DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
2. This action is in response to the papers filed August 26, 2025. Applicant’s remarks and amendments have been fully and carefully considered but are not found to be sufficient to put the application in condition for allowance. Any new grounds of rejection presented in this Office Action are necessitated by Applicant's amendments. Any rejections or objections not reiterated herein have been withdrawn. This action is made FINAL.
Claims 1-12, 16, and 18-23 are currently pending and have been examined herein.
Claim Rejections - 35 USC § 103
3. 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.
4. Claims 1-4, 9, and 11 are rejected under 35 U.S.C. 103 as being unpatentable over Kishi (SABER enables highly multiplexed and amplified detection of DNA and RNA in cells and tissues. bioRxiv 401810; doi: https://doi.org/10.1101/401810 preprint posted 8/27/2018) in view of Chen (Science April 2025 Vol 348 Issue 6233).
Regarding Claim 1 Kishi teaches a method called SABER-FISH. Kishi teaches a modular version of this FISH workflow (Fig 1).
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As shown above, this method uses a 42 base pair (42mer) ‘bridge’ sequence domain to hybridize concatemers to FISH probes. For each new target, relatively short probe sequences with 30-50 bp homology to their targets can be designed using standard approaches, and then a 42 mer bridge sequence can be appended to the end of the sequence. Then, the complementary 42mer sequence can be concatemerized in vitro from a PER primer on its 3’ end and hybridized together with oligopaint FISH probes. This strategy allows the same bulk sets of extensions to be re-deployed for different targets and samples as needed and further reduces the cost by allowing the same sets of concatemers to be re-used for each different application. Complementary fluorescent ‘fluor’ imagers that have 20 bases of homology to the concatemer are typically used for imaging. Optionally, these 20 nt imager oligos can also be stripped from bound concatemers to reset the signal, enabling subsequent use of that fluorescence color on a distinct target (pages 2-4).
Thus Kishi teaches a method comprising the steps of:
(1) providing a set of analyte-specific probes (the bridge probes), each analyte-specific probe comprising:
- a binding element (S) (the grey part of the bridge probe) that specifically interacts with one of the analytes to be encoded, and
- an identifier element (T) (the red part of the bridge probe) comprising a nucleotide sequence which is unique to said set of analyte-specific probes (unique identifier sequence);
(2) incubating the set of analyte-specific probes (the bridge probes) with the sample, thereby allowing a specific binding of the analyte-specific probes to the analyte to be encoded (see Fig 1D);
(3) removing non-bound probes from the sample (see washing steps in Fig 1C);
(4) providing a set of decoding oligonucleotides (the PER concatemer probes), each decoding oligonucleotide comprising:
- a first connector element (t) (the red part of the PER concatemer probe) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence, and
- a translator element (c) (the blue part of the PER concatemer probe) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide;
(5) incubating the set of decoding oligonucleotides (the PER concatemer probes) with the sample, thereby allowing a specific hybridization of the decoding oligonucleotides to the unique identifier sequence (see Fig 1D);
(6) removing non-bound decoding oligonucleotides from the sample (see washing steps in Fig 1C);
(7) providing a set of signal oligonucleotides (the fluor imagers), each signal oligonucleotide comprising:
- a second connector element (C) (the 20bp sequence of the fluor imager) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of the translator element (c), and
- a signal element (the fluorophore on the fluor imagers); and
(8) incubating the set of signal oligonucleotides with the sample, thereby allowing a specific hybridization of the signal oligonucleotides to the translator element (c) (Fig 1C and 1D);
(9) removing non-bound signal oligonucleotides from the sample (see washing in Fig 1C);
(10) detecting the signal (see Fig 1C).
Kishi does not teach a method wherein the translator element (c) is not unique to an analyte to be encoded (clm 1 step 4). Kishi does not teach a method comprising detecting the signal at a plurality of points in the sample, at least some of which do not correspond to the analyte (clm 1 step 10).
However Chen teaches MERFISH. Chen teaches that each RNA species is first labeled with ~192 encoding probes that convert the RNA into a specific combination of readout sequences (Encoding hyb).These encoding probes each contain a central RNA-targeting region flanked by two readout sequences, drawn from a pool of N different sequences, each associated with a specific hybridization round. Encoding probes for a specific RNA species contain a particular combination of four of the N readout sequences, which correspond to the four hybridization rounds in which this RNA should read 1. N subsequent rounds of hybridization with the fluorescent readout probes are used to probe the readout sequences (hyb 1, hyb 2, …, hyb N). The bound probes are inactivated by photobleaching between successive rounds of hybridization. Figure 1 E of Chen is shown below in grayscale. A color version of this Figure is available online.
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Here Chen teaches the use of a readout probe that is NOT unique to an analyte to be encoded (for example the pink readout probe hybrdizes to RNA species 1 and 4, the blue readout probe hybridizes to RNA species 1 and 3, etc.). The readout probe hybridizes to a encoding probe comprising a targeting sequence and first and second read out sequences that are NOT unique to an analyte to be encoded (for example RNA Species 1 and 4 must have the same readout sequence 1 because they both hybridize to the pink readout probe). Further it is noted that when the pink signal is detected RNA species 1 and 4 would both light up. If RNA species 1 is interpreted to be the analyte, then Chen teaches a method comprising detecting the signal at a plurality of points in the sample (on RNA species 1 and 4), at least some of which do not correspond to the analyte (RNA species 4).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Kishi by modifying the concatemer region (the translator element c) of the bridge probe such that it comprises a sequence that is NOT unique to the analyte to be encoded and hybridizes to a fluor imager that is NOT unique to the analyte to be encoded as suggested by the labeling scheme of Chen. As discussed above, Chen discloses readout sequences and readout probes that are NOT unique to the analyte to be encoded. Chen teaches that this allows for each RNA species to be encoded with a particular combination of readout sequences and readout probes. One of skill in the art would have been motivated to use this combinatorial labeling scheme since this would allow for the number of detectable RNA species to grow exponentially with the number of imaging rounds (page 412, col 2-3).
Regarding Claim 2 Kishi teaches SABER effectively amplifies the signal of probes targeting nucleic acids in fixed cells and tissues (abstract). Thus Kishi teaches a method wherein the sample is a biological sample.
Regarding Claim 3 Kishi teaches SABER effectively amplifies the signal of probes targeting nucleic acids in fixed cells and tissues (abstract). Thus Kishi teaches a method wherein the biological sample is fixed.
Regarding Claim 4 Kishi teaches that for each new target, relatively short probe sequences with 30-50bp homology to their targets can be designed using standard approaches, and then one of 84 designed 42mer bridge sequences can be appended to the end of the sequence (page 3). Thus Kishi teaches method wherein within the set of analyte-specific probes (the bridge probes) the individual analyte-specific probes comprise binding elements (at least Sl and S2) which specifically interact with different sub-structures of one of the analytes to be encoded (Fig 1 shows multiple bridge primers hybridizing along the length of the target), and wherein within the set of analyte-specific probes the individual analyte- specific probes share a copy of a common identifier element (T).
Regarding Claim 9 Kishi teaches that for each new target, relatively short probe sequences with 30-50bp homology to their targets can be designed using standard approaches, and then one of 84 designed 42mer bridge sequences can be appended to the end of the sequence (page 3). Thus Kishi teaches a method wherein the binding element (S) (the grey part of the bridge probe which is 30-50bp) comprises a nucleic acid comprising a nucleotide sequence allowing a specific binding to the analyte to be encoded.
Regarding Claim 11 Kishi teaches that SABER enables highly multiplexed and amplified detecting of DNA and RNA (title). Thus Kishi teaches a method wherein the analyte to be encoded is a nucleic acid.
5. Claims 5-8, 16, and 18-23 are rejected under 35 U.S.C. 103 as being unpatentable over Kishi (SABER enables highly multiplexed and amplified detection of DNA and RNA in cells and tissues. bioRxiv 401810; doi: https://doi.org/10.1101/401810 preprint posted 8/27/2018) in view of Chen (Science April 2025 Vol 348 Issue 6233) as applied to claims 1 and 5 above, and in further view of Chee (US 2005/0266407 Pub 12/1/2005) and Shah (Neuron Vol 92 pages 342-357 10/19/2016).
The teachings of Kishi and Chen are presented above.
It is noted that Kishi teaches selectively removing the signal oligonucleotides from the sample (Fig 1E). Kishi teaches that the imager oligos can be stripped from bound concatemers to reset the signal, enabling subsequent use of that fluorescence color on a distinct target (page 4). Kishi teaches that by modeling the melting temperatures of 20mer imagers, 42mer bridge sequences, and FISH probe sequences, we determined that 50-60% formamide in 1×PBS should effectively and rapidly de-stabilize imagers without significantly affecting the underlying probe and 42mer bridge sequence stability (page 8).
The combined references do not teach a method further comprising selectively removing the decoding oligonucleotides (clm 5 step 11). The combined references do not teach a method wherein the oligonucleotides are removed from the sample by heating to break hydrogen bonding among base pairs (clm 5 step 11).
However Chee teaches a method wherein a first set of decoding probes are allowed to hybridize for some period of time, and the excess (non-hybridized) probes are washed off. Detection of the fluorophore then proceeds. Following detection of the first set of probes, the probes are removed, for example by heating, and a second set of decoder probes is added (para 0137).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Kishi in view of Chen by removing the decoding oligonucleotides (the PER concatemer probes) and signal oligonucleotides (the fluor imagers) using heat in order to maintain the binding of the analyte specific probes (the bridge probes) to the analyte. The claim would have been obvious because the substitution of one method of removing the probes (50-60% formamide in 1×PBS) for another (heat) would have yielded predictable results to one of ordinary skill in the art at the time of the invention. Further the skilled artisan would have been motivated to remove the decoding oligonucleotides in addition to the signal oligonucleotides because Chee teaches that this allows for a second set of decoder probes to hybridize.
The combined references do not teach a method further comprising: (12) repeating steps (4)-(11) at least once as steps (42)-(112) to generate an encoding scheme from the repetition of steps (4) - (11) or (42)-112) (clm 5, step 12). The combined references do not teach a method wherein the encoding scheme is predetermined and allocated to the analyte to be encoded (clm 6). The combined references do not teach a method wherein signal oligonucleotides are used in repeated steps (42)-(112) that comprise a signal element which is different from the signal element used in previous steps (4)-(11) (clm 8). The combined references do not teach a method wherein signal oligonucleotides are used in repeated steps (42)-(112) comprising a signal element which is identical to the signal element used in previous steps (4)-(11) (clm 16). The combined references do not teach a method wherein the signal element is not unique to an analyte to be encoded (clm 18). The combined references do not teach a method wherein the number of analytes that can be uniquely encoded doubles with each repeating (clm 19). The combined references do not teach a method wherein the encoding scheme is unique to the analyte to be encoded (clm 20). The combined references do not teach a method wherein the encoding scheme is not limited by an identifier element sequence (clm21). The combined references do not teach a method wherein the encoding scheme correlates to decoding oligonucleotide order of addition to the sample (clm 23).
However Shah teaches that a temporal barcoding scheme was developed that uses a limited set of fluorophores and scales exponentially with time. Specifically, sequential probe hybridizations on the mRNAs in fixed cells impart a unique pre-defined temporal sequence of colors, generating in situ mRNA barcodes. The multiplex capacity scales as Fn, where F is the number of fluorophores and n is the number of rounds of hybridization. Thus, one can increase the multiplex capacity by increasing the number of rounds of hybridization with a limited pool of fluorophores. We called this approach sequential fluorescence in situ hybridization (seqFISH) (page 342-343). Fig 1 is shown below.
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Shah teaches that in Fig 1B, the same points are re-probed through a sequence of 4 hybridizations (numbered). The sequence of colors at a given location provides a barcode readout for that mRNA (“barcode composite”). The genes represented by these barcodes are identified through referencing a lookup table abbreviated in Fig 1D and quantified to obtain single-cell expression levels. As shown in Fig 1D Shah teaches a method wherein the signal element (fluorophore) is different from the signal element used in previous steps (see Adcy4 wherein the signal is blue, purple, green, purple). As shown in Fig 1D Shah teaches a method wherein the signal element (fluorophore) is identical to the signal element used in previous steps (see Gm15688 where the signal is red, red, blue, blue). Shah teaches the signal element (fluorophore) is not unique to the analyte to be encoded but the encoding scheme of fluorophores used is unique to the analyte. The encoding scheme is limited by the number of fluorophores and the number of hybridizations.
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Kishi, Chen, and Chee by repeating steps (4)-(11) at least once to generate an encoding scheme that is predetermined and allocated to each analyte as suggested by Shah. In the instant case Shah teaches they developed a method call seqFISH comprising sequential probe hybridizations on mRNAs in fixed cells to impart a unique pre-defined temporal sequence of colors, generating in situ mRNA barcodes. Shah teaches they developed a temporal barcoding system that uses a limited set of fluorophores to overcome the scalability problem in the prior art that only allows detection of 20-30 genes due to the limited number of fluorophores. Shah teaches using seqFISH, they were able to detect up to 249 genes. One of skill in the art would have been motivated to modify the method of Kishi, Chen, and Chee in view of the teachings of Shah for the benefit of being able to scale up the detection of greater than 20-30 genes.
The combined references do not teach a method wherein the decoding oligonucleotides that are used in the repeated steps comprise a translator element that is different from the translator element of the previous step (clm 7). The combined references do not teach a method wherein the encoding scheme is generated by iterative selection among translator element (c1) and translator element (c2) (clm 22).
However based on the teachings of the combined references one of skill in the art would have been motivated to use decoding oligonucleotides comprising different translator elements for the benefit of being able to hybridize to different signal oligonucleotides in instances where each signal element (fluorophore) was attached to a different connector element (C) since this would result in barcoding. Further the skilled artisan would recognize that since the translator element of the decoding probe hybridizes to the connector element of the signal probe, the translator element is not unique to the analyte. The skilled artisan would have been motivated to base the encoding scheme on the translator element sequences as this would be considered to be an obvious variation of the methods known in the prior art.
6. Claims 10 and 12 are rejected under 35 U.S.C. 103 as being unpatentable over Kishi (SABER enables highly multiplexed and amplified detection of DNA and RNA in cells and tissues. bioRxiv 401810; doi: https://doi.org/10.1101/401810 preprint posted 8/27/2018) in view of Chen (Science April 2025 Vol 348 Issue 6233) as applied to claim 1 above, and in further view of Saka (Highly multiplexed in situ protein imaging with signal amplification by Immuno-SABER. bioRxiv 507566; doi: https://doi.org/10.1101/507566 preprint posted 12/28/2018).
The teachings of Kishi and Chen are presented above.
The combined references do not teach a method wherein the binding element (S) comprises an amino acid sequence allowing a specific binding to the analyte to be encoded, preferably the binding element is an antibody (clm 10). The combined references do not teach a method wherein the analyte to be encoded is a peptide or a protein (clm 12).
However Saka discloses a method called Immuno-SABER which utilizes DNA-barcoded antibodies to image proteins. As shown in Figure 1B, (1a) antibodies are conjugated with DNA bridge strands (sequences) and used to simultaneously stain multiple targets in biological samples, (1b) primer sequences (green line) are extended to a controlled length using PER. (2) The concatemers are hybridized to the bridge sequence (blue) on the antibody. (3) Fluorophore (depicted as purple star)-labeled 20-mer DNA “imagers” strands hybridize to the repeated binding sites on the long PER concatemers. Each imager is designed to bind to a dimer of the unit primer sequence.
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Thus Saka teaches a method wherein the binding element (S) comprises an antibody that specifically binds to the analyte to be encoded, wherein the analyte to be encoded is a peptide or a protein.
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Kishi and Chen by using a set of analyte specific probes wherein the binding element (S) comprises an antibody that specifically binds to a protein to be encoded as suggested by Saka. One of skill in the art would have been motivated to adapt the SABER method of Kishi by using antibodies as the binding elements for the benefit of being able to use this technology for encoding protein targets. Further, the claim would have been obvious because the substitution of one binding element (the nucleic acids of Kishi) for another binding element (the antibodies of Saka) would have yielded predictable results to one of ordinary skill in the art at the time of the invention.
Response To Arguments
7. In the response the Applicants traversed the rejection under 35 USC 102 over the prior art of Kishi. The Applicants argue that Kishi does not anticipate claim 1 as amended because Kishi does not teach (i) that the translator element (c) is not unique to an analyte to be encoded and (ii) detecting the signal at a plurality of points in the sample, at least some of which do not correspond to the analyte.
This argument has been fully considered and is persuasive. It is noted that the rejection has been modified to address the claim as amended. Claim 1 is now rejected under 35 USC 103 over the prior art of Kishi in view of Chen.
In the response the Applicants traversed the rejections under 35 USC 103 over the prior art of Kishi in view of Chee and Shah and the prior art of Kishi in view of Saka. It is noted that the rejections under 35 USC 103 have been modified to address the claims as amended. Applicants arguments have been considered to the extent that they apply to the new rejections set forth herein. In particular Applicants argue that Kishi teaches away from using a nonspecific translator element, instead teaching using a specific translator element that recruits fluorophore labeled imager oligos to one analyte probe only. Similarly, Kishi teaches away from detection of fluorophore labeled imager oligos at a plurality of points in the sample, at least some of which do not correspond to the analyte. Rather, Kishi teaches delivery of fluorophore labeled imager oligos to specific analyte probes indicative of one particular analyte in a particular detection round. This teaching away of Kishi is consistent with the goal of Kishi to increase not throughput or generality of detection (Kishi is comfortable with detection limit of 17 analytes, after all), but to increase signal amplification.
This argument has been fully considered but is not persuasive. The Examiner does not agree that Kishi teaches away. Kishi’s mere disclosure of a different way of labeling a analyte does not constitute a teaching away from labeling of Chen because such disclosure does not criticize, discredit, or otherwise discourage the labeling of Chen.
8. 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 extension fee 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 date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to AMANDA HANEY whose telephone number is (571)272-8668. The examiner can normally be reached Monday-Friday, 8:15am-4:45pm EST.
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/AMANDA HANEY/Primary Examiner, Art Unit 1682