejection have been presented above, making this Office Action Non-Final.
Double Patenting
All previous double patenting rejections outlined in the Office Action of 9/8/2025 are withdrawn in light of Applicant’s amendments to the claims and arguments presented in the Remarks of 12/8/2025. New grounds of Double Patenting rejection are presented below.
Although the claims at issue are not identical, they are not patentably distinct from each other because both sets of claims are drawn to the same limitations. Any additional limitations of conflicting claims are encompassed by the open claim language “comprising” found in the instant claims.
Copending Application No. 18/640,819
Claims 3, 53, 54-56, 58-60, 62-65, and 67 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 3, 6-8, 10, 12-13, 15-17, 19-21, and 23-24 of copending Application No. 18/640,819 in view of Liu (Liu et al., Accounts of Biochemical Research 2013) according to citations and rationales provided above.
Claims 66 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 3, 6-8, 10, 12-13, 15-17, 19-21, and 23-24 of copending Application No. 18/640,819 in view of Liu (Liu et al., Accounts of Biochemical Research 2013) as applied to claims 3, 53, 54-56, 58-60, 62-65, and 67 above and further in view of Frisen I (Frisen et al., WO 2018091676 A1; cited on IDS of 1/19/2023).
Regarding claim 66: The claims of ‘819 in view of Liu do not teach previously staining the biological sample using immunofluorescence, immunohistochemistry, or hematoxylin and eosin.
Frisen I teaches imaging the biological specimen after using a staining technique known in the art (pg 56, ln 1-8) and particularly teaches imaging using fluorescence microscopy, therefore indicating that the tissue was stained using immunofluorescence. Frisen teaches that imaging the biological sample can be accomplished before the nucleic acid is processed (pg 53, ln 31-34).
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 ‘819 in view of Liu with the teachings of Frisen I. One would be motivated to stain the biological sample given the teaching by Frisen I that this allows one to orient the location of the biological sample to a position on the array (pg 53). One would have a reasonable expectation of success given that Frisen I assert that staining of biological specimens is well known in the art (pg 56, ln 1-8).
This is a provisional nonstatutory double patenting rejection.
Conclusion
No claims are allowed.
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/KAILEY ELIZABETH CASH/Examiner, Art Unit 1683
/STEPHEN T KAPUSHOC/Primary Examiner, Art Unit 1683