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 of Group I in the reply filed on 10/08/2025 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
The Applicant also elects the following species without traverse:
SEQ ID NO: 25, in claim 2
SEQ ID NO: 4, in claim 3
MCP, in claim 5
SEQ ID NO: 13, in claim 7
SEQ ID NO: 13, in claim 8
APOBEC1 deaminase, in claim 14
SEQ ID NO: 29, in claim 16
However, SEQ ID NO: 26 is rejoined.
The election is made FINAL.
Claim Status
Claims 1-36 are pending.
Claims 28-36 are withdrawn from examination as being part of nonelected invention.
Claims 1-27 are being examined.
Claim Objections
Claims 2 and 12 are objected to because of the following informalities:
Claim 2 recites “Ca9 nickase” in line 1. It appears that the Applicant implies “Cas9 nickase”
Claim 12, recites, “the second scan” in line 2. It appears that the Applicant implies “scRNA”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
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 2, 14-15, 19 and 21-23 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.
Regarding claims 2, 19 and 21-22; the phrase "for example" renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Regarding claims 14-15, the claims recite, “… functional variant(s)…” of cytosine deaminases comprising APOBEC1 deaminase, activation-induced cytidine deaminase (AID), APOBEC3G, CDA1, human APOBEC3A deaminase. The Applicant does not define the term. It is not clear what the Applicant imply by “functional variants”. Do such “variants” include any deaminase including adenine deaminase found in any organism including bacteria and fungus, for example? As recited in the claims, the term is also not limited to any percentage of homology/identity to any specific SEQ ID NO. Therefore, the metes and bounds of the recited expression of “functional variant(s)” is not clear in the claims.
Regarding claims 23 the phrase "preferably" renders the claim indefinite because use of a narrow range that falls within a broader range in the same claim may render the claim indefinite when the boundaries of the claim are not discernible. See MPEP § 2173.05(c).
Claim Rejections - 35 USC § 112(a)
Written Description
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 7-9 and 12-15 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claims contain subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The Applicant describes, “studies have demonstrated that scRNA formed by adding two MS2 hairpins to the 3′ end of (e)sgRNA can efficiently mediate CRISPRa in human cells, wherein MS2 is a commonly used RNA aptamer. Therefore, the scRNA vector pOsU3-esgRNA-2×MS2 driven by the OsU3 promoter was first constructed” (page 19, line 25-28). Such “scaffold system” of gene editing using scRNA is known in the art (Shakirova et al., Cell Reprogramming With CRISPR/Cas9 Based Transcriptional Regulation Systems, 2020, Front. Bioeng. Biotechnol., 8:882; page 4, left column, para 6, line 1-8). The sequences of 2xMS2 aptamer and the (MS2) aptamer-specific binding protein MCP are standard and long well known in the art ((Zalatan et al. (US 2017/0233762 A1) describes 2xMS2 aptamer sequence (SEQ ID NO: 9) and MCP protein sequence (SEQ ID NO: 2)).
Regarding claims 7-9, following the description of the instant invention and fusing the 2xMS aptamer sequence (small letters below) to the 3’end of the gRNA (SEQ ID NO: 4) (capital letters below) would result in the following sequence: GTTTAAGAGCTATGCTGGAAACAGC ATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTGTTTTTTATGTCT gggagcacatgaggatcacccatgtgccacgagcgacatgaggatcacccatg tcgctcgtgttccc. Sequence alignment of the fused sequence with instant SEQ ID NO: 13 shows an extra sequence, as highlighted in grey, between the gRNA (SEQ ID NO: 4) and the 2xMS2 sequence, as shown below.
Title: US-17-909-309A-13
Perfect score: 173
Sequence: 1 gtttaagagctatgctggaa..........tttttttgttttttatgtct 173
Searched: 1 seqs, 172 residues
Database : NASEQ2_10222025_121824.seq:*
RESULT 1
NASEQ2_10222025_121824
Query Match 71.7%; Score 124; DB 1; Length 172; Best Local Similarity 89.0%;
Matches 153; Conservative 0; Mismatches 0; Indels 19; Gaps 1;
Qy 1 GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAAC 60
Qy 61 TTGAAAAAGTGGCACCGAGTCGGTGC-------------------GGGAGCACATGAGGA 101
|||||||||||||||||||||||||| |||||||||||||||
Db 61 TTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTGTTTTTTATGTCTGGGAGCACATGAGGA 120
Qy 102 TCACCCATGTGCCACGAGCGACATGAGGATCACCCATGTCGCTCGTGTTCCC 153
||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 TCACCCATGTGCCACGAGCGACATGAGGATCACCCATGTCGCTCGTGTTCCC 172
The Applicant does not describe if the grey highlighted sequence in critical in the context of the invention. The Applicant also does not describe where the grey highlight sequence came from and why it is used while performing the function of editing the plant genome. A skilled artisan would not be able to design a suitable scRNA to edit a target genome as provided in the instant description.
Regarding claims 12-13, sequence alignment between instant SEQ ID NO: 22 (as recited in claim 12) and the (second) gRNA comprising the sequence ID NO: 3 (as recited in claim 3) fused with the 2xMS2 sequence (as described above) is shown below.
CLUSTAL 2.1 multiple sequence alignment
SEQ22 GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAAC
2xMS2+gRNA3 GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAAC
************************************************************
SEQ22 TTGAAAAAGTGGCACCGAGTCGGTGCG---------------------------------
2xMS2+gRNA3 TTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGTTTTTTATGTCTGTTTTAGAGCTAGA
**************************
SEQ22 -GGAGCGCCCTGAAGAAGGGCG---CCTGCTGCGGCCCTGAAGAAGGGCCGCAGCAGTTC
2xMS2+gRNA3 AATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGT
. ***.. *.**.:*.*** ** : *..* .**.*. ***. *.. :
SEQ22 CCTTTTTTTGTTTTTTATGTCT
2xMS2+gRNA3 GCTTTTTTTGTTTTTTATGTCT
*********************
In this case, the grey highlighted (TTTTTTGTTTTTTATGTCT) sequence (which does not arise from either the 2xMS2 sequence or the gRNA sequence) is also present. Moreover, in this case, the (second) gRNA sequence comprising instant SEQ ID NO: 3 is not aligning with the second scRNA comprising SEQ ID NO: 22. Further, the second RNA aptamer specific binding protein comprising SEQ ID NO: 36 (as recited in claim 13) is not MCP and would not bind to the 2xMS2 sequence present in the scRNA comprising the sequence of SEQ ID NO: 22.
Regarding claims 14-15, the instant description teaches several examples of specific cytosine deaminases comprising APOBEC1 deaminase, activation-induced cytidine deaminase (AID), APOBEC3G, CDA1, and human APOBEC3A deaminase. However, the Applicant does not describe or define the term “functional variant(s)” of cytosine deaminase.
An invention described solely in terms of a method of making and/or its function would lack written descriptive support where there is no described (in the specification) or art-recognized correlation between the disclosed function and the structure(s) responsible for the function. See MPEP § 2163.
Considering the breadth of the claims and lack of structure function relationship based on the instant description, the Applicant does not appear to have been in possession of the claimed genus at the time this application was filed.
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 1, 4-6, 14-15 and 20 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Brezgin et al. (Dead Cas Systems: Types, Principles, and Applications, 2019, Int. J. Mol. Sci., 20, 6041).
Claim 1 is drawn to a multiplex genome editing system in a plant comprising a CRISPR nickase along with one or more scRNAs wherein each scRNA contains an aptamer, a fusion protein that comprises an aptamer-specific binding protein, and a nucleotide base (adenine or cytosine) deamination domain.
Brezgin et al. describes a multiplex genome editing system in various organisms including plants (page 1, para 1, line 3) using nickase nCas9 (nCas9) which increases efficiency of base editing (page 12, para 6). Brezgin et al. also describes the “scaffold technique” (page 7, Fig. 2) where modified gRNAs comprising scaffold RNA (scRNA) containing two MS2 aptamers (as recited in claims 4 and 6) (page 3, para 9, line 5-6) and an aptamer-specific binding protein (MCP) (as recited in claim 5) fused to effector molecule(s) resulting in fusion proteins (of aptamer-specific binding proteins and effector proteins). Effectors proteins can be a deaminase, either for adenine or cytosine.
It is well known in the art that aptamers are part of scRNAs (i.e., the scaffold sequence in a gRNA). Brezgin et al. describes simultaneously editing or modulating many genes to control complex biological processes with unprecedented accuracy. This multiplexing is done by a Cas endonuclease that cuts a single RNA transcript into many gRNAs (i.e., paired gRNAs) which would read on to targeting individual targets (page 15, para 4), which can be more than three, and would read on to “first”, “second”, and “third” scRNA and corresponding “first”, “second”, and “third” target regions, as recited in claim 1. Brezgin et al. describes various effector proteins comprising cytidine/cytosine deaminases like APOBEC1 (page 13, para 2, line 1) (as recited in claims 14-15); and adenine deaminases including TadA adenosine deaminase (page 12, para 5, last 2 lines) and ADAR2 adenosine deaminase (page 13, para 4), which are known to comprise adenine deamination domain. TadA is a DNA dependent adenine deaminase (page 12, para 5, line 9-10), as recited in claim 20, that specifically targets DNA (page 15, table 4).
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 2-3 and 21-26 are rejected under 35 U.S.C. 103 as being unpatentable over Brezgin et al. as applied to claims 1, 4-6, 14-15 and 20 above (see rejections under 35 USC §102), and further in view of Gao et al. (WO2019120283A1).
Claim 2 is drawn to a Cas9 nickase comprising the amino acid sequence of SEQ ID NO: 25, while claim 3 is drawn to a gRNA sequence having 100% sequence identity to instant SEQ ID NO: 3.
Brezgin et al. describes a multiplex genome editing system comprising a Cas9 nickase (nCas9) along with one or more scRNAs wherein each scRNA contains an aptamer, a fusion protein that comprises an aptamer-specific binding protein, and a nucleotide base (adenine or cytosine) deamination domain, as described above. Brezgin et al. also describes use of peptide linkers (as recited in claims 23 and 25) to further enhance efficacy of the fusion proteins (page 3, para 6, line 3-5; page 4, para 4, line 3-5)
However, Brezgin et al. does not describe a Cas9 nickase comprising the amino acid sequence of SEQ ID NO: 25 or a polynucleotide sequence set forth in SEQ ID NO: 4.
Gao et al. describes a method for performing efficient base editing to a target sequence in a plant genome by a Cas9-cytidine deaminase fusion protein. It describes simultaneous editing three homoalleles in hexaploid bread wheat conferring heritable resistance to powdery mildew (page 23, para 2, line 2-4). All the proteins including the specific fusion proteins needed for base editing especially for editing genomic DNA need to be present in the nucleus in the cell. Gao et al. describes Nuclear Localization Sequence (NLS) (page 15, line 1-3), as recited in claim 26. It teaches one or more NLSs in the base-editing fusion proteins that are sufficient to drive the base-editing fusion protein into the nucleus of a plant cell to achieve base editing (page 15, line 3-6).
Gao et al. teaches a nuclease-inactivated Cas9 nickase (SEQ ID NO: 3) (page 5, para 0060) comprising 100% identity to instant SEQ ID NO: 25, as shown below.
RESULT 1
BGM50953
ID BGM50953 standard; protein; 1367 AA.
AC BGM50953;
DT 22-AUG-2019 (first entry)
DE Streptococcus pyogenes Cas9 protein, SEQ ID 3.
KW CRISPR associated 9; CRISPR-Cas9 system; Cas9 protein; crop improvement;
KW genome editing; herbicide resistance; plant breeding; transgenic plant.
OS Streptococcus pyogenes.
CC PN WO2019120283-A1.
CC PD 27-JUN-2019.
CC PF 21-DEC-2018; 2018WO-CN122640.
PR 21-DEC-2017; 2017CN-11393160.
PR 28-APR-2018; 2018CN-10402244.
CC PA (CAGD ) INST GENETICS & DEV BIOL CAS.
CC PI Gao C, Li C, Zong Y, Wang Y;
DR WPI; 2019-53605Q/53.
CC PT System useful for base editing target sequence in plant genome, comprises base-editing fusion protein, and guide RNA, and expression construct comprising nucleotide sequence encoding base-editing fusion protein, and guide RNA.
CC PS Claim 4; SEQ ID NO 3; 60pp; English.
CC PT System useful for base editing target sequence in plant genome, comprises
base-editing fusion protein, and guide RNA, and expression construct comprising nucleotide sequence encoding base-editing fusion protein, and guide RNA.
CC PS Example 5; Fig 7; 60pp; English.
CC The present invention relates to a novel system useful for base editing of a target sequence in a plant genome. The system comprises: base-editing fusion protein, and a guide RNA; an expression construct comprising a nucleotide sequence encoding a base-editing fusion protein, and a guide RNA; a base-editing fusion protein corresponds to SEQ ID NO: 4, 5 or 18-25 or 33-39 (see BGM50954-BGM50955 or BGM50968-BGM50975 or BGM50983-BGM50989), and an expression construct comprising a nucleotide sequence encoding the guide RNA; an expression construct comprising a nucleotide sequence corresponds to SEQ ID NO: 6, 7 or 10-17 or 26-32 (see BGM50956-BGM50957 or BGM50960-BGM50967 or BGM50976-BGM50982) encoding the base-editing fusion protein, and an expression construct comprising a nucleotide sequence encoding the guide RNA; and an expression construct comprising a nucleotide sequence encoding the base-editing fusion protein and a nucleotide sequence encoding the guide RNA, where the base-editing fusion protein comprises a nuclease-inactivated clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated 9 (Cas9) effector protein corresponds to SEQ ID NO: 3 (see BGM50953) and a DNA-dependent adenine deaminase (tRNA adenine deaminase or TadA) corresponds to SEQ ID NO: 1 (see BGM50951). The invention further relates to: (1) a method for producing a genetically modified plant; (2) a genetically modified plant or its progeny, or a part, where the plant; and (3) a method for plant breeding. The system of the invention is also used for producing a plant having herbicide resistance.
Query Match 100.0%; Score 6998; Length 1384; Best Local Similarity 100.0%;
Matches 1367; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 2 DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA 61
Qy 61 TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGN 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 62 TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGN 121
Qy 121 IVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDV 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 122 IVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDV 181
Qy 181 DKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNL 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 182 DKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNL 241
Qy 241 IALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 242 IALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL 301
Qy 301 LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAG 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 302 LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAG 361
Qy 361 YIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHA 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 362 YIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHA 421
Qy 421 ILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEV 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 422 ILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEV 481
Qy 481 VDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 482 VDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS 541
Qy 541 GEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 542 GEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII 601
Qy 601 KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGR 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 602 KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGR 661
Qy 661 LSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 662 LSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH 721
Qy 721 EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERM 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 722 EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERM 781
Qy 781 KRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHI 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 782 KRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHI 841
Qy 841 VPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 842 VPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT 901
Qy 901 KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 902 KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK 961
Qy 961 LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 962 LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM 1021
Qy 1021 IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFA 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1022 IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFA 1081
Qy 1081 TVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAY 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1082 TVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAY 1141
Qy 1141 SVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY 1200
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1142 SVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY 1201
Qy 1201 SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ 1260
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1202 SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ 1261
Qy 1261 HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP 1320
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1262 HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP 1321
Qy 1321 AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 1367
|||||||||||||||||||||||||||||||||||||||||||||||
Db 1322 AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 1368
Regarding claim 3, Gao et al. describes an gRNA having 100% sequence identity to instant SEQ ID NO: 4, as shown below.
RESULT 3
BGM51116
ID BGM51116 standard; DNA; 487 BP.
AC BGM51116;
DT 22-AUG-2019 (first entry)
DE Wheat U6 promoter target sequence (pTaU6-esgRNA).
KW CRISPR-Cas9 system; U6 gene; crop improvement; ds; genome editing; herbicide resistance; plant; plant breeding; transgenic plant.
OS Triticum aestivum.
OS Synthetic.
CC PN WO2019120283-A1.
CC PD 27-JUN-2019.
CC PF 21-DEC-2018; 2018WO-CN122640.
PR 21-DEC-2017; 2017CN-11393160.
PR 28-APR-2018; 2018CN-10402244.
CC PA (CAGD ) INST GENETICS & DEV BIOL CAS.
CC PI Gao C, Li C, Zong Y, Wang Y;
DR WPI; 2019-53605Q/53.
CC PT System useful for base editing target sequence in plant genome, comprises
base-editing fusion protein, and guide RNA, and expression construct comprising nucleotide sequence encoding base-editing fusion protein, and guide RNA.
CC PS Example 5; Fig 7; 60pp; English.
The present invention relates to a novel system useful for base editing of a target sequence in a plant genome. The system comprises: base-editing fusion protein, and a guide RNA; an expression construct comprising a nucleotide sequence encoding a base-editing fusion protein, and a guide RNA; a base-editing fusion protein corresponds to SEQ ID NO: 4, 5 or 18-25 or 33-39 (see BGM50954-BGM50955 or BGM50968-BGM50975 or BGM50983-BGM50989), and an expression construct comprising a nucleotide sequence encoding the guide RNA; an expression construct comprising a nucleotide sequence corresponds to SEQ ID NO: 6, 7 or 10-17 or 26-32 (see BGM50956-BGM50957 or BGM50960-BGM50967 or BGM50976-BGM50982) encoding the base-editing fusion protein, and an expression construct comprising a nucleotide sequence encoding the guide RNA; and an expression construct comprising a nucleotide sequence encoding the base-editing fusion protein and a nucleotide sequence encoding the guide RNA, where the base-editing fusion protein comprises a nuclease-inactivated clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated 9 (Cas9) effector protein corresponds to SEQ ID NO: 3 (see BGM50953) and a DNA-dependent adenine deaminase (tRNA adenine deaminase or TadA) corresponds to SEQ ID NO: 1 (see BGM50951). The invention further relates to: (1) a method for producing a genetically modified plant; (2) a genetically modified plant or its progeny, or a part, where the plant; and (3) a method for plant breeding. The system of the invention is also used for producing a plant having herbicide resistance.
SQ Sequence 487 BP; 116 A; 110 C; 125 G; 136 T; 0 U; 0 Other;
Query Match 100.0%; Score 106; Length 487; Best Local Similarity 100.0%;
Matches 106; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAAC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 382 GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAAC 441
Qy 61 TTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGTTTTTTATGTCT 106
||||||||||||||||||||||||||||||||||||||||||||||
Db 442 TTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGTTTTTTATGTCT 487
Regarding claims 21-23, Gao et al. describes a TadA DNA-dependent adenine deaminase (SEQ ID NO: 2) (page 13, line 14-16) comprising 100% sequence identity to instant SEQ ID NO: 33, as recited in claim 21, as shown below.
RESULT 1
BGM50952
ID BGM50952 standard; protein; 166 AA.
AC BGM50952;
DT 22-AUG-2019 (first entry)
DE Escherichia coli tRNA adenine deaminase (TadA) variant, SEQ ID 2.
KW CRISPR-Cas9 system; DNA-dependent adenine deaminase; TadA protein;
KW crop improvement; genome editing; herbicide resistance; mutein;
KW plant breeding; tRNA adenine deaminase; transgenic plant.
OS Escherichia coli.
FT /note= "Wild type Trp is substituted by Arg"
FT Misc-difference 35
FT /note= "Wild type His is substituted by Leu"
FT Misc-difference 47
FT /note= "Wild type Pro is substituted by Ala"
FT Misc-difference 50
FT /note= "Wild type Arg is substituted by Leu"
FT Misc-difference 83
FT /note= "Wild type Leu is substituted by Phe"
FT Misc-difference 105
FT /note= "Wild type Arg is substituted by Val"
FT Misc-difference 107
FT /note= "Wild type Asp is substituted by Asn"
FT Misc-difference 122
FT /note= "Wild type His is substituted by Tyr"
FT Misc-difference 145
FT /note= "Wild type Ser is substituted by Cys"
FT Misc-difference 146
FT /note= "Wild type Asp is substituted by Tyr"
FT Misc-difference 151
FT /note= "Wild type Arg is substituted by Pro"
FT Misc-difference 154
FT /note= "Wild type Glu is substituted by Val"
FT Misc-difference 155
FT /note= "Wild type Ile is substituted by Phe"
FT Misc-difference 156
FT /note= "Wild type Lys is substituted by Asn"
CC PN WO2019120283-A1.
CC PD 27-JUN-2019.
CC PF 21-DEC-2018; 2018WO-CN122640.
PR 21-DEC-2017; 2017CN-11393160.
PR 28-APR-2018; 2018CN-10402244.
CC PA (CAGD ) INST GENETICS & DEV BIOL CAS.
CC PI Gao C, Li C, Zong Y, Wang Y;
DR WPI; 2019-53605Q/53.
CC PT System useful for base editing target sequence in plant genome, comprises
CC PT base-editing fusion protein, and guide RNA, and expression construct
CC PT comprising nucleotide sequence encoding base-editing fusion protein, and
CC PT guide RNA.
CC PS Claim 3; SEQ ID NO 2; 60pp; English.
CC The present invention relates to a novel system useful for base editing
CC of a target sequence in a plant genome. The system comprises: base-
CC editing fusion protein, and a guide RNA; an expression construct
CC comprising a nucleotide sequence encoding a base-editing fusion protein,
CC and a guide RNA; a base-editing fusion protein corresponds to SEQ ID NO:
CC 4, 5 or 18-25 or 33-39 (see BGM50954-BGM50955 or BGM50968-BGM50975 or
CC BGM50983-BGM50989), and an expression construct comprising a nucleotide
CC sequence encoding the guide RNA; an expression construct comprising a
CC nucleotide sequence corresponds to SEQ ID NO: 6, 7 or 10-17 or 26-32 (see
CC BGM50956-BGM50957 or BGM50960-BGM50967 or BGM50976-BGM50982) encoding the
CC base-editing fusion protein, and an expression construct comprising a
CC nucleotide sequence encoding the guide RNA; and an expression construct
CC comprising a nucleotide sequence encoding the base-editing fusion protein
CC and a nucleotide sequence encoding the guide RNA, where the base-editing
CC fusion protein comprises a nuclease-inactivated clustered regularly
CC interspaced short palindromic repeat (CRISPR)-CRISPR associated 9 (Cas9)
CC effector protein corresponds to SEQ ID NO: 3 (see BGM50953) and a DNA-
CC dependent adenine deaminase (tRNA adenine deaminase or TadA) corresponds
CC to SEQ ID NO: 1 (see BGM50951). The invention further relates to: (1) a
CC method for producing a genetically modified plant; (2) a genetically
CC modified plant or its progeny, or a part, where the plant; and (3) a
CC method for plant breeding. The system of the invention is also used for
CC producing a plant having herbicide resistance. Note: The mutation
CC positions for this sequence as given in the claim 2 of the specification
CC do not correctly match. Therefore, the mutation position given in the
CC feature table have been deduced by comparing this sequence with the
CC parent sequence (see BGM50951).
SQ Sequence 166 AA;
Query Match 100.0%; Score 869; Length 166; Best Local Similarity 100.0%;
Matches 166; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIM 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIM 60
Qy 61 ALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVL 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 ALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVL 120
Qy 121 HYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD 166
||||||||||||||||||||||||||||||||||||||||||||||
Db 121 HYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD 166
Gao et al. describes an adenine base editing system comprising SEQ ID NO: 24, which is suitable for plant cell editing (page 27, line 10-18), besides a DNA dependent adenine deaminase from E. coli (page 12, line 11-14), as recited in claim 23. SEQ ID NO: 24 of Gao et al. is codon optimized for plant expression and having 100% sequence identity to instant SEQ ID NO: 32 (as recited in claim 22), as shown below.
RESULT 31
BGM50974
ID BGM50974 standard; protein; 809 AA.
AC BGM50974;
DT 22-AUG-2019 (first entry)
DE E. coli TadA-Cas9 fusion protein (SanCas9-ABE-1), SEQ ID 24.
KW CRISPR associated 9; CRISPR-Cas9 system; Cas9 protein;
KW DNA-dependent adenine deaminase; TadA protein; chimeric protein;
KW crop improvement; fusion protein; genome editing; herbicide resistance;
KW plant breeding; tRNA adenine deaminase; transgenic plant.
OS Escherichia coli.
CC PN WO2019120283-A1.
CC PD 27-JUN-2019.
CC PF 21-DEC-2018; 2018WO-CN122640.
PR 21-DEC-2017; 2017CN-11393160.
PR 28-APR-2018; 2018CN-10402244.
CC PA (CAGD ) INST GENETICS & DEV BIOL CAS.
CC PI Gao C, Li C, Zong Y, Wang Y;
DR WPI; 2019-53605Q/53.
DR N-PSDB; BGM50966.
CC PT System useful for base editing target sequence in plant genome, comprises base-editing fusion protein, and guide RNA, and expression construct comprising nucleotide sequence encoding base-editing fusion protein, and guide RNA.
CC PS Claim 10; SEQ ID NO 24; 60pp; English.
CC The present invention relates to a novel system useful for base editing
CC of a target sequence in a plant genome. The system comprises: base-
CC editing fusion protein, and a guide RNA; an expression construct
CC comprising a nucleotide sequence encoding a base-editing fusion protein,
CC and a guide RNA; a base-editing fusion protein corresponds to SEQ ID NO:
CC 4, 5 or 18-25 or 33-39 (see BGM50954-BGM50955 or BGM50968-BGM50975 or
CC BGM50983-BGM50989), and an expression construct comprising a nucleotide
CC sequence encoding the guide RNA; an expression construct comprising a
CC nucleotide sequence corresponds to SEQ ID NO: 6, 7 or 10-17 or 26-32 (see
CC BGM50956-BGM50957 or BGM50960-BGM50967 or BGM50976-BGM50982) encoding the
CC base-editing fusion protein, and an expression construct comprising a
CC nucleotide sequence encoding the guide RNA; and an expression construct
CC comprising a nucleotide sequence encoding the base-editing fusion protein
CC and a nucleotide sequence encoding the guide RNA, where the base-editing
CC fusion protein comprises a nuclease-inactivated clustered regularly
CC interspaced short palindromic repeat (CRISPR)-CRISPR associated 9 (Cas9)
CC effector protein corresponds to SEQ ID NO: 3 (see BGM50953) and a DNA-
CC dependent adenine deaminase (tRNA adenine deaminase or TadA) corresponds
CC to SEQ ID NO: 1 (see BGM50951). The invention further relates to: (1) a
CC method for producing a genetically modified plant; (2) a genetically
CC modified plant or its progeny, or a part, where the plant; and (3) a
CC method for plant breeding. The system of the invention is also used for
CC producing a plant having herbicide resistance.
SQ Sequence 809 AA;
Query Match 100.0%; Score 872; Length 809; Best Local Similarity 100.0%;
Matches 166; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIM 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 2 SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIM 61
Qy 61 ALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVL 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 62 ALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVL 121
Qy 121 HHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD 166
||||||||||||||||||||||||||||||||||||||||||||||
Db 122 HHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD 167
Before the effective filing date, it would have been obvious to an ordinarily skilled artisan to use Cas9 nickase or a functional equivalent of a nuclease inactivated Cas9 nickase, and a gRNA to edit target gene(s)/allele(s) in a plant, as described by Gao et al. Designing gRNA(s) for specific purpose is a well-known standard process that depends on specific target sequence. Using a functional equivalent of Cas9 nickase, a gRNA, and a DNA-dependent adenine deaminase, as described by Gao et al., having specific structures/sequences is an experimental design choice of an ordinarily skilled artisan without having any realistic possibility of changing the outcome.
Before the effective filing date of the invention, one ordinarily skilled in the art would have been motivated to use specific Cas9 nickase, gRNA, and a DNA-dependent adenine deaminase (as described by Gao et al.) to edit target gene(s)/allele(s) in a plant.
Regarding claims 23-25, All the fused proteins (DNA or RNA dependent aptamer specific binding proteins fused with deaminase protein/domain) acts in trans in a cell. It is an experimental design choice of an ordinarily skilled artisan to make fusion proteins by fusing the aptamer-specific binding proteins (e.g. MCP) with either N-terminal or the C-terminal (as recited in claims 23-24) of an effector protein like adenine deaminase (TadA) or the corresponding adenine deamination domain (as recited in claim 24) using a linker protein. C-terminal deamination domain of TadA proteins including in E coli (as recited in claim 23) and in plants are known in the art since long1. Using a linker (as recited in claims 23 and 25) while making fusion protein(s) by fusing two or more proteins and/or domains is a standard practice in the art, because it offers several advantages such as improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles2 and also as described by Brezgin et al.
It is "the normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine” the exact experimental design that works more efficiently in a specific experiment, unless there is evidence indicating a specific experimental design used by the Applicant is critical. Such “critical” feature(s) need(s) to be described by the instant description. See MPEP § 2144.05.
Claims 2 and 21-26 are rejected under 35 U.S.C. 103 as being unpatentable over Brezgin et al. as applied to claims 1, 4-6, 14-15 and 20 above, and further in view of Liu et al. (WO2018027078 A1).
Brezgin et al. describes a multiplex genome editing system comprising a Cas9 nickase (nCas9) along with one or more scRNAs wherein each scRNA contains an aptamer, a fusion protein that comprises an aptamer-specific binding protein, and a nucleotide base (adenine or cytosine) deamination domain, as described above.
However, Brezgin et al. does not describe a Cas9 nickase comprising the amino acid sequence of SEQ ID NO: 25, an Escherichia coli tRNA adenine deaminase TadA (ecTadA) comprising the amino acid sequence shown in SEQ ID NO: 32 or SEQ ID NO: 33.
Liu et al. describes a method of targeted gene editing by using fusion proteins using Cas9 nickase and different adenosine deaminases that are capable of deaminating adenosine in DNA. The fusion proteins comprises one or more linkers (as recited in claim 23) (page 4, para 0009, line 6-7) and one or more nuclear localization sequences (NLS) (as recited in claim 26) (page 4, para 0009, last 2 lines).It describes a Cas9 comprising the amino acid sequence (SEQ ID NO: 35) (page , para 202; para 280) having 100% sequence identity to instant SEQ ID NO: 25, as shown below.
BLM40833
ID BLM40833 standard; protein; 167 AA.
AC BLM40833;
DT 08-SEP-2022 (first entry)
DE E. coli TadA mutant protein (pNMG-619/620/624).
KW CRISPR-Cas9 system; TadA; cytostatic; genetic disorder;
KW genetic-disease-gen.; genome editing; lysosome storage disease;
KW metabolic disorder; metabolic-gen.; neoplasm; neuroprotective;
KW recombinant protein; tRNA adenosine deaminase A; therapeutic; mutein.
OS Escherichia coli.
OS Synthetic.
CC PN WO2018027078-A1.
CC PD 08-FEB-2018.
CC PF 03-AUG-2017; 2017WO-US045381.
PR 03-AUG-2016; 2016US-0370684P.
PR 02-FEB-2017; 2017US-0454035P.
PR 20-MAR-2017; 2017US-0473714P.
CC PA (HARD ) HARVARD COLLEGE.
CC PI Gaudelli N, Liu DR;
DR WPI; 2018-11292C/13.
CC PT New adenosine deaminase capable of deaminating adenine of deoxyadenosine
CC PT in DNA useful for editing nucleobase pair of double-stranded DNA
CC PT sequence.
CC PS Claim 113; Page; 1450pp; English.
CC The invention relates to a novel adenosine deaminase capable of
CC deaminating adenine of deoxyadenosine in DNA useful for editing
CC nucleobase pair of double-stranded DNA sequence. The invention claims: 1)
CC a fusion protein; 2) a complex comprising the fusion protein and a guide
CC RNA (gRNA) bound to the napDNAbp of the fusion protein, where the
CC napDNAbp is a clustered regularly interspaced short palindromic repeat
CC (CRISPR) associated protein 9 (Cas9) domain, a Cpfl, a CasX, a CasY, a
CC C2c1, a C2c2, or a C2c3.; 3) a method involving (a) contacting a nucleic
CC acid molecule with the fusion protein and a gRNA, where the gRNA is 15-
CC 100 nucleotides long and comprises a sequence of at least 10 contiguous
CC nucleotides that is complementary to a target sequence, or (b) contacting
CC a nucleic acid molecule with the complex; 4) a method for editing a
CC nucleobase pair of a double-stranded DNA (dsDNA) sequence; 5) a nucleic
CC acid-guided adenosine deaminase coupled to an inhibitor of base excision
CC repair; 6) a kit comprising a nucleic acid construct; 7) a polynucleotide
CC encoding the adenosine deaminase, or the fusion protein; 8) a vector
CC comprising the polynucleotide; and 9) a cell comprising (a) the adenosine
CC deaminase, or the fusion protein, (b) the complex, or (c) the nucleic
CC acid molecule encoding the adenosine deaminase, or the fusion protein.
CC The adenosine deaminase is used in pharmaceutical composition for editing
CC a nucleobase pair of a dsDNA sequence; for modifying a polynucleotide,
CC and treating a proliferative disease, genetic disease, neoplastic
CC disease, metabolic disease, and lysosomal storage disease. The adenosine
CC deaminase has excellent stability. The present sequence represents an
CC Escherichia coli tRNA adenosine deaminase A (TadA) W23R/H36L/P48A/R51L/L8
CC 4F/A106V/D108N/H123Y/S146C/D147Y/R152P/E155V/I156F/K157N mutant protein
CC (pNMG-619 / pNMG-620 / pNMG-624) used for editing nucleobase pair of
CC double-stranded DNA sequence. Note: The present sequence is not shown in
CC the specification but is created based on the information given in table
CC 4 of the specification from the E. coli TadA protein shown as SEQ ID NO:
CC 1 (see BFB10528).
SQ Sequence 167 AA;
Query Match 100.0%; Score 6998; Length 1367;
Best Local Similarity 100.0%;
Matches 1367; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA 60
Qy 61 TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGN 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGN 120
Qy 121 IVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDV 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 IVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDV 180
Qy 181 DKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNL 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 DKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNL 240
Qy 241 IALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 IALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL 300
Qy 301 LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAG 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAG 360
Qy 361 YIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHA 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 YIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHA 420
Qy 421 ILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEV 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 ILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEV 480
Qy 481 VDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 VDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS 540
Qy 541 GEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 GEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII 600
Qy 601 KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGR 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGR 660
Qy 661 LSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 LSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH 720
Qy 721 EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERM 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERM 780
Qy 781 KRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHI 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 KRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHI 840
Qy 841 VPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 841 VPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT 900
Qy 901 KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 901 KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK 960
Qy 961 LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 961 LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM 1020
Qy 1021 IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFA 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1021 IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFA 1080
Qy 1081 TVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAY 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1081 TVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAY 1140
Qy 1141 SVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY 1200
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1141 SVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY 1200
Qy 1201 SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ 1260
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1201 SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ 1260
Qy 1261 HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP 1320
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1261 HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAP 1320
Qy 1321 AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 1367
|||||||||||||||||||||||||||||||||||||||||||||||
Db 1321 AAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 1367
Liu et al. teaches a E coli TadA adenine deaminase comprising SEQ ID NO: 64 which has 100% sequence identity to instant SEQ ID NO: 33 (as recited in claim 21), as shown below.
BLM40833
ID BLM40833 standard; protein; 167 AA.
AC BLM40833;
DE E. coli TadA mutant protein (pNMG-619/620/624).CRISPR-Cas9 system; TadA; cytostatic; genetic disorder; genetic-disease-gen.; genome editing; lysosome storage disease; metabolic disorder; metabolic-gen.; neoplasm; neuroprotective; recombinant protein; tRNA adenosine deaminase A; therapeutic; mutein.
OS Escherichia coli.
OS Synthetic.
CC PN WO2018027078-A1.
CC PD 08-FEB-2018.
CC PF 03-AUG-2017; 2017WO-US045381.
PR 03-AUG-2016; 2016US-0370684P.
PR 02-FEB-2017; 2017US-0454035P.
PR 20-MAR-2017; 2017US-0473714P.
CC PA (HARD ) HARVARD COLLEGE.
CC PI Gaudelli N, Liu DR;
DR WPI; 2018-11292C/13.
CC PT New adenosine deaminase capable of deaminating adenine of deoxyadenosine in DNA useful for editing nucleobase pair of double-stranded DNA sequence.
CC PS Claim 113; Page; 1450pp; English.
CC The invention relates to a novel adenosine deaminase capable of
CC deaminating adenine of deoxyadenosine in DNA useful for editing
CC nucleobase pair of double-stranded DNA sequence. The invention claims: 1)
CC a fusion protein; 2) a complex comprising the fusion protein and a guide
CC RNA (gRNA) bound to the napDNAbp of the fusion protein, where the
CC napDNAbp is a clustered regularly interspaced short palindromic repeat
CC (CRISPR) associated protein 9 (Cas9) domain, a Cpfl, a CasX, a CasY, a
CC C2c1, a C2c2, or a C2c3.; 3) a method involving (a) contacting a nucleic
CC acid molecule with the fusion protein and a gRNA, where the gRNA is 15-
CC 100 nucleotides long and comprises a sequence of at least 10 contiguous
CC nucleotides that is complementary to a target sequence, or (b) contacting
CC a nucleic acid molecule with the complex; 4) a method for editing a
CC nucleobase pair of a double-stranded DNA (dsDNA) sequence; 5) a nucleic
CC acid-guided adenosine deaminase coupled to an inhibitor of base excision
CC repair; 6) a kit comprising a nucleic acid construct; 7) a polynucleotide
CC encoding the adenosine deaminase, or the fusion protein; 8) a vector
CC comprising the polynucleotide; and 9) a cell comprising (a) the adenosine
CC deaminase, or the fusion protein, (b) the complex, or (c) the nucleic
CC acid molecule encoding the adenosine deaminase, or the fusion protein.
CC The adenosine deaminase is used in pharmaceutical composition for editing
CC a nucleobase pair of a dsDNA sequence; for modifying a polynucleotide,
CC and treating a proliferative disease, genetic disease, neoplastic
CC disease, metabolic disease, and lysosomal storage disease. The adenosine
CC deaminase has excellent stability. The present sequence represents an
CC Escherichia coli tRNA adenosine deaminase A (TadA) W23R/H36L/P48A/R51L/L8
CC 4F/A106V/D108N/H123Y/S146C/D147Y/R152P/E155V/I156F/K157N mutant protein
CC (pNMG-619 / pNMG-620 / pNMG-624) used for editing nucleobase pair of
CC double-stranded DNA sequence. Note: The present sequence is not shown in
CC the specification but is created based on the information given in table
CC 4 of the specification from the E. coli TadA protein shown as SEQ ID NO:
CC 1 (see BFB10528).
SQ Sequence 167 AA;
Query Match 100.0%; Score 869; Length 166; Best Local Similarity 100.0%;
Matches 166; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIM 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIM 60
Qy 61 ALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVL 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 ALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVL 120
Qy 121 HYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD 166
||||||||||||||||||||||||||||||||||||||||||||||
Db 121 HYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD 166
Liu et al. also teaches a E coli TadA adenine deaminase comprising SEQ ID NO: 64 which has 100% sequence identity to instant SEQ ID NO: 32 (as recited in claim 22), as shown below.
RESULT 1
BFB10591
AC BFB10591;
DT 08-SEP-2022 (revised)
DT 05-APR-2018 (first entry)
DE E. coli TadA protein SEQ 64.
KW CRISPR-Cas9 system; TadA; cytostatic; genetic disorder;
KW genetic-disease-gen.; genome editing; lysosome storage disease;
KW metabolic disorder; metabolic-gen.; neoplasm; neuroprotective;
KW recombinant protein; tRNA adenosine deaminase A; therapeutic.
OS Escherichia coli.
CC PN WO2018027078-A1.
CC PD 08-FEB-2018.
CC PF 03-AUG-2017; 2017WO-US045381.
PR 03-AUG-2016; 2016US-0370684P.
PR 02-FEB-2017; 2017US-0454035P.
PR 20-MAR-2017; 2017US-0473714P.
CC PA (HARD ) HARVARD COLLEGE.
CC PI Gaudelli N, Liu DR;
DR WPI; 2018-11292C/13.
CC PT New adenosine deaminase capable of deaminating adenine of deoxyadenosine in DNA useful for editing nucleobase pair of double-stranded DNA sequence.
CC PS Claim 110; SEQ ID NO 64; 1450pp; English.
CC The invention relates to a novel adenosine deaminase capable of deaminating adenine of deoxyadenosine in DNA useful for editing nucleobase pair of double-stranded DNA sequence. The invention claims: 1)a fusion protein; 2) a complex comprising the fusion protein and a guide RNA (gRNA) bound to the napDNAbp of the fusion protein, where the napDNAbp is a clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (Cas9) domain, a Cpfl, a CasX, a CasY, a C2c1, a C2c2, or a C2c3.; 3) a method involving (a) contacting a nucleic acid molecule with the fusion protein and a gRNA, where the gRNA is 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence, or (b) contacting a nucleic acid molecule with the complex; 4) a method for editing a nucleobase pair of a double-stranded DNA (dsDNA) sequence; 5) a nucleic acid-guided adenosine deaminase coupled to an inhibitor of base excision repair; 6) a kit comprising a nucleic acid construct; 7) a polynucleotide encoding the adenosine deaminase, or the fusion protein; 8) a vector comprising the polynucleotide; and 9) a cell comprising (a) the adenosine deaminase, or the fusion protein, (b) the complex, or (c) the nucleic acid molecule encoding the adenosine deaminase, or the fusion protein.
CC The adenosine deaminase is used in pharmaceutical composition for editing a nucleobase pair of a dsDNA sequence; for modifying a polynucleotide, and treating a proliferative disease, genetic disease, neoplastic disease, metabolic disease, and lysosomal storage disease. The adenosine deaminase has excellent stability. The present sequence represents an Escherichia coli TadA protein used for editing nucleobase pair of double-stranded DNA sequence.
CC Revised record issued on 05-SEP-2022 : Correction to DE line and comments.
SQ Sequence 166 AA;
Query Match 100.0%; Score 872; Length 166; Best Local Similarity 100.0%;
Matches 166; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIM 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIM 60
Qy 61 ALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVL 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 ALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVL 120
Qy 121 HHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD 166
||||||||||||||||||||||||||||||||||||||||||||||
Db 121 HHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD 166
Before the effective filing date, it would have been obvious to an ordinarily skilled artisan to use Cas9 nickase or a functional equivalent of a nuclease inactivated Cas9 nickase, and a gRNA to edit target gene(s)/allele(s) in a plant, as described by Liu et al. Designing gRNA(s) for specific purpose is a well-known standard process3 that depends on specific target sequence. Using a functional equivalent of Cas9 nickase, a gRNA, and a DNA-dependent adenine deaminase, as described by Liu et al., having specific structures/sequences is an experimental design choice of an ordinarily skilled artisan without having any realistic possibility of changing the outcome.
Before the effective filing date of the invention, one ordinarily skilled in the art would have been motivated to use specific Cas9 nickase, gRNA, and a DNA-dependent adenine deaminase to edit target gene(s)/allele(s) in a plant.
Regarding claims 23-25, All the fused proteins (DNA or RNA dependent aptamer specific binding proteins fused with deaminase protein/domain) acts in trans in a cell. It is an experimental design choice of an ordinarily skilled artisan to make fusion proteins by fusing the aptamer-specific binding proteins (e.g. MCP) with either N-terminal or the C-terminal (as recited in claims 23-24) of the effector protein like adenine deaminase (TadA) or the corresponding adenine deamination domain (as recited in claim 24) using a linker protein. C-terminal deamination domain of TadA proteins including in E coli (as recited in claim 23) and in plants are known in the art since long1. Using a linker (as recited in claims 23 and 25) while making fusion protein(s) by fusing two or more proteins and/or domains is also standard practice in the art because it offers several advantages such as improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles2.
It is "the normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine” the exact experimental design that works more efficiently in a specific experiment, unless there is evidence indicating a specific experimental design used by the Applicant is critical. Such “critical” feature(s) need(s) to be described by the instant description. See MPEP § 2144.05.
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Brezgin et al. as applied to claims 1, 4-6, 14-15 and 20 above, and further in view of McConnell et al. (US20190151456A1).
Brezgin et al. describes a multiplex genome editing system in various organisms including plants using CRISPR-Cas9 technique comprising nickase nCas9 (nCas9), scaffold RNA (scRNA) containing aptamer sequence(s) (e.g. MS2), aptamer-specific binding proteins (e.g. MCP that binds to MS2 sequence), effector proteins (e.g. cytidine/cytosine deaminases like APOBEC1), as discussed above. Brezgin et al. also describes aptamer-specific binding proteins (e.g. MCP) fused to effector proteins including transcriptional activators (page 6, para 4; page 7, Fig. 2) resulting in fusion proteins (of aptamer-specific binding proteins and effector proteins). Effectors proteins can be a deaminase (either for adenine or cytosine).
However, Brezgin et al. does not teach any protein having 100% sequence identity to either SEQ ID NO: 34.
McConnell et al. teaches that MCP protein recognizes and binds to an RNA stem loop structure of MS2 (aptamer) (page 19, para 0196, line 13-15). It also teaches mutational analysis that identified two positions in MCP that increases affinity to MS2 aptamer (page 19, para 0196, line 19-20). McConnell et al. describes the specific aptamer binding protein, MCP (SEQ ID NO: 111), having 100% sequence identity to instant SEQ ID NO: 34, as shown below.
US-16-194-230-111
Filing date in PALM: 2018-11-16
Sequence 111, US/16194230
GENERAL INFORMATION
APPLICANT: CODIAK BIOSCIENCES, INC.
TITLE OF INVENTION: COMPOSITIONS OF ENGINEERED EXOSOMES AND METHODS OF LOADING
TITLE OF INVENTION: LUMINAL EXOSOME PAYLOADS
FILE REFERENCE: 32724-41406/US
CURRENT APPLICATION NUMBER: US/16/194,230
CURRENT FILING DATE: 2018-11-16
PRIOR APPLICATION NUMBER: 62/634,750
PRIOR FILING DATE: 2018-02-23
PRIOR APPLICATION NUMBER: 62/587,767
PRIOR FILING DATE: 2017-11-17
NUMBER OF SEQ ID NOS: 150
SEQ ID NO 111
LENGTH: 167
TYPE: PRT
ORGANISM: Artificial Sequence
OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide
Query Match 100.0%; Score 595; Length 167; Best Local Similarity 100.0%;
Matches 116; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 ASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTI 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 52 ASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTI 111
Qy 61 KVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIA ANSGIY 116
||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 112 KVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIA ANSGIY 167
Before the effective filing date, it would have been obvious to an ordinarily skilled artisan to use the MS2 aptamer binding protein MCP, as described by McConnell et al. to recognize and bind to the MS2 aptamer, as described by Brezgin et al., with a realistic goal edit specific target sequence(s) in a plant genome.
Before the effective filing date, one with ordinary skill in the rat would have been motivated to use the MS2 aptamer binding protein MCP having a specific amino acid sequence that recognizes and binds to MS2 aptamer with a realistic goal edit specific target sequence(s) in a plant genome.
Claim 11 is rejected under 35 U.S.C. 103 as being unpatentable over Brezgin et al. as applied to claims 1, 4-6, 14-15 and 20 above, and further in view of Zalatan et al. (US20190151456A1).
Brezgin et al. describes a multiplex genome editing system in various organisms including plants using CRISPR-Cas9 technique comprising nickase nCas9 (nCas9), scaffold RNA (scRNA) containing aptamer sequence(s), aptamer-specific binding proteins, effector proteins, as discussed above.
However, Brezgin et al. does not teach any protein having 100% sequence identity to either SEQ ID NO: 37.
Zalatan et al. teaches making and using various scaffold RNAs (abstract). It describes a scaffold region comprising the com sequence which binds to a COM polypeptide (page 1, para 0010, line 1-8) comprising the amino acid sequence of SEQ ID NO: 4, which is having 100% identity to instant SEQ ID NO: 37, as shown below.
RESULT 2
US-15-514-892-4
Sequence 4, US/15514892
Publication No. US20170233762A1
GENERAL INFORMATION
APPLICANT: The Regents of the University of California
APPLICANT: Zalatan, Jesse
APPLICANT: Qi, Lei
APPLICANT: Lim, Wendell
TITLE OF INVENTION: SCAFFOLD RNAS
FILE REFERENCE: 081906-1039785-218610US
CURRENT APPLICATION NUMBER: US/15/514,892
CURRENT FILING DATE: 2017-03-28
PRIOR APPLICATION NUMBER: PCT/US2015/053034
PRIOR FILING DATE: 2015-09-29
PRIOR APPLICATION NUMBER: US 62/057,120
PRIOR FILING DATE: 2014-09-29
NUMBER OF SEQ ID NOS: 55
SEQ ID NO 4
LENGTH: 62
TYPE: PRT
ORGANISM: Artificial Sequence
OTHER INFORMATION: synthetic peptide construct
Query Match 100.0%; Score 344; Length 62; Best Local Similarity 100.0%;
Matches 61; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 KSIRCKNCNKLLFKADSFDHIEIRCPRCKRHIIMLNACEHPTEKHCGKREKITHSDETVR 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 2 KSIRCKNCNKLLFKADSFDHIEIRCPRCKRHIIMLNACEHPTEKHCGKREKITHSDETVR 61
Qy 61 Y 61
|
Db 62 Y 62
Before the effective filing date, it would have been obvious to an ordinarily skilled artisan to use the com scaffold binding protein COM, as described by Zalatan et al. to recognize and bind to the com sequence, with a realistic goal edit specific target sequence(s) in a plant genome.
Before the effective filing date, one with ordinary skill in the rat would have been motivated to use the com scaffold binding protein COM to recognize and bind to the com sequence, with a realistic goal edit specific target sequence(s) in a plant genome.
Claim 16-18 are rejected under 35 U.S.C. 103 as being unpatentable over Brezgin et al. as applied to claims 1, 4-6, 14-15 and 20 above, and further in view of Wang et al. (1) (CRISPR-Cas9 and CRISPR-Assisted Cytidine Deaminase Enable Precise and Efficient Genome Editing in Klebsiella pneumoniae, 2018, Appl. Environ. Microbiol., 84:e01834-18).
Brezgin et al. describes a multiplex genome editing system in various organisms including plants using CRISPR-Cas9 technique comprising nickase nCas9 (nCas9), scaffold RNA (scRNA) containing aptamer sequence(s) (e.g. MS2), aptamer-specific binding proteins (e.g. MCP that binds to MS2 sequence), effector proteins (e.g. cytidine/cytosine deaminases like APOBEC1), as discussed above. Brezgin et al. also describes aptamer-specific binding proteins (e.g. MCP) fused to effector proteins including transcriptional activators (page 6, para 4; page 7, Fig. 2) resulting in fusion proteins (of aptamer-specific binding proteins and effector proteins). Effectors proteins can be a deaminase (either for adenine or cytosine). The fusion proteins are made by fusing the aptamer-specific binding proteins with the N-terminal of the effector protein like cytosine deaminase or cytosine deamination domain (as recited in claim 17), as discussed above. Brezgin et al. also describes use of peptide linkers (as recited in claim 18) to further enhance efficacy of the fusion proteins (page 3, para 6, line 3-5; page 4, para 4, line 3-5)
However, Brezgin et al. does not teach any protein having 100% sequence identity to either SEQ ID NO: 26 (which has been rejoined for compact prosecution) or SEQ ID NO: 29 (the elected species).
Wang et al.(1) describes precise and efficient genome editing in industrial microorganism Klebsiella pneumoniae using cytidine deaminase APOBEC1 and a Cas9 nickase (title; abstract, line 14-15). It also describes a rat APOBEC1 (rAPOBEC1) (page 7, para 2, line 11) (GenBank Accession No. AYA60272) cloned in pBECKP-km vector (page 13, last para, line 3; Fig. 3), which is having 100% sequence identity to instant SEQ ID NO: 26.
Title: US-17-909-309A-26
Perfect score: 1275
Sequence: 1 SSETGPVAVDPTLRRRIEPH..........LQSCHYQRLPPHILWATGLK 228
Searched: 1 seqs, 1154 residues
Total number of hits satisfying chosen parameters: 1
Post-processing: Minimum Match 0%
Maximum Match 100%
Listing first 1 summaries
Database : AASEQ2_10222025_130526.pep:*
RESULT 1
AASEQ2_10222025_130526
Query Match 100.0%; Score 1275; DB 1; Length 1154; Best Local Similarity 100.0%;
Matches 228; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKH 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 12 SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKH 71
Qy 61 VEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYH 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 72 VEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYH 131
Qy 121 HADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLE 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 132 HADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLE 191
Qy 181 LYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK 228
||||||||||||||||||||||||||||||||||||||||||||||||
Db 192 LYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK 239
Before the effective filing date, it would have been obvious to an ordinarily skilled artisan to use a functional equivalent of cytidine deaminase APOBEC1 including the rAPOBEC1, as described by Wang et al.(1), with a realistic goal to edit a genome by precisely converting specific C to T in a gene. Using any specific cytidine deaminase which is functionally equivalent to instant SEQ ID NO: 26 or SEQ ID NO: 29 is an experimental design choice of an ordinarily skilled artisan which would not have expected to change the outcome. Similarly, it also an experimental design choice of an ordinarily skilled artisan to make fusion proteins by fusing the aptamer-specific binding proteins (e.g. MCP) with the N-terminal of the effector protein like cytosine deaminase or cytosine deamination domain (as recited in claim 17) using a linker protein (as recited in claim 18), as discussed above. Using a linker to make fusion protein(s) by fusing two or more proteins/domains is a standard practice in the art that offers several advantages such as improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles2.
Before the effective filing date, an ordinarily skilled artisan would have been motivated to use any specific cytidine deaminase rAPOBEC1 fused with an aptamer-specific binding protein using a linker with a realistic goal to edit a genome by precisely converting specific C to T.
Claim 19 is rejected under 35 U.S.C. 103 as being unpatentable over Brezgin et al. as applied to claims 1, 4-6, 14-15 and 20 above, and further in view of Grunewald et al. (WO2018218206-A1).
Claim 19 depends from claim 1 and is drawn to the first fusion protein comprising an uracil DNA glycosylase inhibitor (UGI), for example, the UGI comprises the amino acid sequence shown in SEQ ID NO: 31.
Brezgin et al. describes a multiplex genome editing system in various organisms including plants using CRISPR-Cas9 technique comprising nickase nCas9 (nCas9), scaffold RNA (scRNA) containing aptamer sequence(s) (e.g. MS2), aptamer-specific binding proteins (e.g. MCP that binds to MS2 sequence), effector proteins, as discussed above. Brezgin et al. teaches that efficacy of cytosine base editors is fairly low due to repair of edited nucleotides by endogenous DNA repair systems like the UNG factor. Improved efficacy of base editing is shown for nickase nCas9 (nCas9) proteins linked to base-editing factors and co-expressed with UGI, an UNG inhibitor. Blocking UNG by UGI transiently impairs DNA repair so that deaminated nucleotides are not corrected.
However, Brezgin et al. does not explicitly describe an uracil DNA glycosylase inhibitor (UGI) comprising of the amino acid sequence shown in SEQ ID NO: 31.
Grunewald et al. describes a Base Editing (BE) technology which is based on the fusion of a DNA binding domain (e.g. catalytically inactive dCas9, nCas9, or nickase) to a cytidine or adenine deaminase and a uracil glycosylase inhibitor (UGI) (page 1, line 14-16). The system comprises: (i) a first fusion protein comprising a programmable DNA binding domain, preferably a transcription-activator-like effector (TALE) or zinc finger array (ZF), fused to a deaminase enzyme, or an active portion thereof, optionally with a linker therebetween, or (ii) a fusion protein comprising a DNA binding domain of Cas9 protein that lacks nuclease activity or is a nickase, but can interact with a guide RNA and target DNA, fused to a Uracil glycosylase inhibitor (UGI), optionally using a linker (abstract). The method also describes targeted deamination of one or more selected cytosines in a nucleic acid (abstract). It describes a UGI protein sequence (SEQ ID NO: 7; Uniprot: P14739) (page 15, line 18-22) having 100% identity to instant SEQ ID NO: 31, as shown below.
BFW14151
ID BFW14151 standard; protein; 83 AA.
AC BFW14151;
DT 24-JAN-2019 (first entry)
DE Bacteriophage uracil glycosylase inhibitor polypeptide, SEQ ID 7.
KW CRISPR-Cas system; CRISPR-Cas9 system; Deamination; UGI protein; Uracil glycosylase inhibitor; genome editing; recombinant protein.
OS unidentified phage.
PN WO2018218206-A1.
CC PD 29-NOV-2018.
CC PF 25-MAY-2018; 2018WO-US034742.
PR 25-MAY-2017; 2017US-0511296P.
PR 04-AUG-2017; 2017US-0541544P.
PR 26-JAN-2018; 2018US-0622676P.
CC PA (GEHO ) GEN HOSPITAL CORP.
CC PA (GRUN/) GRUNEWALD J.
CC PI Grunewald J, Joung JK, Angstman J, Gehrke JM;
DR WPI; 2018-94915V/82.
DR UNIPROT; P14739.
CC PT New fusion protein useful in bipartite cytosine base editor system for targeting deamination of selected cytosines in nucleic acid, comprises fusion protein containing non-R-loop-forming programmable DNA binding domain.
CC PS Disclosure; SEQ ID NO 7; 61pp; English.
CC The present invention relates to a novel fusion protein, useful in a bipartite cytosine base editor system for targeting deamination of selected cytosines in nucleic acid. The fusion protein comprises (i) a first fusion protein comprising a non-R-loop-forming programmable DNA binding domain, preferably a transcription-activator-like effector (TALE)or zinc finger array (ZF), fused to a deaminase enzyme, or its active portion, optionally with a linker, or (ii) a second fusion protein comprising an R-loop-forming clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) protein that lacks nuclease activity or is a nickase, but can interact with a guide RNA and target DNA, fused to a uracil glycosylase inhibitor (UGI), optionally with a linker. The invention further relates to: (1) a composition comprising a purified fusion protein; (2) a nucleic acid encoding the fusion protein; (3) a vector comprising the nucleic acid; (4) an isolated host cell expressing the fusion protein; (5) a method for targeting deamination of selected cytosines in a nucleic acid; (6) a method for targeting deamination of a nucleic acid in a cell; and (7) a monomeric fusion protein. The present sequence is a bacteriophage uracil glycosylase inhibitor polypeptide, used in the method for targeting deamination of selected cytosines in a nucleic acid.
SQ Sequence 83 AA;
ALIGNMENT:
Query Match 100.0%; Score 418; Length 83; Best Local Similarity 100.0%;
Matches 83; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSD 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSD 60
Qy 61 APEYKPWALVIQDSNGENKIKML 83
|||||||||||||||||||||||
Db 61 APEYKPWALVIQDSNGENKIKML 83
Before the effective filing date, it would have been obvious to an ordinarily skilled artisan to use an UGI polypeptide, as taught by Grunewald et al., to improve efficacy of base editing by expressing nickase nCas9 (nCas9) proteins linked to base-editing factors like cytosine deaminase and co-expressed with UGI, as described by Brezgin et al.
Before the effective filing date of the invention, one with ordinarily skilled would have been motivated to use an UGI polypeptide (Uniprot: P14739) to improve efficacy of base editing by expressing a nickase nCas9 (nCas9) protein linked to base-editing factors like cytosine deaminase and co-expressed with UGI.
Claim 27 is rejected under 35 U.S.C. 103 as being unpatentable over Brezgin et al. as applied to claims 1, 4-6, 14-15 and 20 above, and further in view of Wang et al. (2) (A novel CRISPR/Cas9 system for efficiently generating Cas9-free multiplex mutants in Arabidopsis, 2020, aBIOTECH (2020) 1:6–14; Published online on 20 Nov. 2019).
Claim 27 depends from claim 1 and is drawn to the CRISPR nickase, the first fusion protein, and/or the second fusion protein interlinked by a "self-cleavage" peptide.
Brezgin et al. describes a multiplex genome editing system in various organisms including plants using CRISPR-Cas9 technique comprising nickase nCas9 (nCas9), scaffold RNA (scRNA) containing aptamer sequence(s) (e.g. MS2), aptamer-specific binding proteins (e.g. MCP that binds to MS2 sequence), effector proteins, and various fusion proteins, as discussed above.
However, Brezgin et al. does not describe any fusion protein containing a “self-cleavage” peptide.
Wang et al.(2) describes a novel CRISPR/Cas9 system that can generate Cas9-free multiplex mutants efficiently in plants. The system comprises the “most efficient self-cleaving peptide”, 2A, fused to Cas9 and GFP, and then the fused protein Cas9-P2A-GFP is used to generate Cas9-free multiplex mutants efficiently in plants (abstract).
Before the effective filing date, it would have been obvious to an ordinarily skilled artisan to use a polynucleotide sequence encoding a “self-cleaving peptide”, as described by Wang et al. (2), to interlink Cas nickase and any other fusion protein(s) (as described by Brezgin et al.) with a realistic goal to produce transgene-free genome edited plants, as described by Wang et al.
Before the effective filing date, one with ordinary skill in the art would have been motivated to use a polynucleotide sequence encoding a “self-cleaving peptide” to interlink Cas nickase and any other protein(s) including any fusion protein(s), with the realistic goal to produce transgene-free genome edited plants.
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 14-15 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of copending Application No. US20230313234A1 (reference application, hereafter referred to as ‘234A) in evidence of Grunewald et al. (WO 2018/218206 Al). Although the claims at issue are not identical, they are not patentably distinct from each other because claim 1 of ‘234A recites a base editing fusion protein comprising an APOBEC3B deaminase or a APOBEC3B deaminase, which is a functional variant of APOBEC3A, APOBEC3G, and APOBEC1, as recited in instant claims 14-15.
Claim 1 of ‘234A recites “…a CRISPR effector protein…”. The description in ‘234A recites, “The term (effector protein) covers any effector protein based on the CRISPR system and capable of achieving gene targeting (such as gene editing and targeted gene regulation) in cells”. Being part of a CRISPR system, claim 1 of ‘234A implies comprising a scaffold RNA (as part of gRNA) which is critical to bind to any other protein (besides Cas nuclease) including base editing proteins. The base editing fusion protein comprising an APOBEC3B deaminase or a APOBEC3B deaminase mutant, as recite din claim 1 of ‘234A, is implied to be fused to a Cas nickase (Grunewald et al., abstract; page 8, line 30-32 and page 9 line 1-2) and all these proteins (as recited in instant claim 14) are apolipoprotein B mRNA editing cytosine deaminase (Grunewald et al. page 3, line 14-18).
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Conclusion
No claim is allowed.
Although claims 7-8 are rejected under 35 USC § 112(a) (written description), claims 7-8 and 10 are free of prior art.
Claim 10 is objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
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Jay Chatterjee
Patent Examiner
Art Unit 1662
/Jay Chatterjee/ Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661& 1662
1 Delannoy et al. (Arabidopsis tRNA Adenosine Deaminase Arginine Edits the Wobble Nucleotide of Chloroplast tRNAArg(ACG) and Is Essential for Efficient Chloroplast Translation, 2009, Plant Cell, 21:2058–2071) provides the evidence that deamination domain of TadA proteins in E coli (page 2062, left column, para 3, line 7; page 2069, right column, para 3, line 5-7) and in plants (abstract, line 6-8) are known long before the effective filing date of the invention.
2 Chen et al. (Fusion Protein Linkers: Property, Design and Functionality, 2013, Adv Drug Deliv Rev. 2013 October 15; 65:1357–1369) provides the evidence that linkers offers several advantages such as improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles in fusion proteins.
3Cui et al. (Review of CRISPR/Cas9 sgRNA Design Tools, Interdisciplinary Sciences: Computational Life Sciences (2018) 10:455-465) provides the evidence that designing gRNA(s) (or sgRNAs) is a well-known standard process (abstract).
Prosecution Insights