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 .
Claims 1, 2, 4, 6, 7-9, 14, 17-21, 26, 29, 33, 35, 43-45 are pending in the application.
This office action is in response to the amendment filed on 9/23/2025.
All previous rejection not reiterated in this office action are withdrawn.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, 2, 4, 6, 7-9, 14, 17-21, 26, 29, 33, 35 and 45 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. This is a new ground of rejection necessitated by amendment.
Regarding claims 1, 20 and 35, the recitation of “the first/second fusion protein comprising an RNA-guided nuclease fused to a large/small fragment derived from Oplophorus gracilirostris luciferase comprising amino acid 32-190 of SEQ ID NO: 1 or amino acids 1450-1607 of SEQ ID NO:3” renders the claim indefinite because it is unclear whether it is the large/small fragment that comprises the recited amino acid sequence or the full length Oplophorus gracilirostris luciferase comprises said amino acid sequence.
Dependent claims 2, 4, 6, 7-9, 14, 17-19, 21, 26, 29, 33 and 45 are rejected for same reason because they depend on claims 1, 20 and 35 but does not remedy the indefiniteness.
Regarding claim 45, the recitation of “wherein signal to noise ratio of relative fluorescence units or relative luminescence units is at least 2.5 fold above background” renders the claim indefinite because the fusion protein of claim 35 comprises an RNA guided nuclease and fragments derived from Oplophorus gracilirostris luciferase, wherein the fragments does not produce any fluorescence or luminescence. It is unclear how the signal noise ratio is being determined.
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, 17-22, 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).
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, 22, Zhang teaches the RNA guided nuclease is dCas9 (page 212, 1st col., lines 7-11).
Regarding claim 8, the “wherein” clause is not given weight because it simply expresses the intended result of a process step positively recited. In other words, there is no additional step(s) required to be performed to have signal noise ratio of 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 argues that the claimed fusion protein can produce high signal to noise ratio when used as a biosensor according to the claimed methods, and this technically advantageous and surprising feature is due to the specific structure of the fusion protein, which cannot be gleaned from combined teaching of Zhang, Dixon, and Mout. Applicant asserts that the inventors compared signal: noise of the fusion protein to those monomeric dCas9-EGFP fusion protein and monomeric NLuc-dCas9 in live cells under similar condition and are far superior than those monomeric fusions. Applicant argues that the far superior results is attributed to a combination of LgBiT on N-terminus and smBiT on the C-terminus of a fusion to dCas9 structure. Applicant asserts that demonstrated sensitivity of the method through specific binding of gRNA to respective wild type and mutant sequence.
The above argument has been considered but deemed unpersuasive. As discussed in the rejection, the structure of both dCas9 and LgBiT and smBiT are known in the art at the time of filing. Zhang has demonstrated a split enzyme system comprising fusion of dCas9 and NFluc and CFluc. The motivation to use NanoLuc comprising fragments of LgBiT and smBiT comes from Dixon, and the sequences of BGI97653 and BGI97464, which teaches making fusion with said fragment. As such, since the structure of LgBiT and smBiT and dCas9 are known in the art, making a fusion with dCas9 would have been routine experimentation rather than method of innovation. Therefore, for reason discussed in previous office action and set forth above, this rejection is still considered proper and thus maintained.
Allowable Subject Matter
Claims 43 and 44 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Claim 44 is objected to under 37 CFR 1.75 as being a substantial duplicate of claim 43. When two claims in an application are duplicates or else are so close in content that they both cover the same thing, despite a slight difference in wording, it is proper after allowing one claim to object to the other as being a substantial duplicate of the allowed claim. See MPEP § 608.01(m). In the present case, claims 43 and 44 are drawn to fusion protein that have same amino acid sequence.
Conclusion
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.
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/CELINE X QIAN/ Primary Examiner, Art Unit 1637