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 .
The amendment filed on 04/06/2026 has been entered.
Claims 1, 14 and 48 were amended in the claim set filed on 04/06/2026.
Applicant’s election without traverse of the specie, a single affinity reagent (Claims 1-5, 8-10, 13-15, 17, 19, 22-24, 26-27, 34-36, 38, 40-44 and 51) in the reply filed on 10/31/2025 is acknowledged.
Applicant is reminded that upon the cancelation of claims to a non-elected invention, the inventorship must be corrected in compliance with 37 CFR 1.48(a) if one or more of the currently named inventors is no longer an inventor of at least one claim remaining in the application. A request to correct inventorship under 37 CFR 1.48(a) must be accompanied by an application data sheet in accordance with 37 CFR 1.76 that identifies each inventor by his or her legal name and by the processing fee required under 37 CFR 1.17(i).
Claims 6-7 and 48 are withdrawn drawn to a nonelected species.
Claim 13 was canceled in the claim set filed on 04/06/2026.
Claims 1-5, 8-10, 14-15, 17, 19, 22-24, 26-27, 34-36, 38, 40-44 and 51 in the claim set filed on 04/06/2026 are currently under examination.
Response to the Arguments
Arguments for the priority date of claim 44 are persuasive. The priority date of claims the instant claims, are determined to be the filling date of U.S. Provisional Application No. 63/077,496, filed on 09/11/2020.
Objections to the Specification in the previously mailed non-final have been withdrawn in light of applicants Specification amendments and arguments.
Applicant’s arguments regarding previous rejection(s) of claim(s) 13-14 under 35 U.S.C. 112 have been fully considered but are moot since claim 13 was canceled. However, as necessitated by amendment to claim 1, new grounds of rejection are made under 35 U.S.C. 112 in light of applicants claim amendments and arguments on Pg. 11 as documented below in this office action on Pg. 4-5.
Applicant’s arguments regarding previous rejection(s) of claim(s) 26 under 35 U.S.C. 112 have been fully considered and are persuasive. The 35 U.S.C. 112 rejection of claim 26 documented in the previously mailed non-final has been withdrawn in light of applicants claim amendments and arguments on Pg. 11-12.
The 35 U.S.C. 102 rejections of claim(s) 1- 2, 4, 9, 15, 17, 19, 22, 24, 26-27, 34-36, 38, 41-44, 48 and 51 documented in the previously mailed non-final have been withdrawn; and a new grounds of 103 rejection as necessitated by amendment is made in this Final in light of applicants claim amendments and arguments. Applicant' s argument on Pg. 12, states that “claim 1 has been amended to recite ''activating the at least one transposase under low ionic conditions, wherein the low ionic conditions are characterized by monovalent ionic concentration of less than 10 mM''. Applicant submits that Henikoff does not disclose all the elements of instant amended claim 1”. The new grounds of rejection under 103 is necessitated by amendment, are made as documented below in the 35 U.S.C. 103 rejection in this office action on Pg. 4-12.
Applicant’s arguments regarding previous rejection(s) of claim(s) 1-5, 8-10, 14-15, 17, 19, 22-24, 26-27, 34-36, 38, 40-44 and 51 under 35 U.S.C. 103 over Chen et al. have been fully considered and are persuasive. The 35 U.S.C. 103 rejections documented in the previously mailed non-final have been withdrawn in light of applicants claim amendments and arguments on Pg. 14-15.
The new grounds of rejections under 35 U.S.C. 112; and revised rejections under 35 U.S.C. 103 for claims 1-5, 8-10, 14-15, 17, 19, 22-24, 26-27, 34-36, 38, 40-44 and 51 over Henikoff et al. (“Henikoff”; Patent App. Pub. WO 2019060907 A1, March 28, 2019) are documented below in this Final Office Action are necessitated by claim amendments filed on 04/06/2026.
Priority
This application is a 371 national phase application of PCT/US21/49944, filed on 09/10/2021, which claims benefit of U.S. Provisional Application Nos. 63/077,496, filed on 09/11/2020, and 63/196,953, filed on 06/04/2021.The priority date of claims the instant claims, are determined to be the filling date of U.S. Provisional Application No. 63/077,496, filed on 09/11/2020.
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 1-5, 8-10, 14-15, 17, 19, 22-24, 26-27, 34-36, 38, 40-44 and 51 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 1 is indefinite over the limitation “activating the at least one transposase under low ionic conditions, wherein the low ionic conditions are characterized by monovalent ionic concentration of less than 10 mM”. It is unclear from the language of claim 1 whether the low ionic conditions which are characterized by monovalent ionic concentration is meant to claim that the monovalent ionic concentration is required for activating the at least one transposase. As the specification reads “the method also comprises activating the at least one transposase under low ionic conditions, such as with the addition of Mg++” (Pg. 15, ln 31-32). Mg2+ is a divalent ion, thus one of skill in the art would expect the limitation of activating conditions of a transposase to be drawn to the divalent ionic conditions in addition to any other conditions such as the monovalent ionic conditions. The limitation as is makes it unclear as to the low ionic conditions to activate the transposase. Claims 2-5, 8-10, 14-15, 17, 19, 22-24, 26-27, 34-36, 38, 40-44 and 51 depend on claim 1.
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 1-5, 8-10, 14-15, 17, 19, 22-24, 26-27, 34-36, 38, 40-44 and 51 are rejected under 35 U.S.C. 103 as being unpatentable over Henikoff et al. (“Henikoff”; Patent App. Pub. WO 2019060907 A1, March 28, 2019).
Interpretation: Mg2+ solution in claim 14 is interpreted as a solution used to dilute the monovalent ions in a different solution, contains Mg2+, and the monovalent ion is interpreted as any monovalent ion containing solution. The limitation “optionally”, recited in claims 10, 15, 17, 23, 27, 36, 38, and 44, is interpreted as rendering the phrase thereafter as an optional limitation to the claim.
Henikoff discloses “A method for detecting the binding of a chromatin-associated factor of interest to a sequence of chromatin DNA in a cell, including: contacting a permeabilized cell or nucleus with a specific binding agent that specifically recognizes the chromatin-associated factor of interest, wherein the specific binding agent is linked to a nuclease that is inactive or an activatable transposome; activating the nuclease or transposase, thereby excising the sequence of chromatin DNA bound to the chromatin- associated factor of interest; isolating the excised DNA; and determining the sequence of the excised DNA, thereby detecting binding of a chromatin-associated factor of interest to a sequence of chromatin DNA in the cell…” (Abstract).
Regarding claim 1, Henikoff teaches a method comprising “In some embodiments the method is an in situ method for determining the binding site of a chromatin-associated factor of interest to DNA sequences in a cell. In some embodiments the method includes contacting a permeabilized cell with a first antibody that specifically binds the chromatin-associated factor interest wherein the first antibody is coupled to a plurality of transposomes, … In embodiments, each of the plurality of transposomes includes at least one transposase, and a transposon including a first DNA molecule comprising a first transposase recognition site, and a second DNA molecule comprising a second transposase recognition site. In embodiments, the method includes activating the transposase, for example with a divalent cation, such as Mg2+, thereby excising and tagging the sequence of DNA bound to the chromatin-associated factor of interest with the DNA tag, wherein the at least one transposase integrates the first and second DNA molecules into chromatin DNA, thereby cleaving and tagging chromatin DNA with the first and second DNA molecules. In embodiments, the method includes isolating the excised DNA. In embodiments, the method includes determining the sequence of the excised DNA, thereby mapping binding of a chromatin-associated factor of interest to one or more sequences of DNA in the cell. In embodiments, the antibody is indirectly coupled to the at least one transposase. In embodiments, the transposase is linked to a specific binding agent that specifically binds the first antibody” (Para. 155). Henikoff teaches a method comprising “We first compared ITIS to CUT&RUN for profiling of RNAPII. With CUT&RUN, the length of digestion determines the yield of fragments. Under digestion results in low signal with only a fraction of sites within the population of cells undergoing cleavages on both sides of the targeted particle to release fragments into the supernatant. Over digestion releases pA-MN-bound fragments that can result in untargeted digestion, a problem that becomes serious for highly abundant epitopes such as RNAPII and H3K27ac.To reduce untargeted digestion with CUT&RUN, we modified the protocol such that digestion is performed using low-salt and high-divalent cation concentrations … Indeed, using 3.5 mM HEPES pH 7.5 and 10 mM CaCl2 for CUT&RUN digestion we detected no release of H3K27ac-targeted chromatin during digestion, but quantitative release upon addition of 150 mM NaCl. This procedure greatly reduced variation between time points by correlation matrix analysis, and we have since adopted this improved CUT&RUN protocol for all applications” (Para. 884). Henikoff suggests a method comprising “omitting high-salt treatments” (Para. 885) which allows for the sample to be used with other epigenomic profiling methods as well. Henikoff further teaches “it will be appreciated by those of ordinary skill in the art that a wide variety of alternate and/or equivalent embodiments or implementations calculated to achieve the same purposes may be substituted for the embodiments shown and described without departing from the scope. Those with skill in the art will readily appreciate that embodiments may be implemented in a very wide variety of ways” (Para. 938). Hence, one of ordinary skill in the art would be motivated to further optimize the conditions of the method to further improve targeted digestion as it was already known in the prior art that the ionic concentration can affect the size distribution and transposase hypersensitivity (Para. 885-886). Furthermore, the MPEP states, "Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." (MPEP 2144.05). “3.5 mM HEPES pH 7.5 and 10 mM CaCl2” reads on wherein the low ionic conditions are characterized by monovalent ionic concentration of less than 10 mM. Thus, Henikoff suggests an in situ method for detecting a site of DNA accessibility in the chromatin of a cell, comprising: contacting a permeabilized cell with a first affinity reagent that specifically binds a nucleosome depleted region (NDR) marker, wherein the first affinity reagent is coupled to at least one transposome comprising: at least one transposase; and a transposon comprising: a first DNA molecule comprising a first transposase recognition site; and a second DNA molecule comprising a second transposase recognition site; activating the at least one transposase under low ionic conditions, wherein the low ionic conditions are characterized by monovalent ionic concentration of less than 10 mM, thereby cleaving and tagging chromatin DNA with the first and second DNA molecules and excising a tagged DNA segment associated with the NDR marker; isolating the excised tagged DNA segment; and determining the nucleotide sequence of the excised tagged DNA segment, thereby detecting the site of DNA accessibility in the chromatin of the cell.
The teachings of Henikoff are documented above in the rejection of claim 1 under 35 U.S.C. 103. Claims 2, 4, 9, 14-15, 17, 19, 22, 24, 26-27, 34-36, 38, 41-44, 48 and 51 depend on claim 1. Claim 3 depends on claim 2, which depends on claim 1. Claim 3 depends on claim 2, which depends on claim 1. Claim 8 depends on claim 5. Claims 5-7 depend on claim 4, which depend on claim 1. Claim 10 depend on claim 9, which depends on claim 1. Claim 23 depends on claim 22, which depends on claim 1. Claim 40 depends on claim 38, which depends on claim 1.
Regarding claim 2-3, Henikoff teaches a method wherein the first affinity reagent is directly coupled to at least one transposase; and wherein the first affinity reagent and transposase are disposed in a fusion protein. (Para. 67)
Regarding claim 4, Henikoff teaches a method wherein the first affinity reagent is indirectly coupled to the at least one transposase. (Para. 139)
Regarding claim 5, Henikoff teaches a method wherein the transposase is linked to a specific binding agent that specifically binds the first affinity reagent. (Para. 139)
Regarding claim 8, Henikoff teaches a method wherein the specific binding agent comprises protein A or protein G that specifically binds the first affinity reagent, the second affinity reagent and/or the third affinity reagent. (Para. 139)
Regarding claim 9-10, Henikoff teaches a method wherein the first, second, and/or third affinity reagents independently is or comprises an antibody, an antibody- like molecule, a DARPin, an aptamer, a chromatin-binding protein, other specifically binding molecule, or a functional antigen-binding domain thereof; and wherein the antibody-like molecule is an antibody fragment and/or antibody derivative, optionally a single-chain antibody, a bispecific antibody, an Fab fragment, an F(ab)2fragment, a VHH fragment, a VNAR fragment, or a nanobody, optionally wherein the single-chain antibody is a single chain variable fragment (scFv), or a single-chain Fab fragment (scFab). (Para. 143; Para. 92)
Regarding claim 14, Henikoff teaches a method wherein the low ionic conditions are characterized by monovalent ionic concentration of less than about 10 mM; and wherein the low ionic conditions are obtained by diluting liquid conditions of the transposase with a Mg++ solution, removing liquid supernatant from the transposase and replacing it with a low ionic strength solution, and/or conducting a stringent (e.g., 300 mM) wash followed by adding a low ionic strength solution. (Para. 884-886; Para.877)
Regarding claim 15, Henikoff teaches a method further comprising contacting the permeabilized cell with a polar compound prior to or during the step of activating the transposase under low ionic conditions. (Para. 223)
Regarding claim 17, Henikoff teaches a method wherein the cell is immobilized on a solid surface, optionally wherein the solid surface comprises a bead or wall of a microtiter plate. (Para. 132; Para. 150)
Regarding claim 19, Henikoff teaches a method wherein the first and/or second DNA molecule further comprises a barcode, a sequencing adaptor, and/or a universal priming site. (Para. 152 -Para.154; Para. 157)
Regarding claim 22, Henikoff teaches a method wherein the at least one transposase comprises a Tn5 transposase. (Para. 158)
Regarding claim 23, Henikoff teaches a method wherein activating the transposase under low ionic conditions comprises contacting the transposase with Mg++, optionally with about 0.1 mM Mg++ to about 10 mM Mg++. (Para. 155; Para. 223)
Regarding claim 24, Ref teaches a method wherein the at least one transposase comprises a Mu transposase, an IS5 transposase, or an IS91 transposase. (Para. 159)
Regarding claim 26, Henikoff teaches a method wherein the least one transposome comprises at least two different transposases, and wherein the different transposases integrate different DNA sequences into the chromatin DNA. (Para. 130; Para. 158 ; and Para. 156)
Regarding claim 27, Henikoff teaches a method wherein the method is performed with a plurality of first affinity reagents, thereby producing a plurality of excised tagged DNA segments, and wherein the method further comprises isolating a plurality of excised tagged DNA segments, and optionally further comprising analyzing the isolated tagged DNA segments, optionally wherein analyzing the isolated tagged DNA segments comprises determining the nucleotide sequence of the tagged DNA segments, optionally using sequencing or hybridization techniques with or without amplification.
Regarding claim 34, Henikoff teaches a method wherein the cell and/or the nucleus of the cell is permeabilized by contacting the cell with digitonin. (Para. 29; Para. 130)
Regarding claim 35, Henikoff teaches a method further comprising subjecting the excised DNA to salt fractionation. (Para. 134; Para. 318)
Regarding claim 36, Henikoff teaches a method wherein the NDR marker is a histone modification, optionally methylated H3K4, optionally wherein methylated H3K4 is bi-methylated or tri-methylated, optionally wherein the NDR marker is an initiating form of RNA Polymerase II, optionally serine 5-phosphorylated RNA Polymerase II (RNAPIIS5P) or serine 2-phosphorylated RNA Polymerase II (RNAPIIS2P). (Para. 4; Para. 36)
Regarding claim 38, Henikoff teaches a method further comprising contacting the permeabilized cell with a known amount of spike-in DNA configured to facilitate calibration, optionally wherein the spike-in DNA is or comprises exogenous DNA, exogenous chromatin, or recombinant nucleosomes. (Para. 221)
Regarding claim 40, Henikoff teaches a method wherein the first affinity reagent is coupled to a plurality of transposomes, a fraction of the plurality of transposomes comprising a known amount of spike-in DNA, and wherein the spike-in DNA can be used for calibration. (Para. 887)
Regarding claim 41, Henikoff teaches a method wherein the at least one transposome comprises a fusion protein comprising a first domain comprising a Tn5 transposase domain and second domain comprising a protein A domain, a protein G domain, or a protein A/G hybrid domain. (Para. 67)
Regarding claim 42-43, Henikoff teaches a method wherein the method is performed for a plurality of cells and the method further comprises mapping the determined sequences of one or more excised tagged DNA segments to a consensus genome of the plurality of the cells; and further comprising mapping the determined sequence of the excised tagged DNA segment to the genome of the cell. (Para. 223)
Regarding claim 44, Henikoff teaches a method wherein the method is performed for a plurality of cells, wherein the excised tagged DNA segments of each of the plurality of cells is tagged with a cell-specific barcode or combination of barcodes that is unique to each cell, optionally wherein the method further comprises application of combinatorial indexing to provide the cell-specific barcode or combination of barcodes to the excised tagged DNA segments of each of the plurality of cells and/or wherein the plurality of cells is disposed in a three-dimensional arrangement and the cell- specific barcode or combination of barcodes is unique to a location in the three-dimensional arrangement, optionally wherein the three-dimensional arrangement is a tissue slice or tissue culture array. (Para. 152)
Regarding claim 51, Henikoff teaches a method for preparing a library of excised chromatin DNA comprising the method of claim 1. (Para. 6; Para. 223)
Therefore, the invention as recited in claims 1-5, 8-10, 14-15, 17, 19, 22-24, 26-27, 34-36, 38, 40-44 and 51 are prima facie obvious over the prior art Henikoff et al. One of ordinary skill in the art would have had a reasonable expectation of success given the obviousness of the claims in view of the teachings of Henikoff. It would have been obvious to provide an in-situ method for detecting a site of DNA accessibility in the chromatic of a cell, according to the limitations of the instant application claims 1-5, 8-10, 14-15, 17, 19, 22-24, 26-27, 34-36, 38, 40-44 and 51 based on Henikoff et al. (Patent App. Pub. No. WO 2019060907 A1).
Response to Arguments
Applicant's arguments filed 04/06/2026 (Pg. 12-14) with respect to claims 1-5, 8-10, 14-15, 17, 19, 22-24, 26-27, 34-36, 38, 40-44 and 51 have been fully considered but they are not persuasive. To clarify some instances argued in the response filed 04/06/2026 see responses to each argument made by Applicant below:
Applicants’ argument: “First, Henikoff does not teach activating the at least one transposase under low ionic conditions characterized by monovalent ionic concentrations of less than 10 mM.” (Pg. 12)
Response: Applicant’s arguments have been fully considered and found unpersuasive because as stated above in the revised rejection, “Henikoff teaches a method comprising “We first compared ITIS to CUT&RUN for profiling of RNAPII…To reduce untargeted digestion with CUT&RUN, we modified the protocol such that digestion is performed using low-salt and high-divalent cation concentrations… Indeed, using 3.5 mM HEPES pH 7.5 and 10 mM CaCl2 for CUT&RUN digestion we detected no release of H3K27ac-targeted chromatin during digestion, but quantitative release upon addition of 150 mM NaCl. This procedure greatly reduced variation between time points by correlation matrix analysis (Figure 57), and we have since adopted this improved CUT&RUN protocol for all applications” (Para. 884) “3.5 mM HEPES pH 7.5 and 10 mM CaCl2” reads on wherein the low ionic conditions are characterized by monovalent ionic concentration of less than 10 mM. In addition, it was known that hypersensitivity results from untethered transpose could be reduced or eliminated by adjusting the conditions (Para. 75; Para. 886). Furthermore, "Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." (MPEP 2144.05). Thus, Henikoff does suggest activating the at least one transposase under low ionic conditions characterized by monovalent ionic concentrations of less than 10 mM.”
Applicants’ argument: “Second, Henikoff teaches away from low ionic conditions.” (Pg. 13)
Response: Applicant’s arguments have been fully considered and found unpersuasive because the applicant is referring to some embodiments taught by Henikoff, Henikoff also suggests monovalent ionic concentrations of less than 10 mM. Furthermore, "Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." (MPEP 2144.05). , Thus, Henikoff does not teach away from low ionic conditions.
Applicants’ argument: “Third, the low ionic conditions in the instant Application produce superior unexpected results over Henikoff.” (Pg. 13)
Response: In response to applicant's argument stated above, the fact that the inventor has recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious. See Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985).
Conclusion of Response to Arguments
In view of the amendments, revised rejections and above responses to arguments, no claims are in condition for allowance.
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's
disclosure: Kaya-Okur et al. (2019). CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nature communications, 10(1), 1930. (pg.8-9; entire document) Claim 1; and Meers et al. (2019). Improved CUT&RUN chromatin profiling tools. eLife, 8, e46314. (Pg. 2, Para. 2; Pg. 4, Preventing premature release during CUT&RUN digestion; and pg.8, conclusion) Claim 1.
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 KENDRA R VANN-OJUEKAIYE whose telephone number is (571)270-7529. The examiner can normally be reached M-F 9:00 AM- 5:00 PM.
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/KENDRA R VANN-OJUEKAIYE/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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