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 .
Claim Status
Claims 5-7 have been cancelled. Claims 1-4 and 8-17 are pending and under examination. Claims 1, 4, 8-13, and 15 have been amended. Claim 1 is the only independent claim.
Drawings
The drawings were received on 05/11/2026. These drawings are accepted. The objection to the drawings is withdrawn.
Response to Arguments
Rejections withdrawn
The rejection of claims 1-4 and 8-17 under 35 U.S.C. 103 as being unpatentable over Oki et al. (US 2022/0251639 A1, listed on IDS as translation of WO 2020/235563 A1, published Nov. 26 2020) in view of Honda et al. (Nat. Comm., (2021) 12:4416, listed on IDS as bioRxiv published Mar. 20, 2020) is withdrawn following Applicant’s amendments to claim 1.
Rejections maintained
The rejection of claims 1-17 are rejected under 35 U.S.C. 103 as being unpatentable over Oki et al. (US 2022/0251639 A1, listed on IDS as translation of WO 2020/235563 A1, published Nov. 26 2020) in view of Honda et al. (Nat. Comm., (2021) 12:4416) as applied to claims 1-4, 8-17 above and included here for reasons supra, and further in view of Eberwine et al. (US 2017/0253876 A1, published Sep. 7, 2017) and Pirrung et al. (Bioconjugate Chem., 1996, 7, 317-321) is maintained.
Applicant's arguments filed 05/11/2026 have been fully considered but they are not persuasive. The applicant argues that “Pirrung… does not discloses or suggest ‘wherein the first amplification templates and the second amplification templates are capable of being separated based on the first affinity reagent and the second affinity reagent, respectively, via affinity chromatography,’ as recited in amended claim 1.” Applicant’s argument attacks Pirrung individually with respect to a limitation that the rejection does not rely on Pirrung to teach.
As set forth in the rejection, “Eberwine further teaches that such affinity tagged oligonucleotide constructs may be used to recover nucleic acid templates corresponding to defined regions of a biological sample (see Abstract, Figs. 4, 17, 19-20, & 22, [0049], [0195]-[0196], and throughout).” Eberwine discloses that released materials may be isolated via affinity purification using an affinity column specific to an affinity tag on the caged construct [0288], and further discloses that a plurality of caged molecules bearing distinct, individually identifiable affinity tags may be used such that “the index can be used as an affinity tag in order to purify or sort capture nucleic acid molecules based on the presence and/or identity of the index” (see Eberwine [0170], see also [0296]). This is a direct teaching of differential separation of pooled multiply-tagged amplification templates from one another based on the identity of the respective affinity reagent each carries, via affinity based isolation.
Pirrung’s role in the stated rejection is limited to its teaching that a photoremovable cage molecule may be bound directly to an affinity reagent (biotin) so as to gate the affinity reagent’s binding function until photo-removed, as recited in the “photoremovable cage molecule bound to the… affinity reagent” limitation of amended claim 1. The rejection does not rely on Pirrung, alone or in combination to teach that the resulting amplification templates are separable from one another via affinity chromatography; that limitation is expressly attributed to Eberwine.
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).
Claim Rejections - 35 USC § 103
Claims 1-4, and 8-17 are rejected under 35 U.S.C. 103 as being unpatentable over Oki et al. (US 2022/0251639 A1, listed on IDS as translation of WO 2020/235563 A1, published Nov. 26 2020) in view of Honda et al. (“High-depth spatial transcriptome analysis by photo-isolation chemistry”, of record) further in view of Eberwine et al. (US 2017/0253876 A1, published Sep. 7, 2017) and Pirrung et al. (“A General Method for the Spatially Defined Immobilization of Biomolecules on Glass Surfaces Using ‘Caged’ Biotin”, of record).
The present rejection restates and applies against claim 1 as amended, the same combination of Oki, Honda, Eberwine, and Pirrung previously applied in the prior Office Action against then-pending claims 5-7. Amended claim 1 incorporates the subject matter of cancelled claims 5-7, as acknowledged by Applicant’s Remarks dated 05/11/2026, citing paragraphs [0042], [0050], and [0062] of the published specification as support.
Claim Construction:
For purposes of examination, certain claim terms that are broad or otherwise require construction have been interpreted in accordance with their broadest reasonable interpretation consistent with the specification, as is required during prosecution. See MPEP 2111; In re Am. Acad. of Sci. Tech. Ctr., 367 F.3d 1359, 1369, 70 USPQ2d 1827, 1834 (Fed. Cir. 2004). The following claim constructions have been applied in this office action.
Affinity Reagent. The claims recite “a first affinity reagent” and “a second affinity reagent” without further structural limitation. Paragraph [0019] of the specification states that “[t]he affinity reagent may be an antibody, an aptamer, or a Fab fragment, for example” (emphasis added). This is exemplary, non-limiting language. Paragraph [0042], cited by Applicant as support for the instant amendment, likewise states that “[t]he affinity reagent 202 is, for example, an antibody 202… Alternatively, the affinity reagent 202 may be an aptamer or a Fab fragment” (emphasis added). Neither passage restricts “affinity reagent” to any enumerated species, nor does the claim itself recite any structural limitation narrowing the term. Under the broadest reasonable interpretation, “affinity reagent” encompasses any molecule capable of specific binding to a corresponding capture or detection moiety, including small-molecule affinity tags such as biotin, digoxigenin, or dinitrophenyl.
“Capable of being separated based on the first affinity reagent and the second affinity reagent, respectively, via affinity chromatography.” This limitation is construed as requiring that the first amplification templates and the second amplification templates be differentially separable from one another, i.e., resolvable into distinct populations based on which of the first or second affinity reagent each respective template carries, rather than being merely, individually extractable in bulk from the biological sample. This construction is supported by paragraph [0062] of the specification, which states that amplification templates “may be extracted from the biological sample and separated based on the affinity of the first and second affinity reagents”, i.e., separated from one another, in a manner that serves as an alternative means of assigning each template to a specific one of the regions of interest in place of sequence based assignment via the identifying region.
In regards to claim 1, Oki and Honda are acknowledged by the instant specification as disclosing “a transcriptomics method for determining the transcriptome of a region of interest of a biological sample based on photo-caged oligodeoxynucleotides” (see [0006] of the instant specification). Oki discloses an oligonucleotide comprising a promoter sequence, an adapter sequence, and a discrimination (barcode) sequences, unique molecular identifier, and target sequence (see Oki Fig. 1, 4, [0046]). At least one nucleotide of which is modified with a photolabile protecting group such as 2-(2-nitrophenyl)propyl group, 2-(2-nitrophenyl)propyloxymethyl group, 1-(2-nitrophenyl)ethyl group, and 6-nitropiperonyloxymethyl group (NPOM) (see Oki Figs. 1 & 4, [0049], and throughout). Oki also teaches scanning the sample with different light parameters that corresponding to different protecting groups used (see [0060]), and teaches using different parameters for different regions of sample (see [0065]-[0067], and throughout). Oki discloses independent, sequential irradiation of at least two distinct regions of interest of a single tissue section (neural tube and hindlimb) with separate analysis of each (see Oki [0128], [0137]). Oki further teaches synthesizing a complementary first strand from a template bound to the target binding regions of the oligonucleotide constructs (see [0058], [0065], and throughout).
Honda, co-authored by Oki and disclosing the same core photo-caged-oligonucleotide chemistry, additionally discloses independent, sequential focused light irradiation of three distinct regions of interest of a single tissue section (dorsolateral, mediomedial, and ventromedial; see Honda pg. 5 rt. Col. 1st para.), each separately profiled by RNA-seq. To the extent Oki’s two-region embodiment is deemed insufficient standing alone to teach the claimed “first region of interest… second region of interest,” limitation scanned with “a first focused light beam… and a second focused light beam,” Honda’s three region embodiment confirms that region specific, sequential focused light irradiation of multiple distinct regions of interest of a single sample, using Oki-type caged oligonucleotide chemistry, was known in the art.
Neither Oki nor Honda discloses an affinity reagent was part of the disclosed oligonucleotide construct. Eberwine remedies this deficiency. Eberwine discloses a caged oligonucleotide molecule for capturing nucleic acid targets corresponding to defined regions of biological sample, comprising a photo-linker and an antisense capture strand, wherein the caged molecule further comprises “a label or affinity tag that allows for isolation of the antisense oligonucleotide-target nucleic acid molecule complex” (see Eberwine [0145], see also Fig. 4, 17, 19-20, & 22, [0049], [0195]-[0196]), with disclosed affinity tags including biotin, digoxigenin, and dinitrophenyl (see Eberwine [0195]). Eberwine further discloses that released material may be isolated via affinity purification using an affinity column specific to the affinity tag (see Eberwine [0267]), and that a plurality of caged molecules bearing distinct, individually identifiable affinity tags may be used such that “the index can be used as an affinity tag, in order to purify or sort captured nucleic acid molecules based on the presence and/or identity of the index” (see Eberwine [0170], [0269]). Under the claim construction set forth above, this is a direct teaching of differential separation of pooled, multiply-tagged amplification templates from one another , based on the identity of the respective affinity reagent each carries, via affinity-based isolation.
It would have been obvious to a person of ordinary skill in the art before the effective filling date of the claimed invention to modify the Oki/Honda oligonucleotide constructs to further include an affinity tag as taught by Eberwine, with a reasonable expectation of success, because Oki, Honda, and Eberwine are drawn to the same field of endeavor, caged-oligonucleotide based capture and amplification of nucleic acid targets from biological samples, and address the same general problem of multiplexed transcript capture and resolution, such that a person of ordinary skill would have looked to Eberwine’s affinity-tag teachings as a known alternative or supplement to Oki/Hondas sequence based discrimination scheme, for the predictable benefit of enabling isolation of capture material by physical/chemical separation method in addition to, or in lieu or, sequence based discrimination.
Neither Oki, Honda, nor Eberwine discloses a photoremovable cage molecule bound to the affinity reagent itself, as distinguished from being bound elsewhere on the oligonucleotide construct. Pirrung remedies this deficiency. Pirrung discloses a caged binding member system in which the affinity reagent is “biotin derivative is protected from its normal binding to avidin by a photoremovable nitrobenzyl group. When exposed to light through a mask, this group is lost, the patterned, irradiated sites gain affinity for avidin,” (see Pirrung Introduction). This directly teaches “a photoremovable cage molecule bound to the… affinity reagent” as claimed, under the construction of “affinity reagent” set forth above.
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to further modify the Oki/Honda/Eberwine construct such that the photoremovable cage taught by Oki and Honda is instead bound to the affinity tag taught by Eberwine, in the manner taught by Pirrung, with a reasonable expectation of success, because (i) Oki and Honda already establish that photoremovable caging of a construct element controllably gates a downstream function of that element (hybridization/extension) until light-triggered uncaging; (ii) Pirrung teaches, in the same general field of photo-controlled biomolecular capture chemistry, that this same design principle applies directly to an affinity reagent controllably gating its binding function; and (iii) relocating the cage from the oligonucleotide backbone onto the affinity tag itself would have predictably yielded the benefit of region-specific, light-gated control over which affinity tag becomes available for downstream affinity based isolation, a benefit consistent with the shared design principle common to Oki, Honda, Eberwine, and Pirrung, that photo-caging is used to impose spatial or temporal selectivity of a molecular capture or binding function.
The combination of Oki, Honda, Eberwine, and Pirrung, as set forth above, discloses every limitation of claim 1, considered as a whole:
“A method for generating region-specific amplification templates of a biological sample, the method comprising: adding at least a plurality of first oligonucleotide constructs and a plurality of second oligonucleotide constructs to the biological sample, wherein each first oligonucleotide construct comprises a first promoter region, a first adapter region, a first target binding region, a first identifying region, (see Oki Figs. 1 & 4, [0047]-[0049]) a first affinity reagent (see Eberwine [0145], see also Fig. 4, 17, 19-20, & 22, [0049], [0195]-[0196]), and a first photoremovable cage molecule bound to the first affinity reagent (see Pirrung Intro.), and wherein each second oligonucleotide construct comprises a second promoter region, a second adapter region, a second target binding region, a second identifying region (see Oki Figs. 1 & 4, [0047]-[0049]), a second affinity reagent (see Eberwine [0145], see also Fig. 4, 17, 19-20, & 22, [0049], [0195]-[0196]), and a second photoremovable cage molecule bound to the second affinity reagent (see Pirrung Intro), synthesising a complementary first strand from a template bound to the first target binding regions of the first oligonucleotide constructs or the second target binding regions of the second oligonucleotide constructs (see Oki [0058], [0065], and throughout). scanning at least a first region of interest of the biological sample with a first focused light beam and a second region of interest of the biological sample with a second focused light beam to form uncaged first oligonucleotide constructs in the first region of interest and uncaged second oligonucleotide constructs in the second region of interest (see Oki [0054]-[0060], [0128], Honda’s multi-region confirmation), and synthesising a complementary second strand to the uncaged first oligonucleotide constructs to form first amplification templates originating from the first region of interest, and to the uncaged second oligonucleotide constructs to form second amplification templates originating from the second region of interest (see Oki [0061]-[0062]), wherein the first amplification templates and the second amplification templates are capable of being separated based on the first affinity reagent and the second affinity reagent, respectively, via affinity chromatography (see Eberwine [0049], [0170], [0195]-[0196], [0269]).
Claims 2-4 and 8-17 depend from claim 1 and stand or fall with claim 1 for at least the reasons set forth above. No separate patentable weight has been identified for additional limitations of these claims that is not otherwise addressed by the cited combination, and Applicant’s remarks do not separately argue the patentability of the clams 2-4 or 8-17 apart from their dependency on claim 1.
Conclusion
No claim is allowed.
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.
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/MATTHEW HAROLD RAYMONDA/ Examiner, Art Unit 1684
/AARON A PRIEST/ Primary Examiner, Art Unit 1681
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