DETAILED ACTION
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 6/24/2026 has been entered.
Claim Status
As of the Final Office Action mailed 2/24/2026, claims 17, 20-21, 23-24, 26-29, 31-34, 54-56, 58-59, 62, 64, and 67-68, and 70 were pending.
In Applicant's Response filed on 6/24/2026, claims 17, 31, 55-56, 64, 67-68, and 70 were amended and claim 30 was canceled. Claims 71-72 were newly added.
The 103 rejection of record has been amended to address the new limitation regarding a crypt-villus architecture (recited in canceled claim 35).
As such, claims 17, 20-21, 23-24, 26-29, 31-34, 54-56, 58-59, 62, 64, and 67-68, and 70-72 are pending and have been examined herein.
Withdrawn Objections/Rejections
The objections and rejections presented herein represent the full set of objections and rejections currently pending in this application. Any objections or rejections not specifically reiterated are hereby withdrawn.
Claim Rejections - 35 USC § 112(d) – New Grounds of Rejections
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 71 and 72 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claims 71 and 72 recite, respectively, “wherein the isolating comprises isolating cells based on expression of ERBB4.” However, claim 17 and 70 (which claims 71 and 72 respectively depend on) recite “isolating from the MGE-type precursor cells, cells expressing ERBB4.” Claim 71 and 72 do not further limit the isolation step as instantly claimed in claims 17 and 70. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
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.
Claim(s) 17, 20-21, 23-24, 26, 28-29, 54, 58-59, 64, 67-68, and 70-72 is/are rejected under 35 U.S.C. 103 as being unpatentable over Studer et al (WO2014176606A1, 4/28/2014; published 10/30/2014; in IDS filed 11/21/2023) and Chen et al (PLOS One, May 2013 p.1-21; in IDS filed 8/18/2020) as evidenced by Nicholas et al (Cell Stem Cell. 2013 May 2; 12(5):573-86; Ref. 73 of NPL in IDS filed 8/18/2020).
Regarding claims 17, 54, and 70, Studer teaches an in vitro method for generating neurons through differentiation of human pluripotent or multipotent cells, said neurons comprising one or more markers indicating a cortical interneuron or a cortical interneuron precursor, the method comprising: contacting one or more starting cells selected from the group consisting of pluripotent cells, multipotent cells, and mixtures thereof with at least two inhibitors of SMAD and with at least one Wnt antagonist to favor generation of neuronal precursors from said pluripotent or multipotent cells or mixtures thereof; after the passage of a predetermined period of time, contacting the neuronal precursors with at least one activator of SHH signaling to favor generation from said precursors of neurons having at least one marker indicating a cortical interneuron or a cortical interneuron precursor; and optionally subjecting said generated interneurons or interneuron precursors to conditions favoring their maturation.
After permitting the isolated population of pluripotent cells to differentiate to immature interneuron precursors, a purified and enriched population of the immature interneuron precursors is recovered (para 226). The recovery procedure is preferably carried out by the promoter based separation procedure, utilizing an enhancer/promoter which functions only in the immature interneuron precursor cells and not other cell types, with cortical interneurons and precursors ascertained by molecular markers (para 226-227).
Studer continues to teach that cell type can be purified so that there is at least 90% reduction in amount of undesired cell types, resulting in an enriched population of cells (para 110-111) (i.e., less than 10% of enriched cells have undesired marker expression). The method can be used to produce cortical interneurons and their precursors in an amount and purity that cannot be obtained by the state of the art (para 220) (i.e., at least 50% of the cells of the invention express cortical interneuron or cortical interneuron precursor markers).
Regarding claims 17, 23, 55 and 56 in-part, the neuronal precursors comprise one or more markers selected from the group consisting of NKX2.1, LHX6, DLX1, DLX2, DLX5, DLX6, SOX6, MAFB, NPAS1, ASCL1, SIX6, OLIG2, and NKX6.2 (claim 31 of Studer).
Regarding claim 21, the immature interneuron precursor cells mature following transplantation and migration within the cerebral cortex of a mammalian subject (e.g., a human subject) (para 264 of Studer).
Regarding claim 20 and 24, Studer teaches a composition comprising in vitro differentiated neuronal cells comprising one or more markers indicating a cortical interneuron or a cortical interneuron precursor cell, wherein at least 50% of the interneurons are GABAergic interneurons (claim 35 and 38 of Studer).
Regarding claims 58-59, Studer teaches that the pluripotent stem cells are induced pluripotent stem cells and that the iPSCs are created by somatic cells reprogrammed to enter an embryonic stem cell-like fate (claim 6 of Studer and para 92).
Studer does not teach:
(i) isolating cells based on ERBB4 expression (relevant for instant claims 17 in-part, 70 in-part, and 71-72),
(ii) reduced LHX8 expression (relevant for instant claims 17 in-part, 64, 68 in-part, and 70 in-part).
(i) and (ii) Regarding isolating cells based on ERBB4 expression and MAF/MAFB expression, Chen teaches use of MGE enhancers for labeling and selection of MGE progenitors. MGE-GFP+ and ES-derived MGE-GFP+ cells both had relatively high expression (>10 arbitrary units) of MGE progenitor markers (Dlx1, Lhx6, Lhx8, Nkx2-1 and Sox6) and markers of immature MGE-derived pallial (i.e., cortical) interneurons (ErbB4, GAD1, Lhx6, MafB, Sox6, and Sst). A supervised heatmap (Figure 3; Table S9 in File S2) and an unsupervised clustering (Figure S12 in File S3) were developed to compare differences in global mRNA levels. The supervised heatmap showed that while the MGE Lhx6-GFP+ and ES Lhx6-GFP+ cells share commonalities (Table 1; Table S2 and Table S7 in File S2) in their RNA expression profiles, they also have differences. Importantly, both highly expressed genes that are known to be required for the development of MGE-derived cortical interneurons, including Arx, Dlx1, Dlx2, Dlx5, ErbB4, Foxg1, GAD1, Lhx6, Lhx8, Maf, MafB, Mef2C, Nkx2-1, Nrp1,and Sox6. (“Comparing RNA expression profiles between Lhx6-GFP+ MGE cells and ES-derived Lhx6-GFP+ cells” para 3).
On D10, about 50% of Nkx2-1+ cells were Lhx6-GFP+ (mean ± SEM: 48.9±3.8%, n = 3), whereas 70% of Lhx6-GFP+ cells were Nkx2-1+ (72.1±15.0%). On D12, 75% of Nkx2-1+ cells were Lhx6-GFP+ (75.3±12.9%), and 63% of Lhx6-GFP+ cells were Nkx2-1+.
Regarding claims 26, 28-29, 71, and 72, Table S2 shows select marker genes from MGE cells and the specific cell types and regions. MGE interneurons can be selected for expression of, e.g., CXCR4, CXCR7, ErbB4, Lhx6, Maf, MAFB, Sox6, etc. This shows that ErbB4+ cells are also at least Lhx6 and Maf/MafB+.
Chen continues to teach that since the ES-Lhx6-GFP+ cells expressed Nkx2-1 and Lhx8 RNAs (Table 1), they probably correspond to cells that can differentiate into several lineages of MGE-derived neurons, including pallial interneurons, striatal interneurons and the globus pallidus neurons. However, the gene expression array data showed lower expression of markers of globus pallidus neurons (e.g. ER81; Table 1; Table S2 in File S2); therefore, Chen postulates that the ES-Lhx6-GFP+ cells are most similar to bi-potential (pallial and striatal) immature interneurons. Furthermore, Chen suggests that these cells do not differentiate into subpallial cholinergic neurons because they have low expression of Islet1 and Gbx2 based on immunofluorescence (Figure 2H) and array data (Table S2 in File S2). Finally, they found higher expression of MGE-derived cortical interneuron markers MafB and cMaf (Table 1) in the ES-Lhx6-GFP+ cells, providing evidence that they are biased towards pallial (i.e., cortical) GABAergic interneurons.
Regarding claim 68 in-part, Chen shows the cells derived from embryonic stem cells (isolated and expressing ErBB4) became cortical interneurons after transplantation.
(iii) Regarding reduced LHX8 expression, the teachings of Nicholas are relied upon as providing evidence that most cortical interneurons are born in the MGE of the developing ventral telencephalon midgestation and, subsequently, undergo tangential migration into the neocortex (Introduction, para 1). The reference evidences that interneuron maturation occurs with the progressive acquisition of features that include increasing cell size and branching of processes, expression of subtype markers, formation of GABAergic synapses, ability to fire high-frequency trains of action potentials (APs), and development of more mature electrophysiological properties (same para). Fig. 1A shows schematic of hPSC-derived MGE-like progenitors and the pathways of differentiation into different neuronal subtypes. Of importance, Fig. 1A describes that cortical GABAergic interneurons develop from MGE and initially express NKX2.1, LHX6/8, and DLX, for example, and gradually express only LHX6, CXCR4/7, ARX, etc., while losing NKX2.1 expression. This shows that LHX8 and NKX2.1 expression in MGE cells is transient (also relevant for instant claim 64).
Regarding claim 67, Nicholas evidences that NKX2.1 is transiently expressed in MGE-like progenitor cells.
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to produce cortical interneuron precursor cells from human pluripotent stem cells that are isolated for particular cortical markers as taught by Studer, wherein the marker is ErBB4 as taught by Chen, to arrive at the instantly claimed invention. As Chen shows that cortical interneuron precursor cells can be isolated using a variety of markers, including ErbB4, one of ordinary skill would have been motivated to modify the isolation protocol of Studer to include isolation via ErbB4 expression with a reasonable expectation of advantageously obtaining cells that can become GABAergic interneurons when implanted into the central nervous system as taught by the prior art.
Claim(s) 27 and 32-33 is/are rejected under 35 U.S.C. 103 as being unpatentable over Studer et al in view of Chen et al as applied to claims 17, 20-21, 23-24, 26, 28-29, 31, 54, 58-59, 64, 66-68, and 70-72 above, and further in view of Cameron et al (BMC Neuroscience, 2012; 13:90, 1-23; previously cited).
The teachings of Studer and Chen were recited in the above 35 U.S.C. 103 rejection as applied to claim 17 of which claim 27 and 32-33 depend. The teachings will not be repeated here.
Neither Studer nor Chen teach:
that the neural precursor cells express PLEXINA4 (instant claim 27),
further depleting the population of NPCs with low expression of cortical interneuron markers upregulated in NPCs (claim 32), and
depleting the population of NPCs of cells expressing ATP1A2, BCAN, CD271, CD98, CNTFR, EGFR, FGFR1, FGFR2, FGFR3, GJA1, MLC1, NOTCH1, NOTCH3, PDGFRB, PDPN, PLXNA4, PROM1, PTPRZ1, SLC1A3, SLC1A5, TMEM158, or TTYH1.
Regarding (i), (ii) and (iii), Cameron teaches Clustered QISPs of transcripts expressed in excitatory cortical neurons and discloses expression of plexin A4 (PLXNA4) (see figure 6).
Cameron continues to teach that the study provides a rigorously validated list of transcripts expressed by immature excitatory neurons that have been grouped by in situ expression pattern for understanding how specific genes, and functional networks of such genes, control neuronal development and contribute to pathology (pg. 19, col. 2, last par.). Cameron teaches Plxna4 mRNA expression functional groupings of QISPs based on Gene Ontology molecular function and biological process annotations (graphical representation in Figure 4). Following transcriptional profiling, genes are sorted by RMA (robust multichip average) and their level of fold up- or down-regulation are compared to the FACS GFP-population. Verification of up-regulated genes is accomplished with quantified in situ hybridization (QISP), and the verified genes hierarchically clustered (see Methods) (Figure 1). This shows that excitatory cortical neurons express PLXNA4, which have opposite function to GABAergic cortical neurons (i.e, inhibitory cortical neurons), which provides one of ordinary skill motivation to perform the depleting step as claimed in instant claim 32 and 33 with a reasonable expectation of success to ensure production of inhibitory cortical neurons.
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to create cortical interneuron precursor cells as taught by Studer and Chen in combination, where the population is depleted of cells expressing PLXNA4 as taught by Cameron, to arrive at the instantly claimed invention. As Cameron shows PLXNA4 is expressed by excitatory cortical interneurons, one of ordinary skill would have been motivated to further isolate the cells of Studer and Chen in combination to exclude cells expressing PLXNA4 (depleting PLXNA4+ cells) as taught by Cameron with a reasonable expectation of advantageously having only cells capable of becoming cortical inhibitory neurons (i.e., GABAergic cortical neurons) as taught by the prior art.
Claim(s) 34 and 62 is/are rejected under 35 U.S.C. 103 as being unpatentable over Studer et al in view of Chen et al as applied to claims 17, 20-21, 23-24, 26, 28-29, 31, 54, 58-59, 64, 66-68, and 70-72 above, and further in view of Nicholas et al (WO2014153230 A1, 3/14/2014; published 9/25/2014; previously cited).
The teachings of Studer and Chen were recited in the above 35 U.S.C. 103 rejection as applied to claim 17 of which claim 34 and 62 depend. The teachings will not be repeated here.
Neither Studer nor Chen teach:
cryopreserving the neural precursor cells, and
not using enhancer-promoter reporter gene to generate the population of neural precursors.
Regarding (i) and (ii), Nicholas teaches a method of producing medial ganglionic eminence precursor cells from primate pluripotent stem cells comprising culturing the cells in a serum free medium comprising an activator of sonic hedgehog pathway and a neural inducing supplement to generate the MGE precursor cells (see claim 1 of Nicholas) where the PS cells are human pluripotent stem cells (see claim 5 of Nicholas).
Regarding claim 34, Nicholas teaches that the cells can be cryopreserved for a period of 1 day to 10 years (para 205).
Regarding claim 62, MGE precursors can be isolated and enriched using an affinity tag that is specific for the cell, where the affinity tags can be binding agents that are specific to a marker molecule (para 187). The affinity reagent can recover a substantially pure population of cells of interest (para 190).
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to create cortical interneuron precursor cells as taught by Studer and Chen in combination, where binding agents are used to enrich for specific cell type as taught by Nicholas, to arrive at the instantly claimed invention. Nicholas shows that affinity tags can successfully be used to sort MGE precursor cells, one of ordinary skill would have been motivated to simply substitute that reporter gene sorting of Studer with the affinity tag binding of Nicholas with a reasonable expectation of advantageously obtaining a substantially pure population of cells of interest as taught by the prior art. One of ordinary skill in the art would recognize that the improvements caused by the use of affinity tags would improve similar purification methods in the same way.
Response to Arguments
Applicant arguments and the Declaration by Dr. Nicholas (“Nicholas Declaration” herein) have been fully considered but are not persuasive. The arguments related to previously cited references Miyoshi and Mei have been considered but are moot because the new ground of rejection does not rely on these references applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
On p. 9-12 of Remarks, Applicant argues, in sum, that the examiner utilizes impermissible hindsight and that one of ordinary skill would not look to the recited combination of LHX6 and MAF and/or MAFB. Applicant also argues that Nicholas and Nicholas 2 disclose that the cells have multi-lineage potential as purportedly evidenced by continued LHX8 expression. Applicant argues that ErbB4 expression alone is not specific to MGE cortical interneurons and one of ordinary skill would not find a reason to look to ErbB4 for generating the instantly claimed population of cells. Applicant argues that a combination of markers together specifically identify neural precursor cells that give rise to cortical interneurons. Applicant presents the Nicholas Declaration to support the contention that the present claims address technical solution to obtain neural precursor cells from MGE-type culture to produce cortical interneurons via ERBB4 expression, co-expression of LHX6 and MAF/MAFB and depleted LHX8 that successfully gave rise to GABAergic cells in vitro and when transplanted into a host brain.
While Applicant’s argument has been fully considered, it is not found persuasive. First, it should be noted that the claims merely require “isolating . . . cells expressing ERBB4” to result in the in vivo population of instantly claimed cells. The LHX6/MAF/MAFB expression as claimed is a resultant of the isolation step. As described above, Nicholas already describes isolation of cells based on expression of particular surface markers. Chen identifies a multitude of markers, including ERBB4, that can be used to label and select MGE-progenitors to “follow and purify MGE-like derivatives . . ., including GABAergic cortical interneurons.” The cells capable of becoming GABAergic interneurons as described by Chen also expressed LHX6, Nkx2-1, CXCR4, CXCR7, DLX1, DLX2, and others (see Chen disclosure; please note that the ones here listed are required by the instant claims). These teachings provide sufficient teachings, suggestions, and motivations to use any known markers as taught by Chen, including ERBB4, for MGE precursors capable of becoming cortical interneurons when transplanted. Nicholas 2 evidences that Lhx8 is a transiently expressed marker (i.e., depending on when the cells are isolated, Lhx8 may or may not be expressed within a particular population of cells). Applicant’s claimed isolation based on particular markers was known prior to the effective filing date, especially in view of the combination of Nicholas and Chen.
On p. 12 of Remarks, Applicant argues that the rebuttal evidence provided in the previous Office action (Cunningham et al) does not show that the transplanted MGE cells developed into cortical interneurons. Applicant argues that the NKX2.1 expressed by the isolated cells of Cunningham is not an MGE marker on its own. Applicant argues that Cunningham does not provide adequate guidance as to the type of GABAergic interneurons arise from the transplanted MGE cells. Applicant also argues that the contribution of cortical interneurons to the therapeutic effect of transplanting the MGE cells in treating epilepsy is not clear and that the cells of Cunningham develop slower after transplantation than the instantly claimed cells.
This argument is also not found persuasive. The Cunningham reference was not relied upon for rejecting the instant claims; rather, it was cited to rebut Applicant’s purported unexpected results of engraftment, migratory ability, and fast development (which, the examiner notes is not commensurate with the scope of the instant claims). Cunningham acknowledges that MGE cells derived from human embryonic stem cells can become GABAergic interneurons when transplanted, “migrate extensively, and integrate into dysfunctional circuitry.” It is important to note that GABAergic interneurons are one of two major class of cortical interneurons (the other being excitatory neurons that utilize glutamate). However, even if Applicant disagrees that GABAergic interneurons are cortical interneurons, the Chen reference above also shows that the MGE precursor cells, when transplanted, develop into cortical interneurons. The relevancy of the therapeutic effect of the transplanted MGE cells in treating epilepsy as argued to the instant claims is unclear as Applicant is only claiming a method of generating cortical interneurons via transplantation. Thus, Applicant’s arguments are not persuasive.
Applicant states that because Cameron was cited for teaching PLEXINA4 expression, it fails to cure the purported deficiencies of the disclosures of Nicholas and Nicholas 2. As stated above, the instant claims are rendered prima facie obvious over Studer and Chen (as evidenced by Nicholas 2). The Cameron reference provides sufficient teachings and motivations to isolate cells not expressing PLEXINA4 (which is expressed in excitatory interneurons, not GABAergic interneurons). Thus, this argument is not persuasive.
Conclusion
No claim is allowed.
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/G.R./Examiner, Art Unit 1632
/MARCIA S NOBLE/Primary Examiner, Art Unit 1632
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