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 .
Application Status
This action is written in response to applicant' s correspondence received October 30, 2025. Claims 1-9, 12 and 13 are currently pending. Claim 8, 9, and 13 are withdrawn from prosecution as being drawn to non-elected subject matter. Accordingly, claims 1-7 and 12 are examined herein. The restriction requirement mailed June 2, 2025 is still deemed proper. Applicant's elected Group I without traverse in the reply filed July 14, 2025.
Any rejection or objection not reiterated herein has been overcome by amendment. Applicant' s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
Claim Rejections - 35 USC § 112(b)
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 1-5 and 12 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 recites the limitation "the at least seven biomarkers" in (d), line 2. There is insufficient antecedent basis for this limitation in the claim. Claim 1 does not recite biomarkers elsewhere in the limitation, and it is unclear which biomarkers are being referenced. Biomarkers are a broad class of detectable molecules including proteins and metabolites . While a list of regulatory elements is provided in the claim which may be the biomarkers, the biomarkers may be other proteins or metabolites in the cell.
Regarding Claims 2-5 and 12, the dependent claims do not further define or reference biomarkers, and therefore fail to overcome the rejection of Claim 1.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 3-6 and 12 are rejected under 35 U.S.C. 103 as being unpatentable over Couillard-Despres S., et. al., BMC Neuroscience, Vol. 9, Issue 31, p. 1-9, Feb. 29, 2008; Kirchhof, N., et. al., Biology of Reproduction, Vol. 63, Issue 6, p. 1698-1705, Dec. 1, 2000; Tanwar, V., et. al., Stem Cells, Vol. 32, Issue 7, p. 1774-1788, Jun. 17, 2014; Suter, D., et. al., Stem Cells, Vol. 27, Issue 1, p. 49-58, Jan. 05, 2009; and USCS Genome Browser, Kent, W., et. al., Genome Research, Vol. 12, Issue 6, p. 996-1006, published in advance May 16, 2002.
Regarding Claim 1, Claim 1 rejected under 112(b) for improper antecedent. For compact prosecution, “detecting alterations in the expression pattern of the at least seven biomarkers” is interpreted as detecting alterations in the expression pattern of each gene which has a regulatory element of that gene operably linked to a reporter sequence.
Couillard-Despres teaches an in vitro method in which “[f]ive neuronal-like cell lines were tested for their capacity to mimic early events of neuronal determination and differentiation taking place during development” in which the cells include NTERA-2, a human embryonic carcinoma cell line, and “[t]o differentiate cell lines towards a neuronal phenotype, retinoic acid (10 μM) was added to the cultures” (p. 2, col. 2), which reads as a method for determining the effect of an agent, retinoic acid, on a mammalian embryonic development using multiple reporter cells and contacting the cells with the agent.
Couillard-Despres further teaches the (a) five reporter cells comprise a regulatory sequence, firefly luciferase, to a regulatory element of a gene expressed early embryonic development, DCX: “We … generated stable NTERA-2 cell lines bearing the EGFP or the firefly luciferase reporter gene under the control of the DCX promoter sequences” (p. 4, col. 2) and “Doublecortin (DCX) constitutes a marker for neuronal precursors throughout developmental … neurogenesis” (p. 2, col. 1).
Couillard-Despres further teaches (b) contacting the cells with retinoic acid, (c) comparing the expression of the reporter sequence across reporter cells NTERA-2 and HeLa with or without retinoic acid: “Whereas undifferentiated NTERA-2DCX-EGFP and HeLaDCX-EGFP [cells not contacted with retinoic acid] did not express the EGFP reporter, retinoic acid treatment strongly and specifically induced the expression of EGFP in the NTERA-2DCX-EGFP cells but not in the HeLaDCX-EGFP cells” (p. 5, col. 1).
Couillard-Despres further teaches (d) determining the effect of an agent on mammalian embryonic development by comparing DCX expression to determine whether differentiation occurred: “expression of DCX can be detected upon induction of neuronal differentiation” in NTERA-2 embryonic cells with retinoic acid (p. 5, col. 9). Retinoic acid is a teratogenic compound as defined by the instant specification (p. 27, line 22), and therefore Couilard-Despres teaches determining the teratogenic effect by detecting alterations in the expression pattern of a biomarker that indicate stem cell differentiation.
While Couillard-Despres teaches the use of the DCX promoter, a regulatory element of a marker in early development, they do not teach the in vitro method with regulatory elements from OCT4, BMO4, MYH6, PAX6, FOXA2, AFP, or SOX1. Couillard-Despres does not teach the use of mouse embryonic stem cells, and detecting alterations in the expression pattern of at least seven biomarkers that indicate perturbation of stem cell differentiation across endoderm, mesoderm and ectoderm lineages.
Kirchhof teaches a OCT4 reporter “produced by inserting the EGFP reporter gene between a modified 9-kb promoter fragment of the murine Oct-4 and its structural gene” (p. 1699, col. 2), which reads on a regulatory element of the OCT4 gene. The instant specification identifies SEQ ID NO: 21 as the promoter region of murine OCT4 (p. 22, Table 1), and therefore the “promoter fragment of murine Oct-4” reads on SEQ ID NO: 21.
Tanwar studied ESC differentiation in pluripotent, germline competent mouse CGR8, which is a mouse embryonic stem cell line (p. 1775, ESC Culture and Embryoid Body Formation). Markers investigated include BMP4 (p. 1778, col. 2), MYH6 (p. 1777, col. 2), FOXA2 (p. 1777, col. 2), and AFP (p. 1777, col. 2). Tanwar also created a construct using the “Myh6 promoter-polyA hGH cloning vector” to create a Myh6-DsRed2-Nuc construct, which were expressed in CGR8 mouse cells (p. 1775, col. 2); this reads as a reporter cell with a MYH6 regulatory element. The instant specification identifies SEQ ID NO: 23 as the mouse MYH6 promoter region, and therefore, Tanwar teaches SEQ ID NO: 23 in a reporter construct. Tanwar teaches a method of detecting alterations in the expression pattern of 13 biomarkers that indicate perturbations of stem cell differentiation across endoderm and mesodermal lineages: “Differentiating CGR8 cells closely recapitulate events of early development … mesodermal (Bmp2, Foxf1), … and endodermal (Foxa2, Afp) genes” (p. 1777, Figure 1(B)). Tanwar also teaches a method of detecting alterations in the expression patter of at least seven biomarkers that indicate perturbations of stem cell differentiation across ectoderm or neuronal lineages (p. 1780, Figure 3).
Suter teaches, “Sox1 is a member of the SoxB1 transcription factor family and is involved in very early steps of neurogenesis” (p. 49, col. 1) and “Pax6 is a key transcription factor in the development of the central nervous system” (p. 49, col. 2), which reads as Sox1 and Pax6 are important markers for embryonic development. In addition Suter teaches, “In this study, we used mouse embryonic stem cells to investigate the role of SOX1 and PAX6 during early developmental steps of neurogenesis” (p. 49, col. 2), which shows a need to study SOX1 and PAX6 in embryonic development. Suter teaches detecting alterations in the expression pattern of biomarkers that indicate perturbation of stem cell differentiation for ectoderm lineages: “neuroectoderm is characterized by the expression of .. Sox 1” (p. 49, col. 1).
A USCS Genome Browser BLAST search for the POU51 (NM_002701.6), BMP4 (NM_001202.6), MYH6 (NM_002471.4), PAX6 (NM_001368894.2), FOXA2 (NM_021784.5), AFP (NM_001134.3), and SOX1 (NR_120392.1) genes reveals adjacent promoter sequences aligned with SEQ ID NO. 1-5, 8, and 101 respectively with 100% identity. POU51 is also identified as OCT4.
Regarding Claim 1, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have performed a simple substitution with the teachings of Tanwar, Suter, and USCS Genome Browser into the method taught by Couillard-Despres to create the method of Claim 1. Couillard-Despres teaches an in vitro method of determining teratogenic effects of an agent on a mammalian embryonic cell with NTERA-2 cells, and Tanwar teaches a method of studying cell differentiation with (a) mouse embryonic stem cells. One skilled in the art would have a reasonable expectation of success with substituting the cells because both methods involve stem cell differentiation and a reporter construct. Couillard-Despres teaches a method detecting alterations in the expression pattern of one biomarker, and Tanwar teaches a method detecting alterations in at least seven biomarkers that indicated perturbation of stem cell differentiation across endoderm, mesoderm, and ectoderm lineages. One skilled in the art would have a reasonable expectation of success with this substitution because both methods involve detecting alterations in the expression pattern of a biomarker, and Tanwar teaches detecting multiple biomarkers. Couillard-Despres teaches a method with one regulatory element operably linked to a reporter sequence in mammalian embryonic cells, DCX; Kirchof teaches detecting OCT4 expression in mammalian embryonic cells; Tanwar teaches detecting BMP4, MYH6, FOXA2, and AFP expression in mammalian embryonic cells; and Suter teaches detecting PAX6 and SOX1 expression in mammalian embryonic cells. One skilled in the art would have a reasonable expectation of success substituting the regulatory element of other genes into the method of Couillard-Despres because the genes are well known as described in Kirchof, Tanwar, and Suter, and the sequences are known as found in USCS Genome Browser. Therefore, Claim 1 is obvious over Couillard-Despres, Kirchof, Tanwar, Suter, and the USCS Genome Browser.
Regarding Claim 3, Couillard-Despres teaches a reporter sequence that encodes EGFP, a protein (p. 5, col. 1). Therefore, Claim 3 is obvious over Couillard-Despres, Kirchhof, Tanwar, Suter and the USCS Genome Browser.
Regarding Claim 4, retinoic acid, taught by Couillard-Despres, is a chemical agent. Therefore, Claim 3 is obvious over Couillard-Despres, Kirchhof, Tanwar, Suter and the USCS Genome Browser.
Regarding Claim 5, retinoic acid, taught by Couillard-Despres, is a small molecule. Therefore, Claim 3 is obvious over Couillard-Despres, Kirchhof, Tanwar, Suter and the USCS Genome Browser.
Regarding Claim 6, a “kit” is interpreted as a collection of items and does not change the bounds of the claimed invention. Couillard-Despres, Kirchhof, Tanwar, Suter and the USCS Genome Browser combined teach a method that would construct a set of seven reporter cells that each express one regulatory element selected from OCT4, BMP4, MYH6, PAX6, FOXA2, AFP and the SOX1 genes operably linked to a reporter gene as shown in Claim 1, including the use of SEQ ID NO. 1-5, 8 and 101. Tanwar and Suter teach the use of mouse embryonic stem cells and markers for differentiation for ectoderm, mesoderm, and endoderm lineages. Therefore, Claim 6 is obvious over Couillard-Despres, Kirchhof, Tanwar, Suter and the USCS Genome Browser.
Regarding Claim 12, the instant specification teaches, “a reporter sequence encodes a protein that is readily detectable… by its activity that results in the generation of a detectable signal. … a reporter sequence encodes a fluorescent protein … Examples of fluorescent proteins which may be used in accord with the invention include … enhanced green fluorescent protein (EGFP)” (p. 14). Couillard-Despres teaches the use of EGFP, a readily detectable protein (p. 5, col. 1). Therefore Claim 12 is obvious over Couillard-Despres, Kirchhof, Tanwar, Suter and the USCS Genome Browser.
Claims 2 and 7 are rejected under 35 U.S.C. 103 as being unpatentable over Couillard-Despres S., et. al., BMC Neuroscience, Vol. 9, Issue 31, p. 1-9, Feb. 29, 2008; Kirchhof, N., et. al., Biology of Reproduction, Vol. 63, Issue 6, p. 1698-1705, Dec. 1, 2000; Tanwar, V., et. al., Stem Cells, Vol. 32, Issue 7, p. 1774-1788, Jun. 17, 2014; Suter, D., et. al., Stem Cells, Vol. 27, Issue 1, p. 49-58, Jan. 05, 2009; and USCS Genome Browser, Kent, W., et. al., Genome Research, Vol. 12, Issue 6, p. 996-1006, published in advance May 16, 2002 as applied to Claims 1 and 6 above, and further in view of Ghosheh, N., et. al., Stem Cells International, Vol. 2016, Article ID 8648356, p. 1-22, Feb. 01, 2016.
Regarding Claim 2, Couillard-Despres, Kirchhof, Tanwar, Suter and the USCS Genome Browser teach Claim 1.
Couillard-Despres, Kirchhof, Tanwar, Suter and the USCS Genome Browser do not teach the use of the SOX17, ALB or Ck18 genes.
Ghosheh teaches, “Towards gaining better understanding of the differentiation and maturation process, we employed a standardized protocol to differentiate six hPSC lines into hepatocytes and investigated the synchronicity of the hPSC lines by applying RT-qPCR to assess the expression of lineage-specific genes (OCT4, … SOX17, … AFP, … KRT18 [Ck18], ALB, …) which serve as markers for different stages during liver development” (p. 1, Abstract), which reads an in vitro method to investigate the development of mammalian embryonic cells using the SOX17, ALB, and Ck18 genes. Ghosheh teaches the markers can indicate development along the endoderm lineage (p. 2, Materials and Methods).
A USCS Genome Browser BLAST search for SOX17, ALB, and KRT18 genes reveals adjacent promoter sequences aligned with SEQ ID NO. 6, 7, and 9 respectively with 100% identity. KRT18 is also identified as Ck18.
Regarding Claim 2, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have performed a simple substitution with the teachings of Tanwar, Suter, and USCS Genome Browser into the method taught by Couillard-Despres to create the method of Claim 1 and to further substitute the teachings of Ghosheh into the invention of Couillard-Despres to create the method of Claim 2. Couillard-Despres teaches a method with one regulatory element operably linked to a reporter sequence in mammalian embryonic cells, DCX, and Ghosheh teaches SOX17, ALB, and Ck18 expression in embryonic stem cells. One skilled in the art would have a reasonable expectation of success substituting the regulatory element of other genes into the method of Couillard-Despres because the genes are well known as described in Ghosheh, and the sequences are known as found in USCS Genome Browser. Therefore, Claim 2 is obvious over Couillard-Despres, Kirchhof, Tanwar, Suter and the USCS Genome Browser in further view of Ghosheh.
Regarding Claim 7, a “kit” is interpreted as a collection of items and does not change the bounds of the claimed invention. Couillard-Despres, Kirchhof, Tanwar, Suter and the USCS Genome Browser combined teach Claim 6. Ghosheh teaches SOX17, ALB, and Ck18 are important genes to monitor for liver development and the USCS Genome Browser. One skilled in the art would be motivated to add additional reporter cells expressing constructs with the SOX17, ALB and Ck18 promoter to investigate embryonic liver cell development, and it would be predictable as substituting regulatory elements is known in the art. Therefore, Claim 7 is obvious over Couillard-Despres, Kirchhof, Tanwar, Suter and the USCS Genome Browser in further view of Ghosheh.
Response to Applicant’s Arguments – 35 USC 103
In the response dated September 10, 2025, the applicant argues, “Claim 1 has been amended, it has been specified the in vitro method is for determining a teratogenic effect. Claim 1 has further been amended to specify that the method comprises determining that the agent has a teratogenic effect on mammalian embryonic development by detecting alterations in the expression pattern of the at least seven biomarkers that indicate perturbation of stem cell differentiation across endoderm, mesoderm, and ectoderm lineages.” This argument is not persuasive because the instant specification provides in an embodiment: “To measure the effect of the teratogenic compound retinoic acid” (p. 27, Example 2: Effect of teratogenic compounds on hiPSCs) . Couillard-Despres teaches a method using retinoic acid to detect alterations in the expression pattern of a biomarker, DCX, for stem cell differentiation, and therefore is determining a teratogenic effect on mammalian embryonic development by detecting alterations in the expression pattern of a biomarker as described above in the rejection of Claim 1.
Applicant further argues, “These references address different aspects of development (neural, cardiac, hepatic) and come from different contexts. There was no suggestion in the prior art to combine reporter systems from different developmental pathways into a single comprehensive teratogenicity screening method covering all three embryonic germ cell lineages. … The prior art teaches individual markers in isolation for specific differentiation studies, not the coordinate multi-marker approach recited by claim 1.” This argument is not persuasive because Tanwar teaches detecting alterations in the expression pattern of at least seven biomarkers for differentiation in Figure 1(B)on page 1777 and in Figure 3 on p. 1780 including endoderm, mesoderm and ectoderm lineages as described in the rejection of Claim 1 above, therefore covering different developmental pathways.
Conclusion
No claims are allowed.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Krishna Nuggehalli Ravindra whose telephone number is (571)272-2758. The examiner can normally be reached M-Th, alternate F, 8a-5p est.
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/K.N.R./Examiner, Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636