DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
This application is a National-Stage entry of PCT/US2021/52504, filed 09/29/2021, which claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application, No. 63/084,829, filed 09/29/2020.
Status of Claims
Claims 1-15, 32-33, and 36-38 are currently pending in the instant application. Applicant’s election without traverse of Group I, claims 1-15 in the reply filed on October 22, 2025 is acknowledged. Claims 32-33 and 36-38 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Accordingly, claims 1-15 are under examination on the merits in the instant application.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on October 6, 2023 have been considered by the examiner, except those lined through as they are improperly listed.
Drawings
The drawings are objected to because FIG. 3E does not identify the black/dark filled circles. Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-15 are rejected under 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 1 is drawn to a method utilizing a genus of enzymes defined as “double stranded DNA deaminases (DddA).” Claims 5 and 6 recite the DddA domain has “at least about 85% identity” to SEQ ID NO 1. Claim 8 recites any fusion proteins comprising any target domain and a DddA domain wherein the fusion proteins have at least about 85% identity to SEQ ID NO 7. Claims 14 recites any “inhibitor” of DddA while claim 15 limits the inhibitor to “DddA immunity protein (DddIA)” .
Adequate written description support for a claimed genus may be provided by describing sufficient identifying characteristics, describing a representative number of species, actual reduction to practice, disclosure of drawings or structural chemical formulas, complete or partial structure, physical and/or chemical properties, functional characteristics when coupled with a known or disclosed correlation between function and structure and any working examples, method of making the claimed invention, level of skill and knowledge in the art as well as predictability in the art are other determinants that are used to analyze whether applicants had possession of the claimed genus. The present specification fails to meet these requirements for several reasons.
The specification states on page 28, line 11, that “the bacterial toxin-derived cytosine deaminase, DddA, is unique as the only deaminase known to act preferentially on double-stranded DNA (dsDNA).” The admission underscores the unpredictability of the field. The specification describes the DddA domain as comprising SEQ ID NO 1, which is 138 amino acids long. The specification does not provide any guidance as to which of these 138 amino acids are critical to the DddA domain functionality. For any DddA domains that are at least 15% identical to SEQ ID NO 1, there is no guidance in the specification which 20 amino acids could be deleted, added, or modified such that the DddA domain will maintain the deaminase activity.
While the prior art teaches a Burkholderia deaminase (Iyer et al. ((2011) NAR, Vol. 39, No. 22), and that it is delivered to cause toxicity in competing organisms via genome modification, there is no guidance regarding which amino acid residues in the toxin-derived deaminase taught in Iyer are critical for the preferential functionality on a dsDNA. Thus, the amino acids required for such an atypical enzyme remains highly unpredictable. Since this dsDNA activity is an atypical “exception” to the general rule of deaminases such as APOBEC or AID, which act mostly but not exclusively on single-stranded DNA (ssDNA), the structural-functional correlation of these enzymes could not be extrapolated to DddA. Therefore, in such an unpredictable and unique field of art, the disclosure of a single species, SEQ ID NO 1, is insufficient to convey possession of a genus of any DddA proteins. The specification provides no description of which amino acids residues are critical to retain the “unique” function of deaminating dsDNA.
Additionally, the teachings from the art (e.g., Mok et al, Nature Communication 13, 4038 (2022) and Guo et al, Mol Cell. 2023 May 18;83(10):1710-1724.e7) highlight that the DddA protein is highly sensitive to mutation. For example, Mok et al. attempted to obtain nontoxic, full-length DddAtox variants useful for base editing using both structure-based, site-directed mutagenesis (Fig. 1) and random mutagenesis (Page 2, Column 1). However, the E1347A variant combined with the quintuple AAAAA mutation, failed to induce base editing (Page 2, Column 1). Furthermore, Mok replaced positively charged amino-acid residues with alanine to reduce toxicity (Fig. 1a), yet most of the Ala-substituted variants failed to produce E. coli transformants (Page 2, Column 2). Similarly, Guo et al. searched for orthologs using BLAST alignment of the top 100 hits (Page 1712, Column 1 bridging Column 2). Ultimately, they obtained only 13 candidates with conserved amino acids and high similarity, and only 6 candidates possessed the conserved residues necessary for the nuclear DNA editing (Page 1712, Column 2). Guo demonstrates that only a finite number of orthologs with similarity of sequence and function are capable of functioning as a cytosine deaminase for successful base editing with a fusion protein. Consequently, a recitation of “85% identity” encompasses a large number of potential protein sequences (i.e., a 15% divergence), yet the art demonstrates that the vast majority of such variations result in a loss of function or continued toxicity. The specification provides no guidance on how to distinguish the rare functional variants from the non-functional majority within the 85% or greater identity range.
Similarly, given that the specification does not provide guidance on which amino acids are critical to retain functionality of the DddA protein defined by SEQ ID NO 1 as discussed above, one of ordinary skill in the art would not have guidance as to how the fusion protein defined by SEQ ID NO 7 could be modified within the “85% percent” or greater identity window and retain functionality. The fusion protein of SEQ ID NO 7 contains 681 amino acids. The specification does not provide any guidance as to which of these amino acids are critical to the DddA fusion protein functionality. For any DddA fusion proteins that are at least 15% identical to SEQ ID NO 7, there is no guidance in the specification which 102 amino acids could be deleted, added, or modified such that the DddA fusion protein will maintain the critical dsDNA deamination activity.
The specification states on page 15, lines 24-33 that the DddA protein activity can be controlled by “providing a DddA inhibitor,” and in some embodiments the DddA inhibitor can be a “double stranded DNA deaminase A immunity (DddIA) protein” defined by an amino acid sequence with 85% or greater identity to SEQ ID NO 2, which is 123 amino acids long. The specification does not provide any guidance as to which of these 123 amino acids are critical to the DddIA functionality. For any DddIA proteins that are at least 15% identical to SEQ ID NO 2, there is no guidance in the specification which 18 amino acids could be deleted, added, or modified such that the DddIA protein will maintain the DddA activity. While the prior art teaches a Burkholderia deaminase immunity protein (Iyer et al. (2011) NAR, Vol. 39, No. 22), there is no guidance taught in Iyer which amino acids are critical to the inhibitory potential of the immunity protein or any other potential inhibitor of DddA as in claim 14. As the DddA enzyme is admitted to be “unique” and “the only” known deaminase of its kind, the structural requirements for its corresponding inhibitor/immunity proteins are highly specific and unpredictable. The specification fails to provide a representative number of species within this inhibitor genera or identify common structural features (such as conserved binding motif or interface residues) that would allow a person having ordinary skill in the art to distinguish between proteins that will inhibit DddA and those that will not, as the specification provides no guidance on how to distinguish the rare functional variants from the non-functional majority within the 85% or greater identity range. In the absence of a structural-function correlation or a variety of disclosed species showing what variations are tolerated, the disclosure of a single immunity protein does not convey possession of the genus of all DddA inhibitors or DddIA proteins.
Finally, the remaining dependent claims (claims 2-4 and 10-13) add various method steps, such as the timing of contact or the use of affinity reagents, but these do not provide the necessary structural limitations to the underlying DddA or DddIA genera to cure the lack of written description. Since the specification provides no representative number of functional species across the “85% identity” window and no structural data for the inhibitors, one of ordinary skill in the art cannot visualize or recognized the members of the claimed genus.
Claim Interpretation
The terms “target protein” and “double-stranded DNA deaminase”, as used in claim 1, are afforded their Broadest Reasonable Interpretation (B.R.I.), consistent with the specification and the understanding of one of ordinary skill in the art at the time of filing.
The term “target protein” is not limited and is interpreted to encompass any DNA binding protein, protein fragment, or protein domain that localizes to and binds a specific nucleotide sequence or indirectly associates with a nucleic acid molecule through one or more intervening proteins. Examples in the specification include transcription factors (p.9, lines 16-35).
The specification defines “double-stranded DNA deaminase domain” and “DddA” as equivalent terms referring to a protein capable of catalyzing deamination of a target nucleotide, such as cytosine, in a dsDNA molecule (p. 9, lines 3-15).
At the time of filing, the prior art established that cytidine deaminases were capable of catalyzing cytidine deamination within dsDNA contexts, even if such activity occurred under specific structural or sequence-dependent conditions. For example, Yang et al., (Nature Communications, published Nov. 2, 2016) and Shen (Molecular Immunology, published May 11, 2006) teach that activation-induced cytidine deaminase (AID) can deaminate cytosines in dsDNA, notwithstanding that such activity may be context-dependent or less efficient than activity on ssDNA.
The claims impose no minimum efficiency requirement and do not exclude enzymes that also act on ssDNA. Nor do the claims require constitutive or sequence-independent deamination of dsDNA, only that deamination occurs within a dsDNA context at a DPI site. Accordingly, under the broadest reasonable interpretation, the term “double-stranded DNA deaminase” encompasses enzymes that catalyze deamination within dsDNA, including enzymes whose activity is context-dependent.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-7 and 9-15 are rejected under 35 U.S.C. 103 as being unpatentable over Bar (WO 2019/139951 A1, published July 18, 2019) and Mok (Mok et al. Nature 583, 631-637, published July 8, 2020, from IDS submitted 10/06/2023). Mok is a printed publication that qualifies as prior art under 35 U.S.C. 102(a)(1); to the extent applicant asserts that Mok falls within the grace period under 35 U.S.C. 102(b)(1), applicant bears the burden of establishing such exception. It is noted that Liu (US 2025/0011748 A1, priority to January 28, 2020) independently disclose substantially identical subject matter as Mok as set forth below; however, Liu is cited for completeness of the record and is not relied upon to supply any limitations not already taught by Mok.
With respect to claims 1-3, Bar teaches methods for mapping DNA-protein interaction (DPI) sites by locally modifying DNA proximate to a nucleic acid-interacting protein and subsequently detecting those modifications by sequencing (p. 3, ¶ 7; p. 15, ¶ 41; p. 16, ¶ 45-46). Specifically, Bar discloses contacting a sample comprising the nucleic acid-interacting protein, exposing the sample to an engineered nucleic acid-modifying enzyme, allowing coupling of the nucleic acid-modifying enzyme to the nucleic acid-interacting protein, permitting catalysis of a nucleic acid modification reaction, and analyzing the nucleic acid to detect such modifications, wherein the detected modifications indicate proximity to the DPI site (p. 3, ¶ 7; p. 4-6, ¶ 11; p. 29, ¶ 72-76; p. 41-42, claim 8).
With respect to claims 1, 4, and 9 Bar teaches that the disclosed methods are applicable to “single-stranded and double-stranded nucleic acids, including DNA and RNA,” in vitro or in vivo, and that the resulting nucleic acid modifications are detectable by sequencing relative to an unmodified reference sequence (p. 25, ¶ 67). Bar further teaches that suitable nucleic acid-modifying enzymes include, among others, “engineered DNA and RNA deaminases,” and that such modifying domains may be derived from naturally occurring enzymes, engineered de novo, or produced by directed evolution (p. 25, ¶ 67; Table 2).
With respect to claims 1-4, 6-7, 9, and 10-12, Bar teaches that binding sites for transcription factors are exemplary nucleic acid interaction sites suitable for mapping using the disclosed methods, stating that “one example of such sites are the binding sites for nucleic acid-binding proteins, such as binding sites for transcription factors in genomic DNA” (p. 5, ¶ 11; p. 9, ¶ 27). Thus, Bar expressly contemplates transcription factors as target nucleic acid-binding proteins as fusion partners for use in the detection and mapping methods. Bar further teaches that the nucleic acid-modifying enzyme may be coupled to the nucleic acid-binding protein as a “fusion protein” wherein “fusion generally can be an association generated by peptide bond, a chemical linkage, a charge interaction (for example, electrostatic attractions, such as salt bridges, H-bonding, etc.), a ligand-ligand noncovalent interaction, or the like,” and that the order of contacting, coupling, and exposure steps may be varied without departing from the disclosed invention (p. 18, ¶ 51; p. 3, ¶ 7 ). Bar further teaches methods to express the fusion partners via vectors that incorporate the expression cassette in the genomic DNA of the cell, which also serves as the DNA molecule to perform the DPI mapping (p.5, ¶ 11).
With respect to claims 4, 9, and 12-13, Bar also teaches that the disclosed methods may be practiced in both prokaryotic and eukaryotic cells (p. 6, ¶ 11; p. 23, ¶ 60), and that permeabilizing the cells may be performed via routine methodologies to optimize efficiency (p. 17-18, ¶ 49; p. 31, ¶ 77).
Bar does not teach the use of: (1) a double-stranded DNA deaminase (DddA); (2) DddA inhibitors, or a DddA inhibitor that is a DddA immunity protein that inhibits the cytosine deaminase activity and (3) a DddA domain disclosed in SEQ ID NO 1.
Mok teaches a bacterial deaminase, DddAtox, that catalyzes cytosine deamination in dsDNA, resulting in detectable sequence changes at single base pair resolution upon replication and sequencing (Mok, p. 632-633 and Figures 1-2) (Liu, Example 1, p. 155-6, ¶0767-0771). Mok further discloses the amino acid sequence of the functional DddAtox domain (Mok, Supplementary sequences 1, DddAtox) (Liu, p. 427 Seq ID No 338). Mok further describes that DddA belongs to a family of deaminases distinct from, yet mechanistically related to, previously characterized “single-stranded” DNA deaminases, while retaining the same underlying cytosine deamination activity (Mok, p. 632) (Liu, Example 1, p. 155-6, ¶0767-0771; FIG. 23I).
Mok additionally teaches the existence of a cognate immunity protein (DddIA) that inhibits DddA activity and mitigates cytotoxicity, thereby establishing a functional inhibitory relationship between DddA and its immunity protein (Mok, p. 632) (Liu, Example 1, p. 155, ¶0767).
It would have been obvious to one of ordinary skill in the art before the effective filing date to substitute the dsDNA cytosine deaminase, DddAtox, disclosed by Mok for previously contemplated cytosine deaminases in the methods of Bar in order to overcome recognized limitations associated with the suggested cytosine deaminases, AID and APOBEC, as they were understood to exhibit limited activity on dsDNA under specific conditions, including transient DNA breathing, weakened hydrogen bonding, or structural perturbations that permit cytosine extrusion. Thus, the ability of cytosine deaminases to access cytosines within dsDNA through base-flipping mechanisms was already recognized in the art. Furthermore, Mok teaches that DddAtox retains the same fundamental deamination mechanism while exhibiting enhanced and more reliable activity on dsDNA without requiring low GC content or other restrictive conditions. This teaching demonstrates that DddAtox represents an improved member of a known class of cytosine deaminases, providing increased robustness and predictability when acting on dsDNA substrates. Additionally, Mok discloses the peptide sequence of the functional deaminase domain, which aligns 100% to the cytosine deaminase peptide sequence, SEQ ID NO 1, used as a preferred embodiment in the instant application’s claimed methods. One of ordinary skill would therefore have been motivated to employ DddAtox in the methods of Bar as a predicable substitution to improve the efficiency and reliability of cytosine deamination proximal to DPI sites, with a reasonable expectation that the resulting base modification could be detected using the sequencing-based approaches taught by Bar.
It would also have been obvious to one of ordinary skill in the art to provide DddAtox as part of a fusion protein comprising a target protein domain and the DddAtox domain, optionally with a linker, as recited in claims 6 and 7. Bar expressly teaches coupling nucleic acid-modifying enzymes to nuclei acid-binding proteins via fusion proteins, and that such fusion may employ linker peptides. Mok discloses the functional DddAtox domain suitable for incorporating into engineered constructs, as evident by Mok’s own fusion constructs with the DddAtox domain. One of ordinary skill would therefore have been motivated to generate fusion proteins comprising DddAtox and a target protein domain, optionally separated by a linker, using routine protein engineering techniques, with a reasonable expectation that both domains would retain functional activity.
Additionally, it would have been obvious to one of ordinary skill in the art to modulate DddAtox activity using an inhibitor, including DddAtox immunity protein, as recited in claims 14 and 15, when performing the methods of Bar. Mok teaches that DddAtox activity is cytotoxic and is specifically inhibited by a cognate immunity protein, DddIA, establishing a known biological regulatory mechanism. One of ordinary skill would therefore be motivated to reduce background editing or toxicity by providing the DddA immunity protein to suppress deamination activity and to remove or deplete the inhibitor to permit controlled deamination during DPI mapping. Routine molecular biology techniques available prior to the effective filing date, including controlled expression systems and timed removal of inhibitory proteins, would have enabled predictable implementation of such regulation without undue experimentation.
In view of the foregoing, claims 1-7 and 9-15 taken as a whole would have been prima facie obvious before the effective filing date
Claim 8 is rejected under 35. U.S.C. 103 as being unpatentable over Bar (WO 2019/139951 A1, published July 18, 2019) and Mok (Mok et al. Nature 583, 631-637, published July 8, 2020), as applied to claims 1-7 and 9-15 above, and further in view of Rubenfield (US 2007/0020624 A1, published January 25, 2007, from IDS submitted 10/06/2023), Sarwar (Sarwar et al., mSphere, published April 27, 2016; from IDS submitted 10/06/2023) and Liu (Hereinafter “Gaudelli” US 2018/0073012 A1, published March 15, 2018).
The teachings of Bar and Mok are discussed above. As applied to claim 8, Bar teaches coupling a nucleic acid-modifying enzyme to a nucleic acid-binding protein, including transcription factors, via fusion proteins for mapping DPI sites. Mok teaches a dsDNA cytosine deaminase (DddAtox) suitable for fusion constructs and for generating detectable sequence modifications proximate to a DNA binding fusion partner.
Bar and Mok do not teach DddAtox domain fusion via a linker peptide to the GcsR transcription factor DNA binding domain defined by SEQ ID NO 7.
With respect to SEQ ID NO 5, Rubenfield teaches isolated Pseudomonas aeruginosa polypeptides and nucleic acids, including the peptide sequence, SEQ ID NO 23551, which aligns 100% to SEQ ID NO 5 and corresponds to the GcsR protein (see abstract, opening description, and sequence listings). Sarwar teaches that GcsR is a transcription factor and provides a sequence and domain analysis confirming its identity and function, as well a consensus sequence of the GcsR binding site (see abstract, results, FIG 6D).
With respect to SEQ ID NO 6, Gaudelli teaches flexible linker sequences used in base editing (particularly deamination and related fusion constructs) and teaches that such linkers preserve the independent activity of the fused domains while allowing spatial proximity necessary for function (¶s 0337-0340). Gaudelli further teaches the specific linker sequence, SEQ ID NO 385, which aligns 100% with SEQ ID NO 6 in the instant application.
It is noted that SEQ ID NO 7 is a combination of SEQ ID NO 5, SEQ ID NO 6, and SEQ ID NO 1. While the art does not teach the three domains/sequences as one combined sequence, as discussed below, creating such a sequence, would have been obvious.
It would have been obvious to one of ordinary skill in the art to apply the GcsR transcription factor as the nucleic acid-interacting partner when performing the DPI mapping methods of Bar because Bar identified transcription factors are ideal candidates for the mapping methods. One of ordinary skill would have been motivated to apply Bar’s mapping methodology using a known transcription factor such as GcsR as the targeting protein, because the peptide sequence was known and readily obtainable using routine molecular biology techniques, and the known GcsR consensus binding sequence would allow validation of mapping results through identification of expected editing sites and assessment of false positives. Additionally, it would have been obvious to one of ordinary skill in the art to couple the GcsR target protein to the DddA modifying enzyme in the DPI mapping methods of Bar. Bar teaches that such coupling may be achieved by fusion, optionally via a linker peptide. One of ordinary skill in the art would have been motivated to tether the GcsR transcription factor to the DddA domain using the linker disclosed by Gaudelli, with a reasonable expectation of success in generating a fusion protein that retained the functional activity of both fusion partners, thereby enabling mapping of nucleic acid-protein interactions at genomic loci bound by the target protein.
It further would have been obvious to arrive at and use the particular fusion construct required by SEQ ID NO 7 when performing the DPI mapping methods of Bar, in view of Bar and Mok as discussed and as applied to claims 1-7 and 9-15.
Claims 1-7 and 9-15 are rejected under 35 U.S.C. 103 as being unpatentable over Bar (WO 2019/139951 A1, published July 18, 2019) and Fauser (US 2024/0043829 A1, priority to September 25, 2020).
With respect to claims 1-4, 6-7, and 9-13, the teachings of Bar regarding methods for mapping DNA–protein interaction (DPI) sites are incorporated herein by reference from the prior rejection and are not repeated in full for brevity. Briefly, Bar teaches contacting nucleic acids with target proteins, coupling nucleic acid-modifying enzymes, permitting modification, sequencing-based detection, applicability to double-stranded DNA in cells, fusion-based and non-fusion-based coupling strategies, affinity-mediated tethering, expression from nucleic acids in cells, and optional permeabilization.
Bar does not teach the use of: (1) a double stranded DNA deaminase (DddA); (2) a specific DddA immunity protein that inhibits the cytosine DddA activity; and (3) a specific cytosine DddA domain defined by SEQ ID NO 1 in the instant application.
With respect to claims 5-7, and 9, Fauser teaches a double-stranded DNA cytosine deaminase (DddA) derived from bacterial toxin systems that catalyzes cytosine-to-uracil deamination in dsDNA, producing detectable sequence changes upon DNA replication and sequencing (Summary of the invention ¶0006, FIG 1 and 2B. Fauser discloses the amino acid sequence of the functional DddA domain, which aligns 100% with the DddA sequence recited as SEQ ID NO:1 in the instant application (Summary of the invention ¶0006 and 0015). Fauser further teaches that DddA may be fused via linker peptides to DNA-targeting proteins, including zinc finger proteins, to direct deamination to specific genomic loci in dsDNA. Fauser discloses that such fusion constructs retain both targeting and deamination activity (Summary of the invention ¶0007).
With respect to claims 9 and 14-15, Fauser additionally teaches a cognate DddA immunity protein (DddIA) that specifically inhibits DddA activity and discloses the peptide sequence of the immunity protein, which aligns 100% with the immunity protein disclosed in the instant application. Fauser teaches use of the immunity protein to temporally regulate deaminase activity, including reducing background editing and mitigating toxicity (Summary of the invention ¶0009). Fauser further teaches expression of DddA, DddIA, and DddA fusion constructs from nucleic acids in cells and used to edit genomic DNA in prokaryotic or eukaryotic cells, and describes routine molecular biology techniques for co-expression, inducible expression, removal, or depletion of the immunity protein to permit controlled deamination (Summary of the invention ¶0006 and 0014).
It would have been obvious to one of ordinary skill in the art before the effective filing date to substitute the dsDNA cytosine deaminase disclosed by Fauser for previously known cytosine deaminases in the DPI mapping methods of Bar. Cytosine deaminases were known to act primarily on ssDNA but to exhibit limited activity on dsDNA under conditions permitting cytosine extrusion. Fauser teaches that DddA operates on dsDNA with enhanced robustness while retaining the same base-flipping deamination mechanism. One of ordinary skill in the art would therefore have been motivated to employ DddA in the methods of Bar as a predictable substitution, with a reasonable expectation that cytosine deamination events proximal to DPI sites would be detectable using the sequencing-based detection methods taught by Bar.
It would have been obvious to one of ordinary skill in the art to couple DddA to a target protein via fusion when practicing the methods of Bar. Bar teaches fusion-based coupling of nucleic acid-modifying enzymes to nucleic acid-interacting proteins, and Fauser teaches fusion of DddA to DNA-targeting proteins via linker peptides. One of ordinary skill in the art would therefore have been motivated to implement DddA in a fusion paradigm, optionally including a linker domain, with a reasonable expectation that both targeting and deamination functions would be retained.
It would have been obvious to one of ordinary skill in the art to modulate DddA activity using the cognate immunity protein disclosed by Fauser. Fauser teaches that uncontrolled DddA activity causes toxicity and background editing and that these effects are mitigated by the DddA immunity protein. One of ordinary skill in the art would therefore have been motivated to provide and subsequently remove or deplete the immunity protein to permit controlled deamination, using routine expression and regulation techniques, with a reasonable expectation of success.
In view of the foregoing, claims 1-7 and 9-15 taken as a whole would have been prima facie obvious before the effective filing date
Claim 8 is rejected under 35. U.S.C. 103 as being unpatentable over Bar (WO 2019/139951 A1, published July 18, 2019) and Fauser (US 2024/0043829 A1, priority to September 25, 2020), applied to claims 1-7 and 9-15 above, and further in view of Rubenfield (US 2007/0020624 A1, published January 25, 2007), Sarwar (Sarwar et al., mSphere, published April 27, 2016; from IDS submitted 10/06/2023) and Liu (Hereinafter “Gaudelli” US 20180073012 A1, published March 15, 2018).
The teachings of Bar and Fauser are discussed above. As applied to claim 8, Bar teaches coupling a nucleic acid-modifying enzyme to a nucleic acid-binding protein, including transcription factors, via fusion proteins for mapping DPI sites. Fauser teaches a dsDNA cytosine deaminase (DddA) suitable for fusion constructs and for generating detectable sequence modifications proximate to a DNA binding fusion partner.
Bar and Fauser do not teach DddA fusion to the GcsR transcription factor defined by Seq ID No. 7.
The teachings of Rubenfield, Sarwar, and Gaudelli regarding the identity of GcsR as a transcription factor, its peptide sequence, DNA-binding activity, and linker-mediated fusion strategies are incorporated herein by reference from the prior rejection of Claim 8.
It is noted that SEQ ID NO 7 is a combination of SEQ ID NO 5, SEQ ID NO 6, and SEQ ID NO 1. While the art does not teach the three domains/sequences as one combined sequence, as discussed below, creating such a sequence, would have been obvious.
It would also have been obvious to one of ordinary skill in the art, prior to the effective filing date, to apply the GcsR transcription factor as the nucleic acid-interacting partner when performing the DNA–protein interaction (DPI) mapping methods of Bar. Bar expressly identifies transcription factors as exemplary nucleic acid-interacting proteins that are particularly suitable for use in its DPI mapping methods. One of ordinary skill in the art would have been motivated to apply Bar’s mapping methodology using a known transcription factor such as GcsR as the targeting protein, as the peptide sequence was known and readily obtainable using routine molecular biology techniques, and the known GcsR consensus binding sequence would allow validation of mapping results through identification of expected editing sites and assessment of false positives. It would further have been obvious to one of ordinary skill in the art to couple the GcsR target protein to the DddA modifying enzyme when performing the DPI mapping methods of Bar. Bar teaches that coupling between the nucleic acid-modifying enzyme and the nucleic acid-interacting protein may be achieved by fusion, optionally via a linker peptide, and Fauser teaches that the double-stranded DNA cytosine deaminase DddA can be incorporated into fusion proteins with DNA-targeting domains to localize deaminase activity to specific genomic loci. One of ordinary skill in the art would therefore have been motivated to tether the GcsR transcription factor to the DddA domain using the linker disclosed by Gaudelli, with a reasonable expectation of success in generating a fusion protein, because this represents the substitution and combination of known elements according to established fusion paradigms contemplated by both Bar and Fauser, yielding predicable results while retaining the functional activity of both fusion partners.
It further would have been obvious to arrive at and use the particular fusion construct required by SEQ ID NO 7 when performing the DPI mapping methods of Bar, in view of Bar and Fauser as discussed and as applied to claims 1-7 and 9-15.
Claims 1-7 and 9-15 are rejected under 35 U.S.C. 103 as being unpatentable over Bar (WO 2019/139951 A1, published July 18, 2019), Yang (Yang et. al., Nature Communications, published Nov. 2, 2016), Shen (Shen, Molecular Immunology, May 11, 2006), Zhang (Zhang et. al., Nucleic Acid Research, available online February 8, 2011), and Iyer (Iyer et. al., Nucleic Acid Research, available online September 3, 2011; from IDS submitted 10/06/2023), as evidenced by publicly available UniProtKB database entries (A0A1V2VU04_9BURK and A0A1V6L5G6_9BURK, release date June 7, 2017; UniProtKB; /www.uniprot.org/).
The following analysis applies the broadest reasonable interpretation set forth above and addresses whether the claimed subject matter would have been obvious in view of the state of the art at the time of filing.
Regarding claims 1-4, 6-7, and 9-13, Bar is discussed in detail above and is incorporated herein by reference. Briefly, Bar teaches methods for mapping DNA–protein interaction (DPI) sites by coupling a nucleic acid-modifying enzyme to a nucleic acid-interacting protein and detecting resulting nucleic acid modifications by sequencing. Bar further teaches applicability of the methods to single- and double-stranded DNA or RNA, transcription factors as exemplary target proteins, fusion-based or non-fusion-based coupling strategies, and implementation in prokaryotic or eukaryotic cells with permeabilization. Bar expressly contemplates substitution among functionally equivalent nucleic-acid modifying enzymes suitable for generating detectable sequence alterations at protein-bound DNA sites, without limiting the methods to any specific deaminase species, expressly identifying APOBEC and AID as examples.
Bar does not explicitly teach the use of: (1) a double-stranded DNA deaminase (DddA); (2) DddA inhibitors, or a DddA inhibitor that is a DddA immunity protein that inhibits the cytosine deaminase activity and (3) a DddA domain disclosed in SEQ ID NO 1.
At the time of the filing of the instant application, the prior art recognized that the enzymes contemplated by Bar, APOBEC and AID, had dsDNA activity as shown by Yang et. al., and Shen, but under limited context as discussed below.
Yang discloses programmable cytidine deaminases fused to targeted DNA-binding proteins via linker sequences (Title, pg. 2, “Design of targeted deaminases”; Figure 1a), supporting the fusion paradigm expressly contemplated by Bar. Yang demonstrates that linker lengths and sequence compositions affect site-specific cytosine deamination efficiency in genomic DNA (see paragraph one of “Optimization of targeted deaminases.”, starting on pg. 2, right col), and that fusion cytidine deaminases such as APOBEC or AID to zinc-finger or TALE-DNA binding modules enables targeted deamination of genomic loci in both prokaryotic and eukaryotic cells (pg. 2, left col, 4th para).
Yang further teaches that such fusion constructs can exhibit processive activity extending beyond the immediate DNA binding, resulting in off-target mutations. Yang explains that this behavior reflects the inherent catalytic properties of cytosine deaminase once engaged with dsDNA, and proposes regulatory strategies, such as split deaminase systems, to restrict activity until desired. These teachings established that although fusion-based targeting of cytidine deaminases was routine and predictable, controlling deaminase activity remained a recognized challenge.
In support of Yang, Shen teaches that AID is capable of deaminating cytosines within dsDNA under specific structural conditions, such as at blunt-ended double-strand breaks, super-coiled DNA, or within AT-rich regions exhibiting transient helix destabilization (see abstract, results pg. 976 and 979, and discussion pg. 980). Shen identifies mechanistic constraints limiting efficient deamination of intact dsDNA, thereby reinforcing that known cytidine deaminases exhibited context-dependent activity and limited robustness on double-stranded substrates.
Taken together, Bar, Yang, and Shen establish that cytidine deaminases were known and contemplated for use in DPI mapping methods, that fusion of deaminases to DNA-binding proteins using linkers was routine and predictable, and that existing deaminases capable of acting on dsDNA suffered from recognized limitation. In view of these teachings, one of ordinary skill in the art would have been motivated to identify alternative cytidine deaminases with improved or more robust activity on double-stranded DNA to enhance the reliability and resolution of DPI mapping methods such as those taught by Bar.
Furthermore, at the time of the filing of the instant application, Zhang further reinforces this motivation. Zhang teaches that analysis of bacterial toxin systems reveals previously uncharacterized nuclease and deaminase domains operating on nucleic acids and that such toxins include deaminases belonging to the superfamily as APOBEC and AID. Zhang expressly teaches that the availability of extensive genomic sequence data enables computational identification of novel toxin-derived deaminases and associated immunity proteins, and explicitly proposes such enzymes as potential reagents for diverse biotechnological applications, including nucleic acid modification (pg. 4533 left column first and second paragraph). Zhang thus provides explicit motivation to search genomic databases for novel bacterial toxin-derived deaminase analogous to APOBEC/AID but with distinct substrate properties similar to nucleases (pg. 4544 second paragraph).
Regarding claims 5-7, 9, and 14-15, Iyer teaches that cytidine deaminases arose through a broad evolutionary radiation from the bacterial toxin systems, resulting in multiple distinct clades characterized by conserved catalytic motifs and substrate-binding features shared with eukaryotic AID/APOBEC enzymes. Of particular relevance, Iyer identifies the SCP1.201 clade as a toxin-associated deaminase family that is phylogenetically related to the BURPS668 lineage, and which include toxic cytidine deaminases delivered between bacteria. Iyer further teaches that bacterial toxin-derived deaminases are naturally associated with cognate immunity proteins that bind and inhibit deaminase activity to prevent host toxicity. Iyer specifically teaches that predicted immunity protein families are primarily associated with deaminase domains of the SCP1.201 clade. Iyer further teaches that members of the SCP1.201 clade area associated with Type VI secretion system (TS66), a pathway well known in the art to deliver nucleic-acid targeting toxins, including enzymes that act on double-stranded DNA in recipient cells. Thus, Iyer establishes that deaminases within the SCP1.201 clade were recognized prior to the effective filing date as toxic enzymes to act on intact cellular DNA, rather than being limited to single-stranded substrates, and offers “multiple testable hypotheses regarding the activities of deaminases” (See Functional inference for the newly identified versions of the deaminase superfamily, Evolutionary implications and general conclusion and Table 1, and rest of reference).
In view of Yang and Shen’s recognition of limitations of known deaminases, and Zhang’s explicit teaching that bacterial toxin-derived deaminases are discoverable and suitable for biotechnological applications, one of ordinary skill in the art would have been motivated to search for alternative cytidine deaminases exhibiting improved activity on double-stranded DNA. Iyer identifies a finite and defined set of toxin-derived cytidine deaminase clades, rendering selection from among these candidates an obvious-to-try substitution with a reasonable expectation of success. Such evaluation would have involved routine public database searching and screening without undue experimentation.
Publicly available UniProtKB database entries disclose, prior to the effective filing date, an amino acid sequence containing the DddA deaminase domain recited in SEQ ID NO 1 and a corresponding immunity protein that aligns with SEQ ID NO 2, DddIA, and annotate such proteins as predicted cytidine deaminase and immunity protein, respectively. UniProtKB entries are curated, publicly searchable biological sequence records routinely relied upon by those of ordinary skill in the art. These entries confirm that the claimed deaminase sequence and DddIA protein were publicly accessible and identifiable as members of the SCP1.201 deaminase lineage taught by Iyer. The annotated association of the deaminase with the cognate immunity protein reflects known structure-function relationships characteristic of toxin-derived deaminases, reinforcing predictability.
It would therefore have been obvious to one of ordinary skill in the art, prior to the effective filing date, to substitute a bacterial toxin-derived cytidine deaminase from the SCP1.201 lineage taught by Iyer for previously known deaminases in the DPI mapping methods of Bar, motivated by the desire to improve double-stranded DNA deamination efficiency and reliability on dsDNA in its native state. It would further have been obvious to provide such a deaminase as part of a fusion protein, optionally including a linker, as taught by Bar and Yang, to ensure proximity-based modification at DNA–protein interaction sites. One of ordinary skill would have had a reasonable expectation of success in implementing such substitutions within Bar’s sequencing-based DPI mapping framework.
It would have also been obvious to one of ordinary skill in the art to include, remove, or deplete a cognate immunity protein to modulate deaminase activity during the DPI mapping methods of Bar, with a reasonable expectation of success. As taught by Yang, cytidine deaminase fusion proteins can exhibit processive and potentially off-target activity on genomic DNA, and Yang expressly recognizes the need for strategies to restrict or regulate deaminase activity in experimental contexts. Independently, Iyer teaches that bacterial toxin-derived cytidine deaminases, including those of the SCO1.201 clade, are naturally regulated by the cognate immunity proteins (e.g., Imm1) that bind and inhibit deaminase activity to prevent host toxicity. Publicly available UniProtKB database entries corresponding to both DddA and its cognate Imm1 immunity protein confirm that this inhibitory protein was known, identifiable, and associated with the claimed deaminase prior to the effective filing date. In view of Yang’s recognition of the need to control deaminase processivity and off-target effects, and Iyer’s teaching of immunity proteins as a native, reversible, and specific inhibitory mechanism for toxin-associated deaminases, one of ordinary skill in the art would have been motivated to employ a cognate immunity protein to regulate deaminase activity in the methods of Bar to mitigate off-target effects reducing false positives. Such a use represents the predictable application of a known regulatory mechanism to an analogous experimental problem and would have been expected to successfully restrict deaminase activity according to the experimental design.
Implementation of the foregoing substitutions would have involved routine molecular cloning, expression, and screening techniques well withing the ordinary skill in the art, without requiring inventive or undue experimentation. One of ordinary skill in the art would have had a reasonable expectation of success in implementing such substitutions within Bar’s sequencing-based DPI mapping framework. The claimed methods therefore represent a predictable application of known elements and regulatory mechanisms to an analogous experimental problem.
Claim 8 is rejected under 35. U.S.C. 103 as being unpatentable over Bar (WO 2019/139951 A1, published July 18, 2019), Yang (Yang et. al., Nature Communications, published Nov. 2, 2016), Shen (Shen, Molecular Immunology, May 11, 2006), Zhang (Zhang et. al., Nucleic Acid Research, available online February 8, 2011), and Iyer (Iyer et. al., Nucleic Acid Research, available online September 3, 2011; from IDS submitted 10/06/2023), as evidenced by publicly available UniProtKB database entries (A0A1V2VU04_9BURK and A0A1V6L5G6_9BURK, release date June 7, 2017; UniProtKB; /www.uniprot.org/), as applied to claims 1-7 and 9-15 above, and further in view of Rubenfield (US 20070020624 A1, published January 25, 2007), Sarwar (Sarwar et al., mSphere, published April 27, 2016) and Liu (Hereinafter “Gaudelli” US 20180073012 A1, published March 15, 2018).
The teachings of Bar and Iyer are discussed above and are herein incorporated by reference. As applied to claim 8, Bar teaches coupling a nucleic acid-modifying enzyme to a nucleic acid-binding protein, including transcription factors, via fusion proteins for mapping DPI sites. Iyer teaches bacterial toxin-associated deaminases that are secreted via T6SS system to target other bacterial genomes. Lastly, the deaminases taught by Iyer have corresponding UniProtKB entries.
Bar and Iyer do not teach DddA fusion to the GcsR transcription factor defined by SEQ ID NO 7.
The teachings of Rubenfield, Sarwar, and Gaudelli regarding the identity of GcsR as a transcription factor, its peptide sequence, DNA-binding activity, and linker-mediated fusion strategies are incorporated herein by reference from the prior rejections of Claim 8.
It is noted that SEQ ID NO 7 is a combination of SEQ ID NO 5, SEQ ID NO 6, and SEQ ID NO 1. While the art does not teach the three domains/sequences as one combined sequence, as discussed below, creating such a sequence, would have been obvious.
It would also have been obvious to one of ordinary skill in the art, prior to the effective filing date, to apply the GcsR transcription factor as the nucleic acid-interacting partner when performing the DNA–protein interaction (DPI) mapping methods of Bar. Bar expressly identifies transcription factors as exemplary nucleic acid-interacting proteins that are particularly suitable for use in its DPI mapping methods. One of ordinary skill in the art would therefore have been motivated to apply Bar’s mapping methodology using a known transcription factor such as GcsR as the targeting protein, as the peptide sequence was known and readily obtainable using routine molecular biology techniques, and the known GcsR consensus binding sequence would allow validation of mapping results through identification of expected editing sites and assessment of false positives. It would further have been obvious to one of ordinary skill in the art to couple the GcsR target protein to the DddA modifying enzyme when performing the DPI mapping methods of Bar. Bar teaches that coupling between the nucleic acid-modifying enzyme and the nucleic acid-interacting protein may be achieved by fusion, optionally via a linker peptide. One of ordinary skill in the art would therefore have been motivated to tether the GcsR transcription factor to the DddA domain using the linker disclosed by Gaudelli, with a reasonable expectation of success in generating a fusion protein, because this represents the substitution and combination of known elements according to established fusion paradigms contemplated by Bar, yielding predicable results while retaining the functional activity of both fusion partners.
It further would have been obvious to arrive at and use the particular fusion construct required by SEQ ID NO 7 when performing the DPI mapping methods of Bar, in view of Bar, Yang, Shen, Zhang and Iyer as discussed and as applied to claims 1-7 and 9-15.
Conclusion
No claims are allowed.
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/COREY LANE BRETZ/ Patent Examiner, Art Unit 1635
/RAM R SHUKLA/Supervisory Patent Examiner, Art Unit 1635