DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Status of the Application
Receipt is acknowledged of Applicants’ preliminary amendment, filed on 03/14/2022, in which claims 5-8, 10, 12-13, 15, 18-21, 23-25, and 28-30 are amended and claims 33-56 are cancelled.
Claims 1-32 are pending and are examined on the merits herein.
Priority
The instant application claims domestic benefit to 63/161,330 filed on 03/15/2021.
Information Disclosure Statement
The information disclosure statements (IDS) dated 03/14/2022, 08/11/2022, and 12/22/2023 comply with the provisions of 37 CFR 1.97, 1.98 and MPEP § 609. Accordingly, the information disclosure statements have been considered by the examiner.
Claim Rejections
Claim 5 objected to because of the following informalities: the claim recites wherein the first methyltransferase enzyme is selected from … CpG (M. Sssl). CpG itself is not an enzyme. However, this is an obvious typo because M. SssI is a recognized CpG methyl transferase enzyme.. Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 5, 7, and 9are 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 recites “and CpG (M.SssI).” It is not clear whether the parenthetical is used to define the term or provide an optional example. Thus, it is unclear whether the limitation “M.SssI” in the parenthetical is part of the claimed invention. See MPEP § 2173.05(d). For purposes of compact prosecution, under broadest reasonable interpretation, the claim will be interpreted as “and a CpG methyltransferase enzyme.”
Claims 5 and 9 recite the limitation "the first methyltransferase enzyme" in line 1 of each of claims 5 and 9. There is insufficient antecedent basis for this limitation in the claims. For purposes of compact prosecution, under broadest reasonable interpretation, the claims will be interpreted as depending from claim 3.
Claim 7 recites the limitation "the methyl group" in line 1. There is insufficient antecedent basis for this limitation in the claim. For purposes of compact prosecution, under broadest reasonable interpretation, the claim will be interpreted as depending from claim 1.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 12 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 12 recites “and step (b) comprises deaminating the hmC in the target polynucleotide to form hydroxythymine (hT).” Thus dependent claim 12 provides a different set of limitations for step (b) than what is stated in independent claim 1, which requires deamination of mC in the target polynucleotide to form thymine. For purposes of compact prosecution, under broadest reasonable interpretation, the claims will be interpreted as requiring both deamination of mC in the target polynucleotide to form thymine and deamination of hmC in the target polynucleotide to form hydroxythymine. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
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)(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 1-4, 8, and 10-11 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Kohli et al. (US 2023/0183793 A1; PTO-892).
Kohli discusses methods of carboxymethylating cytosine containing DNA sequences (abstract). These methods are useful for gene sequencing and identification of epigenetic modifications in target nucleic acids and enable differentiation of cytosine, 5-methylcytosine and 5-hydroxymethylcytosine in DNA [0004]. In the embodiment of claim 10, Kohli discloses the method comprising:
(a) reacting a polynucleotide containing C, 5mC, and/or 5hmC with a variant methyltransferase having carboxymethyltransferase activity in the presence of carboxy-S-adenosyl-L-methionine (CxSAM) substrate, thereby labeling any unmodified C in said polynucleotide and rendering it resistant to deaminase action; wherein said 5hmC is also optionally glucosylated;
(b) contacting the polynucleotide of step (a) with a deaminase which deaminates 5mC and/or 5hmC, with minimal damage to said target polynucleotide present in said sample;
(c) analyzing said polynucleotide sample, to identify each of unmodified C, 5mC, and 5hmC present in said polynucleotide;
wherein said variant methyl transferase having carboxymethylase activity is a recombinant M.MpeI N374K and said deaminase enzyme is APOBEC3A.
The chemical structure of CxSAM is a chemical structure of instant claim 4 in which the instant protective group coupled to the sulfonium ion comprises a carboxyl group, and the cytosine is protected at the 5 position (Figure 3C). M.MpeI is a CpG methyltransferase enzyme [0034]. Thus, Kohli anticipates the instant claims.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1, 5, 7, 9, 12-20, 24, and 29-32 are rejected under 35 U.S.C. 103 as being unpatentable over Kohli et al. (US 2023/0183793 A1; PTO-892).
Kohli discusses methods of carboxymethylating cytosine containing DNA sequences (abstract). These methods are useful for gene sequencing and identification of epigenetic modifications in target nucleic acids and enable differentiation of cytosine, 5-methylcytosine and 5-hydroxymethylcytosine in DNA [0004]. In claim 7, Kohli discloses the method comprising:
(a) reacting a polynucleotide containing C, 5mC, and/or 5hmC with a variant methyltransferase having carboxymethyltransferase activity in the presence of carboxy-S-adenosyl-L-methionine (CxSAM) substrate, thereby labeling any unmodified C in said polynucleotide and rendering it resistant to deaminase action; wherein said 5hmC is also optionally glucosylated;
(b) contacting the polynucleotide of step (a) with a deaminase which deaminates 5mC and/or 5hmC, with minimal damage to said target polynucleotide present in said sample;
(c) analyzing said polynucleotide sample, to identify each of unmodified C, 5mC, and 5hmC present in said polynucleotide.
In this method, a neomorphic CpG methyltransferase (MTase) enzyme uses CxSAM as a substrate to protect unmodified cytosine bases from deamination via their conversion to 5cxmC. As in traditional ACE-Seq, the 5hmC bases are protected from deamination by glucosylation using βGT. Subsequent treatment with the DNA deaminase therefore only leaves 5mC subject to deamination, resulting in a C to T transition in sequencing for bases that were originally 5mC [0028]. The teaching that conversion of C into 5cxmC protects the unmodified base from deamination is interpreted as teaching that it inhibits the activity of the deaminase enzyme. Kohli further teaches that the product of the MTase catalyzed reaction of cytosine and CxSAM is 5-carboxymethylcytosine [0010]. In figures 7C and 7D, Kohli shows the chemical mechanism of DNA carboxymethylation by the particular MTase M.MepI using CxSAM as a substrate. It teaches that the carboxymethyl group of the CxSAM fits into the active site of M.MpeI and is expected to form a salt bridge with the mutated active site residue N374K [0021]. Kohli demonstrates that the chemical structure of CxSAM is a chemical structure of instant claim 4 in which the instant protective group coupled to the sulfonium ion comprises a carboxyl group, and that the cytosine is protected at the 5 position (Figure 3C). Kohli further discloses that the deaminase enzyme in this method would be APOBEC3A (claim 10). Kohli teaches that APOBEC3A is A3A [0137]. The APOBEC family of enzymes can proficiently deaminate 5mC and C, which are deaminated into uracil [0137]. Kohli teaches that the polynucleotide is genomic DNA (claim 11).
Kohli discloses an ideal workflow for the method, which involves in part reacting DNA with the M.MepI MTase enzyme and CxSAM, followed by purification [0082]. Next the DNA is glucosylated using 14-βGT and UDP-glucose and after this A3A is added to the reaction mixture [0083]. Kohli elsewhere teaches that βGT generates 5-glucosylhydroxymethylcytosine (5ghmC) [0135]. It is interpreted that βGT accomplishes protection of 5hmC bases from deamination by glucosylation and the name 5-glucosylhydroxymethylcytosine indicates that the glucose functionalized the 5-hydroxymethyl of hmC. Thus, this exemplary method of Kohli teaches protecting hmC from deamination using βGT and glucose after protection with CxSAM and before deamination. Furthermore, Kohli teaches that sequence adapters may be linked to the polynucleotide prior to the reaction with CxSAM (claim 8).
Kohli also teaches that the method may be used in conjunction with third generation sequencing. After treatment of the genomic DNA, long amplicons can be generated and subjected to third-generation sequencing. The treatment includes treatment with the deaminase. Formation of the amplicons includes ligating to hairpin adapters [0153]. Thus, Kohli teaches addition of adapters after deamination of the polynucleotide.
Fig 15B shows that without protection, about 75% deamination of hmC occurs upon reaction with A3A. Deaminated hmC forms the hydroxythymine. The method of Kohli indicates that 5hmC is optionally glucosylated, indicating that the method encompasses procedures in which the 5hmC is not glucosylated and hmC is deaminated by A3A.
Kohli teaches that oxidized forms of 5mC (ox-mCs) are intermediates in active DNA methylation and have independent epigenetic functions. They include 5-formylcytosine (5fC) and 5-carboxymethylcytosine (5caC) [0134]. Although A3A can proficiently deaminate C and 5mC, it sterically discriminates against ox-mCs [0137]. The addition of a 3-4 atom substituent is sufficient to protect cytosine bases from A3A-mediated deamination. For example, 5-carboxylcytosine (5caC) is resistant to A3A deamination while C and 5mC are readily deaminated [0141]. Thus, Kohli teaches that the structural difference between cytosine and ox-mCs, such as formyl or carboxyl groups at the 5 position, result in inhibiting deamination by A3A of 5fC and 5caC.
Kohli does not expressly disclose methods comprising inhibition of the addition of X by the methyl group of 5mC (instant claim 7), inhibition of the cytidine deaminase enzyme by X (instant claim 9), modification of a target polynucleotide comprising hmC (instant claims 12 and 13), fC (instant claim 20), caC (instant claim 24), DNA (instant claim 29) or a target polynucleotide comprising adapters (instant claim 30). However, as shown above Kohli expressly teaches a method of doing so such that one of ordinary skill before the effective filing date of the claimed invention would immediately envision the experiment of the instantly claimed methods and have a reasonable expectation of success in doing so based on the disclosed teachings.
Regarding claim 19, Kohli teaches that localizing each DNA cytosine modification at base resolution is critical to understanding their function in cellular fate and function. ACE-Seq is an enzymatic method for localizing 5hmC comprising protection by glucosylation, followed by deamination. Traditional sequencing approaches can localize 5mC+5hmC or 5hmC alone, but depend upon chemical deamination with bisulfite which is destructive. DM-Seq (which is the method disclosed by Kohli) is a novel method that newly allows for specific recognition of 5mC alone [0026]. Kohli teaches that the results of DM-seq steps may be compared with the results obtained from bisulfite dependent 5mC localization or ACE-seq 5hmC localization ([0011] and claim 16). Figure 12 demonstrates that this comparison will contrast a sequence from DM-seq in which 5mC (read as T) is distinguished from C and 5hmC (read as C) versus a sequence from ACE-seq in which 5hmC (read as C) is distinguished from C and 5mC (read as T). It is interpreted that, because 5hmC is the only nucleoside that is read as C in both sequences, this comparison would provide the benefit of allowing localization of each DNA cytosine modification at base resolution.
Kohli also teaches that glucosylation of 5hmC is optional (claim 7). As discussed above, Kohli shows that the majority of unprotected 5hmC is deaminated and read as T. One of ordinary skill in the art would then expect that the sequence of protection using CxSAM followed by deamination will produce sequences in which 5mC and 5hmC (read as T) are distinguished from C (read as C). Comparison of this sequence to that obtained from DM-seq would allow one of ordinary skill in the art to distinguish C, 5mC, and 5hmC present in a polynucleotide because C s the only nucleoside that is read as C in both sequences.
Although Kohli does not expressly disclose a method comprising comparing the sequences of a target polynucleotide protected using CxSAM, protected by glucosylation, followed by deamination (DM-seq) versus that of a target polynucleotide protected only using CxSAM followed by deamination, one of ordinary skill in the art would have been motivated to do so because Kohli teaches that that localization of cytosine modifications is desirable and such a comparison would distinguish between C, mC, and hmC. One of ordinary skill in the art would have a reasonable expectation of success in doing so because Kohli provides example methods which teach comparison of polynucleotide sequences in which C, 5mC, and 5hmC can be distinguished.
Claims 21-23 and 25-28 are rejected under 35 U.S.C. 103 as being unpatentable over Kohli et al. (US 2023/0183793 Al; PTO-892), as applied to claim 1, further in view of Liutkevičiu̅te et al (JACS, 2014; IDS filed 03/14/2022).
Kohli teaches as above. Kohli further teaches that in nature MTases modify cytosine by adding a methylgroup [0007]. Variants of natural MTases are used to modify polynucleotides [0010] and are used in methods to sequence polynucleotides and obtain information about cytosine modifications [0011].
Kohli does not teach the additional steps of conversion of fC or caC into unprotected C which is deaminated to form U (instant claims 21-23 and 25-28) or that the conversion is caused by a thymine deglycosylase enzyme (instant claims 22 and 27) or a second methyl transferase enzyme (instant claim 26).
Liutkevičiu̅te discloses a method for the base selective analysis of caC in genomic DNA (abstract and paragraph bridging pages 5886-5887). In vivo, C5-MTases remove the carboxyl group of caC and regenerate unmodified C as part of the biological DNA demethylation pathway (scheme 2). Liutkevičiu̅te teaches that C5-MTases also promote the in vitro removal of the 5-carboxyl group from caC in DNA yielding unmodified C, and demonstrates that fC was inert to a similar MTase treatment under a variety of reaction conditions (paragraph bridging pages 5884-5885). Liutkeviciute also demonstrates that this treatment is specific for caC and does not affect hmC and 5mC (Figure 2b). The C5-MTase em.SssI converts caC sites into unmodified cytosine sites (paragraph bridging pages 5886-5887). Liutkevičiu̅te demonstrates that the step of decarboxylation of caC by a C5-MTase is suitable for combination with other enzymatic base modification steps, including decarboxylation after protection of hmC sites by glucosylation with BGT (figure 2a).
It would have been prima facie obvious to combine the teachings of Kohli and Liutkevičiu̅te before the effective filing date of the claimed invention by combining the steps of protecting C by CxSAM and deamination, as taught by Kohli, with the step of decarboxylation of caC by an MTase into unmodified cytosine, as taught by Liutkevičiu̅te, to arrive at the instantly claimed invention. One of ordinary skill in the art would have been motivated to decarboxylate the caC of a polynucleotide fragment before deamination in the method of Kohli because Liutkevičiu̅te teaches that decarboxylation can provide a base selective analysis of caC, and Kohli teaches that localizing each DNA cytosine modification at base resolution is critical to understanding their function in cellular fate and function. One of ordinary skill in the art would have a reasonable expectation of success because both references teach the treatment of genomic DNA for sequencing, and Liutkevičiu̅te teaches that the decarboxylation treatment may be combined with other treatment reactions, such as after protection of a nucleotide base. Furthermore, Liutkevičiu̅te teaches that the caC is converted into unmodified C. Kohli teaches that deamination of unmodified C coverts the base into U uracil and the APOBEC family of enzymes can proficiently deaminate 5mC and C, which are deaminated into uracil [0137]. Thus, after decarboxylation the caC into C, any unmodified C in the polynucleotide would be deaminated into U upon treatment with APOBEC.
Regarding instant claim 28, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify a target polynucleotide sequence by the method of Kohli, in which the polynucleotide C is protected using CxSAMs and then deaminated by APOBEC, and compare it to a sequence obtained by protecting the C in the target polynucleotide using CxSAMs and decarboxylating caC followed by deamination, in order to determine which nucleotides in the polynucleotide are caC. For example, APOBEC does not deaminate 5fC or 5caC. Without the decarboxylation step, both fC and caC are not deaminated and are read as C, and so these bases are not distinguishable from each other or from unmodified C. With the decarboxylation step caC is deaminated to U and distinguished from fC and unmodified C. This comparison localizes which bases are caC on a target polynucleotide, and one of ordinary skill in the art would have been motivated to do so because Kohli teaches that localization of each DNA cytosine modification at base resolution is critical to understanding their function in cellular fate and function.
Regarding instant claims 21-23 and 27, Liutkevičiu̅te does not expressly disclose treating a polynucleotide with a thymine deglycosylase enzyme. However, Liutkevičiu̅te discloses that in vivo the replacement of fC and caC with unmodified cytosine occurs by base excision repair (BER) of fC and caC by thymine DNA glycosylase (TDG) (page 5884, paragraph 1 and scheme 2). Thus, Liutkevičiu̅te teaches that the replacement of fC and caC into C occurs by BER of fC and caC by TDG. The TDG of Liutkevičiu̅te is interpreted as the thymine deglycosylase enzyme of instant claims 22 and 27 which is defined by the instant specification as an enzyme that excises the base from fC or caC and replaces the excised base with C, a reaction that may be referred to as base excision repair (BER) (instant specification [0077]). Thus, it is known in the art that fC and caC are transformed into C by treatment with the thymine deglycosylase enzyme TDG.
Because Kohli teaches that localizing each DNA cytosine modification at base resolution is critical to understanding their function in cellular fate and function, one of ordinary skill in the art would have been motivated to treat a target polynucleotide fragment with TDG, which is taught by Liutkevičiu̅te as converting fC and caC into unmodified cytosine, before deamination in the method of Kohli in order to distinguish between unmodified C and the oxidized cytosines fC and caC. For example, APOBEC in the method of Kohli does not deaminate 5fC or 5caC. Without the TDG conversion step, fC and caC are not deaminated and would be read as C, and as a result these bases are not distinguishable from the original unmodified C. With the TDG conversion step, fC and caC are deaminated to U, are read as T, and are thus distinguished from unmodified C, which is read as C. Thus addition of a TDG conversion step would allow determination of which DNA cytosines are unmodified without confounding the oxidized cytosines fC and caC with C. One of ordinary skill in the art would have a reasonable expectation of success because both references demonstrate that it is known in the art to apply enzymes involved in in vivo cellular modification of DNA for the modification of polynucleotides to be used in DNA sequencing, and Liutkevičiu̅te teaches TDG is a known enzyme in the biological pathway to convert fC and caC into unmodified cytosine.
Conclusion
No claims are allowed.
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/S.G.S./Examiner, Art Unit 1693
/SCARLETT Y GOON/Supervisory Patent Examiner
Art Unit 1693