DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicants’ amendments to the claims and arguments filed on November 26, 2025 have been received and entered. Claim 1 has been amended, while claims 2-3, 10-12, 17-19 have been canceled.
Claims 1, 4-9, 13-15 and 16 are pending in the instant application.
Election/Restrictions
Applicant’s election without traverse of claims 1-18 (group I) in the reply filed on November 5, 2024 was acknowledged.
Priority
This application is a continuation of US application no 16/343,057 filed on 04/18/2019 that is a 371 of PCT/US17/57153 filed on 10/18/2017 that claims priority from US provisional application no 62/409,941 filed on 10/19/2016.
Claim Rejections - 35 USC § 101
Claims 1, 4-9, 13-15 and 16 are rejected under 35 U.S.C. 101 because the claimed invention is directed to non-statutory subject matter. Applicant’s argument the combination of (i) specifically engineered low- affinity CaM mutants, (ii) integrated ratiometric reporters is far more than a generic application of fluorescence measurement - it is an inventive concept that ensures the claims do not monopolize the abstract idea itself and (iii) sorting or removing non-viable cells integrates the intermediate calculations into a concrete practical application and cannot be performed mentally is found persuasive. Therefore, previous rejection of claims is hereby withdrawn.
New-Claim Rejections - 35 USC § 112- necessitated by amendments
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1, 4-9, 13-15 and 16 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. In the instant case, the recitation of limitation “..effecting based on the determination in step v a physical manipulation of the cell comprising discarding cells identified as dead or retaining cells identified as alive” (claim 1) is considered new matter. Upon further review of the instant specification, examiner could not find explicit or implicit support for effecting based on the determination in step v a physical manipulation of the cell. There is no explicit or implicit support physical manipulation as broadly recited in the claim. In case if applicants have evidence to support otherwise, applicants are invited to indicate page and line number for the written support specifically for “..effecting based on the determination in step v a physical manipulation of the cell comprising discarding cells identified as dead or retaining cells identified as alive” recited in claim 1 of the instant application. Thus, at the time the application was filed, an Artisan of skill would not recognize from the disclosure that Applicant was in possession of effecting based on the determination in step v a physical manipulation of the cell comprising discarding cells identified as dead or retaining cells identified as alive, as claimed. Claims 4-9, 13-15 and 16 are included in the rejection because they directly or indirectly depend from the rejected base claim. This is a new matter rejection.
New-Claim Rejections - 35 USC § 112- necessitated by amendments
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, 4-9, 13-15 and 16 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.
Claim 1 step vi is vague and indefinite to the extent metes and bounds of the term “effecting” could not be ascertained. The phrase appears to be functional language and not an active method step. A direct recitation of (vi) isolating viable cell from step (v) or alternatively (ii) discarding dead cell from step (v) would obviate the basis of rejection. Claims 4-9, 13-15 and 16 are included in the rejection because they directly or indirectly depend from the rejected base claim. Appropriate amendments/clarification is required.
Maintained-Claim Rejections - 35 USC § 103-in modified form
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, 4-9, 13 and 16 are rejected under 35 U.S.C. 103 as being unpatentable over Liu Ze-Yue. (Basic & Clinical Medicine, April 2016, 36(6): 763-766, IDS)/Addgene plasmid #51085, dated 6/29/2015, wayback machine, IDS) as evidenced by Ai et al (USPGPUB 20150226755, dated 8/13/2015) and Suzuki et al (Nature Communication, 2014, 5, 4153, 1-13, IDS) and Lin et al (Nature Neuroscience, 19, pages1142–1153 (2016).
Claim 1 is broadly interpreted to encompass methos steps that entirely involve the mental steps for measuring signal intensity ratio to determine whether cell is dead or alive. Therefore, recitation of measuring intensity of first and second detectable maker to compare the intensity performed by mental process is not given any patentable weight. Determination of whether cell is dead or alive based on signal intensity ratio is again mental step and there is no requirement to select/remove any dead and/or alive cell based on signal intensity ratio, therefore, is not given any patentable weight.
Recitation of step (vi) is indefinite for the reasons discussed above and interpreted as bring about a cell population retaining cells that are viable.
With respect to claims 1, 4-9, 13-16, Yue et al/addgene teach a method of monitoring calcium level in a neural cells comprising providing a neural cells comprising a vector comprising p-hSyn1 GCaMP6f-P2A-tomato comprising a nucleotide sequence comprising the human synapsin promoter, which is a neuron specific promoter, a calcium binding motif and first detectable marker GFP comprising a modified calcium binding motif and second detectable marker linked via a P2A to the calcium binding motif of calmodulin via a P2A, wherein the nucleotide does not encodes an ER localization or mitochondrial localization sequence (see abstract, page 764, section 1.1, col. 1, para. 2). It is relevant to point out the calcium binding motif GCaMP6f inherently is genetically modified as evident from the teaching of Ai who teaches GCaMP6f as set forth in SEQ ID NO: 9 that has 100% sequence identity to SEQ ID NO: 1 and has over 88% sequence identity to SEQ ID NO: 8 or 10 of the instant application that codes for a fusion protein comprising first detectable marker GFP fused with a calcium binding motif Calmodulin that further comprises M13 (limitation of claim 1, 6-11, 12, 14, 16) (see para. 4, 29, sequence search report). It is noted that said calmodulin is considered as being a genetically modified form of a calcium binding motif because GCaMP6 variants comprises an amino acid substitution, for instance at position 115 and 79 (V115T mutation or I79T) (figure 3, para 26), (b) quantitating changes of calcium levels in cells to compare Ca++ level. In the instant case, the cells comprising the nucleic acid disclosed by Yue et al/addgene and those embraced by the isolated cells comprising an isolated nucleic acid sequence of base claim appear to be structurally same.
With respect to claim 8-10, Yue et al/addgene teach that the first detectable marker is GFP and/or second detectable marker (tdtomato) is a variant thereof, which are distinguishable from teach other (see page 764, section 1.1, col. 1, para. 2 and addgene website).
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With respect to claim 15, Yue et al//addgene teach a method for monitoring Ca+ level in a neural cell, said method comprising providing an isolated primary neuron comprising p-AAV-hSyn1 GCaMP6f-P2A-tomato (see abstract, figure 1 and purpose), the first detectable marker is GFP and/or second detectable marker is a variant thereof, which are distinguishable from teach other (see page 764, section 1.1, col. 1, para. 2 and addgene website).
Yue differ from claimed invention by not disclosing at the modified calcium binding motif comprises at least one genetic modification comprises at least one amino acid E31 ID, E67D, F92W, E104D or D133E substitution.
Before the effective filing date of instant application, Suzuki discloses that for a Ca2+ indicator is useful for intracellular applications, it has to have a reduced affinity for Ca2+ and that E31D/F92W/E104D/D133E mutant had the desired low Ca2+ affinity (Kd=368 microM) (see page 2, col. 2, para. 4) and a large dynamic range (Fmax/Fmin=4.2). Suzuki further teaches amino-acid substitutions of in CaM (see page 9, col. 1, last para). Suzuki teaches capturing the image of the cells using an inverted microscope with CCD camera. Suzuki further teaches analyzing the ratiometric indicators by calculating the fluorescence ratio (F466/F520 for GEM-GECO1 and GEM-CEPIA1er; F340/F365 for fura-2; F535/F480 for D1ER). The modified calcium motif disclosed in Yue in view of Suzuki appears to be structurally similar to one claimed in the instant application and therefore must necessarily show similar functional characteristics of not responding to voltage stimulation by several fold compared to natural calcium binding motif. Lin provided the guidance with respect to genetically encoded calcium indicators (GECIs) that respond to calcium with increased fluorescence to measure Ca2+ level inside and/ or outside a cell (see figure 4).
Therefore, it would have been prima facie obvious for a person of ordinary skill to combine the teachings of prior art to modify the method of prior art nucleic acid disclosed in Yue as evidenced by Ai by introducing substitutions disclosed by Suzuki into GCaMP 6f of Yue, to lower Ca2+ binding affinity of GCaMP6f and measure the intensity of different fluorescent marker as disclosed in Siuzuki, with a reasonable expectation of success, before the effective filing date of the instant invention, in using said construct in quantitating changes of calcium levels and calculating the ratio within cells. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would be motivated to do so in order to study altered calcium regulation of calmodulin mediated activity without affecting calmodulin interaction with the enzyme and determine the insight on the structure–function relationship of CaM-based indicators (see page 10, col. 1, para. 2). Absent of any specific modification of calcium binding motif to reduce calcium binding affinity by at least a factor of five compared to naturally occurring calcium binding motif, it would have been a routine optimization to introduce at least one amino acid E31D, M36L, E67D, F92W, E104D or D133E substitution in the known calcium binding motif to achieve desired low Ca2+ affinity in view of teaching of Suzuki, with reasonable expectation of success. One of skill in the art would have been expected to have a reasonable expectation of success because Suzuki successfully reported introducing substitutions into calcium binding motif to lower Ca2+ binding affinity of GCaMP6f, thereby increasing utility of the Ca2+ sensor encoded by the construct. It should be noted that the KSR case forecloses the argument that a specific teaching, suggestion, or motivation is required to support a finding of obviousness See the recent Board decision Ex parte Smith, —USPQ2d—, slip op. at 20, (Bd. Pat. App. & Interf. June 25, 2007) (citing KSR, 82 USPQ2d at 1396) (www. uspto.gov/web/offices/dcom/bpai/prec/fd071925.pdf).
Claims 1, 14 and 15 are rejected under 35 U.S.C. 103 as being unpatentable over Yue. (Basic & Clinical Medicine, 2016, 36(6): 763-766)/Addgene plasmid #51085, dated 6/29/2015, wayback machine) as evidenced by Ai et al (USPGPUB 20150226755, dated 8/13/2015) Suzuki et al (Nature Communication, 2014, 5, 4153, 1-13, IDS), Lin et al (Nature Neuroscience, 19, pages1142–1153 (2016) as applied above and further in view of Chen et al (Neuron76, 297–308, October 18, 2012, 297-308, IDS).
The teaching of Yue/Addgene, Ai have been described above and relied in same manner here. The combination of reference differs from claimed invention by not disclosing by nucleic acid does not comprises any localization signal.
However, before the effective filing of instant application, it was known that as assay could be developed to measure calcium levels in the cytoplasm of a cells without using any localization signal in the construct of Yue/Addgene. For instance, Chen et al teach an in vitro construct comprising a modified CaMP2.2c operably linked to a coding sequence of tdTomato via a 2A peptide (P2A) sequence (See page 305, col. 1, para. 4). Chen teaches co-expressing GCaMPs and the red fluorescence protein tdTomato in the same construct using the 2A peptide (P2A) sequence. Chen further teaches normalizing for transfection efficiency by calculating the ratio of fluorescence intensity ratio of GCaMPs/tdTomato (See page 298, col.1, para. 3). It is further disclosed that at the single cell level, both GCaMP2.2c and GCaMP3 were homogeneously distributed in the cytoplasm without nuclear localization (Figures 1B, 1Bb1, and 1Bb2; Figures S2B, S2Bb1, and S2Bb2).
Therefore, it would have been prima facie obvious for a person of ordinary skill to combine the teachings of prior art to modify the nucleic acid disclosed in Yue Addgene, Ai by removing the NLS from the construct as suggested by Chen to measure the basal level of cytosolic level of Ca2+ in the cells, with a reasonable expectation of success, before the effective filing date of the instant invention, in using said construct in quantitating changes of calcium levels within cytosol of said cells. One of ordinary skill in the art would be motivated to do so in order to study altered calcium regulation of calmodulin mediated activity without affecting calmodulin interaction with the enzyme and determine the insight on the structure–function relationship of CaM-based indicators (see page 10, col. 1, para. 2). Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would be motivated to do so because prior art emphasized the importance of determining basal levels of fluorescence and stimulation-induced changes in fluorescence while evaluating genetically encoded calcium indicators (See page 298, col. 1, para. 3). One of skill in the art would have been expected to have a reasonable expectation of success because prior art successfully reported using a construct comprising a modified CaMP2.2c operably linked to a coding sequence of tdTomato via a 2A peptide (P2A) sequence as evident from the teaching of Chen. It should be noted that the KSR case forecloses the argument that a specific teaching, suggestion, or motivation is required to support a finding of obviousness See the recent Board decision Ex parte Smith, —USPQ2d—, slip op. at 20, (Bd. Pat. App. & Interf. June 25, 2007) (citing KSR, 82 USPQ2d at 1396) (www. uspto.gov/web/offices/dcom/bpai/prec/fd071925.pdf).
Response to arguments
Applicant disagree with the rejection arguing none of the reference teach or suggest - using a low-affinity Ca²⁺ sensor as an irreversible marker of plasma-membrane failure/cell death, nor do they correlate a fluorescence ratio to viability. To the contrary, Liu expressly relies on the continued viability of neurons to interpret calcium spiking; using that construct to declare the cell dead would defeat its purpose, thereby teaching away from the claimed use. Because the cited references pursue real-time functional Ca²⁺ imaging in living cells, they provide no motivation to repurpose or combine their teachings for the fundamentally different objective of terminal cell-death assessment. Nor is there any reasonable expectation of success. Further, none of the cited documents disclose or suggest the final step vi) which requires a physical manipulation of the assayed cell(s) once the viability state has been determined - for example, discarding or separating cells identified as dead, or selectively retaining live cells. Applicants’ arguments have been fully considered, but are not found persuasive.
In response it should be noted that claimed indicator allows the detection of the presence of calcium in the cytoplasm and thus cell-death. However, independent claim 1 does not exclude the presence of a nuclear localization sequence in the indicator. Thus, the claimed indicator may not be present in the cytoplasm and not be able to detect cell death and therefore combination of prior art references would be obvious to claimed method steps. Further, in response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., low-affinity Ca²⁺ sensor) are not recited in the rejected independent claim. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). In the instant case, Suzuki discloses that for a Ca2+ indicator useful for intracellular applications has a low affinity for Ca2+ and that E31D/F92W/E104D/ D133E mutant had the desired low Ca2+ affinity (Kd=368 microM) (see page 2, col. 2, para. 4) meeting the limitation of the claims. The specification teaches GEDI variants of the present disclosure all have an average predicted Ka greater than 500 µM.(see fig. 8B, example 2). In the instant case, none of the claims recite low affinity for Ca2+ sensor as argued by the applicant. In fact, dependent claims limit the base claim of using high or low affinity Ca²⁺ sensor with a moderately high Ca²⁺ sensor with Kd of 1.25microM. Thus, in view of foregoing issues (i) presence of a nuclear localization sequence and (ii) relatively moderately high affinity Ca²⁺ sensor, all the resulting cells from step (iii-iv) would be live as exemplified by Lin.
Therefore, in view of the fact patterns of the instant case, and the ground of rejection outlined by the examiner, applicants' arguments are not compelling and do not overcome the rejection of record.
Claim Rejections - 35 USC § 112
Claim 1, 4-9, 13-15 and 16 were 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. The rejection is withdrawn in view of applicant’s amendments to the claim 1. Applicants’ arguments with respect to the withdrawn rejections are thereby rendered moot.
Conclusion
No claims allowed.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. Nanki et al teach nucleic acid encoding CaM protein comprising amino acid sequence as set forth in SEQ ID No: 4 that has 100% sequence identity to SEQ IN NO: 5 (see sequence search result).
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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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/ANOOP K SINGH/ Primary Examiner, Art Unit 1632
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