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/Restriction
Applicant’s election of Group I, claims 1, 2, 4-5, 8, 11, 16, 20-21, 23, 25-26, 29, and 31, as well as new claims 72-78 and election of Species I: Type IIIA in the reply filed on 02/05/2026 is acknowledged. Applicant’s statement that “all elected claims encompass this species election” is accepted. 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)).
In response to the Election/Restriction, applicant cancelled claims 3-34, 47-48, 50, 53, and 64-65 without prejudice. Applicant further amended claim 16 and added new claims 72-78. Applicant’s statement that “support for these amendments and new claims is found in the claims as originally filed and throughout the specification (see, e.g., original claims 7, 9- 10, 13, 18-19, and 22) is accepted.
Status of claims
Claims 1-2, 4-5, 8, 11, 16, 20-21, 23, 25-26, 29, 31 and 72-78 are pending and under examination in this office action.
Claim Objections
Claim 31 is objected to because of the following informalities: claim 31 recites “Csm10/Csm1.” The examiner believes this is a misspelling of Cas10 and will consider Csm10 to mean Cas10 for compact prosecution and customer service. Appropriate correction is required.
Claim Rejections - 35 USC § 112
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.
Claim 26 is 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.
Claim 26 is incomplete in itself as it references specific figures, FIG. 7A-7E and FIG. 6A-6F. Where possible, claims are to be complete in themselves. Incorporation by reference to a specific figure or table "is permitted only in exceptional circumstances where there is no practical way to define the invention in words and where it is more concise to incorporate by reference than duplicating a drawing or table into the claim. Incorporation by reference is a necessity doctrine, not for applicant’s convenience." Ex parte Fressola, 27 USPQ2d 1608, 1609 (Bd. Pat. App. & Inter. 1993) (citations omitted). See 2173.05(s) Reference to Figures or Tables [R-10.2019].
Claim 26 recites "to any of the amino acid sequences of the Cas10/Csm1, Csm2, Csm3, Csm4, and Csm5 polypeptides of SEQ ID Nos: 1-5 or FIG. 7A-7E." Thus, the claim as recited requires that the five different polypeptides "each independently comprises" any of the amino acid sequences of SEQ ID NOs: 1-5 or FIG. 7A-7E. The problem pertains to SEQ ID NOs: 1-5 are only Csm1 sequence variants, so SEQ ID NOs: 1-5 do not correspond to "each" of the five polypeptides.
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, 4-5, 8, 11, 16, 20, 23, 25-26, 29, 31, 73-75, and 78 are rejected under 35 U.S.C. 102(a)(1) as being clearly anticipated by Colognori D., et. al., (bioRxiv 496908, published 2022-06-20, provided in IDS, and supplementary materials).
Regarding claim 1, Colognori teaches a method for modifying a target RNA in a eukaryotic cell (Discussion: “we have shown that the Type III-A Csm complex from Streptococcus thermophilus is a powerful tool for eukaryotic RNA knockdown;” Introduction: “we demonstrate the utility of the Csm system as a highly efficient, specific, and versatile RNA knockdown tool in eukaryotes;” Abstract: “these results establish the feasibility and efficacy of multi-protein CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes.”), the method comprising:
introducing into the eukaryotic cell:
a) one or more nucleic acids comprising nucleotide sequences encoding a multi-subunit Type Ill CRISPR-Cas effector polypeptide, wherein the multi-subunit Type Ill CRISPR-Cas effector polypeptide comprises at least 5 subunits (Introduction: “The multi-protein Csm complex comprises five subunits (Csm1-5) in varying stoichiometries and relies on an additional protein, Cas6, for processing the precursor crRNA;” Methods: “1×10^6 HEK293T cells were transfected with 2.5-5 ug plasmid DNA using 7.5-15 ul FuGENE HD transfection reagent;” Abstract: “Using single-vector delivery of the S. thermophilus Csm complex, we observe high-efficiency RNA knockdown (90-99%) and minimal off-target effects in human cells, outperforming existing technologies including shRNA- and Cas13-mediated knockdown;” see Fig. 1B; see Fig. 1J; Results: “seven plasmids individually expressing Csm1-5, Cas6, and either a GFP-targeting or non-targeting crRNA from a U6 promoter were co-transfected into cells;” ); and
b) one or more guide RNAs, wherein each of the one or more guide RNAs comprises: i) a targeting region that comprises a nucleotide sequence that is complementary to a target sequence in the target RNA; and ii) a protein-binding region that binds to the multi-subunit Type Ill CRISPR-Cas effector polypeptide; or a nucleic acid comprising a nucleotide sequence encoding the guide RNA (Results: “seven plasmids individually expressing Csm1-5, Cas6, and either a GFP-targeting or non-targeting crRNA from a U6 promoter were co-transfected into cells.”), wherein the multi-subunit Type Ill CRISPR-Cas effector polypeptide is produced in the cell and forms a complex with the guide RNA, and wherein the complex binds to the target RNA and results in modification of the target RNA in the cell (Introduction: “ the crRNA lies at the core of the complex, with Csm1 and Csm4 binding the 5’ end, Csm5 binding the 3’ end, and multiple copies of Csm2 and Csm3 wrapping around the center;” Results: “HEK293T cells were transfected, transfected (RFP-positive) cells were isolated by FACS after 48 hr, total cell RNA extracted, and RNA KD assayed by RT-qPCR (Fig. S1C,S2A). To our surprise, we achieved >90% KD for all eleven RNAs with at least one crRNA, compared to non-targeting crRNA control (Fig. 2A). These results demonstrate the Csm system to be a highly robust and efficient RNA KD tool for not only cytoplasmic but also nuclear RNAs, which are typically recalcitrant to KD by conventional RNAi methods.”).
Regarding claims 4-5, Colognori teaches the method of claim 1, wherein the nucleotide sequences encoding the at least 5 subunits are operably linked to a single promoter and/or wherein the nucleotide sequences encoding the at least 5 subunits are operably linked to two or more different promoters (Results: “with the Csm system up and running, we sought to simplify its delivery by consolidating all components into a single vector. For this, we pursued two approaches concurrently: 1. expression of each protein from separate promoters, or 2. expression of all proteins from a single bidirectional promoter separated by 2A peptides (Fig. 1J),” and “the single-promoter design is well-equipped for promoter-swapping and thus use in specific cell types or other eukaryotic systems, while the modular design of the separate-promoter vector allows for easy swapping or modification of individual Csm components.”).
Regarding claims, 8, 11, 16, 20, 23, 73-75, and 78 Colognori teaches the method of claim 1, wherein the one or more nucleic acids comprising nucleotide sequences encoding the multi-subunit Type Ill CRISPR-Cas effector polypeptide comprise a nucleotide sequence encoding the one or more guide RNAs, wherein the target RNA is a coding RNA, wherein the target RNA is an endogenous RNA, wherein the modifying comprises cleavage of the target RNA, wherein the target RNA is present in the nucleus or in an organelle, wherein the target RNA is present in the cytoplasm, wherein the target RNA is a non-coding RNA, wherein the target RNA is an exogenous RNA, and wherein the eukaryotic cell is a mammalian cell, a plant cell, an insect cell, a reptile cell, an amphibian cell, a protozoan cell, an arachnid cell, an avian cell, or a fish cell (Results: “seven plasmids individually expressing Csm1-5, Cas6, and either a GFP-targeting or non-targeting crRNA from a U6 promoter were co-transfected into cells;” Abstract: “we show that the CRISPR-Csm complex, a multi-protein effector from type III CRISPR immune systems in prokaryotes, provides surgical RNA ablation of both nuclear and cytoplasmic transcripts;” Results: “we chose to target a panel of three nuclear noncoding RNAs (XIST, MALAT1, NEAT1) and eight cytoplasmic mRNAs (BRCA1, TARDBP, SMARCA1, CKB, ENO1, MECP2, UBE3A, SMAD4) (Fig. 2A) of varying abundances (Fig. 2B), testing three individual crRNAs for each…immortalized human embryonic kidney HEK293T cells were transfected…we achieved >90% KD for all eleven RNAs with at least one crRNA…” Methods: “cells were grown for 48 hr post-transfection to allow protein expression and RNA KD to occur.”).
Regarding claims 25-26, Colognori teaches the method of claim 1, wherein the multi-subunit Type III CRISPR-Cas effector polypeptide is a Type III-A CRISPR-Cas effector polypeptide comprising Cas10/Csm1, Csm2, Csm3, Csm4, and Csm5 polypeptides; or is a Type III-B CRISPR-Cas effector polypeptide comprising Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6 subunits (Abstract: “we show that the CRISPR-Csm complex, a multi-protein effector from type III CRISPR immune systems in prokaryotes, provides surgical RNA ablation of both nuclear and cytoplasmic transcripts;” Introduction: “the multi-protein Csm complex comprises five subunits (Csm1-5) in varying stoichiometries and relies on an additional protein, Cas6, for processing the precursor crRNA;” Results: “we chose the Type III-A Csm complex from Streptococcus thermophilus for several reasons: 1. it has been extensively characterized biochemically, structurally, and in bacteria, 2. functions optimally at 37C, 3. has been demonstrated to work in zebrafish upon ribonucleoprotein (RNP) microinjection, and 4. has fewer components than the analogous Type III-B Cmr complex;” and “ seven plasmids individually expressing Csm1-5, Cas6, and either a GFP-targeting or non-targeting crRNA from a U6 promoter were co-transfected into cells.”); and wherein the Cas10/Csm1, Csm2, Csm3, Csm4, and Csm5 polypeptides each independently comprise an amino acid sequence having at least 50% amino acid sequence identity to any of the amino acid sequences of the Cas10/Csm1, Csm2, Csm3, Csm4, and Csm5 polypeptides of SEQ ID Nos: 1-5 or FIG. 7A-7E; and wherein the Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6 polypeptides each independently comprise an amino acid sequence having at least 50% amino acid sequence identity to any of the amino acid sequences of the Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6 polypeptides depicted in FIG. 6A-6F (See Supplemental Material Fig. 1 and Fig. S1-4, which disclose the vector/complex compositions, and Table S2, which discloses the polynucleotide sequence of the vectors comprising the nucleic acid coding sequence of each of the Csm subunits 1-5. Absent evidence to the contrary, the examiner is considering that the polynucleotide sequence encoding each Csm protein inherently encodes an open reading frame that when translated corresponds to polypeptide sequences of SEQ ID NOs: 1-5 or FIG. 7A-7E of the instant case with at least 50% identity score).
Regarding claims 29 and 31, Colognori teaches the method of claim 1, wherein the multi-subunit Type III CRISPR-Cas effector polypeptide comprises one or more amino acid substitutions that reduce DNAse activity, and wherein the multi-subunit Type III CRISPR-Cas effector polypeptide comprises one or more amino acid substitutions that reduce polymerization of ATP into a cyclic oligoadenylate (cA) molecule, wherein the one or more amino acid substitutions that reduce polymerization of ATP to cA comprise a substitution of D577, a substitution of D578, or a substitution of both D577 and D578 of a Csm10/Csm1 polypeptide (Results: “ablating DNase (H15A, D15A) and cA synthase (D577A, D578A) activities in Csm1 did not affect GFP KD, ablating RNase activity (D33A) in Csm3 completely abolished GFP KD (Fig. 1G), indicating that RNase activity is necessary and sufficient for KD.”).
Claims 1-2, 4, 8, 11, 20, 23, 25, 72, 74, and 76-78 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by and Lin P. et. al., (Nucleic Acids Research, Volume 50, Issue 8, Page e47, published Feb. 15, 2022, provided in IDS, and supplementary materials).
Regarding Claims 1-2, 4, 8, 11, 20, 23, 25, 72, 74, and 76-78 , Lin teaches that “Type III CRISPR endonuclease antivirals for coronaviruses (TEAR-CoV) derived from Streptococcus thermophilus type III CRISPR-Cas system to limit SARS-CoV-2 replication and infection by directly targeting and degrading the viral RNA genome and reduction of viral mRNAs” (i.e., coding RNA). See Title, Abstract, Introduction, Fig 1(A), Figure 1 legend, results and Discussion. Regarding claims 1, 11, and 76-77, Lin demonstrates “that TEAR-CoV-based RNA engineering approach leads to RNA-guided transcript degradation both in vitro and in eukaryotic cells, which could be used to broadly target RNA viruses.” See abstract. Regarding claim 20 and 23, while Lin performed strictly biochemical cleavage assays in vitro, Lin also performed assays in vitro with Eukaryotic cells: “Human Embryonic Kidney 293 plus T cell antigen (HEK293T cells, CRL-3216, ATCC) cells, Vero E6 cells and A549 cells were cultured,” and “HEK293T cells were transfected using LipofectAmine™ CRISPRMAX™ Transfection Reagent (Thermo Fisher Scientific) according to the instruction with 0–6.0 μg of StCsm complex and 2 μg gRNA.” See Cell Lines and TEAR-CoV transfection of HEK293T cells - Materials and Methods. Regarding claim 1 and 4, Lin teaches generating “a Csm complex expression vector, coding sequence of Csm1–2–3–4–5 was codon optimized and the whole sequence was synthesized and inserted into pcDNA3.1(+) vector…forming the pcDNA-CMV-CSM1–2–3–4–5 plasmid.” See methods. Lin further teaches creating a “gRNA expression vector, U6 promoter sequence, gRNA sequence and terminator T was also synthesized and inserted into pcDNA3.1/Zeo(+) … forming pcDNA/Zeo-U6-Type III-gRNA plasmid.” Regarding claim 1, 11, 20, 74 and 76-78, Lin further teaches experiments in vivo: “mice were treated with TEAR-CoV plasmids (pcDNA-CMV-CSM1–2–3–4–5 and pcDNA/Zeo-U6-Type III-gRNA) through TurboFect in vivo Transfection Reagent.” See IAV infection in mice – Materials and Methods. See methods. Lin teaches “TEAR-CoV is capable of inhibiting SARS-CoV-2 gene expression in human cells.” See results. Regarding claim 1, 11, 23, and 76-78, Lin teaches delivering “TEAR-CoV with gRNA5 targeting CTD sequence,” which is a viral coding sequence. See Fig. 3 and Fig. 3 legend. Lin reports “that TEAR-CoV not only cleaves SARS-CoV-2 genome and mRNA transcripts, but also degrades live influenza A virus (IAV), impeding viral replication in cells and in mice.” See abstract. Lin teaches “the fast design and broad targeting of TEAR-CoV may represent a versatile antiviral approach for SARS-CoV-2 or potentially other emerging human coronaviruses.” See abstract. Lin teaches “that further testing could determine the suitable delivery for TEAR-CoV to control infections and enhance human health.” See discussion ¶5. Regarding claims 1-2, 4, 8, 11, 20, 25, 72, 74, and 76-78, Lin teaches “TEAR-CoV may be an antiviral option for controlling SARS-CoV-2 to benefit the suffering human populations, when the CRISPR-based therapeutics may be directly delivered into human respiratory tract cells through a viral vector (i.e. AAV).” See discussion ¶5 and Supplementary Figure S5A. Lin teaches a schematic for AAV design carrying TEAR-CoV, wherein an AAV vector comprising both a single regulatable tissue-specific or cell-type-specific promoters operably linked to Cas10-csm2/3/4/5 and a Type III-gRNA operably linked to another promoter is delivered to a human subject. See (Supplementary Figure S5B) and below:
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Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 4-5, 8, 11, 16, 20-21, 23, 25-26, 29, 31 and 72-78 are rejected under 35 U.S.C. 103 as being unpatentable over Colognori D., et. al., (bioRxiv 496908, published 2022-06-20, provided in IDS, and supplementary materials) in view of Lin P. et. al., (Nucleic Acids Research, Volume 50, Issue 8, Page e47, published Feb. 15, 2022, provided in IDS, and supplementary materials) and Wilson C. et. al., (Nat Biotechnol.; 38(12):1431-1440, published Dec. 29, 2020).
The teaching of Colognori regarding claims 1, 4-5, 8, 11, 16, 20, 23, 25-26, 29, 31, 73-75, and 78 are incorporated herein by reference to the corresponding 102 rejection above.
Colognori teaches claim 1; however, Colognori does not teach limitations of claims 2, 21, 72, and 76-77, namely: wherein the one or more nucleic acids comprises one or more recombinant expression vectors selected from a recombinant adeno-associated virus vector, a recombinant lentivirus vector, a recombinant adenovirus vector, and a recombinant retro viral vector; wherein the modifying comprises methylation or adenylation; wherein the promoter is a regulatable promoter; and wherein the target RNA is a viral RNA.
Lin teaches that “Type III CRISPR endonuclease antivirals for coronaviruses (TEAR-CoV) derived from Streptococcus thermophilus type III CRISPR-Cas system to limit SARS-CoV-2 replication and infection by directly targeting and degrading the viral RNA genome and reduction of viral mRNAs” (i.e., coding RNA). See Title, Abstract, Introduction, Fig 1(A), Figure 1 legend, results and Discussion. Lin demonstrates “that TEAR-CoV-based RNA engineering approach leads to RNA-guided transcript degradation both in vitro and in eukaryotic cells, which could be used to broadly target RNA viruses.” See abstract. Lin teaches that “for Type I and II CRISPR-Cas systems, carrying a single-nucleotide mutation in the protospacer adjacent motif (PAM) or seed causes immune failure and results in viral escape;” whereas “Type III-A CRISPR-Cas targeting tolerates sequence changes with protospacer or upstream of the protospacer, and single-nucleotide substitutions do not impair Type III-A CRISPR-Cas immunity.” See Discussion ¶4. While Lin performed biochemical cleavage assays in vitro, Lin also performed assays in vitro with Eukaryotic cells: “Human Embryonic Kidney 293 plus T cell antigen (HEK293T cells, CRL-3216, ATCC) cells, Vero E6 cells and A549 cells were cultured,” and “HEK293T cells were transfected using LipofectAmine™ CRISPRMAX™ Transfection Reagent (Thermo Fisher Scientific) according to the instruction with 0–6.0 μg of StCsm complex and 2 μg gRNA.” See Cell Lines and TEAR-CoV transfection of HEK293T cells - Materials and Methods. Lin teaches generating “a Csm complex expression vector, coding sequence of Csm1–2–3–4–5 was codon optimized and the whole sequence was synthesized and inserted into pcDNA3.1(+) vector…forming the pcDNA-CMV-CSM1–2–3–4–5 plasmid.” See methods. Lin further teaches creating a “gRNA expression vector, U6 promoter sequence, gRNA sequence and terminator T was also synthesized and inserted into pcDNA3.1/Zeo(+) … forming pcDNA/Zeo-U6-Type III-gRNA plasmid.” Lin further teaches experiments in vivo: “mice were treated with TEAR-CoV plasmids (pcDNA-CMV-CSM1–2–3–4–5 and pcDNA/Zeo-U6-Type III-gRNA) through TurboFect in vivo Transfection Reagent.” See IAV infection in mice – Materials and Methods. See methods. Lin teaches “TEAR-CoV is capable of inhibiting SARS-CoV-2 gene expression in human cells.” See results. Lin teaches delivering “TEAR-CoV with gRNA5 targeting CTD sequence,” which is a viral coding sequence. See Fig. 3 and Fig. 3 legend. Lin reports “that TEAR-CoV not only cleaves SARS-CoV-2 genome and mRNA transcripts, but also degrades live influenza A virus (IAV), impeding viral replication in cells and in mice.” See abstract. Lin teaches “the fast design and broad targeting of TEAR-CoV may represent a versatile antiviral approach for SARS-CoV-2 or potentially other emerging human coronaviruses.” See abstract. Lin teaches “that further testing could determine the suitable delivery for TEAR-CoV to control infections and enhance human health.” See discussion ¶5. Lin teaches “TEAR-CoV may be an antiviral option for controlling SARS-CoV-2 to benefit the suffering human populations, when the CRISPR-based therapeutics may be directly delivered into human respiratory tract cells through a viral vector (i.e. AAV).” See discussion ¶5 and Supplementary Figure S5A. Lin teaches a schematic for AAV design carrying TEAR-CoV, wherein an AAV vector comprising both a single regulatable tissue-specific or cell-type-specific promoters operably linked to Cas10-csm2/3/4/5 and a Type III-gRNA operably linked to another promoter is delivered to a human subject. See (Supplementary Figure S5B) and below:
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However, Lin does not teach that the type III-A CRISPR-Cas system may programmed to effect methylation or adenylation of RNA.
Wilson teaches “a programmable RNA-binding protein such as dCas13 when fused to an appropriate methyltransferase complex mediates the guide RNA-specified methylation of A to m6A site-specifically in a target transcript.” See Fig. 1 and Fig. 1 description. Wilson teaches that “RNA has recently been shown to tune gene expression through its own set of post-transcriptional modifications, including pseudouridine (Ψ), 5-methylcytosine (m5C), N1-methyladenosine (m1A), and N6-methyladenonsine (m6A),” and that “METTL3 and METTL14 form a “writer” complex that catalyzes S-adenosyl methionine (SAM)-dependent methylation of the N6 of adenine in cellular mRNA.” See Fig. 1 description and Introduction. Wilson teaches that in contrast to the M3M14–dCas9, which functions on cytoplasmic RNA, the “nucleus-localized dCas13–M3nls… enables 5’ UTR targeting and the ability to affect m6A-dependent nuclear processing events, such as alternative splicing, microRNA maturation, nuclear export, and chromatin accessibility.” See discussion ¶3. Together, Wilson teaches the dCas13-RNA methyltransferase complexes are termed TRM editors and anticipates “that the TRM editors…will illuminate functional relationships between m6A and phenotype…” and “enable site-specific control of the epitranscriptome in cell culture and in living organisms.” See discussion ¶3.
Regarding claims 2, 72, and 76-77, it would have been obvious to one of ordinary skill in the art before the effective filing date to deliver the polynucleotide sequence comprising a nucleic acid sequence encoding the type III-A CRISPR-Cas system and a crRNA taught by Colognori by way of a viral vector such as an AAV and to operably link the type III-A CRISPR-Cas system of Colognori to a regulatable promoter. It would have further been obvious to target an exogenous RNA such as a viral RNA with the type III-A CRISPR-Cas complex taught by Colognori. One would have had a reasonable expectation of success because introducing nucleic acid comprising the type III-A CRISPR-Cas system operably linked to a regulatable promoter into eukaryotic cells using AAV vectors was expressly taught by Lin. Furthermore, Lin explicitly teaches using the type III-A CRISPR-Cas system to target viral RNA. One would have been motivated to do so in order to efficiently introduce the type III-A CRISPR-Cas system into eukaryotic cells, express the complex in a regulatable manner, and achieve rapid editing. One would have further been motivated to target exogenous viral RNA in order to prevent the completion of the viral infectious cycle, thereby clearing viral infections more efficiently.
Regarding claim 21, it would have been obvious to one of ordinary skill in the art before the effective filing date to modify the type III-A CRISPR-Cas system taught by Colognori such that the RNA modification comprises methylation or adenylation. One would have had a reasonable expectation of success because, as taught by Wilson, Cas13, which also modifies ssRNA, was routinely fused to epigenetic RNA methyltransferases to successfully methylate cytoplasmic and nuclear RNAs. Thus, CRISRP-Cas systems were routinely fused to nucleic acid modifying enzyme as demonstrated by Wilson to introduce site specific epigenetic modifications that result in a desired mutational outcome. One would have been motivated to fuse the type III-A CRISPR-Cas system to, for example, an RNA methyltransferase in order to introduce a site-specific methylation in a target RNA because modifying the epitranscriptome using CRISPR-Cas systems fused to an appropriate methyltransferase complex in cell culture and in living organisms was an art-recognized goal in order to illuminate functional relationships between RNA modifications and phenotype as taught by Wilson.
Claims 1-2, 4-5, 8, 11, 16, 20-21, 23, 25-26, 29, 31 and 72-78 are rejected under 35 U.S.C. 103 as being unpatentable over Lin P. et. al., (Nucleic Acids Research, Volume 50, Issue 8, Page e47, published Feb. 15, 2022, provided in IDS, and supplementary materials) in view of Kriz A. et. al., (Nature Communications volume 1, Article number: 120, published Nov. 16, 2010), Fricke T. et. al., (US-20180105835-A1), Sorek R. et. al., (US-20140113376-A1), Kazlauskiene M, et. al., (Science; 357(6351):605-609, published Aug 11, 2017, provided in IDS), and Wilson C. et. al., (Nat Biotechnol.; 38(12):1431-1440, published Dec. 29, 2020).
The teaching of Lin regarding Claims 1-2, 4, 8, 11, 20, 23, 25, 72, 74, and 76-78 are incorporated herein by reference to the corresponding 102 rejection above.
Regarding claim 31, Lin further teaches “Csm6 RNase is activated for non-specific cleavage of RNAs upon the production of cyclic oligoadenylates (cOAs) that are synthesized by the activated Cas10. This nonspecific RNA targeting can be toxic to cells (Supplementary Figure S1), which may inflict off-target effects and potentially causing host cell transcriptome degradation (Supplementary Figure S1). See See introduction ¶3.
Overall, Lin teaches that “applications for targeting mammalian RNA with Type III CRISPR will be realized at an enhanced scale in the coming years for programmable RNA targeting.” See discussion ¶2.
Lin teaches claim 1; however, Lin does not teach limitations of claims 5, 16, 21, 26, 29, 31, 73, and 75, namely: wherein the nucleotide sequences encoding the at least 5 subunits are operably linked to two or more different promoters; wherein the target RNA is an endogenous RNA; wherein the modifying comprises methylation or adenylation; wherein the Cas10/Csm1, Csm2, Csm3, Csm4, and Csm5 polypeptides each independently comprise an amino acid sequence having at least 50% amino acid sequence identity to any of the amino acid sequences of the Cas10/Csm1, Csm2, Csm3, Csm4, and Csm5 polypeptides of SEQ ID Nos: 1-5 or FIG. 7A-7E; wherein the multi-subunit Type Ill CRISPR-Cas effector polypeptide comprises one or more amino acid substitutions that reduce DNAse activity; wherein the multi-subunit Type Ill CRISPR-Cas effector polypeptide comprises one or more amino acid substitutions that reduce polymerization of ATP into a cyclic oligoadenylate (cA) molecule, wherein the one or more amino acid substitutions that reduce polymerization of ATP to cA comprise a substitution of D577, a substitution of D578, or a substitution of both D577 and D578 of a Cas10/Csm1 polypeptide; wherein the target RNA is present in the nucleus or in an organelle; and wherein the target RNA is a non-coding RNA.
Kriz teaches “homogenous expression of five proteins from a single plasmid.” See results. Kriz teaches “ MultiLabel, a novel and highly efficient modular plasmid-based eukaryotic expression system.” See abstract. Kriz teaches that “independent expression vectors are assembled by a Cre/LoxP reaction into a plasmid with multiple expression cassettes” to create MultiLabel. See Abstract and Fig. 1. Kriz shows that each of the five proteins are operably linked to a promoter independent of the other proteins, see figure 1 and results ¶1 : “one acceptor vector yielding a plasmid with up to five expression cassettes (Supplementary Fig. S5).”
Fricke teaches “a method of assembling a Type III-A Csm complex in an animal, the method comprising administering to the animal a first nucleic acid sequence encoding a first Csm protein, a second nucleic acid sequence encoding a second Csm protein, and a third nucleic acid sequence encoding a crRNA, wherein the first and second Csm proteins and the crRNA interact to form the Type III-A complex.” See claim 20. Fricke teaches the “target RNA molecule is expressed from an endogenous gene in the animal.” See claim 18. Fricke teaches “a Type III-A CRISPR-Cas (StCsm) complex of Streptococcus thermophilus comprising crRNA, Csm4, and Csm3 and use for cleavage of RNA bearing a nucleotide sequence complementary to the crRNA, in vitro or in vivo.” See [0012]. Frick teaches “a ternary StCsm complex of Cas10, Csm2, Csm3, Csm4, and Csm5 proteins and a crRNA complementary to the target RNA transcript was employed.” See [0018]. Fricke teaches “the invention is not limited to the complex used in the exemplary descriptions and other Cas10-containing complexes may be used similarly to Csm complex described.” See [0018]. Fricke teaches that the Type III-A Csm complex “is substantially devoid of DNase activity.” See claim 15. Fricke teaches “that with the addition of a nuclear localization signal, the dual DNase and RNase activities of the Csm complexes could be exploited.” See [0146]. Fricke teaches “the crRNA comprises a nucleotide sequence capable of binding to a substantially complementary nucleotide sequence of the RNA,” and is used “for cleavage of RNA bearing a nucleotide sequence complementary to the crRNA, in vitro or in vivo.” See [0012] and claim 2. Fricke teaches “The inventors determined that Streptococcus thermophilus Type III-A Csm (StCsm) complex targets RNA, and that multiple cuts are introduced in the target RNA at 6 nt intervals. Target RNA that is complimentary to crRNA is cleaved at multiple sites at regular 6 nt intervals, also termed shredding.” See [0013]. Fricke teaches a “schematic organization of the Type III-A CRISPR-Cas systems of Streptococcus thermophilus DGCC8004 (GenBank KM222358), which GenBank KM222358 annotates the peptide sequences for each of Cas10/Csm1, Csm2, Csm3, Csm4, and Csm5 with greater that 50% homology to each SEQ ID NOs: 1-5 or FIG 7A-7E of the instant case. For example, SEQ ID NO: 2, Cas10/csm1, aligns with 96% identity, SEQ ID NO: 8, Csm2, aligns with 71.9% identity, SEQ ID NO: 17, Csm3, aligns with 98.9% identity, SEQ ID NO: 27, Csm4, aligns with 98.4% identity, SEQ ID NO: 34, Csm5, aligns with 98.9% identity.
Sorek teaches “compositions and methods for downregulating prokaryotic genes” (see title) comprising “a specific CRISPR subtype, called the RAMP module (or cmr module),” [0087]. Sorek teaches “that the RAMP module (or cmr module) is unique, and performs its action by silencing RNA rather than DNA.” Sorek teaches “polynucleotides of the CRISPR system which encode a CRISPR array and at least one CRISPR associated (CAS) polypeptide of a repeat associated mysterious protein (RAMP) family so that its spacers will target endogenous genes,” (see [0087]) which “results in degradation of the targeted mRNA, thus allowing selective silencing of specific genes of choice…” (see [0088]). Sorek further teaches “RAMP modules were defined as a cas array + CRISPR array, where the cas array contains at least 4 genes, and where at least one gene belongs to the RAMP subtype” see [0307]. Sorek teaches “CAS polypeptides of the RAMP subtype include Csm3-5, Cmrl, Cmr2, Cmr3, Cmr4, Cmr6 and Csx7” see [0156]. Sorek performed a bioinformatic analysis to characterize RAMP modules in bacterial genomes and identified 73 RAMP modules, one of which is Streptococcus thermophilus. Sorek teaches “the sequences of the 73 identified RAMP modules, as well as their associated repeat arrays and cas gene sequences, are set forth in SEQ ID NOs: 1-1339.” See Example 1 [0311]. Analysis of the cas gene sequences disclosed by Sorek reveal sequence identities of greater than or equal to 50% for each Csm as claimed in the instant case. For example, alignment of SEQ ID NOs: 2, 8, 17, 27, and 34 of the instant case corresponding to Csm1/2/3/4/5, respectively, to the sequences taught by Sorek, align with greater than or equal to 97.9% identity. Specifically, SEQ ID NO: 2 aligns with 97.9% identity to SEQ ID NO: 386 of Sorek; SEQ ID NO: 8 aligns with 99.7% identity to SEQ ID NO: 388 of Sorek; SEQ ID NO: 17 aligns with 98.6% identity to SEQ ID NO: 390 of Sorek; SEQ ID NO: 27 aligns with 98.4% identity to SEQ ID NO: 392 of Sorek; and SEQ ID NO: 34 aligns with 99.3% identity to SEQ ID NO: 394 of Sorek. Sorek teaches implementing the disclosed cas sequences in “a method of treating a bacterial infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an isolated polynucleotide, comprising a clustered, regularly interspaced short palindromic repeat (CRISPR) system nucleic acid sequence, the CRISPR system encoding a CRISPR array and at least one CRISPR associated (CAS) polypeptide of a repeat associated mysterious protein (RAMP) family wherein at least one spacer of the CRISPR array is sufficiently complementary to a portion of at least one bacterial gene so as to down-regulate expression of the bacterial gene.” See SUMMARY OF THE INVENTION, [0021].
Kazlauskiene teaches “a cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems.” See title. Kazlauskiene teaches “the Cas10 subunit (called Csm1 and Cmr2 in the type III-A and III-B systems, respectively) harbors an N-terminal HD domain, two small α-helical domains, and two Palm domains that share a ferredoxin-like fold with the core domain of nucleic acid polymerases and nucleotide cyclases,” and that “the HD domain of Cas10 is responsible for ssDNA degradation in vitro” and “the conserved GGDD motif in one of the two Palm domains has been hypothesized to generate cyclic nucleotides.” See ¶2. Kazlauskiene teaches “upon target recognition, the Cas10 subunit of the effector complex synthesizes cyclic oligoadenylates, which act as second messengers to initiate and amplify the nuclease activity of Csm6.” See Bacterial defense amplification. Kazlauskiene teaches that the two aspartates in the GGDD motif correspond to positions D575 and D576, and that “the double D575A+D576A mutation in the GGDD motif of the Cas10 Palm domain abrogates ATP conversion into” cyclic oligoadenylates (cOAs). See ¶3 and Fig. 1 and fig. S4. Kazlauskiene teaches “The D575A+D576A mutation in the Cas10 GGDD motif disrupts ATP binding (fig. S4C), implying that ATP is bound by the Palm domain.” Whereas, Kazlauskiene teaches that “the D16A mutation that compromises ssDNase activity has no effect on the ATP reaction.” See ¶3. Kazlauskiene teaches that “signaling pathways involving cOAs (i) provide an additional level of control for the antiviral defense system, potentially inducing dormancy to buy time for the host to destroy the invader or promote programmed cell death of the host; (ii) ensure a mechanism for signal amplification; and (iii) allow robust discrimination from other signaling pathways in the cell.” See last ¶.
Wilson teaches “a programmable RNA-binding protein such as dCas13 when fused to an appropriate methyltransferase complex mediates the guide RNA-specified methylation of A to m6A site-specifically in a target transcript.” See Fig. 1 and Fig. 1 description. Wilson teaches that “RNA has recently been shown to tune gene expression through its own set of post-transcriptional modifications, including pseudouridine (Ψ), 5-methylcytosine (m5C), N1-methyladenosine (m1A), and N6-methyladenonsine (m6A),” and that “METTL3 and METTL14 form a “writer” complex that catalyzes S-adenosyl methionine (SAM)-dependent methylation of the N6 of adenine in cellular mRNA.” See Fig. 1 description and Introduction. Wilson teaches that in contrast to the M3M14–dCas9, which functions on cytoplasmic RNA, the “nucleus-localized dCas13–M3nls… enables 5’ UTR targeting and the ability to affect m6A-dependent nuclear processing events, such as alternative splicing, microRNA maturation, nuclear export, and chromatin accessibility.” See discussion ¶3. Together, Wilson teaches the dCas13-methyltransferase complexes are termed TRM editors and anticipates “that the TRM editors…will illuminate functional relationships between m6A and phenotype…” and “enable site-specific control of the epitranscriptome in cell culture and in living organisms.” See discussion ¶3.
Regarding claim 5, it would have been obvious to one of ordinary skill in the art before the effective filing date to modify the AAV vector comprising the Cas10/Csm1, Csm2, Csm3, Csm4, and Csm5 polypeptides operably linked to a single promoter as taught by Lin such that the Cas10/Csm1, Csm2, Csm3, Csm4, and Csm5 polypeptides are operably link to two or more different promoters. One would have had a reasonable expectation of success because, as taught by Kriz, modular expression vectors comprising multiple polypeptide coding rejoins each operably linked to a different promoter was well known with well-known methodologies (also taught by Kriz) to generate such vectors. One would have been motivated to operably link each Csm unit to a different promoter to generate a modular plasmid system that provides the investigator with the convenience of employing a single vector with increased control over expression of each unit.
Regarding claims 16, 20, 29, 73 and 75, it would have been obvious to one of ordinary skill in the art before the effective filing date to employ the Type Ill CRISPR-Cas system taught by Lin against endogenous coding and non-coding RNAs whether found in the cytoplasm, organelle, or nucleus. It would have further been obvious to abrogate the DNase activity of the system. One would have had a reasonable expectation of success because, as taught by Fricke, both cytoplasmic and nuclear RNAs are targetable by the Type Ill CRISPR-Cas system and Kazlauskiene teaches specific mutations to abrogate the ssDNase activity of the Type III CRISPR-Cas system. One of ordinary skill would have been motivated to abrogate the DNase activity to reduce deleterious effects of off target DNA damage especially when targeting nuclear or mitochondrial RNA. One would have further been motivated to target endogenous transcripts whether coding or non-coding in either the nucleus or organelles to, as taught by Fricke, cleave an RNA product of a gene of interest, for example a mutated gene’s RNA product..
Regarding claim 21, it would have been obvious to one of ordinary skill in the art before the effective filing date to modify the type III-A CRISPR-Cas system taught by Lin such that the RNA modification comprises methylation or adenylation. One would have had a reasonable expectation of success because, as taught by Wilson, Cas13, which also modifies ssRNA, was routinely fused to epigenetic RNA methyltransferases to successfully methylate cytoplasmic and nuclear RNAs. Thus, CRISRP-Cas systems were routinely fused to nucleic acid modifying enzyme as demonstrated by Wilson to introduce site specific modifications that result in a desired mutational outcome. One would have been motivated to fuse the type III-A CRISPR-Cas system to, for example, an RNA methyltransferase in order to introduce a site-specific methylation in a target RNA because modifying the epitranscriptome using CRISPR-Cas systems fused to an appropriate methyltransferase complex in cell culture and in living organisms was an art-recognized goal in order to illuminate functional relationships between RNA modifications and phenotype as taught by Wilson.
Regarding claim 26, it would have been obvious to one of ordinary skill in the art before the effective filing date to employ Cas10/Csm1, Csm2, Csm3, Csm4, and Csm5 polypeptides with at least 50% identity to polypeptides of SEQ ID NOs: 1-5 or FIG. 7A-7E in the Type Ill CRISPR-Cas system taught by Lin. One would have had a reasonable expectation of success because the peptide sequences were annotated, publicly available, and explicitly disclosed by Sorek. One would have been motivated to select polypeptides of SEQ ID NOs: 1-5 or FIG. 7A-7E as they were vetted sequences with known performance within the Type III-A CRISPR-Cas system to knockdown RNA in vitro and in vivo in both prokaryotic and eukaryotic cells.
Regarding claim 31, it would have been obvious to one of ordinary skill in the art before the effective filing date to modify the type III-A CRISPR-Cas system taught by Lin to comprise mutations at the aspartate residues of the GGDD motif corresponding to D577A and D578A. Kazlauskiene teaches the aspartate residues of the GGDD motif are at positions 575 and 576, which is consistent with the positions of these residues in the publicly available GenBank KM222358 record. Furthermore, SEQ ID NO: 2 of the instant case is also consistent with the public records and annotates these residues at positions 575 and 576. Thus, the examiner is considering the positions claimed 577 and 578 are analogous to positions 575 and 576. Therefore, one would have had a reasonable expectation of success because it was well known in the art per Kazlauskiene that mutating the aspartate residues in the GGDD motif reduces polymerization of ATP into a cyclic oligoadenylate (cA) molecule. One would have been motivated to substitute the aspartates in the GGDD motif with, for example, as in the prior art with alanine in order to minimize bystander RNA decay, prevent cellular toxicity and quiescence, improve signal-to-noise ratio, and avoid the indiscriminate nuclease response.
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.
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Claims 1-2, 4-5, 8, 11, 16, 20-21, 23, 25-26, 29, 31 and 72-78 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-33 of U.S. Patent No. US-11661599-B1 in view of Lin P. et. al., (Nucleic Acids Research, Volume 50, Issue 8, Page e47, published Feb. 15, 2022, provided in IDS, and supplementary materials), Kriz A. et. al., (Nature Communications volume 1, Article number: 120, published Nov. 16, 2010), Fricke T. et. al., (US-20180105835-A1), Sorek R. et. al., (US-20140113376-A1), Kazlauskiene M, et. al., (Science; 357(6351):605-609, published Aug 11, 2017, provided in IDS), and Wilson C. et. al., (Nat Biotechnol.; 38(12):1431-1440, published Dec. 29, 2020).
Although the claims at issue are not identical, they are not patentably distinct from each other because the instant claims would have been obvious over the ‘599 claims.
The ‘599 claims teach a method of conducting a cleavage, the method comprising:
incubating a synthetic guiding component with a nuclease and a single-stranded target sequence, the synthetic guiding component including a targeting portion configured to bind and/or cleave the single-stranded target sequence; and
cleaving the single-stranded target sequence without a short DNA oligomer containing a proto-spacer adjacent motif (PAM) sequence (PAMmer);
wherein the synthetic guiding component comprises a structure having the formula (I):W—X—Y-L-Z or a salt thereof, wherein:
W is an optional third portion comprising a nucleic acid sequence of from about 1 to 20 nucleic acids;
X is the targeting portion comprising a nucleic acid sequence configured to bind to a target site of the single-stranded target sequence;
Y is a first portion comprising a nucleic acid sequence configured to interact with a nuclease configured to cleave the single-stranded target sequence;
L is a linker; and
Z is a second portion comprising a nucleic acid sequence configured to interact with the nuclease and the first portion;
wherein the nuclease is a SauCas9 or CjeCas9 protein and the single-stranded target sequence is a single-stranded ribonucleic acid sequence (Claim 18).
The methods further comprise limitations wherein the single stranded target sequence recited for X is a single-stranded human mRNA target sequence or a single-stranded pathogen target sequence (claim 5), and
wherein the single-stranded ribonucleic acid sequence is the RNA sequence of a virus, the virus having a lifecycle consisting of solely RNA molecules (claim 31).
‘599 defines a “synthetic guiding component” as comprising “a nucleic acid sequence configured to bind to a target site of the single-stranded target sequence” and “a nucleic acid sequence configured to interact with a nuclease configured to cleave the single-stranded target sequence” see claim 18, which is analogous to guide RNA(s) in the instant case.
‘599 defines a “single stranded target sequence” as “coding RNA or non-coding RNA, e.g., messenger RNA (mRNA, including elements thereof, such as a riboswitch, an untranslated region, a coding sequence, a start codon, a 5′ cap, or a poly-adenine tail), transfer-messenger RNA (tmRNA), ribosomal RNA (rRNA, such as the 30S, 40S, 50S, 60S, 80S, 5S, 5.8S, 12S, 16S, 18S, 23S, and 28S subunits), ribozyme, transfer RNA (tRNA), heterogeneous nuclear RNA (hnRNA), circular RNA, signal recognition particle RNA (SRP RNA, such as 4.5S, 6S, 7SL, or ffs RNA), X-inactive specific transcript (Xist), microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA, such as small Cajal body-specific RNA (scaRNA)), small nuclear RNA (snRNA), extracellular RNA (exRNA), long non-coding RNA (lncRNA), large intergenic non-coding RNA (lincRNA), intergenic RNA, enhancer RNA (eRNA), satellite RNA (satRNA), and promoter-associated RNA (PAR).” See Summary of Invention ¶29.
The major difference between the ‘599 claims and the claims of the instant case is the use of a Type III CRISPR-Cas system with five subunits instead of the Type II Cas system. To the extent that there are limitation of the instant claims that are not taught by the ‘599 claims, the teachings that Lin, Kriz, Fricke, Sorek, Kazlauskiene, and Wilson are discussed above. Given the substantially similar subject matter between the ‘599 claims and the teachings of Lin, Kriz, Fricke, Sorek, Kazlauskiene, and Wilson, it would have been obvious to have modified the subject matter of the ‘599 claims in a manner discussed below to arrive at the instant claims.
It would have been obvious to one skilled in the art to use a Type III CRISPR-Cas complex comprising five polypeptides in place of the Cas9 variants, SauCas9 or CjeCas9, of the ‘599 patent claims, thereby arriving at instant independent claim 1. Incubating as recited in ‘599 does not exclude doing so in a eukaryotic cell; therefore, as claimed it reads on introducing nucleic acids encoding the Type III CRISPR-Cas system and guide RNA into a eukaryotic cell and allowing for the complex to form with and modify the target RNA. Eukaryotic cells were routinely used in the art to test CRISPR systems as evident by Lin and Fricke. One would have had a reasonable expectation of success to introduce the Type III CRISPR-Cas system and gRNA in eukaryotic cells to induce a modification to RNA as this was already successfully performed by Lin and Fricke. One would have been motivated to employ the Type III CRISPR-Cas in place of SauCas9 or CjeCas9 in order to, for example, increase the robustness of targeting viral RNA in eukaryotic cells.
Regarding claim 2, 4-5, 8, and 72, it would have been obvious to one of ordinary skill in the art deliver the Type III CRISPR-Cas system and gRNA for incubation in eukaryotic cells using AAV vectors that comprise either a single regulatable promoter or multiple regulatable promoters and either on the same or different vectors. One would have a reasonable expectation of success because the AAV vector comprising the 5 subunits of the Type III CRISPR-Cas system and gRNA was expressly disclosed by Lin and Kriz provides the framework to generate modular vectors with up to 5 coding regions each under control of a different promoter. One would have been motivated to do so in order to increase efficiency of delivering the system to eukaryotic cells and to generate a highly regulatable modular vector system to achieve spaciotemporal control over the incubation in eukaryotic cells.
Regarding claim 21, it would have further been obvious to one of ordinary skill in the art before the effective filing date to employ a modify the type III-A CRISPR-Cas system such that the RNA modification comprises methylation or adenylation. One would have had a reasonable expectation of success because, as taught by Wilson, Cas13, which also modifies ssRNA, was routinely fused to epigenetic RNA methyltransferases to successfully methylate cytoplasmic and nuclear RNA. Thus, CRISRP-Cas systems were routinely fused to nucleic acid modifying enzyme as demonstrated by Wilson to introduce site specific modifications that result in a desired mutational outcome. One would have been motivated to fuse the type III-A CRISPR-Cas system to, for example, an RNA methyltransferase in order to introduce a site-specific methylation in a target RNA because modifying the epitranscriptome using CRISPR-Cas systems fused to an appropriate methyltransferase complex in cell culture and in living organisms was an art-recognized goal in order to illuminate functional relationships between RNA modifications and phenotype as taught by Wilson.
Regarding claim 23, it would have further been obvious to one of ordinary skill in the art before the effective filing date to target the eukaryotic cell in vitro. One would have had a reasonable expectation of success because, for example, Lin teaches the type III-A CRISPR-Cas system is effective in Eukaryotic cells in vitro. One would have been motivated to employ the system in eukaryotic cells in vitro for example to study the downstream effects of a certain RNA knockdown on the survivability of a certain cancer cell line as a proof-of-concept before moving into more costly and time-consuming in vivo models.
Regarding claims 25-26, it would have further been obvious to one of ordinary skill in the art before the effective filing date to employ Cas10/Csm1, Csm2, Csm3, Csm4, and Csm5 polypeptides with at least 50% identity to polypeptides of SEQ ID NOs: 1-5 or FIG. 7A-7E in the Type Ill CRISPR-Cas system. One would have had a reasonable expectation of success because the peptide sequences were annotated, publicly available, and explicitly disclosed by Sorek. One would have been motivated to select polypeptides of SEQ ID NOs: 1-5 or FIG. 7A-7E as they were vetted sequences with known performance within the Type III-A CRISPR-Cas system to knockdown RNA in vitro and in vivo in both prokaryotic and eukaryotic cells.
Regarding claims 29, it would have been obvious to one of ordinary skill in the art before the effective filing date to abrogate the DNase activity of the Type III-A CRISPR-Cas system. One would have had a reasonable expectation of success because Kazlauskiene teaches specific mutations to abrogate the ssDNase activity of the Type III CRISPR-Cas system. One of ordinary skill would have been motivated to abrogate the DNase activity to reduce deleterious effects of off target DNA damage especially when targeting nuclear or mitochondrial RNA.
Regarding claim 31, it would have been obvious to one of ordinary skill in the art before the effective filing date to modify the Type III-A CRISPR-Cas system to comprise mutations at the aspartate residues of the GGDD motif corresponding to D577A and D578A. Kazlauskiene teaches the aspartate residues of the GGDD motif are at positions 575 and 576, which is consistent with the positions of these residues in the publicly available GenBank KM222358 record. Furthermore, SEQ ID NO: 2 of the instant case is also consistent with the public records and annotates these residues at positions 575 and 576. Thus, the examiner is considering the positions claimed 577 and 578 are analogous to positions 575 and 576. Therefore, one would have had a reasonable expectation of success because it was well known in the art per Kazlauskiene that mutating the aspartate residues in the GGDD motif reduces polymerization of ATP into a cyclic oligoadenylate (cA) molecule. One would have been motivated to substitute the aspartates in the GGDD motif, for example, with alanine, as in the prior art in order to minimize bystander RNA decay, prevent cellular toxicity and quiescence, improve signal-to-noise ratio, and avoid the indiscriminate nuclease response.
Conclusion
No claims are allowed.
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/COREY LANE BRETZ/ Patent Examiner, Art Unit 1635
/DANA H SHIN/ Primary Examiner, Art Unit 1635