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 1-5, 25, 28, 43, 58-61, 63, 67, 70, 74, 80, and 84 are pending and under examination. Claim 84 has been amended. Claims 1, 74, and 84 are independent claims.
Response to Arguments
Rejections Withdrawn
The rejection of claims 1-5, 25, 28, 43,58-61 under 35 U.S.C. 103 as being unpatentable over Bernitz et al. (US 2013/0004953 A1, published Jan. 3, 2013, on IDS 8/8/2022 as US 8551710 B2) in view of Cai et al. (US 2014/0031243 A1, published Jan. 30, 2014) and Chen (Science, 2015, on IDS 8/8/2022) has been withdrawn following the discussion in the videoconference interview on April 20, 2026.
New Rejections
Claim Interpretation
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.
The instant specification defines “barcode” broadly at [0291]: “A ‘barcode’ is a label, or identifier, that conveys or is capable of conveying information… ‘Barcodes’ can have a variety of different formats.” No structural limitation (e.g. requiring detection independent of any primer-binding function) narrows “probe-resolution barcode sequence” beyond this genus definition. Under the broadest reasonable interpretation consistent with the specification, a nucleic acid sequence that identifies which probe (and thus which location) produced a signal, including a primer binding sequence used for that purpose, falls within the scope of “probe-resolution barcode sequence.”
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1-5, 25, 43, 58, 63, 67, and 70 are rejected under 35 U.S.C. 103 as being unpatentable over Glezer et al. (US 2022/0042083 A1, which is prior art under 35 USC 102(a)(2) by virtue of its priority to US provisional Application No. 63/062,054, filed Aug. 6, 2020).
In regards to claim 1, Glezer discloses a method for identifying the location and identity of multiple RNA molecules in a given sample (see Fig. 5).
In regards to the limitation “(a) contacting a biological sample with a plurality of gene-specific probes each comprising a gene-specific barcode sequence, wherein a first probe and a second probe of the plurality of gene-specific probes hybridize to RNA transcripts, or cDNA molecules thereof, of the same target gene, and comprise the same gene-specific barcode sequence corresponding to the target gene, wherein the first probe of the plurality of gene-specific probes comprises a first probe- resolution barcode sequence that is not comprised by the second probe, and the second probe of the plurality of gene-specific probes comprises a second probe-resolution barcode sequence that is not comprised by the first probe and that is different from the first probe-resolution barcode sequence, and wherein the first probe hybridizes to a first RNA transcript, or cDNA molecule thereof, of the target gene at a first location in the biological sample, and the second probe hybridizes to a second RNA transcript, or cDNA molecule thereof, of the target gene at a second location in the biological sample,” Glezer teaches contacting a biological sample with a plurality of gene-specific padlock probes each comprising a gene-specific barcode sequence, (“Transcript 1” and “Transcript 2” probes, extendable to a larger multiplexed probe pool per [0220],[0277]), each comprising a shared gene-identity barcode (“Barcode 1”), which Glezer confirms is used “to identify which gene the transcript is a copy of” (see Glezer [0278]). Glezer further discloses that “two overlapping transcripts in a voxel are targeted by a set of 3 primers” (see Glezer [0221]), i.e., the first probe hybridizes to a first RNA transcript of the target gene, the second probe hybridizes to a second, physically distinct RNA transcript of the same target gene, necessarily at a different locations, consistent with the claimed “first location”/”second location” limitation. The Transcript 1 probe carries a primer-2 binding site not present in the Transcript 2 probe, the Transcript 2 probe carries a primer 3 binding site not present in the Transcript 1 probe, i.e., a first probe-resolution identifier not comprised by the second probe and a second, different probe-resolution identifier not comprised by the first probe.
In regards to the limitation “(b) detecting a plurality of signals associated with the gene-specific barcode sequence of the first probe and second probe,” Glezer disclosing detecting a plurality of signals associated with the shared gene barcode “during the first round of sequencing, using primer 1, the two transcripts are unresolvable”, i.e., a signal associated with Barcode 1 is detected for both probes collectively (see Glezer [0221]).
In regards to the limitations “(cl) detecting a signal associated with the first probe-resolution barcode sequence at the first location; and(c2) detecting a signal associated with the second probe-resolution barcode sequence at the second location, wherein the signals of steps (cl) and (c2) are associated with the target gene,” Glezer further discloses in paragraph [0221] that “in the second round of sequencing, primer 2 can hybridize and sequence the second instance of Barcode 1 in [Transcript 1],” resolving the signal in Transcript 1’s location, and “in the third round of sequencing primer 3 can hybridize and sequence the second instance of Barcode 1 in [Transcript 2],” resolving the signal at Transcript 2’s location, i.e., detecting a signal associated with the first probe-resolution sequence at the first location, and a signal associated with the second probe-resolution sequence at the second location, both of which are associated with the target gene by virtue of confirming the shared Barcode 1 identity at each location.
To the extent applicant argues that a primer binding sequence is not itself a “probe resolution barcode sequence” because the sequence read out downstream of primer binding has the same value (Barcode 1) regardless of which primer bound, Glezer expressly teaches an alternative padlock probe architecture in which “the padlock probe includes two different oligonucleotide barcodes and two different sequencing primer binding sequences”, a genuinely distinct second barcode sequence (independent of and in addition to a primer binding site) used specifically to differentiate probes. A person of ordinary skill in the art, motivated by Glezer’s own stated goal of resolving overlapping/optically crowded transcript signals (see Glezer [0003], [0217], [0219]), would have found it obvious to implement the Fig. 5 “shared gene barcode + probe differentiating element” scheme using the disclosed second discrete barcode sequence ins place of (or in addition to) the primer site differentiator, with a reasonable expectation of success, since Glezer already discloses both architectures as interchangeable design options of the same purpose within a single specification. This combination amounts to nothing more than the predictable application of a known technique (a second discrete barcode as a probe-resolution element (see Glezer [0109]), to a known method (see Glezer [0221]) to yield predictable results, namely resolving individual transcript signals by location.
In regards to claims 2 and 3, resolving signal overlap is the stated purpose of Glezer (see Glezer [0007]), and as such Glezer teaches methods for resolving “overlapping signals that are not spatially resolved into individual puncta in step (b)” (see Glezer [0218], [0221]). The use of the different primer sites in different versions of probes targeting the same gene product allows for detection wherein “an overlapping signal is associated with the signal of step (c1) or the signal of step (c2) but not both,” as recited in the claim 3.
In regards to claim 4, Glezer discloses the multi-location structure explicitly, both at the level of the imaged sample generally and at the level of the specific probe pair relied on in claim 1. Glezer establishes that gene-specific barcode signals of the type detected in step (b) are not confined toa single spot but occur at numerous location throughout the imaged sample (see Glezer [0210]-[0211]). Glezer further discloses that within this multi-location fields, Transcript 1 probe and the Transcript 2 probe occupy different, physically distinct locations (see Glazer [0210]-[0211], [0221], [0224]).
In regards to claim 5, Glezer discloses using hybridization-based detection of barcodes. In paragraph [0157] Glazer discloses a variety of sequencing methodologies can be used such as sequencing-by-synthesis (SBS), pyrosequencing, sequencing by ligation (SBL), or sequencing by hybridization (SBH) and further that in both SBL and SBH methods, target nucleic acids, and amplicons thereof, are subjected to repeated cycles of oligonucleotide delivery and detection. This discloses detectable oligonucleotide probes directly binding to a target sequence, here the RCA product borne barcode sequence or its complement, as an express alternative embodiment to primer extension based readout, reading on the “detectable probes that directly or indirectly bind to the gene-specific barcode sequence or a complement thereof” of claim 5.
In regards to claim 25, Glezer discloses this cross-gene shared probe-resolution identifier directly in Fig. 4 and [0009] disclosing a first, second, third, etc. probe in which each probe includes the unique barcode, labeled as Barcode 1, Barcode 2, and Barcode 3 for Probe 1, Probe 2, and Probe 3, respectively, which correspond with individual “gene-specific” barcodes of the instant application. These probes further comprise, in Probes 1, 2, and 3, a first primer binding sequence, corresponding to the probe-resolution sequence of the instant application. The passage continues to describe a fourth, fifth and sixth probe, which include different primer binding sequence, Primer 2 site. Which is then incorporated into Probes 4, 5, and 6 with their unique barcodes Barcode 4, Barcode 5, and Barcode 6, respectively.
Here the Primer 1 binding site functions as the probe-resolution identifier, and is common to probes 1, 2, and 3, three probes each carrying their own distinct gene-specific barcode and therefore each targeting a different target transcript. This reads directly on “the first probe-resolution barcode sequence is common among two or more probes each targeting a different target gene RNA transcript.” Symmetrically, the Primer2 binding site is common to probes 4, 5 and 6, each again carrying distinct gene-specific barcodes and thus targeting a different transcript, reading on “the second probe- resolution barcode sequence is common among two or more probes each targeting a different target gene RNA transcript.”
Example 3 of Glezer confirms this is a deliberate multiplexing strategy rather than an incidental arrangement, explaining the purpose of grouping unrelated targets under a shared primer-site identifier (see Glezer [0220]). Each probe contains either primer binding site 1 or primer binding site 2, as well as its own respective barcode, if all six targets are present in an optically resolved volume, only half will be detected when initiating sequencing from a primer binding site, thereby increasing detection efficiently, while still allowing for overlapping target detection.
In regards to claim 43, Glezer discloses the use of the padlock probe circularization mechanism (see Glezer [0084]). Glezer further discloses performing ligation using the target sequence as a template (see Glezer [0188]), and teaches embodiments in which the ligation is performed with direct ligation (see Glezer [0188]), or “with gap filling” (see Glezer [0011], Fig. 6).
In regards to claim 58, Glezer teaches cyclic detection/imaging/and removal seps (i)-(vii),including detecting (c1) and (c2) in two different, simultaneously available fluorescent channels (see Glezer [0157] and [0221] for steps (i)-(iv), (vii). Steps (v) and (vi) are taught through the combination of Glezer’s fixed four-color chemistry (see Glezer [0228] disclosing “using 4-color detection… a set of 10 sequencing cycles provides information in 40 dimensions”), such that sequencing distinct probe-resolution barcode sequences for the first and second probes withing the same cycle necessarily produces two different, simultaneously detected colors.
In regards to claim 63, Glezer teaches using a shared four-dye set every round for each probe (see Glezer [0228]) with removal/cycle-termination steps between rounds (see Glezer [0222]).
In regards to claim 67, Glezer teaches repeating steps (i)-(vii) with different pluralities of detectable probes in Example 3 (see Glezer [0277]-[0279]).
In regards to claim 70, Glezer teaches registering images across imaging steps and associating signals using these registered images (see Glezer Fig. 13, [0018]-[0019] disclosing result images requiring per-cycle image alignment and composite output).
Claims 1-5, 25, 28, 43, 58, 63, 67, 70, and 84 are rejected under 35 U.S.C. 103 as being unpatentable over Glezer as applied to Claims 1-5, 25, 43, 58, 63, 67, and 70 above, and included here for reasons supra, in view of Krzywkowski and Nilsson. (“Padlock Probes to Detect Single Nucleotide Polymorphisms”, Methods in Molecular Biology, Vol. 1649, pgs. 209-229, further ‘Krzywkowski’).
In regards to claim 28, Glezer teaches the methods of claim 1, for which claim 28 depends, including the base architecture of a first and second probe sharing a common gene-specific barcode and each carrying a distinct, probe-resolution identifier (see Glezer [0221]). Glezer does not teach associating those resolution identifiers with different species of organism.
Krzywkowski teaches padlock probes differentiating human and mouse transcripts of the conserved ACTB gene. The target binding arms of the human-specific and mouse-specific probes differ at the species discriminating terminal base (see Krzywkowski pg. 213, Fig. 2 (displaying the probe sequences for human and mouse ACTB gene). Each probe additionally carries a distinct internal decorator motif/backbone sequence, read out by a distinctly fluorophore conjugated decorator probe such that when applied together cocultured cells, species-specific signals were exclusively generated in either human or mouse cells (see Krzywkowski pg. 210 Fig. 1 legend “Different DNA backbones of PLP for human and mouse allele allow efficient discrimination of RCP using unique, differently fluorophore-conjugated decorator probes (green and red)”, pgs. 220-221 § 3.4, Anticipated Results, Fig. 3).
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to combine these teachings because Krzywkowski’s own stated purpose, differentiating the same gene transcripts by species of origin in a mixed or cocultured sample, is independently motivated and directly analogous to recognized applications such as PDX/xenograft models that are stated sample types by Glezer (see Glezer [0253], [0256]). A person of ordinary skill implementing Glezer’s probe-resolution architecture would have found it obvious to assign the first and second probe-resolution identifiers to different species of the same target gene as Krzywkowski expressly teaches doing.
In regards to claim 84, recites similar limitations to claim 28 including gene-specific probes hybridizing to homologs of the same target gene, with the caveat that the first/second probe resolutions barcodes are now termed ‘species-resolution barcodes’, which are associated with respective species, and species specific target binding sequences present in the probe. Glezer teaches the shared gene-barcode between probes and distinct probe identifying sequences as stated above (see Glezer [0221]). Glezer does not expressly describe using the probes to interrogate different homologs of the same gene across species.
Krzywkowski teaches both the species-associated barcode limitation (as above, see Krzywkowski pgs. 210, 213, 220, and throughout) and species-specific target binding sequences corresponding to human and mouse ACTB probes with different ligation arms that differ at the species discriminating terminal base, directly reading on “the first probe hybridizes to a first species specific nucleic acid sequence of the target gene of the first species of organism” (and correspondingly for the second probe/species; see Krzywkowski pg. 212-213, Fig. 2).
As stated above in relation to claim 28, It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to combine Krzywkowski’s teaching with the probe architecture of Glezer to arrive at the claimed invention. One would be motivated to combine these teachings in order to differentiating the same gene transcripts by species of origin in a mixed or cocultured sample, which directly analogous to recognized applications such as PDX/xenograft models that are stated sample types by Glezer (see Glezer [0253], [0256]). Furthermore, the probes design of Krzywkowski is directly compatible with the probe design of Glezer and one of ordinary skill would have a reasonable chance of success implementing the two designs to identify homologs of the same target genes in a sample.
Claims 1-5, 25, 28, 43, 58-61, 63, 67, 70, 74, and 80 and 84 are rejected under 35 U.S.C. 103 as being unpatentable over Glezer in view of Krzywkowski, as applied to claims 1-5, 25, 28, 43, 58, 63, 67, 70, and 84 above, and included here for reasons supra, in further view of Gyllborg et al. (“Hybridization-based in situ sequencing (HybISS) for spatially resolved transcriptomics in human and mouse brain tissue," Nucleic Acids Res. (2020) 48(19), on IDS 08/08/2022).
In regards to claims 59 and 60, as discussed above, Glezer teaches the limitations of claim 58 for which claims 59 and 60 depend, however Glezer does not teach the two-component sandwich architecture in which an intermediate probe binds to the identifier regions of the probe (neither gene-specific barcode, as in claim 59, nor probe-resolution sequences, as in claim 60).
Gyllborg teaches a two-component “bride-probe”/”detection-probe” sandwich in which an unlabeled bridge-probe hybridizes directly to a repeated barcode (“ID”) sequence on a rolling circle product, and a separate, fluorophore-conjugated detection probe hybridizes to a non-complementary tail on the bridge-probe (not to the barcode itself), and further teaches that up to four such bridge-probe/detection probe pairs, each carrying a different fluorophore, can be applied and imaged simultaneously withing a single cycle, before being stripped away for the next cycle (see Gyllborg Fig. 1).
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to combine the bridge-probes/sandwich architecture of Gyllborg with Glezer’s multi-barcode probes. Glezer’s own specification supplies the motivation to combine as Glezer expressly relies on Gyllborg’s own probe library for the padlock probes used in Glezer’s Examples 3 and 14 (see Glezer [0277]). A person of ordinary skill in the art implementing Glezer’s disclosed method would accordingly have looked to Gyllborg’s teachings as directly pertinent and directly compatible detection chemistry, which could be used as an alternative or supplement to Glezer’s SBS-based readout. The routine substitution of Gyllborg’s hybridization-sandwich barcode readout for Glezer’s SBS-based readout would be highly likely to succeed given that both are disclosed as interchangeable barcode-detection mechanisms for RCA products in probes of the same general padlock-probe architecture.
In regards to claim 61, Gyllborg further teaches simultaneous application of up to four distinct bridge-probe/detection-probe pairs, each carrying a different fluorophore, within a single cycle, before being stripped away for the next cycle (see Gyllborg Fig. 1, pg. 4, RESULTS, “Currently, as presented here, HybISS is set up with four readout detection probes per cycle… allow[ing] for a target panel of genes… measured in a combinatorial manner with four fluorophores…per cycle”), which teaches the simultaneous detection of multiple barcodes at once, motivated by Gyllborg’s own stated efficiency benefit of consolidating detection into fewer cycles.
In regards to claim 74, Glezer teaches the probe architecture, locational limitation, and RCA amplification steps (see Glezer [0084], [0113] disclosing circular/circularizable probes, [0221], [0109] disclosing shared gene-specific barcode, distinct probe-resolution identifiers, including the discrete second barcode variant, [0221] disclosing the first probe hybridizes to a first, physically distinct transcript at necessarily different location than the second probe’s transcript, [0142]-[0146], [0188] disclosing RNA product generation). Glezer does not teach steps (c) and (e) detailing detection via probes hybridizing directly to the RCA product at the complement of the barcode sequence.
Gyllborg teaches this direct-hybridization mechanism (bridge-probe/detection probe sandwich, as discussed in claims 59-61, including simultaneous, multi-channel readout of multiple barcodes positions within a single cycle (see Gyllborg pg. 4, RESULTS, “four readout detection probes per cycle”) while inherently preserving and reporting each signal’s spatial location, as confirmed by the location-mapped multiplexed output shown across the reference mouse and human tissue examples (see Gyllborg Fig. 3-4).
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to combine the bridge-probes/sandwich architecture of Gyllborg with Glezer’s multi-barcode probes and RCA products. Glezer’s own specification supplies the motivation to combine as Glezer expressly relies on Gyllborg’s own probe library for the padlock probes used in Glezer’s Examples 3 and 14 (see Glezer [0277]). A person of ordinary skill in the art implementing Glezer’s disclosed method would accordingly have looked to Gyllborg’s teachings as directly pertinent and directly compatible detection chemistry, which could be used as an alternative or supplement to Glezer’s SBS-based readout. The routine substitution of Gyllborg’s hybridization-sandwich barcode readout for Glezer’s SBS-based readout would be highly likely to succeed given that both are disclosed as interchangeable barcode-detection mechanisms for RCA products in probes of the same general padlock-probe architecture.
In regards to claim 80, resolving signal overlap is the stated purpose of Glezer (see Glezer [0007]), and as such Glezer teaches methods for resolving “overlapping signals that are not spatially resolved into individual puncta” (see Glezer [0218], [0221]).
Conclusion
No claim is allowed.
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/MATTHEW HAROLD RAYMONDA/ Examiner, Art Unit 1684 /AARON A PRIEST/Primary Examiner, Art Unit 1681
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