Office Action 1: Non-Final Rejection (2024-06-20)
Rejection types: 112 - Written Description/Enablement, Double Patenting

CLAIMS:
AMENDMENTS TO THE CLAIMS

This listing of the claims will replace all prior versions, and listings, of claims in the application:

Application No.: 18/588,987 Page 2

1.-30. (CANCELLED)229

31\. (NEW) A method comprising contacting a nucleic acid molecule with an isolated enzyme in an amount effective to convert 5-methylcytosine of said nucleic acid molecule to an oxidized derivative thereof.

32\. (NEW) The method of claim 31, wherein said isolated enzyme is present in an amount effective to convert substantially all of said 5-methylcytosine of said nucleic acid molecule to said oxidized derivative thereof.

33\. (NEW) The method of claim 31, wherein said isolated enzyme comprises an isolated dioxygenase.

34\. (NEW) The method of claim 33, wherein said isolated dioxygenase comprises a recombinantly expressed dioxygenase.

35\. (NEW) The method of claim 31, wherein said isolated enzyme comprises a TET1 polypeptide, a TET2 polypeptide, a CXXC4 polypeptide, a catalytically active fragment thereof, a variant thereof, or any combination thereof.

36\. (NEW) The method of claim 35, wherein said TETl polypeptide, TET2 polypeptide, or CXXC4 polypeptide comprises a polypeptide having at least 90% sequence identity to any one of SEQ ID NOs:

27, 28, or 29.

37\. (NEW) The method of claim 36, wherein said TETl polypeptide, said TET2 polypeptide, or said CXXC4 polypeptide further comprises SEQ ID NO: 1.

38\. (NEW) The method of claim 31, wherein said isolated enzyme comprises a TET2 polypeptide or a catalytically-active fragment thereof.

39\. (NEW) The method of claim 38, wherein said TET2 polypeptide or said catalytically active fragment of said TET2 polypeptide comprises a polypeptide having at least 90% sequence identity to SEQ ID NO: 28, or a catalytically active fragment of SEQ ID NO: 28.

40\. (NEW) The method of claim 39, wherein said polypeptide having at least 90% sequence identity to SEQ ID NO: 28 or said catalytically active fragment of SEQ ID NO: 28 comprises SEQ ID NO: 1.

41\. (NEW) The method of claim 40, wherein said polypeptide having at least 90% sequence identity to SEQ ID NO: 28 comprises a conservatively-substituted variant of SEQ ID NO: 28 or a conservatively- substituted catalytically active fragment of SEQ ID NO: 28.

42\. (NEW) The method of claim 41, wherein said TET2 polypeptide comprises a polypeptide substantially identical to SEQ ID NO: 28.

43\. (NEW) The method of claim 31, wherein said nucleic acid molecule comprises isolated eukaryotic genomic DNA.

44\. (NEW) The method of claim 43, wherein said isolated eukaryotic genomic DNA is from a somatic cell.

45\. (NEW) The method of claim 31, further comprising adding a sugar residue to said oxidized derivative of said 5-methylcytosine in said nucleic acid molecule.

46\. (NEW) The method of claim 45, wherein adding said sugar residue to said oxidized derivative of said 5-methylcytosine in said nucleic acid molecule comprises adding a glucose molecule or a glucose- derivative donor substrate to said oxidized derivative of said 5-methylcytosine in said nucleic acid molecule.

47\. (NEW) The method of claim 46, wherein said adding a glucose molecule or a glucose- derivative donor substrate to said 5-hydroxymethylcytosine residue in said product nucleic acid comprises contacting said product nucleic acid with an a-glucosyltransferase (AGT) polypeptide or a variant thereof.

48\. (NEW) The method of claim 46, wherein said adding said glucose molecule or said glucose- derivative donor substrate to said 5-hydroxymethylcytosine residue in said product nucleic acid comprises contacting said product nucleic acid with said 3-glucosyltransferase (BGT) polypeptide or a conservatively-substituted variant of said BGT polypeptide.

49\. (NEW) The method of claim 48, wherein said BGT polypeptide comprises the polypeptide sequence of RCSB Accession No. 1J39.

Application No.: 18/588,987 Preliminary Amendment filed May 9, 2024 Page 4

50\. (NEW) The method of claim 49, wherein said BGT polypeptide comprises residues 100-103, 231, and 311 relative to RCSB Accession No. 1J39.

51\. (NEW) The method of claim 47, wherein said AGT polypeptide comprises the polypeptide sequence of RCSB Accession No. 1YA6.

52\. (NEW) The method of claim 51, wherein said AGT polypeptide comprises residues 114-116 of AGT relative to RCSB Accession No. 1YA6.

OFFICE ACTION:
DETAILED ACTION
Notice of Pre-AIA  or AIA  Status
The present application is being examined under the pre-AIA  first to invent provisions.
Priority
This application is a continuation application (CON) of U.S. Application Serial Number 17/675,502, filed 2/18/2022; which is a CON of U.S. Application Serial Number 17/350,181, filed 6/17/2021; which is a CON of U.S. Application Serial Number 16/380,846, filed 4/10/2019; which is a CON of U.S. Application Serial Number 15/440,815, filed 2/23/2017; which is a CON of U.S. Application Serial Number 15/341,344, filed 11/02/2016; which is a CON of U.S. Application Serial Number 15/193,796, filed 6/27/2016; which is a CON of U.S. Application Serial Number 13/795,739, filed 3/12/2013; which is a CON of U.S. Application Serial Number 13/120,861, filed 6/07/2011.  13/120,861 is a 371 of PCT/US2009/058562, filed 9/28/2009.  This application claims benefit to U.S. Provisional Application Serial Numbers 61/121,844, filed 12/11/2008, 61/100,995, filed 9/29/2008 and 61/100,503, filed 9/26/2008.  Claims 31-52 are pending and have been examined on the merits.
Information Disclosure Statement
The information disclosure statements submitted on 2/29/2024 have been considered by the examiner.  NPL cite 28 of the 6-page IDS has been lined through because the document has not been received.
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.
The term “substantially all” in claim 32 is a relative term which renders the claim indefinite. The term “substantially all” 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. Appropriate correction is required.
The term “substantially identical” in claim 42 is a relative term which renders the claim indefinite. The term “substantially identical” 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. Appropriate correction is required.
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 31-34 and 43-52 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 claims contain subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA  35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Independent claim 31 encompasses contacting a nucleic acid with an effective amount of any isolated enzyme that converts 5-methylcytosine to an oxidized derivative thereof which would include not only the TET family of enzymes recited by Applicants, but also all enzymes not yet discovered which can convert 5-methylcytosine to 5-hydroxymethylcytosine or any oxidized derivative of 5-methylcytosine.  Applicants are not necessarily in possession of every enzyme which can convert 5-methylcytosine to an oxidized derivative of 5-methylcytosine; hence, the scope of the broadly drawn independent claim is not commensurate with the subject matter for which the original disclosure conveys possession, i.e., the broadly drawn claim 31 encompasses subject matter for which the disclosure does not provide support, namely, the method wherein the enzyme that converts 5-methylcytosine to an oxidized derivative of 5-methylcytosine is an enzyme other than the TET family enzymes disclosed by Applicants.
Dependent claim 33 restricts the enzyme to a dioxygenase and dependent claim 34 restricts the enzyme to a recombinant dioxygenase, but the argument is the same as for independent claim 31 - the scope of the claims are not commensurate with the subject matter for which the original disclosure conveys possession, i.e., claims 33-34 encompass subject matter for which the disclosure does not provide support, namely, the method wherein the enzyme that converts 5-methylcytosine to an oxidized derivative of 5-methylcytosine is an enzyme other than the TET family enzymes disclosed by Applicants.
The law is clear that the first paragraph of 35 USC § 112 requires more than the disclosure of a theoretical possibility.  As described in Genentech Inc. v. Novo Nordisk A/S (CAFC) 42 USPQ2d 1001, 1005: “Patent protection is granted in return for an enabling disclosure of an invention, not for vague intimations of general ideas that may or may not be workable. See Brenner v. Manson, 383 U.S. 519, 536, 86 S.Ct. 1033, 1042-43, 16 L.Ed.2d 69, 148 USPQ 689, 696 (1966) (stating, in context of the utility requirement, that 'a patent is not a hunting license. It is not a reward for the search, but compensation for its successful conclusion.') Tossing out the mere germ of an idea does not constitute enabling disclosure. While every aspect of a generic claim certainly need not have been carried out by an inventor, or exemplified in the specification, reasonable detail must be provided in order to enable members of the public to understand and carry out the invention.”
See also Rasmusson v. SmithKline Beecham Corp., 75 USPQ2d 1297 (Fed. Cir. 2005) (In response to Rasmusson’s argument that the enablement requirement of section 112 does not mandate a showing of utility or, if it does, it mandates only showing that it is not implausible” that the invention will work for its intended purpose, the Court states, “As we have explained, we have required a greater measure of proof, and for good reason. If mere plausibility were the test for enablement under section 112, applicants could obtain patent rights to inventions consisting of little more than respectable guesses as to the likelihood of their success. When one of the guesses later proved true, the inventor would be rewarded the spoils instead of the party who demonstrated that the method actually worked. That scenario is not consistent with the statutory requirement that the inventor enable an invention rather than merely proposing an unproved hypothesis).  In view of the broad scope of claims 31-34 and 43-52, it must be concluded that the inventors are not in possession of the scope of the invention as claimed, rather, the possession of the subject matter disclosed in the original filing is limited to the method wherein the enzyme that converts 5-methylcytosine to an oxidized derivative of 5-methylcytosine is a member of the TET family of enzymes; hence, claims 31-34 and 43-52 are rejected for failing to comply with the written description requirement.  Claims 35-42 are not rejected because the scope of the claims is commensurate with the subject matter for which Applicants show possession in the original disclosure.
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.
Claims 31-52 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 31-38, 41-42, 45-46 and 49-58 of copending Application No. 18/588930 (reference application; herein “’930”).
Although the claims at issue are not identical, they are not patentably distinct from each other because the method for detecting a methylated cytosine residue in a nucleic acid set forth in claims 31-38 of ‘930 comprises contacting a nucleic acid molecule with an isolated enzyme, which is a dioxygenase, which is a TET1, TET2 or CXXC4 polypeptide which is identical to SEQ ID NOs: 27, 28 or 29 which comprises SEQ ID NO: 1 in an amount effective to convert 5-methylcytosine of said nucleic acid molecule to an oxidized derivative thereof; therefore, instant claims 31, 33 and 35-42 are prima facie obvious over claims 31-38 of ‘930.
Regarding instant claim 32, it would be prima facie obvious to convert all of said 5-methylcytosine of said nucleic acid molecule to said oxidized derivative thereof in view of claims 31-38 and 41 of ‘930 because ‘930 is drawn to detecting methylated cytosine residues in a nucleic acid, claim 41 of ‘930 is drawn to sequencing the nucleic acid and it would be prima facie obvious to detect all of the methylated cytosine residues because sequencing the nucleic acid would require detecting all of the methylated cytosine residues; therefore, instant claim 32 is prima facie obvious.
Regarding instant claim 34, it would be prima facie obvious for the dioxygenase to be recombinantly expressed in view of claims 31-38 of ‘930 because the sequences of TET1, TET2 and CXXC4 are known, i.e., SEQ ID NOs: 27-29, and recombinantly expressed enzymes would give more control over the production, amount, cost and purity of the enzyme; therefore, instant claim 34 is prima facie obvious over claims 31-38 of ‘930.
Regarding instant claims 43-44, claims 31-38 and 45-46 of ‘930 teach oxidizing methylated cytosine residues wherein the nucleic acid is genomic DNA and somatic cells comprise genomic DNA; therefore, instant claims 43-44 are prima facie obvious over claims 31-38 and 45-46 of ‘930.
Regarding instant claims 45-52, claims 49-58 of ‘930 recite further derivatizing the nucleic acids which have had 5-methylcytosine residues oxidized to a derivative residue by contacting the nucleic acids with glycosyltransferases which meet the limitations of instant claims 45-52; therefore, instant claims 45-52 are prima facie obvious over claims 31-38 and 49-48 of ‘930.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 31-52 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 5-6 and 16-29 of copending Application No. 18/588956 (reference application; herein “’956”).
Although the claims at issue are not identical, they are not patentably distinct from each other because claims 5-6 of ‘956 recite a method wherein nucleic acid molecules, inside adult somatic cells, are contacted with an isolated enzyme, comprising TET1 or TET2, in an amount effective to convert 5-methylcytosine of said nucleic acid molecule to an oxidized derivative thereof; therefore, instant claims 31, 33 and 35-44 are prima facie obvious over claims 5-6 of ‘956.
Regarding instant claim 32, it would be prima facie obvious to convert all of said 5-methylcytosine of said nucleic acid molecule to said oxidized derivative thereof in view of claims 5-6 of ‘956 because ‘956 is drawn to improving the efficiency of producing induced pluripotent stem cells by delivering an effective 5-methylcytosine to 5-hydroxymethylcytosine converting amount of at least one catalytically active TET family enzyme; hence, it would be prima facie obvious to convert all of the methylated cytosine residues to maximize improving the efficiency of producing induced pluripotent stem cells; therefore, instant claim 32 is prima facie obvious.
Regarding instant claim 34, it would be prima facie obvious for the dioxygenase to be recombinantly expressed in view of claims 5-6 of ‘956 because the sequences of TET1, TET2 and CXXC4 are known and recombinantly expressed enzymes would give more control over the production, amount, cost and purity of the enzyme; therefore, instant claim 34 is prima facie obvious over claims 5-6 of ‘956.
Regarding instant claims 45-52, claims 16-29 of ‘956 recite a kit for the detection and purification of methylcytosine and 5-hydroxymethylcytosine, wherein the kit comprises alpha-glucosyltransferases and beta-glucosyltransferases meeting the limitations of instant claims 47-52 and suitable for practicing the methods of instant claims 45-52 as seen at [0164] of the disclosure of ‘956; therefore, instant claims 45-52 are prima facie obvious over claims 5-6 and 16-29 of ‘956.
NOTE: It is proper to consult the disclosure for defining the claim (MPEP 1504.06).  Claims 18 and 19 of ‘956 recite alpha-glucosyltransferases encoded by T2, T4, and T6 bacteriophages and beta-glucosyltransferases encoded by T4 bacteriophages.  Paragraph [0164] of the disclosure of ‘956 defines these glucosyltransferases.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 31-46 and 48-50 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-8 of U.S. Patent No. 9816986 (herein “’986”).
Although the claims at issue are not identical, they are not patentably distinct from each other because the method for detecting a methylated cytosine residue in a nucleic acid set forth in claims 1 and 7-8 of ‘986 comprises contacting a nucleic acid molecule with a TET enzyme, i.e., which is a dioxygenase, which can a TET1, TET2 or CXXC4 polypeptide which can be identical to SEQ ID NOs: 27, 28 or 29 which comprises SEQ ID NO: 1 (see [0093-94] of disclosure) in an amount effective to convert 5-methylcytosine of said nucleic acid molecule to an oxidized derivative thereof; therefore, instant claims 31, 33 and 35-42 are prima facie obvious over claims 1 and 7-8 of ‘986.
Regarding instant claim 32, it would be prima facie obvious to convert all of said 5-methylcytosine of said nucleic acid molecule to said oxidized derivative thereof in view of claims 1 and 7-8 of ‘986 because claims 1 and 7-8 of ‘986 are drawn to detecting a methylated cytosine residue in a nucleic acid comprising contacting the nucleic acid with at least one catalytically active TET family enzyme; hence, it would be prima facie obvious to convert all of the methylated cytosine residues to maximize detection of methylated cytosine residues; therefore, instant claim 32 is prima facie obvious.
Regarding instant claim 34, it would be prima facie obvious for the dioxygenase to be recombinantly expressed in view of claims 1 and 7-8 of ‘986 because the sequences of TET1, TET2 and CXXC4 are known and recombinantly expressed enzymes would give more control over the production, amount, cost and purity of the enzyme; therefore, instant claim 34 is prima facie obvious over claims 1 and 7-8 of ‘986.
Regarding instant claims 43-44, claims 1, 5 and 7-8 of ‘986 are drawn to detecting methylated cytosine residues in a mammalian nucleic acid comprising contacting the nucleic acid with at least one catalytically active TET family enzyme wherein the mammalian nucleic acid comprises mammalian DNA and somatic cells comprise mammalian DNA; therefore, instant claims 43-44 are prima facie obvious over claims 1, 5 and 7-8 of ‘986.
Regarding instant claims 45-46, 48-50, claims 1, 3-4 and 7-8 of ‘986 recite a method for detecting methylated cytosine residues in a mammalian nucleic acid comprising contacting the nucleic acid with at least one catalytically active TET family enzyme and further comprising adding a glucose molecule to the produced 5-hydroxymethylcytosine with beta-glucosyltransferases meeting the limitations of instant claims 48-50 as seen at [0164] of the disclosure of ‘956; therefore, instant claims 45-46 and 48-50 are prima facie obvious over claims 1, 3-4 and 7-8 of ‘986.
NOTE: It is proper to consult the disclosure for defining the claim (MPEP 1504.06).  Claim 1 of ‘986 recites a TET enzyme or a catalytically active fragment thereof comprising SEQ ID NO: 1.  Paragraphs [0093-94] of the disclosure of ‘986 defines these TET enzymes.  Claim 1 recites a beta-glucosyltransferase and claim 3 of ‘986 discloses the beta-glucosyltransferase comprises an Asp-Ile-Arg-Leu sequence.  Paragraph [0164] of the disclosure of ‘986 defines these glucosyltransferases.
Claims 31-42 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 6-8 and 10-15 of U.S. Patent No. 10323269 (herein “’269).
Although the claims at issue are not identical, they are not patentably distinct from each other because claims 1, 6-8 and 10-15 of ‘269 are drawn to delivering an effective 5-methylcytosine to 5-hydroxymethylcytosine converting amount of an enzyme that oxidizes at least one methylated DNA base in a cell wherein the enzyme is TET1, TET2, TET3 or CXXC4 which constitutes contacting a nucleic acid molecule, in the cell, with an isolated enzyme, which is a dioxygenase, which is a TET1, TET2 or CXXC4 polypeptide which is identical to SEQ ID NOs: 27, 28 or 29 which comprises SEQ ID NO: 1 in an amount effective to convert 5-methylcytosine of said nucleic acid molecule to an oxidized derivative thereof; therefore, instant claims 31, 33 and 35-42 are prima facie obvious over claims 1, 6-8 and 10-15 of ‘269.
Regarding instant claim 32, it would be prima facie obvious to convert all of said 5-methylcytosine of said nucleic acid molecule to said oxidized derivative thereof in view of claims 1, 6-8 and 10-15 of ‘269 because claim 8 of ‘269 teaches the conversion increases expression of FOXP3 in the human T cell and it would be prima facie obvious to maximally increase expression of FOXP3 in the human T cell by converting all of the 5-methylcytosines in the nucleic acid molecule; therefore, instant claim 32 is prima facie obvious.
Regarding instant claim 34, it would be prima facie obvious for the dioxygenase to be recombinantly expressed in view of claims 1, 6-8 and 10-15 of ‘269 because the sequences of TET1, TET2 and CXXC4 are known, i.e., SEQ ID NOs: 27-29, and recombinantly expressed enzymes would give more control over the production, amount, cost and purity of the enzyme; therefore, instant claim 34 is prima facie obvious over claims 1, 6-8 and 10-15 of ‘269.
Claims 31-52 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-13 of U.S. Patent No. 10443091 (herein “’091”).
Although the claims at issue are not identical, they are not patentably distinct from each other because the method for detecting a 5-hydroxymethylcytosine residue in a nucleic acid set forth in claims 1-3, 5 and 7-13 of ‘091 comprises contacting a nucleic acid molecule with a TET enzyme, i.e., which is a dioxygenase, which can a TET1, TET2 or CXXC4 polypeptide which can be identical to SEQ ID NOs: 27, 28 or 29 which comprises SEQ ID NO: 1 (see [0093-94] of disclosure) in an amount effective to convert 5-methylcytosine of said nucleic acid molecule to an oxidized derivative thereof, i.e. 5-hydroxymethylcytosine; therefore, instant claims 31, 33 and 35-42 are prima facie obvious over claims 1-3, 5 and 7-13 of ‘091.
Regarding instant claim 32, it would be prima facie obvious to convert all of said 5-methylcytosine of said nucleic acid molecule to said oxidized derivative thereof in view of claims 1-3, 5 and 7-13 of ‘091 because claims 1-3, 5 and 7-13 of ‘091 are drawn to detecting 5-hydroxymethylcytosine residues in a nucleic acid comprising contacting the nucleic acid with at least one catalytically active TET family enzyme; hence, it would be prima facie obvious to convert all of the methylated cytosine residues to 5-hydroxymethylcytosine residues to maximize detection; therefore, instant claim 32 is prima facie obvious.
Regarding instant claim 34, it would be prima facie obvious for the dioxygenase to be recombinantly expressed in view of claims 1-3, 5 and 7-13 of ‘091 because the sequences of TET1, TET2 and CXXC4 are known and recombinantly expressed enzymes would give more control over the production, amount, cost and purity of the enzyme; therefore, instant claim 34 is prima facie obvious over claims 1-3, 5 and 7-13 of ‘091.
Regarding instant claims 43-44, claims 1-3, 5 and 7-13 of ‘091 are drawn to detecting 5-hydroxymethylcytosine residues in a nucleic acid comprising contacting the nucleic acid with at least one catalytically active TET family enzyme wherein the nucleic acid is contacted in a cell; therefore, instant claims 43-44 are prima facie obvious over claims 1-3, 5 and 7-13 of ‘091.
Regarding instant claims 45-52, claims 1-3, 5 and 7-13 of ‘091 further recites utilizing a glucose donor substrate or a glucose-derivative donor substrate that traps covalent enzyme-DNA intermediates to detect 5-hydroxymethylcytosine residues, wherein the enzyme can comprise an alpha-glucosyltransferase or a beta-glucosyltransferase which meets the limitations of instant claims 47-52 as seen at [0164] of the disclosure of ‘091; therefore, instant claims 45-52 are prima facie obvious over claims 1-3, 5 and 7-13 of ‘091.
NOTE: It is proper to consult the disclosure for defining the claim (MPEP 1504.06).  Claim 5 of ‘091 recites a catalytically active TET family enzyme.  Paragraphs [0093-94] of the disclosure of ‘091 defines these TET enzymes.  Claim 1 recites alpha-glucosyltransferase and beta-glucosyltransferase.  Paragraph [0164] of the disclosure of ‘091 defines these glucosyltransferases.
Claims 31-42 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 10533213 (herein “’213”).
Although the claims at issue are not identical, they are not patentably distinct from each other because the method for detecting a 5-hydroxymethylcytosine residue in a nucleic acid set forth in claim 1 of ‘213 comprises contacting a nucleic acid molecule with a TET1, TET2, TET3 or CXXC4 enzyme which can be identical to SEQ ID NOs: 27, 28 or 29 and which comprises SEQ ID NO: 1 (see [0093-94] of disclosure) in an amount effective to oxidize 5-methylcytosine of said nucleic acid molecule to an oxidized derivative thereof; therefore, instant claims 31, 33 and 35-42 are prima facie obvious over claim 1 of ‘213.
Regarding instant claim 32, it would be prima facie obvious to convert all of said 5-methylcytosine of said nucleic acid molecule to said oxidized derivative thereof in view of claim 1 of ‘213 because claim 1 of ‘213 is drawn to detecting 5-methylcytosine residues in a nucleic acid comprising contacting the nucleic acid with a TET1, TET2, TET3 or CXXC4 enzyme; hence, it would be prima facie obvious to convert all of the 5-methylcytosine residues to 5-hydroxymethylcytosine residues to maximize detection; therefore, instant claim 32 is prima facie obvious.
Regarding instant claim 34, it would be prima facie obvious for the dioxygenase to be recombinantly expressed in view of claim 1 of ‘213 because the sequences of TET1, TET2 and CXXC4 are known and recombinantly expressed enzymes would give more control over the production, amount, cost and purity of the enzyme; therefore, instant claim 34 is prima facie obvious over claim 1 of ‘213.
NOTE: It is proper to consult the disclosure for defining the claim (MPEP 1504.06).  Claim 1 of ‘213 recites a catalytically active TET family enzyme.  Paragraphs [0093-94] of the disclosure of ‘213 defines these TET enzymes.
Claims 31-42 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 13 and 16-17 of U.S. Patent No. 10612076 (herein “‘076”).
Although the claims at issue are not identical, they are not patentably distinct from each other because claims 1, 13 and 16-17 of ‘076 recite a method of regulating the methylation status of cytosine residues in an adult cell comprising contacting the cell with an effective amount of a 5-methylcytosine to 5-hydroxymethylcytosine converting enzyme wherein the enzyme can comprise TET1, TET2, TET3 or CXXC4, which can be identical to SEQ ID NOs: 27, 28 or 29 and which comprises SEQ ID NO: 1 (see [0093-94] of disclosure); therefore, instant claims 31, 33 and 35-42 are prima facie obvious over claims 1, 13 and 16-17 of ‘076.
Regarding instant claim 32, it would be prima facie obvious to convert all of said 5-methylcytosine of said nucleic acid molecule inside the cell, to said oxidized derivative thereof in view of claims 1, 13 and 16-17 of ‘076 because it would maximally regulate the methylation status of the cell; therefore, instant claim 32 is prima facie obvious.
Regarding instant claim 34, it would be prima facie obvious for the dioxygenase to be recombinantly expressed in view of claims 1, 13 and 16-17 of ‘076 because the sequences of TET1, TET2 and CXXC4 are known and recombinantly expressed enzymes would give more control over the production, amount, cost and purity of the enzyme; therefore, instant claim 34 is prima facie obvious over claims 1, 13 and 16-17 of ‘076.
NOTE: It is proper to consult the disclosure for defining the claim (MPEP 1504.06).  Claim 16 of ‘076 recites TET1, TET2, TET3 and CXXC4.  Paragraphs [0093-94] of the disclosure of ‘076 defines these TET enzymes.
Claims 31-52 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-3, 6-10, 13-15 and 19 of U.S. Patent No. 10731204 (herein “’204”).
Although the claims at issue are not identical, they are not patentably distinct from each other because claims 1-3, 6-10, 13-15 and 19 of ‘204 are drawn to converting methylated cytosine residues of an isolated nucleotide sequence to hydroxymethylcytosine residues with TET1, TET2, TET3 or CXXC4 enzymes, which can be identical to SEQ ID NOs: 27, 28 or 29 and which comprises SEQ ID NO: 1 (see [0093-94] of disclosure); therefore, instant claims 31, 33 and 35-42 are prima facie obvious over claims 1-3, 6-10, 13-15 and 19 of ‘204.
Regarding instant claim 32, it would be prima facie obvious to convert all of said methylcytosine of said nucleic acid to said oxidized derivative thereof, i.e., hydroxymethylcytosine, in view of claim 3 of ‘204 which is drawn to detecting the methylation status of the nucleotide sequence, because it would most effectively provide accurate detection if all the methylcytosine was converted to hydroxymethylcytosine; therefore, instant claim 32 is prima facie obvious.
Regarding instant claim 34, it would be prima facie obvious for the dioxygenase to be recombinantly expressed in view of claims 1-3, 6-10, 13-15 and 19 of ‘204 because the sequences of TET1, TET2, TET3 and CXXC4 are known and recombinantly expressed enzymes would give more control over the production, amount, cost and purity of the enzyme; therefore, instant claim 34 is prima facie obvious over claims 1-3, 6-10, 13-15 and 19 of ‘204.
Regarding instant claims 45-52, claims 1-3, 6-10, 13-15 and 19 of ‘204 further recite utilizing a glucose donor substrate or a glucose-derivative donor substrate to label hydroxymethylcytosine residues using alpha-glucosyltransferase or a beta-glucosyltransferase which meets the limitations of instant claims 47-52 as seen at [0164] of the disclosure of ‘204; therefore, instant claims 45-52 are prima facie obvious over claims 1-3, 6-10, 13-15 and 19 of ‘204.
NOTE: It is proper to consult the disclosure for defining the claim (MPEP 1504.06).  Claims 1-2, 13-15 and 19 of ‘204 recite catalytically active TET family enzymes.  Paragraphs [0093-94] of the disclosure of ‘204 defines these TET enzymes.  Claims 6 and 8-9 of ‘204 recite alpha-glucosyltransferases and beta-glucosyltransferases.  Paragraph [0164] of the disclosure of ‘204 defines these glucosyltransferases.
Claims 31-52 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-3, 8-11, 13 and 16 of U.S. Patent No. 10767216 (herein “’216”).
Although the claims at issue are not identical, they are not patentably distinct from each other because the method for detecting a 5-hydroxymethylcytosine residue in a nucleic acid set forth in claims 1-3, 8-11, 13 and 16 of ’216 comprises contacting a nucleic acid molecule with a TETI, TET2, TET3 or CXXC4 enzyme, i.e., which is a dioxygenase, which can a TET1, TET2 or CXXC4 polypeptide which can be identical to SEQ ID NOs: 27, 28 or 29 which comprises SEQ ID NO: 1 (see [0093-94] of disclosure) in an amount effective to convert 5-methylcytosine of the nucleic acid sequence to an oxidized derivative thereof, i.e. 5-hydroxymethylcytosine; therefore, instant claims 31, 33 and 35-42 are prima facie obvious over claims 1-3, 8-11, 13 and 16 of ’216.
Regarding instant claim 32, it would be prima facie obvious to convert all of said 5-methylcytosine of said nucleic acid molecule to said oxidized derivative thereof in view of claims 1-3, 8-11, 13 and 16 of ’216 because claims 1-3, 8-11, 13 and 16 of ’216 are drawn to detecting 5-hydroxymethylcytosine residues in a nucleic acid comprising contacting the nucleic acid with at least one catalytically active TET family enzyme; hence, it would be prima facie obvious to convert all of the methylated cytosine residues to 5-hydroxymethylcytosine residues to maximize detection; therefore, instant claim 32 is prima facie obvious.
Regarding instant claim 34, it would be prima facie obvious for the dioxygenase to be recombinantly expressed in view of claims 1-3, 8-11, 13 and 16 of ’216 because the sequences of TET1, TET2 and CXXC4 are known and recombinantly expressed enzymes would give more control over the production, amount, cost and purity of the enzyme; therefore, instant claim 34 is prima facie obvious over claims 1-3, 8-11, 13 and 16 of ’216.
Regarding instant claims 43-44, claims 1-3, 8-11, 13 and 16 of ’216 are drawn to detecting 5-hydroxymethylcytosine residues in a nucleic acid comprising contacting the nucleic acid with at least one catalytically active TET family enzyme wherein the nucleic acid can be mammalian genomic DNA; therefore, instant claims 43-44 are prima facie obvious over claims 1-3, 8-11, 13 and 16 of ’216.
Regarding instant claims 45-52, claims 1-3, 8-11, 13 and 16 of ’216 further recite glycosylating 5-hydroxymethylcytosine residues with an alpha-glucosyltransferase or a beta-glucosyltransferase which meets the limitations of instant claims 47-52 as seen at [0164] of the disclosure of ‘216; therefore, instant claims 45-52 are prima facie obvious over claims 1-3, 8-11, 13 and 16 of ’216.
NOTE: It is proper to consult the disclosure for defining the claim (MPEP 1504.06).  Claim 11 of ‘216 recites a catalytically active TETI, TET2, TET3 and CXXC4 enzymes.  Paragraphs [0093-94] of the disclosure of ‘216 defines these TET enzymes.  Claim 16 of ‘216 recites alpha-glucosyltransferase and beta-glucosyltransferase.  Paragraph [0164] of the disclosure of ‘216 defines these glucosyltransferases.
Claims 31-44 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-9 of U.S. Patent No. 11072818 (herein “’818”).
Although the claims at issue are not identical, they are not patentably distinct from each other because claims 1-9 of ‘818 are drawn to contacting a nucleic acid sequence with an enzyme which oxidizes 5-methylcytosine to 5-hydroxymethylcytosine wherein the enzyme is a TET family enzyme, i.e., which is a dioxygenase, which can be a TET1, TET2 or CXXC4 polypeptide which can be identical to SEQ ID NOs: 27, 28 or 29 which comprises SEQ ID NO: 1 (see [0093-94] of disclosure); therefore, instant claims 31, 33 and 35-42 are prima facie obvious over claims 1-9 of ‘818.
Regarding instant claim 32, it would be prima facie obvious to convert all of said 5-methylcytosine of said nucleic acid molecule to said oxidized derivative thereof in view of claims 1-9 of ‘818; therefore, instant claim 32 is prima facie obvious.
Regarding instant claim 34, it would be prima facie obvious for the dioxygenase to be recombinantly expressed in view of claims 1-9 of ‘818 because the sequences of TET1, TET2 and CXXC4 are known and recombinantly expressed enzymes would give more control over the production, amount, cost and purity of the enzyme; therefore, instant claim 34 is prima facie obvious over claims 1-9 of ‘818.
Regarding instant claims 43-44, claim 9 of ‘818 is drawn to wherein the nucleic acid sequence is from a somatic cell; therefore, instant claims 43-44 are prima facie obvious over claims 1-9 of ‘818.
NOTE: It is proper to consult the disclosure for defining the claim (MPEP 1504.06).  Claim 3 of ‘818 recites a catalytically active TET family enzyme.  Paragraphs [0093-94] of the disclosure of ‘818 defines these TET enzymes.
Claims 31-42 and 45-52 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-13 of U.S. Patent No. 12018320 (issues 6/25/2024 from application 17/675502; herein “’320”).
Although the claims at issue are not identical, they are not patentably distinct from each other because claims 1-13 of ‘320 are drawn to a method comprising detecting epigenetically modified residues in a nucleic acid comprising contacting a nucleic acid molecule with a TET1, TET2, TET3 or CXXC4 enzyme, i.e., which is a dioxygenase, which can a TET1, TET2 or CXXC4 polypeptide which can be identical to SEQ ID NOs: 27, 28 or 29 which comprises SEQ ID NO: 1 (see [0093-94] of disclosure) in an amount effective to convert 5-methylcytosine of the nucleic acid sequence to an oxidized derivative thereof, i.e. 5-hydroxymethylcytosine; therefore, instant claims 31, 33 and 35-42 are prima facie obvious over claims 1-13 of ‘320.
Regarding instant claim 32, it would be prima facie obvious to convert all of said 5-methylcytosine of said nucleic acid molecule to said oxidized derivative thereof in view of claims 1-13 of ‘320 because claims 1-13 of ‘320 are drawn to detecting epigenetic modifications in a nucleic acid comprising contacting the nucleic acid with at least one catalytically active TET family enzyme; hence, it would be prima facie obvious to convert all of the methylated cytosine residues to 5-hydroxymethylcytosine residues to maximize detection; therefore, instant claim 32 is prima facie obvious.
Regarding instant claim 34, it would be prima facie obvious for the dioxygenase to be recombinantly expressed in view of claims 1-13 of ‘320 because the sequences of TET1, TET2 and CXXC4 are known and recombinantly expressed enzymes would give more control over the production, amount, cost and purity of the enzyme; therefore, instant claim 34 is prima facie obvious over claims 1-13 of ‘320.
Regarding instant claims 45-52, claims 1-13 of ‘320 further recite glycosylating the oxidized epigenetic modification, i.e., 5-hydroxymethylcytosine residues, with an alpha-glucosyltransferase or a beta-glucosyltransferase which meets the limitations of instant claims 47-52 as seen at [0164] of the disclosure of ‘320; therefore, instant claims 45-52 are prima facie obvious over claims 1-13 of ‘320.
NOTE: It is proper to consult the disclosure for defining the claim (MPEP 1504.06).  Claims 7-10 of ‘320 recite TET1, TET2, TET3 and CXXC4 enzymes.  Paragraphs [0093-94] of the disclosure of ‘320 defines these TET enzymes.  Claim 13 of ‘320 recites alpha-glucosyltransferase and beta-glucosyltransferase.  Paragraph [0164] of the disclosure of ‘320 defines these glucosyltransferases.
Conclusion
No claims are allowed.

APPLICANT REBUTTAL: REMARKS
=======

Claims 31-52 were pending prior to entry of the abovementioned amendments. In this

response, claims 31, 32, 36, 37, 38, and 42 have been amended, and claim 35 has been cancelled. Accordingly, claims 31-34 and 36-52 are pending. The amendments to claim 31 are fully supported by the application as originally filed, U.S. Patent App. No. 18/588,987 (the "Application"), such as at paragraph \[0013\]. Claims 32, 36, 37, 38, and 42 have been amended for clarity or for antecedent basis. The amendments to the claims do not add new matter.

Reconsideration is respectfully requested in light of the abovementioned amendments and following remarks.

35 U.S.C. & 112(b)

Claims 32 and 42 stand rejected under 35 U.S.C. § 112(b) as indefinite.

Without conceding to the validity of the rejection, and solely to expedite prosecution, claims 32 and 42 have been amended. Applicant submits these amendments fully address the § 112(b) rejections and requests withdrawal of the rejections under § 112(b).

35 U.S.C. 4 112(a)

Claims 31-34 and 43-52 stand rejected under 35 U.S.C. § 112(a) as failing to comply with the written description requirement.

Without conceding to the validity of the rejection, and solely to expedite prosecution,

claim 31 has been amended to recite subject matter from claim 35, which was not rejected under § 112(a). Applicant submits that the amendment to claim 31 fully addresses the § 112(a) rejections and requests their withdrawal.

Double Patenting
================

Various claims stand rejected on the ground of double patenting as being unpatentable over one or more of claims 31-38, 41-42, 45-46 and 49-58 of copending Application No. 18/588930 (reference application; herein "'930"), claims 5-6 and 16-29 of copending Application No. 18/588956 (reference application; herein "'956"), claims 1-8 of U.S. Patent No. 9816986

(herein "'986"), claims 1, 6-8 and 10-15 of U.S. Patent No. 10323269 (herein "'269), claims 1-13 of U.S. Patent No. 10443091 (herein "'091"), claim 1 of U.S. Patent No. 10533213 (herein "'213"), claims 1, 13 and 16-17 of U.S. Patent No. 10612076 (herein "'076"), claims 1-3, 6-10, 13-15 and 19 of U.S. Patent No. 10731204 (herein "'204"), claims 1-3, 8-11, 13 and 16 of U.S. Patent No. 10767216 (herein "'216"), claims 1-9 of U.S. Patent No. 11072818 (herein "'818"), and/or claims 1-13 of U.S. Patent No. 12018320 (issues 6/25/2024 from application 17/675502; herein "'320").

Applicant respectfully disagrees, but solely for the purpose of advancing prosecution, Applicant submits herewith a terminal disclaimer over each of the above-cited patents. Applicant respectfully submits that the double patenting rejections are obviated in view of the terminal disclaimers.

CONCLUSION
==========

Applicant respectfully submits that the indefiniteness, written description, and double patenting rejections have been fully addressed herein. Applicant submits that where no other rejections are raised, the claims are now in allowable form.

Applicant does not acquiesce in the Examiner's characterizations of the art including the alleged motivation to combine references. For brevity and to advance prosecution, however, applicants may have not addressed all characterizations of the art and reserve the right to do so in further prosecution of this or a subsequent application. The absence of an explicit response by the applicants to any of the Examiner's positions does not constitute a concession of the Examiner's positions. The fact that applicants' comments have focused on particular arguments does not constitute a concession that there are no other arguments for patentability of the claims. All of the dependent claims are patentable for at least the reasons given with respect to the claims on which they depend.

Accordingly, Applicant respectfully requests early and favorable action finding the

claims allowable. If the Office has additional constructive comments or suggestions to expedite prosecution, Applicant sincerely encourages the Examiner to contact the undersigned attorney of record by telephone so that prosecution of the application can be expedited.

AUTHORIZATION TO CHARGE DEPOSIT ACCOUNT
=======================================

The Director of the USPTO is hereby authorized to charge any fee deficiencies associated with the instant filing except excess claims fees and credit any overpayments to NIXON PEABODY LLP Deposit Account No. 50-4181, from which the undersigned is authorized to withdraw.

Date: September 11, 2024 Respectfully submitted,

/Mark J. FitzGerald/

Customer No.: 50828 David S. Resnick (Reg. No. 34,235)

Mark J. FitzGerald (Reg. No. 45,928)Nixon Peabody LLP Exchange Place, 53 State Street Boston, MA 02109 Phone: 617-345-6057 / 1058 Fax: 617-345-1300 patentdocketing(anixonpeabody.com

NEXT OFFICE ACTION: EXAMINER'S AMENDMENT
An examiner’s amendment to the record appears below. Should the changes and/or additions be unacceptable to applicant, an amendment may be filed as provided by 37 CFR 1.312. To ensure consideration of such an amendment, it MUST be submitted no later than the payment of the issue fee.
Authorization for this examiner’s amendment was given in an interview with Mark FitzGerald on 9/20/2024 followed up by voicemail on 9/23/2024.
The application has been amended as follows:
42.	The method of claim [41]]38, wherein said TET2 polypeptide comprises a polypeptide comprising SEQ ID NO: 28.
48.	The method of claim 46, wherein said adding said glucose molecule or said glucose-derivative donor substrate to said oxidized derivative of said 5-methylcytosine in said nucleic acid molecule comprises contacting said oxidized derivative of said 5-methylcytosine in said nucleic acid molecule with
Information Disclosure Statement
The information disclosure statement submitted on 7/3/2024 has been considered by the examiner.
Reasons for Allowance
The following is an examiner’s statement of reasons for allowance: The claims are novel and non-obvious over the prior art and are enabled for the scope of the claims as evidenced by the original disclosure (for example, [0308-333]). The terminal disclaimer filed and approved on 9/11/2024 disclaiming the terminal portion of any patent granted on this application which would extend beyond the expiration date of U.S. Patents 9816986, 10443091, 10533213, 10731204, 10767216, 12018320, 10612076 and 11072818 and co-pending applications 18/588930 and 18/588956.  With the filing and approval of the terminal disclaimer, the claims are now in condition for allowance.
Any comments considered necessary by applicant must be submitted no later than the payment of the issue fee and, to avoid processing delays, should preferably accompany the issue fee.  Such submissions should be clearly labeled “Comments on Statement of Reasons for Allowance.”
Conclusion
Claims 31-34 and 36-52 are allowed.

CITED REFERENCE SUMMARIES: Ref US2009058562: Claims:
1. A method for improving the generation of stable human Foxp3+ T cells, the method comprising contacting with, or delivering to, a human T cell an effective 5- methylcytosine to 5-hydroxymethylcytosine converting amount of at least one catalytically active TET family enzyme, functional TET family derivative, TET family catalytically active fragment thereof, or combination thereof.
2. The method of claim 1, wherein the human T cell is a purified human CD4+ T cell.
3. The method of claim 1, further comprising contacting with, or delivering to, the human T cell a composition comprising at least one cytokine, growth factor, activating reagent, or combination thereof.
4. The method of claim 3, wherein said composition comprises TGF-β.
5. A method for improving efficiency or rate with which an induced pluripotent stem (iPS) cell is produced from an adult somatic cell, the method comprising contacting with, or delivering to a somatic cell, an effective 5-methylcytosine to 5- hydroxymethylcytosine converting amount of at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active thereof, or combination thereof.
6. The method of claim 5, wherein the catalytically active TET family enzyme is TETl or TET2.
7. The method of claim 5, further comprising comprising contacting with, or delivering to, the somatic cell, an effective amount of a TET family inhibitor.
8. The method of claim 7, wherein the TET family inhibitor is a TET3 inhibitor.
9. The method of claim 5, further comprising contacting the somatic cell with or delivering to the somatic cell a combination of nucleic acid sequences encoding Oct- 4, Sox2, c-MYC, and KIf 4.
10. The method of claim 5, wherein the combination of nucleic acid sequences encoding Oct-4, Sox2, c-MYC, and Klf4 are delivered in a viral vector.
11. The method of claim 5, wherein the somatic cell is a fibroblast.
12. A method for improving efficiency of cloning a mammal by nuclear transfer or nuclear transplantation, the method comprising contacting a nucleus extracted from a cell to be cloned with an effective 5-methylcytosine to 5-hydroxymethylcytosine converting amount of at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment thereof, or combination thereof, during a nuclear transfer protocol.
13. The method of claim 12, wherein the catalytically active TET family enzyme is TETl or TET2.
14. The method of claim 12, further comprising contacting the nucleus from the cell to be cloned with a TET family inhibitor.
15. The method of claim 14, wherein the TET family inhibitor is a TET3 inhibitor.
16. A method for detecting a 5-hydroxymethylcytosine nucleotide in a biological sample, the method comprising contacting a biological sample with a detectably labeled antibody or a binding portion thereof, labeled intrabody, or labeled protein, that specifically binds to 5-hydroxymethylcytosine, and detecting the amount of bound label, wherein the presence of the bound label is indicative of the 5-methylcytosine being converted to 5-hydroxymethylcytosine.
17. A kit for modulating gene transcription via hydroxylation of 5-methylcytosine to 5- hydroxymethylcytosine, the kit comprising the following separate components: (a) at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment thereof, or combination thereof, or nucleic acid molecule that comprises a sequence encodingat least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment, or combination thereof in an appropriate buffer or solution; and (b) packaging materials and instructions therein to use said kit to hydroxylate 5- methylcytosine to 5-hydroxymethylcytosine, for the purpose of modulating gene transcription.
18. The kit of claim 17, further comprising a composition comprising at least one cytokine, growth factor, activating reagent, or combination thereof for the purposes of generating stable human Foxp3+ regulatory T cells.
19. The kit of claim 18, wherein the composition comprises TGF-β.
20. The kit of claim 17, further comprising at least one nucleic acid sequence selected from the group consisting of nucleic acid sequences encoding Oct-4, Sox2, c-MYC, and Klf4, or any combination thereof.
21. The kit of claim 20, wherein the nucleic acid sequences encoding Oct-4, Sox2, c- MYC, and KIf 4 are incorporated into a viral vector.
22. The kit of claim 17, further comprising at least one reagent suitable for the detection of 5-hydroxymethylcytosine.
23. The kit of claim 22, wherein the reagent suitable for the detection of 5- hydroxymethylcytosine is an antibody, an antigen-binding portion thereof, an intrabody, or a protein, that specifically binds to 5-hydroxymethylcytosine.
24. The kit of claim 22, wherein the reagent suitable for the detection of 5- hydroxymethylcytosine is an antibody, an antigen-binding portion thereof, an intrabody, or a protein that is specific for cytosine-5-methylsulfonate.
25. A method for improving stem cell therapies, the method comprising contacting with, or delivering to, a stem cell an effective 5-methylcytosine to 5- hydroxymethylcytosine converting amount of at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment thereof, or combination thereof, or at least one nucleic acid molecule that comprises a sequence encoding a catalytically active TET family enzyme, a functional TET family derivative, TET catalytically active fragment thereof, or combination thereof.
26. A method for treating an individual affected with or at risk for cancer, the method comprising administering to an individual affected with, or at risk for, cancer, an effective amount of at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment, or a combination thereof, involved in transforming 5-methylcytosine into 5-hydroxymethylcytosine.
27. The method of claim 26, wherein the agent that specifically modulates hydroxylase activity is an inhibitor.
28. The method of claim 26, wherein the agent that specifically modulates hydroxylase activity is an activator.
29. The method of claim 26, wherein the cancer is a leukemia.
30. The method of claim 29, wherein the leukemia is an acute myeloid leukemia comprising the t(10:ll)(q22:q23) Mixed Lineage Leukemia translocation of TETl.
31. A method for screening for an agent with TET family enzyme modulating activity, the method comprising the steps of: a) providing a cell comprising at least one TET family enzyme, functional TET family derivative, TET catalytically active fragment, recombinant TET family enzyme thereof, or combination thereof; b) contacting said cell with a test agent, thereby creating a test sample; and c) detecting the level of 5-hydroxymethylated cytosine in the cell in the test sample with the level expressed in a control sample; and (d) determining whether or not the test agent increases or decreases the level of 5- hydroxymethylated cytosine, wherein a statistically significant decrease in the level of 5-hydroxymethylated cytosine indicates the test agent is an inhibitor, and a statistically significant increase in the level of 5-hydroxymethylated cytosine indicates the test agent is an activator.
32. The method as in any one of claims 1, 5, 12, 17, 25, 26, and 31, in which the catalytically active TET family enzyme is selected from the group consisting of TETl, TET2, TET3, and CXXC4.
33. The method as in any one of claims 1, 5, 12, 17, 25, 26, and 31, in which the functional TET family derivative comprises SEQ ID NO: 1.
34. The method as in any one of claims 1, 5, 12, 17, 25, 26, and 31, in which the TET family catalytically active fragment comprises SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.
35. A method for covalent tagging of 5-hydroxymethylcytosine in a nucleic acid, the method comprising contacting a nucleic acid molecule with an enzyme that adds one or more glucose molecules to a 5-hydroxymethylcytosine residue to generate gluco s ylated- 5 -hydroxymethylcyto sine or gentibio se-containing- 5 - hydroxymethylcytosine, wherein the enzyme is an alpha-glucosyltransferase, beta- glucosyltransferase, or a beta-glucosyl-alpha-glucosyl-transferase.
36. The method of claim 35, wherein the 5-hydroxymethylcytosine is naturally occurring.
37. The method of claim 35, further comprising the step of first contacting said nucleic acid with at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment thereof, or combination thereof, thereby converting 5-methylcytosine to hydroxymethylcytosine.
38. The method of claim 35, wherein the nucleic acid is contacted in vitro, in a cell, or in vivo.
39. A method for detecting 5-hydroxymethylcytosine in a nucleic acid, the method comprising contacting a nucleic acid with an enzyme that utilizes labeled glucose or glucose-derivative donor substrates to add one or more labeled glucose molecules or glucose-derivatives to a 5-hydroxymethylcytosine residue to generate glucosylated-5- hydroxymethylcytosine or gentibiose-containing-5-hydroxymethylcytosine, wherein the enzyme is an alpha-glucosyltransferase, a beta-glucosyltransferase, or a beta- glucosyl-alpha-glucosyl-transferase.
40. The method of claim 39, wherein the glucose or glucose-derivative donor substrate is a uridine diphosphate glucose.
41. The method of claim 39, wherein the labeled glucose or glucose-derivative donor substrates is radioactively labeled.
42. The method of claim 39, wherein the 5-hydroxymethylcytosine is naturally occurring.
43. The method of claim 39, further comprising the step of first contacting said nucleic acid with at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment thereof, or combination thereof, thereby converting 5-methylcytosine to 5-hydroxymethylcytosine.
44. The method of claim 39, wherein the nucleic acid is contacted in vitro, in a cell, or in vivo.
45. A method for detecting 5-hydroxymethylcytosine in a nucleic acid, the method comprising contacting the covalently tagged 5-hydroxymethylcytosine of claim 35 with a protein that recognizes a glucose molecule, glucose-derivative or gentibiosyl molecule.
46. The method of claim 45, wherein the protein recognizes only the glucose molecule, glucose-derivative, or gentibiosyl.
47. The method of claim 45, wherein the protein recognizes the glucose molecule, glucose-derivative, or gentibiosyl only in the context of 5-hydroxymethylcytosine.
48. The method of claim 45, wherein the protein is a lectin.
49. The method of claim 48, wherein the lectin is Musa acuminata lectin.
50. The method of claim 45, wherein the protein is an antibody or antigen-binding fragment thereof.
51. The method of claim 50, wherein the antibody or antibody fragment thereof is modified with a tag.
52. The method of claim 51, wherein the tag is a biotin molecule a bead, a gold particle, or a fluorescent molecule.
53. The method of claim 45, wherein the protein is an enzyme.
54. The method of claim 53, wherein the enzyme is hexokinase or beta-glucosyl-alpha- glucosyl-transferase.
55. A method for detecting 5-hydroxymethylcytosine in a nucleic acid, the method comprising contacting a nucleic acid with an enzyme and utilizing glucose or glucose-derivative donor substrates that trap covalent enzyme-DNA intermediates to detect 5-hydroxymethylcytosine residues, wherein the enzyme is an alpha- glucosyltransferase, a beta-glucosyltransferase, or a beta-glucosyl-alpha-glucosyl- transferase.
56. The method of claim 55, wherein the glucose donor substrate is a uridine diphosphate glucose analog.
57. The method of claim 56, wherein the uridine diphosphate glucose analog is uridine-2- deoxy-2-fluoro-glucose.
58. The method of claim 55, wherein the 5-hydroxymethylcytosine is naturally occurring.
59. The method of claim 55, further comprising the step of first contacting said nucleic acid with at least one catalytically active TET family enzyme, functional TET family derivative, TET catalytically active fragment thereof, or combination thereof, thereby converting 5-methylcytosine to 5-hydroxymethylcytosine.
60. The method of claim 55, wherein the enzyme is tagged.
61. The method as in any one of claims 35, 39, and 55, in which the alpha- glucosyltransferase is encoded by a bacteriophage selected from the group consisting of T2, T4, and T6 bacteriophages.
62. The method as in any one of claims 35, 39, and 55, in which the beta- glucosyltransferase is encoded by a bacteriophage selected from T4 bacteriophages.
63. The method as in any one of claims 35, 39, and 55, in which the beta-glucosyl-alpha- glucosyl-transferase is encoded by a bacteriophage selected from the group consisting of T2 and T6 bacteriophages.
64. The method of claim 55, wherein the nucleic acid is contacted in vitro, in a cell, or in vivo.
65. A method to detect 5-hydroxymethylcytosine in a nucleic acid, the assay comprising contacting a nucleic acid with sodium hydrogen sulfite to convert a 5- hydroxymethylcytosine in a nucleic acid to cytosine-5-methylsulfonate, and contacting the sodium hydrogen sulfite contacted nucleic acid with a protein specific for cytosine-5-methylsulfonate.
66. The method of claim 65, wherein the protein is an antibody, an antigen binding fragment thereof, an enzyme, or an intrabody.
67. The method of claim 66, wherein the antibody comprises an antiserum.
68. The method of claim 66, wherein the antibody, antigen binding fragment thereof, enzyme, or intrabody is modified with a tag.
69. The method of claim 68, wherein the tag is a biotin molecule, a bead, a gold particle, or a fluorescent molecule.
70. The method of claim 65, further comprising isolating the 5-hydroxymethylcytosine containing nucleic acid with the protein specific for cytosine-5-methylsulfonate.
71. The method of claim 65, wherein the nucleic acid is in vitro, a cell, or in vivo.
72. A kit for the detection and purification of methylcytosine and 5- hydroxymethylcytosine for use in downstream applications, the kit comprising: (a) one or more catalytically active TET family enzymes, functional TET family derivatives, or TET catalytically active fragments thereof for the conversion of methylcytosine to 5-hydroxymethylcytosine; (b) one or more enzymes encoded by bacteriophages of the "T even" family; (c) one or more glucose or glucose-derivative donor substrates; (d) one or more proteins to detect glucose or glucose-derivative modified nucleotides; (e) standard DNA purification columns, buffers, and substrate solutions; and (f) packaging materials and instructions therein to use said kits.
73. The kit of claim 72, wherein the enzyme encoded by bacteriophages of the "T even" family is selected from the group consisting of alpha-glucosyltransferases, beta- glucosyltransferases, and beta-glucosyl-alpha-glucosyl-transferases.
74. The kit of claim 73, wherein the alpha-glucosyltransferase is encoded by a bacteriophage selected from the group consisting of T2, T4, and T6 bacteriophages.
75. The kit of claim 73, wherein the beta-glucosyltransferase is encoded by a bacteriophage selected from T4 bacteriophages.
76. The kit of claim 73, wherein the beta-glucosyl-alpha-glucosyl-transferase is encoded by a bacteriophage selected from the group consisting of T2 and T6 bacteriophages.
77. The kit of claim 72, wherein the glucose or glucose-derivative donor substrate is uridine diphosphate glucose (UDPG).
78. The kit of claim 72, wherein the glucose or glucose-derivative donor substrate is radiolabeled.
79. The kit of claim 77, wherein the uridine diphosphate glucose is radiolabeled with 14C or 3H.
80. The kit of claim 72, wherein the protein to detect glucose or glucose-derivative modified nucleotides is a lectin, an antibody or an antigen binding fragment thereof, or an enzyme.
81. The kit of claim 80, wherein the protein recognizes only the glucose or glucose- derivative.
82. The kit of claim 80, wherein the protein recognizes the glucose or glucose-derivative only in the context of 5-hydroxymethylcytosine.
83. The kit of claim 80, wherein the antibody or antigen binding fragment thereof is modified with at least one tag.
84. The kit of claim 83, wherein the tag is a biotin molecule, a bead, a gold particle, or a fluorescent molecule.
85. The kit of claim 80, wherein the enzyme is a hexokinase or a beta-glucosyl-alpha- glucosyl-transferase.
86. The kit of claim 80, wherein the lectin is Musa acuminata lectin (BanLec).
87. The kit of claim 80, wherein the lectin is modified with a gold particle or a fluorescent tag.
88. A method for diagnosing a myelodysplastic syndrome, a myeloproliferative disorder, acute myelogenous leukemia, systemic mastocytosis, or chronic myelomonocytic leukemia in an individual in need thereof, the method comprising the steps of (i) determining a level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, in a tissue or a cell sample from an individual in need thereof, and (ii) comparing the level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, in the tissue or cell sample from the individual with a level of 5- methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, in a tissue or cell sample from a normal control, wherein a difference in the level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, between the sample from the individual in need and the control sample is indicative of the individual having a myelodysplastic syndrome, a myeloproliferative disorder, acute myelogenous leukemia, systemic mastocytosis, or chronic myelomonocytic leukemia.
89. The method of claim 88, further comprising a step of comparing the level of 5- methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, in a tissue or cell sample of the individual to the level of 5-methylcytosine, 5- hydroxymethylcytosine, or a combination thereof, in at least one sample from a diseased tissue or a diseased cell, wherein if the level of 5-methylcytosine, 5- hydroxymethylcytosine, or a combination thereof, in the tissue or cell sample from the individual in need is similar to the level of 5-methylcytosine, 5- hydroxymethylcytosine, or a combination thereof, from at least one of the samples from the diseased tissue or cell then the individual is diagnosed with a myelodysplastic syndrome, a myeloproliferative disorder, acute myelogenous leukemia, systemic mastocytosis, or chronic myelomonocytic leukemia.
90. A method for monitoring progression of or an effect of a therapy on a a myelodysplastic syndrome, a myeloproliferative disorder, acute myelogenous leukemia, systemic mastocytosis, or chronic myelomonocytic leukemia, the method comprising the steps of (i) determining a level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, in a tissue or cell sample from an individual in need thereof to establish a baseline level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, in the tissue or cell sample; (ii) determining a level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, in a tissue or cell sample from the individual at least one time following the establishment of the baseline level of 5-methylcytosine, 5- hydroxymethylcytosine, or a combination thereof in the tissue or cell sample thereby etsablishing at least one follow-up level of 5-methylcytosine, 5- hydroxymethylcytosine, or a combination thereof, wherein a difference in the follow- up level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, relative to the baseline level of 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof, in the tissue or cell sample is indicative of the progressi
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