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
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Acknowledgment is made of applicant's claim for foreign priority based on the applications filed as KR 10-2021-0114750 on 08/30/2021, KR 10-2021-0092056 on 07/14/2021, KR 10-2021-0085474 on 06/30/2021, KR 10-2021-0085473 on 06/30/2021, KR 10-2021-0050497 on 04/19/2021, KR 10-2021-0049348 on 04/15/2021, KR 10-2021-0016788 on 02/05/2021, KR 10-2021-0013263 on 01/29/2021, KR 10-2020-0159920 on 11/25/2020 and KR 10-2020-0120399 on 09/18/2020.
It is noted, however, that applicant has not filed an English translation of the certified copies as required by 37 CFR 1.55.
All claims are given the filing date of 09/17/2021.
Application Status
Receipt is acknowledged of amendment, filed 09/24/2024, in which claims 1-54 were cancelled, and claims 55-61 and 64-98 were added.
Information Disclosure Statement
Receipt of acknowledgment of the information disclosure statements filed on 03/09/2023, 07/22/2024, 12/05/2024 and 03/13/2025 have been received and all references have been considered.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code (Paragraphs [931], [966], [975] and [999]). Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Claim Objections
The numbering of claims is not in accordance with 37 CFR 1.126 which requires the original numbering of the claims to be preserved throughout the prosecution. When claims are canceled, the remaining claims must not be renumbered. When new claims are presented, they must be numbered consecutively beginning with the number next following the highest numbered claims previously presented (whether entered or not).
Misnumbered claims 64-98 have been renumbered as 62-96.
Claim 65 objected to because of the following informalities:
Claim 65 shows the first instance of the abbreviations “CRISPR” and “TALE”. It would be remedial to replace the abbreviations with “Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR)” and “Transcription Activator-Like Effector (TALE)” respectively.
Appropriate correction is required.
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 55-57, 59-79 and 81-96 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 55 is drawn to a genus of cytosine deaminase. The rejected claims thus comprise a genus of cytosine deaminase that encompass the “first split derived from a cytosine deaminase or a variant thereof,” and “a second split derived from a cytosine deaminase or a variant thereof”. The number and type of mutations relative to the recited sequences is not limited in any way, yet the variant must be capable of functioning as a cytidine deaminase within a fusion protein for genome editing within a cell.
Claim 57 depends from claim 55 and recites, “wherein the cytosine deaminase is derived from a double-stranded DNA deaminase (DddA) or an orthologue thereof.” Thus, the claim encompasses any cytosine deaminase sequence that has been derived by any number of changes relative to any DddA, as well as any orthologs of DddA or the derivatives. The derivatives and orthologues thereof must be capable of functioning within a fusion protein for genome editing within a cell.
Claim 59 depends from claim 55 and recites, “wherein the first split comprises the amino acid sequence of SEQ ID NO: 23, wherein at least one amino acid selected from the group consisting of amino acids at positions 3, 5, 10, 11, 13, 14, 15, 16, 17, 18, 19, 28, 30 and 31 of SEQ ID NO: 23 is substituted with a different amino acid, and wherein the second split comprises the amino acid sequence of SEQ ID NO: 24, wherein at least one amino acid selected from the group consisting of amino acids at positions 13, 16, 17, 20, 21, 28, 29, 30, 31, 32, 33, 56, 57, 58 and 60 of SEQ ID NO: 24 is substituted with a different amino acid.” Claim 61 depends from claim 59 and specifies that the one or more substitutions are alanine residues. The claim encompasses variants where one or more of the recited amino acid positions is substituted with any other amino acid or alanine. The variant must be capable of functioning as a cytidine deaminase within a fusion protein for genome editing within a cell.
Claim 60 depends from claim 55 and recites, “wherein the first split comprises the amino acid sequence of SEQ ID NO: 25, wherein at least one amino acid selected from the group consisting of amino acids at positions 87, 88, 91, 92, 95, 100, 101, 102 and 103 of SEQ ID NO: 25 is substituted with a different amino acid, and wherein the second split comprising the amino acid sequence of SEQ ID NO: 26, wherein at least one amino acid selected from the group consisting of amino acids at positions 13, 14, 15 and 16 of SEQ ID NO: 26 is substituted with a different amino acid.” The claim encompasses variants where one or more of the recited amino acid positions is substituted with any other amino acid. The variant must be capable of functioning as a cytidine deaminase within a fusion protein for genome editing within a cell.
Claim 56 is drawn to a genus of cytosine deaminase. The rejected claims thus comprise a genus of cytosine deaminase that encompass “a non-toxic full-length variant of a cytosine deaminase”. The number and type of mutations relative to the recited sequences is not limited in any way, yet the variant must be capable of functioning as a cytidine deaminase within a fusion protein for genome editing within a cell while remaining non-toxic.
Claim 78 depends from claim 56 and recites, “wherein the cytosine deaminase is derived from a double-stranded DNA deaminase (DddA) or an orthologue thereof.” Thus, the claim encompasses any cytosine deaminase sequence that has been derived by any number of changes relative to any DddA, as well as any orthologs of DddA or the derivatives. The derivatives and orthologues thereof must be capable of functioning within a fusion protein for genome editing within a cell while remaining non-toxic.
Claim 79 depends from claim 56 and recites, “wherein the non-toxic full-length variant of a cytosine deaminase comprises the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid is selected from the group of amino acids at positions 37, 59, 109 and 129 is substituted with a different amino acid. The claim encompasses variants where any one or more of the recited amino acid positions is substituted with any other amino acid. The variant must be capable of functioning as a cytidine deaminase within a fusion protein for genome editing within a cell while remaining non-toxic.
Claims 65, 66, 84 and 85 drawn to a genus of cytosine deaminase. The rejected claims thus comprise a genus of cytosine deaminase that encompass “a cytosine deaminase comprising the amino acid sequence of SEQ ID NO: 1, or an ortholog thereof”. The claims encompass genes in various species related by a common ancestor to the sequence of SEQ ID NO: 1, where the ortholog must be capable of functioning for C to T base editing.
Claims 68 and 87 are drawn to a genus of adenine deaminase. The rejected claims thus comprise a genus of adenine deaminase that encompass “variant of E. coli TadA”. The number and type of mutations relative to the recited sequences is not limited in any way, yet the variant must be capable of functioning as an adenine deaminase capable of functioning within a fusion protein for genome editing within a cell.
Claims 69 and 88 are drawn to a genus of cytosine deaminase and adenine deaminase. The rejected claims thus comprise a genus of cytosine deaminase and adenine deaminase that encompass “an amino acid sequence of SEQ ID NO: 1, or an ortholog thereof,” and “an amino acid sequence of SEQ ID NO: 458, or variant thereof”. The claims encompass genes in various species related by a common ancestor to the sequence of SEQ ID NO: 1, where the ortholog must be capable of functioning as a cytosine deaminase. With regard to SEQ ID NO: 458, the number and type of mutations relative to the recited sequences is not limited in any way, yet the variant must be capable of functioning as an adenine deaminase capable of functioning within a fusion protein for genome editing within a cell.
To provide adequate written description and evidence of possession of a claimed genus, the specification must provide sufficient distinguishing identifying characteristics of the genus. The factors to be considered include disclosure of a complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, and any combination thereof. The specification describes specific variants of the full-length cytosine deaminase as SEQ ID NOs: 12-22 that were proved to be non-toxic variants of the wild-type sequence for use in the fusion protein with a programmable DNA binding protein [0230-0243, 0251-0252 and 0255-0261]. The sequences provided in the specification as SEQ ID NOs: 12-22 are variants of SEQ ID NO: 1 with specific residue mutations at different amino acid positions such as Al341D KRKKA (SEQ ID No: 12), AAAAA (SEQ ID No: 13), AAAAK (SEQ ID No: 14), AAKAA (SEQ ID No: 15), AAKAK (SEQ ID No: 16), KAAAA (SEQ ID No: 17), El347A (SEQ ID No: 18), GSVG (SEQ ID No: 19), SSVG (SEQ ID No: 20), GSAG (SEQ ID No: 21) and GSVS (SEQ ID No: 22), respectively [230, 232, 234, 236, 238, 240, 242, 251, 256 258, and 260]. The specification also envisions specific adenine deaminases such as APOBEC1, AID and tadA, specifically the sequence of ABE 8e is provided as SEQ ID NO: 458 [0319 and 0468-0469]. In regards to the split-DddAtox halves, the specification teaches DddAtox is cytotoxic, and thus, in order to avoid toxicity in host cells, DddAtox is split into two inactive halves, each of which is fused to a DNA-binding protein in a DddA-derived cytosine base editor (DdCBE) and wherein a functional deaminase is reassembled at a target DNA site, when two inactive halves are brought together by the DNA-binding protein [7]. The instant specification also teaches the DddAtox split system has many limitations in experiments such as many Gl397-split DddAtox variants, containing interface mutations such as C1376A, Ml390A and Fl412A, failed to induce C-to-T conversions in the spacer region between the two TALE-binding sites, even when combined with the wildtype partner, suggesting that these mutants cannot interact with other wildtype DddAtox half nearby at the target site [8 and 1007]. In regards to the full-length toxicity of DddA, the specification describes that since DNA is negatively charged, it binds to the positively charged amino acid of a protein and by substituting the positively charged amino acid with an amino acid that is not charged, binding force of DddAtox to DNA is weakened, thus reducing or eliminating cytotoxicity which results in a non-toxic combination resulting from substitution of a positively charged amino acid with a non-polar amino acid enables cloning using E. coli to afford full-length DddA [220]. The specification teaches that the results showed a limited number of non-toxic full-length DddA variants with reduced affinity for dsDNA (AAAAA), attenuated deaminase activity (E1347A and possibly GSVG), or reduced cytotoxicity (GSVG) that can be fused to dCas9 or nCas9 to create novel base editors with altered editing windows [1040]. Although a description is provided of specific variants that were capable of functioning comparable to the wild-type versions of the cytosine and adenine deaminases, no description is provided of all the variants that retain the original enzymatic function (e.g., adenosine deaminase or cytosine deaminase) or that switch from adenosine deaminase to cytosine deaminase function, or vice versa. The specification also provides no description on what orthologs of the cytosine deaminases would be capable of the claimed function of the claims.
Even if one accepts that the examples described in the specification meet the claim limitations of the rejected claims with regard to structure and function, the examples are only representative of the specific sequences listed within the SEQ ID NOs 12-22, listed in the claims, as well as specific substitutions of a charged amino acid with a non-polar amino acid and not the broad variants currently claimed. The results are not necessarily predictive of a cytidine or adenosine deaminase variant that is capable of functioning for the specific functions. Thus, it is impossible for one to extrapolate from the few examples described herein those variants that would necessarily meet the structural/functional characteristics of the rejected claims.
The prior art does not appear to offset the deficiencies of the instant specification in that it does not describe a set of variants of cytosine and adenine deaminases. Mok et al (Nat Commun 13, 4038 (2022); Supplemental Information Pages 1/30-30/30) teaches to obtain nontoxic, full-length DddAtox variants useful for base editing, they took two different approaches: structure-based, site-directed mutagenesis (Fig. 1) and random mutagenesis (Page 2, Column 1). the E1347A variant combined with the quintuple AAAAA mutation, however, failed to induce base editing (Page 2, Column 1). Mok teaches subcloning synthetic DNA segments encoding DddAtox variants, in which positively charged amino-acid residues were replaced with alanine (Fig. 1a), in an expression vector, reasoning that these variants would bind to negatively charged dsDNA with reduced affinity, potentially avoiding toxicity, however most of the Ala-substituted variants failed to produce E. coli transformants (Page 2, Column 2).
Guo et al (Mol Cell. 2023 May 18;83(10):1710-1724.e7) teaches that they subjected the protein sequences of DddAtox (1,264–1,427 amino acids [aa]) from Burkholderia cenocepacia to BLAST alignment to search for orthologs from the top 100 hits according to the following criteria: (1) 30%– 99.9% sequence similarity with DddAtox; ( 2) the presence of an SCP1.201-like deaminase domain; (3) unbiased selection of one DddAtox ortholog from a single subspecies; and (4) the presence of a conserved catalytic activation site ‘‘E’’ (E1347 for DddAtox) (Page 1712, Column 1 bridging Column 2). Gou teaches ultimately they obtained 13 candidates, with 10 conserved amino acids within the 1,343–1,395 aa region (including E1347) and high peptide similarity with DddAtox ranging from 36.5% to 90.6% (1,264–1,427 aa); however, considering the potential toxicity of these DddAtox orthologs, they were split into two parts referring to the G1333 or G1397 split site for DddAtox18; thus, 6 candidate deaminases possessing conserved amino acids at the G1333, G1397, and/or A1398 sites were further selected for the nuclear DNA editing test (Page 1712, Column 2). Gou teaches that there are a finite number of orthologs with similarity of sequence and function that would be capable of functioning as a cytosine deaminase for successful base editing with the fusion protein.
Therefore, the skilled artisan would have reasonably concluded applicants were not in possession of the claimed invention for claims 55-57, 59-79 and 81-96.
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 77, 86-88 and 96 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 77 and 96 contains the trademark/trade name “TALEN”. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify a transcription activator-like effector nuclease and, accordingly, the identification/description is indefinite. It would be remedial to amend the claim to replace the term “TALEN” with the phrase “TALE nuclease.”
Claim 86 recites the limitation "The composition for base editing according to claim 56, wherein at least one of the fusion proteins comprising the first split and the fusion protein comprising the second split further comprises an adenine deaminase" in Lines 1-2. Claim 56, for which claim 86 relies, does not require a fusion protein comprising a split.
There is insufficient antecedent basis for this limitation in the claim. It would be remedial to amend the claim to rely on claim 55 instead of claim 56.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 55, 57, 58, 60, 62-73, 76, 77 and 86-88 are rejected under 35 U.S.C. 102(a)(1)/(a)(2) as being anticipated by Liu et al (WO 2021/155065 A1) as evidenced by Liu et al (US 2018/0073012 A1).
Regarding claims 55, 57, Liu teaches engineered split-DddA halves that are
non-toxic and inactive until brought together on target DNA by adjacently bound
programmable DNA-binding proteins (Page 1, Abstract). Liu teaches the sequence of the DddAtox as SEQ ID NO: 338 which is 100% identical to instant SEQ ID NO: 1 (See Appendix I). Liu teaches DddAtox (G1333 DddAtox-N and G1333 DddAtox-C) halves split with the split at G44 for the N-terminal and P45 for the C-terminal and fused to a TALE nuclease ([0090, 0343 and 0344] and Page 605, Fig 26A).
Regarding claim 58 Liu teaches engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins (Page 1, Abstract). Liu teaches DddAtox (G1397 DddAtox-N and G1397 DddAtox-C) halves split with the split at G108 for the N-terminal and A109 for the C-terminal and fused to a TALE nuclease ([0089, 0345 and 0346] and Page 605, Fig 26A).
Regarding claim 60, Liu teaches the sequence of SEQ ID NO: 380 which comprises the residue substitutions of T1380I and T1413I which aligns with an amino acid residue substitution in instant SEQ ID NO: 25 at position 91 and instant SEQ ID NO: 26 at position 16 (See Appendices II and III) and [0150 and 0384]. Liu teaches the use of SEQ ID NO: 380 in both the G1333 and G1397 split orientation for fusion to a TALE nuclease [0767].
Regarding claims 62, 64 and 65, Liu teaches DddAtox (G1333 DddAtox-N and G1333 DddAtox-C) halves split with the split at G44 for the N-terminal and P45 for the C-terminal and fused to a TALE nuclease ([0090, 0343 and 0344] and Page 605, Fig 26A). Liu teaches the sequence of the DddAtox as SEQ ID NO: 338 which is 100% identical to instant SEQ ID NO: 1 (See Appendix I).
Regarding claims 63 and 66, Liu teaches the structure of the fusion protein as [mitoZFP]-[DddA half A] and [mitoZFP]-[DddA half B] [0031-0034]. Liu teaches the sequence of the DddAtox as SEQ ID NO: 338 which is 100% identical to instant SEQ ID NO: 1 (See Appendix I). Liu teaches a pair of mtDNA base editors each comprising a mitoZFP (mitoZFP A and mitoZFP B) fused to an inactive fragment of DddA (DddA halfA and DddA halfB) [0063].
Regarding claim 67, Liu teaches engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins (Page 1, Abstract). Liu teaches the sequence of the DddAtox as SEQ ID NO: 338 which is 100% identical to instant SEQ ID NO: 1 (See Appendix I). Liu teaches DddAtox (G1333 DddAtox-N and G1333 DddAtox-C) halves split with the split at G44 for the N-terminal and P45 for the C-terminal and fused to a TALE nuclease ([0090, 0343 and 0344] and Page 605, Fig 26A).
Regarding claims 67-69, the claim is interpreted as any two or more consecutive amino acids due to the claim language reciting “an amino acid sequence of SEQ ID NO: 458”.
Liu teaches the fusion protein comprising the first split and the fusion protein comprising the second split further comprises an adenine deaminase (TadA) derived from E. coli [0159, 0532 and 0709]. Liu teaches the sequence for the ecTadA used was taken from U.S. Patent 2018/0073012 A1. Liu et al (US 2018/0073012 A1) is only cited to show that the sequence of SEQ ID NO: 1 which represents the sequence of the ecTadA used in prior art Liu reference is 89% identical to instant SEQ ID NO: 458 (See Appendix IV).
Regarding claims 86-88, the claims are interpreted as being dependent upon claim 55 as previously described and applied above due to lack of antecedent basis. The claim is interpreted as any two or more consecutive amino acids due to the claim language reciting “an amino acid sequence of SEQ ID NO: 458”.
Liu teaches the fusion protein comprising the first split and the fusion protein comprising the second split further comprises an adenine deaminase (TadA) derived from E. coli [0159, 0532 and 0709]. Liu teaches the sequence for the ecTadA used was taken from U.S. Patent 2018/0073012 A1. Liu et al (US 2018/0073012 A1) is only cited to show that the sequence of SEQ ID NO: 1 which represents the sequence of the ecTadA used in prior art Liu reference is 89% identical to instant SEQ ID NO: 458 (See Appendix IV).
Regarding claims 70 and 71, the claims provide an intended use but do not further modify the structure of the base editing composition. See MPEP 2111.02(II).
Liu teaches the use of the split fusion proteins within HEK293T cells [0067].
Regarding claim 72, the claims provide an intended use but do not further modify the structure of the base editing composition. See MPEP 2111.02(II).
Liu teaches the use of the invention within cells originating from organisms such as animals, plants and fungi [0594].
Regarding claims 73 and 74, Liu teaches engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins (Page 1, Abstract). Liu teaches the composition is for nuclear DNA base editing and comprises a nuclear localization signal (NLS) peptide [0099]. Liu teaches mitochondrial DNA base editing and further comprising a mitochondrial targeting signal (MTS) [0098].
Regarding claim 76, Liu teaches the architecture of each monomer of a mitoTALE-split-DddAtox pair (in N- to C-terminus order): an MTS, a TALE array, a 2-amino acid linker, a DddAtox half from the G 1333 or G 1397 split, and one or two UGI proteins [0070].
Regarding claim 77, Liu teaches the TALE nuclease is capable of cleaving the wild-type but not the edited base sequence [0011]. Liu teaches the use of TC31 and TC32 which are FokI-based TALENs that target nuclear CCR5 [0072].
Claims 55, 57, 58, 62, 64, 65 and 72-76, are rejected under 35 U.S.C. 102(a)(1) as being unpatentable over Baek et al (Plants 7, 899–905 (July 1, 2021); Supplemental Information Pages 1/23-23/23).
Applicant cannot rely upon the certified copy of the foreign priority application to overcome this rejection because a translation of said application has not been made of record in accordance with 37 CFR 1.55. When an English language translation of a non-English language foreign application is required, the translation must be that of the certified copy (of the foreign application as filed) submitted together with a statement that the translation of the certified copy is accurate. See MPEP §§ 215 and 216.
Regarding claims 55 and 57, Baek teaches engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound
programmable DNA-binding proteins (Page 1, Abstract). Baek teaches DddAtox (G1333 DddAtox-N and G1333 DddAtox-C) halves split with the split at G44 for the N-terminal and P45 for the C-terminal and fused to a TALE nuclease (Supplemental Information Page 20).
Regarding claim 58, Baek teaches engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins (Page 1, Abstract). Baek teaches DddAtox (G1397 DddAtox-N and G1397 DddAtox-C) halves split with the split at G108 for the N-terminal and A109 for the C-terminal and fused to a TALE nuclease (Supplemental Information Page 20).
Regarding claims 62, 64 and 65, Baek teaches plasmids encode fusion proteins composed of a chloroplast transit peptide (CTP) or a mitochondrial targeting sequence (MTS), the TALE N- or C-terminal domains, split-DddAtox halves (G1333N, G1333C, G1397N and G1397C) and UGI, which are codon-optimized for expression in dicot plants, under the control of the parsley ubiquitin (PcUbi) promoter and pea3A terminator (Page 899, Column 2). Baek teaches DddAtox (G1397 DddAtox-N and G1397 DddAtox-C) halves split with the split at G108 for the N-terminal and A109 for the C-terminal and fused to a TALE nuclease (Supplemental Information Page 20). Baek teaches DddAtox (G1333 DddAtox-N and G1333 DddAtox-C) halves split with the split at G44 for the N-terminal and P45 for the C-terminal and fused to a TALE nuclease (Supplemental Information Page 20).
Regarding claims 72 and 75, Baek teaches a Golden Gate assembly system to construct chloroplast-targeting DdCBE (cp-DdCBE) plasmids or mitochondrial-targeting DdCBE (mt-DdCBE) plasmids (Page 899, Column 2). Baek teaches plasmids encode fusion proteins composed of a chloroplast transit peptide (CTP), the TALE N- or C-terminal domains, split-DddAtox halves (G1333N, G1333C, G1397N and G1397C) and UGI, which are codon-optimized for expression in dicot plants, under the control of the parsley ubiquitin (PcUbi) promoter and pea3A terminator (Page 899, Column 2).
Regarding claims 73 and 74, Baek teaches plasmids encode fusion proteins composed of a mitochondrial targeting sequence (MTS), the TALE N- or C-terminal domains, split-DddAtox halves (G1333N, G1333C, G1397N and G1397C) and UGI, which are codon-optimized for expression in dicot plants, under the control of the parsley ubiquitin (PcUbi) promoter and pea3A terminator (Page 899, Column 2).
Regarding claim 76, Baek teaches plasmids encode fusion proteins composed of a mitochondrial targeting sequence (MTS), the TALE N- or C-terminal domains, split-DddAtox halves (G1333N, G1333C, G1397N and G1397C) and UGI, which are codon-optimized for expression in dicot plants, under the control of the parsley ubiquitin (PcUbi) promoter and pea3A terminator (Page 899, Column 2).
Conclusion
No claims are allowed.
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/ALEXANDRA ROSE LIPPOLIS/Examiner, Art Unit 1637
/Jennifer Dunston/Supervisory Patent Examiner, Art Unit 1637