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 . Applicants’ preliminary amendment filed 5/2/2024 is acknowledged. Claims 1-55 have been canceled. Claims 56-75 have been added and are pending.
Priority
This application claims benefit of 63/463,527 filed 05/02/2023.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 5/15/2026 and 5/2/2024 is acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Claim Objections
Claim 72 is objected to because of the following informalities:
(a) Claim 72 is objected for the abbreviation “NHS” because it does not recite the full terminology for the acronym (or abbreviation). Claims are more concise when the first time an acronym (or abbreviation) is presented the full terminology is also presented. Additionally, an acronym (abbreviation) may have alternative meanings to an artisan. Appropriate correction is required.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 56-75 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Independent claim 56 is drawn to a method of analyzing a biological sample, comprising:(a) contacting the biological sample with a primary immobilizable probe comprising: (1) a first nucleic acid molecule comprising (i) a first part of a split hybridization region and (ii) an overhang region comprising a primary barcode sequence; and (2) a second nucleic acid molecule comprising (i) a second part of the split hybridization region and (ii) an attachment moiety, wherein: the first part of the split hybridization region comprises a first nucleotide sequence complementary to a first portion of a region of interest in a target nucleic acid, and the second part of the split hybridization region comprises a second nucleotide sequence complementary to a second portion of the region of interest in the target nucleic acid;(b) ligating the first part of the split hybridization region to the second part of the split hybridization region using the region of interest as a template, thereby forming a ligated primary immobilizable probe; (c) using the attachment moiety, attaching the ligated primary immobilizable probe to the biological sample or to a matrix embedding the biological sample; and (d) detecting the ligated primary immobilizable probe at a position in the biological sample and/or matrix.
The claims thus encompass any biological sample, any part of a split hybridization, any portion of any region of a target nucleic acid, any barcode sequence and any attachment moiety and any matrix.
The Federal Circuit discussed claim interpretation by the PTO in In re Morris, where the Federal Circuit noted “[A]s an initial matter, the PTO applies to the verbiage of the proposed claims the broadest reasonable meaning of the words in their ordinary usage as they would be understood by one of ordinary skill in the art, taking into account whatever enlightenment by way of definitions or otherwise that may be afforded by the written description contained in the applicant's specification.” In re Morris, 44 USPQ2d 1023, 1029 (Fed. Cir. 1997). The decision of the court in In re Bigio, 72 USPQ2d 1209 (Fed. Cir. 2004) strongly supports the breadth of interpretation. That court noted “[T]his court counsels the PTO to avoid the temptation to limit broad claim terms solely on the basis of specification passages.” In concert with Morris and Bigio is the decision in In re American Academy of Science Tech Center, 70 USPQ2d 1827, 1834 (Fed. Cir. 2004), where the Federal Circuit noted “We have cautioned against reading limitations into a claim from the preferred embodiment described in the specification, even if it is the only embodiment described, absent clear disclaimer in the specification.”
In this case, the specification provides no limiting definition of a biological sample, rather the claims provide exemplary disclosure (e.g., a cell or tissue sample, para. [0020]).
Thus, the claims encompass any sample from any species. This includes any bodily fluid, isolated nucleic acid, isolated proteins, cells, tissues, fixed samples, frozen samples, other biofluids, etc.
The specification provides no standard or definition of what is required of a matrix. Thus, this is an enormous genus.
The specification teaches that the target nucleic acid encompasses DNA, cDNA, RNA, mRNA or RNA fragment with or without a polyA tail ([0010]). The specification provides no guidance as to any part of or region of any of the recited target nucleic acid sequences that are functionally required to perform the intended purpose of analyzing a biological sample as instantly claimed. Thus, this encompasses an enormous genus.
The specification provides exemplary disclosure for a barcode (e.g., a gene-specific sequence) ([0033]). Thus, this encompasses an enormous genus.
The specification provides exemplary disclosure of a linker (e.g., disulfide linker [0096], or phosphoramidite [0098] or exemplary cross-linker(s) [0122]). Thus, this encompasses an enormous genus.
The specification provides no standard or definition of what is required of a matrix. Thus, this is an enormous genus.
The claims provide no specific guidance on how to selectively perform the claimed method in view of the large plethora of species encompassed by the enormous genus supported by the specification and claims. Merely attaching a probe(s) to a sample will not result in analyzing any biological sample as different conditions, specificity, affinity and compatibility must be taken into consideration to achieve the desired results. Thus, given the large genus encompassed by the claims, the claims fail to provide adequate written description commensurate fully in scope.
Claim Rejections - 35 USC § 112(b)
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 56-75 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.
(a) Claims 56-75 are indefinite in the claim 56 at the recitation “primary immobilizable probe” and “primary barcode sequence” because it cannot be de determined from the claims or specification how the limitation “primary” defines or limits the “immobilizable probe or barcode sequence. It is unclear how a primary immobilizable probe or a primary barcode sequence differs structurally or function from any other probe or barcode sequence respectively. Clarification is required.
Claim Rejections - 35 USC § 102
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.
(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.
Claim(s) 56-75 is/are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Bava et al (US 20230037182, effective filing date July 2021).
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.
Regarding claims 56-75, Bava teaches [0004] a method for analyzing a biological sample, the method comprising: a) contacting the biological sample with a circularizable probe, wherein the circularizable probe comprises: (i) a hybridization region HR1′ that is complementary to a target sequence HR1 in a target nucleic acid comprised by the sample, and (ii) a first end and a second end that do not hybridize to the target nucleic acid, wherein the circularizable probe hybridizes to the target nucleic acid comprising the target sequence via HR1′; b) dissociating molecule(s) of the circularizable probe that do not hybridize via HR1′ from the target nucleic acid, under conditions in which molecule(s) of the circularizable probe that hybridize via HR1′ remain hybridized to the target nucleic acid; c) ligating the first and second ends of the circularizable probe, thereby generating a circularized probe; d) performing rolling circle amplification of the circularized probe to generate a rolling circle amplification product; and e) detecting the rolling circle amplification product in the biological sample. The target sequence HR1 can alternately be referred to as the “hybridization region” HR1.
[0005] A circularizable probe can be any nucleic acid molecule or set of nucleic acid molecules that hybridizes to another one or more other nucleic acids such that the ends of the nucleic acid molecule or nucleic acid molecules are juxtaposed or are in proximity for ligation to form a circularized probe (e.g., by ligation with or without gap filling). For example, a circularizable probe may be a padlock probe with ends that can be ligated to form a circularized padlock probe. Examples of circularizable probe designs according to the present application are provided in FIGS. 1-3.
[0006] In some embodiments, hybridization region HR1′ is a split hybridization region comprising a first portion HR1a′ and a second portion HR1b′, wherein HR1a′ hybridizes to a first portion of HR1 (HR1a) and HR1b′ hybridizes to a second portion of HR1 (HR1b), and wherein the first end and the second end of the circularizable probe that do not hybridize to the target nucleic acid are positioned between HR1a and HR1b upon hybridization of the circularizable probe. In some embodiments, HR1a′ and HR1b′ are not connected directly connected by a phosphodiester linkage. In some embodiments, HR1a′ and HR1b′ are two hybridizing regions separated by a non-hybridizing region.
[0007] In any of the preceding embodiments, the circularizable probe can comprise, from 5′ to 3′: a first end that does not hybridize to the target nucleic acid, a first portion HR1a′ of the hybridization region HR1′, one or more sequences for hybridization of additional probes or primers, a second portion HR1b′ of the hybridization region HR1′ and a second end that does not hybridize to the target nucleic acid.
[0008] In any of the preceding embodiments, the first end can comprise a first splint hybridization sequence and the second end can comprise a second splint hybridization sequence, and the method can further comprise contacting the sample with a splint that hybridizes to the first and second splint hybridization sequences and ligating the first and second ends of the circularizable probe using the splint as a template.
[0009] In any of the preceding embodiments, the combined length of the first splint hybridization sequence and second splint hybridization sequence can be shorter than the combined length of HR1a′ and HR1b′. Alternatively, in any of the preceding embodiments, the combined length of the first splint hybridization sequence and second splint hybridization sequence can be greater than or equal to the combined length of HR1a′ and HR1b′.
[0010] In any of the preceding embodiments, the melting temperature of the splint hybridized to the circularizable probe can be less than the melting temperature of the circularizable probe hybridized to the target nucleic. Alternatively, in any of the preceding embodiments, the melting temperature of the splint hybridized to the circularizable probe can be greater than or equal to the melting temperature of the circularizable probe hybridized to the target nucleic acid.
[0011] In any of the preceding embodiments, the ligating can be enzymatic ligation or chemical ligation.
[0012] In any of the preceding embodiments, the ligating can be template independent ligation. Alternatively, in any of the preceding embodiments, the ligating can be template dependent ligation. In some embodiments, the ligating is splint-templated ligation with or without gap-filling. In some embodiments, the method can further comprise contacting the sample with a splint. In some embodiments, the splint does not hybridize to the target nucleic acid.
Bava additionally teaches at paragraphs [0028] – [0034] the following:
[0028] In any of the preceding embodiments, the probe or the target nucleic acid comprises a toehold region adjacent to the interrogatory region or the region of interest, respectively. In some embodiments, in the dissociating step, the toehold region can hybridize to the target nucleic acid or the probe, thereby allowing displacement of the blocking strand. In any of the preceding embodiments, the toehold region can be between about 5 and about 50 nucleotides in length. In some embodiments, the toehold region is between about 5 and about 10, between about 10 and about 15, or between about 15 and about 20 nucleotides in length.
[0029] In any of the preceding embodiments, the dissociating step can comprise removing probe molecules that are bound to the target nucleic acid but comprise in the interrogatory region one or more mismatches with the region of interest, and/or allowing probe molecules or portions thereof comprising one or more mismatches to dissociate from the target nucleic acid while probe molecules comprising no mismatch in the interrogatory region remain bound to the target nucleic acid. In some embodiments, under the same conditions, probe molecules comprising one or more mismatches in the interrogatory region are less stably bound to the target nucleic acid than probe molecules comprising no mismatch in the interrogatory region.
[0030] In any of the preceding embodiments, the dissociating step can comprise one or more stringency washes.
[0031] In any of the preceding embodiments, the circularizable probe can be a first circularizable probe, and the method can comprise contacting the sample with a second circularizable probe, wherein the second circularizable probe comprises: (i) a hybridization region HR2′ that is complementary to a second target sequence HR2, and (ii) a first end and a second end that do not hybridize to the second target sequence. In some embodiments, the dissociating step can comprise dissociating molecule(s) of the first and/or second circularizable probe that do not hybridize via HR1′ or HR2′ to the first and/or second target sequence, under conditions in which molecule(s) of the circularizable probes that hybridize via HR1′ or HR2′ remain hybridized to their respective target sequence.
[0032] In any of the preceding embodiments, the ends of the first circularizable probe and the second circularizable probe can comprise common sequences.
[0033] In any of the preceding embodiments, the method can comprise contacting the sample with a splint, wherein the splint hybridizes to the common sequences. In some embodiments, the splint is used to template the ligating in step c).
[0034] In some aspects, provided herein is a method for analyzing a biological sample, the method comprising: a) contacting the biological sample with a plurality of circularizable probes, wherein each circularizable probe of the plurality of circularizable probes comprises: (i) a hybridization region HRn′ that is complementary to a target sequence HRn in a target nucleic acid, and (ii) a first end and a second end that do not hybridize to the target nucleic acid; b) hybridizing molecule(s) of the circularizable probes that comprise a hybridization region HRn′ complementary to the target sequence HRn to the target nucleic acid; c) ligating the first and second ends of the circularizable probes that are hybridized to the target nucleic acid(s) in the sample, thereby generating circularized probes; d) performing rolling circle amplification of the circularized probes to generate a plurality of rolling circle amplification products; and e) detecting the rolling circle amplification products in the biological sample. In some embodiments, the method comprises removing molecule(s) of the circularizable probes that do not comprise a hybridization region HRn′ complementary to a target sequence HRn from the sample, under conditions in which molecule(s) of the circularizable probes that comprise a hybridization region HRn′ complementary to a target sequence remain hybridized to the target nucleic acid(s) in the sample.
At paragraph [0054] – [0061], [0063], [0066] –[0074], Bava teaches the following:
[0054] In any of the preceding embodiments, the first nucleic acid molecule, second nucleic acid molecule, and third nucleic acid molecule can be a first set of probes (first probe A, second probe A, and third probe A), and the first set of probes can comprise a sequence complementary to a first sequence of the region of interest, and the method can further comprise: a) contacting the target nucleic acid with a second set of probes comprising a first probe B, a second probe B, and a third probe B that each hybridize to the target nucleic acid, wherein the second set of probes comprises a sequence complementary to a second sequence of the region of interest, and wherein the first and second sequences of the region of interest are different; b) forming a second circularized probe using the first probe B and the target nucleic acid to template a first and second ligation between the second probe B and third probe B, wherein the first and second ligations are performed simultaneously or in any order; c) generating one or more amplification products using the circularized second probe; and d) detecting the presence or absence of the amplification product of the second circularized probe.
[0055] In some embodiments, the first and second sequences of the region of interest can be different at one, two, three, four, five, or more nucleotide positions.
[0056] In any of the preceding embodiments, the first and/or second sequences of the region of interest can comprise a single nucleotide of interest, an alternatively spliced region, a deletion, and/or a frameshift.
[0058] In any of the preceding embodiments, the first set of probes can comprise a first barcode sequence corresponding to the first sequence of the region of interest, the second set of probes can comprise a second barcode sequence corresponding to the second sequence of the region of interest, and the first and second barcode sequences can be different.
[0059] In any of the preceding embodiments, the sequence complementary to the region of interest can be located at the 3′ end of the second nucleic acid molecule or third nucleic acid molecule.
[0060] In any of the preceding embodiments, the target nucleic acid can be in a biological sample and the circularized probe and/or the amplification product thereof can be generated in situ in the biological sample.
[0061] In any of the preceding embodiments, the amplification product can be generated using a linear rolling circle amplification (RCA), a branched RCA, a dendritic RCA, or any combination thereof.
[0063] In any of the preceding embodiments, the circularized probe and/or the amplification product thereof can be immobilized in the biological sample and/or crosslinked to one or more other molecules in the biological sample.
[0066] In any of the preceding embodiments, the signal can be amplified in situ in the biological sample. In some embodiments, the signal amplification in situ comprises rolling circle amplification (RCA) of a probe that directly or indirectly binds to the circularized probe and/or the amplification product thereof; hybridization chain reaction (HCR) directly or indirectly on the circularized probe and/or the amplification product thereof; linear oligonucleotide hybridization chain reaction (LO-HCR) directly or indirectly on the circularized probe and/or the amplification product thereof; primer exchange reaction (PER) directly or indirectly on the circularized probe and/or the amplification product thereof; assembly of branched structures directly or indirectly on the circularized probe and/or the amplification product thereof; hybridization of a plurality of detectable probes directly or indirectly on the circularized probe and/or the amplification product thereof, or any combination thereof.
[0067] In any of the preceding embodiments, the circularized probe and/or the amplification product thereof can be analyzed by sequential hybridization, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof.
[0068] In any of the preceding embodiments, the circularized probe and/or the amplification product thereof can comprise one or more barcode sequences or complements thereof.
[0069] In any of the preceding embodiments, the one or more barcode sequences or complements thereof can correspond to the target nucleic acid and/or the region of interest.
[0070] In any of the preceding embodiments, the one or more barcode sequences or complements thereof can be detected by: i) contacting the biological sample with one or more detectably-labeled probes that directly or indirectly bind (e.g., hybridize) to the one or more barcode sequences or complements thereof, ii) detecting signals associated with the one or more detectably-labeled probes, and iii) removing (e.g., dehybridizing) the one or more detectably-labeled probes. In some embodiments, the contacting, detecting, and removing (e.g., dehybridizing) steps are repeated with the one or more detectably-labeled probes and/or one or more other detectably-labeled probes that directly or indirectly bind to the one or more barcode sequences or complements thereof.
[0071] In any of the preceding embodiments, the one or more barcode sequences or complements thereof can be detected by: i) contacting the biological sample with one or more intermediate probes that directly or indirectly bind (e.g., hybridize) to the one or more barcode sequences or complements thereof, wherein the one or more intermediate probes are detectable using one or more detectably-labeled probes, detecting signals associated with the one or more detectably-labeled probes, and ii) removing (e.g., dehybridizing) the one or more intermediate probes and/or the one or more detectably-labeled probes. In some embodiments, the contacting, detecting, and removing (e.g., dehybridizing) steps are repeated with the one or more intermediate probes, the one or more detectably-labeled probes, one or more other intermediate probes, and/or one or more other detectably-labeled probes.
[0072] In any of the preceding embodiments, the biological sample can be a fixed and/or permeabilized biological sample.
[0073] In any of the preceding embodiments, the biological sample can be a tissue sample. In any of the preceding embodiments, the biological sample can be a formalin-fixed, paraffin-embedded (FFPE) tissue sample, a frozen tissue sample, or a fresh tissue sample. In some embodiments, the tissue sample can be a tissue slice between about 1 μm and about 50 μm in thickness. In some embodiments, the tissue slice is between about 5 μm and about 35 μm in thickness.
[0074] In any of the preceding embodiments, the biological sample can be crosslinked. In any of the preceding embodiments, the biological sample can be embedded in a matrix. In some embodiments, the matrix is a hydrogel. In any of the preceding embodiments, the biological sample can be cleared.
Bava teaches at paragraph [0099], [0129] - [0131], Bava teaches the following: [0099] Biological samples can include analytes (e.g., protein, RNA, and/or DNA) embedded in a 3D matrix. In some embodiments, amplicons (e.g., rolling circle amplification products) derived from or associated with analytes (e.g., protein, RNA, and/or DNA) can be embedded in a 3D matrix. In some embodiments, a 3D matrix may comprise a network of natural molecules and/or synthetic molecules that are chemically and/or enzymatically linked, e.g., by crosslinking. In some embodiments, a 3D matrix may comprise a synthetic polymer. In some embodiments, a 3D matrix comprises a hydrogel.
[0129] In some embodiments, a biological sample embedded in a matrix (e.g., a hydrogel) can be isometrically expanded. Isometric expansion methods that can be used include hydration, a preparative step in expansion microscopy, as described in Chen et al., Science 347(6221):543-548, 2015.
[0130] Isometric expansion can be performed by anchoring one or more components of a biological sample to a gel, followed by gel formation, proteolysis, and swelling. In some embodiments, analytes in the sample, products of the analytes, and/or probes associated with analytes in the sample can be anchored to the matrix (e.g., hydrogel). Isometric expansion of the biological sample can occur prior to immobilization of the biological sample on a substrate, or after the biological sample is immobilized to a substrate. In some embodiments, the isometrically expanded biological sample can be removed from the substrate prior to contacting the substrate with probes disclosed herein.
[0131] In general, the steps used to perform isometric expansion of the biological sample can depend on the characteristics of the sample (e.g., thickness of tissue section, fixation, cross-linking), and/or the analyte of interest (e.g., different conditions to anchor RNA, DNA, and protein to a gel). See also paragraph [0222] – [0226] and Figures 1-3.
Thus, Bav meets the limitations of the claims 56-75 as recited herein.
Conclusion
No claims are allowed. Any inquiry concerning this communication or earlier communications from the examiner should be directed to CYNTHIA B WILDER whose telephone number is (571)272-0791. The examiner can normally be reached Flexible.
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/CYNTHIA B WILDER/Primary Examiner, Art Unit 1681
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