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 .
Claims 1-3, 7-16, 19-29 are pending in the application.
This office action is in response to the amendment filed on 7/17/2026.
All previous rejection not reiterated in this office action are withdrawn.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 7/17/2026 has been considered by the examiner.
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 119(e) as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed application, Application No. 63/062,054, 63/140,700 and 63/209,886 fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. Claim 1 and claim 14 of present application recites a method for detecting a plurality of targets in a cell comprising a plurality of detecting cycles, wherein each cycle comprises contacting at least two primers with a sequencing solution that comprises three labeled nucleotide types with reversible terminator, and a fourth nucleotide type without label. The claimed method is not disclosed in the provisional applications 63/062,054, 63/140,700 and 63/209,886. Therefore, the priority date of the present application is 8/6/2021, the filing date of application 17/396,575.
Response to Arguments
In response, Applicant argues that the’054 provisional describes the same multiplexed in-cell sequencing framework now claimed, as evidenced by paragraph [0147], which states “each sequencing cycle produces information about the magnitude of the signal in all 4 channels or a subset of 3 or 2 could be used.” Applicant states paragraph [00166] also teach that the sequencing cycle produce information in “all channel used,” including “4-channel-4-color, 3 channel-3-color, or 2-channel-2-color” implementation, paragraph [0031] provides “one or more of the above moieties can be absent from a nucleotide used in the methods and compositions set forth herein” and “a nucleotide can lack a label moiety or a blocking moiety or both.” Applicant concludes that ‘054 provisional discloses both the use of a three color sequencing scheme and the use of nucleotides lacking a labeling moiety. Applicant states paragraph [0052] states “absence of signal is understood to be a signal level of zero or a signal level that is not meaningfully distinguished from noise,” and paragraph [0053] explains that a signal may be binary, ternary, quaternary, or otherwise, gives “presence of absence of signal detected at particular wavelength” as an example of signal state.
The above arguments are fully considered but deemed unpersuasive. The amended claim 1 is drawn to a method of detecting a plurality of targets in a cell comprising amplifying a first circular polynucleotide bound to a first nucleic acid molecule in the cell and amplifying a second circular molecule bound to a second nucleic acid molecule in the cell, wherein the first and second nucleic acid is covalently attached to a first protein binding reagent; contacting a first primer hybridized to a first amplification product in the cell and a second primer hybridized to a second amplification product in the cell with a sequencing solution, wherein the sequencing solution comprises three labeled nucleotide types comprising a reversible terminator and a distinguished label, and a fourth nucleotide type without distinguished label, incorporating one of the labeled nucleotide into the first primer and non-labeled nucleotide into the second primer; removing the detectable reversible terminator from the incorporated nucleotides; and incorporating the non-labeled primer into the first primer and incorporating the labeled nucleotide into the second primer. Claim 14 is drawn to a method of detecting a plurality of targets in a cell comprising the amplification of circular polynucleotide; contacting a plurality of primers with a sequencing solution that comprises three labeled nucleotide types with reversible terminator, and a fourth nucleotide type without label. The teaching cited by Applicant in ‘054 provisional does not teach the claimed method that comprises each step in sequential order as recited. They are from different sections of the specification that either defines certain terms “signal state” (paragraph [0053]), “label” “label moiety” (paragraph [0039]), or explaining what is considered to be absence of signal in general (paragraph [0052]). The mere mention of a subset of 3 or 2 of channels could also be used in parenthesis does not support the method specifically claimed in claim 1 or 14 because there is no teaching regarding how the signal is generated, detected or obtained for the purpose of detecting a plurality of targets in a cell as claimed. The entire paragraph of [0147] and [0166] explains how the signal from 4 color detection is deconstructed. Therefore, for reason discussed in previous office action and set forth above, the priority date of present application is 8/6/2021, the filing date of application 17/396,575.
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.
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.
Claim(s) 1-3, 7, 9-16, 19, 20-24, 28 and 29 is/are rejected under 35 U.S.C. 103 as being unpatentable over Feldman (Cell, 2019, Vol.179, pages 787-799), in view of Hanselmann (EP 3702474A1) and Bernitz (US2014/0120534). This rejection has been rewritten to address the amendment.
Amended claim 1 added an amplification step prior to the sequencing step. Claim 1 is drawn to a method of detecting a plurality of targets in a cell comprising amplifying a first and a second circular polynucleotide bound to a first and second nucleic acid in the cell, wherein the nucleic acid is covalently attached to a first and second protein binding moiety, contacting a first primer hybridized to a first amplification product in the cell and a second primer hybridized to a second amplification product in the cell with a sequencing solution, wherein the sequencing solution comprises three labeled nucleotide types comprising a reversible terminator and a distinguished label, and a fourth nucleotide type without distinguished label, incorporating one of the labeled nucleotide into the first primer and non-labeled nucleotide into the second primer; removing the detectable reversible terminator from the incorporated nucleotides; and incorporating the non-labeled primer into the first primer and incorporating the labeled nucleotide into the second primer. Claim 14 is drawn to a method of detecting a plurality of targets in a cell comprising contacting an amplification product with a plurality of primers attached to it, with a sequencing solution that comprises three labeled nucleotide types with reversible terminator, and a fourth nucleotide type without label, wherein the amplification product is a product by process product, resulting from amplification of a circular polynucleotide.
Feldman teaches a method of targeted in situ sequencing to readout multiple RNA barcodes following amplification by rolling circle amplification (see page 788, Figure 1B and legend, page 789, 1st col., 2nd paragraph). Feldman teaches the in situ sequence comprising using primers with dye terminators that are removed following each cycle (page e4, 6th paragraph), which meets the limitation of primers with reversible terminator and a distinguishable label. The rolling circle amplification meets the newly added limitation that amplifying a circular polynucleotide.
Feldman’s teaching differs from the claimed invention that the sequencing is performed by 4-color sequencing by synthesis chemistry, not 3-color sequencing as claimed in claim 1 and 14. Feldman does not teach the amplification of circular polynucleotide is performed with a bound nucleic acid to said polynucleotide, wherein the nucleic acid is covalently attached to protein binding reagent.
Hanselmann teaches a sequencing method that uses three detectable label based system, which is a three-color-system, which shows equivalence to the current four-color-system (paragraph [0008]). Hanselmann teaches a method of determining the sequence of a nucleic acid template comprising contacting the nucleic acid sequence with nucleotide analogues with 3 different label and one type without label, and identifying the nucleotide analogue incorporated into the primer based on the detectable label of the 3 different label, and the fourth type based on the absence of a detectable label (paragraph [0010]). Hanselmann teaches the three detectable label sequencing system allows for a higher system throughput by reducing the number of images to be taken per sequencing cycle/step to identify the different fluorescent dyes that were incorporated in the sequencing reaction (paragraph [0021]). Hanselmann teaches the sequencing method comprises using reaction mixture that comprises a polymerase, a primer, wherein the nucleotide analogue comprises a reversible terminating group and a detectable label, the detectable label(s) incorporated during the sequencing cycle (or the absence thereof) can then be detected and the type of nucleotide attached to the primer can then be identified, removing the incorporated detectable label and capping moiety and the next synthesis cycle may be carried out in which a new nucleotide analogue is attached to the growing chain (paragraph [0057]).
Bernitz teaches detection of RNA in a sample of cells by converting the RNA to cDNA prior to the targeting of the cDNA with a padlock probe, circularize the padlock probe and rolling circle amplification of the circularized padlock, and detecting the amplified product (abstract). Bernitz describes labeling detection probe complementary to the backbone sequence of the padlock probe for detecting the amplification product, wherein the detection probe has a substance attached to it, and the substance is an antibody or hapten, or an epitope recognized by an antibody, wherein the antibody may be detected by directly or indirectly (paragraph [0047]).
It would have been obvious to an ordinary skilled in the art that the three-color sequencing system taught by Hanselmann can be used in the in situ sequencing method following amplification of the targets in a cell as taught by Feldman. The ordinary skilled in the art would have been motivated to do so because Hanselmann teaches the three color system has the advantage of generating a higher system throughput by reducing the number of images to be taken per sequencing cycle/step to identify the different fluorescent dyes that were incorporated in the sequencing reaction. An ordinary skilled in the art would have reasonable expectation of success to replace the four color sequencing method taught by Feldman with the three color sequencing method taught by Hanselmann following combined teaching from Feldman and Hanselmann. The inclusion or a labeled detection probe would also have been obvious to an ordinary skilled in the art based on the teaching from Bernitz because Bernitz teaches such probe may be used to detect the amplification product of RCA. Therefore, the claimed invention of claims 1, 7, 14, 19 and 20 would have been prima facie obvious to an ordinary skilled in the art at the time the application was filed.
Regarding claims 2 and 15, Feldman teaches the cells are seeded onto glass-bottom dishes and permeabilized (page e4, 5th paragraph).
Regarding claim 3, Feldman teaches amplification of the barcodes or sgRNA (page 789, 2nd col., 1st and 2nd paragraph), which meets the limitation of designed sequences from a known set of sequences.
Regarding claims 9-12, Hanselmann teaches a nucleotide of first type, second type and third type that comprises a label, wherein said nucleotide comprising nucleobase A, T or U, G, and the fourth type comprises nucleobase C (paragraph [0010]). It would have been obvious to an ordinary skilled in the art that any one of the nucleotide A, T or U, G and C may be the unlabeled nucleotide and the other three being the labeled nucleotides would work the same way in the 3-color sequencing method taught by Hanselmann.
Regarding claim 13, it is inherent that a cell would form a tissue, and Feldman also suggests existing protocols for in situ sequencing in tissue sample (page 796, 1st col., last 4 lines).
Regarding claim 16, Hanselmann teaches the nucleotide analogue comprises a reversible terminating group and a detectable label, the detectable label(s) incorporated during the sequencing cycle (or the absence thereof) can then be detected and the type of nucleotide attached to the primer can then be identified, removing the incorporated detectable label and capping moiety and the next synthesis cycle may be carried out in which a new nucleotide analogue is attached to the growing chain (paragraph [0057]).
Regarding claim 21 and 22, Feldman teaches the circularized padlock probe comprises a barcode sequence that is 5 nucleotide in length (Figure 1 A and B and legend).
Regarding claim 23, Feldman teaches detecting an RNA molecule in the cell (Figure 3 and legend).
Regarding claim 24, Feldman teaches determining spatial locations of the amplification products (Figure 5 and legend, page 796, 1st col., last paragraph
Regarding claims 28 and 29, Feldman teaches using different sequencing primers for sequencing different target sequences following multiple rounds of sequencing (Figure S1 and legend, and page 796, 1st col., last paragraph).
Claim(s) 25-27 is/are rejected under 35 U.S.C. 103 as being unpatentable over Feldman, Hanselmann and Bernitz, as applied to claim 1 and 24 above, and further in view of Ried (PNAS, 1992, Vol.89, pp. 1388-1392). This is a new rejection necessitated by amendment.
The teaching from Feldman, Hanselmann and Bernitz have been discussed above. However, none of the reference teaches generating an image overlay comprising the determined spatial locations of the first and second amplification products over an image of the cell.
Feldman teaches the optical pooled screening approach is broadly applicable across many settings, including the possibility of perturbing cells in vivo and measuring the resulting phenotypes within the native spatial context using existing protocols for in situ sequencing in tissue samples (page 796, 1st col., last paragraph).
Reid teaches a method of simultaneous visualization of different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy (title). Reid teaches digital images is overlayed and merged with a cell image (DAPI counterstain) for gene mapping (page 1389, 2nd col., last paragraph, page 1390, 2nd col., 4th paragraph, and Figure 1 and legend).
It would have been obvious to an ordinary skilled in the art to use image overlay method for identifying spatial location of the gene of interest because Reid teaches probes may be detected by overlaying and merge images to obtain the spatial location of the probe within a cell. The ordinary skilled in the art practicing the method rendered obvious by Feldman, Hanselmann and Bernitz for determining perturbation in native spatial context of a cell would have been motivated to use such image analysis tool for determining the cellular location of the probe within the cell. Using prior art known method to determining the cellular location of images generated by in situ sequencing would have been within the capability of an ordinary skilled in the art. Therefore, the claimed invention of claims 25-27 would have been prima facie obvious to an ordinary skilled in the art at the time the application was filed.
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.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1, 7, 19, 20, 21, 22, 28 and 29 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 31, 33, 34, 38-40 and 47 of copending Application No. 17/938,329 in view of Hanselmann. This is a new ground of rejection necessitated by amendment.
Claims 31 and 41 of ‘329 application recites a method of detecting a protein and a nucleic acid molecule in a cell comprising: 1) contacting the cell with a specific binding reagent and binding the specific binding reagent to the protein, wherein the binding reagent comprises a oligonucleotide; 2) contacting the cell with a first polynucleotide probe that comprise barcode sequence and bind said polynucleotide to the first nucleic acid molecule; 3) contact the cell with a second polynucleotide probe that comprise a barcode sequence and bind said polynucleotide to the second oligonucleotide sequence; 4) amplifying both probe and forming an amplification product; 5) sequencing the first and second amplification product using 1st and 2nd primer to incorporate modified nucleotides into the sequencing primer to generate optically resolvable feature.
The claims from ‘329 application do not specifically recite whether a four-color sequencing or three-color sequencing method is being used.
The teaching from Hanselmann has been discussed above.
It would have been obvious to an ordinary skilled in the art that the three-color sequencing system taught by Hanselmann can be used in the in situ sequencing method following amplification of the targets in a cell in situ as claimed in claims 31 and 41 of the ‘329 application. The ordinary skilled in the art would have been motivated to do so because Hanselmann teaches the three color system has the advantage of generating a higher system throughput by reducing the number of images to be taken per sequencing cycle/step to identify the different fluorescent dyes that were incorporated in the sequencing. Therefore, the claimed invention of claims 1 and 14 would have been obvious variant of the method claimed in claims 31 and 41 of the ‘329 application.
Regarding claims 7, 19 and 20, claim 33 and 34 of the ‘329 application recites the same limitation that the specific binding reagent is an antibody.
Regarding claim 21 and 22, claims 38-40 of ‘329 application recites the barcode sequence is 1, or 5-15 nucleotides.
Regarding claims 28-29, claim 47 of the ‘329 application recites hybridizing multiple primer to multiple amplification product and perform multiple detection cycle.
This is a provisional nonstatutory double patenting rejection.
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 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 23 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 23 recites a further step that detects an RNA molecule in the cell. It is unclear when this step is performed in the context of the claim steps recited in claim 1, before, after or during the amplification and/or sequencing detection.
This is a new ground of rejection necessitated by amendment.
Allowable Subject Matter
Claim 8 is objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Conclusion
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CELINE X QIAN whose telephone number is (571)272-0777. The examiner can normally be reached M-F (8-4:00).
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/CELINE X QIAN/ Primary Examiner, Art Unit 1637
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