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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 5/13/2026 has been entered.
Claims 1, 2, 6-9, 14, 17-21, 26, 29, 33, 35, 43 and 44 are currently under examination.
All previous rejection not reiterated in this office action are withdrawn.
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.
Claims 1, 20 and 35 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Regarding claims 1, 20 and 35, the recitation of “wherein a) signal to noise ratio of relative fluorescence units or luminescence units (FRU/RLU) is at least 2.5, b) RFU/RLU signal is measured when the first and second fusion proteins, the first and second guide RNAs, and the genomic sequence of interest are present; and c) RFU/RLU noise is measured when one or more of the first and second fusion proteins, the first and second nucleotide sequences are absent” renders the claim 1 indefinite because there is no prior step(s) for measuring a signal and noise in the same living cell. Steps i)-iii) only introduces fusion proteins, gRNAs into the cell. As such, it is unclear if this “wherein” clause is part of the method step. Such recitation also renders claim 20 indefinite because it is unclear whether the claimed cell comprises all components from (i)-(iv), or missing one of them so noise may be measured. Such recitation also renders claim 35 indefinite because it is unclear how the fusion proteins may be brought together without a genomic sequence of interest.
Dependent claims 2, 4, 6-9, 14, 17-19, 21, 26, 29, 33, 43 and 44 are rejected for same reason because they depend on claims 1, 20 and 35 but do not remedy the indefiniteness of these claims.
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.
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.
Claim(s) 1, 2, 7, 8, 9, 14, 17-21, 26, 29, 33 and 35 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (IDS), in view of Dixon et al (ACS Chem. Biol. 2016, Vol. 11, pages 400-408), Mout (ACS NANO 2017, Vol.11, pages 2452-2458) and sequence BGI97653 and sequence BGI97646. This rejection is rewritten to address the amendment.
Zhang teaches a paired dCas9 reporter system, in which dCas9 is linked to N- and C-terminal half of the firefly luciferase enzyme (page 212, 1st col., 1st paragraph, and Figure 1a and legend). Zhang teaches sgRNAs directs dCas9-luc fusion to upstream and downstream segments of a target DNA sequence, and when substrate DNA containing the two segments in proximity are detected by a pair of dCas9, luminescence is generated from the catalytic activity of luciferase in its entirety (Figure 1a and legend). Zhang teaches that gains in luminescence upon target recognition ranged from 2 to 35 fold (page 212, 2nd col., 2nd paragraph, and Figure 2a and legend). As such, Zhang’s teaching demonstrates signal to noise ratio of the luminescence being at least 2.5.
Zhang does not teach the paired reporter system using NanoLuc luciferase, and the detection of genomic sequence of interest in a living cell.
Dixon teaches NanoLuc is the most recently developed commercially available luciferase enzyme, which was derived from the naturally occurring luciferase present in deep sea shrimp O. gracilirostris and has been optimized to produce a luciferase enzyme subunit with improved luminescence and stability (page 401, bridging paragraph of 1st-2nd col). Dixon teaches the reporter is designed to be quantifiable within living cells (bridging paragraph of page 401) teaches through random mutagenesis, an optimized Nluc is generated that has greatly increased luminescence (~150x that of Fluc or Rluc) and stability (half-life >2h) when compared to the wild type luciferase (page 403, 1st col., 3rd paragraph). Dixon teaches NanoBit fulfills the general expectation of a complementation reporter in mammalian cells (page 406, 2nd col., last paragraph).
Mout teaches a method of direct cytosolic delivery of CRISPR ribonucleoprotein (RNP) for efficient gene editing through engineering RNP with carrier nanoparticles (Figure 3 and legend). Mout teaches said delivery system successfully delivered RNP and resulted in gene editing in multiple cell lines (page 2455, 2nd col., last paragraph).
Sequence BGI97653 is 100% identical to 32-190 of SEQ ID NO:1, and sequence BGI97646 is 100% identical to 32-43 of SEQ ID NO: 2 (see attached alignment). Both sequences are known in the art for being part of NanoLuc luciferase from O. gracilirostris, LgBiT, SmBiT and a linker for making fusion protein.
It would have been obvious to an ordinary skilled in the art that the commercially available NanoLuc produces improved luminescence and stability compared to firefly luciferase based on the teaching from Dixon. The ordinary skilled in the art reading Zhang would thus be motivated to substitute fusion protein of dCas9-Nluc and dCas9-Cluc with dCas9-LgBiT and dCas-smBiT pair for detecting nucleic acid of interest because of the advantage the NanoLuc offers. The sequences encoding LgBiT and smBiT are already known in prior art for making fusion proteins as evidenced by sequence having accession number BGI97653 and BGI97646, which are from O. gracilirostris luciferase. Since NanoLuc has already been demonstrated of bioluminescence imaging in vivo, the ordinary skilled in the art would recognize that the nucleic acid of interest can also be detected in a living cell using NanoLuc. Following the teaching from Mout, the ordinary skilled in the art would have reasonable expectation of success to make RNP that comprises sgRNA, dCas9-LgBiT and smBiT fusion and deliver said RNP into living cells. Therefore, the claimed invention of claims 1, 7, 20 and 35 would have been prima facie obvious to an ordinary skilled in the art at the time the application was filed.
Regarding claim 2, 21, Zhang teaches the RNA guided nuclease is dCas9 (page 212, 1st col., lines 7-11).
Regarding claim 8, Zhang teaches that gains in luminescence upon target recognition ranged from 2 to 35 fold (page 212, 2nd col., 2nd paragraph, and Figure 2a and legend). As such, Zhang’s teaching demonstrates signal to noise ratio of the luminescence being at least 10.
Regarding claim 9 and 26, Zhang teaches the first and second nucleotide sequence are arrayed in inverse and 20 nucleotides apart (see Figure 1 and legend, page 212, 2nd col., line 1).
Regarding claim 17-19, 33, Mout teaches introducing RNP into HEK293T cells (page 2455, 2nd col., last paragraph), which is eukaryotic, mammalian and human cells.
Regarding claims 14 and 29, introducing the second fusion protein, SmBiT and dCas9, is introduced at molar excess relative to LgBiT and dCas9, would have been routine optimization to achieve the most sensitive reporter sensor.
Claim(s) 4 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zhang, Dixon and Mout, as applied to claims above, and further in view of the sequence having accession number BGP88093 (see attached alignment)
The teaching from Zhang, Dixon and Mout has been discussed above. However, none of the references teaches a fusion protein have sequence at least 80% identical to SEQ ID NO:1.
The sequence of BGP88093 has 88.6% sequence identity to SEQ ID NO:1. BGP88903 is a fusion of dCas9 and KRAB.
It would have been obvious to an ordinary skilled in the art to make a fusion between dCas9-LgBiT and/or dCas9-SmBiT because all sequences encoding dCas9 and NanoLuc are known in the art. As shown in BGP88093, a fusion between dCas9-KRAB shows 88.6% identity with SEQ ID NO: 1, replacing KRAB with LgBiT or SmBiT such as BGI97653 (100% identical with 32-190 of SEQ ID NO: 1) or BGI97464 (100% identical with 32-43 of SEQ ID NO: 2) would result in fusion having at least 80% sequence identity with SEQ ID NO:1. Therefore, the claimed invention would have been prima facie obvious to an ordinary skilled in the art at the time the application was filed.
Claim(s) 6 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zhang, Dixon and Mout, as applied to claims above, and further in view of the sequence having accession number BGP88100 (see attached alignment)
The teaching from Zhang, Dixon and Mout has been discussed above. However, none of the references teaches a fusion protein have sequence substantially identical to SEQ ID NO:4.
The sequence of BGP88100 has 98.4% sequence identity to SEQ ID NO:4. BGP88100 is a fusion of dCas9 and KRAB.
It would have been obvious to an ordinary skilled in the art to make a fusion between dCas9-LgBiT and/or dCas9-SmBiT because all sequences encoding dCas9 and NanoLuc are known in the art. As shown in BGP88100, a fusion between dCas9-KRAB shows 98.4% identity with SEQ ID NO: 4, replacing KRAB with LgBiT or SmBiT such as BGI97653 (100% identical with 32-190 of SEQ ID NO: 1) or BGI97464 (100% identical with 32-43 of SEQ ID NO: 2) would result in fusion having substantially sequence identity with SEQ ID NO:4. Therefore, the claimed invention would have been prima facie obvious to an ordinary skilled in the art at the time the application was filed.
Response to Arguments
In response to the rejection, Applicant states that the high signal to noise ratio is a technically advantageous and surprising feature that is due to the specific structure of the fusion proteins as recited in claims 1, 20 and 35. Applicant states that high signal to noise ratio is maintained even when different RNA-guided catalytically inactive nucleases are used in the fusion proteins. Applicant also submitted a Rule 132 Declaration by Dr. Segal to establish this point. The declaration describes an experiment in which a series of fusion proteins with dSpCas9 and dSaCas9 were constructed and tested, and the fusion produce comparable, high signal to noise ratio as long as the domain configurations of the fusion proteins are (1) a N-terminus LgBit with a C-terminus catalytically inactive nuclease and (2) a N-terminus catalytically inactive nuclease with a C-terminus SmBit. Applicant argues this surprisingly high signal to noise ratio is an entirely unexpected finding.
The above arguments have been fully considered but deemed unpersuasive. As stated by Applicant, the specific structure of the fusion protein recited in claims 1, 20 and 35, is directed to (1) a N-terminus LgBit with a C-terminus catalytically inactive nuclease and (2) a N-terminus catalytically inactive nuclease with a C-terminus SmBit, and result in the high signal to noise ratio is at least 2.5. The teaching from Zhang already demonstrated that using conventional firefly luciferase fusion in four different configuration has resulted in between 2-30 fold signal to noise ratio. It would have been obvious to an ordinary skilled in the art that replacing the firefly luciferase with the NanoLuc, which was already recognized in prior art that has optimized to produce a luciferase enzyme subunit with improved luminescence and stability (page 401, bridging paragraph of 1st-2nd col), would result in at least 2.5 signal to noise ratio as claimed. Dixon teaches the reporter is designed to be quantifiable within living cells (bridging paragraph of page 401) teaches through random mutagenesis, an optimized Nluc is generated that has greatly increased luminescence (~150x that of Fluc or Rluc) and stability (half-life >2h) when compared to the wild type luciferase (page 403, 1st col., 3rd paragraph). Based on such teaching, the superior result of being at least 2.5 signal to noise ratio would have been within the expectation of ordinary skilled in the art. Moreover, according to the teaching from Zhang, the specific structure of having N-terminal half luciferase + dCas and dCas + C terminal half luciferase was one of the four combination being tested (Figure 2a), and it would have been routine optimization to find the optimal structure of the fusion protein as claimed.
The Declaration under 37 CFR 1.132 filed on 5/13/2026 is insufficient to overcome the rejection of claims 1, 2, 4, 6, 7-9, 14, 17-21, 26, 29, 33, 35, 43 and 44 based upon Zhang, Dixon, Mout and sequence BGI97653 and sequence BGI97646 as set forth in the last Office action for following reason. Although 8 different combination of fusion were tested, the finding of only two pairs showed high signal to noise ratio is not surprising for reasons discussed above. Especially when Zhang already demonstrated signal to noise ratio ranged from 2-30 when 4 different structure combination was tested. Therefore, for reason discussed in previous rejection and set forth above, this rejection is still considered proper and thus maintained.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CELINE X QIAN whose telephone number is (571)272-0777. The examiner can normally be reached M-F (8-4:00).
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/CELINE X QIAN/Primary Examiner, Art Unit 1637
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