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 16-23, 40, 63-69 are pending.
Claims 1-15, 24-39, 41-62, 70-93 are cancelled by the Applicant.
Claims 63-69 are withdrawn as part of non-elected groups of inventions.
Claims 16-23 and 40 are being examined.
All previous objections and rejections not set forth below have been withdrawn in view of applicant’s amendments to the claims. However, the claim amendments by the Applicant by adding new issues, which was not present in any of the claims before, necessitated new prior art references and new grounds of rejections, as discussed below.
Claim Rejections - 35 USC § 112(b)
Response to Applicant’s arguments: Amendments made to the claims filed in Applicant’s response submitted on 10/27/2025 overcame the rejection of record under 35 USC § 112(b).
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 16-18, 23 and 40 are rejected under 35 U.S.C. 103 as being unpatentable over
Ye et al. (Programmable DNA repair with CRISPRa/I enhanced homology-directed repair efficiency with a single Cas9, 2018, Cell Discovery, 4:46) in view of Selma et al. (Strong gene activation in plants with genome-wide specificity using a new orthogonal CRISPR/Cas9-based programmable transcriptional activator, 2019, Plant Biotechnology Journal, 17:1703–1705) and Lowder et al. (Multiplexed Transcriptional Activation or Repression in Plants Using CRISPR-dCas9-Based Systems, 2017, in Kerstin Kaufmann and Bernd Mueller-Roeber (eds.), Plant Gene Regulatory Networks: Methods and Protocols, Methods in Molecular Biology, vol. 1629, DOI 10.1007/978-1-4939-7125). This is a new rejection necessitated by the claim amendments.
Claim 16 is drawn to a system for activating expression of a target nucleic acid, the system comprising:
a nuclease active Cas polypeptide, or a polynucleotide encoding the nuclease active Cas polypeptide;
a first dead guide polynucleotide comprising a 14 to 16 nucleotide spacer sequence that mediates increased expression of the first target nucleic acid, wherein the first dead guide polynucleotide comprises an aptamer;
a polypeptide comprising an adapter domain and a multimerized epitope, wherein the adapter domain binds the aptamer, or a polynucleotide encoding the polypeptide;
a polypeptide comprising an affinity domain and a transcriptional activation domain, wherein the affinity domain binds the multimerized epitope, or a polynucleotide encoding the polypeptide; and
a second dead guide polynucleotide comprising a 14 to 16 nucleotide spacer sequence that mediates reduced expression of the second target nucleic acid and/or a guide polynucleotide that mediates sequence-specific cleavage at a target site in the genome.
Ye et al. describes an effective, flexible, and a safer strategy to perform both CRISPRa/i (“a” refers to activation, while “i” refers to interference or repression) and precise gene editing enhance mediated by catalytically dead guide RNAs (dgRNAs) and a catalytically active Cas9 (abstract). During gene editing process, Cas9-sgRNA complex is known to induce double stranded breaks (DSBs) which is then repaired by either non-homologous DNA end joining (NHEJ) or homology directed repair (HDR) (page 2, left column, para 2, line 7). The error prone repairs via NHEJ pathway often introduce unpredictable indel mutations in the target gene(s) (page 2, left column, para 2, line 7-9). NHEJ has been considered the major pathway to repair the DNA in mammals (page 1, right column, para 1, line 10-11) and plants. NHEJ is known to be more error prone in plants than any other organism1. HDR is more desirable to reduce or eliminate off-target gene editing, gene activation, and/or gene repression/interference.
Ye et al. describes dead guide RNAs (dRNA or dgRNA) of 14-15 nucleotide long retaining its binding ability to a target DNA sequence (page 2, left column, para 2, line 23-25) but does not allow catalytically active Cas9 polypeptide and, thus, with the associated CRISPR activation (CRISPRa) and interference (CRISPRi) modules can be deployed to achieve HDR enhancement using a single active Cas9 (as recited in claim 17). The 14-15 nucleotide long dgRNA reads on to “comprising a 14 to 16 nucleotide long” (as recited in instant claim 16) and is a functional equivalent of a dgRNA 14-16 nucleotide long dgRNA. Ye et al. also describes two dead guide RNAs (dgRNAs or dRNAs) by- i) the “first dead guide polynucleotide” sequence is made by fusing the dgRNA scaffold sequence to Com binding loop (aptamer) to repress the NHEJ-related gene(s) like KU80 (Fig. 3a-b), and ii) the “second dead guide polynucleotide” sequence is made by fusing the dgRNA scaffold sequence to MS2 binding loop (aptamer) for recruiting (MS2 binding protein) MCP (as recited in claim 18) to activate HDR-related gene(s) (page 2, right column, para 1, line 1-5) like CDK1 (Fig. 3a-b). Transcription activation of CDK1 gene is achieved by P65 transcription activation domain (as part of the MCP-P65-HSF1 fusion protein) while transcriptional repression is achieved by KRAB (transcription repression) domain (as part of the COM-KRAB fusion protein (page 2, right column, para 1, line 1-5).
However, Ye et al. does not describe a multimerized epitope comprising 10 copies of a GCN4 epitope.
Selma et al. describes strong gene activation in plants using CRISPR/Cas9 based system (title and abstract). It teaches the MS2 aptamer (page 1703, right column, line 11) and the adapter domain (MS2 viral Coat Protein, MCP) that binds to the MS2 aptamer (page 1703, right column, lines 10-11); a polypeptide comprising multimerized or multiepitope tags, as recited in claims 16 and 19 (page 1703, left column, para 1, line 18) such as GCN4, which is attached to an affinity domain (ScFv) (page 1703, right column, para 1, lines 8-9); and transcriptional activation domains like VP64 fused to the GCN4-ScFv fusion polypeptide (page 1703, right column, line 8-9).
Lowder et al. describes multiplexing transcriptional activation and/or repression in plants using CRISPR-dCas9-based systems (title and abstract) by expressing different gRNAs simultaneously (page 169, last para, last 2 lines). It teaches activation and repression of two different genes using different guide RNAs (gRNAs) (page 167, Abstract). Lowder et al. also describes two different sets of gRNAs to target two different genes (AtPAP1 and AtCSTF64) in the same genome in Arabidopsis (page 167, Abstract). The first group of gRNAs are used to activate AtPAP1 gene (page 172, Table 1) while the second group of gRNAs are used to repress AtCSTF64 gene (page 172, Table 2). Lowder et al. teaches transcriptional activation by using the transcriptional activation domain (VP64) as well as transcriptional repression by using the transcriptional repression domain (SRDX) (page 167, abstract, line 4-6; page 168, para 3, line 1-5; page 169, Fig. 1).
Before the effective filing date of this application, it would have been obvious to one of ordinary skill in the art to develop a system of simultaneous transcriptional activation and repression by using two different sets of dead guide RNAs (dgRNAs), as described by Ye et al.; an activation domain and a repressor domain, as taught by Lowder et al. The modified system would further comprise an MS2 aptamer sequence; an adapter domain polypeptide (MCP) (that binds to the MS2 aptamer at one end) fused to a multimerized epitope (GCN4), as described by Selma et al. In the modified system, the multimerized epitope binds to an affinity domain (ScFv) which, in turn, binds to either a transcriptional activation domain (VP64) as taught by Selma et al. and Lowder et al., or a transcriptional repression domain (SRDX) as taught by Lowder et al., with a realistic expectation to simultaneously activate specific gene(s) while repressing different gene(s) as directed by two different sets of gRNAs, as taught by Lowder et al. and Ye et al. Thus, activation and repression can be achieved simultaneously in a single individual and/or in the same cell. The first dgRNA (“dead guide polynucleotide”) would activate specific gene(s) while the second dgRNA would repress separate gene(s) in the same individual and/or in the same cell.
Before the effective filing date of this application, an ordinarily skilled artesian would have been motivated to develop a system by using Cas9 polypeptide based technique that includes: two sets of dead guide RNAs each comprising aptamer(s), adapter domain binding specific aptamer sequence, multimerized epitope, an affinity domain, and a transcription effector which includes an activator and a repressor, as discussed above. The nucleotide-protein complex would have the ability to simultaneously active and repress transcription of two different set of target genes, and, thus, would regulate in-vivo gene expression.
Regarding claim 23, Ye et al (Fig. 1) and Lowder et al. (page 171, Fig. 2) describe several vectors containing different components of the system. It used specific vector for different steps of the method (i.e., the system of instant claim 16 wherein components are located on one or more vectors).
Regarding claim 40, Selma et al. uses tobacco plant (Nicotiana benthamiana) (page 1703, left column, para 2, line 2-3) to activate gene using the CRISPR-Cas based transcriptional activation system, as described before (i.e., a plant comprising the system of instant claim 16).
Claims 19-21 are rejected under 35 U.S.C. 103 as being unpatentable over Ye et al. in view of Selma et al. and Lowder et al., as applied to claims 16-18, 23 and 40 above, and further in view of Jacobsen et al. (US 20200017869A1) and Zhang et al. (US20160153005A1).
Claim 19 depends from claim 16, wherein the multimerized epitope comprises 10 copies of a GCN4 epitope. Claim 20 also depends from claim 16, wherein the affinity domain comprises scFv.
Ye et al. in view of Selma et al. and Lowder et al. describes a nuclease active Cas9 polypeptide based system of transcriptional activation and repression by using two different sets dead guide RNAs that contain an aptamer (MS2), an adapter domain polypeptide (MCP) that binds to the (MS2) aptamer at one end and is fused to a multimerized epitope (GCN4) at the other end. The multimerized epitope binds to an affinity domain (ScFv), which, in turn, binds to either a transcriptional activation domain (VP64) or a transcriptional repression domain (SRDX) to simultaneously activate specific gene(s) while repressing different gene(s) as directed by two different sets of gRNAs, as described above. Selma et al. describes the ScFv affinity domain, as recited in claim 20, attached to the GCN4 epitope (page 1703, right column, para 1, line 9-10) as recited in claim 19.
However, Ye et al. in view of Selma et al. and Lowder et al do not specify the number (10) of GCN4 epitopes used, as recited in claim 19.
Jacobsen et al. (US 20200017869A1) describes using at least 2 to 24 copies of the GCN4 epitope (page 11, para 0127). Jacobsen et al. also describes specifically using 10 copies of the GCN4 epitope (page 27, para 0323, line 4, 10; para 0324, line 3, 9). Zhang et al. teaches that using multiple epitope tags are preferred for the Cas9 to improve detection (page 24, para 0329, line 1-3). Using any specific number of GCN4 epitopes in a multimerized form is within the design choice of a person having ordinary skill in the art without affecting the outcome.
Regarding claim 21, the Applicant describes TAD as TAL Activation Domain (Spec, page 6, line 23). Jacobsen et al. describes TAL Activation Domain (TAD) as one of the transcriptional activators which can be used (page 8, para 0090) to regulate gene expression using the Cas9 based system (page 1, para 0006). Ye et al describes P65 activation domain, which is also known as Transactivation Domain (TAD)2. Moreover, Selma et al. describes using several transcriptional activation domains including VP64 to achieve higher activation rate (page 1703, left column, para 1, line 14-16). 2xTAD, as described by Jacobsen et al., would be a functional equivalent of transcriptional activators (VP64 and EDLL), as described by Selma et al. (page 1703, right column, para 1, line 8-11). Using any specific activator(s) is within the design choice of a person having ordinary skill in the art without affecting the outcome.
Claim 22 is rejected under 35 U.S.C. 103 as being unpatentable over Ye et al. in view of Selma et al. and Lowder et al., as applied to claims 16-18, 23 and 40 above, and further in view of Brezgin et al. (Dead Cas Systems: Types, Principles, and Applications, 2019, Int. J. Mol. Sci., 20:6041).
Claim 22 depends from claim 16, wherein the Cas polypeptide is fused to a deaminase domain.
Ye et al. in view of Selma et al. and Lowder et al. describes a nuclease active Cas9 polypeptide based system of transcriptional activation and repression by using two different sets dead guide RNAs that contain an aptamer (MS2), an adapter domain polypeptide (MCP) that binds to the (MS2) aptamer at one end and is fused to a multimerized epitope at the other end. The multimerized epitope binds to an affinity domain (ScFv), which, in turn, binds to either a transcriptional activation domain or a transcriptional repression domain to simultaneously activate specific gene(s) while repressing different gene(s) as directed by two different sets of gRNAs, as described above.
However, Ye et al. in view of Selma et al. and Lowder et al. do not describe any deaminase domain.
Brezgin et al. describes systems comprising a Cas protein fused to cytidine or adenosine deaminases (page 12, para 5, line 1). The use of adenosine deaminases or adenine base editors effectively convert A-T base pairs to G-C base pairs in DNA (page 12, para 5, line 6-10). It also describes that specific Cas polypeptide (Cas type VI) directly interacts with target RNA molecules and, thus, allowing the Cas-bound deaminase to edit RNA molecules (page 13, para 3, line 1-2). Compared to DNA editing, RNA editing has several important advantages, including a wider range of potential sites and direct RNA editing by deaminases without the assistance of endogenous repair systems (page 13, para 5, line 1-3). The RNA based gene editing system is more specific and does not exhibit significant off-target binding or RNA editing (page 13, para 4, line 5-7).
Before the effective filing date, it would have been obvious to one of ordinary skill in the art to further modify the system described by Ye et al. in view of Selma et al. and Lowder et al. by replacing the transcription activation/repression domain with deaminase protein or a deaminase domain of a deaminase protein as described by Brezgin et al. Fusing the deaminase protein or a deaminase domain of a deaminase protein with a nuclease active Cas9 polypeptide (as described by Ye et al.) and using dead guide RNAs would have enabled an ordinarily skilled artisan to perform targeted mutagenesis by converting A-T base pairs to G-C base pairs in DNA or in RNA sequences, as taught by Brezgin et al. Besides DNA editing (as described in this invention), the RNA-based gene editing system has its own advantages as it is more specific and does not exhibit significant off-target binding or RNA editing, as taught by Brezgin et al. Specific change(s) in the polynucleotide sequence(s) would influence expression of the target gene(s).
Before the effective filing date, an ordinary skilled artisan would be motivated to fuse the nuclease active Cas polypeptide with a deaminase domain while using dead guide RNAs with the realistic goal to introduce specific substitution mutation(s) by converting specific A-T base pairs to G-C base pairs in a DNA or in a RNA sequence, and reducing off-target mutation(s) while editing RNA sequences directly.
Response to Applicant’s Arguments
The argument set forth in the Applicant’s replies on 10/27/2025 has been fully considered but is not found persuasive.
The Applicant argues that the amendments to the claims 16-17 and 23 would nullify the rejections under 35 U.S.C. 103 (response, page 7, para 2; page 7, last para, last 2 lines and page 8, first 3 lines).
The Examiner disagrees. The claim amendments by adding new limitations, which were not present in any of the claims before, necessitated new prior art references and new grounds of rejections, as discussed above. Ye et al. describes a catalytically active Cas9 polypeptide (abstract, line 11; page 2, left column, para 2, line 26-29). Ye et al. also describes dead guide RNAs (dRNA or dgRNA) that retain its binding ability to a target DNA sequence (page 2, left column, para 2, line 23-25) but does not allow catalytically active Cas9 polypeptide to cut (making a double stranded break, DSB) in the DNA strand(s) in the target DNA sequence, and, thus, fulfills the limitations of the amended claims and also the definition of “dead guide RNA” by the Applicant. The Applicant defines the term “dead guide RNA” as “a guide RNA, which is catalytically inactive yet maintains target-site binding capacity” (page 25, line 29-30).
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Contact Detail
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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Jay Chatterjee
Patent Examiner
Art Unit 1662
/Jay Chatterjee/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662
1 Kumaran et al. (Gene technologies in weed management: a technical feasibility analysis, 2020, Current Opinion in Insect Science, 38:6–14) provides the evidence that NHEJ has been considered the dominant or major pathway to repair mechanism in plants and is more error prone in plants than any other organism (page 10, left column, para 3, line 4-6).
2 Lecoq et al. (Structural characterization of interactions between transactivation domain 1 of the p65 subunit of NF-κB and transcription regulatory factors, 2017, Nucleic Acids Research, 45:5564–5576) provides the evidence that transactivation domain in P65 is also known as TAD (page 5565, left column, para 2, line 14-17.
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