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 .
Application Status
This action is written in response to applicant’s correspondence received on April 30, 2026. Claims 1-12, 14, 23, 33, 46, 55, and 65-68 are currently pending. Applicant’s election of KRAB as the single specific transcription inhibitor (claims 9 and 10) is acknowledged on the correspondence received on December 19, 2025. Claims 11, 12, 23, and 25 are withdrawn from prosecution as being drawn to non-elected subject matter. Accordingly, claims 1-10, 14, 33, 45, and 65-68 are examined herein.
Priority
Acknowledgment is made of applicant's claim for priority based on a US Provisional Application
No. 63/002,576 filed on 31 March 2020.
Withdrawn Claim Objections
Claim 14 objected to under 37 CFR 1.75 as being a substantial duplicate of claim 1. Claim 46 objected to under 37 CFR 1.75 as being a substantial duplicate of claim 33. Applicant’s arguments, see pg. 7 under section “Claim Objections”, filed on April 30, 2026, with respect to claims 14 and 46 reciting catalytically active CRISPR/Cas effector polypeptide have been fully considered and are persuasive. The claim objections of claims 14 and 16 has been withdrawn.
Claim Objections
Claim 33 is objected to because of the following informalities: the recitation of “under the control a viral RNA translation element” is grammatically incorrect. It is recommended to amend claim to recite “under the control of a viral RNA translation element” (emphasis added). Appropriate correction is required.
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 1-5,7-10, and 33 are rejected under 35 U.S.C. 103 as being unpatentable over Jaafar et al (Translation initiation by the hepatitis C virus IRES requires eIF1A and ribosomal complex remodeling; eLife, 2016, 5:e21198) in view of Dasgupta et al (US 6,833,254 B2; Published Date: 21 December 2004) and further in view of Gersbach et al (US 2019/0127713 A1; Published Date: 02 May, 2019)
Regarding claim 1, Jaafar teaches a method for testing whether eukaryotic initiation factor eIF1A (i.e., a target protein) is necessary for IRES-based translation (i.e., regulates viral RNA translation) (pg. 3, para. 2). Jaafar teaches the method comprises:
a) introducing into rabbit reticulocyte lysate:
i) a reporter nucleic acid comprising a nucleotide sequence encoding a bicistronic reporter (i.e., bicistronic translation monitor) comprising (1) a Renilla luciferase gene (Rluc) under the control of a Cap-driven promoter (i.e., a first reporter protein translated under the control of a Cap-dependent translation element) and (2) a chloramphenicol antibiotic gene (CAT) under the control of an IRES element (i.e., a second reporter protein translated under the control of a Cap-independent translation element) (pg. 5, para. 3; Figure 2C);
ii) an RNA aptamer α-eIF1A that binds to eIF1A (i.e., a regulatory nucleic acid comprising a nucleotide sequence that specifically binds to the target protein) (pg. 5, para. 3; Figure 2A); and
b) detecting expression of Rluc and CAT from lysates treated with α-eIF1A to lysates treated with no RNA or a randomized 40 nucleotide RNA (i.e., lysates treated with α-eIF1A is the test host cell comprising the regulatory nucleic acid, whereas lysates treated with no RNA or randomized RNA is the control host cell that does not comprise the regulatory nucleic acid) (pg. 5, para. 3; Figure 2C).
Jaagar further teaches the IRES requires eIF1A for full function (i.e., wherein the target protein is considered to regulate translation via the Cap-independent element) because expression of Rluc and CAT in lysate with the aptamer α-eIF1A are reduced in comparison to their expression in lysate with no RNA of a randomized RNA (pg. 5, para. 3; Fig. 2C). Thus, Jaagar identifies eIF1A as a relevant translation initiation that regulates viral RNA translation via IRES-mediated translations. Jaagar further demonstrates that inhibiting the eIF1A changes expression of proteins in a bicistronic reporter.
However, Jaagar teaches a method comprising introducing a bicistronic reporter into rabbit reticulocyte lysate, which is a cell-free system used for in vitro protein synthesis, rather than introducing the bicistronic reporter into a host cell.
Dasgupta teaches a method to identify trans-acting translation factors, e.g., eIF1A, (i.e., target protein) using a bicistronic reporter in cells (abstract; FIG. 1A). The method of Dasgupta comprises: a) introducing into a host cell: i) a reporter nucleic acid comprising a nucleotide sequence encoding a bicistronic reporter comprising (1) an enhanced blue fluorescent protein under the control of a CMV promoter (i.e., a first reporter protein translated under the control of a Cap-dependent translation element) and (2) an enhanced green fluorescent protein under the control of viral IRES (i.e., a Cap-independent translation element) (FIG. 1A). Dasgupta teaches a bicistronic reporter architecture substantially the same as Jaagar’s (a cap-dependent reporter and an IRES-driven reporter) is suitable for expression and high-throughput screening in living cells (Example 5; col. 10). Dasgupta further teaches the expression of the bicistronic reporter observed cell lysates is consistent with in vivo testing for representative IRES including PS3 and PS4 (Example 7; col. 12). Thus, Dasgupta’s teachings positively support that this bicistronic reporter architecture remains functional in both cellular and lysates context.
Thus, it would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to have recognized the bicistronic reporter of Jaagar could be implemented in cells using the expression procedure taught by Dasgupta (Examples 2 and 4) in order to evaluate the reporter activity in cells. The introduction of the bicistronic reporter into cells would have merely amounted to a simple substitution of prior art elements according to known methods to yield predictable results. The substitution is merely introducing a bicistronic reporter of a known architecture from one known environment (e.g., in lysates) to another known environment (intracellular) where prior art has demonstrated predictable outcomes. Thus, this substitution is merely swapping similar features (cell lysates and intact cells) that serve the same purpose (expression of proteins encoded in the bicistronic reporter). One would have had a reasonable expectation of success in doing so because Dasgupta demonstrates that the same type of bicistronic reporter comprising a Cap-driven promoter and an IRES is suitable for cellular expression, and the bicistronic reporter remains functional after transferring from lysates to cells (Example 4).
However, Jaagar does not teach generating a test host cell comprising a catalytically inactive CRISPR/Cas effector polypeptide and a regulatory nucleic acid that comprises a nucleotide sequence encoding a single guide RNA (sgRNA) that comprises a targeting sequence that specifically binds to a target sequence within a nucleic acid encoding the target protein.
Gersbach teaches a method of modulating expression of endogenous genes in cells, comprising (i) a fusion protein comprising a catalytically inactive CRISPR Cas9 effector and a transcriptional inhibitor, and (ii) a nucleic acid encoding a sgRNA that specifically binds to the target gene (claim 1; FIG. 3A).
Thus, it would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to have modified Jaagar's method to substitute the RNA aptamer for the target protein eIF1A with CRISPR dCas9 effector and sgRNA to silence transcription of the target protein as taught by Gersbach because it would have merely amounted to a simple substitution of prior art elements according to known methods to yield predictable results. The substitution is merely swapping one known means of inhibiting a target protein with another to serve the same purpose. One would have had a reasonable expectation of success in doing so because Gersbach establishes that CRISPR dCas9 and transcriptional inhibitor fusion proteins can reduce expression of endogenous genes in a site-specific manner. One of ordinary skill in the art would have recognized that CRISPR-mediated transcription repression as an alternative technique for inhibiting activity of a target protein that Jaagar accomplished with an aptamer.
Regarding claims 2 and 3, Jaafar teaches the Cap-dependent translation element is a CMV promoter from cytomegalovirus (pg. 17, "pDBS" subsection), and the Cap-independent translation element is from hepatitis C virus (pg. 5, third paragraph).
Regarding claim 4, Jaagar recites "cap-driven (Rluc) and HCV IRES-driven (CAT) ORFs of a bicistronic reporter" (pg. 5, third paragraph), thus Jaagar teaches a method wherein the first reporter protein is luciferase and the second reporter protein is a protein that confers antibiotic resistance.
Regarding claim 5, the obviousness to modify Jaagar's method with teachings of Dasgupta and Gersbach is discussed above as applied to claim 1. Both Dasgupta (Col. 3, line 31) and Gersbach ([0511]) teach wherein the host cell is HEK293 cells.
Regarding claim 7, the obviousness to modify Jaagar's method with teachings of Dasgupta and Gersbach is discussed above as applied to claim 1. Gersbach further teaches the sgRNA (i.e., regulatory nucleic acid) and the dCas9 fusion protein are packaged in the same or different AAV vectors ([0061]).
Regarding claims 8-10, the obviousness to modify Jaagar's method with teachings of Dasgupta and Gersbach is discussed above as applied to claim 1. Gersbach further teaches dCas9 is fused to a transcription inhibitor, KRAB (FIG. 3A).
Regarding claim 33, the obviousness to to modify Jaagar's method with teachings of Dasgupta and Gersbach is discussed above as applied to claim 1. Jaagar further teaches the IRES translation element is from hepatitis C virus (pg. 5, para. 3). Accordingly, Jaagar’s bicistronic reporter (i.e., reporter nucleotide sequence) encodes a reporter protein translated under the control of a viral RNA translation element. In addition, Jaagar also teaches the method comprises a monocistronic reporter that comprises a firefly luciferase gene (Fluc) under the control of a viral IRES translation element (Figure 2D and 2H; pg. 5, para. 3; pg. 7, para. 1). Jaagar further compares expression of Fluc from lysates treated with α-eIF1A aptamer (i.e., in the test host cell) to lysates treated with a negative control antisense aptamer (AS) (i.e., in the control host cell that comprises the reporter nucleic acid but not the regulatory nucleic acid (pg. 7, para. 1). Jaagar further teaches "eIF1A depletion reduced translation initiation by the HCV and CSFV IRES" (Figure 2H), where expression of Fluc in lysates with α-eIF1A aptamer is different compared to the expression of Fluc in lysates with negative control AS aptamer (pg. 7, para. 1).
Regarding claim 65, Jaagar teaches the Cap-independent translation element is HCV IRES (pg. 5, third paragraph; Figure 2C).
Claims 6, 14, 46, and 66-68 are rejected under 35 U.S.C. 103 as being unpatentable over Jaafar et al (Translation initiation by the hepatitis C virus IRES requires eIF1A and ribosomal complex remodeling; eLife, 2016, 5:e21198) in view of Dasgupta et al (US 6,833,254 B2; Published Date: 21 December 2004) and Chavez et al (WO 2019/079462 A1; Published Date: April 25, 2019) as applied to claims 1 and 33, and further in view of Zhang (US 8,697,359 B1; Published Date: Apr 15, 2014).
Regarding claim 14, the teachings of Jaagar, Dasgupta, and Gersbach are discussed above as applied to claim 1. However, Dasgupta does not teach wherein the CRISPR Cas effector is catalytically active.
Zhang teaches a method for altering expression of endogenous target gene sequences comprising introducing into a host cell: (i) a first regulatory element encoding a catalytically active CRISPR Cas9 protein, and (ii) a second regulatory element encoding a sgRNA that hybridizes with the target sequence (i.e., a regulatory nucleic acid) (claim 1). Zhang teaches the Cas9 protein directs cleavage of one or two strands of the target sequence (col. 6, line 27) to induce non-homologous end-joining in cells to knockout a specific gene (col. 19, para. 1; col. 62).
The obviousness to substitute Jaagar’a aptamer to a fusion protein comprising a dCas9 and transcriptional inhibitor as taught by Gersbach is discussed above as applied to claim 1. Similarly, it would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to have modified Jaagar's method to substitute the RNA aptamer for the target protein eIF1A with a catalytically active Cas9 protein and sgRNA to silence transcription of the target protein as taught by Zhang because it would have merely amounted to a simple substitution of prior art elements according to known methods to yield predictable results. The substitution is merely swapping one known means of inhibiting a target protein with another to serve the same purpose. One would have had a reasonable expectation of success in doing so because Zhang establishes that catalytically active Cas9 can knockout endogenous genes leading to modulation of expression. One of ordinary skill in the art would have recognized that CRISPR-mediated gene knockout as an alternative technique for inhibiting activity of a target protein that Jaagar accomplished with an aptamer.
Regarding claim 46, the teachings of Jaagar, Dasgupta, and Gersbach are discussed above as applied to claim 33. Further, the obviousness to substitute Jaagar’s aptamer to a catalytically active Cas9 as taught by Zhang is discussed above as applied to claim 14.
Regarding claims 66 and 68, Zhang further teaches the Cas9 protein and sgRNA are encoded on a single vector (brief description of FIG 22A-B), a U6 promoter driving the expression of sgRNA and a CBh promoter (i.e., polII promoter) driving the expression of the Cas9 protein (Example 4, col. 51). Further, Zhang teaches that Cas9 protein is fused to a green fluorescent protein (i.e., selectable marker) (col. 22, para. 2; FIG 2B).
Although Zhang does not explicitly state that the sgRNA and selectable marker are encoded on a single vector as required by instant claims, it would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to have combined the vectors that separately encode (i) sgRNA and Cas9, and (ii) Cas9 and selectable marker because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. Each element in the combination merely performs the same function as it does separately (sgRNA binds to the target sequence, Cas9 protein induces cleavage of the target sequence, and the fluorescent protein serves as a selectable marker). One would have been motivated to have done so for the advantage of including a fluorescent tag to monitor expression of Cas9, transfection of vector encoding all components necessary for the gene editing method, and select for cells post-transfection. One would have had a reasonable expectation of success in doing so because Zhang already teaches vectors encoding the sgRNA, Cas9, and selectable marker in different combinations.
Regarding claims 6 and 67, the obviousness to modify Jaagar's method with teachings of Dasgupta and Gersbach is discussed above as applied to claims 1 and 33. Gersbach teaches wherein a U6 promoter is operably linked to the nucleotide sequence encoding the sgRNA ([0180]; FIG. 3A), and wherein a CMV promoter (i.e., polII promoter) is operably linked to the dCas9-KRAB fusion protein (FIG. 3A).
However, Gersbach does not teach the same vector comprises polII promoter operably linked to a selectable marker gene.
The teachings of Zhang’s method are discussed above as applied to claim 14, and the teachings of Zhang’s vector encoding the Cas9 protein, sgRNA, and selectable marker are discussed above as applied to claims 66 and 68.
Thus, it would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to have modified the dCas9-KRAB fusion protein of Gersbach to further comprise a selectable marker, specifically a fluorescent protein, as taught by Zhang because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results. Each element in the combination merely performs the same function as it does separately (dCas9-KRAB modulated expression of target genes while the fluorescent protein serves as a selectable marker). One would have been motivated to have done so for the advantage of including a fluorescent tag to monitor expression of dCas9-KRAB, cellular state, and select for cells post-transfection. One would have had a reasonable expectation of success in doing so because Zhang demonstrates that Cas9 protein fused to selectable markers are functional in cells to modulate expression of target genes.
Response to the Arguments
Applicant argues that “Jaafar teaches that the in vitro translations (using rabbit reticulocyte and human cell lysates) are reliable, dependable, and even “quantitative”. One of ordinary skill in the art would immediately understand that the ability to perform ‘quantitative’ assays means that ‘promiscuous translations were clearly of no concern” (pg. 9, para. 2).
Applicant’s arguments have been fully considered but are not persuasive because the prima facie case of obviousness does not rely on avoiding promiscuous in vitro translation, rather, it relies on obvious to substitute the lysates environment in Jaagar’s method to intracellular expression of the bicistronic reporter as taught by Dasgupta. Further, Dasgupta demonstrates that a bicistronic reporter comprising the same architecture (cap-dependent promoter and an IRES-translation element) functions in cells with results consistent with those obtained in vitro, thereby supporting the reasonable expectation of success that Jaagar’s bicistronic reporter would likewise function when implemented in a cellular context.
Applicant argues that “Gersbach expressly refers to the “GFP/KAN” as “Stuffer”” (pg. 9, para. 6).
Applicant’s arguments have been fully considered but are not persuasive because claim 6 has since been amended, thereby changing the scope of the claim. Therefore, applicant’s argument is moot with respect to the rejections presented in instant Office Action.
Conclusion
No claims are allowable.
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/QIWEN SU-TOBON/
Examiner
Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636
Prosecution Insights