DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant's election with traverse of Group I of invention (claims 1-2, 6-7, 9, 11, 13-14, 17-26, and 37) in the reply filed on May 1, 2026 is acknowledged. The traversal is on the ground(s) that “neither Lu nor Jankervicius, considered individually or in combination, teaches…a composition that includes a gap editor…” and that “combining a DNA-recognition domain (e.g., Cas protein) and a DNA-modifying domain (e.g., a DarT enzyme) in a single gap editor complex is not a matter of simple substitution…but amounts to a novel and non-obvious combination of two separate proteins capable of successfully modifying a genome in a manner that reduces toxicity and/or enhances cell viability” (pg. 6 of Remarks received on May 1, 2026) (pg. 6 of Remarks received on May 1, 2026).
This is not found persuasive because Applicant’s arguments are unsupported by specific evidence or technical reasoning. Upon further reconsideration, the provisional restriction no longer relies on substituting the cytidine deaminase of Lu with a DarT enzyme disclosed by Jankervicius to satisfy the limitation “wherein the DNA-modifying domain induces formation of a replication blocking moiety on at least one nucleotide in the genome” recited in previously presented claim 1. Lu et al teach the editing efficiency of cytidine deaminase can be improved by fusing an uracil DNA glycosylate that generates an abasic site in the DNA target sequence ([0206]; Table 6). As explained in instant 35 USC 102 rejection below, Cuniasse et al (cited infra) teach an abasic site in DNA is a mutagenic base lesion that act as strong blocks for replication either by preventing binding of the DNA polymerase to the DNA template or blocking the progression of the DNA polymerase as it travels up the helix (pg. 303, col. 2, para. 1; pg. 313, col. 2, para. 4). Accordingly, Lu’s cytidine deaminase meets the claimed limitation. Thus, Applicant’s argument regarding the alleged lack of motivation to substitute the cytidine deaminase is not responsive to the basis of this rejection. Therefore, the common technical feature of a composition comprising a gap editor of claim 1 is not a special technical feature as it does not make a contribution over the prior art.
In regards to the obviousness of substituting a DNA-modifying domain to a DarT enzyme, the instant 35 USC 103 rejection below specifically addresses why one ordinary skill in the art would have had a reasonable expectation of success in doing so.
The requirement is still deemed proper and is therefore made FINAL.
Applicant's election with traverse of DarT as the single specific DNA-modifying domain, SEQ ID NO: 18 as the single specific catalytic domain, and LIDDR as the single specific amino acid sequence in the catalytic domain motif in the reply filed on May 1, 2026 is acknowledged. The traversal is on the ground(s) that “the elected species are mutually consistent and compatible, as each is a component that may be present in the same composition defined by independent claim 1” (pg. 6 of Remarks received on May 1, 2026).
This is not found persuasive because as stated in previous Office Action, mailed on 02/04/2026, the common technical feature among the claimed species is a gap editor complex of claim 1 comprising a DNA-modifying domain that induces formation of a replication blocking moiety on at least one nucleotide in the genome. As set forth in the election requirement, the prior art demonstrates that this common technical feature is not a special technical feature as it does not make a contribution over the prior art (also see discussion above as applied to response to applicant’s arguments regarding restriction requirements). The elected and non-elected species are distinguished by different DNA-modifying domains (e.g., DarT that installs ADP-ribosylation on thymine, Scabin that installs ADP-ribosylation on guanosine, methylcarbamoylase Mom that installs methylcabamoylation on adenine), each of which are structurally different and performs a different modification on nucleotides. Further, whether different embodiments are compatible and may be present in the same composition is not determinative of whether the species share a special technical feature for purposes of unity of invention. The election requirement was based on the absence of a special technical feature over the prior art, not on whether the claimed species are capable of coexisting in the same composition.
The requirement is still deemed proper and is therefore made FINAL.
Claims 20-26, and 55 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species and Group II of invention, respectively, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on May 01, 2026.
Claims 1-2, 6-7, 9, 11, 13-14, 17-19, and 37 are pending and under examination.
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 119(e) as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed application, Application No. 63/149,419, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. The following claims are supported in the Provisional Application No. 63/149,419: instant claims 1 and 6 (claim 1), instant claim 2 (claim 2), instant claim 7 (claim 6), instant claim 9 (claim 8), instant claim 11 (claim 10), instant claim 13 (claim 12), instant claim 14 (claim 13), instant claim 17 ([0067]), instant claim 19 ([0088]), instant claim 37 (claim 27). However, support for instant claim 18 is not found in the disclosure of Provisional App. No. ‘419. The specification does not disclose a catalytic domain comprising at least 70% amino acid sequence identity with any of SEQ ID NO: 18-21. The specification fails to disclose catalytic domain for any DNA-modifying domain.
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55. Claims 1-2, 6-7, 11, 13-14, 17, 18-19, and 37 are granted priority to the International Patent Application PCT/US22/16313 filed on 02/14/2022.
Accordingly, instant claims 1-2, 6-7, 11, 13-14, 17, 19, and 37 have an effective filling date of 02/15/2021, which is the filling date of Provisional App. No. ‘419. Claim 18 have an effective filling date of 02/14/2022, which is the filling date of PCT/US22/16313.
Drawings
Color photographs and color drawings are not accepted in utility applications unless a petition filed under 37 CFR 1.84(a)(2) is granted. Any such petition must be accompanied by the appropriate fee set forth in 37 CFR 1.17(h), one set of color drawings or color photographs, as appropriate, if submitted via the USPTO patent electronic filing system or three sets of color drawings or color photographs, as appropriate, if not submitted via the via USPTO patent electronic filing system, and, unless already present, an amendment to include the following language as the first paragraph of the brief description of the drawings section of the specification:
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Color photographs will be accepted if the conditions for accepting color drawings and black and white photographs have been satisfied. See 37 CFR 1.84(b)(2).
The following descriptions for FIGs in the specification recite colors in the FIGs: “the edited nucleotides are highlighted in red” ([0161], “highlighted in red are mutations” ([0163].
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency - The incorporation by reference paragraph required by 37 CFR 1.834(c)(1), 1.835(a)(2), or 1.835(b)(2) is missing, defective or incomplete. The Sequence Listing, in ASCII text file is designated 39212-601_SEQUENCE_LISTING_ST25.txt, 144908 B, and received on 08-14-2023 while the Sequence Listing disclosed in the specification’s incorporation by reference statement is 39212-601_SEQUENCE_LISTING_ST25.txt, 144908 B, and created on February 14, 2022. The date of the ASCII text file does not match.
Required response - Applicant must:
• Provide a substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3), and 1.125 inserting the required incorporation by reference paragraph, consisting of:
• A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
• A copy of the amended specification without markings (clean version); and
• A statement that the substitute specification contains no new matter.
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.
Claim 18 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
MPEP 2163.II.A.3.(a).i) states, “Whether the specification shows that applicant was in possession of the claimed invention is not a single, simple determination, but rather is a factual determination reached by considering a number of factors. Factors to be considered in determining whether there is sufficient evidence of possession include the level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the method of making the claimed invention”.
In making a determination of whether the application complies with the written description requirement of 35 U.S.C. 112, first paragraph, it is necessary to understand what Applicant has possession of and what Applicant is claiming. Claim 1, from which claim 18 depends, is drawn to a gap editor complex comprising (i) a DNA-recognition domain that binds a DNA target sequence in the genome, and (ii) a DNA-modifying domain that induces formation of a replication block moiety on at least one nucleotide in the genome. The specification and the state of art provide adequate written description support for such DNA-modifying domains, including representative DarT enzymes demonstrated in working examples ([0147], [0151], [0154], [0157], [0160], [0163], [0165]). In contrast, claim 18 further limits the DNA-modifying domain of claim 1 to comprise a catalytic domain having at least 70% amino acid sequence identity to LIDDR (i.e., the elected sequence of SEQ ID NO: 18). The additional limitation in claim 18 modified the breadth of the claim to encompass a genus of DNA-modifying domain characterized by the presence of the recited catalytic domain LIDDR, while retaining the catalytic function of a DNA-modifying domain.
The specification identifies the recited catalytic domain LIDDR as a catalytic domain of DarT enzyme ([0202]), but no working example or actual reduction to practice of a DarT enzyme comprising a catalytic domain LIDDR is disclosed. The embodiments exemplified are a wild-type catalytic domain LIGKR from the enteropathogenic Escherichia coli DarT enzyme, and a mutant catalytic domain LIGAR that comprises a K56A mutation to reduce cytotoxicity effect while maintaining catalytic activity of the DarT enzyme to install ADP-ribosylation ([0147]). The specification fails to disclose any DNA-modifying domain other than DarT that comprises the LIDDR sequence, or sufficient relevant identifying characteristics including common structural characteristics demonstrating that DNA-modifying domains other than the disclosed DarT would possess the claimed catalytic domain having the LIDDR sequence while retaining DNA-modifying function.
Further, the state of the art recognizes that residues in the recited LIDDR are functionally significant. Schuller et al (Molecular basis for DarT ADP-ribosylation of a DNA base; Nature, 2021, 596:597-602) teach that arginine at position 51 is essential for polarization of the NAD+ molecule and proton abstraction from N3 of the thymidine base (pg. 599, col. 1, para. 2). Further, arginine at position 51 is the last residue in recited catalytic domain having the LIDDR sequence, also referred as to position 57 in DarT enzymes in the specification ([0195]). This arginine is the active site of DarT enzyme and lysine at position 50 is essential for DNA-binding (Extended Fig. 4b); thus, these two residues are functionally constrained. However, the state of the art does not establish the recited catalytic domain having the LIDDR sequence is catalytically active. The state of the art does not recognize LIDDR to be a representative sequence of catalytic domains for DNA-modifying domains.
Based on the preponderance of the evidence, including the relevant teachings of the specification, the absence of working examples, and the state of prior art including the knowledge of DarT active site and catalytic domains having the sequence LIDDR, one skilled in the art would conclude that Applicant was not in possession of the claimed genus of DNA-modifying domains that comprises a catalytic domain having at least 70% amino acid sequence identity to LIDDR.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-2, 6-7, 9, 11, 13-14, and 37 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Liu et al (WO 2018/165629 A1; Published Date Sept 13, 2018) as evidenced by Cuniasse et al (The Abasic Site as a Challenge to DNA Polymerase; J. Mol. Biol., 1990, 213:303-314).
Regarding claim 1, Liu et al teach a composition for targeted genome modification, the composition comprising base editing fusion protein (i.e., a gap editor complex) comprising (i) a DNA binding protein (i.e., a DNA-recognition domain) that binds a DNA target sequence in the genome, and (ii) a cytidine deaminase (i.e., DNA-modifying domain) that converts a first nucleobase in the DNA target sequence to a second nucleobase, which is then excised, thereby creating an abasic site ([0002], [0003], [00253]).
Liu et al does not explicitly state that the DNA-modifying domain induces formation of a replication blocking moiety on at least one nucleotide in the genome as required by the claim.
Cuniasse et al teach an abasic site in DNA is a mutagenic base lesion that act as strong blocks for replication either by preventing binding of the DNA polymerase to the DNA template or blocking the progression of the DNA polymerase as it travels up the helix (pg. 303, col. 2, para. 1; pg. 313, col. 2, para. 4).
Thus, based on the teachings of Cuniasse et al, the abasic site induced by the cytidine deaminase in the gap editor complex of Liu et al is considered as a replication blocking moiety. Accordingly, the gap editor complex of Liu et al comprises a DNA-recognition domain that binds a DNA target sequence in a genome, and a DNA-modifying domain that induces formation of a replication blocking moiety on at least one nucleotide in the genome.
Regarding claim 6, Liu et al teach wherein the composition comprises at least one guide RNA molecules (FIG. 6, [00249]).
Regarding claim 7, Liu et al teach wherein the DNA-recognition comprises at least one Cas protein (e.g., Cas9 domain) or fragment thereof lacking deoxyribonuclease activity (e.g., nuclease inactive Cas9) (claims 37 and 43).
Regarding claim 9, Liu et al teach wherein the DAN-recognition domain comprises a Cas protein (e.g., Cas9 domain) or fragment thereof having nickase activity (e.g., Cas9 nickase) (claims 37 and 40).
Regarding claim 11, Liu et al teach wherein the DNA-recognition domain and the DNA-modifying domain are functionally coupled (e.g., fused together via a linker) (claim 35).
Regarding claim 13, Liu et al teach wherein the DNA-modifying domain blocks DNA replication by adding the replication blocking moiety (e.g., abasic site) to (i) at least one nucleotide in the DNA strand complementary to the DNA target sequence (FIG. 2).
Regarding claim 14, Liu et al teach wherein the DNA-recognition domain induces a single stranded break (e.g., Cas9 nickase) in the DNA target strand, and wherein the DNA-modifying domain adds the replication blocking moiety to at least one nucleotide in the DNA strand complementary to the DNA target sequence (claims 37 and 40; FIG. 2).
Regarding claim 37, Liu et al teach a kit comprising the gap editor complex of claim 1 ([00267], claim 147).
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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 17 and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Liu et al (WO 2018/165629 A1; Published Date Sept 13, 2018) as evidenced by Cuniasse et al (The Abasic Site as a Challenge to DNA Polymerase; J. Mol. Biol., 1990, 213:303-314) as applied to claim 1 above, and in view of Doudna et al (US20140068797 A1; Published Date: March 06, 2021) and Lawaree et al (DNA ADP-Ribosylation Stalls Replication and Is Reversed by RecF-Mediated Homologous Recombination and Nucleotide Excision Repair; Cell Reports, 2020, 30:1373-1384).
Regarding claim 17, the teachings of Liu et al regarding a composition comprising a DNA-recognition domain and a DNA-modifying domain are discussed above as applied to claim 1. Liu et al teach wherein the DNA-modifying domain comprises a cytidine deaminase that introduces a targeted DNA lesion that is subsequently processed by endogenous DNA repair pathways to yield desired genetic modification ([0002], [0003], [00253]).
However, Liu et al do not teach wherein the DNA-modifying domain comprises a DarT enzyme or a functional fragment, derivative, or variant thereof.
Doudna et al teach a chimeric site-directed modifying polypeptide (i.e., a gap editor complex) comprising (i) an RNA-binding domain (i.e., CRISPR Cas protein) that interacts with a DNA-targeting RNA (i.e., guide RNA), and (ii) an activity portion that exhibits site-directed enzymatic activity (i.e., DNA-modifying domain), wherein the site of enzymatic activity is determined by the DNA-targeting RNA (claim 18). Further, Doudna et al teach the activity portion has enzymatic activity that modifies target DNA, including deamination activity, methyltransferase activity, demethylase activity, alkylation activity, depurination activity, and etc. ([0234]). Accordingly, Doudna et al teach the activity portion or DNA-modifying domain recited in instant claims are substitutable to modify nucleotides of the target DNA.
In addition, Lawaree et al teach a toxin DarT from enteropathogenic Escherichia coli catalyzes ADP-ribosylation on single-stranded DNA, stalling DNA replication and increasing RecA levels as cells perceive the ADP-ribosylation modification as DNA damage (pg. 1373, col. 2, para. 2 and para. 3; pg. 1376, col. 1, para. 5; Figure 3A and 3B). Lawaree et al further teach that DarT preferentially ADP-ribosylates the sequences TTT or TCT (Figure 1B and Suppl. Figure 2B; pg. 1376, coo. 1, para. 3). Lawaree et al further teach that ADP-ribosylation installed by DarT is a DNA lesion that blocks replication forks, and removal of DNA ADP-ribose is completed via RecF-mediated homologous recombination and nucleotide excision repair pathway (NER) (Figure 7; paragraph bridging pg. 1380 and pg. 1381; paragraph bridging col. 1 and 2 on pg. 1381). Specifically, “initiation of Okazaki fragment synthesis from a new primer downstream of the lesion leaves a single-stranded DNA gap that is filled via RecF-mediated homologous recombination” (paragraph bridging pg. 1380 and pg. 1381), and NER acts after RecF-mediated homologous recombination to remove the DNA ADP-ribose installed by DarT (Figures 6A and 6C; paragraph bridging col. 1 and 2 on pg. 1381). Additionally, Lawaree et al teach that abasic sites is also a DNA lesion dealt with by NER (pg. 1381, col. 2).
Thus, it would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to have modified the DNA-modifying domain of Liu et al with the DarT enzyme of Lawaree et al because it would have merely amounted to a simple substitution of prior art elements according to known methods to yield predictable results. The substituted components (cytidine deaminase and DarT) and its function (generating a localized DNA lesion that initiates endogenous DNA repair pathways including NER) were known in the art. One would have been motivated to have done so for the advantage of expanding genome editing tools and targetable sites with additional DNA-modifying domains that recognize different sequence motifs. One would have had a reasonable expectation of success in doing so because Doudna et al demonstrate the DNA-modifying domain, particularly a deaminase, can be substituted to alternative domains exhibiting diverse enzymatic activity to install desirable modifications on site-specific nucleotides in the target DNA, and Lawaree et al teach that lesions induced by DarT are repaired through homologous recombination and NER, a repair pathway that Liu’s DNA-modifying domain also relies on. One would have reasonably expected DarT to function as an alternative DNA-modifying domain capable of inducing formation of a replication block moiety on a desired target DNA sequence localized by the DNA-recognition domain.
Regarding claim 19, the obviousness to modify the DNA-modifying domain in the gap editor complex of Liu et al with a DarT enzyme is discussed above as applied to claim 17. Lawaree et al further teach wherein the DarT enzyme comprises a G49D amino acid substitution as the substituted glycine is not a conserved residue in DarT from Thermus aquaticus and this single point mutation allows DarT to be less lethal to cells while retaining ADP-ribosylation activity (pg. 1374, col. 2, para. 2).
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Liu et al (WO 2018/165629 A1; Published Date Sept 13, 2018) as evidenced by Cuniasse et al (The Abasic Site as a Challenge to DNA Polymerase; J. Mol. Biol., 1990, 213:303-314), in view of Doudna et al (US20140068797 A1; Published Date: March 06, 2021) and Lawaree et al (DNA ADP-Ribosylation Stalls Replication and Is Reversed by RecF-Mediated Homologous Recombination and Nucleotide Excision Repair; Cell Reports, 2020, 30:1373-1384) as applied to claim 17 above, and further in view of Schuller et al (Molecular basis for DarT ADP-ribosylation of a DNA base; Nature, 2021, 596:597-602).
Regarding claim 18, the teachings of Liu et al regarding a composition comprising a DNA-recognition domain and a DNA-modifying domain are discussed above as applied to claim 1. Lawaree et al teach a DarT enzyme from enteropathogenic Escherichia coli comprises a catalytic domain having the sequence LIGKR from positions 47 to 51, as evidenced by Schuller et al (Extended Fig. 4b).
However, neither Liu et al nor Lawaree et al teach a DNA-modifying domain that comprises a catalytic domain having at least 70% amino acid sequence identity to LIDDR (elected sequence).
Schuller et al teach DarT enzyme from Thermus sp. 2.9 transfer ADP-ribose from NAD+ onto thymidine bases in single-stranded DNA, specifically at the four-base motif TNTC (pg. 597, col. 1, para. 1). Further, arginine at position 51 is essential for polarization of the NAD+ molecule and proton abstraction from N3 of the thymidine base (pg. 599, col. 1, para. 2). The arginine at position 51 is the last residue in recited catalytic domain having the LIDDR sequence, also referred to as position 57 in DarT enzymes in the specification ([0195]). This arginine is the active site of DarT enzyme across multiple bacteria species (Extended Fig. 4b). Notably, DarT from Enterobacter hormaechei contains the sequence LIDKR at positions 47 to 51, which is only one amino acid different than instantly recited sequence LIDDR (residue highlighted for emphasis). Schuller et al recognizes the positively-charged side chain of this lysine residue interacts with the DNA fragment to stabilize the negatively-charged phosphate-ribose backbone (pg. 598, col. 2, para. 2; Fig. 2a and 2b); this lysine residue is equivalent to arginine at position 50 of DarT from Thermus sp. 2.9 discussed in text. Additionally, Schuller et al discloses that expression of wild-type DarT is lethal to cells, but DarT comprising mutation G49D is a characterized DarT mutant that retains ssDNA ADP-ribosylation activity but is less toxic for recombinant expression (caption of Extended Fig. 6b).
The obviousness to modify the DNA-modifying domain of Liu et al with a DarT enzyme as taught by Lawaree et al is discussed above as applied to claim 17. Further, it would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to have modified the lysine residue at position 50 in DarT from Enterobacter hormaechei as taught by Schuller et al because it would have merely amounted to a simple use of known technique to improve similar devices in the same way. Schuller et al demonstrates that specific mutations in DarT enable its recombinant expression in cells while retaining ADP-ribosylation activity, and lysine at position 50 is known to interact with the phosphate-ribose backbone of the DNA which DarT installs the ADP-ribose to. Thus, one would have been motivated to have mutated this lysine residue to an amino acid residue without positively-charged side chain groups, including amino acids with negatively-charged side chains such as aspartic acid and glutamic acid, or amino acids with uncharged side chains. In addition, the recited sequence, LIDDR, contains an aspartic acid residue in place of lysine represents a finite number of identified predictable potential outcomes. One would have been motivated to have done so for the advantage of expressing active DarT enzymes in cells via rationally designed mutations in regions known to participate in the process of ADP-ribosylation. One would have had a reasonable expectation of success in doing so because Schuller et al recognizes the role of lysine at position 50 and demonstrates DarT enzymes containing rationally-designed mutations can be expressed in cells while retaining catalytic activity.
Claim 2 is rejected under 35 U.S.C. 103 as being unpatentable over Liu et al (WO 2018/165629 A1; Published Date Sept 13, 2018) as evidenced by Cuniasse et al (The Abasic Site as a Challenge to DNA Polymerase; J. Mol. Biol., 1990, 213:303-314) ), in view of Doudna et al (US20140068797 A1; Published Date: March 06, 2021) and Lawaree et al (DNA ADP-Ribosylation Stalls Replication and Is Reversed by RecF-Mediated Homologous Recombination and Nucleotide Excision Repair; Cell Reports, 2020, 30:1373-1384) as applied to claims 1 and 17 above, and further in view of Ran et al (Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity; Cell, 2013, 154 (6): 1380-1389).
Regarding claim 2, the teachings of Liu et al regarding a composition comprising a gap editor complex that comprises a DNA-recognition domain and a DNA-modifying domain are discussed above as applied to claim 1. Liu et al further teach that the DNA-modifying domain is a cytidine deaminase, which deaminates a cytosine within the DNA target sequence to a uridine that can then be excised by uracil DNA glycosylase, thereby generating an abasic site within the DNA target sequence ([0002]). The nucleobase opposite the abasic site can then be replaced with another base by an endogenous translesion polymerase ([0003]). Thus, the composition of Liu et al does not require a donor nucleic acid template because DNA lesions or replication blocking moiety induced by cytidine deaminase are resolved through endogenous base excision or mismatch repair pathways that directly convert one base to another.
The teachings of Lawaree et al regarding DarT and its ability to install ADP-ribosylation on single-stranded DNA are discussed above as applied to claim 17. Lawaree et al teach that ADP-ribosylation installed by DarT is a DNA lesion that blocks replication forks, and removal of DNA ADP-ribose is completed via RecF-mediated homologous recombination and nucleotide excision repair pathway (NER) (Figure 7; paragraph bridging pg. 1380 and pg. 1381; paragraph bridging col. 1 and 2 on pg. 1381). Specifically, “initiation of Okazaki fragment synthesis from a new primer downstream of the lesion leaves a single-stranded DNA gap that is filled via RecF-mediated homologous recombination” (paragraph bridging pg. 1380 and pg. 1381), and NER acts after RecF-mediated homologous recombination to remove the DNA ADP-ribose installed by DarT (Figures 6A and 6C; paragraph bridging col. 1 and 2 on pg. 1381).
Further, Ran et al teach a composition comprising two DNA-recognition domains that binds a DNA target sequence in the genome, and wherein the DNA-recognition domains comprise Cas9 proteins having nickase activity (Figure 2; pg. 1382). Ran et al also teach this composition enables precise editing of genomic target sites via homology-directed repair (HDR). Specifically, two single-guide RNAs designed with an offset by -3 and +18 base pairs localize two separate Cas9 nickases to the human EMX1 locus to introduce single-stranded breaks at opposite strands of the DNA (pg. 1384, col. 2, para. 1). The single-stranded breaks (SSB) induce endogenous HDR to incorporate a single-stranded oligodeoxynucleotide (i.e., donor nucleic acid template) bearing modified sequences at the site of SSB. Ran et al further teach that this strategy successfully induced HDR at frequencies higher than those of single-guide Cas9 nickase and comparable to those of wild-type Cas9 that introduced double-stranded breaks.
The obviousness to substitute the DNA-modifying domain of Liu et al to a DarT enzyme as taught by Lawaree et al is discussed above as applied to claim 17. Considering the teachings of Ran et al, it would have also been obvious to one of ordinary skill in the art before the effective filling date of the invention to incorporate a donor nucleic acid temple into the composition because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. Each component in the combination performs the same function as they do separately: the DNA-recognition domain (e.g., Cas protein having nickase activity) binds a DNA target sequence in the genome, the DNA-modifying domain (e.g., DarT enzyme) installs a replication blocking moiety on at least one nucleotide in the genome (e.g., ADP-ribosylation) that is repaired via homologous recombination pathways, and the donor nucleic acid template provides predetermined sequences for incorporation into the DNA target sequence also via endogenous homology recombination pathways. One would have been motivated to do so for the advantage of inserting donor nucleic acid templates encoding desirable genomic sequences, including protein tags, recombination sites, mutations, and exogenous genes, into an endogenous locus. One would have had a reasonable expectation of success in doing so because Ran et al demonstrates successful homologous recombination of a donor nucleic acid template into a genomic site bearing two DNA lesions (e.g, (i) two single-stranded breaks, or (ii) one single-stranded break and one replication blocking moiety generated by a gap editor complex comprising a Cas9 nickase and a DarT enzyme), and Lawaree et al teach that the DNA lesion generated by ADP-ribosylation from DarT is also repaired via homologous recombination.
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 1-2, 6-7, 9, 11, 13-14, 17-19, and 37 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-2, 4, 6-7, 11, 17, and 35 of U.S. Patent No. 12, 649,917 in view of Cuniasse et al (The Abasic Site as a Challenge to DNA Polymerase; J. Mol. Biol., 1990, 213:303-314), Lawaree et al (DNA ADP-Ribosylation Stalls Replication and Is Reversed by RecF-Mediated Homologous Recombination and Nucleotide Excision Repair; Cell Reports, 2020, 30:1373-1384), and Schuller et al (Molecular basis for DarT ADP-ribosylation of a DNA base; Nature, 2021, 596:597-602).
Regarding instant claim 1, ‘917 recites a composition for targeted editing of a nucleic acid (i.e., targeted genome modification, the composition comprising (a) a gap editor complex comprising a DNA-recognition domain and a DNA-modifying domain (claim 1). ‘917 further recites wherein the DNA-recognition domain comprises a Type II Cas9 protein lacking deoxyribonuclease activity (claim 4); thus, ‘917 recites wherein the DNA-recognition domain binds a DNA target sequence in the genome as it is an inherent property of Type II Cas9 protein. Further, ‘917 recites wherein the gap editor complex induces formation of at least one abasic site in a target sequence of a DNA (claim 1), and wherein DNA-modifying domain is a cytidine deaminase (claim 11). As supported by Cuniasse et al, an abasic site is a mutagenic DNA lesion that blocks replication (pg. 303, col. 2, para. 1; pg. 313, col. 2, para. 4). Accordingly, ‘917 recites a DNA-modifying domain that induces formation of a replication blocking moiety on at least one nucleotide in the genome.
Regarding instant claim 2, ‘917 recites wherein the composition comprises a donor nucleic acid template (claim 1).
Regarding instant claim 6, ‘917 recites wherein the composition comprises at least one guide RNA molecule (claim 1).
Regarding instant claims 7 and 9, ‘917 recites wherein the DNA-recognition domain comprises at least one Cas protein lacking deoxyribonuclease activity (claim 2), or a Cas protein having nickase activity (claim 6).
Regarding instant claim 11, ‘917 recites wherein the DNA-recognition domain and the DNA-modifying domain comprise a functional coupling (claim 7).
Regarding instant claims 13-14, ‘917 recites wherein the DNA-recognition domain comprises a Cas protein having nickase activity (claim 6); thus, ‘917 recites a DNA-recognition domain that induces single-stranded break in the DNA target strand. Further, 917 recites the DNA-modifying domain adds an abasic site (i.e., replication blocking moiety) to at least one nucleotide in the DNA strand complementary to the DNA target sequence (claim 17).
Regarding instant claim 17, ‘917 recites wherein the DNA-modifying domain comprises a cytidine deaminase (claim 11). ‘917 does not recite wherein the DNA-modifying domain comprises a DarT enzyme or a functional fragment, derivate, or variant thereof. ‘917 does not recite wherein the DarT enzyme comprises the recited amino acid substitutions.
Lawaree et al teach a toxin DarT from enteropathogenic Escherichia coli catalyzes ADP-ribosylation on single-stranded DNA, stalling DNA replication and increasing RecA levels as cells perceive the ADP-ribosylation modification as DNA damage (pg. 1373, col. 2, para. 2 and para. 3; pg. 1376, col. 1, para. 5; Figure 3A and 3B). Lawaree et al further teach that DarT preferentially ADP-ribosylates the sequences TTT or TCT (Figure 1B and Suppl. Figure 2B; pg. 1376, coo. 1, para. 3). Lawaree et al further teach that ADP-ribosylation installed by DarT is a DNA lesion that blocks replication forks, and removal of DNA ADP-ribose is completed via RecF-mediated homologous recombination and nucleotide excision repair pathway (NER) (Figure 7; paragraph bridging pg. 1380 and pg. 1381; paragraph bridging col. 1 and 2 on pg. 1381). Specifically, “initiation of Okazaki fragment synthesis from a new primer downstream of the lesion leaves a single-stranded DNA gap that is filled via RecF-mediated homologous recombination” (paragraph bridging pg. 1380 and pg. 1381), and NER acts after RecF-mediated homologous recombination to remove the DNA ADP-ribose installed by DarT (Figures 6A and 6C; paragraph bridging col. 1 and 2 on pg. 1381). Additionally, Lawaree et al teach that abasic sites is also a DNA lesion dealt with by NER (pg. 1381, col. 2).
Thus, it would have been obvious to one of ordinary skill in the art to have modified the DNA-modifying domain of ‘917 with the DarT enzyme of Lawaree et al because it would have merely amounted to a simple substitution of prior art elements according to known methods to yield predictable results. The substituted components (cytidine deaminase and DarT) and its function (generating a localized DNA lesion that initiates endogenous DNA repair pathways including NER) were known in the art. One would have been motivated to have done so for the advantage of expanding genome editing tools and targetable sites with additional DNA-modifying domains that recognize different sequence motifs. One would have had a reasonable expectation of success in doing so because Lawaree et al teach that lesions induced by DarT are repaired through homologous recombination and NER. One would have reasonably expected DarT to function as an alternative DNA-modifying domain capable of inducing formation of a replication block moiety on a desired target DNA sequence localized by the DNA-recognition domain.
Regarding instant claim 18, ‘917 does not recite wherein the DNA-modifying domain comprises a catalytic domain having at least 70% amino acid sequence identity to LIDDR (elected sequence).
Schuller et al teach DarT enzyme from Thermus sp. 2.9 transfer ADP-ribose from NAD+ onto thymidine bases in single-stranded DNA, specifically at the four-base motif TNTC (pg. 597, col. 1, para. 1). Further, arginine at position 51 is essential for polarization of the NAD+ molecule and proton abstraction from N3 of the thymidine base (pg. 599, col. 1, para. 2). The arginine at position 51 is the last residue in recited catalytic domain having the LIDDR sequence, also referred as to position 57 in DarT enzymes in the specification ([0195]). This arginine is the active site of DarT enzyme across multiple bacteria species (Extended Fig. 4b). Notably, DarT from Enterobacter hormaechei contains the sequence LIDKR at positions 47 to 51, which is only one amino acid different than instantly recited sequence LIDDR (residue highlighted for emphasis). Schuller et al recognizes the positively-charged side chain of this lysine residue interacts with the DNA fragment to stabilize the negatively-charged phosphate-ribose backbone (pg. 598, col. 2, para. 2; Fig. 2a and 2b); this lysine residue is equivalent to arginine at position 50 of DarT from Thermus sp. 2.9 discussed in text. Additionally, Schuller et al discloses that expression of wild-type DarT is lethal to cells, but DarT comprising mutation G49D is a characterized DarT mutant that retains ssDNA ADP-ribosylation activity but is less toxic for recombinant expression (caption of Extended Fig. 6b).
The obviousness to modify the DNA-modifying domain of ‘917 with a DarT enzyme as taught by Lawaree et al is discussed above as applied to instant claim 17. Further, it would have been obvious to one of ordinary skill in the art to have modified the lysine residue at position 50 in DarT from Enterobacter hormaechei as taught by Schuller et al because it would have merely amounted to a simple use of known technique to improve similar devices in the same way. Schuller et al demonstrates that specific mutations in DarT enable its recombinant expression in cells while retaining ADP-ribosylation activity, and lysine at position 50 is known to interact with the phosphate-ribose backbone of the DNA which DarT installs the ADP-ribose to. Thus, one would have been motivated to have mutated this lysine residue to an amino acid residue without positively-charged side chain groups, including amino acids with negatively-charged side chains such as aspartic acid and glutamic acid, or amino acids with uncharged side chains. In addition, the recited sequence, LIDDR, contains an aspartic acid residue in place of lysine represents a finite number of identified predictable potential outcomes. One would have been motivated to have done so for the advantage of expressing active DarT enzymes in cells via rationally designed mutations in regions known to participate in the process of ADP-ribosylation. One would have had a reasonable expectation of success in doing so because Schuller et al recognizes the role of lysine at position 50 and demonstrates DarT enzymes containing rationally-designed mutations can be expressed in cells while retaining catalytic activity.
Regarding instant claim 19, the obviousness to modify the DNA-modifying domain in the gap editor complex of ‘917 with a DarT enzyme is discussed above as applied to instant claim 17. Lawaree et al further teach wherein the DarT enzyme comprises a G49D amino acid substitution as the substituted glycine is not a conserved residue in DarT from Thermus aquaticus and this single point mutation allows DarT to be less lethal to cells while retaining ADP-ribosylation activity (pg. 1374, col. 2, para. 2).
Regarding instant claim 37, ‘917 recites a kit comprising the gap editor complex of instant claim 1 (claim 35).
Claims 1-2, 6-7, 9, 11, 13-14, 17-19, and 37 provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 4, 6-7, 9, 11, 13, 16-19, and 37 of copending Application No. 19/521,759 (reference application) in view of Lawaree et al (DNA ADP-Ribosylation Stalls Replication and Is Reversed by RecF-Mediated Homologous Recombination and Nucleotide Excision Repair; Cell Reports, 2020, 30:1373-1384) and Ran et al (Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity; Cell, 2013, 154 (6): 1380-1389). Although the claims at issue are not identical, they are not patentably distinct from each other because the copending claims recite the instantly claimed composition comprising the same gap editor complex, and the copending claims recite the same subject matter in instant claims.
Instant claim
Copending claim
1
1
6
4
7
7
9
9
11
11
13
6, 13,
14
14
17
16, 17
18
18
19
19
37
37
Regarding instant claim 2, ‘759 recite a composition of instant claim 1 (claim 1). Further, ‘759 recites the composition of claim 1, wherein the DNA-modifying domain comprises a DarT enzyme (claim 17).
However, ‘759 does not recite wherein the composition further comprises a donor nucleic acid template.
Lawaree et al teach a toxin DarT from enteropathogenic Escherichia coli catalyzes ADP-ribosylation on single-stranded DNA, stalling DNA replication and increasing RecA levels as cells perceive the ADP-ribosylation modification as DNA damage (pg. 1373, col. 2, para. 2 and para. 3; pg. 1376, col. 1, para. 5; Figure 3A and 3B). Lawaree et al further teach that DarT preferentially ADP-ribosylates the sequences TTT or TCT (Figure 1B and Suppl. Figure 2B; pg. 1376, coo. 1, para. 3). Lawaree et al further teach that ADP-ribosylation installed by DarT is a DNA lesion that blocks replication forks, and removal of DNA ADP-ribose is completed via RecF-mediated homologous recombination and nucleotide excision repair pathway (NER) (Figure 7; paragraph bridging pg. 1380 and pg. 1381; paragraph bridging col. 1 and 2 on pg. 1381). Specifically, “initiation of Okazaki fragment synthesis from a new primer downstream of the lesion leaves a single-stranded DNA gap that is filled via RecF-mediated homologous recombination” (paragraph bridging pg. 1380 and pg. 1381), and NER acts after RecF-mediated homologous recombination to remove the DNA ADP-ribose installed by DarT (Figures 6A and 6C; paragraph bridging col. 1 and 2 on pg. 1381).
Further, Ran et al teach a composition comprising a gap editor complex comprising two DNA-recognition domains that binds a DNA target sequence in the genome, and wherein the DNA-recognition domains comprise Cas9 proteins having nickase activity (Figure 2; pg. 1382). Ran et al also teach this composition enables precise editing of genomic target sites via homology-directed repair (HDR). Specifically, two single-guide RNAs designed with an offset by -3 and +18 base pairs localize two separate Cas9 nickases to the human EMX1 locus to introduce single-stranded breaks at opposite strands of the DNA (pg. 1384, col. 2, para. 1). The single-stranded breaks (SSB) induce endogenous HDR to incorporate a single-stranded oligodeoxynucleotide (i.e., donor nucleic acid template) bearing modified sequences at the site of SSB. Ran et al further teach that this strategy successfully induced HDR at frequencies higher than those of single-guide Cas9 nickase and comparable to those of wild-type Cas9 that introduced double-stranded breaks.
Considering the teachings of Lawaree et al that cells rely on homologous recombination to repair the DNA lesion generated by DarT, it would have been obvious to one of ordinary skill in the art to incorporate a donor nucleic acid temple into the composition of ‘759 comprising a DarT because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. Each component in the combination performs the same function as they do separately: the DNA-recognition domain (e.g., Cas protein having nickase activity) binds a DNA target sequence in the genome, the DNA-modifying domain (e.g., DarT enzyme) installs a replication blocking moiety on at least one nucleotide in the genome (e.g., ADP-ribosylation) that is repaired via homologous recombination pathways, and the donor nucleic acid template provides predetermined sequences for incorporation into the DNA target sequence also via endogenous homology recombination pathways. One would have been motivated to do so for the advantage of inserting donor nucleic acid templates encoding desirable genomic sequences, including protein tags, recombination sites, mutations, and exogenous genes, into an endogenous locus. One would have had a reasonable expectation of success in doing so because Ran et al demonstrates successful homologous recombination of a donor nucleic acid template into a genomic site bearing two DNA lesions (e.g, (i) two single-stranded breaks, or (ii) one single-stranded break and one replication blocking moiety generated by a gap editor complex comprising a Cas9 nickase and a DarT enzyme), and Lawaree et al teach that the DNA lesion generated by ADP-ribosylation from DarT is also repaired via homologous recombination.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Conclusion
No claims are allowable.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to QIWEN SU-TOBON whose telephone number is (571)272-0331. The examiner can normally be reached Monday - Friday, 9:30am - 5:00pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Neil Hammell can be reached at 571-270-5919. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/QIWEN SU-TOBON/
Examiner
Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636
Prosecution Insights