DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicant's amendments to the claims filed on 09-17-2025 have been received and entered. Claims 1-2, 4, 6, 9-15, 21, 27 have been amended. Claims 3, 7-8, 16-20, 22, 25-26, 28-34, 37-38 have been canceled. Claim 39 is added. Claims 1-2, 4-6, 9-15, 21, 23-24, 27, 35-36, 39 are pending.
Election/Restrictions
Applicant’s election of Group I: claim(s) 1-2, 4-6, 9-16, 20-21, 23-24, 27 in the reply filed on 01-23-2025 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claims 35-36 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected subject matter there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 01-23-2025.
Claims 1-2, 4-6, 9-15, 21, 23-24, 27, 39 are under consideration.
Priority
This application is a 371 of PCT/AU2019/051296 filed on 11/26/2019.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 06-17-2025 in compliance with the provisions of 37 CPR 1.97. Accordingly, the information disclosure statements have been considered by the examiner.
Claim Objections
Claims 21 and 27 are objected to because of the following informalities:
Claim 21 recites “one or more” and, in the second bullet, says that wherein a primed pluripotent state comprises expression of up to “15” or “all” of the markers selected from the group consisting of 15 different markers. The two terms “15” and “all” are redundant here because there are 15 markers recited in the Markush group.
Claim 27, line 6 recites “…(KLF4) and MYC of variants thereof…”. It should read “…(KLF4) and MYC, or variants thereof…”.
Claim 27, line 8 lacks an “are” between the words “factors” and “OCT4”.
Appropriate correction is required.
Maintained in modified form -Claim Rejections - 35 USC § 112- necessitated by amendments
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-2, 4-6, 9-15, 21, 23-24, 27, and 39 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
In analyzing whether the written description requirement is met for the genus claim, it is determined whether a representative number of species have been sufficiently described by other relevant identifying characteristics, specific features and functional attributes that would distinguish different members of the claimed genus. To satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention. See, e.g., Moba, B. V. v. Diamond Automation, Inc., 325 F.3d 1306, 1319, 66 USPQ2d 1429, 1438 (Fed. Cir. 2003); Vas-Cath, Inc. v. Mahurkar, 935 F.2d at 1563, 19 USPQ2d at 1116. An applicant shows possession of the claimed invention by describing the claimed invention with all of its limitations using such descriptive means as words, structures, figures, diagrams, and formulas that fully set forth the claimed invention. Lockwood v. Amer. Airlines, Inc., 107 F.3d 1565, 1572, 41 USPQ2d 1961, 1966 (Fed. Cir. 1997). Possession may be shown in a variety of ways including description of an actual reduction to practice, or by showing that the invention was "ready for patenting" such as by the disclosure of drawings or structural chemical formulas that show that the invention was complete, or by describing distinguishing identifying characteristics sufficient to show that the applicant was in possession of the claimed invention. See, e.g., Pfaffv. Wells Elecs., Inc., 525 U.S. 55, 68, 119 S.Ct. 304,312, 48 USPQ2d 1641, 1647 (1998); Eli Lilly, 119 F.3d at 1568, 43). USPQ2d at 1406; Amgen, Inc. v. Chugai Pharm., 927 F.2d 1200, 1206, 18 USPQ2d 1016, 1021 (Fed. Cir. 1991).
The claims encompass a genus of a method of producing any induced pluripotent stem (iPSC), the method comprising (a) culturing any somatic cell in any first culture condition adapted to promote the reprogramming of the cell towards a pluripotent state; (b) culturing the cell in any second culture condition adapted to promote any hypomethylated DNA state, wherein the culturing in the second culture condition is for a period of time that is insufficient to allow the cell to achieve an established naive pluripotent state; and (c) culturing the cell in any third culture condition adapted to promote a primed pluripotent state, thereby producing an iPSC from a somatic cell. Additionally, the method further comprise: (a) increasing the protein expression or amount of any factors in the somatic cell, wherein the factors are for reprogramming the somatic cell towards a pluripotent state; (b) culturing the cell in any first culture medium, for a sufficient time and under conditions to allow the reprogramming of the cell towards a pluripotent state; (c) culturing the cell in any second medium adapted to induce any hypomethylated DNA state, for a sufficient time and under conditions to reset any epigenetic profile of the cell; and (d) culturing the cell in any third culture medium adapted to induce a primed pluripotent state, for a sufficient time and under conditions to convert the cell to a primed pluripotent state.
The instant disclosure exemplifies the reprogramming of primary adult human dermal fibroblasts to iPSCs by transducing viruses consisting of four transcription factors, OCT4, SOX2, cMYC, KLF4 to generate dome-shaped colonies of iPSCs and media switching from naive to primed can be performed without splitting the cells (see example 1, page 46). Example 2 describes “Correction of epigenome in established iPSCs ("naive-to-primed protocol")”, the steps in example 1 are essentially repeated in example 2 with additional final step of harvesting Naive iPSCs and the medium was switched to Essential 8; however, there is no further description of “Correction of epigenome” (see example 2, page 47-48). Example 3 describes the use of whole genome bisulfite sequencing (by MethylC-seq) and DNA methylation analyses for epigenetic profiling of iPSCs at different stages of reprogramming (see example 3, page 48-50). Example 4 describes the differentiation of naïve to primed iPSCs to generate neural stem cell (NSC) (see example 4, page 51-54)
1. While somatic cell types can be reprogramed into induced pluripotent stem cells, certain cell exhibit resistance to reprograming due to various intrinsic and extrinsic factors:
Tang et al (F1000Research 2014, 3:102, doi: 10.12688/f1000research.4092.1) teach transient acid treatment cannot induce neonatal somatic cells to become pluripotent stem cells (Title). Tang et al discuss that it was possible to induce adult fibroblasts into pluripotent stem cells using four factors: Oct3/4, Sox2, c-Myc and Klf4. However, the method for generating iPS cells involves complex genetic manipulation and it is beset by low efficiency of conversion and yield (Page 3, left column, 1st para). Tang et al have attempted to create stimulus-triggered acquisition of pluripotency (STAP) stem cells using most updated protocol: Tang et al isolated CD45 splenocytes from five-day-old Oct4-GFP mice and treated the cells with acidified (pH 5.7) Hank’s Balanced Salt Solution (HBSS) for 25 min. Tang et al found that this method did not induce the splenocytes to express the stem cell marker Oct4-GFP when observed under a confocal microscope three to six days after acid treatment. Tang et al concluded that they have not been able to produce stimulus-triggered acquisition of pluripotency (STAP) stem cells from neonatal splenocytes or lung fibroblasts using the acid-based treatment (Abstract).
Nefzger et al (Cell Reports 21, 2649–2660, 2017, Doi: 10.1016/j.celrep.2017.11.029) teach cell type of origin dictates the route to pluripotency (title). Nefzger et al reveal limitations for the use of fibroblasts as a universal model for the study of the reprogramming process and provide crucial insights about iPSC generation from alternative cell sources (Abstract). Nefzger et al discuss that the molecular events that underpin the reprogramming process of different cell types are still largely unknown, and to what extent aspects of reprogramming are universal or cell-type specific has not been properly addressed yet. For example, although a variety of human cell types pass through a primitive streak-like state late during reprogramming, whether this applies across species boundaries and to other cell types is still unclear. Nefzger et al show that the nuclear reprogramming process of somatic cells into the induced pluripotent state has a universal and cell-type-specific component, underscoring the complexity of such processes (Page 2649, bridging 1st para left column to right column). Nefzger et al unveils a large cell-type-specific component to the reprogramming process, which is partially the consequence of a restricted developmental reversion determined by the cell type of origin. Accordingly, early reprogramming intermediates have distinct cell-type-specific molecular signatures, a finding with possible implications for direct reprogramming strategies using transient OKSM expression. By revealing universal and cell-type-specific components of the reprogramming process, their findings show limitations for the use of only fibroblasts as a sole model for its study (Page 2657, right column, last para).
2. The claims also read on using any culture condition/medium to promote any hypomethylated DNA state. However, remodeling DNA methylation during the reprogramming of somatic cells by using any culture condition/medium containing various factors to induce pluripotent stem cell presents several challenges:
Planello et al (Cell Regeneration 2014, 3:4, doi:10.1186/2045-9769-3-4) discuss that the conversion of somatic cells into pluripotent stem cells via overexpression of reprogramming factors involves epigenetic remodeling. DNA methylation at a significant proportion of CpG sites in induced pluripotent stem cells (iPSCs) differs from that of embryonic stem cells (ESCs). Whether different sets of reprogramming factors influence the type and extent of aberrant DNA methylation in iPSCs differently remains unknown. In order to help resolve this critical question, we generated human iPSCs from a common fibroblast cell source using either the Yamanaka factors (OCT4, SOX2, KLF4 and cMYC) or the Thomson factors (OCT4, SOX2, NANOG and LIN28), and determined their genome-wide DNA methylation profiles. In addition to shared DNA methylation aberrations present in all our iPSCs, we identified Yamanaka-iPSC (Y-iPSC)-specific and Thomson-iPSC (T-iPSC)-specific recurrent aberrations. Strikingly, not only were the genomic locations of the aberrations different but also their types: reprogramming with Yamanaka factors mainly resulted in failure to demethylate CpGs, whereas reprogramming with Thomson factors mainly resulted in failure to methylate CpGs. Differences in the level of transcripts encoding DNMT3b and TET3 between Y-iPSCs and T-iPSCs may contribute partially to the distinct types of aberrations. Finally, de novo aberrantly methylated genes in Y-iPSCs were enriched for NANOG targets that are also aberrantly methylated in some cancers. Our study thus reveals that the choice of reprogramming factors influences the amount, location, and class of DNA methylation aberrations in iPSCs (Abstract).
Bar et al (The EMBO Journal 38: e101033 | 2019, DOI 10.15252/embj.2018101033) teach that DNA methylation aberrations were also observed to be highly induced upon reprogramming of human somatic cells to iPSCs. This process encompasses substantial epigenetic changes, transforming the chromatin landscape of a differentiated cell to that of an undifferentiated one by inducing the expression of key pluripotency genes, thus leading to broad changes in the overall transcription pattern. Accordingly, the efficiency of reprogramming was found to be inversely correlated with the extent of differences in CpG methylation between the somatic cell-of-origin and hPSCs. Correspondingly, many reported hiPSC lines were insufficient in completely erasing their somatic identity, thus carrying residual methylation of their source cells in various regions. This resulted in an epigenetic memory at distinct regions which bear differential methylation between hESCs and hiPSCs. Additional methylation alterations, which are not found in either the normal somatic source cells or hESCs, are acquired during reprogramming and vary between different hiPSC lines (Page 3, bridging last paragraph in left column to right column).
3. The claims read on any culture condition/medium that can be used to produce an iPSC from a somatic cell. However, there are various reprogramming barriers to iPSC generation:
Haridhasapavalan et al (Stem Cell Reviews and Reports (2020) 16:56–81, Doi: 10.1007/s12015-019-09931-1) stated that achieving a pluripotent state in most of the reprogramming studies is marred by serious limitations such as low reprogramming efficiency and slow kinetics. These limitations are mainly due to the presence of potent barriers that exist during reprogramming when a mature cell is coaxed to achieve a pluripotent state. Several studies have revealed that intrinsic factors such as non-optimal stoichiometry of reprogramming factors, specific signaling pathways, cellular senescence, pluripotency-inhibiting transcription factors and microRNAs act as a roadblock. In addition, the epigenetic state of somatic cells and specific epigenetic modifications that occur during reprogramming also remarkably impede the generation of iPSCs (Abstract). Transcription factors play a crucial role in the induction and maintenance of pluripotency in mouse and human cells. On the contrary, expression of specific transcription factors can either decrease the reprogramming efficiency or even block the reprogramming process (Page 58, left column, 3rd para): c-Jun completely blocked, rather than promoted, reprogramming of mouse embryonic fibroblasts (MEFs) to iPSCs (Page 58, right column, 2nd para); Tcf3 has been identified as a negative regulator of reprogramming, and genetic ablation of this gene in neuronal precursor cells strongly augmented the reprogramming efficiency (Page 58, right column, 4th para); Similarly, Gata4, Zfp281 and Patz1 were also reported to inhibit somatic cell reprogramming (Page 60, left column, 3rd para.); In addition to these, other transcription factors that act as reprogramming roadblocks were also identified by performing knockdown experiments but their biological function from a reprogramming perspective is not yet investigated (Table 2) (Page 60, right column, 3rd para.)
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Haridhasapavalan et al also teach senescence serves as another major barrier to cellular reprogramming as cells lose the capacity to proliferate and divide. It occurs as a result of oxidative stress, DNA damage, telomere shortening and the derepression of Ink4a/ Arf locus by chromatin remodeling in somatic cells (Fig. 2) (Page 62, right column, 2nd para.)
Haridhasapavalan et al also teach there are specific miRNAs that impede the reprogramming process (Fig. 3). Various miRNAs highly expressed in specialized cells post-transcriptionally regulate proteins that serve as reprogramming barriers. Therefore, depletion of these miRNAs in specialized cells in a reprogramming set-up can result in improvement in reprogramming efficiency and kinetics (Page 64, right column, last para).
Haridhasapavalan et al also teach the conversion of a differentiated cell to a pluripotent cell involves resetting of the global epigenome. The epigenetic state in somatic cells and the specific epigenetic modifications that occur during reprogramming specify lineage-specific programs rather than the induction of pluripotency, and thereby act as a roadblock to efficient reprogramming (Page 66, left column, 2nd para). DNA methylation act as a barrier and DNA demethylation of pluripotency-related genes is indispensable for the generation of iPSCs (Fig. 4). In addition to DNA methylation, specific chromatin modifications disturb the ability of the reprogramming factors to bind to their target sites and affect iPSC formation (Fig. 5) (Page 66, right column, para 2-3).
4. The amended claims read on any culture condition/medium that can convert cells in any pluripotent state to primed pluripotent state. However, converting any pluripotent stem cells such as naïve pluripotent stem cells to primed state or vice versa using any culture condition/medium presents several challenges primarily due to differences in signaling dependencies, epigenetic landscapes and metabolic profiles between these states:
Kim et al (Stem Cell Research & Therapy (2022) 13:329, Doi: 10.1186/s13287-022-02976-z) teach dichotomous role of Shp2 for naïve and primed pluripotency maintenance in embryonic stem cells (title). Shp2 serves as a negative regulator for naïve pluripotency due to its dual roles in Jak/Stat3 and Ras-to-Erk signaling, whereas the pluripotency of primed cells depended on bFGF/Activin, which could also be modulated by Shp2- dependent signaling. Allosteric inhibition of Shp2 with an inhibitor to disrupt these dual functions can therefore be used to improve naïve pluripotency and replace the use of iMek1 (Page 10, left column, 3rd para). Thus, culture condition/medium that leads to suppression/inhibition of Shp2 would not be suitable to promote a primed pluripotent state.
Takahashi et al (Cellular and Molecular Life Sciences (2018) 75:1191–1203, Doi: 10.1007/s00018-017-2703-x) teach that major epigenetic marks as we know them today can be subdivided into two types: histone modifications and DNA methylation. Histone modification patterns are distinct between naïve and primed cells. However, it is difficult to describe all of the differences in histone modifications concisely and pinpoint the ones that are critical. Moreover, it remains controversial whether histone modifications are a cause or a consequence of gene expression patterns. Distinct histone modification patterns on gene promoters may simply reflect their distinct transcription states (Page 1193, left column, 2nd para). DNA methylation also varies across the naïve and primed states; surprisingly however, this difference is observed only in vitro and not in the in vivo counterparts of these cell types; it has been shown that the mouse epiblast cells are globally DNA hypomethylated, both pre- and post-implantation. Thus, any epigenetic difference between mESCs and mEpiSCs may not readily translate to a difference between naïve and primed states in vivo. It is also important to note that a cause–effect relationship has not been established. DNA methylation is enriched in repressed genes in mEpiSCs, but this could merely reflect gene repression (Page 1193, left column, 4th para). Therefore, specific culture condition/medium would be needed for specific epigenetic landscapes of between naïve and primed states.
Takahashi et al also teach that naïve mESCs rely on both anaerobic (glycolytic) and aerobic (mitochondrial) respiration, while primed mEpiSCs rely almost exclusively on glycolysis that lead to the discovery of the role of α-ketoglutarate, a TCA (tricarboxylic acid) cycle intermediate, in maintaining naïve pluripotency through promoting histone/DNA demethylation, while accelerating differentiation of primed mouse EpiSCs and human ESCs (Page 1197, right column, bridging last paragraph to page 1198). Thus, various culture condition/medium would need to be tested in order to confirm the conversion of naïve pluripotent stem cells to primed state.
The specification lacks sufficient variety of species of any somatic cell and any culture condition/medium to reflect this variance in the genus showing any somatic cell and any culture condition/medium can be used in reprogramming of the somatic cell towards a naïve pluripotent state and primed pluripotent state. The specification does not provide sufficient descriptive support for the myriad of variant embraced by the claims.
Overall, what these statements indicate is that the Applicant must provide adequate description of such various somatic cells and compositions/functions of various culture condition/medium related to reprogramming of the various somatic cell towards a pluripotent state such that the Artisan of skill could determine the desired effects/functions of the claimed various culture condition/medium on various somatic cells. Hence, the analysis above demonstrates that Applicant has not determined the desired effects/functions of the broadly claimed various culture condition/medium on various somatic cells for full scope of the claims.
The skilled artisan cannot envision the detailed compositions/functions of the encompassed any culture condition/medium on various somatic cells other than those described in the specification, and therefore conception is not achieved until reduction to practice has occurred, regardless of the complexity or simplicity of the method of preparation. Adequate written description requires more than a mere statement that it is part of the invention and reference to a potential method of preparing/using it. See Fiers v. Revel, 25 USPQ2d 1601, 1606 (Fed. Cir. 1993) and Amgen lnc. v.Chugai Pharmaceutical Co. Ltd., 18 USPQ2d 1016 (Fed. Cir. 1991). Thus, it is concluded that the written description requirement is not satisfied for the claimed genus of any culture condition/medium and any somatic cells.
Response to Arguments
Applicant's arguments filed on 09-17-2025 have been fully considered but they are not persuasive.
Applicants argue that the transient naive treatment (TNT) method can be applied to any cell that can be reprogrammed. In support of this, we submit Buckberry et al. (Nature 2023) (herein after "Buckberry") - which is the article, by the inventors, that describes the claimed invention - that shows that not only fibroblasts can be reprogrammed by the claimed method, but also keratinocytes, mesenchymal stem cells, and in vitro generated fibroblasts, thereby showing that the claimed method is generalizable to multiple distinct cell types originating from different germ layers. See Figure 5 (replicated below).
In particular part (a) which describes the experimental design for multi-lineage primed and TNT reprogramming and differentiation into five cell types. The top panel describes the four somatic cell lines reprogrammed into primed-hiPS cells and TNT-hiPS cells with three independent reprogrammings (rl-r3) performed per group, and with each subsequently differentiated into five different cell types, with independent replication. In the bottom panel, the number of independent differentiation replicates performed for origin cell types (rows) and differentiated cell types (columns). Coloured circles represent primed-hiPS cell (green), TNT-hiPS cell (yellow) and hES cell (grey), 2° fibroblasts, secondary fibroblasts (Remarks, page 8).
Response to Arguments:
Applicants asserts that any cell that can be reprogrammed and submitted Buckberry et al to state that the four somatic cell lines reprogrammed into primed-hiPS cells and TNT-hiPS cells with three independent reprogramming (rl-r3) performed per group. However, the claimed invention and the cited reference Buckberry et al as a whole is not adequately described to be commensurate with the scope of the claim: The cited reference Buckberry et al teach that “hiPS cell lines that were reprogrammed from primary human dermal fibroblasts (HDFs), keratinocytes (NHEK cells), mesenchymal stem cells (MSCs) and our hES cell-derived isogenic secondary fibroblasts to comprehensively test for differences in primed and TNT-hiPS cell differentiation capacity (Fig. 5a).” (see Buckberry et al page 871, left column, 1st para. ) . Thus, there are 3 types of somatic cells and 1 mesenchymal stem cells, and among somatic cells, 2 of them are fibroblasts. The base claim 1 reads on any somatic cells which encompass all the cells in body that aren't sperm or egg cells (gametes), making up your tissues and organs, with examples including skin cells, muscle cells (myocytes), nerve cells (neurons), blood cells (red, white), bone cells, hair cells, and lung cells. Applicants argue that any cell that can be reprogrammed; however, somatic cells are diverse functionally and structurally. For example, red blood cells do not have nucleus and function to deliver oxygen while neurons are cell body (soma) containing the nucleus, branch-like dendrites to transmit information throughout the body using electrical and chemical signals. There is no evidence on the record that the any somatic cells have structural relationships to each other or have the same functions or behave/require the same culture medium composition. Thus, the claimed invention as a whole is not adequately described to be commensurate with the scope of the claim.
Additionally, Buckberry et al teach a very specific transient-naive-treatment (TNT) reprogramming strategy with specific timing to reprogram cells: “By combining our new understanding of epigenomic reconfiguration during reprogramming, we hypothesized that we could avoid somatic cell epigenetic memory and aberrant DNA methylation by reprogramming through a transient naive-like state, similar to the demethylation observed during embryonic development. Thus, we devised two experimental systems. In the first system, we reprogrammed fibroblasts with a transient naive culture treatment for 5 days after the initial 7 days of culturing in fibroblast medium, followed by culturing in primed medium for the remainder of the reprogramming (Fig. 3a), to give rise to transient-naive-treatment hiPS cells (TNT-hiPS cells). In the second system, we first established naive-hiPS cell colonies by extended naive culturing and then transitioned the cells to a primed pluripotent state to give rise to naive-to-primed hiPS cells (NTP-hiPS cells) (Fig. 3a).”. Additionally, Buckberry et al teach a very specific medium composition (see below and first page of the method).
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Thus, the cited reference Buckberry et al still lacks sufficient variety of species to reflect variance in the genus of any somatic cell and the genus of any first, second and third culture conditions showing contemplated biological activity and outcomes of (a) culturing a somatic cell in a first culture condition adapted to promote the reprogramming of the cell towards a pluripotent state; (b) culturing the cell in a second culture condition adapted to promote a hypomethylated DNA state, wherein the culturing in the second culture condition is for a period of time that is insufficient to allow the cell to achieve a pluripotent state; and (c) culturing the cell in a third culture condition adapted to promote a primed pluripotent state, thereby producing an iPSC from a somatic cell.”. The skilled artisan cannot envision the detailed medium composition other than those described in the specification, and therefore conception is not achieved until reduction to practice has occurred.
Also, it is noted that the features upon which applicant relies in the arguments (i.e., the transient naive treatment (TNT) method) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
2. Applicants argue that the claims only define culture conditions/media that can support a naive or primed state, and only somatic cell types that can be reprogrammed to iPSCs. i.e. conditions/media that cannot sustain primed or naive states are not relevant to our claims, and cells that cannot be reprogrammed are not relevant to our claims
In the inventors' paper (Buckberry) they demonstrate that a broader set of conditions/medium and cell types are compatible with their epigenetic reset method. Specifically, the inventors showed that the TNT method works for multiple primed media formulations (E8 and KSR), both media described and defined in the specification as filed (see for example, Table 2 last row - "Primed media")(Remarks, page 10).
Response to Arguments:
Base claim 1 recites “A method of producing an induced pluripotent stem cell (iPSC), the method comprising the following steps in order: (a) culturing a somatic cell in a first culture condition adapted to promote the reprogramming of the cell towards a pluripotent state; (b) culturing the cell in a second culture condition adapted to promote a hypomethylated DNA state, wherein the culturing in the second culture condition is for a period of time that is insufficient to allow the cell to achieve a pluripotent state; and (c) culturing the cell in a third culture condition adapted to promote a primed pluripotent state, thereby producing an iPSC from a somatic cell.”. Thus, the claim recites 3 different generic culture medium/condition that are expected to have 3 specific outcomes: (i) the first medium is “adapted to promote the reprogramming of the cell towards a pluripotent state”; (ii) the second medium is “to promote a hypomethylated DNA state” and “to allow the cell to achieve a pluripotent state”; (iii) the third medium is to “promote a primed pluripotent state”. Thus, these culture conditions/media play important role to achieve functional outcome required by the claim. There is no evidence in record that any somatic cell can be reprogramed by any first, second and third culture conditions to achieve functional outcomes as recited by the claim.
Applicant also argue that the inventors showed that the TNT method works for multiple primed media formulations (E8 and KSR), both media described and defined in the specification as filed. However, there is no recitation of any specific media and/or TNT method steps.
3. Applicants arguments attaching individual cited references Tang, et al; Nefzger, et al; Planello, et al, Bar et al; Haridhasapavalan et al; Kim et al; Takahashi et al are addressed below.
The Applicant's disclosure meets the written description requirement by providing sufficient representative examples, mechanistic insight, and predictable extensions enabling the skilled artisan to practice the invention without undue experimentation. the specification provides a general teaching that any somatic cell, any culture medium (meeting the defined requirements) and any combination of factors for reprogramming can be used (Remarks, page 10-14).
Response to Arguments:
While somatic cell types can be reprogramed into induced pluripotent stem cells, certain cell exhibit resistance to reprograming due to various intrinsic and extrinsic factors: Tang et al teach transient acid treatment cannot induce neonatal somatic cells to become pluripotent stem cells (Title). Tang et al discuss that it was possible to induce adult fibroblasts into pluripotent stem cells using four factors: Oct3/4, Sox2, c-Myc and Klf4. However, the method for generating iPS cells involves complex genetic manipulation and it is beset by low efficiency of conversion and yield (Page 3, left column, 1st para). Tang et al concluded that they have not been able to produce stimulus-triggered acquisition of pluripotency (STAP) stem cells from neonatal splenocytes or lung fibroblasts using the acid-based treatment (Abstract). Additionally, Nefzger et al reveal limitations for the use of fibroblasts as a universal model for the study of the reprogramming process and provide crucial insights about iPSC generation from alternative cell sources (Abstract). Nefzger et al discuss that the molecular events that underpin the reprogramming process of different cell types are still largely unknown, and to what extent aspects of reprogramming are universal or cell-type specific has not been properly addressed yet. For example, although a variety of human cell types pass through a primitive streak-like state late during reprogramming, whether this applies across species boundaries and to other cell types is still unclear. (Page 2649, bridging 1st para left column to right column). By revealing universal and cell-type-specific components of the reprogramming process, their findings show limitations for the use of only fibroblasts as a sole model for its study (Page 2657, right column, last para).
Remodeling DNA methylation during the reprogramming of somatic cells by using any culture condition/medium containing various factors to induce pluripotent stem cell presents several challenges: Planello et al discuss that “the conversion of somatic cells into pluripotent stem cells via overexpression of reprogramming factors involves epigenetic remodeling. DNA methylation at a significant proportion of CpG sites in induced pluripotent stem cells (iPSCs) differs from that of embryonic stem cells (ESCs). Whether different sets of reprogramming factors influence the type and extent of aberrant DNA methylation in iPSCs differently remains unknown …… Our study thus reveals that the choice of reprogramming factors influences the amount, location, and class of DNA methylation aberrations in iPSCs” (Abstract). Bar et al teach that DNA methylation aberrations were also observed to be highly induced upon reprogramming of human somatic cells to iPSCs. This process encompasses substantial epigenetic changes, transforming the chromatin landscape of a differentiated cell to that of an undifferentiated one by inducing the expression of key pluripotency genes, thus leading to broad changes in the overall transcription pattern. Accordingly, the efficiency of reprogramming was found to be inversely correlated with the extent of differences in CpG methylation between the somatic cell-of-origin and hPSCs. Correspondingly, many reported hiPSC lines were insufficient in completely erasing their somatic identity, thus carrying residual methylation of their source cells in various regions. This resulted in an epigenetic memory at distinct regions which bear differential methylation between hESCs and hiPSCs. Additional methylation alterations, which are not found in either the normal somatic source cells or hESCs, are acquired during reprogramming and vary between different hiPSC lines (Page 3, bridging last paragraph in left column to right column).
The claims read on any culture condition/medium that can be used to produce an iPSC from a somatic cell. However, there are various reprogramming barriers to iPSC generation. Haridhasapavalan et al stated that achieving a pluripotent state in most of the reprogramming studies is marred by serious limitations such as low reprogramming efficiency and slow kinetics. These limitations are mainly due to the presence of potent barriers that exist during reprogramming when a mature cell is coaxed to achieve a pluripotent state. Several studies have revealed that intrinsic factors such as non-optimal stoichiometry of reprogramming factors, specific signaling pathways, cellular senescence, pluripotency-inhibiting transcription factors and microRNAs act as a roadblock. In addition, the epigenetic state of somatic cells and specific epigenetic modifications that occur during reprogramming also remarkably impede the generation of iPSCs (Abstract).
The amended claims read on any culture condition/medium that can convert cells in any pluripotent state to primed pluripotent state. However, converting any pluripotent stem cells such as naïve pluripotent stem cells to primed state or vice versa using any culture condition/medium presents several challenges primarily due to differences in signaling dependencies, epigenetic landscapes and metabolic profiles between these states: Kim et al teach dichotomous role of Shp2 for naïve and primed pluripotency maintenance in embryonic stem cells (title). Shp2 serves as a negative regulator for naïve pluripotency due to its dual roles in Jak/Stat3 and Ras-to-Erk signaling, whereas the pluripotency of primed cells depended on bFGF/Activin, which could also be modulated by Shp2- dependent signaling. Allosteric inhibition of Shp2 with an inhibitor to disrupt these dual functions can therefore be used to improve naïve pluripotency and replace the use of iMek1 (Page 10, left column, 3rd para). Thus, culture condition/medium that leads to suppression/inhibition of Shp2 would not be suitable to promote a primed pluripotent state. Takahashi et al teach that major epigenetic marks as we know them today can be subdivided into two types: histone modifications and DNA methylation. Histone modification patterns are distinct between naïve and primed cells. However, it is difficult to describe all of the differences in histone modifications concisely and pinpoint the ones that are critical. Moreover, it remains controversial whether histone modifications are a cause or a consequence of gene expression patterns. Distinct histone modification patterns on gene promoters may simply reflect their distinct transcription states (Page 1193, left column, 2nd para). DNA methylation also varies across the naïve and primed states; surprisingly however, this difference is observed only in vitro and not in the in vivo counterparts of these cell types; it has been shown that the mouse epiblast cells are globally DNA hypomethylated, both pre- and post-implantation. Thus, any epigenetic difference between mESCs and mEpiSCs may not readily translate to a difference between naïve and primed states in vivo. It is also important to note that a cause–effect relationship has not been established. DNA methylation is enriched in repressed genes in mEpiSCs, but this could merely reflect gene repression (Page 1193, left column, 4th para). Therefore, specific culture condition/medium would be needed for specific epigenetic landscapes of between naïve and primed states. Takahashi et al also teach that naïve mESCs rely on both anaerobic (glycolytic) and aerobic (mitochondrial) respiration, while primed mEpiSCs rely almost exclusively on glycolysis that lead to the discovery of the role of α-ketoglutarate, a TCA (tricarboxylic acid) cycle intermediate, in maintaining naïve pluripotency through promoting histone/DNA demethylation, while accelerating differentiation of primed mouse EpiSCs and human ESCs (Page 1197, right column, bridging last paragraph to page 1198). Thus, various culture condition/medium would need to be tested in order to confirm the conversion of naïve pluripotent stem cells to primed state.
In summary, the specification lacks sufficient variety of species of any somatic cell and any culture condition/medium to reflect this variance in the genus showing any somatic cell and any culture condition/medium can be used in reprogramming of the somatic cell towards any pluripotent state to primed pluripotent state. The specification does not provide sufficient descriptive support for the myriad of variant embraced by the claims. Applicant’s arguments regarding the inventors’ findings in the specification are not commensurate with the scope of the claims. The skilled artisan cannot envision the detailed compositions/functions of the encompassed any culture condition/medium on various somatic cells other than those described in the specification, and therefore conception is not achieved until reduction to practice has occurred, regardless of the complexity or simplicity of the method of preparation.
Maintained in modified form and New - Claim Rejections - 35 USC § 103- necessitated by amendments
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.
Claims 1-2, 4-6, 9-15, 21, 23-24, 27 are rejected under 35 U.S.C. 103 as being unpatentable over Shalom-Feuerstein et al (WO 2019/135238 A1, International Publication Date: 11 July 2019) in view of Smith et al (WO 2018/138281 A1, International Publication Date: 02 August 2018).
Regarding to claim 1, Shalom-Feuerstein et al teach methods of generating pluripotent stem cells in a naive state (Abstract) (For the preamble).
Shalom-Feuerstein et al teach that to generate iPSCs, somatic cells are provided with reprogramming factors (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) known in the art to reprogram the somatic cells to become pluripotent stem cells ([047], page 11-12) (For claim 1(a)).
Shalom-Feuerstein et al teach the method of the invention further comprises culturing the cell that is not a pluripotent stem cell in a naive state in media comprising a RAS inhibitor for an amount of time sufficient for the cell to acquire at least one characteristic of a pluripotent stem cell in a naive state ([017], page 4). The characteristic is decreased expression relative to expression before the culturing, of DNA methyltransferase 3b (Dnmt3b) ([018], page 4). It is noted that Dnmt3b is a DNA methyltransferase responsible for methylation on DNA; thus, decreased expression of Dnmt3b and being in pluripotent stem cell in a naive state would promote a hypomethylated DNA state (For claim 1(b)).
Shalom-Feuerstein et al do not teach “culturing in the second culture condition is for a period of time that is insufficient to allow the cell to achieve an established a pluripotent state”. Smith et al cure the deficiency.
Smith et al teach propagating pluripotent stem cells in the "formative" state (abstract). Smith et al teach in one strategy for obtaining a fully competent human pluripotent stem cell, the precursor cell is transiently reset to naive status, then converted to formative status before erasure of imprints occurs in the naive state (Page 13, lines 11-13). The period in the naive state culture conditions may be transient but should be sufficient to achieve epigenetic erasure (hypomethylated DNA state) (Page 9, lines 17-19). Smith et al teach that “one advantageous embodiment of the invention for obtaining a fully competent human pluripotent stem cell involves transient derivation or resetting to naive status then conversion to formative status before erasure of imprints occurs in the naive state. FS cells may therefore be a superior or alternative source material for chimaera formation or directed differentiation compared with other forms of pluripotent stem cell. Formative pluripotent stem cells may be a desirable intermediate stage for population expansion prior to directed differentiation of human naive pluripotent stem cells.” (Page 6, lines 36-44). Since FS cells were “transient derivation or resetting to naive status”, “conversion to formative status before erasure of imprints occurs in the naive state” and “intermediate stage for population expansion prior to directed differentiation of human naive pluripotent stem cells” (Page 6, lines 36-44), the culture condition is insufficient for formative pluripotent stem cells to become Naïve cells (For claim 1(b)).
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the rejected claims to combine the teachings of prior art to modify the method of Shalom-Feuerstein et al by using pluripotent stem cells in the "formative" state with epigenetic erasure (hypomethylated DNA state) as taught by Smith et al as instantly claimed, with a reasonable expectation of success. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to do so because Smith et al teach that formative pluripotent stem cells is an attractive option for obtaining robust and unbiased cultures with imprints maintained (Page 13, lines 13-17) and the formative stem (FS) cells described have many utilities and potential advantages over existing pluripotent stem cell sources (Page 16, lines 15-16). One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Smith et al were successful in generation of formative stem cells that can be used to create primed cells (page 17 lines 29-40) and germ cells (page 17, last para).
Shalom-Feuerstein et al teach mESCs that were grown in naive (ground state) conditions (2i/LIF) were switched into primed state by cultivation the cells for 10 passages in the presence of knockout serum and basic fibroblast growth factor (KSR/bFGF) (Page 29, example 2). Additionally, Smith et al teach the uses of formative stem (FS) cells to create primed cells: In one aspect the invention provides a process for producing a primed pluripotent cell from an FS cell by withdrawal from FS cell culture medium and transfer to primed cell medium such as E8 for human primed cells or AhiF with or without XAV for mouse EpiS cells (Page 17, lines 29-32). Thus, both Shalom-Feuerstein et al and Smith et al teach conditions that promote a primed pluripotent state (For claim 1(c)).
Regarding to claim 2, Shalom-Feuerstein et al teach culturing the cell that is not a pluripotent stem cell in a naive state in media for an amount of time sufficient for the cell to acquire at least one characteristic of a pluripotent stem cell in a naive state: increased expression relative to expression before the culturing, of a naivety marker selected from octamer-binding transcription factor 4 (Oct4), Nanog, stage-specific embryonic antigen 1 (Sseal), kruppel-like factor 4 (Klf4), Stella and E-Cadherin (E-Cad) ([017]-[018], page 4) (For claim 2(a) and 2(b)).
Shalom-Feuerstein et al teach culturing the cell for an amount of time sufficient for the cell to acquire at least one characteristic of decreased expression relative to expression before the culturing, of a DNA methyltransferase 3b (Dnmt3b) ([017]-[018], page 4). In some embodiments, euchromatin is characterized by a decrease or low levels of DNA methylation ([058], page 4). Additionally, Smith et al teach methods for obtaining a fully competent human pluripotent stem cell: the precursor cell is transiently reset to naive status, then converted to formative status before erasure of imprints occurs in the naive state (Page 13, lines 11-13) . The period in the naive state culture conditions may be transient but should be sufficient to achieve epigenetic erasure (hypomethylated DNA state) (Page 9, lines 17-19). Thus, a person of ordinary skill in the art would be able to reset epigenetic profile of the cell during reprogramming of the cell towards a pluripotent state.
Smith et al teach propagating pluripotent stem cells in the "formative" state (abstract). Smith et al teach in one strategy for obtaining a fully competent human pluripotent stem cell, the precursor cell is transiently reset to naive status, then converted to formative status before erasure of imprints occurs in the naive state (Page 13, lines 11-13). The period in the naive state culture conditions may be transient but should be sufficient to achieve epigenetic erasure (hypomethylated DNA state) (Page 9, lines 17-19). Smith et al teach that “one advantageous embodiment of the invention for obtaining a fully competent human pluripotent stem cell involves transient derivation or resetting to naive status then conversion to formative status before erasure of imprints occurs in the naive state. FS cells may therefore be a superior or alternative source material for chimaera formation or directed differentiation compared with other forms of pluripotent stem cell. Formative pluripotent stem cells may be a desirable intermediate stage for population expansion prior to directed differentiation of human naive pluripotent stem cells.” (Page 6, lines 36-44). Since FS cells were “transient derivation or resetting to naive status”, “conversion to formative status before erasure of imprints occurs in the naive state” and “intermediate stage for population expansion prior to directed differentiation of human naive pluripotent stem cells” (Page 6, lines 36-44), the culture condition is insufficient for formative pluripotent stem cells to become Naïve cells (For claim 2(c)).
Shalom-Feuerstein et al teach mESCs that were grown in naive (ground state) conditions (2i/LIF) were switched into primed state by cultivation the cells for 10 passages in the presence of knockout serum and basic fibroblast growth factor (KSR/bFGF) (Page 29, example 2). Additionally, Smith et al teach the uses of formative stem (FS) cells to create primed cells: In one aspect the invention provides a process for producing a primed pluripotent cell from an FS cell by withdrawal from FS cell culture medium and transfer to primed cell medium such as E8 for human primed cells or AhiF with or without XAV for mouse EpiS cells (Page 17, lines 29-32). Thus, both Shalom-Feuerstein et al and Smith et al teach conditions that promote a primed pluripotent state (For claim 2(d)).
It should be noted that Shalom-Feuerstein et al provides tissue culture media for reprograming stem cells into a pluripotent and naive state ([037] page 9) including (i) media provided with reprogramming factors (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) known in the art to reprogram the somatic cells to become pluripotent stem cells ([047], page 12); media for naïve (ground state) conditions (2i/LIF) in which naive cells grown in 2i/LIF express very low levels of DNMT3B (hypomethylated DNA state) ([0103], page 29), and media for switching into primed state in the presence of knockout serum and basic fibroblast growth factor (KSR/bFGF) ([0103], page 29). Thus, a person of ordinary skill in the art would be able to use different media at different state for reprogramming the somatic cell towards a pluripotent state.
Regarding to claim 4, an iPSC obtained from a naive stem cell as taught by Shalom-Feuerstein et al and Smith et al using the same method would inherently produce an epigenetic profile similar to that which is claimed in claim 4. Additionally, Shalom-Feuerstein et al teach a more uniform "ground state" culture that mirrors better the undifferentiated transcriptional and epigenetic landscape of pre-implantation epiblast cells can be achieved in the presence of a combination of LIF and inhibitors of MEK and GSKβ (2i/LIF) ([003], page 1). Thus, optimization by using culture conditions as taught by Shalom-Feuerstein et al could result in uniform culture that mirrors epigenetic landscape of epiblast cells (epiblast cells are a source of embryonic stem cells).
Regarding to claim 5, Shalom-Feuerstein et al teach reprogramming human cells into naive state ([029], last line in 1st para on page 8) and the transition between naive and primed PSCs in human cells were examined ([0103], page 29).
Regarding to claim 6, Smith et al teach during early stages of reprogramming, for example after around 10 days of reprogramming with Yamanaka factors, cultures may be switched to naive resetting medium (Page 13, lines 3-5).
Regarding to claim 9, Shalom-Feuerstein et al teach culturing the cell that is not a pluripotent stem cell in a naive state in media comprising a RAS inhibitor for an amount of time sufficient for the cell to acquire at least one characteristic of a pluripotent stem cell in a naive state ([017], page 4).
Regarding to claim 10-11, Smith et al teach a resetting medium which comprises a HDAC inhibitor, a MEK inhibitor, and optionally a STAT3 activator, and optionally one or more further inhibitors e.g., a PKC inhibitor, a GSK3 inhibitor, or a Wnt inhibitor (Page 11, lines 42-45). A stable native culture can be established by 3-5 passages in T2ilGo with ROCK inhibitor (Page 12, lines 8-9). FS cells were cultured with hLIF for 4 days in the presence of Rock inhibitor (Page 17, last para., line 46). It is noted that T2iLGoY is T2ilGo with ROCK inhibitor Y-27632.
Regarding to claim 12-13, Shalom-Feuerstein et al teach mESCs that were grown in naive (ground state) conditions (2i/LIF) were switched into primed state by cultivation the cells for 10 passages in the presence of knockout serum and basic fibroblast growth factor (KSR/bFGF) ([0103], page 29). Examples of media that may be used include TeSR medium, mTeSR medium, KSR and Essential 8 media ([051], page 13).
Regarding to claim 14-15, Smith et al teach that following 7-21 days resetting naive cells can be distinguished by expression of naive markers using a reporter line such as KLF4:GFP (Page 12, lines 22-23).
Regarding to claim 21, Shalom-Feuerstein et al teach the primed marker is selected from fibroblast growth factor 5 (Fgf5), DNA methyltransferase 3b (Dnmt3b), Histone 3 tri-methyl lysine 9 (H3K9me3) and NCadherin (N-Cad) ([055], page 14). the transition to the primed state was accompanied by morphological changes (Fig. 3A), and the increase in expression of markers of the primed state (Fgf5 and Dnmt3b) (Fig. 3B) ([0103], page 29).
Regarding to claim 23, Smith et al teach that naive cells are directly transferred to A10X with/without RARi, or left initially for 6-10 days in basal medium (N2B27) to exit from the naive state (Page 27, lines 22-24). Additionally, Smith et al state that those skilled in the art are generally familiar with the stages of pluripotency lineage in different mammalian embryos, from naive to primed phases. Thus, a person of ordinary skill in the art would be able to culture and induce a primed pluripotent state for different periods of time.
Regarding to claim 24 and 27, Shalom-Feuerstein et al teach that, to generate iPSCs, somatic cells are provided with reprogramming factors (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) known in the art to reprogram the somatic cells to become pluripotent stem cells ([047], page 12). Smith et al also teach reprogramming somatic cells to naive induced pluripotent stem cells (iPSCs) with Yamanaka factors (a set of four transcription factors Oct4, Sox2, Klf4, and c-Myc) cultures, and the dome-shaped naive colonies expressing naive markers including KLF17, KLF4 or DNMT3L can be further expanded in t2iIGo medium (Bridging paragraph between page 12-13). Thus, a person of ordinary skill in the art would be able to contacting the cell with an agent for increasing expression level of OCT4, SOX2, KLF4 and MYC for reprogramming the somatic cell towards a pluripotent state.
Response to Arguments
Applicant's arguments filed on 09-17-2025 have been fully considered but they are not persuasive.
1. Applicants argue that Shalom-Feuerstein does not teach or suggest culturing cells in media for converting a cell to a primed pluripotent state after resetting the epigenetic profile of the cell; or culturing cells in media for converting a cell to a primed pluripotent state after reprogramming the cell towards a hypomethylated DNA state. Therefore Shalom-Feuerstein does teach or suggest all of the claim limitation of the pending claims and does not render the invention defined in claim 1 obvious (remarks, page 15).
Response to Arguments:
It is noted that Shalom-Feuerstein et al teach resetting the epigenetic profile of the cell: culturing the cell that is not a pluripotent stem cell in a naive state in media comprising a RAS inhibitor for an amount of time sufficient for the cell to acquire at least one characteristic of a pluripotent stem cell in a naive state ([017], page 4), and the characteristic is decreased expression relative to expression before the culturing, of DNA methyltransferase 3b (Dnmt3b) ([018], page 4). It is noted that Dnmt3b is a DNA methyltransferase responsible for methylation on DNA; thus, decreased expression of Dnmt3b and being in pluripotent stem cell in a naive state would promote a hypomethylated DNA state. Additionally, Shalom-Feuerstein et al teach that to generate iPSCs, somatic cells are provided with reprogramming factors (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) known in the art to reprogram the somatic cells to become pluripotent stem cells ([047], page 11-12). Also, Shalom-Feuerstein et al teach that mESCs (naive state) that were grown in naïve (ground state) conditions (2i/LIF) were switched into primed state by cultivation the cells for 10 passages in the presence of knockout serum and basic fibroblast growth factor (KSR/bFGF) ([0103], page 28). Thus, culturing cells in media for converting a cell to a primed pluripotent state after resetting the epigenetic profile of the cell as required by the base claim 1.
2. Applicants argue that Shalom-Feuerstein does disclose methods for generating pluripotent stem cells in a naive state using RAS inhibition, the reference requires culturing conditions sufficient for cells to achieve naive pluripotency, with decreases in DNMT3B expression. The reference does not contemplate or suggest transient exposure insufficient to establish the naive state. To the contrary, their goal is to stabilize naive pluripotency. Moreover, there is nothing in Shalom-Feuerstein which indicates to the skilled person that it is possible to achieve the improved characteristics of primed iPSCs by contacting cells during the reprogramming process, with nai:ve media for a short period of time and then placing cells in primed media (remarks, page 15).
Response to Arguments:
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 this case, Shalom-Feuerstein et al teach the method of culturing the cell that is not a pluripotent stem cell in a naive state in media comprising a RAS inhibitor for an amount of time sufficient for the cell to acquire at least one characteristic of a pluripotent stem cell in a naive state ([017], page 4). Shalom-Feuerstein et al do not teach “culturing in the second culture condition is for a period of time that is insufficient to allow the cell to achieve an established a pluripotent state”. Smith et al cure the deficiency. Smith et al teach propagating pluripotent stem cells in the "formative" state (abstract). Smith et al teach in one strategy for obtaining a fully competent human pluripotent stem cell, the precursor cell is transiently reset to naive status, then converted to formative status before erasure of imprints occurs in the naive state (Page 13, lines 11-13). The period in the naive state culture conditions may be transient but should be sufficient to achieve epigenetic erasure (hypomethylated DNA state) (Page 9, lines 17-19) (For claim 1(b)). Therefore, a person of ordinary skill in the art before the effective filing date of the rejected claims would be motivated to combine the teachings of prior art to modify the method of Shalom-Feuerstein et al by using pluripotent stem cells in the "formative" state with epigenetic erasure (hypomethylated DNA state) as taught by Smith et al as instantly claimed. One of ordinary skill in the art would have been motivated to do so because Smith et al stated that formative pluripotent stem cells is an attractive option for obtaining robust and unbiased cultures with imprints maintained (Page 13, lines 13-17) and the formative stem (FS) cells described have many utilities and potential advantages over existing pluripotent stem cell sources (Page 16, lines 15-16). One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Smith et al were successful in generation of formative stem cells that can be used to create primed cells (page 17 lines 29-40) and germ cells (page 17, last para).
3. Applicants argue that the deficiencies in the teachings of Shalom-Feuerstein, are not cured by the addition of the teaching of Smith. Smith relates neither to methods for generating primed PSCs nor for generating naive PSCs. Rather, Smith relates to methods for generating so-called "formative stem cells" which are said to be distinct from both primed and naive pluripotent stem cells. Formative stem cells can be derived from primed PSCs by resetting primed PSCs to a naive state (page 9, lines 15 to 19 of Smith). Inherent in their definition as a distinct state of pluripotency, formative stem cells are not in a primed or nai:ve state of pluripotency (remarks, page 15)
Response to Arguments:
It is noted that the base claim 1 requires “the cell to achieve a pluripotent state” and subsequently “to promote a primed pluripotent state”. In this case, Shalom-Feuerstein et al teach the transition from naive ground state to primed state of pluripotency ([0103], page 29, Example 2), and Smith et al teach formative cells quickly become primed in mouse embryos (page 4, line 8) and formative cells can be derived from primed pluripotent stem cells by resetting to a naive state (Page 9, line 15). Thus, formative cells are transient state between naive and primed state, and since the base claim 1 requires “culturing in the second culture condition is for a period of time that is insufficient to allow the cell to achieve a pluripotent state”, the Smith et al ’s teachings exactly satisfy the requirement of the claim because formative pluripotent cells are transient state between naive and primed state, and it is not in the culture sufficiently to be either.
4. Applicants argue that Smith discloses a distinct paradigm involving formative stem cells, which are proposed intermediates between two pluripotent states, naive and primed pluripotency, not somatic and pluripotent states as in the claimed invention. Crucially, Smith emphasizes that the naive phase should be of sufficient duration to allow "major epigenome remodeling", i.e., exposure that achieves epigenetic erasure. Smith therefore also teaches sufficient time in naive culture, not the claimed insufficient time. In contrast, claim 1 explicitly recites: "culturing in the second culture condition is for a period of time that is insufficient to allow the cell to achieve a pluripotent state.". Neither Shalom-Feuerstein, nor Smith, teach, suggest, or make obvious at least this limitation of the pending claims. Shalom-Feuerstein teaches culturing to achieve naive; Smith teaches transient naive exposure sufficient to achieve epigenetic erasure. In contrast, the present method deliberately avoids establishment of naive pluripotency by limiting the culture period, while nonetheless promoting a hypomethylated state. This distinction is critical: the claimed method achieves beneficial hypomethylation without incurring the genomic instability and imprint erasure associated with prolonged naive culture (remarks, page 16).
Response to Arguments:
As applicant admitted, claim 1 explicitly recites: "culturing in the second culture condition is for a period of time that is insufficient to allow the cell to achieve a pluripotent state.", and the cells at the end of step b of base claim 1 is in a pluripotent state. In this case, Smith et al teach processes for producing or propagating a formative stem (FS) cell line which is pluripotent stem cells in transient state between naive and primed state (see abstract and page 4, line 8 and Page 9, line 15). Shalom-Feuerstein et al teach the transition from naive ground state to primed state of pluripotency ([0103], page 29, Example 2), and Smith et al teach uses of formative stem cells to create primed cells (Page 17, lines 27-40) and formative cells quickly become primed in mouse embryos (page 4, line 8). Thus, a person of ordinary skill in the art would combine the references to use formative stem cells which is pluripotent stem cell in a pluripotent state that is not in naive and primed state
Applicants further argue that “the present method deliberately avoids establishment of naive pluripotency by limiting the culture period” which is not recited in the claim. The base claim 1 recites "culturing in the second culture condition is for a period of time that is insufficient to allow the cell to achieve a pluripotent state.". Nevertheless, as applicant admitted Smith et al teach formative stem cells are not in a primed or naive state of pluripotency but a transient state between primed and naive states which satisfy the limitation “a pluripotent state”. One of ordinary skill in the art would have been motivated to combine the references because Smith et al stated that formative pluripotent stem cells is an attractive option for obtaining robust and unbiased cultures with imprints maintained (Page 13, lines 13-17) and the formative stem (FS) cells described have many utilities and potential advantages over existing pluripotent stem cell sources (Page 16, lines 15-16). One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Smith et al were successful in generation of formative stem cells that can be used to create primed cells (page 17 lines 29-40) and germ cells (page 17, last para).
Lastly, as describe above, the prior art Smith et al provide evidence for controlling pluripotent state between a primed and a naive state to obtain robust and unbiased cultures with imprints maintained (Page 13, lines 13-17). Thus, it is indicating that the controlling pluripotent state between a primed and a naive state was recognized in the prior art to be a result-effective variable. A person of ordinary skill in the art would have been motivated to perform the controlling pluripotent state between a primed and a naive state a plurality of times out of the course of routine optimization.
5. Applicants argue that neither reference provides motivation to deliberately prevent establishment of naive pluripotency. To the contrary, both stress the importance of achieving stable naive or achieving sufficient remodeling during naive exposure. Shortening exposure below these thresholds would have been expected to fail, not to yield the advantageous hypomethylated yet non-naive state described in the present claims. The art thus teaches away from the claimed invention. the claimed method refers to reprogramming from a somatic cell, such as a fibroblast, into iPSCs (and also other somatic cell types to iPSCs), and not to the conversion of a pluripotent stem cell, in a given pluripotent state, into a different pluripotent state, which is what Smith discloses. Furthermore, with respect to Smith, this document relates neither to methods for generating primed PSCs, nor for generating naive PSCs. Rather, Smith specifically relates to methods for generating so-called "formative stem cells" which are said to be distinct from both primed and naive pluripotent stem cells. Formative stem cells can be derived from primed PSCs by resetting primed PSCs to a naive state (page 9, lines 15 to 19 of Smith). Inherent in their definition as a distinct state of pluripotency, formative stem cells are not in a primed or naive state of pluripotency. Accordingly, there is nothing in the disclosure of Smith that would motivate the skilled person to arrive at the present invention since Smith is related to achieving an entirely different goal. (Remarks, page 16-17)
Response to Arguments:
Applicant argue that neither reference provides motivation to deliberately prevent establishment of naive pluripotency which is not recited in the claim. Nevertheless, Smith et al teach processes for producing or propagating a formative stem (FS) cell line which is pluripotent stem cells in transient state between naive and primed state (see abstract and page 4, line 8 and Page 9, line 15). Shalom-Feuerstein et al teach the transition from naive ground state to primed state of pluripotency ([0103], page 29, Example 2), and Smith et al teach uses of formative stem cells to create primed cells (Page 17, lines 27-40) and formative cells quickly become primed in mouse embryos (page 4, line 8). Thus, a person of ordinary skill in the art would combine the references to use formative stem cells which is pluripotent stem cell in a pluripotent state that is not in naive and primed state.
Applicant argument regarding “advantageous hypomethylated yet non-naive state” is not persuasive because there is no evidence in record the cells from applicant efforts are superior as compared with the teaching of prior art: Smith et al teach that “FS cells are believed to retain imprinted gene status. Current human naive pluripotent stem cells do not stably retain imprints. Thus, one advantageous embodiment of the invention for obtaining a fully competent human pluripotent stem cell involves transient derivation or resetting to naive status then conversion to formative status before erasure of imprints occurs in the naive state. FS cells may therefore be a superior or alternative source material for chimaera formation or directed differentiation compared with other forms of pluripotent stem cell. Formative pluripotent stem cells may be a desirable intermediate stage for population expansion prior to directed differentiation of human naive pluripotent stem cells.” (Page 6, lines 36-44).
Applicant arguments regarding “reprogramming from a somatic cell, such as a fibroblast, into iPSCs (and also other somatic cell types to iPSCs), and not to the conversion of a pluripotent stem cell” is not persuasive because Shalom-Feuerstein et al teach to generate iPSCs, somatic cells are provided with reprogramming factors (e.g. Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) known in the art to reprogram the somatic cells to become pluripotent stem cells ([047], page 12). Also, Smith et al teach FS cells can be derived from naive pluripotent cells either taken directly from embryos, or established as naïve embryonic stem cells, or generated as induced pluripotent stem cells by somatic cell reprogramming (Page 9, lines 4-6). Thus, both cited references teach reprograming somatic cells.
Applicant arguments regarding “with respect to Smith, this document relates neither to methods for generating primed PSCs, nor for generating naive PSCs” is not persuasive because Smith et al teach “Uses of FS cells to create primed cells” (see page 17 lines 27-40), and in another embodiment naive cells are provided by reprogramming somatic cells to naive induced pluripotent stem cells (iPSCs) (Page 12, lines 36-39).
Applicant arguments regarding “inherent in their definition as a distinct state of pluripotency, formative stem cells are not in a primed or naive state of pluripotency” is exactly what the base claim 1 requires and what is described in the instant disclosure that “insufficient to allow the cell to achieve an established naive pluripotent state” (see the instant disclosure Page 2, lines 18-19).
Conclusion
No claim is 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.
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/KHOA NHAT TRAN/Examiner, Art Unit 1632
/PETER PARAS JR/Supervisory Patent Examiner, Art Unit 1632