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 .
Claim Status
Claims 1-14 are pending and being examined.
Claim Objections
Claim 1 is objected to because of the following informalities:
Claim 1 recites, “…6-nucleotide length target sequence… or a 16- or 17- nucleotide length target sequence…” (in line 2). It is suggested to replace the term “length” with “long”.
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 1-14 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 1 and 14, it is apparent to the Examiner that the target sequence (in the host genomic DNA) comprises the mismatch sequence present in the crRNA. It is, however, not clear to the Examiner what is/are the difference(s) between the “target sequence” and the “mismatch sequence” in the context of the Casϕ cleavage.
It is also not clear to the Examiner if the Casϕ protein cleaves the target sequence at a separate site (than the site with mismatch) as well as at the site of the mismatch sequence present in the crRNA and host genomic DNA, OR Casϕ protein cleaves the target sequence at or near the mismatch sequence (with the crRNA).
To provide better customer service and compact prosecution, the Examiner interprets that Casϕ protein cleaves the target sequence in the host genome at or near the mismatch sequence.
Claim 13 recites, “… wherein the expression vector is a transient or a constitutive expression vector”. It is not clear to the Examiner what the Applicant means by “a transient or a constitutive expression vector”. Vectors are not known to be a transient or a constitutive vector. It is the promoter(s), which is/are used to control the expression of one or more polynucleotide sequences cloned in a vector, that are referred to as “transient” or a “constitutive”. There can be vectors comprising both transient and constitutive promoters controlling expression of different genes in a single expression vector.
It is suggested to amend the claim in line to include or imply expression vector(s) comprising a transient or a constitutive promoter.
Claim Rejections - 35 USC § 112(a)
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.
Written Description
Claim 3 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim 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 inventors, at the time the application was filed, had possession of the claimed invention.
Claim 3 recites “… Casϕ protein includes an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3 and 4”.
The Applicant describes four different Casϕ proteins- Casϕ1 (SEQ ID NO: 2), Casϕ2 (SEQ ID NO: 1), Casϕ3 (SEQ ID NO: 3) (spec, p.5, Fig. 5A) and vCasϕ (SEQ ID NO: 4) (spec, p6, Fig. 5J).
The Applicant does not describe any species of Casϕ protein that has less than 100% sequence identity to instant SEQ ID NOs: 1-4 while claiming the broad genus of a Casϕ protein having at least 80% sequence identity to SEQ ID NOs: 1-4. In other words, there is no example of Casϕ1 variant with less than 100% sequence identity to SEQ ID NO: 2 or a Casϕ2 variant with less than 100% sequence identity to SEQ ID NO: 1.
The Applicant also does not provide any structure function relationship between the structure (i.e., protein sequence) of the Casϕ protein and its function as an endonuclease so that a skilled artisan can mutate up to 80% of the amino acid residues in the protein along the entire length of the polypeptide while retaining its endonuclease functions; e.g., mutating up to 151 amino acids in the 757 amino acid long Casϕ2 protein (SEQ ID NO: 1) while retaining the endonuclease functions of Casϕ2.
It is known in the art that the RuvC domain in Casϕ proteins is responsible for its endonuclease activity (Pausch et al., CRISPR-CasΦ from huge phages is a hypercompact genome editor, 2020, Science, 369:333–337; p.3, para 3, line 12-13). The claim includes mutation(s) in the RuvC domain (and other domains). Moreover, it is also known in the art that change in even one amino acid in the polypeptide can modify the secondary and/or tertiary structure of the protein and, thus, can affect its function. Alanine substitution of the lid residues 610-614, or replacement of the lid-loop structure (aa 610-638) by a short glycine-serine linker (GSSG), abolished CasΦ’s ability to cut dsDNA (Pausch et al., DNA interference states of the hypercompact CRISPR-CasΦ effector, 2021, Nat. Struct. Mol. Biol., 28:652–661).
Considering the breadth of the claims, lack of representative species of the broad genus claimed, lack of structure function relationship of the broad genus claimed, and unpredictability of the art, the Applicant does not appear to have been in possession of the claimed genus at the time this application was filed.
Scope of Enablement
Claim 3 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for Casϕ protein having 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4, does not reasonably provide enablement for Casϕ protein having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
Claim 3 recites “… Casϕ protein includes an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3 and 4”.
The Applicant describes four different Casϕ proteins- Casϕ1 (SEQ ID NO: 2), Casϕ2 (SEQ ID NO: 1), Casϕ3 (SEQ ID NO: 3) (spec, p.5, Fig. 5A) and vCasϕ (SEQ ID NO: 4) (spec, p6, Fig. 5J).
The Applicant does not provide any example of a Casϕ protein that has less than 100% sequence identity to corresponding sequence among instant SEQ ID NOs: 1-4. For example, there is no example of a Casϕ1 protein with less than 100% sequence identity to SEQ ID NO: 2 or a Casϕ2 with less than 100% sequence identity to SEQ ID NO: 1 and still able to perform all the functions of a Casϕ endonuclease.
The Applicant does not provide guidance to enable a skilled artisan to make a Casϕ endonuclease having at least 80% sequence identity to SEQ ID NOs: 1-4 along the entire length of the polypeptide, e.g., mutating up to 151 amino acids in the 757 amino acid long Casϕ2 protein (SEQ ID NO: 1) while retraining all the functions of a Casϕ endonuclease.
It is known in the art that the RuvC domain in the Casϕ protein is responsible for its endonuclease activity (Pausch et al., CRISPR-CasΦ from huge phages is a hypercompact genome editor, 2020, Science, 369:333–337; p.3, para 3, line 12-13). It is also known in the art that a change in even one critical amino acid in a polypeptide can modify the secondary and/or tertiary structure of the protein and, thus, can affect its function. Alanine substitution of the lid residues 610-614, or replacement of the lid-loop structure (aa 610-638) by a short glycine-serine linker (GSSG), abolish CasΦ’s ability to cut dsDNA (Pausch et al., DNA interference states of the hypercompact CRISPR-CasΦ effector, 2021, Nat. Struct. Mol. Biol., 28:652–661).
Undue trial and error experimentations would be needed to mutate up to 20% of the amino acid residues in SEQ ID NOs: 1-4 while retaining all the functions of the Casϕ protein(s).
Based on breadth of the claims, lack of any working example, lack of guidance in the instant description or in prior art, the specification at the time of the application filed would not have taught one skilled in the art how to make and use the full scope of the claimed invention without performing undue experiments.
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 1-14 are rejected under 35 U.S.C. 103 as being unpatentable over Hyams et al. (CRISPys: Optimal sgRNA Design for Editing Multiple Members of a Gene Family Using the CRISPR System, 2018, J. Mol. Biol., 430:2184–2195) in view of Pausch et al. (CRISPR-CasΦ from huge phages is a hypercompact genome editor, 2020, Science, 369:333–337).
Claim 1 is drawn to a genome editing method for duplicated genes having 16-17 nucleotide long target sequence comprising a mismatch sequence of 1-2 nucleotide, wherein the method comprises a step of expressing a crRNA containing a spacer sequence targeting the target sequence and a Casϕ protein derived from a huge phage which is able to cleave the target sequence.
Hyams et al. teaches that due to large-scale genomic duplications, many eukaryotic genomes harbor homologous gene families of partially overlapping functions, which reads on to “functional redundancy”, as recited in claim 4 (p. 2184, left column, para 1, line 1-4). Hyams et al. describes a CRISPR-Cas9 based genome editing method to simultaneously knockout/edit multiple members of a gene family by using mismatch(es) in the target sequences of the homologous members of the gene family (abstract, line 6-9). The sgRNA used is designed to perfectly match (reads on to “completely match”) one of the target gene (as recited in claim 6) in the duplicated gene family members while the other two homologs possessed one and two mismatches, as recited in claim 1 (p.2185, left column, para 2, line 10-13).
Hyams et al. also describes 17-23 nucleotide long spacer sequence upstream of the PAM motif(s)/sequence(s) in the gRNA, and, thus, also present in the target sequence (which reads on to “a PAM sequence near the target sequence”, as recited in claim 5) can be used (bridging paragraph between left column and right column, p.2186). 17-23 nucleotide long spacer sequence in the gRNA/crRNA would align with similar 17-23 long target sequence, which reads on to “6- or 17- nucleotide length target sequence” as recited in claims 1 and 7-8.
It is noted here that the terms sgRNA, gRNA, and crRNA indicate the same RNA molecule comprising the spacer sequence used in CRISPR-Cas based genome editing technique and are often used interchangeably. The Applicant describes and uses the terms gRNA and crRNA (comprising the spacer sequence) interchangeably and as synonyms (spec, p.1, para 0003, line 1-3).
Hyams et al. describes editing 2-10 members of a gene family (as recited in claims 9-10) among a total of 3697 gene families in a plant cell (as recited in claim 11) of a tomato (S. lycoperesicum) genome (p. 2188, right column, last para, last 4 lines; p.2189, left column, last para, last 4 lines).
However, Hyams et al. does not explicitly describe any Casϕ protein.
Pausch et al. describes a minimal functional CRISPR-Cas system comprising a single ~70 kilodalton protein, CasΦ encoded exclusively in the genomes of huge bacteriophages (title; abstract, line 2-4), as recited in claim 1. Pausch et al. teaches several Casϕ proteins including Casϕ2 (p. 2, para 3, line 3-4; Fig. S3) (as recited in claim 2) which comprises 100% sequence identity to instant SEQ ID NO: 1 (as recited in claim 3), as shown below.
Title: US-19-109-216C-1
Perfect score: 3971
Sequence: 1 MPKPAVESEFSKVLKKHFPG..........KAPPAEREDQTPAQEPSQTS 757
Searched: 1 seqs, 763 residues
Database : AASEQ2_06012026_101326.fasta:*
RESULT 1
AASEQ2_06012026_101326
Best Local Similarity 100.0%; Query Match 100.0%; Score 3971; DB 1; Length 763;
Matches 757; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MPKPAVESEFSKVLKKHFPGERFRSSYMKRGGKILAAQGEEAVVAYLQGKSEEEPPNFQP 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MPKPAVESEFSKVLKKHFPGERFRSSYMKRGGKILAAQGEEAVVAYLQGKSEEEPPNFQP 60
Qy 61 PAKCHVVTKSRDFAEWPIMKASEAIQRYIYALSTTERAACKPGKSSESHAAWFAATGVSN 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 PAKCHVVTKSRDFAEWPIMKASEAIQRYIYALSTTERAACKPGKSSESHAAWFAATGVSN 120
Qy 121 HGYSHVQGLNLIFDHTLGRYDGVLKKVQLRNEKARARLESINASRADEGLPEIKAEEEEV 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 HGYSHVQGLNLIFDHTLGRYDGVLKKVQLRNEKARARLESINASRADEGLPEIKAEEEEV 180
Qy 181 ATNETGHLLQPPGINPSFYVYQTISPQAYRPRDEIVLPPEYAGYVRDPNAPIPLGVVRNR 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 ATNETGHLLQPPGINPSFYVYQTISPQAYRPRDEIVLPPEYAGYVRDPNAPIPLGVVRNR 240
Qy 241 CDIQKGCPGYIPEWQREAGTAISPKTGKAVTVPGLSPKKNKRMRRYWRSEKEKAQDALLV 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 CDIQKGCPGYIPEWQREAGTAISPKTGKAVTVPGLSPKKNKRMRRYWRSEKEKAQDALLV 300
Qy 301 TVRIGTDWVVIDVRGLLRNARWRTIAPKDISLNALLDLFTGDPVIDVRRNIVTFTYTLDA 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 TVRIGTDWVVIDVRGLLRNARWRTIAPKDISLNALLDLFTGDPVIDVRRNIVTFTYTLDA 360
Qy 361 CGTYARKWTLKGKQTKATLDKLTATQTVALVAIDLGQTNPISAGISRVTQENGALQCEPL 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 CGTYARKWTLKGKQTKATLDKLTATQTVALVAIDLGQTNPISAGISRVTQENGALQCEPL 420
Qy 421 DRFTLPDDLLKDISAYRIAWDRNEEELRARSVEALPEAQQAEVRALDGVSKETARTQLCA 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 DRFTLPDDLLKDISAYRIAWDRNEEELRARSVEALPEAQQAEVRALDGVSKETARTQLCA 480
Qy 481 DFGLDPKRLPWDKMSSNTTFISEALLSNSVSRDQVFFTPAPKKGAKKKAPVEVMRKDRTW 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 DFGLDPKRLPWDKMSSNTTFISEALLSNSVSRDQVFFTPAPKKGAKKKAPVEVMRKDRTW 540
Qy 541 ARAYKPRLSVEAQKLKNEALWALKRTSPEYLKLSRRKEELCRRSINYVIEKTRRRTQCQI 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 ARAYKPRLSVEAQKLKNEALWALKRTSPEYLKLSRRKEELCRRSINYVIEKTRRRTQCQI 600
Qy 601 VIPVIEDLNVRFFHGSGKRLPGWDNFFTAKKENRWFIQGLHKAFSDLRTHRSFYVFEVRP 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 VIPVIEDLNVRFFHGSGKRLPGWDNFFTAKKENRWFIQGLHKAFSDLRTHRSFYVFEVRP 660
Qy 661 ERTSITCPKCGHCEVGNRDGEAFQCLSCGKTCNADLDVATHNLTQVALTGKTMPKREEPR 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 ERTSITCPKCGHCEVGNRDGEAFQCLSCGKTCNADLDVATHNLTQVALTGKTMPKREEPR 720
Qy 721 DAQGTAPARKTKKASKSKAPPAEREDQTPAQEPSQTS 757
|||||||||||||||||||||||||||||||||||||
Db 721 DAQGTAPARKTKKASKSKAPPAEREDQTPAQEPSQTS 757
Pausch et al. teaches that the small size of Casϕ which is half that of Cas9 and Cas12a (p.2, para 1, line 4) in combination with its minimal PAM requirement is advantageous for both (expression) vector-based delivery into cells (as recited in claim 12) and a wider range of targetable genomic sequences (abstract; p.4, para 2, last 3 lines). Pausch et al. describes several expression vectors comprising an inducible promoter (tetracycline inducible promoter), which is known to be used to control transient gene expression, and expression vectors comprising constitutive promoters like U6 (RNA Polymerase III promoter) and CMV-CAG promoters (Supplementary material, Table S1), as recited in claim 13.
The Applicant does not define “a kit” used for genome editing of duplicated genes in a cell. The Examiner interprets a kit to include applicable protocol(s) and all the reagents including the expression vector(s) needed for genome editing. Pausch et al. describes the protocol and all the components needed for genome editing including the expression vectors (reads on to “expression cassette”) based on Golden Gate assembly system (Supplementary material, p.3, para 2, line 2) while the expression vectors/cassettes comprising analyzed and validated gRNA/CrRNA sequence (Supplementary material, bridging paragraph between p.2-3) targeting the target sequence and mismatch sequence(s), and expression cassette for CasΦ protein derived from a huge phage (Supplementary material, Table S1), as recited in claim 14.
Before the effective filing date of the invention, it would have been obvious to an ordinarily skilled artisan to modify the CRISPR-Cas9 based genome editing method to simultaneously knockout/edit multiple members of a duplicated genes in a gene family by replacing the Cas9 endonuclease, as described by Hyams et al., with Casϕ endonuclease as described by Pausch et al.
Before the effective filing date of the invention, an ordinarily skilled artisan would have been motivated to use Casϕ endonuclease, instead of Cas9, because the small size of Casϕ in combination with its minimal PAM requirement which is advantageous for expression vector-based delivery of gRNA/crRNA and the endonuclease into the genome of the target cells, as described by Pausch et al.
Conclusion
No claim is allowed.
Communication
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JAY CHATTERJEE whose telephone number is (703)756-1329. The examiner can normally be reached (Mon - Fri) 8.30 am to 5.30 pm..
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Bratislav Stankovic can be reached at (571) 270-0305. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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Jay Chatterjee
/Jay Chatterjee/ Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/ Supervisory Patent Examiner, Art Units 1616 & 1662
Prosecution Insights