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 .
Election/Restrictions
Applicant’s election without traverse of Group I in the reply filed on March 4, 2026 is acknowledged.
The Office acknowledges the cancellation of claims 18-20 directed to a nonelected invention.
Information Disclosure Statement
The IDS received on April 30, 2024 is proper and is being considered by the Examiner.
Drawings
The drawings received on December 12, 2023 are acceptable.
Specification
The disclosure is objected to because of the following informalities:
Under ST.26 rules, in a sequencing listing, SEQ ID Numbers directed to nucleotides that are less than 10 nucleotides in length are skipped and not disclosed therein. Therefore, these sequences should not be referred to by their SEQ ID numbers in the specification. SEQ ID NOs: 1-20 contain nucleotide sequences having less than 10 bases in length, but are referenced in the specification. (see section [0133] for example).
All such references should be removed from the specification.
Appropriate correction is required.
Claim Objections
Claim 17 is objected to for reasons discussed above. All references to SEQ ID numbers being less than 10 ntds in length (thus skipped in the sequence listing) should be removed from the claim (i.e., all of the claims). Appropriate correction is required.
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 13-15 and 17 are 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 13 is indefinite for reciting the phrase, “circular polynucleotide in a cell or tissue by extending a first primer hybridized to said circular polynucleotide …” because it is unclear what the source of this circular polynucleotide in the cell is as cells do not have a circular polynucleotide that can be amplified by a primer and a strand displacement polymerase. For the purpose of prosecution, the claim has been construed to mean that a circular polynucleotide is first added to the cell or tissue.
Claim 15 is indefinite because the claim appears to be disjointed. Claim 15 depends from claim 13 (through claim 14). Claim 13 contains step (iii) that requires that a first extension product is nicked with an endonuclease and removed, and second immobilized extension products (i.e., the single-stranded polynucleotides on the solid support), and these second immobilized extension products are annealed to a sequencing primer.
Claim 15, now redefines this step by reciting that step (iii) nicks the second immobilized extension products with an endonuclease. However, if this step is performed, then step (iv) that sequences the second immobilized extension products cannot be performed.
Claims 14 and 15 are indefinite by way of their dependency on claim 13.
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.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1, 6, 8, and 10-13 are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Witters et al. (WO 2022/015600 A2, published January 2022, priority July 2020).
As regard to the rejection under 102(a)(2), the applied reference has a common assignee with the instant application. Based upon the earlier effectively filed date of the reference, it constitutes prior art under 35 U.S.C. 102(a)(2). This rejection under 35 U.S.C. 102(a)(2) might be overcome by: (1) a showing under 37 CFR 1.130(a) that the subject matter disclosed in the reference was obtained directly or indirectly from the inventor or a joint inventor of this application and is thus not prior art in accordance with 35 U.S.C. 102(b)(2)(A); (2) a showing under 37 CFR 1.130(b) of a prior public disclosure under 35 U.S.C. 102(b)(2)(B) if the same invention is not being claimed; or (3) a statement pursuant to 35 U.S.C. 102(b)(2)(C) establishing that, not later than the effective filing date of the claimed invention, the subject matter disclosed in the reference and the claimed invention were either owned by the same person or subject to an obligation of assignment to the same person or subject to a joint research agreement.
With regard to claim 1, Witters et al. teach a method of forming a single-stranded polynucleotide in situ, the method comprising:
extending a first primer hybridized to a circular polynucleotide with a strand displacing polymerase to generate a first extension product comprising one or more complement of the circular polynucleotide within a cell or tissue (the formation of a circular polynucleotide see Fig. 4B, probe 416 annealing to a target nucleic acid becoming circularized, also “fixed cellular matrix 400 … annealing a target-specific probe 416 to one of nucleic acids 415 … ligation and circularization of probe 416”, section [0010]; annealing of the circularized polynucleotide to a first primer to generate a first extension product, see Fig. 4D, also “annealing of the circularized oligonucleotide to an immobilized primer 425 attached to cellular component (or matrix) 420”, section [0010]);
contacting the first extension product with a second immobilized primer and extending the second immobilized (see Fig. 4F, the process shown in FIG. 1A, step 3); and
nicking the first extension product with an endonuclease, thereby generating one or more polynucleotide fragment, and removing said polynucleotide fragments, thereby forming single-stranded polynucleotides in situ (see Fig. 1B, step 9, also “this strand can then be optionally cleaved …”, section [0007]; also “‘cleavable site’ or ‘scissile linkage’ in the context of polynucleotide is a site which allows a controlled cleavage of the polynucleotide strand (e.g., the linker, the primer, or the polynucleotide) by chemical enzymatic … nicked by a nicking endonuclease enzyme”, section [0038]; also “Bridged template structures may be linearized by cleavage of one or both strands with a restriction endonuclease or by cleavage of one strand with a nicking endonuclease”, section [0200]).
With regard to claim 6, the second immobilized extension product is sequenced (see Fig. 1B, step 8, also “hybridize the first sequencing primer to begin sequencing (step 8)”, section [0007]).
With regard to claim 8, the sequencing is performed by SBS (sequencing-by-synthesis, also “sequencing methodologies can be used such as sequencing-by-synthesis (SBS)”, section [0123]).
With regard to claim 10, the circularization of the probe is made by ligation of two ends of a single-stranded polynucleotide to a target of a cell (“ligating ends of a linear polynucleotide … together to form a circular template polynucleotide”, section [0096]).
With regard to claims 11 and 12, the artisans teach a well-known means of providing a circularized polynucleotide where the two ends of the single-stranded polynucleotide anneal adjacent to each other on a target sequence and ligated, or gapped by 1 or more nucleotides, filled-in, then ligated (“[t]ypically, both ends of the padlock probe hybridize to adjacent sequences and are then ligated together to form a circular oligonucleotide. Oligonucleotide primers hybridize to sequences adjacent to the target nucleic acid sequence resulting in a gap (e.g., a gap spanning the length fo the target nucleic acid sequence) … polymerase then extends the first complementary region to generate a complement to the target nucleic acid sequence, and ligation circularizes the oligonucleotide primer … circular oligonucleotide is then amplified and sequenced”, section [0215]).
With regard to claim 13, the steps discussed above (with reference to claim 1), is further disclosed in conjunction with a sequencing step of the single-stranded polynucleotide with a sequencing primer (see FIG. 1B, steps 8 and 9; and FIG. 1C, steps 10 and 11).
Therefore, the invention as claimed is deemed anticipated by Witters et al.
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 2-5, 7, and 14-16 are rejected under 35 U.S.C. 103 as being obvious over Witters et al. (WO 2022/015600 A2, published January 2022, priority July 2020) in view of Boles et al. (U.S. Patent No. 6,300,070 B1, published October 9, 2001) as evidenced by New England BioLabs Product Catalogue (herein “NEB”, available on-line: www.neb.com).
The applied reference has a common assignee with the instant application. Based upon the earlier effectively filed date of the reference, it constitutes prior art under 35 U.S.C. 102(a)(2).
The teachings of Witters et al. have already been discussed above.
Witters et al. teach the use of additional oligonucleotides (in the form of primers) being annealed to the first immobilized extension product, and therefore evidences the presence of the circularized polynucleotide (see Fig. 1C, steps 10 and 11). However, the additional oligonucleotides (in the form of the primers/probe) are annealed to the first immobilized extension product and not the second immobilized extension product (claim 2).
Witters et al. do not explicitly teach using additional immobilized primers for the generation of immobilized extended products for sequencing (claims 3 and 14), and steps of their cleavage and detection (claims 5, 7, and 15).
Witters et al. while explicitly teaching the incorporation of restriction/endonuclease recognition sites for cleaving means, do not explicitly list the types of endonucleases recited in claim 16.
Consequently Witters et al. do not teach that the circular polynucleotide comprises a sequence of 5’ – CCTCAGC – 3’ (i.e., SEQ ID NO: 1) (claim 17).
NEB evidences that Nb.Bbv.CI1 endonuclease has a recognition sequence 5’ – CCTCAGC – 3’.
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Witters et al. with the teachings of Boles et al. and NEB, thereby arriving at the invention as claimed for the following reasons.
As discussed above, Witters et al. teach a method of generating a circularized template polynucleotide in the presence of a target nucleic acid in situ, whereupon the circularized template is then subsequently annealed to a first primer to generate a first extension product. The artisans further teach the step of annealing the first extension product to a second immobilized primer, wherein the second immobilized primer is extended to form a second immobilized extension product (known in the art as bridge amplification).
While Witters et al. did not explicitly teach that a third immobilized primer was utilized in generating a third immobilized extension product, it is clear from Figure 4D, that the artisans contemplated the use of a plurality of such immobilized primers (figure shows three primers) that can participate in such a reaction.
Indeed, Boles et al. teach that the participation of additional such immobilized primers of Witters et al. would have resulted in the generation of multiple immobilized extension products, all of which could follow the steps of sequencing reaction taught by Witters et al., rendering claims 2-5, 7, 14, and 15 obvious.
With regard to the use of prior art known endonucleases such as Nb.BbvCI, having the recognition site 5’ – CCTCAGC – 3’ (i.e., SEQ ID NO: 1), it would have been prima facie obvious to incorporate such a recognition site into the single-stranded polynucleotide that forms into a circular template polynucleotide, for the cleaving the immobilized extended polynucleotides of Witters et al. Doing so would have been obvious based on the suggestion made by Witters et al. in incorporating a endonuclease nicking site into the template structure:
“Bridged template structures may be linearized by cleavage of one or both strands with a restriction endonuclease or by cleavage of one strand with a nicking endonuclease”, section [0200]).
And in combination of cleaving the immobilized strand that have been sequenced (see Fig. 1B, steps 8-9 and the structure produced after cleavage step in Fig. 1C), one of ordinary skill in the art would have recognized that any prior art known nicking endonucleases, such as Nb.BbvCI, having the recognition site 5’ – CCTCAGC – 3’ (i.e., SEQ ID NO: 1), would have sufficed for this purpose, yielding no more than a predictable outcome.
In KSR, the Supreme Court particularly emphasized “the need for caution in granting a patent based on the combination of elements found in the prior art,” Id. at 415, 82 USPQ2d at 1395, and discussed circumstances in which a patent might be determined to be obvious. Importantly, the Supreme Court reaffirmed principles based on its precedent that “[t]he combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results.” Id. at 415-16, 82 USPQ2d at 1395. The Supreme Court stated that there are “[t]hree cases decided after Graham [that] illustrate this doctrine.” Id. at 416, 82 USPQ2d at 1395. (1) “In United States v. Adams, . . . [t]he Court recognized that when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable result.”
Therefore, the invention as claimed is deemed prima facie obvious over the cited references.
This rejection under 35 U.S.C. 103 might be overcome by: (1) a showing under 37 CFR 1.130(a) that the subject matter disclosed in the reference was obtained directly or indirectly from the inventor or a joint inventor of this application and is thus not prior art in accordance with 35 U.S.C.102(b)(2)(A); (2) a showing under 37 CFR 1.130(b) of a prior public disclosure under 35 U.S.C. 102(b)(2)(B); or (3) a statement pursuant to 35 U.S.C. 102(b)(2)(C) establishing that, not later than the effective filing date of the claimed invention, the subject matter disclosed and the claimed invention were either owned by the same person or subject to an obligation of assignment to the same person or subject to a joint research agreement. See generally MPEP § 717.02.
Claim 9 is rejected under 35 U.S.C. 103 as being obvious over Witters et al. (WO 2022/015600 A2, published January 2022, priority July 2020) in view of Cheng et al. (Adv. Sci., July 2021, vol. 8, pages 1-14).
The applied reference has a common assignee with the instant application. Based upon the earlier effectively filed date of the reference, it constitutes prior art under 35 U.S.C. 102(a)(2).
The teachings of Witters et al. have already been discussed above.
Witters et al. do not teach an additional step of providing a specific binding reagent to a protein in the cell or tissue with an oligonucleotide barcode and sequencing the barcode.
Cheng et al. teach a well-known means of tagging an antibody with a sample-specific oligonucleotide barcode (see Fig. 2).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Witters et al. and Cheng et al., thereby arriving at the invention as claimed because the use of antibody conjugated to a sample-specific barcode had been well-established technique.
“labeling distinct samples with predefined or specific barcodes” (page 2)
“Barcoded antibody is prepared … By sequencing sample barcode in parallel with endogenous mRNA, wherein both have been tagged with identical cel barcode during library construction, the sample identity of each cell can be mapped.” (pages 3 and 5).
Therefore, by combining the teachings of Cheng et al. with Witters et al., one of ordinary skill in the art would have been able to associate the sequence reads with their sample source by utilizing an antibody conjugated to sample-specific barcode.
Therefore, the invention as claimed is deemed prima facie obvious over the cited references.
Conclusion
No claims are allowed.
Inquiries
Any inquiry concerning this communication or earlier communications from the Examiner should be directed to Young J. Kim whose telephone number is (571) 272-0785. The Examiner can best be reached from 7:30 a.m. to 4:00 p.m (M-F). The Examiner can also be reached via e-mail to Young.Kim@uspto.gov. However, the office cannot guarantee security through the e-mail system nor should official papers be transmitted through this route.
If attempts to reach the Examiner by telephone are unsuccessful, the Examiner's supervisor, Gary Benzion, can be reached at (571) 272-0782.
Papers related to this application may be submitted to Art Unit 1681 by facsimile transmission. The faxing of such papers must conform with the notice published in the Official Gazette, 1156 OG 61 (November 16, 1993) and 1157 OG 94 (December 28, 1993) (see 37 CFR 1.6(d)). NOTE: If applicant does submit a paper by FAX, the original copy should be retained by applicant or applicant’s representative. NO DUPLICATE COPIES SHOULD BE SUBMITTED, so as to avoid the processing of duplicate papers in the Office. All official documents must be sent to the Official Tech Center Fax number: (571) 273-8300. Any inquiry of a general nature or relating to the status of this application should be directed to the Group receptionist whose telephone number is (571) 272-1600.
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.
Information regarding the status of an application may be obtained from the Patent Application Information Retrieval (PAIR) system. Status information for published applications may be obtained from either Private PAIR or Public PAIR. Status information for unpublished applications is available through Private PAIR only. For more information about the PAIR system, see http://pair-direct.uspto.gov. Should you have questions on access to the Private PAIR system, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative or access to the automated information system, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/YOUNG J KIM/Primary Examiner
Art Unit 1637 March 20, 2026
/YJK/
1 Nb.Bbv.CI had been well-characterized and known in the prior art as evidenced by Antipova et al. (FEMS Microbiol. Lett., 2014, vol. 357, pages 144-150).