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 .
Status of Application/Amendments/Claims/RCE 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1.114 and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicants’ submission filed on 10/31/2025 has been considered.
Claims 4, 8-9 and 13 have been canceled. Claim 1 has been amended. Claims 1-3, 5-7, 10-12 and 14-39 are pending. Claims 12, 14-19, 24-25 and 31-38 are currently withdrawn from further consideration pursuant to 37 CFR 1.142 (b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Claims 1-3, 5-7, 10-11, 20-23, 26-30 and 39 are the subject of the present Official action. The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Priority
Applicant’s claim for the benefit of a prior-filed application PRO 62/864,612 and 371 of PCT/US2020/038716 filed on 6/21/2019 and 6/19/2020, respectively, under 35 U.S.C 119(e) or under 35 U.S.C 120, 121 or 365(c) is acknowledged.
Accordingly, the effective priority date of the instant application is granted as 6/21/2019.
Withdrawn Rejections
The 35 U.S.C. 103 rejection of claims 1-7, 10-11, 20-23, 26-30 and 39 has been withdrawn light of applicants claim amendments which move the limitations of claims 4 into independent claim 1 describing the first secreted reporter protein as emitting a light signal upon contact with a substrate. All previous 103 rejections have been re-applied in modified form to address applicants claim amendments.
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 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.
Claims 1-3, 10-11, 20, 22, 28-30 and 39 are newly rejected under 35 U.S.C. 103 as being unpatentable over Bito et al. WO 2014/045674A1, published 4/27/2014 (hereinafter Bito, reference of record) in view of Lewis et al. US 2006/0265771, published 11/23/2006 (hereinafter Lewis), Borokov et al. WO 2001/11058A1, published 2/15/2001 (hereinafter Borokov, reference of record) and Xu et al. "A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid." BMC biotechnology 18.1 (2018): 52. This new rejection is necessitated by applicant’s claim amendments dated 10/31/2025.
Claim 1: Bito describes a secreted neuronal activity reporter (SNAR) construct comprising two or more tandem repeats of a core domain of the synaptic activity response element (SARE) of Arc/Arg3.1 (Bito, para 9, 10, 15, 17). Bito provides specific embodiments to four tandem repeats of a core domain of the SARE of Arc/Arg3.1 (Bito, para 15). Bito describes the use of an Arc minimal promoter (Bito, para 18). Bito describes the use of a first secreted reporter protein (Bito, para 25, 26, 29). Bito provides a diagram of the SNAR construct in Fig 1A. Bito describes transfecting nerve cells and detecting the expression with a reporter gene, with preferred embodiments to CAT, GFP and other fluorescent proteins (Bito, para 22). Although Bito describes the expression of reporter genes like fluorescent proteins and describes transcriptional activity assays using luciferase, Bito does not expressly describe a first secreted reporter protein that emits light signal upon contact with a substrate as described in newly amended claim 1. Furthermore, Bito does not describe a loxP site and lox2272 site which are either upstream or downstream of the inverted polynucleotide encoding a first secreted reporter protein.
Claim 2: Bito discloses the core domain of the SARE of Arc/Arg3.1 with 100% sequence similarity to instant SEQ ID NO: 2 as shown below.
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Sequence search results for SEQ ID NO: 2 (Core domain of Arc/Arg3.1 SARE, SEQ ID NO: 2)
Claim 3: Bito discloses an Arc minimal promoter with 100% sequence similarity to instant SEQ ID NO: 3 as shown below.
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Sequence search results for SEQ ID NO: 3 (Arc minimal promoter, SEQ ID NO: 3)
Claim 11: Bito describes the further inclusion of a control construct comprising a constitutive promoter and a polynucleotide encoding a second secreted reporter protein (Bito, para 35, 37, 44, 51). An exemplary embodiment is provided below from Fig 3A, wherein the constitutive promoter E-PGK is used.
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Claim 20: Bito describes the use of a constitutive promoter including a human PGK promoter (Bito, para 35, 37, 44, 51 and Fig 3A).
Claim 22: Bito describes the use of an E-SARE reporter (GFP) and an infection marker (RFP), which have distinct emission signals (Bito, para 35, 37, 44, 51 and Fig 3A).
Claim 39: Bito describes delivery using an adeno-associated viral (AAV) vector (Bito, para 35, 51 and Fig 10).
Although Bito describes the expression of reporter genes like fluorescent proteins and describes transcriptional activity assays using luciferase, Bito does not expressly describe a first secreted reporter protein that emits light signal upon contact with a substrate as described in newly amended claim 1.
Claims 1: Lewis describes how reporter proteins can be expressed in neurons and used to quantify neuronal activity in real-time (Lewis, para 10, 28). Lewis provides preferred embodiments towards the use of secreted reporter proteins like luciferase, which emits light upon contact with a substrate (Lewis, para 28 and example 2).
It would have been prima facie obvious to one of ordinary skill in the art to select a secreted reporter protein like luciferase as disclosed by Lewis in the secreted neuronal activity reporter construct described by Bito. It would have been a matter of simple substitution of one known reporter protein for another to obtain predictable results. One of ordinary skill would be motivated to substitute luciferase since luciferase reporter constructs allow the precise quantification of neuronal activity levels rather than just localization which would be the case for fluorescent proteins like GFP (Lewis, para 28 and example 2). One of ordinary skill would have a reasonable expectation of success given that unlike GFP, luciferase requires a substrate to produce a signal (light), thus results in virtually zero background signal and is routinely used as a reporter in molecular biology.
However, neither Bito nor Lewis describe a loxP site and lox2272 site which are either upstream or downstream of the inverted polynucleotide encoding a first secreted reporter protein as described in claim 1.
Claims 1 and 28-29: Borokov describes the use of loxP and lox2272 sites for selectively regulating site-specific recombination (Borokov, pg 5, 12). Borokov describes loxP and lox2272 sites which are upstream or downstream of a polypeptide-encoding sequence in a transgene (Borokov, pg 9, 18). Although Borokov describes regulating site-specific recombination using a Cre/LoxP systems, Borokov does not expressly describe the use of an inverted polynucleotide encoding a first secreted reporter protein, which us commonly used in the Cre/LoxP system called Double-Floxed Inverted Open Reading Frame (DIO). However, this application of DIO and arrangement using an inverted polynucleotide was known in the art as shown by Xu. Xu describes the development of the Cre/LoxP system called Double-Floxed Inverted Open Reading Frame (DIO) which was invented to turn on gene expression via Cre-mediated recombination (Xu, abstract – background). Xu describes how the DIO method greatly reduces leaky expression in the absence of Cre recombinase, making the DIO switch the preferred method to turn on genes in mammalian cells using AAV vectors (Xu, background para 4).
Claim 10 and 30: Borokov discloses a loxP site and lox2272 site which share 100% sequence similarity to SEQ ID NO: 4 and 5, respectively (sequence search results shown below).
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Sequence search results for SEQ ID NO: 4 (loxP, example 1)
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Sequence search results for SEQ ID NO: 5 (lox2272, SEQ ID NO: 21)
It would have been prima facie obvious to one of ordinary skill in the art at the time of the instant invention to use a Double-Floxed Inverted Open Reading Frame (DIO) Cre/LoxP system as described by Borokov and Xu to selectively express the SNAR construct described by Bito. Xu shows that the DIO method greatly reduces leaky expression in the absence of Cre recombinase, making the DIO switch the preferred method to selectively turn on genes in mammalian cells using AAV vectors (Xu, background para 4). Thus, it would have been a matter of combining prior art elements according to known methods to yield predictable results for one of ordinary skill to achieve selective reporter activity in mammalian cells like neurons. One of ordinary skill would be motivated to make this combination in order to reduce leaky expression in the absence of Cre recombinase using DIO and selectively monitor neuronal activity in heterogeneous cultures. One of ordinary skill would have a reasonable expectation of success given that the DIO Cre/LoxP construct designs using an inverted ORF and loxP and lox2272 sites positioned upstream or downstream of luciferase-based reporter protein are known in the art and have been used “extensively” in rAAV vectors to spatially restrict gene delivery to cells expressing Cre recombinase (Xu, background para 4).
Claims 1-3, 5-7, 10-11, 20, 22, 23, 26-30 and 39 are newly rejected under 35 U.S.C. 103 as being unpatentable over Bito (supra), Lewis (supra), Borokov (supra) and Xu (supra) as applied to claims 1-3, 10-11, 20, 22, 28-30 and 39 above in further view of Inouye et al. US 2015/0184134, published 7/2/2015 (hereinafter Inouye, reference of record) as evidenced by Inouye et al. US 2016/0076052, published 3/17/2016. This new rejection is necessitated by applicant’s claim amendments dated 10/31/2025.
Neither Bito, Borokov nor Xu describe coelenterazine as the substrate for the first and second secreted reporter protein, a first reporter protein comprising Gaussia luciferase disclosed in SEQ ID NO: 7 or a second reporter protein comprising nanoluciferase comprising an N-terminal secretion signal peptide disclosed in SEQ ID NO: 9.
Claims 5 and 23: Inouye discloses novel luciferase variants and assays thereof which use coelenterazine as a substrate (Inouye, para 1, 3, 5 and 14). Inouye also describes the use of coelenterazine derivatives including furimazine (Inouye, para 55).
Claims 6 and 7: Inouye describes the use of luciferase variants including Gaussia luciferase in reporter assays (Inouye, para 5, 180, 198). The exact sequence listing of Gaussia luciferase is disclosed in a subsequent patent filed by Inouye which is provided as an evidentiary reference and shares 100% sequence similarity to SEQ ID NO: 7 (sequence search results shown below).
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Sequence search results for SEQ ID NO: 7 (Gaussia luciferase, SEQ ID NO: 8)
Claims 26 and 27: Inouye describes the use of a reporter proteins including nanoluciferase using a N-terminal secretory signal peptide (Inouye, Example 9). The nanoluciferase peptide disclosed by Inouye shares 100% sequence similarity to SEQ ID NO: 9 (sequence search results shown below).
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Sequence search results for SEQ ID NO: 9 (Gaussia luciferase, SEQ ID NO: 15)
It would have been prima facie obvious to one of ordinary skill in the art to use Gaussia luciferase and nanoluciferase reporter system as described by Inouye in the SNAR construct expressed by the DIO Cre/LoxP system described by Bito in view of Borokov and Xu. It would have been a matter of simply substituting one known reporter protein pair for another to obtain predictable results. For example, Bito describes using GFP and RFP as reporter proteins for labeling active nerve cells (Bito, para 35, 37, 44, 51 and Fig 3A). Although GFP and RFP emit different wavelengths, there is still significant spectral overlap. Thus, one of ordinary skill would be motivated to use a luciferase-based reporter system as described by Inouye to limit spectral overlap and limit autofluorescence interference which is commonly observed in GFP-based reporter systems. One of ordinary skill would have a reasonable expectation of success because the Gaussia luciferase and nanoluciferase reporter system as described by Inouye is a known to function well in a dual-reporter assay and are often used together to measure multiple biological processes simultaneously.
Claims 1-3, 5-7, 10-11, 20-23, 26-30 and 39 are newly rejected under 35 U.S.C. 103 as being unpatentable over Bito (supra), Lewis (supra), Borokov (supra), Xu (supra and Inouye (supra) as applied to claims 1-7, 10-11, 20, 22, 23, 26-30 and 39 above in further view of Wong et al. US 2017/0183654, published 6/29/2017 (hereinafter Wong, reference of record). This new rejection is necessitated by applicant’s claim amendments dated 10/31/2025.
The collection of cited art does not describe the use of a human PGK promoter set forth in SEQ ID NO: 6.
Claim 21: Wong describes the use of constitutive promoters including human PGK in an expression cassette. Wong discloses SEQ ID NO: 1 which shares 100% sequence similarity to SEQ ID NO: 6 (Sequence search results shown below).
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Sequence search results for SEQ ID NO: 6 (human PGK, SEQ ID NO: 1)
It would have been prima facie obvious to one of ordinary skill in the art to use a human PGK promoter as disclosed by Wong in the in the SNAR construct described by Bito in view of Borokov, Xu and Inouye. In fact, Bito describes the use of an E-PGK constitutive promoter, but falls short of providing the promoter sequence (Bito, Fig 3A). Thus, it would have been a matter of combining prior art elements according to known methods to yield predictable results. One of ordinary skill would be motivated to use the human PGK promoter as disclosed by Wong since the PGK promoter is constitutively active in nearly all human cells. One of ordinary skill would have a reasonable expectation of success since the particular human PGK promoter is known in the art as shown by Wong and Bito used a similar PGK promoter in the SNAR construct. Accordingly, in the absence of evidence to the contrary, one of ordinary skill in the art would have considered the claimed invention to have been prima facie obvious to at the time the invention was made.
Response to Traversal
Although the rejections are newly applied, some of applicant’s arguments are relevant and are discussed below.
Applicant argues that Bito, Inouye, Borokov and Wong fail to teach or suggest all elements in currently amended claim 1. Specifically, applicant argues that Bito describes the use of a luciferase assay as a means to evaluate the transcriptional activity of different promoters and conspicuously switches from a luciferase reporter to a fluorescent reporter. Applicant argues that this would lead a person of ordinary skill in the art to select a fluorescent reporter.
This argument has been fully considered, but is not found persuasive since nonpreferred and alternative embodiments constitute prior art. Disclosed examples and preferred embodiments do not constitute a teaching away from a broader disclosure, see MPEP 2123. Furthermore, the current 103 rejection provides Lewis as a reference showing the known advantages of a luciferase-based reporter assay.
Applicant further argues that the half-life of the SARE construct disclosed by Bito is not compatible with the operable duration of the claimed construct. Applicant states that the degradation tag and sequence disclosed in Bito is associated with proteins that have a short half-life.
This argument has been fully considered, but is not found persuasive since the instant claims do not contain any limitations regarding the half-life or duration of the SNAR construct. Attorney arguments do not replace evidence where evidence is necessary, see MPEP 2145.
Conclusion
No claims allowed.
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Alexander Nicol
Patent Examiner
Art Unit 1633
/ALEXANDER W NICOL/Examiner, Art Unit 1634