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 .
Applicant’s response of 09/03/2025, including replacement drawings, has been received and entered into the application file.
Claims 1, 5, 8, 11, and 13 were amended in the claim set filed 09/03/2025.
Claims 14 and 15 were cancelled in the claim set filed 09/03/2025.
Claims 16-22 were added in the claim set filed 09/03/2025.
Accordingly, claims 1-13 and 16-22 are pending and under consideration.
Status of Prior Objections/Rejections
RE: Drawings
The drawings were previously objected to due to the titles of figures 2-4 containing a typographical error. The replacement sheets received on 09/03/2025 have obviated the basis of the prior objections. The objections of record are hereby withdrawn.
RE: Claim Objections
Claim 13 was previously objected to for minor informalities. The amendments to claim 13 have obviated the basis of the prior objection. The objection of record is hereby withdrawn.
RE: Claim Rejections - 35 USC § 112
Claims 5 and 8 were previously rejected under 35 U.S.C. 112(b) as being indefinite due to lack of antecedent basis. The amendments to claims 5 and 8 have obviated the basis of the prior rejections. The rejections of record are hereby withdrawn.
RE: Claim Rejections - 35 USC § 103
►Claims 1, 4, 5, 6, and 10 were previously rejected under 35 U.S.C. 103 as being unpatentable over Cooper et al., 2013 (as cited in the IDS filed 03/22/20221) in view of Iyer and Doktycz, 2013 (as cited in the IDS filed 03/22/2022) and Diagnostic Automation/Cortez Diagnostics, Inc., 2012, as evidenced by Tabor, 1990.
Applicant has traversed the rejection of record, asserting that amended claim 1 is directed to a cell-free test kit comprising a steroid hormone receptor, a double-stranded nucleic acid molecule (comprising a single polypeptide polymerase promoter sequence, a steroid hormone receptor response element, and a reporter construct), and a single polypeptide polymerase. Applicant asserts that Cooper et al., 2013 is a review article that does not motivate the instantly claimed cell-free test kit in which the claimed nucleic acid molecule therein comprises a single polypeptide polymerase promoter sequence.
In response, the argument regarding the limitations of amended instant claim 1 is found persuasive and the rejection of record is hereby withdrawn. However, upon further consideration, new grounds of rejection necessitated by amendment are set forth in greater detail below.
►Claim 2 was previously rejected under 35 U.S.C. 103 as being unpatentable over Cooper et al., 2013 (as cited in the IDS filed 03/22/2022), in view of Iyer and Doktycz, 2013 as cited in the IDS filed 03/22/2022), and Diagnostic Automation/Cortez Diagnostics, Inc., 2012 (of record), as evidenced by Tabor, 1990 as applied to claim 1, and further in view of Smith and Toft, 2008, as evidenced by Echeverria and Picard, 2009.
Applicant has traversed the rejection of record, asserting that the cited references do not sufficiently supplement the disclosures of Cooper et al., 2013 and/or Iyer and Doktycz, 2013 (or the other secondary references).
In response, the argument regarding the limitations of amended instant claim 1, from which instant claim 2 depends, is found persuasive and the rejection of record is hereby withdrawn. However, upon further consideration, new grounds of rejection necessitated by amendment are set forth in greater detail below.
►Claim 3 was previously rejected under 35 U.S.C. 103 as being unpatentable over Cooper et al., 2013 (as cited in the IDS filed 03/22/2022), Iyer and Doktycz, 2013 (as cited in the IDS filed 03/22/2022), Diagnostic Automation/Cortez Diagnostics, Inc., 2012, and Tabor, 1990 in view of Smith and Toft, 2008 (of record), as evidenced by Echeverria and Picard, 2009 as applied to claim 2, and further in view of Kang et al., 1999.
Applicant has traversed the rejection of record, asserting that the cited references do not sufficiently supplement the disclosures of Cooper et al., 2013 and/or Iyer and Doktycz, 2013 (or the other secondary references).
In response, the argument regarding the limitations of amended instant claim 1, from which instant claim 3 indirectly depends, is found persuasive and the rejection of record is hereby withdrawn. However, upon further consideration, new grounds of rejection necessitated by amendment are set forth in greater detail below.
►Claims 7 and 8 were previously rejected under 35 U.S.C. 103 as being unpatentable over Cooper et al., 2013 (as citedin the IDS filed 03/22/2022), in view of Iyer and Doktycz, 2013 (as cited in the IDS filed 03/22/2022), and Diagnostic Automation/Cortez Diagnostics, Inc., 2012, as evidenced by Tabor, 1990 as applied to claim 1, and further in view of Autour et al., 2018.
Applicant has traversed the rejection of record, asserting that the cited references do not sufficiently supplement the disclosures of Cooper et al., 2013 and/or Iyer and Doktycz, 2013 (or the other secondary references).
In response, the argument regarding the limitations of amended instant claim 1, from which instant claims 7 and 8 depend, is found persuasive and the rejection of record is hereby withdrawn. However, upon further consideration, new grounds of rejection necessitated by amendment are set forth in greater detail below.
►Claim 9 was previously rejected under 35 U.S.C. 103 as being unpatentable over Cooper et al., 2013 (as cited in the IDS filed 03/22/2022), in view of Iyer and Doktycz, 2013 (as cited in the IDS filed 03/22/2022), and Diagnostic Automation/Cortez Diagnostics, Inc., 2012, as evidenced by Tabor, 1990 as applied to claim 1, and further in view of Wu et al., 2018.
Applicant has traversed the rejection of record, asserting that the cited references do not sufficiently supplement the disclosures of Cooper et al., 2013 and/or Iyer and Doktycz, 2013 (or the other secondary references).
In response, the argument regarding the limitations of amended instant claim 1, from which instant claim 9 depends, is found persuasive and the rejection of record is hereby withdrawn. However, upon further consideration, new grounds of rejection necessitated by amendment are set forth in greater detail below.
►Claim 11 was previously rejected under 35 U.S.C. 103 as being unpatentable over Cooper et al., 2013 (as cited in the IDS filed 03/22/2022), in view of Iyer and Doktycz, 2013 (as cited in the IDS filed 03/22/2022), and Diagnostic Automation/Cortez Diagnostics, Inc., 2012, as evidenced by Tabor, 1990 as applied to claim 1, and further in view of Helsen et al., 2011.
Applicant has traversed the rejection of record, asserting that the cited references do not sufficiently supplement the disclosures of Cooper et al., 2013 and/or Iyer and Doktycz, 2013 (or the other secondary references).
In response, the argument regarding the limitations of amended instant claim 1, from which instant claim 11 depends, is found persuasive and the rejection of record is hereby withdrawn. However, upon further consideration, new grounds of rejection necessitated by amendment are set forth in greater detail below.
►Claim 12 was previously rejected under 35 U.S.C. 103 as being unpatentable over Cooper et al., 2013 (as cited in the IDS filed 03/22/2022), in view of Iyer and Doktycz, 2013 (as cited in the IDS filed 03/22/2022), and Diagnostic Automation/Cortez Diagnostics, Inc., 2012, as evidenced by Tabor, 1990 as applied to claim 1, and further in view of Helsen et al., 2011 and Autour et al., 2018.
Applicant has traversed the rejection of record, asserting that the cited references do not sufficiently supplement the disclosures of Cooper et al., 2013 and/or Iyer and Doktycz, 2013 (or the other secondary references).
In response, the argument regarding the limitations of amended instant claim 1, from which instant claim 12 depends, is found persuasive and the rejection of record is hereby withdrawn. However, upon further consideration, new grounds of rejection necessitated by amendment are set forth in greater detail below.
►Claim 13 was previously rejected under 35 U.S.C. 103 as being unpatentable over Cooper et al., 2013 (as cited in the IDS filed 03/22/2022) in view of Iyer and Doktycz, 2013 (as cited in the IDS filed 03/22/2022) and Wu et al., 2018.
Applicant has traversed the rejection of record, asserting that the cited references do not sufficiently supplement the disclosures of Cooper et al., 2013 and/or Iyer and Doktycz, 2013 (or the other secondary references).
In response, the argument regarding the limitations of amended instant claim 13 is found persuasive and the rejection of record is hereby withdrawn. However, upon further consideration, new grounds of rejection necessitated by amendment are set forth in greater detail below.
►Claim 14 was previously rejected under 35 U.S.C. 103 as being unpatentable over Cooper et al., 2013 (as cited in the IDS filed 03/22/2022), in view of Iyer and Doktycz, 2013 (as cited in the IDS filed 03/22/2022), and Diagnostic Automation/Cortez Diagnostics, Inc., 2012, as evidenced by Tabor, 1990 as applied to claim 1, and further in view of Anawalt, 2017 (of record).
The cancellation of claim 14 renders the rejection thereof moot.
►Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Cooper et al., 2013 (as cited in the IDS filed 03/22/2022), in view of Iyer and Doktycz, 2013 (as cited in the IDS filed 03/22/2022), and Diagnostic Automation/Cortez Diagnostics, Inc., 2012 (of record), as evidenced by Tabor, 1990 as applied to claim 1, and further in view of Anawalt, 2017 as applied to claim 14, and further in view of the Equine Anti-Doping and Controlled Medication Regulations of the International Federation for Equestrian Sports, 2018.
The cancellation of claim 15 renders the rejection thereof moot.
New/Maintained Grounds of Rejection
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.
Claims 1, 4, 5, 6, and 10 are rejected under 35 U.S.C. 103 as being unpatentable over Cooper et al., 2013 (as cited in the IDS filed 03/22/2022; of record) in view of Iyer and Doktycz, 2013 (as cited in the IDS filed 03/22/2022; of record), WO 01/73434 A2 (hereinafter Klein), and Diagnostic Automation/Cortez Diagnostics, Inc., 2012 (of record), as evidenced by Tabor, 1990 (of record).
With regard to amended claim 1, which recites “a cell-free test kit for screening a test sample for the presence of a ligand capable of eliciting a steroid hormone genomic response…comprising: (i) a steroid hormone receptor that is capable of forming a ligand-receptor complex with a ligand from the test sample; and (ii) a nucleic acid molecule comprising: (a) a single polypeptide polymerase promoter sequence; (b) a steroid hormone receptor response element that is capable of being bound by the ligand-receptor complex; and (c) a reporter construct where the response element (b) is located between the promoter sequence (a) and the reporter construct (c), and (a), (b) and (c) are operably linked; and (iii) a single polypeptide polymerase,” Cooper et al., 2013 reviews in vitro androgen bioassays to detect androgens. Specifically, Cooper et al., 2013 discloses in vitro androgen receptor-reporter gene bioassays in which a cell is transformed with a reporter vector comprising a minimal promoter driven by androgen response elements that regulate the expression of a reporter enzyme or protein (such as luciferase, a fluorescent protein, beta-galactosidase, and others) (page 2155, paragraph 4; figure 2). When androgens bind to their receptor, the ligand-receptor complex binds to the androgen response element, and the reporter enzyme or protein is detectably expressed (page 2155, paragraph 4-page 2156, paragraph 1; figure 2).
Cooper et al., 2013 thus discloses an in vitro assay for detecting a ligand capable of eliciting a steroid hormone genomic response (i.e. an androgen capable of eliciting a corresponding genomic response) comprising a steroid hormone receptor capable of forming a ligand-receptor complex (i.e. an androgen receptor capable of complexing with androgens) and a reporter vector comprising a promoter and a reporter enzyme or protein (i.e. a nucleic acid molecule comprising a polymerase promoter sequence and a reporter construct), the expression of which is driven by the ligand-receptor complex binding to its response element.
However, Cooper et al., 2013 does not disclose the cell-free nature of the claimed test kit, nor does it disclose the response element capable of being bound by the ligand-receptor complex being flanked by the single polypeptide polymerase promoter sequence and the reporter construct, as in amended instant claim 1.
These deficiencies are cured by Klein and Iyer and Doktycz, 2013. Klein discloses methods for detecting and measuring the interaction of co-activators, co-repressors, and other accessory molecules able to directly or indirectly associate with members of the steroid hormone receptor superfamily in a ligand-dependent manner (page 10, lines 20-30; page 18, line 27-page 19, line 8). Similar to the disclosure of Cooper et al., 2013, the methods of Klein involve detection of ligands that bind to a steroid hormone response element by expression of a reporter construct (i.e. firefly luciferase or β-galactosidase) driven by said binding (page 1, line 16-20; page 11, lines 1-4; page 18, line 27-page 19, line 8; page 19, lines 13-24; page 19, line 29-page 20, line 32). As in the in vitro assays disclosed in Cooper et al., 2013, Klein utilizes cellular machinery to facilitate expression of the desired nuclear receptors by cell culture (page 27, lines 15-16). However, unlike Cooper et al., 2013, Klein specifically discloses that following said culturing to express the desired nuclear receptors, the cells may be lysed and a cell-free extract may be used for subsequent experimentation and analysis (page 27, lines 16-24). Given that Klein discloses that a cell-free extract derived from the culturing of cells (such as those disclosed in the assay of Cooper et al., 2013) functions in detecting binding of ligands to a steroid hormone response element, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to practice the assay of Cooper et al., 2013 with the cell-free protocol of Klein to predictably detect ligands capable of eliciting a steroid hormone genomic response. One would have been motivated to make such a modification in order to receive the expected benefit of being able to indefinitely freeze the cell-free extract for later use (KIein: page 27, lines 21-24) rather than requiring constant cell culturing, as in Cooper et al., 2013.
Additionally, as previously set forth, Iyer and Doktycz, 2013 disclose a cell-free method of using DNA aptamers to regulate transcription via ligand-binding in cell-free systems (abstract). Aptamers are defined as single-stranded DNA or RNA molecules engineered to strongly and specifically bind target molecules (i.e. ligands) (page 341, column 1, paragraph 2). While the instantly claimed “response element that is capable of being bound by the ligand-receptor complex” differs structurally from these aptamers, they function in exactly the same manner, meaning both the response element and the aptamers strongly and specifically bind their corresponding ligands. Iyer and Doktycz, 2013 go on to disclose a nucleic acid construct in which a thrombin-binding ssDNA aptamer is placed downstream of a T7 promoter such that when thrombin binds the ssDNA aptamer, transcription of GFP from the T7 promoter via T7 RNA polymerase is repressed (figure 1; page 341, column 2, paragraph 1). The T7 polymerase of Iyer and Doktycz, 2013 thus has established utility in cell-free assays and reads on the instantly claimed single polypeptide polymerase, as previously set forth. Applicant has asserted that the DNA-DNA binding interactions of Iyer and Doktycz, 2013 are significantly distinguishable from a DNA-protein binding interaction. While the Examiner acknowledges that these interactions are different in that the DNA aptamers of Iyer and Doktycz, 2013 are built from nucleic acid residues rather than amino acid residues, this difference is not meaningful regarding the instant invention in that aptamers are special nucleic acid sequences that form elaborate three-dimensional structures and shapes and bind to a wide variety of target molecules with high specificity and affinity, similar to antibodies (reviewed in Rimmele, 2003; see especially: abstract; page 963, column 1, paragraph 1). Thus, for all intents and purposes, the function of the DNA aptamers disclosed in Iyer and Doktycz, 2013 in binding to a specific DNA sequence is identical to proteins binding to a specific DNA sequence, as in both Cooper et al., 2013 and Klein. Additionally, while Applicant has noted that Iyer and Doktycz, 2013 is drawn to thrombin rather than a steroid hormone receptor ligand, as instantly claimed. As previously set forth, the disclosure of Iyer and Doktycz, 2013 has been applied to the instant invention due to its disclosure of reduction of transcription of a reporter construct upon binding of a ligand to its associated sequence.
This arrangement of promoter-binding site-reporter construct, as well as detection of ligand binding by repressing reporter expression broadly read on the instantly claimed nucleic acid molecule comprising a polymerase promoter sequence, a response element, and a reporter construct, which facilitates detection of a ligand by measuring a reduction in transcription of the reporter construct upon ligand-receptor complex binding to the response element, particularly when combined with the disclosure of in vitro androgen receptor-reporter gene bioassays in Cooper et al., 2013 and the cell-free assays disclosed in Klein (set forth above).
Given that Cooper et al., 2013 and Klein both disclose manipulation of reporter expression upon binding of a steroid hormone ligand to its associated response element, as previously set forth, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to apply the principles of Iyer and Doktycz, 2013 regarding the T7 polymerase and reduction of reporter expression taught therein to the assays of Cooper et al., 2013 and Klein to predictably screen for ligands capable of eliciting a steroid hormone genomic response via reduction in reporter expression via T7 RNA polymerase following binding of a ligand-receptor complex (i.e. androgen-androgen receptor) to its associated response element (i.e. androgen response element) between a T7 polymerase promoter and a reporter construct. As set forth in greater detail below regarding instant claims 5 and 6, Tabor, 1990 teaches that not only is T7 RNA polymerase a very active enzyme that synthesizes RNA at a high rate and terminates transcription infrequently, but it is also highly selective for its own promoter sequence (page 16.2.1, paragraph 1), meaning it produces robust and specific transcriptional output, which it clearly advantageous for utility of a transcriptional reporter system. Accordingly, one would have been motivated to make such a modification in order to receive the expected benefit of producing a system capable of robust transcription (as evidenced by Tabor, 1990), in which reduction of reporter expression caused by its interruption due to ligand binding would be clearly noticeable.
With regard to claim 4, which recites “the relative amount of steroid hormone receptor to nucleic acid molecule is y:1, where y is the amount of steroid hormone receptor and is defined as [7.0 < y < 10.0],” Iyer and Doktycz, 2013 disclose altering the concentration of the ssDNA template (which comprises the nucleic acid molecule comprising the polymerase promoter sequence, response element, and reporter construct as set forth above for instant claim 1) relative to thrombin (which functions as the ligand-receptor complex as set forth above for instant claim 1 and therefore effectively comprises the instantly claimed steroid hormone receptor) to maximize fold change in expression of GFP (supplemental data, S5, page 10). Per MPEP 2144.05.II.A, “it is not inventive to discover the optimum or workable ranges by routine experimentation” (In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955)). Thus, given the disclosure of Iyer and Doktycz, 2013 regarding the routine experimentation to discover the optimum or workable ranges of the ratio of thrombin to nucleic acid molecule, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to similarly establish optimum or workable ranges by routine experimentation.
With regard to claims 5 and 6, which recite “the polymerase is T7 RNA polymerase,” and “the polymerase promoter sequence is defined by SEQ ID NO:1,” the system of Iyer and Doktycz, 2013 utilizes a T7 promoter to drive transcription of the reporter via T7 RNA polymerase (figure 1). As shown in the alignment below, SEQ ID NO:1 (top line) is identical to the T7 promoter of pKSGFP of Iyer and Doktycz, 2013 (bottom line) (supplemental data, S6, page 11).
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Furthermore, Tabor, 1990 teaches that not only is T7 RNA polymerase a very active enzyme that synthesizes RNA at a high rate and terminates transcription infrequently, but it is also highly selective for its own promoter sequence (page 16.2.1, paragraph 1), meaning it produces robust and specific transcriptional output, which it clearly advantageous for utility of a transcriptional reporter system.
With regard to claim 10, which recites “the steroid hormone receptor is selected from the group consisting of androgen receptor (AR),” among others, Cooper et al., 2013 discloses in vitro androgen receptor-reporter gene bioassays that utilize the instantly claimed androgen receptor (page 2155, paragraph 4-page 2156, paragraph 1; figure 2), as set forth in detail above.
While neither Cooper et al., 2013 nor Iyer and Doktycz, 2013 disclose the packaging of the above components into a test kit, such test kits to detect steroid ligands were publicly available prior to the effective filing date of the claimed invention. Using the Wayback Machine, it is evident that Diagnostic Automation/Cortez Diagnostics, Inc. sold steroid test kits as early as if not earlier than September 22, 2012. This reference has been applied solely to establish that standardized test kits for detecting steroid ligands were both known in the art and publicly available prior to the effective filing date of the claimed invention.
Given the success of in vitro androgen receptor-reporter gene bioassays in detecting androgens bound to their receptor and subsequently their response element, as disclosed in Cooper et al., 2013, the utility of cell-free extracts to detect such steroid ligand interactions disclosed in Klein, and the success of detecting ligand binding between a T7 polymerase promoter and a reporter via decreased reporter expression, as disclosed in Iyer and Doktycz, 2013, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize the cell-free extracts disclosed in Klein (as set forth above) in the in vitro assays of Cooper et al., 2013 and further to apply the system of Iyer and Doktycz, 2013 to the in vitro androgen receptor-reporter gene bioassays of Cooper et al., 2013 to predictably screen for ligands capable of eliciting a steroid hormone genomic response via reduction in reporter expression via T7 RNA polymerase following binding of a ligand-receptor complex (i.e. androgen-androgen receptor) to its associated response element (i.e. androgen response element) between a T7 polymerase promoter and a reporter construct. One would have been motivated to make such a modification in order to receive the expected benefit of sensitively detecting androgen presence (as taught in Cooper et al., 2013) in a cell-free system capable of indefinite frozen storage (as taught in Klein) via reduction of T7 RNA polymerase-based reporter expression (as taught in Iyer and Doktycz, 2013). T7 RNA polymerase and its associated promoter would predictably produce a system capable of robust transcription, as evidenced by Tabor, 1990, in which reduction caused by its interruption would predictably be clearly noticeable. Additionally, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to package these assay components into a kit as demonstrated by Diagnostic Automation/Cortez Diagnostics, Inc., 2012 to predictably produce an effective test kit for detecting ligands capable of eliciting a steroid hormone genomic response. One would have been motivated to make such a modification in order to receive the expected benefit of standardized, commercialized ligand detection kits.
Claim 2 is rejected under 35 U.S.C. 103 as being unpatentable over Cooper et al., 2013 (as cited in the IDS filed 03/22/2022; of record), in view of Iyer and Doktycz, 2013 (as cited in the IDS filed 03/22/2022; of record), WO 01/73434 A2 (hereinafter Klein), and Diagnostic Automation/Cortez Diagnostics, Inc., 2012 (of record), as evidenced by Tabor, 1990 (of record) as applied to claim 1 above, and further in view of Smith and Toft, 2008 (of record), as evidenced by Echeverria and Picard, 2009 (of record).
The combined disclosures of Cooper et al., 2013, Iyer and Doktycz, 2013, Klein, Diagnostic Automation/Cortez Diagnostics, Inc., 2012, and Tabor, 1990 are described above and applied as before. However, these disclosures do not teach the hormone receptor cofactors of instant claim 2.
With regard to claim 2, which recites “the test kit [of claim 1]…further compris[es] a steroid hormone receptor cofactor selected from heat shock protein 90 (HSP90),” as well as various complexes of HSP90, HSP70, HSP40, p23, Hop, Hip, p60, and/or FKBP52, Smith and Toft, 2008 teach that heat shock proteins in steroid receptor complexes (i.e. Hsp70, Hsp40, and Hsp90), along with cochaperones (i.e. Hsp90-associated cochaperones) are required both to establish steroid receptor binding ability, as well as to maintain its binding ability (Page 2229- Paragraph 1; Section "Hormone Binding Ability and Chaperones"-Paragraph 1). Additionally, Echeverria and Picard, 2009 depict Hsp90 machinery associated with nuclear localization, which includes the species recited in instant claim 5 (i.e. Hsp90, Hsp70, p23, HOP, and Hsp40) in addition to other cofactors (Figure 1).
Given that the test kit recited in instant claim 1 relies upon binding of a receptor-ligand complex to a hormone response element, and that hormone receptor cofactors such as HSP90 are crucial for facilitating and maintaining steroid hormone-receptor binding, as taught by Smith and Toft, 2008 and as evidenced by Echeverria and Picard, 2009, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to apply the teachings of Smith and Toft, 2008 to the test kit of instant claim 1 to predictably promote and enhance receptor-ligand formation and persistence to more accurately detect the ligand of interest. One would have been motivated to make such an addition to the test kit in order to receive the expected benefit of increased detection accuracy.
Claim 3 is rejected under 35 U.S.C. 103 as being unpatentable over Cooper et al., 2013 (as cited in the IDS filed 03/22/2022; of record), Iyer and Doktycz, 2013 (as cited in the IDS filed 03/22/2022; of record), WO 01/73434 A2 (hereinafter Klein), Automation/Cortez Diagnostics, Inc., 2012 (of record), and Tabor, 1990 (of record) in view of Smith and Toft, 2008 (of record), as evidenced by Echeverria and Picard, 2009 (of record) as applied to claim 2 above, and further in view of Kang et al., 1999 (of record).
The combined disclosures of Cooper et al., 2013, Iyer and Doktycz, 2013, Klein, Diagnostic Automation/Cortez Diagnostics, Inc., 2012, Tabor, 1990, Smith and Toft, 2008, and Echeverria and Picard, 2009 are described above and applied as before. However, these disclosures do not teach the defined ratio of HSP90 to steroid hormone receptor of instant claim 3.
With regard to claim 3, which recites “the relative amount of HSP90 to steroid hormone receptor is x:1, where x is the amount of HSP90 and is defined as 1.0 < x < 5.0,” Kang et al., 1999 teaches that the ratio of Hsp90 to glucocorticosteroid receptor impacts both receptor- response element binding as well as ligand responsiveness (abstract). As set forth above regarding instant claim 2, cofactors such as HSP90 are required first to facilitate and maintain hormone-receptor binding, as taught by Smith and Toft, 2008, and also to govern receptor- response element binding and ligand responsiveness, as taught by Kang et al., 1999.
While Kang et al., 1999 does not teach the range of ratios disclosed in instant claim 3, they do teach that these ratios impact receptor-ligand interactions, which in turn impact the accuracy of ligand detection using the instantly claimed test kit. Per MPEP 2144.05.II.A, “it is not inventive to discover the optimum or workable ranges by routine experimentation” (In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955)). Thus, given the teachings of Kang et al., 1999 regarding the importance of HSP90 to glucocorticosteroid receptor ratio, it would have been obvious to someone of ordinary skill in the art before the filing date of the claimed invention to establish optimum or workable ranges by routine experimentation. Such routine experimentation regarding ratios of other, comparable essential components is disclosed in Iyer and Doktycz, 2013, as set forth above for instant claim 4 with regard to manipulation of the receptor:reporter ratio.
Given that the test kit recited in instant claim 1 relies upon binding of a receptor-ligand complex to a hormone response element, which is facilitated by hormone receptor cofactors such as HSP90 (set forth above regarding instant claim 2), and that the ratio of these hormone receptor cofactors to receptors such as the glucocorticosteroid receptor impacts both receptor-response element binding and ligand responsiveness, as taught by Kang et al., 1999, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to apply the teachings of Kang et al., 1999 to routine experimentation to establish an optimum or workable range, as evidenced by Iyer and Doktycz, 2013 to predictably promote receptor-response element binding as well as ligand responsiveness to more accurately detect the ligand of interest. One would have been motivated to make such an addition to the test kit in order to receive the expected benefit of increased detection accuracy.
Claims 7 and 8 are rejected under 35 U.S.C. 103 as being unpatentable over Cooper et al., 2013 (as cited in the IDS filed 03/22/2022; of record), in view of Iyer and Doktycz, 2013 (as cited in the IDS filed 03/22/2022; of record), WO 01/73434 A2 (hereinafter Klein), and Diagnostic Automation/Cortez Diagnostics, Inc., 2012 (of record), as evidenced by Tabor, 1990 (of record) as applied to claim 1 above, and further in view of Autour et al., 2018 (of record).
The combined disclosures of Cooper et al., 2013, Iyer and Doktycz, 2013, Klein, Diagnostic Automation/Cortez Diagnostics, Inc., 2012, and Tabor, 1990 are described above and applied as before. However, these disclosures do not teach the construct encoding an RNA aptamer capable of binding to a fluorophore of instant claim 7 or the scaffold of instant claim 8.
With regard to claim 7, which recites “the reporter construct [of the test kit of instant claim 1] comprises a sequence encoding an RNA aptamer capable of binding to a fluorophore,” Autour et al., 2018 teaches that fluorogenic RNA aptamers (i.e. those that enhance the fluorescence of an unbound fluorophore, which read on the instantly claimed RNA aptamer capable of binding to a fluorophore) enable RNA visualization (page 2, paragraph 1), meaning they are useful for tracking RNA produced by transcription (and in fact Autour et al., 2018 tests these aptamers with in vitro transcription assays (figure 2)), as is required for ligand detection in the instantly claimed system.
With regard to claim 8, which recites “the RNA aptamer is Mango II, and optionally comprises an F30 scaffold,” Autour et al., 2018 teaches the production of Mango aptamer variants I, II, III, and IV (figure 1). These Mango aptamer variants are taught to have a number of advantages over other fluorogenic RNA aptamers, such as their small size, their ability to fold correctly into monomers at physiological temperatures, and their ability to withstand formaldehyde fixation (page 9, paragraph 2). Additionally, Autour et al., 2018 discloses that Mango variants such as Mango II are compatible with F30 folding scaffolds, which are known to improve fluorescence and RNA stability (page 5, column 2, paragraph 2; page 8, column 1, paragraph 2).
Given that the test kit for ligand detection of instant claim 1 relies upon detection of transcriptional output, or lack thereof, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to encode the RNA aptamers (specifically the Mango variants) and folding scaffold F30 taught by Autour et al., 2018 within the reporter construct of instant claim 1 to predictably generate reliable, detectable, and robust reporter output to more accurately detect the ligand of interest. One would have been motivated to make such an addition to the test kit in order to receive the expected benefits of increased detection accuracy and sensitivity.
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Cooper et al., 2013 (as cited in the IDS filed 03/22/2022; of record), in view of Iyer and Doktycz, 2013 (as cited in the IDS filed 03/22/2022; of record), WO 01/73434 A2 (hereinafter Klein), and Diagnostic Automation/Cortez Diagnostics, Inc., 2012 (of record), as evidenced by Tabor, 1990 (of record) as applied to claim 1 above, and further in view of Wu et al., 2018 (of record).
The combined disclosures of Cooper et al., 2013, Iyer and Doktycz, 2013, Klein, Diagnostic Automation/Cortez Diagnostics, Inc., 2012, and Tabor, 1990 are described above and applied as before. However, these disclosures do not teach inclusion of nucleoside triphosphates, as in instant claim 9.
With regard to claim 9, which recites “the test kit according to claim 1, further comprising nucleoside triphosphates,” Wu et al., 2018 teaches that T7 RNA polymerase not only utilizes nucleoside triphosphates as the building blocks to synthesize RNA during transcription but also that it is capable of discriminating between correct and incorrect nucleoside triphosphates by free energy (abstract).
Given that the test kit for ligand detection of instant claim 1 relies upon detection of transcriptional output, or lack thereof, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to include nucleoside triphosphates to predictably facilitate successful and accurate transcription of the reporter construct, as taught by Wu et al., 2018. One would have been motivated to make such an addition to the test kit in order to receive the expected benefit of successfully transcribing the reporter construct of the test kit, thereby facilitating increased detection accuracy and sensitivity.
Claim 11 is rejected under 35 U.S.C. 103 as being unpatentable over Cooper et al., 2013 (as cited in the IDS filed 03/22/2022; of record), in view of Iyer and Doktycz, 2013 (as cited in the IDS filed 03/22/2022; of record), WO 01/73434 A2 (hereinafter Klein), and Diagnostic Automation/Cortez Diagnostics, Inc., 2012 (of record), as evidenced by Tabor, 1990 (of record) as applied to claim 1 above, and further in view of Helsen et al., 2011 (of record).
The combined disclosures of Cooper et al., 2013, Iyer and Doktycz, 2013, Klein, Diagnostic Automation/Cortez Diagnostics, Inc., 2012, and Tabor, 1990 are described above and applied as before. However, these disclosures do not teach the response element sequences of instant claim 11.
With regard to claim 11, which recites “the response element is selected from: a. an androgen response element (ARE) including, but not limited to, a sequence comprising 5’-AGAACAnnnTGTTCT-3’ (SEQ ID NO: 4), wherein n is A, T, G or C; b. an estrogen response element (ERE) sequence comprising 5’-AGGTCAnnnTGACCT-3’ (SEQ ID NO: 8), wherein n is A, T, G or C,” as well as progesterone response element (SEQ ID NO: 10), mineralocorticoid response element (SEQ ID NO: 12), and glucocorticoid response element sequences (SEQ ID NO: 12), Helsen et al., 2011 teaches that “steroid receptors bind…to their cognate response elements…that are organized as an inverted repeat of two 5’-AGAACA-3’ [for androgen receptors]…or 5’-AGGTCA-3’ [for estrogen receptors]…motifs” (section 2.2, page 414, column 1, paragraph 3). For clarity, the inverted repeat of 5’-AGAACA-3’, associated with androgen receptors, is 5’-TGTTCT-3’, and the inverted repeat of 5’-AGGTCA-3’, associated with estrogen receptors, is 5’-TGACCT-3’. These sequences each read on SEQ ID NOs 4 and 8, with the exception of the three variable nucleotides joining the motifs. However, Helsen et al., 2011 additionally teaches that “these two hexameric elements are separated by a spacer that is exactly three nucleotides long” (section 2.2, page 414, column 1, paragraph 3), meaning the sequences disclosed in Helsen et al., 2011 read on the instantly claimed sequences.
Given that the test kit for ligand detection of instant claim 1 requires successful ligand-receptor complex binding to its associated response element, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to utilize the known steroid hormone response element sequences taught in Helsen et al., 2011 to predictably produce a test kit capable of facilitating the requisite successful binding of a ligand-receptor complex to its associated response element. One would have been motivated to make such a modification to the test kit in order to receive the expected benefit of ligand-receptor complexes successfully binding to their associated response element(s).
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Cooper et al., 2013 (as cited in the IDS filed 03/22/2022; of record), in view of Iyer and Doktycz, 2013 (as cited in the IDS filed 03/22/2022; of record), WO 01/73434 A2 (hereinafter Klein), and Diagnostic Automation/Cortez Diagnostics, Inc., 2012 (of record), as evidenced by Tabor, 1990 (of record) as applied to claim 1 above, and further in view of Helsen et al., 2011 (of record) and Autour et al., 2018 (of record).
The combined disclosures of Cooper et al., 2013, Iyer and Doktycz, 2013, Klein, Diagnostic Automation/Cortez Diagnostics, Inc., 2012, and Tabor, 1990 are described above and applied as before. However, these disclosures do not teach the nucleic acid molecule sequence of instant claim 12.
With regard to claim 12, which recites “the test kit [of claim 1] is configured to detect a ligand that binds to an androgen receptor, and the nucleic acid molecule comprises a sequence defined by SEQ ID NO: 14, or wherein the test kit is configured to detect a ligand that binds to an estrogen receptor, and the nucleic acid molecule comprises a sequence defined by SEQ ID NO: 15,” SEQ ID NOs 14 and 15 are annotated in the instant application as “T7i-ARE-F30 Mango II” and “T7i-ERE-F30 Mango II,” respectively, meaning these nucleic acid sequences satisfy the limitations of the nucleic acid molecule of claim 1 (i.e. a promoter, a response element, and a reporter). However, all of the functional components of these nucleic acid sequences are disclosed in the prior art, as set forth above regarding the T7 promoter of instant claim 6, disclosed in Iyer and Doktycz, 2013, the androgen and estrogen response elements of instant claim 11, disclosed in Helsen et al., 2011, and the F30 scaffold/Mango II aptamer of instant claim 8, disclosed in Autour et al., 2018. Furthermore, per the disclosure of Iyer and Doktycz, 2013, the construction of non-natural sequences combining several independent sequences is well-known and standard in the art (page 344, column 1, paragraph 3).
Given that the disclosed prior art teaches sequences of the T7 promoter, androgen or estrogen response element, and the scaffolded aptamer of SEQ ID NOs 14 and 15, which in turn satisfy the limitations of the nucleic acid molecule of claim 1 (i.e. a promoter, a response element, and a reporter), it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine these sequence components into a single nucleic acid molecule using well-known and standard methods, as in the disclosure of Iyer and Doktycz, 2013 to predictably produce a single nucleic acid molecule encoding the machinery necessary for the instantly claimed test kit/assay. One would have been motivated to combine these sequences in order to receive the expected benefit of a simplified test kit/assay with the further benefit of the accuracy and sensitivity of all the prior-disclosed components (i.e. the T7 promoter, androgen or estrogen response elements, and the scaffolded aptamer Mango II).
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over Cooper et al., 2013 (as cited in the IDS filed 03/22/2022; of record) in view of Iyer and Doktycz, 2013 (as cited in the IDS filed 03/22/2022; of record), WO 01/73434 A2 (hereinafter Klein), and Wu et al., 2018 (of record), as evidenced by Tabor, 1990 (of record).
With regard to claim 13, which recites “a cell-free assay method for detecting a ligand [capable of eliciting a steroid hormone genomic response] in a sample…comprising the steps of: (i) contacting a sample with: (a) a steroid hormone receptor that forms a ligand-receptor complex with a ligand from the sample; and (b) a nucleic acid molecule comprising: (1) a single polypeptide polymerase promoter sequence; (2) a response element that is bound by the ligand-receptor complex; and (3) a reporter construct where the response element (2) is located between the promoter sequence (1) and the reporter construct (3)…; (c) a single polypeptide polymerase; and (d) nucleoside triphosphates; and (ii) measuring a reduction or inhibition in transcription of the reporter construct caused by binding of the ligand-receptor complex,” Cooper et al., 2013 (set forth above regarding instant claim 1) teaches in vitro androgen receptor-reporter gene bioassays in which a cell is transformed with a reporter vector comprising a minimal promoter driven by androgen response elements that regulate the expression of a reporter enzyme or protein (such as luciferase, a fluorescent protein, beta-galactosidase, and others) (page 2155, paragraph 4; figure 2). Specifically, when androgens bind to their receptor, the ligand-receptor complex binds to the androgen response element, and the reporter enzyme or protein is detectably expressed (page 2155, paragraph 4-page 2156, paragraph 1; figure 2).
Cooper et al., 2013 thus discloses an assay method for detecting a ligand capable of eliciting a steroid hormone genomic response (i.e. an androgen capable of eliciting a corresponding genomic response) comprising a steroid hormone receptor capable of forming a ligand-receptor complex (i.e. an androgen receptor capable of complexing with androgens) and a reporter vector comprising a promoter and a reporter enzyme or protein (i.e. a nucleic acid molecule comprising a polymerase promoter sequence and a reporter construct), the expression of which is driven by the ligand-receptor complex binding to its response element.
However, Cooper et al., 2013 does not disclose the cell-free nature of the claimed assay method, nor does it disclose the response element capable of being bound by the ligand-receptor complex being flanked by the single polypeptide polymerase promoter sequence and the reporter construct, as in amended instant claim 1.
These deficiencies are cured by Klein and Iyer and Doktycz, 2013. Klein discloses methods for detecting and measuring the interaction of co-activators, co-repressors, and other accessory molecules able to directly or indirectly associate with members of the steroid hormone receptor superfamily in a ligand-dependent manner (page 10, lines 20-30; page 18, line 27-page 19, line 8). Similar to the disclosure of Cooper et al., 2013, the methods of Klein involve detection of ligands that bind to a steroid hormone response element by expression of a reporter construct (i.e. firefly luciferase or β-galactosidase) driven by said binding (page 1, line 16-20; page 11, lines 1-4; page 18, line 27-page 19, line 8; page 19, lines 13-24; page 19, line 29-page 20, line 32). As in the in vitro assays disclosed in Cooper et al., 2013, Klein utilizes cellular machinery to facilitate expression of the desired nuclear receptors by cell culture (page 27, lines 15-16). However, unlike Cooper et al., 2013, Klein specifically discloses that following said culturing to express the desired nuclear receptors, the cells may be lysed and a cell-free extract may be used for subsequent experimentation and analysis (page 27, lines 16-24). Given that Klein discloses that a cell-free extract derived from the culturing of cells (such as those disclosed in the assay of Cooper et al., 2013) functions in detecting binding of ligands to a steroid hormone response element, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to practice the assay of Cooper et al., 2013 with the cell-free protocol of Klein to predictably detect ligands capable of eliciting a steroid hormone genomic response. One would have been motivated to make such a modification in order to receive the expected benefit of being able to indefinitely freeze the cell-free extract for later use (KIein: page 27, lines 21-24) rather than requiring constant cell culturing, as in Cooper et al., 2013.
Additionally, as previously set forth, Iyer and Doktycz, 2013 disclose a cell-free method of using DNA aptamers to regulate transcription via ligand-binding in cell-free systems (abstract). Aptamers are defined as single-stranded DNA or RNA molecules engineered to strongly and specifically bind target molecules (i.e. ligands) (page 341, column 1, paragraph 2). While the instantly claimed “response element that is capable of being bound by the ligand-receptor complex” differs structurally from these aptamers, they function in exactly the same manner, meaning both the response element and the aptamers strongly and specifically bind their corresponding ligands. Iyer and Doktycz, 2013 go on to disclose a nucleic acid construct in which a thrombin-binding ssDNA aptamer is placed downstream of a T7 promoter such that when thrombin binds the ssDNA aptamer, transcription of GFP from the T7 promoter via T7 RNA polymerase is repressed (figure 1; page 341, column 2, paragraph 1). The T7 polymerase of Iyer and Doktycz, 2013 thus has established utility in cell-free assays and reads on the instantly claimed single polypeptide polymerase, as previously set forth. Applicant has asserted that the DNA-DNA binding interactions of Iyer and Doktycz, 2013 are significantly distinguishable from a DNA-protein binding interaction. While the Examiner acknowledges that these interactions are different in that the DNA aptamers of Iyer and Doktycz, 2013 are built from nucleic acid residues rather than amino acid residues, this difference is not meaningful regarding the instant invention in that aptamers are special nucleic acid sequences that form elaborate three-dimensional structures and shapes and bind to a wide variety of target molecules with high specificity and affinity, similar to antibodies (reviewed in Rimmele, 2003; see especially: abstract; page 963, column 1, paragraph 1). Thus, for all intents and purposes, the function of the DNA aptamers disclosed in Iyer and Doktycz, 2013 in binding to a specific DNA sequence is identical to proteins binding to a specific DNA sequence, as in both Cooper et al., 2013 and Klein. Additionally, while Applicant has noted that Iyer and Doktycz, 2013 is drawn to thrombin rather than a steroid hormone receptor ligand, as instantly claimed. As previously set forth, the disclosure of Iyer and Doktycz, 2013 has been applied to the instant invention due to its disclosure of reduction of transcription of a reporter construct upon binding of a ligand to its associated sequence.
This arrangement of promoter-binding site-reporter construct, as well as detection of ligand binding by repres