Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Information Disclosure Statement
The information disclosure statement filed 11 February 2026 fails to comply with the provisions of 37 CFR 1.97, 1.98 and MPEP § 609. Non-patent literature documents 5 and 8 are improperly cited as the pre-print version of the manuscripts were provided but the citations refer to the print versions. They have been placed in the application file, but the information referred to therein has not been considered as to the merits. Applicant is advised that the date of any re-submission of any item of information contained in this information disclosure statement or the submission of any missing element(s) will be the date of submission for purposes of determining compliance with the requirements based on the time of filing the statement, including all certification requirements for statements under 37 CFR 1.97(e). See MPEP § 609.05(a).
Claim Objections
Claim 11 is objected to because of the following informalities: the last line of the claim should read as “…label have the same optical properties…”. Appropriate correction is required.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-3, 7, 9-12, and 15 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Lubeck 1 [Lubeck E, et al. Nat Methods. 2014 Apr; 11(4):360-1] as evidenced by Lubeck 2 [Lubeck E and Cai L. Nat Methods Author Manuscript; 2013 Jan: 1-18].
Lubeck 1 teaches a sequential barcoding scheme (seqFISH) which enables multiplex, single cell, in situ profiling of different mRNAs. This scheme was demonstrated by barcoding 12 genes in single yeast cells. Probes were labeled with 4 dyes and designed to be 25 nucleotides long with similar melting temperatures, as described by Lubeck 2 [Lubeck, p360; Lubeck 2, p7]. Cells were immobilized on a glass surface and then subjected to rounds of probe hybridization, imaging, and removal by DNAse I treatment [Lubeck 1, p361]. After treatment with DNAse I, the cells were washed twice and any remaining fluorescent signal was photobleached [Lubeck 1, supplemental methods pdf-p14]. The scheme and the imaging results of the demonstration are described by Figure 1, replicated below.
Therefore, Lubeck 1 anticipates the claimed invention as they:
(1) Provided a biological sample (yeast cells) with a plurality of analytes (mRNAs of 12 genes);
(2) Introduced a first marker (labeled probe) comprising a first affinity reagent (oligonucleotide) specific for a target analyte (mRNA) bound to a first optically detectable label (dye) and configured to be degraded by a degradation agent (DNAse I);
(3) Generated a first optical readout with the first marker (first FISH image);
(4) Applied a degradation agent (DNAse I) to degrade the first affinity reagent;
(5) Introduced a second marker (labeled probe);
(6) Generated a second optical readout with the second marker (second FISH image).
PNG
media_image1.png
666
1060
media_image1.png
Greyscale
Regarding claims 9 and 10, during each round of hybridization, each transcript was targeted by a set of FISH probes labeled with a single type of fluorophore. In a subsequent round, the target was hybridized with the same FISH probe, but labeled with a different dye. This iterative hybridization of differently labeled probes results in a unique barcode for each target mRNA allowing for their identification [Lubeck, p360].
Regarding claim 11, Lubeck’s Figure 1 shows the use of markers wherein the first and second affinity reagents have different targets but are bound to the same label. As an example, in composite images 1 and 2, there are two dots in the upper center of the zoom-in; in the first image, the bottom dot is purple and in the second image, the top dot is purple. This shows that different probes are labeled with the same color dye during different hybridization rounds.
Regarding claim 12, Lubeck used four different markers simultaneously in each round of hybridization [Lubeck 1, p360]. These markers were specific for different targets and labeled with different dyes. This is shown in Lubeck’s Figure 1 which depicts the imaging results of each round of hybridization as a composite image of the different dyes. Each dye channel depicts the results of hybridization with a singular marker.
Claim 5 is rejected under 35 U.S.C. 102(a)(1) as being anticipated by Lubeck 1 and 2, as applied to claim 1 above, and as evidenced by BioNumbers [BioNumbers. “Length of nucleotide”. Harvard.edu. 2019 Oct 17: 1. Obtained from the WayBack Machine on 2026 Aug 20].
Lubeck 1 and 2 applied to the relevant teachings of claim 1 as discussed above and are incorporated herein by reference. BioNumbers teaches that the length of a nucleotide is ~1nm. Therefore, the oligonucleotide probes of Lubeck which are 25 nucleotides long would have a length of ~25nm and anticipate the claimed size of the affinity reagents.
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.
Claims 4-6, 8, 13, and 14 are rejected under 35 U.S.C. 103 as being unpatentable over Lubeck 1 and 2 as applied to claim 1 above, and further in view of Lubeck 2 and Wang [Wang J, et al. bioRxiv; 2020 Feb 28: 1-24].
Lubeck 1 and 2 are applied to the relevant teachings of claim 1 as discussed above and are incorporated herein by reference. Lubeck 1 does not teach targeting proteins using aptamers as the affinity reagents, the application of an electric field during marker introduction, or that the labels are comprised of a degradable nucleic acid backbone which is complementary to a portion of the affinity reagent.
However, Lubeck 1 states that their sequential barcoding scheme can be used in combination with super-resolution FISH which they reported on in Lubeck 2 [Lubeck 1, p361]. In Lubeck 2, they describe a strategy to increase the capacity for multiplex detection of molecules in single cells by using optical super-resolution microscopy, specifically stochastic optical reconstruction microscopy (STORM), and combinatorial labeling via spectral barcoding [Lubeck 2, abstract, p3]. While Lubeck’s study focused on multiplexed identification of mRNA, they state that one advantage of a spectral barcoding scheme is that molecules do not need to be linearized to be faithfully identified which potentially permits other molecules, such as proteins, to be similarly multiplexed [Lubeck 2, p4].
Wang describes existing protein-labeling techniques, such as tagging with fluorescent proteins or enzymatic tags, used for all modes of fluorescence microscopy, including STORM. While genetically encodable fluorescent proteins allow for study of cell dynamics and live-cell imaging, available options have poor photo-stability and low molecular brightness. While enzymatic tags overcome these issues when tagging cell-surface proteins, intercellular labelling efficiency and uniformity is low. Wang also states that antibodies labelled with fluorescent dyes are the main labeling method for STORM imaging of proteins for permeabilized and fixed cells [Wang, p2].
Wang presents RNA aptamers as a class of molecules capable of overcoming these issues. These small nucleic acid oligonucleotides have high binding affinities, are small (2-3nm), and are easy and inexpensive to produce in the laboratory, making them a useful substitute for conventional antibody labeling. While aptamers are not readily cell permeable, they are significantly smaller than antibodies and can diffuse into cells after a permeabilization step, such as electroporation, which allows for intracellular imaging of proteins. Finally, Wang teaches that aptamers can be used to create unique spectral barcodes by hybridizing different color fluorophores to the same aptamer and can be easily removed via RNase treatment [Wang, p2-3, 9-10].
As proof-of-concept, they introduced a method of targeting GFP-labelled proteins in which an aptamer was generated via in vitro transcription using primers to introduce a docking site into the aptamer sequence. This docking site is complementary to the sequence of an imager strand which is labelled with Alexa 647. Wang used this aptamer/imager strand pair to perform a super-resolution technique called PAINT (Points Accumulation for Imaging in Nanoscale Topography) which is able to use a wider selection of fluorophores than STORM and is able to image live cells [Wang, p3-4, 8].
Therefore, one of ordinary skill in the art prior to the effective filing date of the claimed invention looking to develop a method of imaging protein targets via seqFISH, as suggested by Lubeck 2, would have been motivated to adopt aptamers as the affinity reagent as Wang explicitly teaches they are able to be used in this manner and are preferable over antibodies as previously discussed.
Regarding claim 8, Wang teaches that performing electroporation allows aptamers to diffuse into cells allowing for intracellular imaging [Wang, p7]. The skilled artisan would therefore recognize that performing electroporation of cells would allow for the applied markers to more readily and uniformly diffuse into cells which Wang taught was a problem encountered with other protein tagging methods.
Regarding claims 13, the imager strand described by Wang comprises a nucleic acid backbone which could be degraded by a nuclease. This backbone is designed to be complementary to the docking strand of the aptamer [Wang, p3].
Regarding claims 4, 5, 13, and 14, while Wang only uses one marker (i.e., one affinity reagent and one marker), the skilled artisan adopting Lubeck 1’s method for protein identification, would have recognized the need to develop multiple aptamer markers for this purpose.
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 1-15 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 8, 9, 13, 14, and 16 of copending Application No. 18/823,766 in view of Lubeck 1, Lubeck 2, and Wang.
The instant application describes method of analyzing a biological sample comprised of a plurality of target analytes in which a first marker is applied to the sample which is then imaged. The marker is then degraded and the process is repeated with a second marker. Both the first and second markers are comprised of a degradable affinity reagent specific for a target and which is bound to optically detectable labels. The affinity reagents comprise a nucleic acid backbone and may be aptamers. The labels are comprised of a nucleic acid backbone that is complementary to a portion of the affinity reagent and at least one optically detectable moiety.
18/823,766 describes a method for analyzing a biological sample comprising at least one target analyte in which at least a first marker is applied to the sample and a readout is generated. This marker is comprised of an affinity reagent and a first label. The affinity reagent comprises a nucleic acid backbone in a complex structure, such as an aptamer, which is specific for a target analyte and is attached to a barcode oligonucleotide. The label is comprised of an oligonucleotide and at least one labeling moiety and is complementary to the barcode oligonucleotide of the affinity reagent. The labeling moiety may be optically detectable.
Therefore, both methods describe markers which are comprised affinity reagents and labels. In both instances the affinity labels are comprised of a nucleic acid backbone, may be aptamers, are target specific, and attached to a label via complementary sequences. Similarly, both labels are comprised of a nucleic acid backbone and at least one optically detectable moiety. Therefore, the markers are considered equivalent. Additionally, both methods use these markers to identify target analytes in a sample.
The only material difference between the claimed inventions is the instant application’s addition of sequential imaging in which the previously applied marker is degraded before application of a subsequent marker.
However, Lubeck 1 teaches a sequential barcoding scheme which enables multiplex, single cell, in situ profiling of different mRNAs. As described in detail in the previous rejection under 35 U.S.C. 102(a)(1), this method entails the iterative application of fluorescent probes, imaging, and removal of the probes via DNAse I degradation. Lubeck 1 therefore demonstrates that sequentially applying, imaging, and removing nucleic acid-based markers was a known technique for increasing the number of analytes that can be identified in a single sample. Lubeck 2 further recognizes that this iterative process can be applied to other analytes including proteins and Wang makes this step by adapting the process to utilize fluorescent aptamers as affinity reagents to specifically target proteins.
Therefore, one of ordinary skill in the art would have been motivated to modify the single-round imaging method of 18/823,766 to employ the sequential imaging and marker-removal process of Lubeck 1 in order to increase the number of analytes that may be identified in a sample as this amounts to nothing more than using known elements according to their established functions to obtain predictable results. The artisan would have further been motivated to use the aptamer-based affinity reagents of Wang to apply this sequential imaging approach to protein targets as Wang demonstrates this modification to be both successful and an improvement upon current protein imaging methods.
This is a provisional nonstatutory double patenting rejection.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Kara N Kovach whose telephone number is (571)272-8134. The examiner can normally be reached Monday - Friday, 9am - 3pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Gary Benzion can be reached at (571) 272-0782. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/K.N.K./Examiner, Art Unit 1681
/SAMUEL C WOOLWINE/Primary Examiner, Art Unit 1681
Prosecution Insights