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 .
Effective Filing Date
The present application, filed on April 21, 2022 is a 371 of PCT/US2020/065797, filed December 18, 2020 and claims the benefit of priority to 63/060,947, filed August 4, 2020 and 62/954,720, filed December 30, 2019. The priority date of the present application is determined to be December 30, 2019.
Claim Status and Action Summary
This action is in response to the papers filed on December 3, 2025.
Claims 264, 265, 267-269, 271, 273-280, and 282 are pending in the present application.
Claims 266, 270, 272, and 281 were canceled by applicant in the response filed December 3, 2025.
Claims 264, 265, 267-269, 271, 273-280, and 282 are under examination.
Any objections and rejections not reiterated below are hereby withdrawn.
The objections of record to the claims are withdrawn in view of the amendments to claim 264 and the cancellation of claim 281.
The 112(d) rejection of record has been withdrawn in view of the cancellation of previously rejected claim 270.
The 102(a)(1) rejection over Zhuang et al. has been withdrawn in view of the amendment to independent claim 264, now further requiring a step of imaging nascent RNA from genes located in the genomic loci.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 273 is rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention AND under 35 U.S.C. 112(d) as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
This is a new grounds of rejection necessitated by the amendments to the claims.
Claim 272 was canceled in the amendment filed December 3, 2025. Claim 273, as presently written, depends on “the method of claim 272”.
Therefore, claim 273 is of improper dependent form because it depends upon a cancelled claim.
Furthermore, claim 273 is indefinite because it is unclear whether the claim, as presently amended, is intended to depend upon claim 271 (upon which canceled claim 272 depended in the previous version of the claims) or is intended to depend upon independent claim 264.
Claims 274-275, and 282 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
This rejection has been updated as necessitated by the amendments to the claims.
Regarding claim 274, the claim recites the limitation "each population of nucleic acid probes comprises the same readout sequence" in lines 3-4. There is insufficient antecedent basis for this limitation in the claim. Claim 264, upon which claim 274 depends, recites “at least two distinct fluorescent readout probes that hybridize to read sequences of the primary nucleic probes”. Additionally, claim 264 recites “readout probes” and “read sequences” to which the readout probes hybridize.
Therefore, claim 274 lacks antecedent basis for the claim terms: “the same readout sequence”.
It is uncertain whether the claim terms “readout sequence” and “read sequence”, recited as a component of the primary nucleic acid probes in claims 274 and 264, respectively, are intended to be equivalents, or are intended to encompass different structural elements within the primary nucleic acid probes.
Furthermore, it is unclear whether “each population of nucleic acid probes comprises the same readout sequence” recited in claim 274 is intended to require: (a) that each population of distinct primary nucleic acid probes within the plurality of populations, recited by claim 264, comprise the same “read sequence” to which fluorescent readout probes hybridize such that each population targeting a given genomic locus shares a same read sequence, OR (b) each “population” of “at least two distinct fluorescent readout probes” comprises the same readout sequence such that the “read sequence” on the primary nucleic acid probe may be labeled with either probe.
Claim 275 is rejected because it depends on, and therefore includes the indefinite limitation of, claim 274.
Regarding claim 282, the phrase “wherein the assigned codewords have a… code space with a Hamming distance to maximize distance…” does not recite structure or method steps. Furthermore, the specification does not teach method steps “to maximize [Hamming] distance…”. Therefore, it is unclear what values of Hamming distance are intended to be encompassed by the claim as written or what method steps are intended to be covered by the claim.
Applicant is reminded that no new matter may be added.
Response to Arguments
The response does not address or present any arguments relevant to the 112(b) rejection of claim 274 regarding the lack of clarity surrounding the potentially interchangeable terms “readout sequence” and “read sequence” recited in claims 264 and 273. Similarly, the response does not address the 112(b) rejection of claim 282 regarding the lack of clarity as to what method steps for “maximizing distance” or values of “Hamming distance” are intended to be encompassed by the claims.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 264, 265, 267, 268, 271, 273, 274, 278, and 282 are rejected under 35 U.S.C. 103 as being unpatentable over Zhuang et al., US 2017/0220733 A1 (Published August 3, 2017) in view of Wang et al., “Spatial organization of chromatin domains and compartments in single chromosomes” Science, Vol 353 Issue 6299, pages 598-602, and Brown and Buckle, “Detection of Nascent RNA Transcripts by Fluorescence in situ Hybridization” Fluorescence in situ Hybridization (FISH): Protocols and Applications, Methods in Molecular Biology, vol 659, pp. 33-50, 2010.
This rejection has been updated as necessitated by the amendments to the claims.
Regarding claim 264, Zhuang et al. teaches methods of visualizing the localization of nucleic acids in cells (Zhuang et al., abstract) by imaging at least 100 distinct genomic loci in a cell (Zhuang et al., paragraph 0076-0077) comprising: (a) exposing cells (i.e. a sample comprising chromatin) with a plurality of encoding probe (i.e. primary nucleic acid probes) populations, (b) contacting the bound primary nucleic acid probes with at least two distinct fluorescent readout probes that hybridize to readout sequences (i.e. read sequences) of the primary probes, wherein each read sequence corresponds to one position of a codeword, (c) imaging the readout probes, and (d) repeating steps (b) and (c) in one or more sequential hybridization and imaging rounds (Zhuang et al., figures 5A and 5E). Zhuang et al. further teaches that each target nucleic acid (i.e. each genomic locus) is assigned a codeword that forms an error-checking and/or error-correcting code space (Zhuang et al., paragraph 0022-0023). Zhuang et al. names these techniques “Multiplexed Error-Robust Fluorescence in Situ Hybridization or MERFISH” (Zhuang et al., paragraph 0186).
Zhuang does not teach further imaging nascent RNA transcribed from genes located in the genomic loci.
However, Brown and Buckle teach RNA-FISH methods for sensitive detection of specific transcripts within individual cells while preserving the cellular morphology that allow the detection of nascent transcripts within the cell nucleus and place gene transcription in a functional context of the whole cell (Brown and Buckle, abstract). Brown and Buckle further teach that combination of RNA-FISH with DNA-FISH and RNA-immunoFISH (for detection of cellular proteins) allows for visualizing nuclear processes in space and time and allows gene activity to be placed in the context of spatial relationship to specific nuclear proteins or bodies (Brown and Buckle, page 34, paragraph 3 and figure 1).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to modify the MERFISH methods taught by Zhuang et al. directed to highly multiplexed and error-correcting imaging of nucleic acid localization (RNA and/or DNA) in single cells by further imaging nascent RNA transcribed from genes located in the genomic loci, as taught by Brown and Buckle (Brown and Buckle, page 34, paragraph 3 and figure 1). The ordinary artisan would have been motivated to image nascent RNA and corresponding genomic loci probed by the methods taught by Zhuang et al. because of the teachings of Brown and Buckle that imaging nascent RNA in addition to corresponding genomic loci allow placing gene transcription in the context of nuclear processes and organization (i.e. chromatin) (Brown and Buckle, page 34, paragraph 3). The ordinary artisan would have been reasonably confident that the MERFISH probes taught by Zhuang et al. for highly multiplexed and error-correcting detection of nucleic acids (RNA and/or DNA) could have been readily adapted for detection of nascent transcripts in addition to genomic DNA in chromatin because Zhuang et al. and Brown and Buckle teach detecting nucleic acids in preserved single cells (in situ) and Brown and Buckle teach that probes may be designed such that they specifically hybridize to an intronic sequence to detect only the nascent transcript (Brown and Buckle, page 34, paragraph 2). The ordinary artisan would have been reasonably confident that MERFISH probes taught by Zhuang et al., modified such that the target-specific sequence of the probe hybridizes to introns of genes within the targeted loci would have allowed for simultaneous detection of nascent RNA in addition to genomic loci in chromatin.
Regarding claim 265, Zhuang et al. does not teach further classifying the imaged genomic loci as active or inactive chromatin.
However, Wang et al. teaches methods of mapping the spatial organization of genomic loci in chromatin (Wang et al., figure 1) comprising: (a) exposing chromatin to a plurality of primary probe populations that hybridize to spatially separated genomic loci in chromatin, (b) contacting the bound primary probes with multiple distinct fluorescent secondary (i.e. readout) probes, (c) imaging the readout probes bound to the primary probes, and repeating steps (b) and (c) in one or more sequential hybridization and imaging rounds (Wang et al., figure 1). Wang et al. teaches “we exploited a similar hybridization and imaging protocol as… MERFISH”. (Wang et al., page 599, column 1, lines 21-25). Wang et al. further teaches classifying whether the imaged loci are in active or inactive chromatin (Wang et al., figure 4 and page 601, column 2).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the “MERFISH” method of visualizing more than 100 distinct nucleic acid targets in single cells with error-checking/correcting and target-specific “codewords”, taught by Zhuang et al. to further comprise classifying whether the target loci are associated with active or inactive chromatin. The ordinary artisan would have been motivated to further characterize the loci detected by the method taught by Zhuang et al. as comprising active or inactive chromatin, as taught by Wang et al., because of the express teaching of Wang et al. that MERFISH probes can be readily adapted to image multiple genomic loci within intact chromatin. (Wang et al., page 599, column 1, lines 17-29). Additionally, Wang et al. teaches that such methods that directly visualize the conformation of single chromosomes in single cells are needed because other techniques (such as Hi-C) are based on ensemble averaging of many chromosomes and may not reflect structures that exist in individual chromosomes. (Wang et al., page 598, column 3, lines 2-17) The ordinary artisan would have been reasonably confident that the error-checking and/or error correcting encoded MERFISH method taught by Zhuang et al. would have successfully detected genomic loci in intact chromatin because both Wang et al. and Zhuang et al. teach that said probes can detect DNA in cells, and Wang et al. expressly teaches that similar probes allow for localization of DNA target loci in active or inactive chromatin (Wang et al., Figure 4).
Regarding claim 267, the methods of Zhuang et al. and Wang et al. teach imaging mRNA using a plurality of primary nucleic acid probe pools, wherein each pool of primary probes hybridizes to a distinct RNA and each transcript has a valid codeword (Zhuang et al., paragraph 0051) with a Hamming distance of at least 2 (Zhuang et al., paragraph 0107).
Zhuang et al. and Wang et al. do not teach imaging nascent RNAs. However, as discussed above in the rejection of claim 264, it would have been prima facie obvious to one of ordinary skill in the art to modify the multiplexed and error-correcting MERFISH-based probes taught by Zhuang et al. and Wang et al. that hybridize to specific mRNA or genomic DNA targets by designing target-hybridizing sequences that correspond to introns, rather than untranscribed or exonic sequences, as taught by Brown and Buckle (Brown and Buckle, page 34, paragraph 2).
Regarding claim 268, Brown and Buckle teach further imaging specific nuclear proteins or bodies (i.e. structures) (Brown and Buckle, page 324, paragraph 3).
Regarding claim 271, Zhuang et al. and Wang et al. teach at least 1000 genomic loci can be imaged in individual cells (Zhuang et al., paragraph 0077).
Regarding claim 273, Brown and Buckle teach that imaging of nascent RNA alongside detection of DNA or proteins in cells allows placing gene transcription within a functional context of the whole cell (Brown and Buckle, abstract) and correlation of gene transcription with nuclear organization, including spatial relation to specific nuclear proteins or bodies (Brown and Buckle, page 34, paragraph 3) (i.e. imaging nuclear structures wherein the chromatin organization is placed in its native structural and functional context).
Regarding claim 274, Zhuang et al. teaches primary nucleic acid probes comprise a target sequence that hybridizes to the genomic loci and a readout sequence complementary to the readout probes (Zhuang, figure 5E and paragraph 0077) wherein each population of probes corresponding to a genomic locus comprises the same readout sequence (Zhuang et al., paragraph 0071).
Regarding claim 278, Zhuang et al. teaches MERFISH using combinatorial labeling and error-robust encoding using 16-bit encoding wherein the Hamming distance separating codewords is 4 and the number of non-zero bits (i.e. the Hamming weight) is greater than two (Zhuang et al., paragraph 0271). Zhuang et al. teaches increasing the number of bits while maintaining a Hamming distance of 4 increases the number of codewords (i.e. species) that can be identified while constraining the potential for increased misidentification rate (Zhuang et al., figure 5 and paragraph 0271). Finally, Zhuang et al. teaches the total number of nucleic acid target sequences to be detected and the amount of desired error correction determines the length of the codewords (i.e. the number of bits in the binary code) and that “information theory provides several efficient algorithms for assembling error-correcting binary codebooks” (Zhuang et al., paragraph 0150).
Therefore it would have been prima facie obvious prior to the effective filing date of the claimed invention of one of ordinary skill in the art to modify the combinatorial labeling and error-robust encoding using 16 bit encoding and a Hamming weight of 2 or more, taught by Zhuang et al. (Zhuang et al., paragraph 0271), by increasing the number of bits comprising the binary code assigned to each genomic locus while maintaining a Hamming distance separating code words, as taught by Zhuang et al. (Zhuang et al., figure 5 and paragraph 0271). The ordinary artisan would have been motivated to increase the number of bits in the code for each locus because of the teaching of Zhuang et al. that the total number of nucleic acid target sequences to be detected and the amount of desired error correction determines the length of the codewords (i.e. the number of bits in the binary code) and that “information theory provides several efficient algorithms for assembling error-correcting binary codebooks” (Zhuang et al., paragraph 0150). The ordinary artisan would have been reasonably confident that increasing the number of bits in the binary code would have increased the number of distinct loci that could be detected by the method taught by Zhuang et al.
Regarding claim 282, Zhuang et al. teaches the assigned codewords have an error-checking and/or error-correcting code space with a Hamming distance (Zhuang et al., figure 12 and paragraphs 0022-0023).
Claim 269 is rejected under 35 U.S.C. 103 as being unpatentable over Zhuang et al., US 2017/0220733 A1 (Published August 3, 2017) in view of Wang et al., “Spatial organization of chromatin domains and compartments in single chromosomes” Science, Vol 353 Issue 6299, pages 598-602 and Brown and Buckle, “Detection of Nascent RNA Transcripts by Fluorescence in situ Hybridization” Fluorescence in situ Hybridization (FISH): Protocols and Applications, Methods in Molecular Biology, vol 659, pp. 33-50, 2010 as applied to claims 264, 265, 267, 268, 271, 273, 274, 278, and 282 above, and further in view of Brown et al., “Association between active genes occurs at nuclear speckles and is modulated by chromatin environment” Journal of Cell Biology Vol. 182, No. 6 pp. 1083-1097, 2008.
This rejection has been updated as necessitated by the amendments to the claims.
As discussed in the 103 rejections above, the methods taught by Zhuang et al. in view of Wang et al. and Brown and Buckle teach methods of imaging at least 100 distinct genomic loci as well as nascent RNA transcribed from genes located in the genomic loci using MERFISH-based probes imaging nuclear structures to relate the nucleic acid signals in space and time to the location of specific nuclear proteins or bodies.
The methods taught by Zhuang et al. in view of Wang et al. and Brown and Buckle do not specify that the nuclear structures include nuclear speckles and nucleoli. However, Brown et al. teaches methods comprising imaging nascent RNA transcripts, the gene loci from which they are transcribed, nuclear speckles, and nucleoli (Brown et al., Figure 8A). Brown et al. teaches that nuclear speckles are aggregations of splicing-related factors around which many active genes cluster (Brown et al., page 1091, column 1, paragraph 2).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to combine the methods comprising imaging genomic loci and nascent RNA using MERFISH probes as well as nuclear structures, taught by Zhuang et al. in view of Wang et al. and Brown and Buckle, with the methods comprising imaging nascent RNA, active genes, and nuclear speckles, taught by Brown et al. because Brown et al. teaches that active genes (and their nascent transcripts) tend to cluster around nuclear speckles (Brown et al., page 1091, column 1, paragraph 2). The ordinary artisan would have been motivated to image nuclear structures including nuclear speckles and nucleoli in addition to nascent RNA and the gene loci from which they are transcribed because of the teaching of Brown et al. that nuclear speckles are nuclear structures important for the structural organization and function of active genes (Brown et al., page 1091, column 1, paragraph 1-2). The ordinary artisan would have predicted that imaging nuclear structures including nuclear speckles and nucleoli would have provided further information regarding the structural and functional status of a given genic locus and/or nascent RNA signals observed by the methods taught by Zhuang et al. in view of Wang et al. and Brown and Buckle because of the teaching of Buckle et al. that nuclear speckles are spatially associated with actively transcribed genes.
Claim 275 is rejected under 35 U.S.C. 103 as being unpatentable over Zhuang et al., US 2017/0220733 A1 (Published August 3, 2017) in view of Wang et al., “Spatial organization of chromatin domains and compartments in single chromosomes” Science, Vol 353 Issue 6299, pages 598-602 and Brown and Buckle, “Detection of Nascent RNA Transcripts by Fluorescence in situ Hybridization” Fluorescence in situ Hybridization (FISH): Protocols and Applications, Methods in Molecular Biology, vol 659, pp. 33-50, 2010 as applied to claims 264, 265, 267, 268, 271, 273, 274, 278, and 282 above, and further in view of Xia et al., “Multiplexed detection of RNA using MERFISH and branched DNA amplification”, bioRxiv, published December 25, 2018.
This rejection has been updated as necessitated by the amendments to the claims.
As discussed in the 103 rejections above, Zhuang et al. in view of Wang et al. and Brown and Buckle teaches methods comprising detecting nucleic acids in situ in single cells using MERFISH-based probes. Zhuang et al. in view of Wang et al. and Brown and Buckle does not teach methods further comprising the use of adapter oligonucleotide probes. However, Xia et al. teaches branched DNA amplification for MERFISH imaging. Xia teaches so-called “amplifier’ probes that hybridize to the encoding probe and to the readout probes (i.e. an adapter between the encoding/primary probe and the fluorescently labeled readout probes) (Xia et al., Figure 1). Xia et al. further teaches that inclusion of “amplifier”/adapter probes improves the performance of MERFISH because it allows for more sensitive detection of target nucleic acids using fewer encoding probes along the length of the target nucleic acid (Xia et al., page 9, paragraph 3).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to modify the method comprising detection of genic loci in chromatin using MERFISH-based probes to further comprise the use of “amplifier probes” that bridge the association between the primary “encoding” probes and the fluorescently labeled “readout probes”. The ordinary artisan would have been motivated to modify the methods taught by Zhuang et al. in view of Wang et al. and Brown and Buckle to further comprise such adapter probes because of the teaching of Xia et al. that the use of adapters improved the detection efficiency of MERFISH when only ~ 1/6 of the number of encoding probes per target locus were used (Xia et al., page 9, paragraph 4 – page 10, paragraph 1). The ordinary artisan would therefore have been reasonably confident that the use of such “amplifiers” (i.e. adapters) would have improved the detection efficiency of target loci in the MERFISH-based methods taught by Zhuang et al. in view of Wang et al. and Brown and Buckle.
Claim 276, 277, 279, and 280 are rejected under 35 U.S.C. 103 as being unpatentable over Zhuang et al., US 2017/0220733 A1 (Published August 3, 2017) in view of Wang et al., “Spatial organization of chromatin domains and compartments in single chromosomes” Science, Vol 353 Issue 6299, pages 598-602 and Brown and Buckle, “Detection of Nascent RNA Transcripts by Fluorescence in situ Hybridization” Fluorescence in situ Hybridization (FISH): Protocols and Applications, Methods in Molecular Biology, vol 659, pp. 33-50, 2010 as applied to claims 264, 265, 267, 268, 271, 273, 274, 278, and 282 above, and further in view of Bintu et al., “Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells”, Science 362, 419 (published October 26, 2018).
This rejection has been updated as necessitated by the amendments to the claims.
As discussed in the 103 rejections above, Zhuang et al. in view of Wang et al. and Brown and Buckle teaches methods comprising detecting nucleic acids in situ in single cells using MERFISH-based probes. Zhuang et al. in view of Wang et al. and Brown and Buckle does not teach that the hybridization and imaging rounds are performed 50 times or more. However, Bintu et al. teaches a method in which sequential highly multiplexed DNA FISH is used to map chromatin organization across topologically associating domains (TADs) in thousands of individual cells (Bintu et al., page 2, abstract). Bintu et al. further teaches segmenting a large chromatin region into sequential ~30kb segments and performing 65 rounds of sequential fluorescence in situ hybridization and imaging (Bintu et al., page 1, column 2 and figure “Super resolution chromatin tracing reveals TAD-like domain structures in single cells”).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to modify the method of highly multiplex, error-correcting MERFISH to determine the 3D localization of given nucleic acid targets within the nuclei of single cells, taught by Zhuang et al. in view of Wang et al. and Brown and Buckle, to further comprise 65 rounds of sequential hybridization and imaging to trace individual TADs in single cells using the segmented FISH chromatin tracing approach taught by Bintu et al. The ordinary artisan would have been motivated to modify the method taught by Zhuang et al. in view of Wang et al. and Brown and Buckle to further comprise 65 rounds of sequential hybridization and imaging because of the teaching of Bintu et al. that highly multiplexed sequential detection of genomic loci allows for “chromatin conformation tracing… and unbiased determination of both the structural features and their genomic coordinates with high resolution in single cells.” (Bintu et al., page 2, column 3, paragraph 3). The ordinary artisan would have been reasonably confident that such a modification to the method taught by Zhuang et al. in view of Wang et al. and Brown and Buckle would have allowed for high resolution tracing of individual chromosome structural features (i.e. TADs), as taught by Bintu et al.
Regarding claim 277, Bintu et al. teaches the genomic loci are 30 kb in size (Bintu et al., page 2, column 3, paragraph 3).
Regarding claim 279, Bintu et al. teaches each round comprises imaging one or two segments (i.e. targeted genomic loci) (Bintu et al., page 2, column 3, paragraph 3).
Regarding claim 280, Bintu et al. teaches each round comprises imaging one or two segments (i.e. targeted genomic loci) (i.e. about an equal number of genomic loci are imaged in each of the imaging rounds) (Bintu et al., page 2, column 3, paragraph 3).
Response to Arguments
The response traverses the 103 rejections of record on the grounds that the limitations of claim 266, now incorporated into independent claim 264, were not included in the 103 rejection of record of claim 264 over Zhuang et al. in view of Wang et al.
It is noted that the limitations of claim 266 were addressed in the 103 rejection of record over Zhuang in view of Wang and further in view of Brown and Buckle in the office action dated June 3, 2025.
The rejection of claim 264 has been updated to incorporate the limitations of claim 266 that are now incorporated into the independent claim.
The response asserts that the claimed methods “were inspired by [MERFISH]… but with significant modifications…” comprising increasing the length of the binary code assigned to each target nucleic acid to a 100-bit binary code.
This argument has been thoroughly reviewed and is not persuasive.
It is noted that the response (and the specification) reference the teachings of Zhuang (cited in the office action of record) as a starting point for the claimed invention. As is/was noted in the 103 rejection, Zhuang et al. provide clear motivation and guidance for the ordinary artisan to have increased the size of the number of bits while maintaining sufficient Hamming distance: (reproduced here for convenience/clarity)
“
Zhuang et al. teaches MERFISH using combinatorial labeling and error-robust encoding using 16-bit encoding wherein the Hamming distance separating codewords is 4 and the number of non-zero bits (i.e. the Hamming weight) is greater than two (Zhuang et al., paragraph 0271). Zhuang et al. teaches increasing the number of bits while maintaining a Hamming distance of 4 increases the number of codewords (i.e. species) that can be identified while constraining the potential for increased misidentification rate (Zhuang et al., figure 5 and paragraph 0271). Finally, Zhuang et al. teaches the total number of nucleic acid target sequences to be detected and the amount of desired error correction determines the length of the codewords (i.e. the number of bits in the binary code) and that “information theory provides several efficient algorithms for assembling error-correcting binary codebooks” (Zhuang et al., paragraph 0150).
Therefore it would have been prima facie obvious prior to the effective filing date of the claimed invention of one of ordinary skill in the art to modify the combinatorial labeling and error-robust encoding using 16 bit encoding and a Hamming weight of 2 or more, taught by Zhuang et al. (Zhuang et al., paragraph 0271), by increasing the number of bits comprising the binary code assigned to each genomic locus while maintaining a Hamming distance separating code words, as taught by Zhuang et al. (Zhuang et al., figure 5 and paragraph 0271). The ordinary artisan would have been motivated to increase the number of bits in the code for each locus because of the teaching of Zhuang et al. that the total number of nucleic acid target sequences to be detected and the amount of desired error correction determines the length of the codewords (i.e. the number of bits in the binary code) and that “information theory provides several efficient algorithms for assembling error-correcting binary codebooks” (Zhuang et al., paragraph 0150). The ordinary artisan would have been reasonably confident that increasing the number of bits in the binary code would have increased the number of distinct loci that could be detected by the method taught by Zhuang et al.
“
The response further summarizes the teachings of Wang et al. and asserts that the method of Wang et al. is distinct from the claimed subject matter.
This argument has been thoroughly reviewed and is not persuasive.
The rejection of record relies on the combination of the teachings of Zhuang et al. and Wang et al., rather than the teachings of Wang et al. in isolation.
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).
Both Zhuang et al. and Wang et al. teach methods comprising in situ detection of nucleic acid sequences in cells comprising primary probes and secondary probes, and an artisan having ordinary skill in the relevant art would have recognized that methods comprising hybridization of nucleic acid probes to one type of nucleic acid (i.e. RNA) can be readily adapted to another type of nucleic acid (i.e. DNA).
Additionally, the response asserts that “The claimed subject matter provides methods that massively increase the target identification for genome-scale chromatin imaging… and neither Zhuang nor Wang provide any motivation to modify the MERFISH methods of Zhuang to arrive at the claimed invention…”
This argument has been thoroughly reviewed and is not persuasive. As is/was addressed in the 103 rejection and response to arguments above, Zhuang et al. provide clear motivation and guidance for the ordinary artisan to have increased the size of the number of bits while maintaining sufficient Hamming distance.
Finally, the response argues:
“Since each of the dependent claims depends from a base claim that is believed to be in condition for allowance, for the sake of brevity, it is believed that it is unnecessary at this time to argue the further distinguishing features of the dependent claims… [and] the right to specifically address the further patentability of the dependent claims is… reserved.”
This argument/assertion has been reviewed and is not persuasive. See MPEP 804(I)(b)(1) and 37 C.F.R. 1.111(b), which allows that some objections may be held in abeyance but includes no provision for holding rejections in abeyance.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
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Claim 264 is rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 11,098,303 (herein referred to as ‘303). Although the claims at issue are not identical, they are not patentably distinct from each other.
This rejection has been updated as necessitated by the amendments to the claims.
Regarding claim 264, the claims of ‘303 recite a method for detecting a plurality of different nucleic acid targets within a sample by in situ hybridization (i.e. DNA or RNA within individual cells) comprising: (a) exposing a sample to a plurality of encoding (i.e. primary) nucleic acid probes, (b) exposing (i.e. contacting) each of the encoding probes to a first fluorescent readout nucleic acid probe that hybridizes to the encoding probe, (c) imaging the readout probes, and (d) repeating the hybridization and imaging rounds using different readout nucleic acid probes (‘303, claim 1). The claims of ‘303 further recite that the target nucleic acids are each assigned an error-checking and/or correcting codeword (‘303, claim 1)
It is noted that the claims of ‘303 are generic with regard to the type of nucleic acid targets to which the “encoding probes” bind. Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention to one of ordinary skill in the art that the “nucleic acid targets” recited by claim 1 of ‘303 clearly encompass: genomic DNA and cellular RNAs produced from said genomic DNA (i.e. nascent RNAs present in the nucleus of the cell).
Claim 264 is rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 12,209,237 (herein referred to as ‘237).
This rejection has been updated as necessitated by the amendments to the claims.
Regarding claim 264, the claims of ‘237 recite a method for imaging spatial organization of a nucleic acid target (i.e. DNA or RNA) in a sample comprising: (a) contacting the sample comprising a plurality of distinct nucleic acid targets with a plurality of primary nucleic acid probe pools wherein each pool of probes hybridizes to a distinct target and encodes an N-bit codeword that form an error-checking and/or correcting code space, (b) contacting the sample with a plurality of readout probes comprising a fluorescent label, (c) imaging the readout probes, and (d) repeating the readout hybridization and imaging steps for all N positions in the N-bit codeword (‘237, claim 1).
It is noted that the claims of ‘237 are generic with regard to the type of nucleic acid targets to which the “encoding probes” bind. Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention to one of ordinary skill in the art that the “nucleic acid targets” recited by claim 1 of ‘237 clearly encompass: genomic DNA and cellular RNAs produced from said genomic DNA (i.e. nascent RNAs present in the nucleus of the cell).
Claim 264 is rejected on the ground of nonstatutory double patenting as being unpatentable over claim 19 of U.S. Patent No. 12,104,151 (herein referred to as ‘151).
This rejection has been updated as necessitated by the amendments to the claims.
Regarding claim 264, the claims of ‘151 recite a method for determining a plurality of nucleic acid targets in a sample by in situ hybridization (i.e. DNA or RNA in single cells) comprising: (a) contacting a sample comprising a plurality of nucleic acid targets with a plurality of primary nucleic acid probes that each comprise a target sequence and one or more read sequences, (b) contacting the plurality of hybridized primary nucleic acid probes with a plurality of fluorescent secondary nucleic acid probes that hybridize to the read sequences (i.e. readout probes), (c) imaging the readout probes, and (d) repeating the readout hybridization and imaging steps for each position of a codeword read from the pattern of binding of the readout probes to the primary probes, wherein the codeword facilitates error correction (‘151, claim 19).
It is noted that the claims of ‘151 are generic with regard to the type of nucleic acid targets to which the primary nucleic acid probes bind. Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention to one of ordinary skill in the art that the “nucleic acid targets” recited by claim 1 of ‘151 clearly encompass: genomic DNA and cellular RNAs produced from said genomic DNA (i.e. nascent RNAs present in the nucleus of the cell).
Claim 264 is rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 11,959,075 (herein referred to as ‘075) in view of Wang et al., “Spatial organization of chromatin domains and compartments in single chromosomes” Science, Vol 353 Issue 6299, pages 598-602.
This rejection has been updated as necessitated by the amendments to the claims.
Regarding claim 264, the claims of ‘075 recite a method for imaging RNA spatial organization in a sample comprising: (a) contacting a plurality of distinct RNA species in situ (i.e. in single cells) with a plurality of primary nucleic acid probe pools each comprising a target sequence and one or more read sequences, wherein each pool hybridizes to a distinct RNA species and encodes an N-bit error-checking and/or correcting codeword, (b) contacting the bound primary nucleic acid probes with a plurality of fluorescent readout probes, (c) imaging the readout probes, and (d) repeating the readout hybridization and imaging steps for each position of the N-bit codeword (‘075, claim 1).
The claims of ‘075 do not recite nucleic acid targets that are not RNA. However, Wang et al. teaches methods of mapping the spatial organization of genomic loci in chromatin (Wang et al., figure 1) comprising: (a) exposing chromatin to a plurality of primary probe populations that hybridize to spatially separated genomic loci in chromatin, (b) contacting the bound primary probes with multiple distinct fluorescent secondary (i.e. readout) probes, (c) imaging the readout probes bound to the primary probes, and repeating steps (b) and (c) in one or more sequential hybridization and imaging rounds (Wang et al., figure 1). Wang et al. teaches “we exploited a similar hybridization and imaging protocol as… MERFISH”. (Wang et al., page 599, column 1, lines 21-25).
Therefore, it would have been prima facie obvious for one of ordinary skill in the art to modify the method for imaging RNA spatial organization in individual cells, as claimed by ‘075, to image DNA spatial organization in individual cells (i.e. genomic loci in chromatin), as taught by Wang et al., using MERFISH-based nucleic acid probes recited by ‘075 and Wang et al.
The ordinary artisan would have been motivated to utilize the method claimed by ‘075 to visualize DNA spatial organization (i.e. genomic loci in chromatin) because Wang et al. teaches that such methods that directly visualize the conformation of single chromosomes in single cells are needed because other techniques (such as Hi-C) are based on ensemble averaging of many chromosomes and may not reflect structures that exist in individual chromosomes. (Wang et al., page 598, column 3, lines 2-17) The ordinary artisan would have been reasonably confident that the error-checking and/or error correcting encoded MERFISH method claimed by ‘075 would have successfully detected genomic loci in intact chromatin because Wang et al. teaches that MERFISH probes can detect DNA in cells, and Wang et al. expressly teaches that similar probes allow for localization of DNA target loci in active or inactive chromatin (Wang et al., Figure 4).
Claim 264 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 12,522,819 (referred to herein as ‘819). Although the claims at issue are not identical, they are not patentably distinct from each other.
This rejection has been updated as necessitated by the amendments to the claims and because previously pending application number 18/932,378 has now issued as US PATENT NO. 12,522,819.
Regarding claim 264, the claims of 819 recite a method for determining a plurality of nucleic acid targets (DNA and/or RNA) within a sample by in situ hybridization (i.e. in single cells) comprising: (a) contacting a plurality of nucleic acid targets with a plurality of primary nucleic acid probes that each comprise a target sequence and one or more read sequences, wherein each primary probe hybridizes to a nucleic acid target, (b) contacting the bound primary probes with a plurality of fluorescent secondary probes that hybridize to a subset of the read sequences (i.e. readout probes), (c) detecting fluorescent signals (i.e. imaging) each of the hybridized readout probes, and (d) repeating the readout probe hybridization and imaging steps to generate a pattern of binding of the readout probes wherein the pattern forms an error correcting codeword assigned to the identity of each nucleic acid target (‘819, claim 1).
It is noted that the claims of ‘819 are generic with regard to the type of nucleic acid targets to which the primary nucleic acid probes bind. Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention to one of ordinary skill in the art that the “nucleic acid targets” recited by claim 1 of ‘819 clearly encompass: genomic DNA and cellular RNAs produced from said genomic DNA (i.e. nascent RNAs present in the nucleus of the cell).
Claim 264 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 177 of copending Application No. 18/046,409 (referred to herein as ‘409). Although the claims at issue are not identical, they are not patentably distinct from each other.
This rejection has been updated as necessitated by the amendments to the claims.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Regarding claim 264, the claims of ’409 recite a method for determining a plurality of nucleic acid targets (DNA and/or RNA) within a sample by in situ hybridization (i.e. in single cells) comprising: (a) contacting a plurality of nucleic acid targets with a plurality of primary nucleic acid probes that each comprise a target sequence and one or more read sequences, wherein each primary probe hybridizes to a nucleic acid target, (b) contacting the bound primary probes with a plurality of fluorescent secondary probes that hybridize to a subset of the read sequences (i.e. readout probes), (c) detecting fluorescent signals (i.e. imaging) each of the hybridized readout probes, and (d) repeating the readout probe hybridization and imaging steps to generate a pattern of binding of the readout probes wherein the pattern forms an error correcting codeword assigned to the identity of each nucleic acid target (‘409, claim 177).
It is noted that the claims of ‘409 are generic with regard to the type of nucleic acid targets to which the primary nucleic acid probes bind. Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention to one of ordinary skill in the art that the “nucleic acid targets” recited by claim 1 of ‘409 clearly encompass: genomic DNA and cellular RNAs produced from said genomic DNA (i.e. nascent RNAs present in the nucleus of the cell).
Claim 264 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 12,473,546 (herein referred to as ‘546) in view of Wang et al., “Spatial organization of chromatin domains and compartments in single chromosomes” Science, Vol 353 Issue 6299, pages 598-602.
This rejection has been updated as necessitated by the amendments to the claims and because previously pending application number 18/932,298 has now issued as US PATENT NO. 12,473,546 .
Regarding claim 264, the claims of ‘546 recite a method for imaging RNA spatial organization in a sample comprising: (a) contacting a plurality of distinct RNA species in situ (i.e. in single cells) with a plurality of primary nucleic acid probe pools each comprising a target sequence and one or more read sequences, wherein each pool hybridizes to a distinct RNA species and encodes an N-bit error-checking and/or correcting codeword, (b) contacting the bound primary nucleic acid probes with a plurality of fluorescent readout probes, (c) imaging the readout probes, and (d) repeating the readout hybridization and imaging steps for each position of the N-bit codeword (‘546, claim 1).
The claims of ‘546 do not recite nucleic acid targets that are not RNA. However, Wang et al. teaches methods of mapping the spatial organization of genomic loci in chromatin (Wang et al., figure 1) comprising: (a) exposing chromatin to a plurality of primary probe populations that hybridize to spatially separated genomic loci in chromatin, (b) contacting the bound primary probes with multiple distinct fluorescent secondary (i.e. readout) probes, (c) imaging the readout probes bound to the primary probes, and repeating steps (b) and (c) in one or more sequential hybridization and imaging rounds (Wang et al., figure 1). Wang et al. teaches “we exploited a similar hybridization and imaging protocol as… MERFISH”. (Wang et al., page 599, column 1, lines 21-25).
Therefore, it would have been prima facie obvious for one of ordinary skill in the art to modify the method for imaging RNA spatial organization in individual cells, as claimed by ‘546, to image DNA spatial organization in individual cells (i.e. genomic loci in chromatin), as taught by Wang et al., using MERFISH-based nucleic acid probes recited by ‘546 and Wang et al.
The ordinary artisan would have been motivated to utilize the method claimed by ‘546 to visualize DNA spatial organization (i.e. genomic loci in chromatin) because Wang et al. teaches that such methods that directly visualize the conformation of single chromosomes in single cells are needed because other techniques (such as Hi-C) are based on ensemble averaging of many chromosomes and may not reflect structures that exist in individual chromosomes. (Wang et al., page 598, column 3, lines 2-17) The ordinary artisan would have been reasonably confident that the error-checking and/or error correcting encoded MERFISH method claimed by ‘298 would have successfully detected genomic loci in intact chromatin because Wang et al. teaches that MERFISH probes can detect DNA in cells, and Wang et al. expressly teaches that similar probes allow for localization of DNA target loci in active or inactive chromatin (Wang et al., Figure 4).
Claim 264 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 12,460,250 (referred to herein as ‘250). Although the claims at issue are not identical, they are not patentably distinct from each other.
This rejection has been updated as necessitated by the amendments to the claims and because previously pending application number 17/413,148 has now issued as US PATENT NO. 12,460,250.
Regarding claim 264, the claims of ‘250 recite a method for imaging nucleic acids in a sample (i.e. DNA and/or RNA) comprising: (a) exposing target nucleic acids to nucleic acid probes that each comprise a target sequence and one or more read sequences, (b) exposing the bound probes to “primary amplifier nucleic acids” (i.e. adapter oligonucleotide probes) that bind to the read sequences of the bound probes, (c) exposing the “primary amplifier nucleic acids” to “secondary amplifier nucleic acids” that comprise sequences that bind to the primary amplifier and a fluorescent signaling entity (i.e. fluorescent readout probes), (d) repeating fluorescent readout probe hybridization and imaging steps,(e) creating codewords for a target nucleic acid based on the sequential fluorescence imaging within the sample (‘250, claim 1). ‘250 further claims the codewords are separated by a Hamming distance in a code space (i.e. the code is error-checking and/or correcting) (‘250, claim 13).
It is noted that the claims of ‘250 are generic with regard to the type of nucleic acid targets to which the primary nucleic acid probes bind. Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention to one of ordinary skill in the art that the “nucleic acid targets” recited by claim 1 of ‘250 clearly encompass: genomic DNA and cellular RNAs produced from said genomic DNA (i.e. nascent RNAs present in the nucleus of the cell).
Response to Arguments
The response asserts that the incorporation of the subject matter of previously rejected claim 266 into independent claim 264 obviates the rejections. This argument has been thoroughly reviewed and is not persuasive as is detailed in the updated double patenting rejections above.
Furthermore, the response requests that the double patenting rejections be further held in abeyance until notification of allowable subject matter.
This request has been noted and is denied. See MPEP 804(I)(b)(1) and 37 C.F.R. 1.111(b), which allows that some objections may be held in abeyance but includes no provision for holding rejections in abeyance.
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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/Z.M.T./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682