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 (claims 1-26 and 33-38) in the reply filed on 9/18/2025 is acknowledged.
Claims 1-38 are pending.
Claims 27-32 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 9/18/2025.
Claims 1-26 and 33-38 are being examined on the merits.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure statement (e.g., pg 6, ln 13; pg 11, ln 15-17). 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Non-Patent Literature reference 13 has been lined through, as it is a duplicate of NPL reference 6 (Chan et al., 2004).
Abstract
Applicant is reminded of the proper content of an abstract of the disclosure.
A patent abstract is a concise statement of the technical disclosure of the patent and should include that which is new in the art to which the invention pertains. The abstract should not refer to purported merits or speculative applications of the invention and should not compare the invention with the prior art.
If the patent is of a basic nature, the entire technical disclosure may be new in the art, and the abstract should be directed to the entire disclosure. If the patent is in the nature of an improvement in an old apparatus, process, product, or composition, the abstract should include the technical disclosure of the improvement. The abstract should also mention by way of example any preferred modifications or alternatives.
Where applicable, the abstract should include the following: (1) if a machine or apparatus, its organization and operation; (2) if an article, its method of making; (3) if a chemical compound, its identity and use; (4) if a mixture, its ingredients; (5) if a process, the steps.
Extensive mechanical and design details of an apparatus should not be included in the abstract. The abstract should be in narrative form and generally limited to a single paragraph within the range of 50 to 150 words in length.
See MPEP § 608.01(b) for guidelines for the preparation of patent abstracts.
The abstract of the disclosure is objected to because it is 160 words long. A corrected abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text. See MPEP § 608.01(b).
Specification
The disclosure is objected to because of the following informalities: there is an inconsistency in the formatting of the “SEQ ID NO:” disclosures. For example, on page 6 of the specification the designation of sequences is written as “SEQ ID NO: 4” in line 13 and “SEQ ID NO:2” in line 15. “SEQ ID NO: 4” (with a space after “:” and before “#”) is the proper way to format the sequence listing designations. Please ensure that each instance of a SEQ ID NO has the proper formatting throughout the specification.
Appropriate correction is required.
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code (pg 34, ln 10). Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
The use of the term “Texas-Red” (pg 25, ln 26; some instances are properly marked while others are not), which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claims 2, 5, 8, 12, 22, 33, and 36 are objected to because of the following informalities:
Claim 2 reads “SEQ ID NOS:” and should read “SEQ ID NOs:”.
Claim 5 reads “wherein the domain is Protein A, Protein L or Protein G or a combination of one or more of Protein A, Protein L or Protein G in tandem” and should read “wherein the domain is Protein A, Protein L, , or a combination of one or more of Protein A, Protein L, and Protein G in tandem”.
Claim 8(a) reads “adding to the sample a nicking endonuclease fusion protein (NEFP) comprising the nicking endonuclease and a domain that binds to a constant region of an antibody, wherein the antibody is bound directly or indirectly to the target to produce site specific nicked chromosomal DNA in the sample” and should read “adding to the sample a nicking endonuclease fusion protein (NEFP) comprising the nicking endonuclease and a domain that binds to a constant region of an antibody, wherein the antibody is bound directly or indirectly to the target, to produce site specific nicked chromosomal DNA in the sample”. The comma between “target” and “to produce” provides clarity by emphasizing that the wherein clause of direct or indirect binding to the target is a limitation of the antibody.
Claim 9 reads “where in the target is selected from the group consisting of a DNA binding protein a structural feature of the chromosomal DNA and a modified base” and should read “where in the target is selected from the group consisting of a DNA binding protein, a structural feature of the chromosomal DNA, and a modified base” (commas added between the options within the group to clearly delineate).
Claim 12 reads “so as to permit antibody to access the target and binding of the NEFP to the antibody” and should read “so as to permit the antibody to the target and permit binding of the NEFP to the antibody”.
Claim 22 reads “a dye labeled dNTP for visualization the presence and location of the target” and should read “a dye labeled dNTP for visualizing the presence and location of the target”
Claim 33 reads “wherein the NEFP and the heparin may be lyophilized or in a buffer, in separate compartments or combined in the same compartment” and should read “wherein the NEFP and the heparin may be lyophilized or in a buffer, and may be in separate compartments or combined in the same compartment”.
Claim 36 reads “wherein the fusion protein further comprises” and should read “wherein the NEFP further comprises” to remain consistent with how this fusion protein is referred to in the rest of the claims.
Appropriate correction is required.
Claim Rejections - 35 USC § 112b - Indefiniteness
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 5, 10, 12, 15-16, 19, and 33-38 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 5 is directed to the composition of claim 1 “wherein the domain is Protein A, Protein L or Protein G or a combination of one or more of Protein A, Protein L or Protein G in tandem”. However, it is unclear how you can have a combination of “one”. A combination of one of Protein A (for e.g.) does not make sense for a singular domain and furthermore cannot be provided “in tandem” with only a single domain.
Claims 10, 15, and 33: The term “low salt” (10 and 15) and “low inorganic salt” (claim 33) in claims 10, 15, and 33 is a relative term which renders the claims indefinite. The term “low” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. “Low salt” is only provided as embodiments in the specification (“includes a molar concentration of less than 110 mM salt”, “may be no more than 75 mM or no more than 50 mM”, pg 14, ln 21-23). This is contrary to the term “high salt” (as written in claim 15) which is defined in the specification as “refer[ing] to a concentration of 400 mM to 2 M salt” (pg 14, ln 26-27). High salt is defined by the specification in a way that low salt is not. Therefore, the scope of what constitutes a “low salt buffer” is unclear.
Claim 16 depends from claims 10 and 15, inherits this deficiency and is rejected on the same basis. Claims 34-38 depend from claim 33, inherit this deficiency, and are rejected on the same basis.
Claim 12 recites the limitation "the cytoplasmic membrane and nuclear membrane" in line 2. There is insufficient antecedent basis for this limitation in the claim. While the cytoplasmic membrane and nuclear membrane are referring to the cell sample as defined in claim 11 (from which claim 12 depends), the specification indicates that “cell samples containing the chromosomal DNA may be fixed cells, non-fixed cells, or lysates” (emphasis added, pg 4, ln 1-4). In the case of lysates, a cytoplasmic membrane and nuclear membrane are not necessarily present, therefore the cytoplasmic membrane and nuclear membrane lack antecedent basis.
Claim 19 contains the limitation “wherein the one or more modified dNTPs are selected from the group consisting of d5mCTP, biotinylated CTP and a dye labeled dNTP”. Claim 19 depends from claim 8 “wherein one or more dNTPs in the mixture is modified for blocking secondary nicking”. Claim 19 as written encompasses an embodiment in which the one or modified dNTPs are dye labeled dNTPs only. This does not meet the requirement of claim 8 in which one or more of the modified dNTPs has to be modified for blocking secondary nicking (such as with d5mCTP). Further clarification is required.
Claims 35 and 36 are directed to the kit of claim 33 further comprising “one or more modified nucleotides and dNTPs”. It is unclear if it’s one or modified nucleotides, one or more modified dNTPs, or if both nucleotides and dNTPs are modified. Furthermore, it is unclear if this means that there are nucleotides which are different than the dNTPs being added, and if so, how these nucleotides differ. Clarification is required.
Claim 36 contains the limitation “wherein the fusion protein further comprises a polymerase”. It is unclear if the polymerase would be provided within the NEFP (as a tripartite fusion) or if it would be provided in a mixture in addition to the NEFP. The scope of the claim is unclear and further clarification is required.
Claim 37 depends from claim 36, inherits these deficiencies, and is rejected on the same basis.
Claim 38 recites the limitation "the modified nucleotides" in line 3. There is insufficient antecedent basis for this limitation in the claim. The modified nucleotides are defined in claims 35 and 36, but not in claim 33 from which this claim depends.
Claim Rejections - 35 USC § 112a – Written Description
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 1-26 and 33-38 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.
The claims are rejected for failing to describe the genus of structures of the nicking endonuclease fusion proteins as currently claimed. The claims are rejected for failing to describing a genus of any nicking endonuclease fusion protein with any domain that can bind a constant region of any antibody. Claims 5 and 17 are rejected for failing to describe the genus of nicking endonuclease fusion protein comprising a nicking endonuclease and a protein L domain that can bind to a constant region of the antibody. The structure of the protein L that binds to the constant region is not described. The art has shown protein L binding to some kappa light chain region structures (see Nilson et al., cited on IDS of 10/18/2022). The genus of fusion proteins with an additional undescribed polymerase domain is not described (relevant to claims 7 and 36). The structure of the tripartite fusion is not described (relevant to claims 7 and 36). It is art recognized that structure will determine the function of an enzymatic activity.
The MPEP states that the purpose of the written description requirement is to ensure that the inventor had possession, at the time the invention was made, of the specific subject matter claimed. The courts have stated:
"To fulfill the written description requirement, a patent specification must describe an
invention and do so in sufficient detail that one skilled in the art can clearly conclude that
"the inventor invented the claimed invention.” Lockwood y. American Airlines, Inc., 107
F.3d 1565, 1572, 41 USPQ2d 1961, 1966 (Fed. Cir. 1997); In re Gostelli, 872 F.2d 1008,
1012, 10 USPQ2d 1614, 1618 (Fed. Cir. 1989) ("[T]he description must clearly allow persons of ordinary skill in the art to recognize that [the inventor] invented what is claimed."). Thus, an applicant complies with the written description requirement "by describing the invention, with all its claimed limitations, not that which makes it obvious,” and by using "such descriptive means as words, structures, figures, diagrams, formulas, etc., that set forth the claimed invention." Lockwood, 107 F.3d at 1572, 41 USPQ2d at 1966." Regents of the University of California v. Eli Lilly & Co., 43 USPQ2d 1398.
Further, for a broad generic claim, the specification must provide adequate written description to identify the genus of the claim. In Regents of the University of California v. Eli Lilly & Co. the court stated:
"A written description of an invention involving a chemical genus, like a description of a
chemical species, 'requires a precise definition, such as by structure, formula, [or]
chemical name,’ of the claimed subject matter sufficient to distinguish it from other
materials.” Fiers, 984 F.2d at 1171, 25 USPQ2d 1601; Jn re Smythe, 480 F.2d 1376,
1383, 178 USPQ 279, 284985 (CCPA 1973) ("In other cases, particularly but not
necessarily, chemical cases, where there is unpredictability in performance of certain
species or subcombinations other than those specifically enumerated, one skilled in the art may be found not to have been placed in possession of a genus ...") Regents of the
University of California v. Eli Lilly & Co., 43 USPQ2d 1398.
MPEP § 2163 further states that if a biomolecule is described only by a functional characteristic, without any disclosed correlation between function and structure of the sequence, it is "not sufficient characteristic for written description purposes, even when accompanied by a method of obtaining the claimed sequence." MPEP § 2163 does state that for a generic claim the genus can be adequately described if the disclosure presents a sufficient number of representative species that encompass the genus. If the genus has a substantial variance, the disclosure must describe a sufficient variety of species to reflect the variation within that genus. See MPEP §
2163. Although the MPEP does not define what constitute a sufficient number of representative species, the courts have indicated what do not constitute a representative number of species to adequately describe a broad generic. In Gostelli, the courts determined that the disclosure of two chemical compounds within a subgenus did not describe that subgenus. Jn re Gostelli, 872, F.2d at 1012, 10 USPQ2d at 1618.
As stated supra, the MPEP states that written description for a genus can be achieved by a representative number of species within a broad genus. Claims are broadly generic to all possible fusion constructs with any nicking endonuclease domain and any domain that binds a constant region of any antibody encompassed by the claims. The possible variations are enormous. Since the MPEP states that if a biomolecule is described only by a functional characteristic, without any disclosed correlation between function and structure, it is "not sufficient characteristic for written description purposes, even when accompanied by a method of obtaining the claimed sequence." MPEP § 2163. Here, though the claims may recite some functional characteristics, the claims lack written description because there is no disclosure of a correlation between function and structure beyond those disclosed in the examples in the specification. Moreover, the specification lacks sufficient variety of species to reflect this variance in the genus. While having written description of the Nt.CviPII-protein A fusion and protein G-gHNH fusion, the specification is devoid of any genus of any nicking endonuclease with any antibody binding domains as currently pending.
The description requirement of the patent statue requires a description of an invention, not an indication of a result that one might achieve if one made that invention. See Jn re Wilder, 736, F.2d 1516, 1521, 222 USPQ 369, 372-73 (Fed. Cir. 1984) (affirming rejection because the specification does "little more than outlin[e] goals appellants hope the claimed invention achieves and the problems the invention will hopefully ameliorate.") Accordingly, it is deemed that the specification, while providing for the structure/species of fusion proteins of protein A/G-Nt.CviPII/gHNH in the form of SEQ ID NOs: 5, 6, 7, and 8, fails to provide adequate written description for the genus of the claims (any nicking endonuclease domain, any domain that binds to a constant region of an antibody, and any polymerase) and does not reasonably convey to one skilled in the relevant art that the inventor(s), at the time the application was filed, had possession of the entire scope of the claimed invention.
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.
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 and 5 are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Laemmli (Laemmli et al., US 7,790,379 B2; cited on IDS of 10/18/2022).
Laemmli teaches a composition comprising a nicking endonuclease fusion protein that is comprised of a nicking endonuclease (MNase) and a domain, wherein the domain binds to a constant region of an antibody (relevant to claim 1; Fig 1A, col 9, ln 18-32). Laemmli teaches that the domain that binds to a constant region of an antibody can be Protein A or Protein G (relevant to claim 5; col 9, ln 26-32).
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 3 and 4 are rejected under 35 U.S.C. 103 as being unpatentable over Laemmli (Laemmli et al., US 7,790,379 B2; cited on IDS of 10/18/2022) in view of Harrington (Harrington et al., US 20210009974 A1).
As noted above in the rejection of claim 1, Laemmli teaches a composition comprising a nicking endonuclease fusion protein that is comprised of a nicking endonuclease (MNase) and a domain, wherein the domain binds to a constant region of an antibody (relevant to claim 1; Fig 1A, col 9, ln 18-32).
Laemmli does not teach that the composition comprises a heparin salt (claim 3) or an inorganic salt at a concentration of less than 100 mM (claim 4). However, use of heparin in reactions with nucleases and buffers containing less than 100 mM of an inorganic salt is known in the art, as taught by Harrington.
Harrington teaches a composition which comprises a programmable nuclease, heparin, and NaCl (an inorganic salt; paragraph [0246]). Harrington teaches that the NaCl is at a concentration of 1-200 mM, 80-120mM or 100 mM (paragraph [0246]). The range of NaCl in the compositions of Harrington significantly overlaps with the claimed range of less than 100 mM of claim 4. The exact concentrations of NaCl provided in claim 4 could be achieved through “routine optimization” through experimentation commonly employed when optimizing cleavage reaction conditions and thus are not patentably distinct from the prior art (see MPEP 2144.05 II.A. Routine Optimization). Additionally, where Harrington teaches different NaCl concentration for the cleavage by nuclease Cas13a, the prior art recognizes NaCl concentration as a results effective variable (Fig 23A).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the composition of Laemmli with that of Harrington. One would be motivated to do so given the assertion by Harrington that inclusion of heparin and NaCl in the reaction composition “enhances the specificity of the programmable nuclease” (paragraph [0248]). One would have a reasonable expectation of success given that addition of heparin and NaCl to cleavage compositions is performed by Harrington.
Claims 6 and 7 are rejected under 35 U.S.C. 103 as being unpatentable over Laemmli (Laemmli et al., US 7,790,379 B2; cited on IDS of 10/18/2022) in view of Ponnaluri (Ponnaluri et al., Genome Biology 2017; cited on IDS of 10/18/2022).
As noted above in the rejection of claim 1, Laemmli teaches a composition comprising a nicking endonuclease fusion protein that is comprised of a nicking endonuclease (MNase) and a domain, wherein the domain binds to a constant region of an antibody (relevant to claim 1; Fig 1A, col 9, ln 18-32). Laemmli teaches that the nicking endonuclease used in the composition creates either single or double cleavage sites in DNA, and that this cleavage can be sequence-specific or sequence-independent (col 7, ln 62-66 and col 8, ln 1-3).
Laemmli does not teach that the nicking endonuclease is Nt.CviPII or gHNH, or that a polymerase is included in the composition. However, usage of Nt.CviPII as a nicking endonuclease in a reaction composition that also includes polymerase is known in the art, as taught by Ponnaluri.
Ponnaluri teaches a composition that comprises Nt.CviPII nicking endonuclease and a polymerase (“Open chromatin DNA was labeled with biotin by incubating the nuclei in the presence of 2.5 U of Nt.CviPII (NEB R0626S), 10 U of DNA polymerase I (M0209S) and 30 μM of each dNTP including 6 μM of biotin-14-dATP (Invitrogen, 19524-016) and 6 μM of biotin-16-dCTP (ChemCyte, CC-6003-1) in 200 μL of 1× NEB buffer 2”, Methods – Open chromatin labeling).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the composition of Laemmli with that of Ponnaluri. One would be motivated to do so given the teaching of Ponnaluri that Nt.CviPII can travel through the nuclear membrane and nick sites of open chromatin with high sequence specificity (Results - Nicking enzyme-mediated tagging of the open chromatin). One would be motivated to include polymerase given the teaching by Ponnaluri that this, in addition to modified enzymes, allows for nick translation of sites of open chromatin and facilitates downstream methods such as sequencing (Nicking enzyme-mediated tagging of the open chromatin).
Claims 8, 9, 11-13, 17-21, 24 and 25 are rejected under 35 U.S.C. 103 as being unpatentable over Ponnaluri (Ponnaluri et al., Genome Biology 2017; cited on IDS of 10/18/2022) in view of Laemmli (Laemmli et al., US 7,790,379 B2; cited on IDS of 10/18/2022).
Regarding claim 8, 9, and 17: Ponnaluri teaches a method of identifying a location of a target associated with chromosomal DNA (open chromatin; reads on a structural feature of the chromosomal DNA of claim 9) in a sample comprising contacting the sample with a nicking endonuclease (Nt.CivPII), performing nick translation on the nicked chromosomal DNA in the presence of a strand displacing polymerase and a mixture of dNTPs where one or more of the dNTPs in the mixture is modified for blocking secondary nicking, and then identifying the location of the target on the DNA (Results - Nicking enzyme-mediated tagging of the open chromatin, Results - Open chromatin mapping with 250 cells). The modified dNTPs included in the mixture include biotin-14-dATP, which the specification of the instant application lists as a modified nucleotide that would act as an inhibiting moiety to nicking endonuclease cleavage (pg 24, ln 22-23).
Ponnaluri does not teach that the nicking endonuclease is fused to a domain that binds to a constant region of an antibody, which is then bound directly or indirectly to the target. However, fusion of endonucleases to domains that bind the constant regions of antibodies that bind directly/indirectly to targets on chromosomal DNA is known in the art, as taught by Laemmli.
Laemmli teaches a nicking endonuclease fusion protein that is comprised of a nicking endonuclease (MNase) and a domain, wherein the domain binds to a constant region of an antibody (relevant to claim 8 and 17; protein A or protein G, Fig 1A, col 9, ln 18-32).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Ponnaluri with that of Laemmli. One would be motivated to do so given the teaching of Laemmli that by fusing an endonuclease with an antibody binding domain, this allows for direct/indirect tethering of the endonuclease to a protein or genomic feature of interest on the DNA, and thus allows for site-specific nicking at the target site and identification of binding sites of target proteins (col 2, ln 41-56, col 2, ln 65-67, col 3, ln 1-4, col 9, ln 7-12, and col 9, ln 18-24). One would have a reasonable expectation of success given that Laemmli successfully fuse a nicking endonuclease to protein A (a domain that binds a constant region of an antibody) and map the location of a target of interest associated with chromosomal DNA (Example 1).
Regarding claim 11 and 12: Ponnaluri teaches that the sample is a cell sample in which the nuclear membrane is made porous to allow binding of the endonuclease to the target (Methods - Open chromatin labeling).
Regarding claim 13: Ponnaluri teaches contacting the cells with the nicking endonuclease at 37º C (Methods - Open chromatin labeling).
Regarding claim 18: Ponnaluri teaches that the nicking endonuclease is Nt.CviPII (Results - Nicking enzyme-mediated tagging of the open chromatin).
Regarding claim 19: Ponnaluri teaches that the one or more modified dNTPs include biotinylated CTP (biotin-16-dCTP) or a dye labeled dNTP (TexasRed-dATP; Results - Nicking enzyme-mediated tagging of the open chromatin).
Regarding claims 20 and 21: Ponnaluri teaches immobilizing the nick translated DNA from the sample and preparing a library from the immobilized DNA and then sequencing the library to identify the location of the target in the DNA sequence (“The genomic DNA from the biotin labeling reaction was purified, fragmented, and captured using Streptavidin beads for library construction. Streptavidin-captured DNA from putative open chromatin regions was used for high-throughput sequencing.” Results - Open chromatin mapping with 250 cells).
Regarding claims 24: Ponnaluri teaches that differences in the global landscape of closed versus open chromatin is associated with cancers (Results - DNA hypomethylation of HCT116 genome correlates with OCSs).
Regarding claim 25: Ponnaluri teaches that the genome of cancer is globally hypomethylated and thus determination of differences in levels of open chromatin would be associated with a cancer genome (diagnostic; Results - DNA hypomethylation of HCT116 genome correlates with OCSs). Ponnaluri teaches determining the levels of open chromatin through the use of a nicking endonuclease, strand displacement polymerase, and modified dNTPs (as described above). Ponnaluri teaches including biotinylated dNTPs (for sequencing) OR dye labeled dNTPs for visualization. While Ponnaluri does not demonstrate combination of the two, Ponnaluri does teach that combination of NicE-Seq (biotinylated dNTPs in nicking endonuclease mediated nick translation) with dye-labeled dNTPs would facilitate visualization of the open chromatin in the same cell sample (Conclusion).
Claims 10, 15, 16, 33, and 35-38 are rejected under 35 U.S.C. 103 as being unpatentable over Ponnaluri (Ponnaluri et al., Genome Biology 2017; cited on IDS of 10/18/2022) in view of Laemmli (Laemmli et al., US 7,790,379 B2; cited on IDS of 10/18/2022) as applied to claims 8, 9, 11-13, 17-21, 24 and 25 above, and further in view of Harrington (Harrington et al., US 20210009974 A1).
The teachings of Ponnaluri in view of Laemmli as they apply to claims 8, 9, 11-13, 17-21, 24 and 25 are detailed above. Relative to the instantly rejected claims, Ponnaluri in view of Laemmli teach a method of identifying a target associated with chromosomal DNA using a fusion protein comprised of a nicking endonuclease fused to a domain that binds to the constant region of an antibody, performing nick translation with a strand displacement polymerase and modified dNTPs, and then identifying the location of the target on the DNA.
Regarding claims 10, 15, and 16: Ponnaluri in view of Laemmli teach inclusion of a low amount of inorganic salt in the reaction buffer (NEBuffer™ 2 from the method of Ponnaluri comprises 50 mM NaCl). Ponnaluri teaches that the activating ions for Nt.CviPII are magnesium (Methods – Open chromatin labeling). Laemmli teaches keeping the nicking endonuclease inactive until addition of a buffer with activating components (col 2, ln 58-64). Laemmli teaches washing the reaction stringently with a high salt buffer (col 33, ln 46-48) before adding the activating ions into the reaction mixture (Ca2+ ions for MNase activity, col 33, ln 60-63).
Ponnaluri in view of Laemmli do not teach adding heparin to the reaction mixture. However, use of heparin in reactions with nucleases and buffers containing less than 100 mM of an inorganic salt is known in the art, as taught by Harrington.
Harrington teaches a cleavage reaction buffer which comprises a programmable nuclease and heparin (paragraph [0246]).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Ponnaluri in view of Laemmli with that of Harrington. One would be motivated to do so given the assertion by Harrington that inclusion of heparin and NaCl in the reaction composition “enhances the specificity of the programmable nuclease” (paragraph [0248]). One would have a reasonable expectation of success given that addition of heparin to cleavage compositions is performed by Harrington.
Regarding claims 33 and 35-38: Ponnaluri in view of Laemmli teach including reagents necessary for performance of the methodology in a kit (Laemmli, col 19, ln 19-20). Ponnaluri in view of Laemmli teach the components of the kit necessary, including the NEFP and a low inorganic salt buffer (claim 33), a polymerase and one or more modified nucleotides and dNTPs such as biotinylated CTP or dye labeled dNTP (claims 35-37), and magnetic beads coated with an affinity binding molecule for binding at least one of the modified nucleotides (such as streptavidin coated beads for capturing biotinylated dNTPs; claim 38; see citations above).
Ponnaluri in view of Laemmli do not teach inclusion of heparin in the kit, however as noted above, Harrington teaches inclusion of heparin in a nuclease reaction to increase specificity of the nuclease. Therefore, it would have been obvious to include the heparin in the kit in addition to the other reaction reagents taught by Ponnaluri in view of Laemmli. Harrington teaches kits comprising reagents necessary for performing a reaction as well (paragraph [0484-0485]). Harrington teaches that the kit comprises buffers which can be in a container (reads on compartment).
Claims 14 and 26 are rejected under 35 U.S.C. 103 as being unpatentable over Ponnaluri (Ponnaluri et al., Genome Biology 2017; cited on IDS of 10/18/2022) in view of Laemmli (Laemmli et al., US 7,790,379 B2; cited on IDS of 10/18/2022) as applied to claims 8, 9, 11-13, 17-21, 24 and 25 above, and further in view of Chan (Chan et al., Nucleic Acids Research 2004; cited on IDS of 10/18/2022).
The teachings of Ponnaluri in view of Laemmli as they apply to claims 8, 9, 11-13, 17-21, 24 and 25 are detailed above. Relative to the instantly rejected claims, Ponnaluri in view of Laemmli teach a method of identifying a target associated with chromosomal DNA using a fusion protein comprised of a nicking endonuclease fused to a domain that binds to the constant region of an antibody, performing nick translation with a strand displacement polymerase and modified dNTPs, and then identifying the location of the target on the DNA.
Ponnaluri in view of Laemmli do not teach performing each step of the method at a single reaction temperature (claim 14). However, performing multiple reaction steps at a single temperature is known in the art, as taught by Chan.
Chan teaches a method of nicking endonuclease-mediated random DNA amplification in which an endonuclease (Nt.CviPII) is used to nick genomic DNA and a strand displacement polymerase and dNTPs are added to perform nick translation and amplification (Discussion, paragraph 8). Chan teaches that contacting DNA with Nt.CviPII and amplification can be done in a single-step isothermal reaction at the same temperature of 55ºC.
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Ponnaluri in view of Laemmli with that of Chan. One would be motivated to do so given the assertion by this is a single-step reaction. One skilled in the art would recognize the inherent advantage of performing multiple reactions at a single temperature (less room for error, less time needed for temperature adjustment). One would have a reasonable expectation of success given that Chan demonstrates that the same nicking endonuclease taught by Ponnaluri (Nt.CviPII) can perform at the temperature required for polymerase activity and nick translation to occur.
Ponnaluri in view of Laemmli and Chan do not explicitly teach performing this method in a single tube (claim 26). However, Chan teaches that both the nicking activity and the nick translation activity can be performed in a single-step reaction at the same temperature and Ponnaluri teaches that modified dNTPs can be added to nick translation to facilitate visualization. Therefore, it would have been obvious to perform the method in a single tube given the obvious advantage of minimized sample handling and minimized sample loss upon transfer between tubes.
Claims 22 and 23 are rejected under 35 U.S.C. 103 as being unpatentable over Ponnaluri (Ponnaluri et al., Genome Biology 2017; cited on IDS of 10/18/2022) in view of Laemmli (Laemmli et al., US 7,790,379 B2; cited on IDS of 10/18/2022) as applied to claims 8, 9, 11-13, 17-21, 24 and 25 above, and further in view of Krystosek (Krystosek et al., US 5,264,343 A; cited on IDS of 10/18/2022) and Wenger (Wenger et al., Neuro-Oncology 2019).
The teachings of Ponnaluri in view of Laemmli as they apply to claims 8, 9, 11-13, 17-21, 24 and 25 are detailed above. Relative to the instantly rejected claims, Ponnaluri in view of Laemmli teach a method of identifying a target associated with chromosomal DNA using a fusion protein comprised of a nicking endonuclease fused to a domain that binds to the constant region of an antibody, performing nick translation with a strand displacement polymerase and modified dNTPs, and then identifying the location of the target on the DNA. Ponnaluri in view of Laemmli teach performing this methodology on cancer cell samples (colorectal cancer HCT116 cells). Ponnaluri in view of Laemmli teach incorporating modified dye labeled dNTPs for visualizing the presence and location of the target (open chromatin) in the cells (Ponnaluri Fig 1B).
Ponnaluri in view of Laemmli do not teach that the sample is a biopsy sample from a patient. However, use of patient biopsy samples for assessment of open chromatin through nicking endonuclease mediated nick translation is known in the art, as taught by Krystosek.
Krystosek teaches a method of labeling open chromatin by exposing cells to DNA polymerase I, DNase, and dNTPs (at least one of which is biotin labeled; Abstract). Krystosek teaches that this methodology can be performed on fresh biopsy cells (claim 11).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Ponnaluri in view of Laemmli with that of Krystosek. One would be motivated to do so given the assertion by Krystosek that this methodology of labeling open chromatin (similar to that of Ponnaluri) can be used to diagnose cancer through the detection of exposed DNA in cells (col 1, ln 61-68 and col 2, ln 1-4). One would have a reasonable expectation of success given that Krystosek performs nicking endonuclease mediated nick translation in cell samples and using cell samples from patients in a diagnostic assay would predictably yield similar results. Krystosek also demonstrates that the methodology could be successfully performed in a normal human lymphocyte (non-dividing) obtained from a clinical laboratory (col 9, ln 6-15).
Ponnaluri in view of Laemmli and Krystosek do not obtaining a plurality of aliquots from the same biopsy sample (claim 23). However, obtaining multiple aliquots from the same biopsy sample is known in the art, as taught by Wenger.
Wenger teaches performing genome-wide DNA methylation analysis on multiple samples from the same biopsied tumor to study intratumor heterogeneity (Abstract).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Ponnaluri in view of Laemmli and Krystosek to obtain a plurality of aliquots from the same biopsied sample, as taught by Wenger. One would be motivated to do so given the teaching of Wenger that there is significant intratumor heterogeneity of methylation patterns (and thus patterns of open chromatin) which could impact diagnostics and classification of tumor status (Abstract). One would have a reasonable expectation of success given that Wenger demonstrates that multiple samples/aliquots can be obtained from the same tumor biopsy.
Allowable Subject Matter
SEQ ID NOs: 5, 6, 7, and 8 are free of the prior art.
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.
US 11,492,667 B2
Claims 1, 3, 5-7, 8-11, 13-15, 17-21, and 25 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-14 of U.S. Patent No. US 11,492,667 B2. Although the claims at issue are not identical, they are not patentably distinct from each other because both sets of claims are drawn to the same limitations. Any additional limitations of '667 claims are encompassed by the open claim language “comprising” found in the instant claims.
Additionally, though claims 1-14 of ‘667 teach a method, it teaches all reagents that would be present in a composition as taught by the instant claims (1, 3, and 5-7).
Claims 4, 16, 22-24, 26, 33, and 35-38 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-14 of U.S. Patent No. US 11,492,667 B2 in view of Ponnaluri (Ponnaluri et al., Genome Biology 2017; cited on IDS of 10/18/2022), Laemmli (Laemmli et al., US 7,790,379 B2; cited on IDS of 10/18/2022), Harrington (Harrington et al., US 20210009974 A1), Chan (Chan et al., Nucleic Acids Research 2004; cited on IDS of 10/18/2022), Krystosek (Krystosek et al., US 5,264,343 A; cited on IDS of 10/18/2022), and Wenger (Wenger et al., Neuro-Oncology 2019) according to citations and rationales provided above.
Conclusion
No claims are allowed.
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/KAILEY ELIZABETH CASH/Examiner, Art Unit 1683
/STEPHEN T KAPUSHOC/Primary Examiner, Art Unit 1683