DETAILED ACTION
Acknowledgment and entry of the Amendment submitted on 2/17/26 is made.
Claims 9-21 remain withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention.
Claims 1-8 and new claims 22 and 23 are currently under examination.
Claim Rejections - 35 USC § 112-2nd paragraph
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.
Claim 22 is 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 22 is vague and indefinite because it recites a percentage identity to a gene that has not been identified. There is no sequence provided to determine such homology. The mere recitation of a name, i.e., target domain gene, to describe the invention is not sufficient to satisfy the Statute's requirement of adequately describing and setting forth the inventive concept. The claim should provide the nucleic acid sequence of the gene which would allow for one to identify the gene without ambiguity. It is also unclear what proteins encompass “factor arrest proteins.” The metes and bounds of this term are not readily understood. The cell type is not identified in the claim from which it depends so it is unclear how one would determine the factor protein when any type of cell is encompassed in claim 1. The mere recitation of a gene name to determine percent identity does not adequately define the claimed gene in the product claim. While the specification can be used to provide definitive support, the claims are not read in a vacuum. Rather, the claim must be definite and complete in and of itself. Limitations from the specification will not be read into the claims. The claims as they stand are incomplete and fail to provide adequate structural properties to allow for one to identify what is being claimed.
Claim Rejections - 35 USC § 103
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.
Claim(s) 1-4, 6-8 and 22 is/are rejected under 35 U.S.C. 103 as being unpatentable over Dowell et al. Dowell et al (“Yeast Assays for G Protein-Coupled Receptors,” G Protein-Coupled Receptors in Drug Discovery: Methods in Molecular Biology, April 2009, Vol. 552, Pgs. 213-229) in view of Kosuri et al (WO 2019/010270, Jan. 10, 2019).
Regarding claim 1, Dowell discloses a plurality of cells (Pg. 5, sixth paragraph, a panel of engineered yeast strains), wherein each cell comprises (i) one or more of a target domain gene that specifically binds to a binding partner (Pg. 5, sixth paragraph, Previously untested GPCRs are usually transformed into a panel of engineered yeast strains expressing different Gpa1p/Ga chimeras), (ii) one or more of an intracellular chimeric G-protein alpha subunit comprising an endogenous G-protein alpha subunit with a humanized C-terminus (Pg. 5, - sixth paragraph, The GlaxoSmithKline system consists of 11 isogenic host yeast strains, with the prefix MMY, containing different integrated Gpa1p/Ga chimeras (Table 1) in which the C-terminal five amino acids of Gpa1p are replaced with the equivalent sequence from mammalian Ga proteins; Pg. 6, Table 1.Genotypes of yeast strains used for GPCR research at GlaxoSmithKline; wherein Table 1 of Dowell includes strain: MMY14; Genotype: MMY11 TRP1::Gpa1/Gaq(5); see Brown, Pg. 18, left-hand column, third paragraph, We replaced the five C-terminal residues of Gpa1p (468 KIGII-COOH) with the five C-terminal residues of mammalian Ga subunits Gaq (EYNLV-COOH); see Para. [0056] of the instant application, Table 1 includes DI dCyFIRP1 Q, BY4741 farlA sst2A ste2A figlA::mTq2 X-2: Pmnla-UnTS-TcYclb Gpa1 (468-472)(KIGH>EYNLV)); and (iii) one or more of an inducible reporter, wherein the expression of the reporter is dependent on the activation of the target domain encoded by the target domain gene (Pg. 5, seventh paragraph, The MMY strains have two chromosomally integrated reporter genes, FUS1-HIS3 and FUS1-lacZ. The preferred assay is to measure His3p-mediated cell growth, induced by agonism of the GPCR, which can be quantified using a fluorometric indicator of cell number. Alternatively, assays may be configured measuring the 8-galactosidase reporter for which various fluorogenic and chromogenic substrates are available).
Regarding claim 2, Dowell further discloses wherein the one or more of target domain is a membrane channel, a symporter transporter, an antiporter transporter, an ATPase, an enzyme or a receptor (Pg. 5, sixth paragraph, previously untested GPCRs).
Regarding claim 3, Dowell further discloses wherein the receptor is a G-protein coupled receptor (GPCR) (Pg. 5, sixth paragraph, previously untested GPCRs).
Regarding claim 4, modified Dowell discloses the plurality of cells of claim 1. Dowell further discloses wherein the one or more of inducible reporter is a transcriptional reporter (Pg. 5, seventh paragraph, The MMY strains have two chromosomally integrated reporter genes, FUS1-HIS3 and FUS1-lacZ; Pg. 3, second paragraph, several reporter genes have been described, typically utilizing the FUS1 promoter which is induced by activation of the pheromone-response signal transduction pathway. These include HIS3 (His3p), lacZ (B-galactosidase) and EGFP (enhanced green fluorescent protein).
Regarding claim 6, Dowell further discloses wherein the cells are yeast cells (Pg. 5, sixth paragraph, a panel of engineered yeast strains).
Regarding claim 7, Dowell further discloses where the yeast cells are Saccharomyces cerevisiae (Pg. 5, sixth paragraph, a panel of engineered yeast strains; Pg. 1, first paragraph, Saccharomyces cerevisiae).
Regarding claim 8, Dowell further discloses wherein the yeast cells lack an endogenous GPCR (Pg. 5, sixth-seventh paragraph, The GlaxoSmithKline system consists of 11 isogenic host yeast strains, with the prefix MMY ... (Table 1); wherein Table 1 of Dowell includes strain: MMY11; Genotype: MMY9 ste2A::G4184R; see Para. [00108] of the instant application, a strain lacking the endogenous yeast GPCR (Ste2)).
Regarding new claim 22, Dowell teaches that in early developments of an assay system for heterologous GPCRs it was shown that some mammalian GPCRs could couple directly to the yeast heterotrimeric G protein (3, 4). Subsequently, systems were improved through a number of steps including: deletion of SST2 to sensitize the pathway, deletion of FAR1 (factor arrest protein) to prevent cell cycle arrest enabling a positive readout for agonism, and the development of chimeric G proteins (1). Several reporter genes have been described, typically utilizing the FUS1 promoter which is induced by activation of the pheromone-response signal transduction pathway. These include HIS3 (His3p; (3)), lacZ (β-galactosidase; (5)), and EGFP (enhanced green fluorescent protein; (6)). The methods described herein are based on the GlaxoSmithKline system (7, 8) utilizing the HIS3 reporter. See “Introduction.”
However, Dowell fails to explicitly to disclose wherein the target domain gene comprises a barcode.
Kosuri et al teaches a cell comprising a target domain gene that specifically binds to a binding partner; and an inducible reporter, wherein the expression of the reporter is dependent on the activation of the target domain encoded by the target domain gene, and further teaches a barcode (Para. [0010] a cell comprising i) a heterologous receptor gene; and ii) an inducible reporter comprising a receptor-responsive element; wherein expression of the reporter is dependent on the activation of the activity of the receptor encoded by the receptor gene, and wherein the reporter comprises a barcode; Para. [0011] In some embodiments, the receptor gene encodes for a G-protein coupled receptor (GPCR). In some embodiments, the reporter is induced upon signal transduction by the activated receptor protein). The abstract recites: Aspects of the disclosure relate to a population of cells, wherein each cell comprises: i.) a heterologous receptor gene; ii.) an inducible reporter comprising a receptor-responsive element; wherein expression of the reporter is dependent on the activation of the activity of the receptor encoded by the receptor gene, and wherein the reporter comprises a barcode comprising an index region that is unique to the heterologous receptor gene; and wherein the cells express different heterologous receptors and wherein each single cell expresses one or more copies of one specific heterologous receptor and one or more copies of one specific reporter.
It would have been obvious to one of ordinary skill in the art at the time of the invention to modify the plurality of cells of Dowell to include a heterologous target domain gene comprising a barcode, since the general conditions of the claim are disclosed in the prior art (Kosuri, Para. [0010] a cell comprising i) a heterologous receptor gene; and ii) an inducible reporter comprising a receptor-responsive element; wherein expression of the reporter is dependent on the activation of the activity of the receptor encoded by the receptor gene, and wherein the reporter comprises a barcode), including a barcode in the target domain gene involves only routine skill in the art. The motivation of doing so would be to develop plurality of cells using a target domain gene having a barcode or an inducible reporter having a barcode to uniquely identify or distinguish the target domain.
Claim(s) 5 and 23 is/are rejected under 35 U.S.C. 103 as being unpatentable over Dowell et al (“Yeast Assays for G Protein-Coupled Receptors,” G Protein-Coupled Receptors in Drug Discovery: Methods in Molecular Biology, April 2009, Vol. 552, Pgs. 213-229) in view of Kosuri et al (WO 2019/010270) and Goedhart et al. (Nat. Comm. March 2012. 3(751): 1-9).
The teachings of Dowell and Kosuri et al are set forth above. However, they do not explicitly disclose wherein the transcriptional reporter is mTurquoise2.
Goedhart teaches mTurquoise2 (Pg. 1, Abstract, Cyan variants of green fluorescent protein ... mTurquoise2, a brighter variant with faster maturation, high photostability, longer mono-exponential lifetime and the highest quantum yield measured for a monomeric fluorescent protein; Pg. 7, right-hand column, second paragraph, Because of its high quantum yield, mono-exponential fluorescence lifetime, improved maturation rate, and high photostability in vivo, mTurquoise2 is the preferable CFP variant for live-cell imaging).
It would have been obvious to one of ordinary skill in the art at the time of the invention to modify the plurality of cells of Dowell and Kosuri to include wherein the transcriptional reporter is mTurquoise2 for the purpose of developing various cells including various reporters (see Dowell, Pg. 3, second paragraph, several reporter genes have been described, typically utilizing the FUS1 promoter which is induced by activation of the pheromone-response signal transduction pathway. These include HIS3 (His3p), lacZ (B-galactosidase), and EGFP (enhanced green fluorescent protein). One or ordinary skill in the art would have been motivated to use is mTurquoise2 because it has one of the highest quantum yields of any fluorescent protein, i.e., exceptionally bright, mono-exponential fluorescence lifetime, improved maturation rate, and high photostability in vivo, mTurquoise2. The prior art teaches it is the preferable CFP variant for live-cell imaging. A tracer as recited in new claim 23 is broadly defined as a specialized molecule or probe used to track, visualize, or measure specific processes, structures, or molecules within a cell or organism. Tracers are designed to be detectable and often targeted to specific sites, allowing researchers or clinicians to observe biological activity that would otherwise be invisible, e.g., such as mTurquoise2. Dowell also teaches fluorescent detection, i.e., tracers, at item 3 under “Notes.”
Response to Applicants’ arguments:
Applicants argue that:
Dowell teaches measuring GPCR signaling in yeast using transcriptional reporters. More specifically, Dowell teaches engineering Saccharomyces cerevisiae strains to functionally express GPCRs using chimeric G-protein α-subunits. Dowell uses single-strain non-multiplexed yeast, and is silent regarding tracking or methods for distinguishing multiple receptors or target domains. Dowell is further absent of any teaching, suggestion, or motivation to incorporate barcodes into the target domain gene, the reporter, or any other genetic element. In fact, the experimental design of Dowell does not require barcoding, because each strain is evaluated independently. Thus, Dowell provides no basis for modifying its system to include a barcoded target domain gene. Kosuri and Goedhart fail to cure the deficiencies of Dowell. Kosuri teaches high-throughput pooled screening, wherein barcoded reporters are used as a proxy for heterologous receptor activity and are essential for tracking large receptor libraries. A heterologous large receptor library is not a single-strain yeast GPCR assay. Goedhart teaches improvements to mammalian cel fluorescent imaging but does not teach using Turquoise2 as a transcriptional reporter in yeast or in GPCR assays. In view in combination, one of skill in the art would have no reason to combine Dowell, Kosuri, and Goedhart. In contrast, the claimed invention provides a screening platform system for the discovery of novel ligands and drugs that interact with the "target domain gene". In this system, a human GPCR ("target domain gene") is coupled to the yeast Ga subunit using C-terminal Ga chimeras in which the last five residues of the yeast Ga are replaced with the last five residues of a human Ga. Activation of a GPCR-Gα chimera pair stimulates a downstream mitogen-activated protein kinase cascade that drives the expression of pheromone-responsive genes. Replacing a dispensable pheromone-responsive gene with a reporter, such as a fluorescent protein, provides a readout of GPCR signaling that is compatible with high-throughput screening formats. The screening platform is advantageous over the prior art as it allows for simultaneous, cost-effective profiling of ligands and drugs against mixtures of hundreds of cell strains barcoded with "target domain genes", such as GPCR, in a single experiment. See examples section of the current application. The current invention teaches the technological advantage of the "target domain gene" (e.g. GPCR) acting as a unique barcode that can be identified by quantitative polymerase chain reaction, NanoString, sequencing, or similar methods. See paragraph [0041] of the application, as filed. As shown in the examples, the inventors constructed a yeast strain library of 30 human GPCRs and their 300 possible GPCR-Gα coupling combinations. Profiling the 300 strains using parallel (DCyFIRscreen) and multiplex (DCyFIRplex) DCyFIR modes recapitulated known GPCR agonism with 100% accuracy and identified unexpected interactions for the receptors ADRA2B, HCAR3, MTNR1A, S1PR1, and S1PR2. See paragraphs [0114]-[0115] of the application as filed. Moreover, the DCyFIRscreen system and associated method allows the 300 GPCR-Gα strains to be screened for known agonism to determine Ga subunit association.
These arguments have been fully and carefully considered, but are not deemed persuasive. Applicants’ arguments are not commensurate in scope with the claimed invention. 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 are not recited in the rejected claim(s). 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). The claimed invention is drawn to a ‘plurality of cells’, not a method. These are product claims, not method claims. Applicants argue that Dowell uses single-strain non-multiplexed yeast; however, the type of cell is not specified in the claims with the exception of claim 6 which recites yeast cells and claim 7 which recites the yeast cells are Saccharomyces cerevisiae, as is the case for the cells of Dowell. Regarding claim 6, Dowell further discloses wherein the cells are yeast cells (Pg. 5, sixth paragraph, a panel of engineered yeast strains). Regarding claim 7, Dowell further discloses where the yeast cells are Saccharomyces cerevisiae (Pg. 5, sixth paragraph, a panel of engineered yeast strains; Pg. 1, first paragraph, Saccharomyces cerevisiae). Dowell discloses a plurality of cells (Pg. 5, sixth paragraph, a panel of engineered yeast strains), wherein each cell comprises (i) one or more of a target domain gene that specifically binds to a binding partner (Pg. 5, sixth paragraph, Previously untested GPCRs are usually transformed into a panel of engineered yeast strains expressing different Gpa1p/Ga chimeras), (ii) one or more of an intracellular chimeric G-protein alpha subunit comprising an endogenous G-protein alpha subunit with a humanized C-terminus (Pg. 5, - sixth paragraph, The GlaxoSmithKline system consists of 11 isogenic host yeast strains, with the prefix MMY, containing different integrated Gpa1p/Ga chimeras (Table 1) in which the C-terminal five amino acids of Gpa1p are replaced with the equivalent sequence from mammalian Ga proteins; Pg. 6, Table 1.Genotypes of yeast strains used for GPCR research at GlaxoSmithKline; wherein Table 1 of Dowell includes strain: MMY14; Genotype: MMY11 TRP1::Gpa1/Gaq(5); see Brown, Pg. 18, left-hand column, third paragraph, We replaced the five C-terminal residues of Gpa1p (468 KIGII-COOH) with the five C-terminal residues of mammalian Ga subunits Gaq (EYNLV-COOH); see Para. [0056] of the instant application, Table 1 includes DI dCyFIRP1 Q, BY4741 farlA sst2A ste2A figlA::mTq2 X-2: Pmnla-UnTS-TcYclb Gpa1 (468-472)(KIGH>EYNLV)); and (iii) one or more of an inducible reporter, wherein the expression of the reporter is dependent on the activation of the target domain encoded by the target domain gene (Pg. 5, seventh paragraph, The MMY strains have two chromosomally integrated reporter genes, FUS1-HIS3 and FUS1-lacZ. The preferred assay is to measure His3p-mediated cell growth, induced by agonism of the GPCR, which can be quantified using a fluorometric indicator of cell number. Alternatively, assays may be configured measuring the 8-galactosidase reporter for which various fluorogenic and chromogenic substrates are available). Regarding claim 6, Dowell further discloses wherein the cells are yeast cells (Pg. 5, sixth paragraph, a panel of engineered yeast strains). Regarding claim 7, Dowell further discloses where the yeast cells are Saccharomyces cerevisiae (Pg. 5, sixth paragraph, a panel of engineered yeast strains; Pg. 1, first paragraph, Saccharomyces cerevisiae).
With respect to the addition of a barcode to the cells of Dowell, e.g., Applicant argues that Dowell does not require barcoding, because each strain is evaluated independently, is not found persuasive because the motivation of doing so would be to develop plurality of cells using a target domain gene having a barcode or an inducible reporter having a barcode to uniquely identify or distinguish the target domain. Adding a barcode to a biological cell is for each cell is assigned a unique nucleic acid sequence (a “barcode”) enables researchers to track it through space and time. Dowell does teach a ‘plurality of cells’. Adding a barcode to a biological cell is a powerful method for giving each cell a unique, heritable identifier, enabling researchers to trace its origin, fate, and interactions with other cells over time. In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). It would have been obvious to one of ordinary skill in the art at the time of the invention to modify the plurality of cells of Dowell and Kosuri to include wherein the transcriptional reporter is mTurquoise2 for the purpose of developing various cells including various reporters (see Dowell, Pg. 3, second paragraph, several reporter genes have been described, typically utilizing the FUS1 promoter which is induced by activation of the pheromone-response signal transduction pathway. These include HIS3 (His3p), lacZ (B-galactosidase), and EGFP (enhanced green fluorescent protein). One or ordinary skill in the art would have been motivated to use is mTurquoise2 because it has one of the highest quantum yields of any fluorescent protein, i.e., exceptionally bright, mono-exponential fluorescence lifetime, improved maturation rate, and high photostability in vivo, mTurquoise2. The prior art teaches it is the preferable CFP variant for live-cell imaging.
Specification
The disclosure is objected to because of the following informalities:
It is noted that Table 2 does not have the proper SEQ ID Nos. for the sequences provided therein. See pages 21-25 of the instant specification.
It does appear that these sequences were properly submitted in the sequence listing. The nucleotide/amino acid sequences throughout the specification which are encompassed by the definitions for nucleotide/amino acid sequences as set forth in 37 C.F.R. 1.821(a)(1) and (a)(2) must conform with the sequence rules for all applications that include nucleotide/amino acid sequences. The sequence identifiers obtained through conformance (paper submission and CRF/electronic) must be inserted into the body of the specification directly following the sequence.
Appropriate correction is required.
Prior art not presently relied upon:
Kebschull et al (Nature Methods volume 15, pages 871–879 (2018) Cellular barcoding: lineage tracing, screening and beyond).
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.
Correspondence regarding this application should be directed to Group Art Unit 1645. Papers related to this application may be submitted to Group 1600 by facsimile transmission. Papers should be faxed to Group 1600 via the PTO Fax Center located in Remsen. The faxing of such papers must conform with the notice published in the Official Gazette, 1096 OG 30 (November 15,1989). The Group 1645 Fax number is 571-273-8300 which is able to receive transmissions 24 hours/day, 7 days/week.
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Any inquiry concerning this communication or earlier communications from the examiner should be directed to Jennifer E. Graser whose telephone number is (571) 272-0858. The examiner can normally be reached on Monday-Friday from 8:00 AM-4 PM.
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/JENNIFER E GRASER/ Primary Examiner, Art Unit 1645 5/6/26
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