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 .
Priority
The present application filed 05/31/2022 claims the benefit of a provisional application PRO 63/195,613 filed 06/01/2021. The application will receive the benefit of an earlier filing date.
Claim Status
Claims 24 and 85 have been cancelled. Claims 1-5, 25, 28, 43, 58-61, 63, 67, 70, 74, 80, and 84 are pending and under examination. Claims 1, 3, 5, 25, 28, 43, 58, 59, 61, 63, 67, 74, 80, and 84 have been amended. Claims 1, 74, and 84 are independent claims.
Response to Arguments
Rejections Withdrawn
The rejection of claim 43 under 35 USC 112(b) is withdrawn following the applicants’ amendments.
The rejection of claims 1-3, 5, 24, 25, 43, 58-61, 63, 67, 70 and 74 under 35 USC 102 as being anticipated by Deng et al. is withdrawn following the applicants’ amendments.
The rejection of claims 1, 5, 24-25, and 28 under 35 USC 102(a)(1) as being anticipated by Chee et al. is withdrawn following the applicants’ amendments.
The rejection of claims 1, 4, 5, 24, 25, 28, 43, and 84 under 35 U.S.C. 102(a)(1) as being anticipated by Gunderson is withdrawn following the applicants’ amendments.
The rejection of claims 28, 84, and 85 under 35 U.S.C. 103 as being unpatentable over Deng is withdrawn following the applicants’ amendments.
The rejection of claims 59-61, 63, and 67 under 35 U.S.C. 103 as being unpatentable over Deng in further view of Zhuang is withdrawn following the applicants’ amendments.
The rejection of claims 1, 5, 24, 25, 28, 58-61, 63, 70, 74, 84, and 85 under 35 U.S.C. 103 as being unpatentable over Chee in view of Deng and Zhuang is withdrawn following the applicants’ amendments.
The rejection of claims 1, 5, 24, 25, 28, 43, 58, 70, 74, and 84-85 are under 35 U.S.C. 103 as being unpatentable over Gunderson in view of Deng and Zhuang is withdrawn following the applicants’ amendments.
New Rejections
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 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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1-5, 25, 28, 43,58-61, is/are rejected 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).
In regards to claims 1-3, 74, 80, and 84 Bernitz is in the field of in situ RNA detection, and discloses a method of detecting RNA targets in a biological sample using gene specific padlock probes that hybridize to RNA or cDNA targets, are circularized, amplified by rolling circle amplification , and detected in situ, thereby providing spatially resolved detection of RNA transcripts (see Abstract, [0009]-[0014], and throughout). Bernitz further teaches using multiple probes targeting the same gene which have distinct tag regions distinguishing the different probes targeting the same gene (see Fig. 9, [0040]-[0041], [0214]-[0215], [0233]-[0235] and throughout) and discloses that the probes comprise sequence regions encoding target identity, which are detected by hybridization or sequencing to identify the corresponding gene (see [0040]-[0041]).
Thus, Bernitz teaches step (a), including contacting a biological sample with a plurality of gene-specific probes that hybridize to RNA transcripts or cDNA molecules thereof, and that encode gene identity via barcode sequences, as well as detecting signals associated with the probe-specific barcode sequences at different spatial locations within a biological sample. Bernitz does not expressly disclose providing different “gene specific barcode sequence” and “probe-resolution barcode sequence” on each padlock probe that targets the same gene and instead teaches different tags entirely for each probe (see [0040]-[0041]).
However, Cai and Chen teach multiplexed in situ nucleic acid detection using multiple probes per gene and employing combinatorial and multi-part barcode sequences that are decoded over sequential detection cycles to distinguish individual probes or probe subsets targeting the same gene (Cai: Abstract, [0132]-[0133] and throughout; Chen: Abstract, pg. 1 ¶ 1, pg. 2. ¶2 and throughout). These references teach that transcript counting accuracy, signal deconvolution, and multiplexing capacity are limited when multiple transcript molecules of the same gene generate indistinguishable signals, and therefore employ combinatorial and sequential barcoding strategies to enable discrimination of transcript associated signals and to improve molecular counting accuracy (see Cai: Abstract, Fig. 1, [0132]-[0133] and throughout; Chen Abstract, pg. 1 ¶ 1, pg. 2. ¶2 and throughout). Further, Bernitz teaches detecting gene homologs (see [0202], [0215]), while Chen teaches detecting genes from different organisms (see pg. 2, ¶ 1).
Although Cai and Chen employ the same probe set for all copies of a given gene, these references explicitly recognize that transcripts molecules are individually resolved spatial objects and that improved indexing schemes improve transcript discrimination, counting fidelity, and multiplexed detection performance.
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to modify the gene specific probes of Bernitz to include different probe-resolution barcode sequences on different probes targeting the same genes, such that different transcript copies could be distinguished based on probe-specific barcode identity, in order to improve transcript counting accuracy, reduce signal ambiguity in regions of high transcript density, and enable more precise spatial discrimination of individual transcript molecules.
Such modification represents a predictable application of known barcoding and indexing techniques to a known problem in spatial transcriptomics, namely the difficulty of accurately distinguishing and counting multiple transcripts copies of the same gene in close proximity. Assigning different barcode sequence to different barcode sequences to different probes constitutes an obvious indexing design choice that does not alter the underlying chemistry or detection mechanism, but merely applies known barcoding principles for their established purpose of improving molecular discrimination and counting accuracy. Accordingly, the combination of Bernitz, Cai, and Chen renders claims 1-3, 74, 80, and 84 obvious.
In regards to claim 4, Bernitz teaches detecting the signals in multiple location within the biological sample (see [0011], [0042]-[0045], and throughout), as do Cai (see [0038], [0042] and throughout) and Chen (see Figs. 2-5 and throughout).
In regards to claim 5, Bernitz teaches using a detectable probe that binds the tag region of the proximity probes (see [0041]-[0042]).
In regards to claim 25, depends on claim 1 and further recites that a probe-resolution barcode sequence is common among two or more probes targeting different target gene RNA transcripts or cDNA molecules.
As discussed with respect to claim 1, Bernitz, teaches gene-specific probes that hybridize to RNA transcripts and are detected in situ via barcode-associated signals corresponding to spatial locations of transcript molecules (see [0041]-[0042]). Cai and Chen further teach combinatorial and sequential barcoding strategies in which barcode sequences are reused across different genes and decoding is achieved by interpreting combinations or patterns of barcode signals across multiple detection rounds (see Cai: Fig. 1 and throughout; Chen: Fig. 1 and throughout).
In Cai and Chen, individual barcodes elements are not uniquely assigned to a single gene but rather are intentionally reused across multiple genes as part of a combinatorial indexing scheme, such that gene identity is determined by a pattern or set of barcode signals rather than by a single unique barcode. Thus, these references teach that barcode sequences may be shared among probes targeting different genes.
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to
configure the barcode sequences of the probes of Bernitz as modified in view of Cai and Chen, such that a given barcode sequence is common among probes targeting different genes, in order to enable combinatorial decoding, reduce the total number of required barcode sequences, and increase multiplexing capacity while maintaining discrimination among transcript targets.
This modification represents a predictable and routine application of known combinatorial barcoding principle to probe design, and does not require any change to the underlying hybridization, amplification, or detection chemistry, but merely reflects an indexing optimization well established in multiplex in situ transcript detection methods. Accordingly, the combination of Bernitz, Cai, and Chen render claim 25 obvious.
In regards to claim 28, Chen teaches using their methods to identify RNA expression and spatial organization between different organisms (see pg. 2, ¶ 1). It would have been obvious to one of ordinary skill in the art to utilize different barcodes to represent different species of organism corresponding to the probes being used to target the gene in the different organisms.
In regards to claim 43, Bernitz teaches using circularizable probes wherein the ends of the probe are ligated utilizing the nucleic acid sequence of the target gene as a template (see Fig. 1, [0037], and throughout).
In regards to claims 58-61, 63, and 67 the claim depends from claim 1 and further recites sequential detection steps comprising hybridizing detectable probes to barcode sequences, imaging, optionally removing the probes, and sequentially hybridizing detectable probes to another barcode sequence followed by imaging in a detection channel. Bernitz teaches detecting barcode sequences associated with the padlock probes in situ by hybridization of detectable probes and imaging of spatially localize signals (see Fig. 1, [0041]-[0045], [0202]). Cai and Chen further teach sequential rounds of probe hybridization imaging, and optional probe removal or displacement followed by subsequent rounds of probe hybridization to different barcode or readout sequences in order to decode multiplexed barcodes using a limited number of detection channels (see Cai Fig. 1, [0051]-[0053], [0088]-[0101]; Chen Figs. 1-5). Cai and Chen further teach multi-stage probe architectures in which unlabeled or weakly labeled intermediate probes hybridize to target or barcode sequences and fluorescently labeled readout probes subsequently hybridize to the intermediate probes in order to amplify signal intensity, improve specificity and enable modular probe reuse across detection cycles (see Cai [0101]-[0103]; Chen Fig. 1 and throughout).
These references explicitly disclose the steps of contacting a biological sample with detectable probes that hybridize to barcode sequences, imaging to detect barcode associated signals, removing or displacing the detectable probes, and repeating the hybridization and imaging steps for additional barcode or readout sequences (see Cai [0109]-[0110]; Chen pg. 16 last para.). Further, these references explicitly disclose the use of intermediate probe layers to separate target hybridization from signal generation, thereby permitting flexible labeling strategies, improved signal-to-noise ratio, and multiplex detection (see Cai [0102]-[0103] and throughout; Chen Figs. 1-2, 4-5, pg. 3, ¶3 and throughout) .
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to perform the detection of the gene-specific barcode sequences using intermediate probes and labeled readout probes because such probe layering represents a well-established and routine strategy in in situ nucleic acid detection for improving signal amplification, probe modularity, and multiplexing efficiency. The use of intermediate probes and labeled probes does not alter the underlying barcode decoding concept of claim 1, but merely applies a conventional probe architecture to execute the detection steps in a predictable and well-understood manner. Accordingly, the combination of Bernitz with Cai and Chen renders these claims obvious.
In regards to claim 70, Bernitz, Cai and Chen all teach imaging methods in which the images taken for each imaging step are registered so that the signal can be associated with the corresponding analytes (see Bernitz [0202]; Cai [0111]-[0122]; Chen pg. 4, ¶1, and throughout each).
Conclusion
Conclusion
No claim is allowed.
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/MATTHEW HAROLD RAYMONDA/Examiner, Art Unit 1684
/AARON A PRIEST/Primary Examiner, Art Unit 1681