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 .
DETAILED ACTION
Acknowledgement is hereby made of receipt and entry of the communication filed on Aug. 02, 2024. Claims 1-15 are pending and are currently examined.
Claim Objection
Claims 1, 4, 7, 9, 10 and 12 are objected to because of the following informalities:
These claims recite abbreviations, WNT, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49, OCT3/4, KLF4, L-MYC, FGF2, TGF-β1, ROCK, and NANOG, without spelling it out the first time it appears in the claims set.
Appropriate correction is required.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-2, 4 and 7-15 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Takeuchi et al. (US 2023/0094568 A1, published on Mar. 30, 2023, PCT filed on Feb. 12, 2021, hereinafter,” Takeuchi”).
(Note: For the US 2023/0094568 A1, the PCT file is published earlier than the instant patent priority published date at 02/04/2022. Two inventors of US 2023/0094568 A1 are not on the list of the instant patent).
The base claim 1 is directed to a method for producing an induced pluripotent stem cell (iPS cell), comprising the following steps I and II: I. introducing an initialization gene into a somatic cell; and II. performing initialization and amplification culture of the cell into which the gene has been introduced, in a liquid medium comprising at least one selected from the group consisting of a protein kinase C3 (PKCP) inhibitor and a WNT inhibitor under a suspension culture condition.
Takeuchi teaches a production method of producing pluripotent stem cells by suspension culture, and to a production method of producing a pluripotent stem cell population by suspension culture (See [0001]).
Takeuchi teaches the step I by stating that an "iPS cell" refers to a pluripotent stem cell that has become reprogrammable by introducing a few genes encoding initialization factors into a differentiated somatic cell to turn the somatic cell into an undifferentiated state (See [0047]).
Takeuchi teaches a method for producing a pluripotent stem cell population comprising the step of performing suspension culture of a pluripotent stem cell in a liquid medium comprising a PKCβ inhibitor and a TNKS inhibitor (See [0008]) and the iPS cell is reprogrammable by the initialization factors encoded by the initialization gene and is cultured for the amplification (See [0035]). Here the description of Takeuchi teaches the base claim 1 step II because Takeuchi teaches that the TNKS inhibitor used in Example 1 is known to have inhibitor effect on WNT signaling (See [0234]).
Regarding claim 2, Takeuchi teaches that as shown in Table 10 and FIG. 5, the proportions of cells positive for the undifferentiation markers OCT4, SOX2, and NANOG were 90% or higher for Reference Example 3, in which adherent culture was performed; thus, pluripotent stem cell populations with the undifferentiated state maintained were obtained (See [0231]). Fig. 18 of Takeuchi shows transition of the positive rates for the undifferentiation marker OCT4 for Comparative Example 12 and Example 12. It is understood from FIG. 18 that the positive rate for the undifferentiation marker OCT 4 was kept high even in long-term culture for Example 12 (See [0249] and below). Here it teaches the claim 2-IV.
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As for “sorting an undifferentiation marker positive cell” in the claim 2-III, Takeuchi teaches using flow cytometry to detect the undifferentiation markers (See e.g., [0225]), Table 10 and Fig. 5), which teaches sorting an undifferentiation marker positive cell. It is common knowledge in the art that the major application of flow cytometry is sorting cells for analysis.
Regarding claim 4, Takeuchi teaches some pluripotent stem cells in the course of culture can be taken out for checking whether the undifferentiated state is maintained. For example, whether the undifferentiated state is maintained can be checked by measuring expression of a pluripotent stem cell marker expressed on pluripotent stem cells taken out during culture. Examples of pluripotent stem cell markers include, as described above, Alkaline Phosphatase, Nanog, OCT4, SOX2, TRA-1-60, c-Myc, KLF4, LIN28, SSEA-4, and SSEA-1 9See [0161]).
Regarding claim 7, Takeuchi teaches that if the iPS cells to be used in the present specification are newly prepared cells, applicable combinations of genes for initialization factors to be introduced include, but are not limited to, a combination of the OCT3/4 gene, KLF4 gene, SOX2 gene, and c-Myc gene (See [0052]).
Regarding claim 8, Takeuchi teaches that the method according to claim 1, wherein the liquid medium comprises at least one selected from the group consisting of L-ascorbic acid, insulin, transferrin, selenium, and sodium hydrogen carbonate (See page 32, claims 1, 5), where claim 1 here include the liquid medium used in and after the step II as claimed.
Regarding claim 9, Takeuchi teaches that the method according to claim 1, wherein the liquid medium comprises FGF2 and/or TGF-β1 (See page 32, claims 1 and 6), where claim 1 here include the liquid medium used in and after the step II as claimed.
Regarding claims 10 and 11, Takeuchi teaches that the method according to claim 1, wherein the liquid medium comprises a ROCK inhibitor (teach claim 9) and wherein the ROCK inhibitor is Y-27632 (teach claim 10) (See page 32, claims 1, 7-8), where claim 1 here include the liquid medium used in and after the step II as claimed.
Regarding claim 12, Takeuchi teaches the method according to (1 ), in the pluripotent stem cell population, the proportion of cells positive for OCT4 is 90% or higher, the proportion of cells positive for SOX2 is 90% or higher, and the proportion of cells positive for Nanog is 90% or higher (See [0008]-(11)), where these proportion of cells is “in the obtained cell population” as claimed because Takeuchi also teaches that the percentage (proportion) of cells expressing a pluripotent stem cell marker (e.g., OCT4, SOX2, NANOG) and/or being positive for a pluripotent stem cell marker in a pluripotent stem cell population obtained with the method of the present aspect for culturing a pluripotent stem cell population is, for example, 90% or higher (See [0189]).
Regarding claim 13, Takeuchi teaches the method according to (1) (see [0008]-(1), wherein the step of performing suspension culture comprises the step of collecting a cell aggregate (See [0008]-(10)), where the method (1) includes the step II as claimed.
Regarding claim 14, Takeuchi teaches that a "cell aggregate" is a massive cell population formed through cell aggregation in suspension culture. Cell aggregates are generally spherical in typical cases. Cells constituting a cell aggregate are not limited as long as they are one or more types of the aforementioned cells. For example, a cell aggregate composed of pluripotent stem cells such as human pluripotent stem cells and human embryonic stem cells include cells expressing a pluripotent stem cell marker and/or being positive for a pluripotent stem cell marker (See [0054]), where the markers can be the undifferentiation markers such as in Fig. 5 showing a characteristic diagram representing positive rates for the undifferentiation markers OCT4, SOX2, and NANOG in pluripotent stem cells cultured in Reference Example 3, Comparative Examples 6 and 7-1 to 7-4, and Examples 5-1 and 5-2 (See [0026]).
Regarding claim 15, it requires that a cell population comprising iPS cells produced by the method according to claim 1. Takeuchi teaches that the present invention relates to a production method of producing pluripotent stem cells by suspension culture, and to a production method of producing a pluripotent stem cell population by suspension culture (See [0001]) by introducing a few genes encoding initialization factors into a differentiated somatic cell to turn the somatic cell into an undifferentiated state (See [0047]) and in suspension culture of pluripotent stem cells, the undifferentiated state is maintained by the presence of a PKC inhibitor, especially, a PKC~ inhibitor, and a tankyrase inhibitor (TNKS inhibitor) (See Abstract), where the TheTNKS inhibitor used in Example 1 is known to have inhibitor effect on WNT signaling (See [0234]).
Note: Claim 15 is a product-by-process claim, which is drawn to a cell population comprising iPS cells. The method steps recited to produce the cell population are not considered when determining patentability of the product (MPEP § 2113). In In re Thorpe, the court stated, “even though product-by-process claims are limited by and defined by the process, determination of patentability is based on the product itself. The patentability of a product does not depend on its method of production. If the product in the product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process.” In re Thorpe, 777 F.2d 695, 698, 227 USPQ 964, 966 (Fed. Cir. 1985). (Emphasis added)
Accordingly, claims 1-2, 4 and 7-15 are anticipated by Takeuchi.
Claims 1-2, 4, 6-12 and 15 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Hanna et al. (US 2017 /0275593 A1, published on Sep. 28, 2017, hereinafter “Hanna”).
The base claim 1 is directed to a method for producing an induced pluripotent stem cell (iPS cell), comprising the following steps I and II: I. introducing an initialization gene into a somatic cell; and II. performing initialization and amplification culture of the cell into which the gene has been introduced, in a liquid medium comprising at least one selected from the group consisting of a protein kinase C3 (PKCP) inhibitor and a WNT inhibitor under a suspension culture condition.
Hanna teaches an invention for a novel culture media which can be used to generate and expand pluripotent stem cells in general and more particularly naive pluripotent stem cells (See [0001]). The induced pluripotent (iPS) cells can be generated from somatic cells by ectopic expression of different transcription factors, originally Oct4, Sox2, Klf4 and c-Myc, that share all defining features with naïve mouse ESCs (See [0002]). Hanna further discloses that induced pluripotent stem cells (iPS) (embryoniclike stem cells) can be generated from somatic cells by genetic manipulation of somatic cells, e.g., by retroviral transduction of somatic cells such as fibroblasts, hepatocytes, gastric epithelial cells with transcription factors such as Oct-3/4, Sox2, c-Myc, and KLF4 (See 0567]). Here it teaches that the retroviral vector can carry the gene that express the factors of Oct-3/4, Sox2, c-Myc, and KLF4, which are the reprogramming factors (See [0524]). For example, Hanna teaches that it is a non-limiting description of expression vectors and modes of administering thereof into cells which can be used to express a polypeptide-of-interest ( e.g., any of the proteins described hereinabove, e.g., OCT4, c-MYC, SOX2, KLF4, LIF, bFGF, TGF~l, an oncogenic protein such as C-MYC, N-MYC, L-MYC, EDAR (ecto-dysplasin A receptor), MDM2, and ERAS, a reporter protein such as GFP or RFP) in a cell (See [0812]).
Thus, Hanna teaches the base claim 1-I. In addition, the above description also teaches the claim 7 at “the initialization gene to be introduced comprises at least one gene selected from the group consisting of an OCT3/4 gene, a KLF4 gene and an L-MYC gene” as claimed.
For the base claim 1-II, Hanna teaches that the present invention provided a culture medium a STAT3 activator, an ERKl/2 inhibitor, an Axin stabilizer, a PKC inhibitor and a GSK3b inhibitor (See e.g., [0020]), where the PKC inhibitor can be the PKCβ (beta) inhibitor (See 0322]), and Axin stabilizer is XAV930 (See [0317]). Here the XAV930 is thought to alter the PARsylation and ubiquitination of AXIN2 that results in its increased stability and in inhibition of canonical WNT signaling (See [0312]), which teaches the WNT inhibitor as claimed.
Accordingly, Hanna also teaches the base claim 1-II.
Regarding claim 2, Hanna teaches that cells were expanded in the indicated conditions (Table 3 and below), with or without Doxycycline for 10 passages (PIO) on Gelatin/DR4 coated plates, and cultures were immunostained for OCT4 pluripotency marker in surviving cells (See [1284] and Table 3 below), where OCT4 is a undifferentiation marker of stem cell and the cells is obtained from a culture including PKCβ inhibitor, Go6983, and WNT inhibitor, IWR1 (See Table 3 below).
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Furthermore, the immunostained method can be performed by sorting of cells of the EBs via fluorescence activated cell sorter (FACS) (See [0657) as taught by Hanna, which teaches the claim 2-III. Hanna also teaches that cells expanded in the culture medium of some embodiments of the invention (Medium number 138 and 136 retain their pluripotency as is indicated by the high percentage of OCT4 positive cells (93% and 97%) despite the loss of DNMTl expression in this engineered system (upper two examples) (See [1284]), which the “cell expanded” and the “OCT4 positve cells” of Here Hanna teaches the claim 2-IV at “culture of the undifferentiation marker positive cell sorted in the step III to obtain a cell population” as claimed. For example, Hanna teaches that the cells were expanded for 28 days (total of 6 passages) passages and evaluated by FACS analysis for OCT 4-GFP+ reporter (See [0255]) and the phrase "generating lineage specific cells" refers to the enrichment of a mixed population of cells in a culture with cells predominantly displaying at least one characteristic associated with a specific lineage phenotype (See [0645]).
Regarding claim 4, Hanna teaches that the primordial germ cell is characterized by a CD61 +/SSEA4 + expression pattern ( expression signature) (See [0687]).
Regarding claim 6, Hanna teaches that a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell (See [0820]).
Regarding claim 8, Hanna teaches that alternatively, the following materials (substitute the N2 supplement) can be added to a 500 ml culture medium: Recombinant Insulin (Sigma I-1882) at a 12.5 microg/ml (μg/ml) final concentration; Apo-Transferrin (Sigma T-1147) at a 500 μg/ml final concentration (See [0285]).
Regarding claims 9-11, Hanna teaches that according to some embodiments of the invention, the culture medium comprises a STAT3 activator, an ERKl/2 inhibitor, a GSK3β inhibitor, a P38 inhibitor, a JNK inhibitor, FGF2, TGFβ1, a ROCK inhibitor, a SRC family kinase inhibitor and a BMP inhibitor (See [0109]), where the ROCK inhibitor is Y-27632 (See [0337]).
Regarding claim 12, Hanna teaches that a method of improving generation of induced pluripotent stem cells (iPSCs) from a somatic cell, comprising : expressing within the somatic cell a factor selected from the group consisting of NANOG, Oct4, Sox2, Klf4, c-Myc etc. (See [0622]-[0623]). When the somatic cells are subject to de-differentiation using DNA transfection of the growth factors then the method results in e.g., at least about 95%, at least about 99 % , e.g., 100 % more iPSCs (See [0634]).
Regarding claim 15, it requires a cell population comprising iPS cells produced by the method according to claim 1. Hanna teaches that the phrase "generating lineage specific cells" refers to the enrichment of a mixed population of cells in a culture with cells predominantly displaying at least one characteristic associated with a specific lineage phenotype (See [0645]), where the "generating lineage specific cells" can be the naive PSCs or the iPSCs of some embodiments of the invention can be used to generate lineage specific cells (See 0644) that includes expression of Oct4, Nanog and Kif pluripotency genes (See [0004]). Here the culture media includes PKCβ inhibitor and IWR1, an WNT inhibitor (See [1283 and Table 2 and below).
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Note: Claim 15 is a product-by-process claim, which is drawn to a cell population comprising iPS cells. The method steps recited to produce the cell population are not considered when determining patentability of the product (MPEP § 2113). In In re Thorpe, the court stated, “even though product-by-process claims are limited by and defined by the process, determination of patentability is based on the product itself. The patentability of a product does not depend on its method of production. If the product in the product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process.” In re Thorpe, 777 F.2d 695, 698, 227 USPQ 964, 966 (Fed. Cir. 1985). (Emphasis added)
Accordingly, claims 1-2, 4, 6-12 and 15 are anticipated by Hanna.
Claim Rejections - 35 USC § 103
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.
Claim 3 is rejected under 35 U.S.C. 103 as being unpatentable over Hanna as applied to the claims 1-2, 4, 6-12 and 15 above and in view of Faye et al. ( Anal Chem. 2016 Jul 5;88(13):6696-702).
Claim 3 requires the step III is a step of fractionating the sorted cell.
Hanna teaches that the reprogramming process typically requires extensive cell proliferation of a period of at least one week, after which a certain fraction of the cell progeny successfully converts into ES-like state in an unpredictable pattern and with different time latencies (See [0002]), which indicates a fractioning of cells.
Nevertheless, Faye studies a New Method for Sorting Endothelial and Neural Progenitors from Human Induced Pluripotent Stem Cells by Sedimentation Field Flow Fractionation and teaches that they used sedimentation Field Flow Fractionation (SdFFF) to prepare enriched populations derived from hiPSc after only 10 days of culture in a classical medium and the isolation of subpopulations representing various differentiation lineages is of major interest for the production of specialized, cell-enriched fractions and in the preparation of increasingly complex models for the development of new therapeutic tools (See Abstract). Faye teaches that among FFF techniques, Sedimentation FFF (SdFFF) is a gentle, noninvasive, tagless cell sorting method (See page 6697, left column, paragraph 1).
It would have been prima facie obvious for one having ordinary skill in the art before the effective filing date of the claimed invention to introduce the SdFFF cell sorting method into Hanna’s invention to arrive at an invention as claimed. One of skill in the art would have been motivated to do so to use the noninvasive cell sorting method, such as SdFFF, to prepare enriched populations derived from hiPSc. There would be a reasonable expectation of success to develop such a method for producing an induced pluripotent stem cell (iPS cell) with a step of fractioning the sorted cell as claimed.
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Hanna as applied to the claims 1-2, 4, 6-12 and 15 above and in view of Wang et al. (Genomics Proteomics Bioinformatics. 2013 Oct;11(5):304-11).
Claim 5 requires the somatic cell is a blood mononuclear cell.
Relevance of Henna is set forth above, however, it is silent on the blood mononuclear cell.
Wang describes the generation of induced pluripotent stem cells with high efficiency from human umbilical cord blood mononuclear cells and teaches that generating iPSCs from immunologically immature newborn umbilical cord blood mononuclear cells (UCBMCs) is of great significance. They report generation of human iPSCs with great efficiency from UCBMCs using a dox-inducible lentiviral system carrying four Yamanaka factors (See Abstract).
It would have been prima facie obvious for one having ordinary skill in the art before the effective filing date of the claimed invention to introduce the UCBMCs into Hanna’s invention to arrive at an invention as claimed. One of skill in the art would have been motivated to do so to use UCBMCs to generate highly functional human iPSCs that could accelerate the development of cell-based regenerative therapy for patients suffering from various diseases (See Abstract). There would be a reasonable expectation of success to develop such a method for producing an induced pluripotent stem cell (iPS cell) from the blood mononuclear cell as claimed.
Claims 13 and 14 are rejected under 35 U.S.C. 103 as being unpatentable over Hanna as applied to the claims 1-2, 4, 6-12 and 15 above and in view of Lipsitz et al. ( US 2019/0316081 A1, published on Oct. 17, 2019) and Ashok et al. (Methods Mol Biol. 2016;1502:35-52).
Claims 13 and 14 require the step II comprises a procedure of colleting a cell aggregate and the step IV comprises a procedure of collecting a cell aggregate, respectively.
Relevance of Henna is set forth above, however, Hanna does not specifically teach forming a cell aggregate and collecting a cell aggregate as claimed.
Lipsitz teaches that one method for scalable expansion of PSC is culture as cell aggregates in dynamic suspension conditions. The PSC aggregate size distribution and average size is known to affect oxygen and nutrient diffusion throughout the aggregate and the resulting characteristics of the cell product. Surprisingly, while others have used polysulfated compounds to prevent aggregation in the biopharmaceutical industry, the authors describe herein the use of controlled aggregation of pluripotent stem cells (See [0007]). Lipsitz discloses that in suspension, altered state hPSC had improved single cell survival, more efficient aggregate formation, and enhanced shear tolerance compared to primed hPSC, all of which could be related to adhesion signaling (See [0221]) and at the end of the culture period, aggregates were harvested and dissociated using a 5 minute TrypLE™ treatment at 37° C. and the cells were counted on a hemocytometer using a Trypan Blue viability exclusion stain (See [0178]), which indicates a collection of the cell aggregates.
Ashok studies the aggregate and microcarrier cultures of human pluripotent stem cells in stirred suspension systems and teaches that they develop a method of culturing human pluripotent stem cells in spinner flasks either as aggregates or on microcarriers. Techniques for assessing the quality of the culture and characterizing the cells based on the presentation of pertinent markers are also presented. Spinner flask culture with its relatively low capital and operating costs is appealing to laboratories interested in scaling up their production of stem/ progenitor cells (See Abstract). Ashok discloses that cell aggregates are collected from spinner flasks into centrifuge tubes and incubated with Accutase for 10–15 min while gently mixing occasionally (See page 10).
It would have been prima facie obvious for one having ordinary skill in the art before the effective filing date of the claimed invention to introduce the method of PSC aggregates collection of Lipsitz and Ashok into Hanna’s invention to arrive at an invention as claimed, where the iPSC cell can be the OCT4 positive cell and the medium contains PKC inhibitor and WNT inhibitor. One of skill in the art would have been motivated to do so based on the benefit to form aggregates taught by Lipsitz and the benefit used for collecting cell aggregates from the spinner flasks taught by Ashok. There would be a reasonable expectation of success to develop such a method for collecting the cell aggregates at the claimed steps.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to RUIXUE WANG whose telephone number is (571)272-7960. The examiner can normally be reached Monday-Friday 8:00 am to 4:30 pm, EST.
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/RUIXUE WANG/ Examiner, Art Unit 1672
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