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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 02/03/2026 has been entered.
Claim Status
1. The amendment filed 02/03/2026 has been entered. All previously pending claims have been cancelled and new claims 23 – 28 are pending and under consideration.
Election/Restrictions
2. Applicant’s election without traverse of Group I (claims 1 – 12) in the reply filed on 05/13/2025 is acknowledged.
3. Claims 13 – 19 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 05/13/2025.
Priority
4. This application claims priority to Japanese Patent Application No. 2019-201231, filed on November 6, 2019.
5. Should applicant desire to obtain the benefit of foreign priority under 35 U.S.C. 119(a)-(d) prior to declaration of an interference, a certified English translation of the foreign application must be submitted in reply to this action. 37 CFR 41.154(b) and 41.202(e).
Failure to provide a certified translation may result in no benefit being accorded for the non-English application.
Withdrawn Claim Objections and Rejections
6. All claim objections and rejections set forth in the Final Office Action mailed 11/06/2025 are rendered moot in view of Applicant’s cancellation of all previously pending claims (claims 1, 6, 9, 10, 12 – 22, and 78).
Claim Objections
7. Claim 23 is objected to because of the following informalities: in line 13, “p-tau/3R p-tau” should read “phosphorylated tau/3R phosphorylated tau” to clarify that “p-tau” refers to the phosphorylated version of tau. Appropriate correction is required.
8. Claim 28 is objected to because of the following informalities: in line 2, “the culture medium 1” should read “the first culture media” and in line 3, “the culture medium 2” should read “the second culture media” to clarify that the culture media recited in claim 28 are the same culture media recited in claim 23. Appropriate correction is required.
Claim Interpretation
9. For the purpose of applying prior art, “p-tau” is interpreted as phosphorylated tau based on Applicant’s specification at page 46, lines 4 – 15.
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.
10. Claim(s) 23 – 27 are rejected under 35 U.S.C. 103 as being unpatentable over Garcia-Leon (García-León JA, et. al. Alzheimers Dement. 2018 Oct;14(10):1261-1280; previously cited), hereinafter Garcia-Leon which is cited on the IDS filed 05/02/2022 in view of Verheyen (Verheyen A, Stem Cell Reports. 2018 Aug 14;11(2):363-379; previously cited), hereinafter Verheyen which is cited on the IDS filed 05/02/2022 in view of Lancaster (Lancaster, Madeline A., et al. Nature biotechnology 35.7 (2017): 659-666.), hereinafter Lancaster.
Regarding claim 23, Garcia-Leon teaches culturing hiPSCs having mutations N279K, P301L and E10+16 in the MAPT gene (“human pluripotent stem cells comprising a microtubule-associated protein tau (MAPT) gene that comprises an N279K mutation and a P301S mutation in exon 10 and an E10 + 16 mutation in intron 10”) and differentiation to cortical neurons (Figure 1; page 1263, left col. paragraph 1; page 1272, left col. para. 1; page 1275, left col. para. 2; page 1276, left col. paragraph 4) but does not teach “P301S mutation”. Garcia-Leon teaches culturing these triple mutant hiPSCs in a N2B27 culture medium comprising SB431542 (“an inhibitor of transforming growth factor β (TGF-β) family signaling”) and LDN193189 (“an inhibitor of bone morphogenetic (BMP) signaling”) for neural induction (“first culture media”) and then plating the resulting cells in N2B27 medium for formation of neural precursor cells (NPCs) (“second culture media” and “an inhibitor of transforming growth factor β (TGF-β) family signaling”) and culturing the NPCs in N2B27 media for terminal neuronal differentiation (“third culture media that does not comprise extracellular matrix”) (page 1276, left col. para. 4). Garcia-Leon does not teach “a GSK-3β inhibitor” and “an extracellular matrix mixed therein” in the “second culture media”. Garcia-Leon teaches in Figure 2H and 2I that the ratio of 4R phosphorylated tau/3R phosphorylated tau is at least 1 using the antibodies that detect the phosphorylated forms of tau (page 1263, right col. para. 2).
Regarding claim 24, Garcia-Leon teaches SB-431542 (page 1276, left col. para. 4).
Regarding claim 25, Garcia-Leon teaches LDN193189 (page 1276, left col. para. 4).
Regarding claim 27, Garcia-Leon teaches SB-431542 and LDN193189 (page 1276, left col. para. 4) but does not teach “CHIR99021”.
Garcia-Leon does not teach “suspension culturing”, “P301S”, “a GSK-3β inhibitor” and “an extracellular matrix mixed therein” in the “second culture media” of claim 23, or “CHIR99021” of claims 26 and 27. However, Garcia-Leon teaches the method generates a Tau mutant model displaying neurodegenerative disease phenotypes that could be used for disease modeling and drug screening to identify therapeutic targets counteracting Tau pathology excluding the variability associated with patient-derived cells (Abstract; page 1273, right col. last paragraph). Garcia-Leon teaches tauopathies include FTDP-17 and aberrant regulation of exon 10 splicing in the adult brain has been identified as a major cause of tauopathies (page 1262, left col. paragraph 1 – 2). Garcia-Leon teaches more than 50 mutations in the MAPT gene have been described where they are typically associated with FTDP-17 (page 1262, right col. paragraph 1). Garcia-Leon teaches the method of differentiation of the triple mutant hiPSCs to cortical neurons reproduces several neurodegenerative aspects, including modified Tau isoform expression and Tau aggregation among others (page 1262, right col. last paragraph; page 1263, right col. para. 1 and 3; Figure 2). Garcia-Leon teaches mouse models have been generated expressing Tau variants but only partially reproduce human Tau pathology (page 1269, right col. paragraph 4). Garcia-Leon teaches ideally drug discovery and validation platforms should use cell models wherein most of the Tau-associated phenotypes are present and can be compared with wild type control lines to prove the causal relation of the phenotype (page 1272, left col. para. 1).
Regarding “P301S” of claim 23, Verheyen teaches genetically engineered iPSCs comprising the double mutations of IVS10+16 (E10+16) and P301S of the MAPT gene and a method of forming neurons from these iPSCs (page364, left col. para. 3; page 369, right col. paragraph 1; page 376, left col. last paragraph and right col. paragraph 1; Supplemental experimental procedure). Verheyen teaches the double mutant neurons show hyperexcitability due to the P301S mutation, which is in line with studies showing neuronal hyperexcitability and epileptic seizures in P301S animal models and FTDP-17 patients with this mutation (page 374, left col. para. 4). Verheyen teaches a well-recognized characteristic of P301S-related FTDP-17 is tau hyperphosphorylation and the formation of NFTs (page 374, left col. last para.). Verheyen teaches patient-based evidence show the presence of tau aggregates and NFTs in P301S neurons (page 369, right col. last paragraph; page 374, left col. last paragraph). Verheyen teaches the P301S mutation was chosen to generate an aggressive FTDP-17 model (page 372, left col. paragraph 2). Verheyen teaches adding the P301S mutation to the IVS10+16 mutation revealed additional phenotypes such as tau oligomerization and neurodegeneration (Summary; page 364, left col. para. 1). Verheyen teaches the double MAPT mutant could serve as a platform to identify new targets for drug development and to reveal the mechanisms underlying FTDP-17 (page 376, left col. paragraph 3).
Verheyen does not teach “suspension culturing”, “a GSK-3β inhibitor” and “an extracellular matrix mixed therein” in the “second culture media” of claim 23 and “CHIR99021” of claims 26 and 27. However, Verheyen teaches Wnt protein binding pathway is affected in IVS10+16 mutant neurons including decreased transcript levels of Wnt-related genes and upregulation of GSK3B at DIV 65 (page 367, left col. para. 2; page 374, left col. para. 3). Verheyen teaches Wnt signaling is indispensable for normal brain development and neural cell fate commitment and its link with the IVS10+16 mutation suggests that developmental aberrations might underlie the regional neuronal selectivity and vulnerability that are characteristic for FRDP-17 related tauopathies (page 376, left col. para. 2). Verheyen teaches an increasing amount of evidence suggests that neurodegenerative diseases are linked with aberrant Wnt signaling (page 374, left col. para. 3). Verheyen teaches an aberrant Wnt/Shh signaling pathway might cause the observed differences in neuronal cell types in their purely neuronal in vitro model and therefore might exacerbate the phenotypes and mechanisms that are potentially linked with the MAPT IVS10+16 mutation, as additional cell types and cellular interactions are lacking in their model (page 376, left col. para. 2). One would have been motivated to combine the teachings of Garcia-Leon and Verheyen because both teach engineering hiPSCs to express multiple mutations found in FTDP-17 including those at P301 and differentiating these hiPSCs to neurons to elucidate the role of combining these mutations on neuron development and function.
Regarding “suspension culturing”, “a GSK-3β inhibitor” and “an extracellular matrix mixed therein” in the “second culture media” of claim 23 and “CHIR99021” of claims 26 and 27, Lancaster teaches a method of making brain organoids comprising suspension culturing iPSCs in neural induction media (“suspension culturing” of claim 23) to form neuroectoderm and then culturing the neuroectoderm in IDM + A media comprising CHIR99021 and extracellular matrix (“a GSK-3β inhibitor” and “an extracellular matrix mixed therein” in the “second culture media” of claim 23 and “CHIR99021” of claims 26 and 27) (Figure 1a; page 667 Online Methods, right col. para. 2 – 4). Lancaster teaches because Wnt pathway activation has been shown to induce lateral expansion of cortical neuroepithelium, CHIR99021 was added to the media after neuroepithelial budding and the addition resulted in larger lumens with surrounding continuous neuroepithelium (page 663, left col. para. 2). Lancaster teaches suspension culturing with CHIR99021 resulted in more consistent formation of brain lobules that were large (page 663, left col. para. 3). Lancaster teaches exogenous extracellular matrix reconstituted the basement membrane that remained outside the migrating neurons and elicited cortical plate formation (page 663, right col. para. 4 – 5). Lancaster teaches the method produces results in reproducible formation of forebrain tissue (page 663, left col. para. 3). Lancaster teaches organoids have many potential applications in drug discovery and disease research but their utility has been limited by high variability, random tissue identity, and incomplete morphological differentiation (page 659, left col.).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Garcia-Leon regarding culturing hiPSCs comprising three mutations in the human MAPT gene encoding N279K, P301S, and IVS10+16 with the teachings of Verheyen regarding culturing hiPSCs comprising IVS10+16 and P301S mutations in the human MAPT gene with the teachings of Lancaster regarding a method of producing brain organoids from iPSCs to arrive at the claimed method of making a brain organoid, comprising: suspension culturing human pluripotent stem cells comprising a microtubule-associated protein tau (MAPT) gene that comprises an N279K mutation and a P301S mutation in exon 10 and an E10 + 16 mutation in intron 10 in a first culture media comprising an inhibitor of bone morphogenetic protein (BMP) signaling and an inhibitor of transforming growth factor β (TGF-β) family signaling to produce a first culture preparation; suspension culturing the first culture preparation in a second culture media comprising a GSK-3β inhibitor, an inhibitor of transforming growth factor β (TGF-β) family signaling, and an extracellular matrix mixed therein to produce a second culture preparation; and culturing the second culture preparation in a third culture media that does not comprise extracellular matrix to produce a cultured brain organoid comprising a ratio of 4R p-tau/3R p-tau of at least 1. One would have been motivated to combine the teachings of Garcia-Leon, Verheyen, and Lancaster in a method to produce a brain organoid to model tauopathies as Garcia-Leon teaches the method generates a Tau mutant model displaying neurodegenerative disease phenotypes that could be used for disease modeling and drug screening to identify therapeutic targets counteracting Tau pathology excluding the variability associated with patient-derived cells and Verheyen teaches the P301S mutation was chosen to generate an aggressive FTDP-17 model and Lancaster teaches organoids have many potential applications in drug discovery and disease research. One would have a reasonable expectation of success in combining the teachings as Garcia-Leon teaches the method of differentiation of the triple mutant hiPSCs to cortical neurons reproduces several neurodegenerative aspects, including modified Tau isoform expression and Tau aggregation among others and Verheyen teaches adding the P301S mutation to the IVS10+16 mutation revealed additional phenotypes such as tau oligomerization and neurodegeneration and Lancaster teaches the method produces results in reproducible formation of forebrain tissue from iPSCs.
11. Claim(s) 28 is/are rejected under 35 U.S.C. 103 as being unpatentable over Garcia-Leon (García-León JA, et. al. Alzheimers Dement. 2018 Oct;14(10):1261-1280; previously cited), hereinafter Garcia-Leon which is cited on the IDS filed 05/02/2022 in view of Verheyen (Verheyen A, Stem Cell Reports. 2018 Aug 14;11(2):363-379; previously cited), hereinafter Verheyen which is cited on the IDS filed 05/02/2022 as evidenced by Shi (Shi, Yichen, et. al." Nature protocols 7.10 (2012): 1836-1846.), hereinafter Shi in view of Lancaster (Lancaster, Madeline A., et al. Nature biotechnology 35.7 (2017): 659-666.), hereinafter Lancaster as applied to claims 23 – 27 above, and further in view of Qian (Qian, Xuyu, et al. Cell 165.5 (2016): 1238-1254; previously cited), hereinafter Qian.
Garcia-Leon in view of Verheyen and Lancaster make obvious the limitations of claim 23 as set forth above. Lancaster teaches the method produces forebrain tissue expressing the forebrain marker Foxg1 (Abstract; Figure 2; page 663, left col. para. 3). Lancaster teaches the organoids most closely matched the forebrain identities of the human brain at early gestation (page 663, right col. para. 2).
Regarding “the culture medium 1”, Verheyen teaches the differentiation of iPSCs to cortical neurons was performed using an adapted dual SMAD inhibition protocol of Shi (Supplemental experimental procedure, para. 1) where neural induction medium contained dorsomorphin and SB431542 as evidenced by Shi (page 1836, right col. last para.; page 1839, left col. para. 4; page 1845, para. 2). Garcia-Leon, Verheyen, and Lancaster do not teach A83-01.
Regarding “the culture medium 2”, Garcia-Leon teaches a second culture media that comprises SB-431542 (page 1276, left col. para. 4) and Lancaster teaches a second culture media that comprises CHIR99021 and extracellular matrix that is Matrigel (Figure 1a; page 667, Online Methods left col. para. 2 – 4).
Regarding “A83-01”, Qian teaches a method of producing brain organoids from hiPSCs by treating the hiPSCs in an ultra-low attachment plate in a first culture media with dual SMAD inhibitors dorsomorphin and A-8301, followed by culturing in a second culture media containing CHIR99021 and SB431542 where the cells are embedded in Matrigel (page 1240, right col. para. 3; Figure 1B and S2; page S13, para. 2). Qian teaches the method allows for the formation of forebrain-specific organoids from hiPSCs and these organoids recapitulate key features of human cortical development in a reproducible and quantifiable manner (Summary; page 1240, left col. last para.). Qian teaches the forebrain organoids expressed Foxg1 (page 1241, right col. para. 2). Qian teaches the homogeneity of forebrain organoids produced by the method comprise a platform that is amenable to chemical compound testing and teaches treatment of the organoids with bisphenol A affects their function (page 1243, left col. para. 3). Qian teaches there is a critical need to develop an organoid platform with reduced cost, higher throughput, and increased reproducibility and one that better resembles critical aspects of human cortical development (page 1240, left col. para. 3). Qian teaches the method employing spinning cultures enhance cell viability and promote maintenance of the stem cell niche in the forebrain organoids and opens doors for cost-effective generation of and provides accessible and affordable organoid technology to a broader scientific community (page 1240, left col. para. 2 and right col. para. 1). Qian teaches the method is versatile, simple-to-use, cost-effective, and the reproducible brain-region specific organoid platform provides accessible and affordable technology for modeling human disorders and for compound testing (page 1240, left col. last paragraph and right col. paragraph 1; page 1249, left col. last paragraph; page 1253, left col. paragraph 1).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Garcia-Leon regarding culturing hiPSCs comprising three mutations in the human MAPT gene encoding N279K, P301S, and IVS10+16 with the teachings of Verheyen regarding culturing hiPSCs comprising IVS10+16 and P301S mutations in the human MAPT gene with the teachings of Lancaster regarding a method of producing brain organoids from iPSCs with the teachings of Qian regarding a method of producing brain organoids comprising culturing hiPSCs in a first culture media containing A83-01 and dorsomorphin and then culturing in a second culture media containing CHIR99021 and SB431542 to produce forebrain organoids to arrive at the claimed method wherein the culture medium 1 comprises dorsomorphin and A83-01, and the culture medium 2 comprises CHIR99021, SB-431542, and the extracellular matrix. One would have been motivated to combine the teachings of Garcia-Leon, Verheyen, Lancaster, and Qian in a cost-effective and reproducible method to produce a model of FTDP-17 for testing potential drug treatments as Garcia-Leon teaches the method generates a Tau mutant model displaying neurodegenerative disease phenotypes that could be used for disease modeling and drug screening to identify therapeutic targets counteracting Tau pathology and Qian teaches there is a critical need to develop an organoid platform with reduced cost, higher throughput, and increased reproducibility and one that better resembles critical aspects of human cortical development and Qian teaches the reproducible brain-region specific organoid platform provides accessible and affordable technology for modeling human disorders and for compound testing. One would have a reasonable expectation of success in combining the teachings as Garcia-Leon teaches the method of differentiating the triple mutant hiPSCs reproduces several neurodegenerative aspects, including modified Tau isoform expression and Tau aggregation among others and both Lancaster and Qian teach methods of forming forebrain organoids that express Foxg1 and Qian teaches the homogeneity of forebrain organoids produced by the method comprise a platform that is amenable to chemical compound testing and teaches an example using bisphenol A.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
12. Claims 23 – 28 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 3 and 8 of copending Application No. 16984241 in view of Garcia-Leon (García-León JA, et. al. Alzheimers Dement. 2018 Oct;14(10):1261-1280; previously cited), hereinafter Garcia-Leon which is cited on the IDS filed 05/02/2022. Although the claims at issue are not identical, they are not patentably distinct from each other because the instant claims are drawn to a species of the reference claims.
Instant claim 23 is drawn to a method of making a brain organoid, comprising: suspension culturing human pluripotent stem cells comprising a microtubule-associated protein tau (MAPT) gene that comprises an N279K mutation and a P301S mutation in exon 10 and an E10 + 16 mutation in intron 10 in a first culture media comprising an inhibitor of bone morphogenetic protein (BMP) signaling and an inhibitor of transforming growth factor β (TGF-β) family signaling to produce a first culture preparation; suspension culturing the first culture preparation in a second culture media comprising a GSK-3B inhibitor, an inhibitor of transforming growth factor β (TGF-β) family signaling, and an extracellular matrix mixed therein to produce a second culture preparation; and culturing the second culture preparation in a third culture media that does not comprise extracellular matrix to produce a cultured brain organoid comprising a ratio of 4R p-tau/3R p-tau of at least 1.
Reference claim 1 is drawn to a method comprising: three-dimensionally culturing pluripotent stem cells having a mutation in Microtubule Associated Protein Tau (MAPT) gene to form a nerve
organoid; dissociating the nerve organoid into single cells and performing two-dimensional adherent culture of the single cells under conditions suitable for the single cells to differentiate into or continue to grow as neurons; and obtaining a pure population of neurons from the two-dimensional adherent culture of the single cells; wherein the two-dimensional adherent culture of the single cells is performed in a monolayer for 30 days or more; wherein the neurons are a tau-related disease model and the mutation is one or a plurality of mutations present in exons 9, 10, 12, or 13 or intron 10; wherein the one or plurality of mutations present in exons 9, 10, 12, or 13 are a mutation that encodes a mutant tau protein selected from the group consisting of K257T, I260V, G272V, N279K, K28011, L284L, S285R, N296H, P301L, P301S, S305N, S303S, S305S, V337M, E342V, G389R and R406W, and the one or plurality of mutations present in intron 10 are a mutation in any one or a plurality of bases at nucleotide positions 3 (Ex10+3), 12 (Ex10+ 12), 13 (Ex10+ 13), 14 (Ex10+ 14), 16 (Ex10+ 16), and 19 (Ex10+ 19) from the 5'-end of intron 10.
Reference claim 1 differs from instant claim 23 in that reference claim 1 recites “performing two-dimensional adherent culture” of the dissociated nerve organoid “under conditions suitable for the single cells to differentiate into or continue to grow as neurons; and obtaining a pure population of neurons from the two-dimensional adherent culture of the single cells”, while instant claim 23 recites “suspension culturing” and broadly recites “culturing” to produce a “brain organoid comprising a ratio of 4R p-tau/3R p-tau of at least 1”. Therefore, instant claim 23 lacks “adherent culture” and “obtaining a pure population of neurons”. Garcia-Leon teaches a method of adherent culture for obtaining neurons from hiPSCs containing three mutations of MAPT (two in exon 10 and one in intron 1) (page 1275, left col. para. 2; page 1276, left col. last para.). Garcia-Leon teaches the mutant neurons expressed pathogenic 4R and phosphorylated Tau, endogenously triggered Tau aggregation, and had increased electrophysiological activity (Abstract). Garcia-Leon teaches although mouse models have been generated overexpressing different human Tau variants, these only partially reproduce human Tau pathology with significant variability between models depending on the Tau variant expressed and/or promoter used and murine neurons appear to be less sensitive to neurodegeneration compared with human neurons (page 1269, right col. para. 4). Garcia-Leon teaches the mutant neurons could be used for disease modeling and drug screening (Abstract). Garcia-Leon teaches the MAPT-mutant neurons are an ideal candidate platform for the identification of therapeutic targets counteracting Tau pathology excluding the variability associated with patient-derived cells (page 1273, right col. last para.). It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to modify the method of the instant claim to perform adherent culture to obtain a pure population of human neurons with MAPT mutations as taught by Garcia-Leon for disease modeling and drug screening of tauopathies as Garcia-Leon teaches mouse models have been generated overexpressing different human Tau variants only partially reproduce human Tau pathology and murine neurons appear to be less sensitive to neurodegeneration compared with human neurons and Garcia-Leon teaches the mutant neurons could be used for disease modeling and drug screening as the MAPT-mutant neurons are an ideal candidate platform for the identification of therapeutic targets counteracting Tau pathology excluding the variability associated with patient-derived cells.
This is a provisional nonstatutory double patenting rejection.
13. Claims 23 – 28 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 – 9 of copending Application No. 18550496 in view of Garcia-Leon (García-León JA, et. al. Alzheimers Dement. 2018 Oct;14(10):1261-1280; previously cited), hereinafter Garcia-Leon which is cited on the IDS filed 05/02/2022 in view of Verheyen (Verheyen A, Stem Cell Reports. 2018 Aug 14;11(2):363-379; previously cited), hereinafter Verheyen which is cited on the IDS filed 05/02/2022. Although the claims at issue are not identical, they are not patentably distinct from each other because the instant claims are drawn to a species of the reference claims.
Instant claim 23 is drawn to a method of making a brain organoid, comprising: suspension culturing human pluripotent stem cells comprising a microtubule-associated protein tau (MAPT) gene that comprises an N279K mutation and a P301S mutation in exon 10 and an E10 + 16 mutation in intron 10 in a first culture media comprising an inhibitor of bone morphogenetic protein (BMP) signaling and an inhibitor of transforming growth factor β (TGF-β) family signaling to produce a first culture preparation; suspension culturing the first culture preparation in a second culture media comprising a GSK-3B inhibitor, an inhibitor of transforming growth factor β (TGF-β) family signaling, and an extracellular matrix mixed therein to produce a second culture preparation; and culturing the second culture preparation in a third culture media that does not comprise extracellular matrix to produce a cultured brain organoid comprising a ratio of 4R p-tau/3R p-tau of at least 1.
Reference claim 1 is drawn to a method for producing a brain organoid having an aggregated tau protein, the method comprising: (a) culturing pluripotent stem cells in a presence of a SMAD inhibitor to form an embryoid body; (b) embedding the embryoid body in an extracellular matrix and three-dimensionally culturing the embryoid body in a presence of a SMAD inhibitor and a glycogen synthase kinase 3P (GSK3P) inhibitor to form an organoid that includes neural precursor cells; (c) extracting the organoid from the extracellular matrix and suspension-culturing the organoid in a presence of leukemia inhibitory factor (LIP) to form a brain organoid; (d) forcing the brain organoid to express a mutant microtubule associated protein tau (MAPT) gene; and (e) further suspension-culturing the brain organoid after the (d) to obtain a brain organoid having an aggregated tau protein.
The claims differ in that reference claim 1 broadly recites “a mutant microtubule associated protein (MAPT) gene” and “to obtain a brain organoid having an aggregated tau protein” while instant claim 23 recites specific mutations in the MAPT gene and a ratio of 4R p-tau/3R p-tau of at least 1. Garcia-Leon teaches a method of differentiating hiPSCs containing three mutations of MAPT (two in exon 10 and one in intron 1) (page 1275, left col. para. 2; page 1276, left col. last para.). Garcia-Leon teaches the resulting cells expressed pathogenic 4R and phosphorylated Tau, endogenously triggered Tau aggregation, and had increased electrophysiological activity (Abstract). Garcia-Leon teaches although mouse models have been generated overexpressing different human Tau variants, these only partially reproduce human Tau pathology with significant variability between models depending on the Tau variant expressed and/or promoter used and murine neurons appear to be less sensitive to neurodegeneration compared with human neurons (page 1269, right col. para. 4). Garcia-Leon teaches the MAPT-mutant neurons are an ideal candidate platform for the identification of therapeutic targets counteracting Tau pathology excluding the variability associated with patient-derived cells (page 1273, right col. last para.). Verheyen teaches genetically engineered iPSCs comprising the double mutations of IVS10+16 (E10+16) and P301S of the MAPT gene and a method of forming neurons from these iPSCs (page364, left col. para. 3; page 369, right col. paragraph 1; page 376, left col. last paragraph and right col. paragraph 1; Supplemental experimental procedure). Verheyen teaches the double mutant neurons show hyperexcitability due to the P301S mutation, which is in line with studies showing neuronal hyperexcitability and epileptic seizures in P301S animal models and FTDP-17 patients with this mutation (page 374, left col. para. 4). Verheyen teaches a well-recognized characteristic of P301S-related FTDP-17 is tau hyperphosphorylation and the formation of NFTs (page 374, left col. last para.). Verheyen teaches patient-based evidence show the presence of tau aggregates and NFTs in P301S neurons (page 369, right col. last paragraph; page 374, left col. last paragraph). Verheyen teaches the P301S mutation was chosen to generate an aggressive FTDP-17 model (page 372, left col. paragraph 2). Verheyen teaches the double MAPT mutant could serve as a platform to identify new targets for drug development and to reveal the mechanisms underlying FTDP-17 (page 376, left col. paragraph 3). It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to modify the method of reference claim 1 to express a mutant MAPT gene comprising N279K, P3-1S, and E10+16 mutations as taught by Garcia-Leon and Verheyen for modeling FTDP-17 and drug screening of tauopathies as Garcia-Leon teaches mouse models have been generated overexpressing different human Tau variants only partially reproduce human Tau pathology and murine neurons appear to be less sensitive to neurodegeneration compared with human neurons and Garcia-Leon teaches the mutant neurons could be used for disease modeling and drug screening as the MAPT-mutant neurons are an ideal candidate platform for the identification of therapeutic targets counteracting Tau pathology excluding the variability associated with patient-derived cells and Verheyen teaches patient-based evidence show the presence of tau aggregates and NFTs in P301S neurons and the P301S mutation was chosen to generate an aggressive FTDP-17 model and Verheyen teaches the double MAPT mutant could serve as a platform to identify new targets for drug development and to reveal the mechanisms underlying FTDP-17.
This is a provisional nonstatutory double patenting rejection.
Applicant’s Arguments/ Response to Arguments
14. Applicant Argues: On page 6, last para. and page 7, Applicant asserts that the ratio of 4R tau/3R tau is less than 0.8 in Figure 2B and the ratio of 4R p-tau/3R p-tau is less than 1. Applicant asserts that a person of skill in the art would not have had a reason to deviate from Garcia-Leon’s method to instead perform a suspension culture as recited in the new claims and would not have expected success in doing so.
Response to Argument: This is not found persuasive because the claims require a ratio of 4R:3R phosphorylated tau to be at least 1 and Garcia-Leon teaches in Figure 2H and 2I using phospho-specific antibodies (AT270) that the ratio is at least 1 for the triple mutant (page 1263, right col. para. 2). Further, Garcia-Leon teaches the N279K and P301L mutations are characterized by elevated prevalence of 4R tau isoforms and the E10+16 mutation increases expression of 4R tau isoforms (page 1263, left col. para. 1).
Regarding suspension culturing required by new claim 23, Garcia-Leon and Verheyen teach methods of differentiating iPSCs containing MAPT mutations to neurons. However, Verheyen teaches an aberrant Wnt/Shh signaling pathway might cause the observed differences in neuronal cell types in their purely neuronal in vitro model and therefore might exacerbate the phenotypes and mechanisms that are potentially linked with the MAPT IVS10+16 mutation, as additional cell types and cellular interactions are lacking in their model (page 376, left col. para. 2).
Applicant Argues: On page 7, para. 1, Applicant disagrees with the statements and conclusion of nonstatutory double patenting and submits that these rejections do not apply to the new claims and that the new claims are distinguished from the cited claims for at least the reasons state in rebuttal of the 103 rejections.
Response to Argument: New provisional nonstatutory double patenting rejections of the new claims are set forth above over claim 3 of Application No. 16,984,241 and over claims 1 – 9 of Application No. 18/550,496 in view of the teachings of Garcia-Leon and Verheyen. The instant claims are obvious variants of the reference claims in view of the teachings of Garcia-Leon and Verheyen.
Conclusion
No claims allowed.
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/ZANNA MARIA BEHARRY/Examiner, Art Unit 1632