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 4 February 2026. Claims 1-4, and 6-18 are currently pending. Claims 6-18 are withdrawn from prosecution as being drawn to non-elected subject matter. Accordingly, claims 1-4 are examined herein. The restriction requirement mailed 18 January 2023 is still deemed proper. Applicant's elected claims 1-5 without traverse in the reply filed 23 March 2023.
Any rejection or objection not reiterated herein has been overcome by amendment. Applicant' s 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
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-4 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. This is a new rejection necessitated by amendment.
Regarding claim 1, the claim recites “an isolated pluripotent stem cell comprising a DNA encoding a reporter molecule […] wherein the DNA encoding the reporter molecule is located upstream of the endogenous tau gene […] by using a targeting vector that includes homology arms upstream and downstream of a tau insertion site […] wherein the homology arm upstream of the tau gene insertion site is a sequencing having 90% or more identity with a sequence set forth in SEQ ID NO: 3, and the homology arm downstream of the tau gene insertion site is a sequence having 90% or more identity with a sequence set forth in SEQ ID NO: 4.”
However, the instant specification defines the claimed SEQ ID NOs: 3-4 as a “right homology arm (for inserting TagGFP2 at C-terminus)” ([0077]) and a “left homology arm (for inserting TagGFP2 at C-terminus” ([0078]), respectively. Accordingly, it is unclear if the claimed SEQ ID NOs: 3-4 are intended to be utilized to insert the claimed DNA encoding the reporter molecule upstream of the endogenous tau gene, or if the claimed SEQ ID NOs: 3-4 were referenced in error.
Regarding claims 2-4, as the claims do not rectify the currently outstanding 35 USC 112(b) rejections above, the claims are also rejected under 35 USC 112(b).
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.
Claim(s) 1-4 is/are rejected under 35 U.S.C. 103 as being unpatentable over Karch (Stem cell reports 13.5 (2019): 939-955) in view of Lee (Stem cell research 40 (2019): 101554), Boulin (WormBook: The Online Review of C. elegans Biology [Internet] (2006)), Samsonov (Journal of cell science 117.25 (2004): 6129-6141), Fetter (Chromosomal Mutagenesis. New York, NY: Springer New York, 2014. 231-240), NG_007398.2 (NCBI Reference Sequence NG_007398.2, earliest publishing date of 26 August 2019), and NG_007398.2 sequence alignments (accessed 19 June 2026).
Regarding claim 1, Karch is drawn to a study concerned with the expression patterns and detection of tau proteins that are expressed from a MAPT gene (i.e., an endogenous tau gene) (Abstract). Karch teaches that 29 induced pluripotent stem cell lines carrying MAPT mutations were collected from human patients (pg. 949). Karch teaches that the induced pluripotent stem cells can be differentiated into the neuronal and glial subtypes that are affected in primary tauopathies, giving a tool toward understanding the biology of tau in a human cell model that may more faithfully reflect the endogenous condition (pg. 943). Karch teaches that tau proteins are important to detect because they are responsible for frontotemporal lobar degeneration (pg. 939).
However, Karch does not teach expressing tau as a fusion protein with a reporter molecule, wherein the DNA encoding the reporter molecule is located upstream of an endogenous tau gene (Claim 1). In addition, although Karch studies iPSCs having tau mutations and detects pluripotency marker expression in the iPSCs (pg. 946; see Figure 2), Karch does not specifically teach the detection of tau protein in the iPSCs themselves (Claim 1). Karch does not teach or suggest the use of a targeting vector that includes homology arms comprising at least 90% identity to the claimed SEQ ID NOs: 1 and 2 that could be utilized to insert the DNA encoding the reporter molecule (Claim 1). Karch does not teach or suggest that the reporter molecule is a fluorescent protein (Claim 4).
Regarding the detection of endogenous proteins in iPSCs, Lee is drawn to a study concerned with the generation of a Nestin-EGFP reporter in human iPSCs (Abstract). Lee teaches that Nestin is a neuroectodermal marker involved in induced pluripotent stem cell differentiation toward neural lineages (Abstract). Lee teaches that reporter human iPSCs can be created by adding an EGFP fusion tag (i.e., a fluorescent reporter protein), via a CRISPR/Cas9-mediated knock-in system, to the C-terminus of an endogenous Nestin gene in KSCBi005-A cells (i.e., a human iPSC line originating from bone marrow blood cells) (pg. 1-2; see Figure 1A). Lee teaches that vectors encoding Cas9 and an sgRNA targeting the stop codon region of the Nestin gene were co-transfected together with a donor construct containing EGFP (i.e., a DNA encoding the fluorescent reporter molecule), a Puro marker, and two homologous sequences to upstream and downstream of the NESTIN targeting site (pg. 1-2; see Figure 1A). Lee teaches that the endogenous Nestin gene and downstream EGFP were in frame and could be expressed as a fusion protein (pg. 1-2; see Fig. 1A, 1C). Lee teaches that the NESTIN-GFP reporter was able to be visualized in neural cells following differentiation of the iPSCs (pg. 3; see Figure 1J).
Regarding the use of a targeting vector that includes homology arms upstream and downstream of a genomic insertion site of interest, Fetter is drawn towards a review concerned with endogenous gene tagging with fluorescent proteins (Abstract). Fetter teaches the use of a targeting vector utilized to insert a nucleic acid encoding a GFP reporter protein upstream of a target gene of interest in order to express the endogenous protein as a fusion protein with the GFP reporter (pg. 236; see Fig. 1). Fetter teaches that BFP-LMNB1, GFP-TUBA1B, and RFP-ACTB fusion proteins were generated via the use of first and second homology arms present in the targeting vector, wherein a left homology arm matches exactly upstream of an ATG start codon of the endogenous protein and a right homology arm matches, and includes, the ATG start site to insert the fluorescent protein upstream of the endogenous protein (pg. 236; see Fig. 1).
Regarding the DNA encoding the reporter protein being located upstream of the endogenous gene, Boulin is drawn towards a study concerned with reporter gene fusions (pg. 1; Introduction). Boulin teaches that it is known in the art how to generate an N-terminal GFP translational reporter (pg. 7, section 2.3.3; see reproduced Figure 4 on pg. 14). Boulin teaches that the GFP protein is located upstream of a genomic gene of interest (i.e., an endogenous gene) and is able to be expressed as a fusion protein when translated (pg. 7, section 2.3.3; see reproduced Figure 4 on pg. 14).
Regarding the generation of a GFP-tau fusion protein, Samsonov is drawn towards a study concerned with investigating the interaction of tau proteins with microtubules in cultured embryo neurons (Abstract). Samsonov teaches that the embryo neurons were injected with a plasmid encoding either GFP-tau23 of GFP-tau24 (pg. 6130). Samsonov teaches that the GFP-tau fusion constructs were able to be visualized in the embryo cell cultures and their interactions with microtubules studied (pg. 6131; see Fig. 1). Samsonov teaches that a fusion of GFP to either the N-terminal or C-terminal of tau does not significantly affect the functional properties of tau (pg. 6132). Samsonov teaches that tau is implicated in microtubule stabilization and the use of the GFP-tau reporter construct allowed for reliable information concerning tau-microtubule interactions in living cells (pg. 6136-6137).
Regarding the usage of the claimed SEQ ID NOs: 1-2 as homology arms within the claimed targeting vector, NG_007398.2 is drawn towards a known microtubule associated protein tau (i.e., termed MAPT) gene on chromosome 17 (pg. 1). As shown below in the provided sequence alignments, the claimed SEQ ID NO: 1 has 100% identity to base pairs 72076-72875 of the MAPT gene and the claimed SEQ ID NO: 2 has 100% identity to base pairs 72876-73675 of the MAPT gene (see Figures 1 and 2 below).
PNG
media_image1.png
964
614
media_image1.png
Greyscale
Figure 1. SEQ ID NO: 1 location within NG_007398.2.
PNG
media_image2.png
956
612
media_image2.png
Greyscale
Figure 2. SEQ ID NO: 2 location within NG_007398.2.
When base pairs 72076-73675 of the MAPT gene are isolated from the chromosome 17, as shown in NG_007398.2, SEQ ID NO: 1 is represented by base pairs 1-800, the claimed SEQ ID NO: 2 is represented by base pairs 801-1600 (see pg. 2 of NG_007398.2). Therefore, it is noted that the claimed SEQ ID NO: 1 is drawn towards a sequence that has 100% identity to a sequence exactly upstream of an ATG start codon of an endogenous tau protein and the claimed SEQ ID NO: 2 matches and includes the ATG start site present in the endogenous tau protein.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the iPSCs comprising the MAPT mutations described by Karch via a CRISPR/Cas9-mediated knock in of an EGFP reporter protein at the endogenous MAPT gene, as described by Lee, Fetter, and NG_007398.2, in order to generate a fusion construct comprising the EGFP located upstream of the endogenous tau protein, as described by Boulin and Samsonov.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the iPSCs comprising the MAPT mutations described by Karch via a CRISPR/Cas9-mediated knock in of an EGFP reporter protein at a known endogenous MAPT gene through the use of a targeting vector comprising homology arms that are 100% identical to an endogenous MAPT gene, as described by Lee, Fetter, and NG_007398.2, in order to generate a fusion construct comprising the EGFP located upstream of the endogenous tau protein, as described by Boulin and Samsonov, because it would have merely amounted to a combination of prior art elements according to known methods to yield predictable results. Because Lee teaches that knocking-in an EGFP reporter protein at an endogenous gene of interest in iPSCs was a known method of detecting the endogenous protein, one would have expected the knock-in of a fluorescent reporter protein at an endogenous tau gene to have similarly allowed for the detection of the endogenous tau. Because Fetter teaches that utilizing a targeting vector comprising a left homology arms that match the region upstream of an endogenous gene and a right homology arm that matches and includes an ATG start codon of the endogenous gene, alongside the disclosure of NG_007398.2 providing evidence that the claimed SEQ ID NOs: 1-2 fulfill these exact requirements for a known endogenous tau gene sequence, one would have expected utilizing the claimed SEQ ID NOs: 1-2 as the homology arms to have resulted in the successful insertion of a fluorescent reporter upstream of an endogenous tau gene. Additionally, because Samsonov teaches that a GFP reporter protein fused to the N-terminus of a tau protein did not affect the functional properties of tau one would have expected the endogenous tau to have maintained its ability to allow for tau-microtubule interactions in living cells. Further, one would have been motivated to have generated the endogenous tau fusion protein in order to study tau-microtubule interactions, as described by Samsonov, and because mutations in endogenous tau proteins are responsible for frontotemporal lobar degeneration as taught by Karch.
Regarding claims 2-3, Karch teaches the use of human induced pluripotent stem cells (pg. 945; see Table 3).
Response to Arguments
Applicant's arguments filed 4 February 2026 have been fully considered but they are not persuasive.
Applicant alleges the present specification demonstrates that there is a correlation between the cellular function (i.e., neurite length) and the fluorescence intensity of the reporter molecule (see Figure 6 of the present application) (Remarks; pg. 7). Applicant alleges that the correlation between the cellular function and the fluorescence intensity of the reporter molecule is an advantageous effect, which cannot be expected from the cited references (Remarks; pg. 7). Applicant alleges that the disclosure provides a unique and advantageous effect wherein the Tau protein fused with the reporter molecule localizes to neurites in vivo, similar to endogenous Tau (Remarks; pg. 8).
This argument is not found persuasive because, as discussed above, Samsonov teaches that microtubule bundles are present within neurite-like cell processes and that GFP-tau fusion constructs were able to be visualized in the embryo cell cultures and their interactions with microtubules studied, including the correlation between the presence of a fluorescent signal and the length of the microtubules (pg. 6131; see Fig. 1). Thus, from the disclosures of the prior art, one of ordinary skill in the art would have expected that the generation of the claimed endogenous tau fusion protein would have allowed for the fusion protein to localize to neurites in vivo, similar to endogenous tau, in order to allow for the correlation of fluorescent intensity with neurite length.
Applicant alleges that since there is a difference between the localization of Nestin and Tau, one of ordinary skill in the art would have no proper reason, rationale, or motivation to replace Nestin with Tau for the purpose of studying the interaction between endogenous Tau protein and microtubules (Remarks; pg. 8). Applicant alleges that one of ordinary skill in the art, when using Nestin, would have no proper reason, rationale, or motivation to evaluate a correlation between the "cell function of (mature) nerve cell neurite length" (a feature of the claimed invention) and the "fluorescence intensity of the reporter molecule (Nestin)" because Nestin is a marker for neural stem cells and is not expressed in differentiated neurons (Remarks; pg. 8-9).
These arguments are not found persuasive because the disclosure of Lee (i.e., the reference in which endogenous Nestin was labeled with a fluorescent reporter) was only relied upon for the teachings that labeling an endogenous protein in iPSCs was a known method of detecting the endogenous protein. The endogenous Tau of Karch was not substituted for the endogenous Nestin of Lee. Rather, the rejection of claim 1 above relies on Lee for the disclosure that endogenous proteins in iPSCs could be labeled with fluorescent reporters and subsequently detected.
Additionally, 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). In the instant case, it is the combination of Karch, Lee, Boulin, Fetter, and NG_007398.2 that renders the claimed invention obvious.
Conclusion
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 KYLE T REGA whose telephone number is (571)272-2073. The examiner can normally be reached M-R 8:30-4:30, every other F 8:30-4:30 (EDT/EST).
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Neil Hammell can be reached at 571-270-5919. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/KYLE T REGA/Examiner, Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636
Prosecution Insights