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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 8-24-26 has been entered.
Claims 2-6, 8-12, 14-17, 19-21, 35, 37, 39 have been canceled. Claim 40 has been added. Claims 1, 7, 13, 18, 22-34, 36, 38, 40 are pending.
Election/Restrictions
Applicant elected Group I, claims 1, 3, 4, 6, 7, 13, 16, 18, 19, 35, 38 in the reply filed on 8-11-25 with traverse. Claims 22-31, 33, 34, 36 remain withdrawn.
Claims 1, 7, 13, 18, 32, 38, 40 are under consideration.
Applicant's arguments filed 8-24-26 have been fully considered but they are not persuasive.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim objections
Claim 1 can be written more clearly using numbers to describe the structures/functions that define the pluripotent cell.
Claim 1 is missing the fact that the cells are mammalian.
It is wholly unclear how the “process” portion of claim 1 defines and structures or functions of the pluripotent cells.
The phrase “three-dimensional culture” in claim 1 is redundant; all cells and cultures are 3D.
The meaning of “formation medium” in claim 1 cannot be determined.
The meaning of “pre-gastrulation pluripotent cell” in claim 1 cannot be determined.
The phrase “in an amount of 0.1-20% (v/v)” at the end of claim 1 should be put with the serum substitute.
The meaning of a “pre-gastrulation pluripotent cell” that is “derived from a naïve ESC, [iPS cells, epiblast,……]early embryo from post-implantation to pre-gastrulation” in claim 1 cannot be determined. The concept does not make sense because ESC, iPS cells, epiblast, etc. are not “pre-gastrulation” per se. It is unclear how a “pre-gastrulation pluripotent” cell can be “derived from” an ES or iPS cell, i.e. it is unclear how an ES or iPS cell can be turned in to a “pre-gastrulation pluripotent” cell.
The phrase “cell (Epiblast)” in the phrase “epiblast cell (Epiblast)” is redundant; just say epiblast.
A pluripotent cell “induced from a somatic cell” in claim 1 is an induced pluripotent stem cell (iPS) cell which has already been listed, so the pluripotent cell “induced from a somatic cell” is redundant; please delete it.
The step of isolating a “pre-gastrulation pluripotent cell” from a naïve ESC, iPS cell, etc. in lines 18-21 should be before culturing the “pre-gastrulation pluripotent cell” in line 15-17.
The features of the “formation medium” in lines 22-24 should be with the formation medium in line 15.
The term “stem” in “pluripotent stem” in claim 1 is redundant.
Claim 1 can be written more clearly as ---An isolated mammalian pluripotent cell that:
a) expresses at least 2 times more CLDN6, WNT3,… and ZIC5 as compared to a naïve embryonic stem (ES) cell,
b) is capable of being cultured for at least 5 passages while maintaining expression of at least 2 times more CLDN6, WNT3,… and ZIC5 as compared to a naïve ES cell---.
This suggestion is not meant to be construed as allowable because of the numerous rejections below.
Claim Rejections - 35 USC § 112
Written Description
Claims 1, 7, 13, 18, 22-34, 36, 38 remain and claim 40 is 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.
Claim 1 is drawn to an isolated pluripotent stem cell
wherein expression levels of CLDN6, WNT3, MIXL1, LIN28B, EPHA1, MMP25, MOXD1, MYC, NECTIN1, SLC39A8, SLC7A8, TMEM125, TMEM59L, GRHL2, ETV4, and ZIC5 in the isolated pluripotent stem cell are each at least 2 times of that in a naive embryonic stem cell (ESC);
wherein the isolated pluripotent stem cell is capable of being passaged for at least 5 times while maintaining said expression levels;
wherein the isolated pluripotent stem cell is produced by a method comprising carrying out three-dimensional culture of a pre-gastrulation pluripotent stem cell in a formation medium in the presence of a hydrogel;
wherein the pre-gastrulation pluripotent stem cell is derived from a naive ESC, an induced pluripotent stem cell (iPSC), an epiblast cell (Epiblast), a blastocyst inner cell mass, a pluripotent stem cell induced from a somatic cell, or an early embryo from post-implantation to pre- gastrulation;
wherein the formation medium comprises a basal medium supplemented with a serum substitute, Activin A, bFGF, and a Wnt/R-catenin signal transduction inhibitor; and wherein the serum substitute is in an amount of 0.1-20% (v/v).
A) The specification lacks written description for pluripotent cells that express at least 2 times as much CLDN6, WNT3,… …and ZIC5 as a “naïve” ES cell as required in claim 1. The metes and bounds of when an ES cell is “naïve” cannot be determined. The specification uses the term “naïve” to describe ES and iPS cells (pg 1, last partial paragraph) and discusses a “formative” state of pluripotency between a “naïve” state and a “primed” state (pg 2, 1st full paragraph). However, the specification and the art do not define when an ES cell is “naïve”. The concept does not make sense in context of iPS cells because they are somatic cells that have been artificially induced to become pluripotent; they are not “naïve” because they have been manipulated by the hand of man. If there are structures/functions associated with “naïve” pluripotent cells that distinguish them from “formative” or “primed” pluripotent cells, then the specification and the art has failed to provide a definition so that those of skill would be able to make such a determination. Without such guidance, there is no way for those of skill to determine how to use CLDN6, WNT3,… …and ZIC5 expression in “naïve” ES cells as a control as required in claim 1.
It is noted that pg 12, line 8, describes pluripotent cells that can be passages 5 times (pg 12, line 8) as required in claim 1.
B) The specification lacks written description for pluripotent cells that express 2X CLDN6, WNT3,… …and ZIC5 as required in claim 1. Applicants pointed to pg 8-10, pg 11-12, pg 20-21, Examples 3, 4, 6, and 7 (pg 9 of the remarks filed 3-2-26); however, the Markush group cannot be found. Accordingly, the concept lacks written description.
C) The specification lacks written description for pluripotent cells as broadly encompassed by claim 1 other than mammalian pluripotent cells. The claim encompasses plant, insect, fish, amphibian, reptile, bird or mammalian pluripotent cells. However, the specification is limited to mammalian pluripotent cells (examples). The specification does not correlate mammalian pluripotent cells to plant, insect, fish, amphibian, reptile, or bird pluripotent cells. Accoridngly, pluripotent cells in claim 1 lack written description other than mammalian pluripotent cells.
D) The specification lacks written description for wherein the isolated pluripotent stem cell is produced by a method comprising carrying out three-dimensional culture of a pre-gastrulation pluripotent stem cell in a formation medium [comprising a basal medium supplemented with a serum substitute in an amount of 0.1-20% (v/v), Activin A, bFGF, and a Wnt/R-catenin signal transduction inhibitor] in the presence of a hydrogel. It is unclear how/whether the term “formation” in the phrase “formation medium” indicates the medium must be capable of making “formative” pluripotent cells out of “naïve” pluripotent cells. While the components of the medium are clearly set forth, it is unclear whether the “carrying out three dimensional culture of a pre-gastrulation pluripotent stem cell” must result in “formative” pluripotent cells. The metes and bounds of “formative” pluripotent cells as inferred by the term/step cannot be determined because the phrase is not defined in the specification or the art at the time of filing. If there are structures/functions associated with “formative” pluripotent cells that distinguish them from “naive” or “primed” pluripotent cells, then the specification and the art has failed to provide a definition so that those of skill would be able to make such a determination. Without such guidance, there is no way for those of skill to determine how to use medium that is “formation” medium as claimed.
Likewise, the metes and bounds of “pre-gastrulation” pluripotent cells cannot be determined because the phrase is not defined in the specification or the art at the time of filing. If there are structures/functions associated with “pre-gastrulation” pluripotent cells that distinguish them from “naive”, “formative”, or “primed” pluripotent cells, then the specification and the art has failed to provide a definition so that those of skill would be able to make such a determination. Without such guidance, there is no way for those of skill to determine how to determine whether they were starting with a pre-gastrulation” pluripotent cell as claimed.
To further complicate the matter, it is unclear how the process of culturing a pre-gastrulation” pluripotent cell in “formation” medium further limits the structure/function of the pluripotent cells that expresses 2X CLDN6, WNT3,… …and ZIC5 as compared to “naïve” ES cells as required in claim 1. It is wholly unclear how the process claimed alters the structure/function of any pluripotent cell, specifically a “pre-gastrulation” pluripotent cell to arrive at the overexpression claimed.
It is again noted that all tissue culture is 3D, so it is unclear how/why the phrase “tree-dimensional culture” is being used. If there are particular structures that are associated with 3D culture beyond any cell culture technique, then those structures are missing from the claim.
Accordingly, the “carrying out three-dimensional culture” step in claim 1 lacks written description as claimed.
E) The specification lacks written description for deriving a “pre-gastrulation” pluripotent cell from a naive ESC, an iPSC, an epiblast, a blastocyst inner cell mass, a pluripotent stem cell induced from a somatic cell, or an early embryo from post-implantation to pre- gastrulation. It is unclear when/why/how a pluripotent cell is a “pre-gastrulation” pluripotent cell for reasons set forth above. It is unclear how to take a “naive ESC, an iPSC, an epiblast, a blastocyst inner cell mass, a pluripotent stem cell induced from a somatic cell, or an early embryo from post-implantation to pre- gastrulation” and turn it into a “pre-gastrulation” pluripotent cell as claimed. The concept of “naïve” ES cells lacks written description for reasons set forth above. The “pluripotent cell induced from a somatic cell” is equivalent to the iPSC. The concept of an “early embryo from post-implantation to pre- gastrulation” lacks written description because the phrase does not make sense - it is unclear when an embryo is “from post-implantation to pre- gastrulation”. It is also unclear when an embryo is “early”. Accordingly, the specification lacks written description for when they had arrived at a “pre-gastrulation” pluripotent cell or how to derive one from a naive ESC, an iPSC, an epiblast, a blastocyst inner cell mass, a pluripotent stem cell induced from a somatic cell, or an early embryo from post-implantation to pre- gastrulation.
F) The specification lacks written description for mammalian pluripotent cells that have “H3K4me3 and H3K27me3 super bivalency in [a] HOXA, GATA4, GATA6,… …LHX5” promoter region as required in claim 7.
Claim 7 requires “wherein the super bivalency of H3K4me3 and H3K27me3 refers to bivalent modifications that meet both conditions in chromatin immunoprecipitation-sequencing (ChIP- seq) assays: (i) the enrichment signal of H3K4me3 in the promoter region is stronger than that of a house-keeping gene; and (ii) the promoter region has strong H3K27me3 modification with reads per kilobase per million mapped reads (RPKM) of more than 1 after normalization.”
The metes and bounds of “both conditions” or any “conditions” in a ChIP-seq assay cannot be determined. The assay name “chromatin immunoprecipitation-sequencing” is jargon and fails to set forth the reagents or active steps in the assay. The assay name also fails to clearly set forth the result, so it cannot be determined what was sequenced or how the sequencing data was process, how sequences of interest were selected, the desired “conditions,” or when “both conditions” of a ChIP-seq assay are/were met.
The metes and bound of a house-keeping gene in context of the genes listed in claim 7 cannot be determined. Therefore, it cannot be determined when “superbivalency” is “stronger than that of a house-keeping gene”.
The metes and bounds of a “strong H3K27me3 modification” cannot be determined. It is unclear when “H3K27me3 modification” are “strong”. It is unclear when super bivalency is “strong.
The metes and bounds of “RPKM” cannot be determined; it is unclear how it relates to the “strong” H3K27me3 modification or when it is “after normalization” or when it is “more than 1 after normalization”.
Pg 8, 2nd full paragraph, says: “H3K4me3 refers to the trimethylation modification of the 4th lysine of histone H3, and H3K27me3 refers to the trimethylation modification of the 27th lysine on histone H3”. However, the meaning cannot be discerned. The structure of modification of the trimethylation is not disclosed. The meaning and location of “the 4th lysine of histone H3” or “the 27th lysine on histone H3” cannot be discerned because the amino acid sequences in each species, subspecies, variants, and individuals are not the same. It is also unclear when a pluripotent cell has “H3K4me3 and H3K27me3 super bivalency” of any nucleic acid sequence because it is unclear how “super bivalency” further limits the “H3K4me3 and H3K27me3” state or how “H3K4me3 and H3K27me3” further limits “super bivalency”. It is unclear whether the phrase is attempting to say one H3 histone within a promoter has a modification of a trimethylation at the 4th and 27th lysine or if it is attempting to say ALL H3 histones within the promoter must have a modification of a trimethylation at the 4th and 27th lysine. The specification fails to teach any reasonable number of representative structures within the genus. Fig. 3A discusses “H3K4me3 and H3K27me3” but does not teach all H3 histones within a “HOXA, GATA4, GATA6,… …LHX5” promoter have a “trimethylation modification of the 4th lysine of histone H3, and H3K27me3 refers to the trimethylation modification of the 27th lysine on histone H3”. Pg 8 teaches: “It is known in the art that when used to describe epigenetics, "covalent" (also referred to as bivalent/bivalency) refers to an epigenetic modification with both a transcriptional activation marker H3K4me3 and a transcriptional repression marker H3K27me3, wherein, H3K4me3 refers to the trimethylation modification on the 4th lysine of histone H3, and H3K27me3 refers to the trimethylation modification of the 27th lysine on histone H3. A gene in which the above-mentioned covalent modifications exist in the promoter region is called a bivalent gene. "Super bivalent/super bivalency" refers to a bivalent modification that meets the following conditions in ChIP-seq assays: 1) the enrichment signal of H3K4me3 in the promoter region is stronger than that of the house-keeping gene; 2) the promoter region has strong H3K2 7me3 modification (RPKLVl> l after normalization). Gene with the above-mentioned super bivalent modifications in the promoter region is called a super bivalent gene. Detailed description of super bivalent modification can be found in Xiang Y, et al. Nat Genet. 2020 Jan;52(1):95-l05, which is incorporated herein by reference. Herein, when used to describe epigenetics, "bivalent" and "covalent" are two translations of bi valency, both have the same meaning and can be used interchangeably.” However, the only way the promoter is “bivalent” for anything is the trimethylation modification on the 4th lysine of histone H3 and trimethylation modification of the 27th lysine on histone H3 which is missing from the claim. The specification lacks written description for when this “bivalent” trimethylation is “super” as claimed. Xiang (Nature Genetics, 2020, Vol. 52, No. 1, pg 95-105) relies on this ChIP-seq assay without defining when the desired trimethylation is “super”. Xiang discussion of “Super bivalent gene identification” says: “Among all bivalent genes (described above), we sought to search for those with unusually strong H3K4me3 and H3K27me3 by the following criteria: 1) H3K4me3 enrichment (normalized by Z-score) is higher than that of average H3K4me3 at housekeeping genes in each lineage; and 2) H3K27me3 (normalized by Z-score) is also strong (with an arbitrary cutoff, normalized RPKM > 1). We required strong H3K27me3 because bivalent genes can have very low H3K27me3 when they become activated in E6.5 Epi.” (pg 1, col. 2, last full paragraph, of “Methods” of Xiang. The specification, Xiang, and the art at the time of filing do not teach when “bivalency” of the trimethylation of the 4th or 27th lysine on H3 is “super”, so those of skill could not determine when this trimethylation is “super”.
Fig. 3A discusses “H3K4me3 and H3K27me3” but does not teach all H3 histones within all 200 promoters of the “ABLIM2, ADAM22, ADM,… …and ZFP703” genes have a “trimethylation modification of the 4th lysine of histone H3, and H3K27me3 refers to the trimethylation modification of the 27th lysine on histone H3”.
Accordingly, the concept in claim 7 lacks written description.
G) The specification lacks written description for mammalian pluripotent cells that have “super bivalency of H3K4me3 and H3K27me3 in the promoter region of [ ] UTF1, FGF5, ZFP13, ZSCAN10, ZIC2, and ESRP1, wherein the super bivalency of H3K4me3 and H3K27me3 refers to bivalent modifications that meet both conditions in chromatin immunoprecipitation-sequencing (ChIP- seq) assays: (i) the enrichment signal of H3K4me3 in the promoter region is stronger than that of a house-keeping gene; and (ii) the promoter region has strong H3K27me3 modification with reads per kilobase per million mapped reads (RPKM) of more than 1 after normalization” as required in claim 38 for reasons set forth above for claim 7.
H) The specification lacks written description for mammalian pluripotent cells that have “super bivalency of H3K4me3 and H3K27me3 in the promoter region of [ ] HOXA1, HOXA11, HOXA2, HOXA3, HOXA5, HOXA6, GATA4, GATA6, HAND1, PAX2, PAX6, FOXF2, T or LHX, wherein the super bivalency of H3K4me3 and H3K27me3 refers to bivalent modifications that meet both conditions in chromatin immunoprecipitation-sequencing (ChIP- seq) assays: (i) the enrichment signal of H3K4me3 in the promoter region is stronger than that of a house-keeping gene; and (ii) the promoter region has strong H3K27me3 modification with reads per kilobase per million mapped reads (RPKM) of more than 1 after normalization” as required in claim 40 for reasons set forth above for claim 7.
Response to arguments
Applicants argue support is found on claims 22-24, 26 and page 1 last paragraph of the English translation filed (pg 9). Applicants’ argument is not persuasive. There is nothing in the cited claims or paragraph about culturing “pre-gastrulation pluripotent cells” in 3D culture such that new pluripotent cells expressing the markers listed in an amount that is at least 2X greater than “naïve” ES cells.
Applicants point to support for claim 7 in pg 8, 2nd para from the bottom, and pg 38, 2nd para from the bottom through the top of pg 39 (pg 9). Applicants’ argument is not persuasive. Pg 38-39 discusses references for identifying super bivalent genes but not anything that says culturing “pre-gastrulation pluripotent cells” in 3D culture results in new pluripotent cells that express the markers in amounts that is at least 2X greater than “naïve” ES cells as required in claim 1 AND have “super bivalency” in the locations listed in the promoter of the genes listed in claim 7.
Applicants’ discussion on pg 11-15 appears to argue the amendment overcomes the rejection. Applicants’ argument is not persuasive because the specification does not teach the limitations claimed, and the limitations have invoked new indefiniteness rejections. The expression pattern in claim 1 fails to capture anything that indicates the pluripotent cells have a different function than the starting material. In fact, the specification is so confusing, the specification lacks written description that the expression pattern in claim 1 anything within the realm of turning a “naïve” pluripotent cell into a “formative” pluripotent cell (whatever that is). The definitions and lines between “naïve” and “formative” pluripotent cells is not adequately described in the specification or the art at the time of filing.
Applicants discussion of super bivalency in claim 7 cites Example 3 (pg 15-16). The discussion and Examples 3 and 4 not persuasive. Example 3 (pg 33) analyzes genomic RNA of “fPSCs” (whatever they are) without defining what genomic RNA defines a cell as a “fPSC”. Example 3 does not correlate any specific genomic RNA pattern to any specific new function of “fPSCs” vs. “naïve” PSCs. Example 4, specifically pg 38-39, discusses references for identifying super bivalent genes but not anything that says culturing “pre-gastrulation pluripotent cells” in 3D culture results in new pluripotent cells that express the markers in amounts that is at least 2X greater than “naïve” ES cells as required in claim 1.
Applicants discuss Xiang who taught how to determine super bivalency (pg 16). Applicants’ argument is not persuasive. Xiang discusses super bivalency without defining what super bivalency defines a cell as a “naïve” vs. “fPSC”. Xian does not correlate any specific super bivalency pattern to any specific new function of “fPSCs” vs. “naïve” PSCs.
Applicants discuss Kinoshita and Zheng who taught how to determine super bivalency (pg 17). Applicants’ argument is not persuasive. Kinoshita and Zheng discuss super bivalency without defining what super bivalency defines a cell as a “naïve” vs. “fPSC”. Kinoshita and Zheng do not correlate any specific super bivalency pattern to any specific new function of “fPSCs” vs. “naïve” PSCs.
Applicants argue RPKM was known in the art as defined by Chapter 12 of a website (Exhibit A) and Li (Exhibit B) (pg 17-18). Applicants’ argument is not persuasive. The website information was printed in 2026, and there is no evidence that it was available at the time of filing; therefore, it cannot be used to establish what applicants were in possession of at the time of filing. There is nothing in Exhibit A or B that defines when “super bivalent” modifications are “reads per kilobase per million”. It is unclear what “reads” means, and it is unclear when anything (specifically “reads”) is “per kilobase per million”. This phrase appears to be jargon and does not define when promoters have the “super bivalency”.
Applicants’ discussion of claim 7 on pg 18-19 refers to Examples 4 and 7 and Fig. 3. Applicants’ argument and the Examples and Figure are not persuasive. Examples 4 and 7 discuss “fPSCs” (whatever they are) without defining what genomic RNA or what “super bivalency” pattern defines a cell as a “fPSC”. Examples 4 and 7 do not correlate any specific genomic RNA pattern or “super bivalency” pattern to any specific new function of “fPSCs” vs. “naïve” PSCs. Example 4 does not teach culturing “pre-gastrulation pluripotent cells” in 3D culture so that new pluripotent cells with a specific “super bivalency” patter are obtained as compared to “naïve” ES cells. Examples 4 and 7 do not teach the “super bivalency” pattern in claim 7.
Applicants’ discussion of claim 38 on pg 19-20 is noted but is not persuasive.
Enablement
Claims 1, 7, 13, 18, 22-34, 36, 38 remain and claim 40 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for mammalian pluripotent stem cells capable of being passages at least 5 times, does not reasonably provide enablement for any “formative differentiation function, morphological characteristic, genomic characteristic, or epigenomics characteristic” other than pluripotent characteristics, or any “super bivalency” in an ABLIM2, ANKRD33B,… …or VLDLR promoter. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make/use the invention commensurate in scope with these claims.
Claim 1 is summarized above.
A) The specification does not enable making/using pluripotent cells that express at least 2 times as much CLDN6, WNT3,… …and ZIC5 as a “naïve” ES cell as required in claim 1. The metes and bounds of when an ES cell is “naïve” cannot be determined. The specification uses the term “naïve” to describe ES and iPS cells (pg 1, last partial paragraph) and discusses a “formative” state of pluripotency between a “naïve” state and a “primed” state (pg 2, 1st full paragraph). However, the specification and the art do not define when an ES cell is “naïve”. The concept does not make sense in context of iPS cells because they are somatic cells that have been artificially induced to become pluripotent; they are not “naïve” because they have been manipulated by the hand of man. If there are structures/functions associated with “naïve” pluripotent cells that distinguish them from “formative” or “primed” pluripotent cells, then the specification and the art has failed to provide a definition so that those of skill would be able to make such a determination. Without such guidance, there is no way for those of skill to determine how to make/use CLDN6, WNT3,… …and ZIC5 expression in “naïve” ES cells as a control as required in claim 1.
It is noted that pg 12, line 8, describes pluripotent cells that can be passages 5 times (pg 12, line 8) as required in claim 1.
B) The specification does not enable making/using pluripotent cells that express 2X CLDN6, WNT3,… …and ZIC5 as required in claim 1. Applicants pointed to pg 8-10, pg 11-12, pg 20-21, Examples 3, 4, 6, and 7 (pg 9 of the remarks filed 3-2-26); however, the Markush group cannot be found. Accordingly, the concept is not enabled.
C) The specification does not enable making/using pluripotent cells as broadly encompassed by claim 1 other than mammalian pluripotent cells. The claim encompasses plant, insect, fish, amphibian, reptile, bird or mammalian pluripotent cells. However, the specification is limited to mammalian pluripotent cells (examples). The specification does not correlate mammalian pluripotent cells to plant, insect, fish, amphibian, reptile, or bird pluripotent cells. Accordingly, pluripotent cells in claim 1 are not enabled other than mammalian pluripotent cells.
D) The specification does not enable making/using isolated pluripotent stem cell is produced by a method comprising carrying out three-dimensional culture of a pre-gastrulation pluripotent stem cell in a formation medium [comprising a basal medium supplemented with a serum substitute in an amount of 0.1-20% (v/v), Activin A, bFGF, and a Wnt/R-catenin signal transduction inhibitor] in the presence of a hydrogel. It is unclear how/whether the term “formation” in the phrase “formation medium” indicates the medium must be capable of making “formative” pluripotent cells out of “naïve” pluripotent cells. While the components of the medium are clearly set forth, it is unclear whether the “carrying out three dimensional culture of a pre-gastrulation pluripotent stem cell” must result in “formative” pluripotent cells. The metes and bounds of “formative” pluripotent cells as inferred by the term/step cannot be determined because the phrase is not defined in the specification or the art at the time of filing. If there are structures/functions associated with “formative” pluripotent cells that distinguish them from “naive” or “primed” pluripotent cells, then the specification and the art has failed to provide a definition so that those of skill would be able to make such a determination. Accordingly, there is no way for those of skill to determine how to make/use medium that is “formation” medium as claimed.
Likewise, the metes and bounds of “pre-gastrulation” pluripotent cells cannot be determined because the phrase is not defined in the specification or the art at the time of filing. If there are structures/functions associated with “pre-gastrulation” pluripotent cells that distinguish them from “naive”, “formative”, or “primed” pluripotent cells, then the specification and the art has failed to provide a definition so that those of skill would be able to make such a determination. Without such guidance, there is no way for those of skill to determine how to determine whether they were starting with a “pre-gastrulation” pluripotent cell as claimed.
To further complicate the matter, it is unclear how the process of culturing a pre-gastrulation” pluripotent cell in “formation” medium further limits the structure/function of the pluripotent cells that expresses 2X CLDN6, WNT3,… …and ZIC5 as compared to “naïve” ES cells as required in claim 1. It is wholly unclear how the process claimed alters the structure/function of any pluripotent cell, specifically a “pre-gastrulation” pluripotent cell to arrive at the overexpression claimed.
It is again noted that all tissue culture is 3D, so it is unclear how/why the phrase “tree-dimensional culture” is being used. If there are particular structures that are associated with 3D culture beyond any cell culture technique, then those structures are missing from the claim.
Accordingly, the “carrying out three-dimensional culture” step in claim 1 is not enabled.
E) The specification does not enable deriving a “pre-gastrulation” pluripotent cell from a naive ESC, an iPSC, an epiblast, a blastocyst inner cell mass, a pluripotent stem cell induced from a somatic cell, or an early embryo from post-implantation to pre- gastrulation. It is unclear when/why/how a pluripotent cell is a “pre-gastrulation” pluripotent cell for reasons set forth above. It is unclear how to take a “naive ESC, an iPSC, an epiblast, a blastocyst inner cell mass, a pluripotent stem cell induced from a somatic cell, or an early embryo from post-implantation to pre- gastrulation” and turn it into a “pre-gastrulation” pluripotent cell as claimed. The concept of “naïve” ES cells lacks written description for reasons set forth above. The “pluripotent cell induced from a somatic cell” is equivalent to the iPSC. The concept of an “early embryo from post-implantation to pre- gastrulation” lacks written description because the phrase does not make sense - it is unclear when an embryo is “from post-implantation to pre- gastrulation”. It is also unclear when an embryo is “early”. Accordingly, the specification does not enable those of skill to determine when they had arrived at a “pre-gastrulation” pluripotent cell or how to derive one from a naive ESC, an iPSC, an epiblast, a blastocyst inner cell mass, a pluripotent stem cell induced from a somatic cell, or an early embryo from post-implantation to pre- gastrulation.
F) The specification does not enable making/using mammalian pluripotent cells that have “H3K4me3 and H3K27me3 super bivalency in [a] HOXA, GATA4, GATA6,… …LHX5” promoter region as required in claim 7.
Claim 7 requires “wherein the super bivalency of H3K4me3 and H3K27me3 refers to bivalent modifications that meet both conditions in chromatin immunoprecipitation-sequencing (ChIP- seq) assays: (i) the enrichment signal of H3K4me3 in the promoter region is stronger than that of a house-keeping gene; and (ii) the promoter region has strong H3K27me3 modification with reads per kilobase per million mapped reads (RPKM) of more than 1 after normalization.”
The metes and bounds of “both conditions” or any “conditions” in a ChIP-seq assay cannot be determined. The assay name “chromatin immunoprecipitation-sequencing” is jargon and fails to set forth the reagents or active steps in the assay. The assay name also fails to clearly set forth the result, so it cannot be determined what was sequenced or how the sequencing data was process, how sequences of interest were selected, the desired “conditions,” or when “both conditions” of a ChIP-seq assay are/were met.
The metes and bound of a house-keeping gene in context of the genes listed in claim 7 cannot be determined. Therefore, it cannot be determined when “superbivalency” is “stronger than that of a house-keeping gene”.
The metes and bounds of a “strong H3K27me3 modification” cannot be determined. It is unclear when “H3K27me3 modification” are “strong”. It is unclear when super bivalency is “strong.
The metes and bounds of “RPKM” cannot be determined; it is unclear how it relates to the “strong” H3K27me3 modification or when it is “after normalization” or when it is “more than 1 after normalization”.
Pg 8, 2nd full paragraph, says: “H3K4me3 refers to the trimethylation modification of the 4th lysine of histone H3, and H3K27me3 refers to the trimethylation modification of the 27th lysine on histone H3”. However, the meaning cannot be discerned. The structure of modification of the trimethylation is not disclosed. The meaning and location of “the 4th lysine of histone H3” or “the 27th lysine on histone H3” cannot be discerned because the amino acid sequences in each species, subspecies, variants, and individuals are not the same. It is also unclear when a pluripotent cell has “H3K4me3 and H3K27me3 super bivalency” of any nucleic acid sequence because it is unclear how “super bivalency” further limits the “H3K4me3 and H3K27me3” state or how “H3K4me3 and H3K27me3” further limits “super bivalency”. It is unclear whether the phrase is attempting to say one H3 histone within a promoter has a modification of a trimethylation at the 4th and 27th lysine or if it is attempting to say ALL H3 histones within the promoter must have a modification of a trimethylation at the 4th and 27th lysine. The specification fails to teach any reasonable number of representative structures within the genus. Fig. 3A discusses “H3K4me3 and H3K27me3” but does not teach all H3 histones within a “HOXA, GATA4, GATA6,… …LHX5” promoter have a “trimethylation modification of the 4th lysine of histone H3, and H3K27me3 refers to the trimethylation modification of the 27th lysine on histone H3”. Pg 8 teaches: “It is known in the art that when used to describe epigenetics, "covalent" (also referred to as bivalent/bivalency) refers to an epigenetic modification with both a transcriptional activation marker H3K4me3 and a transcriptional repression marker H3K27me3, wherein, H3K4me3 refers to the trimethylation modification on the 4th lysine of histone H3, and H3K27me3 refers to the trimethylation modification of the 27th lysine on histone H3. A gene in which the above-mentioned covalent modifications exist in the promoter region is called a bivalent gene. "Super bivalent/super bivalency" refers to a bivalent modification that meets the following conditions in ChIP-seq assays: 1) the enrichment signal of H3K4me3 in the promoter region is stronger than that of the house-keeping gene; 2) the promoter region has strong H3K2 7me3 modification (RPKLVl> l after normalization). Gene with the above-mentioned super bivalent modifications in the promoter region is called a super bivalent gene. Detailed description of super bivalent modification can be found in Xiang Y, et al. Nat Genet. 2020 Jan;52(1):95-l05, which is incorporated herein by reference. Herein, when used to describe epigenetics, "bivalent" and "covalent" are two translations of bi valency, both have the same meaning and can be used interchangeably.” However, the only way the promoter is “bivalent” for anything is the trimethylation modification on the 4th lysine of histone H3 and trimethylation modification of the 27th lysine on histone H3 which is missing from the claim. The specification lacks written description for when this “bivalent” trimethylation is “super” as claimed. Xiang (Nature Genetics, 2020, Vol. 52, No. 1, pg 95-105) relies on this ChIP-seq assay without defining when the desired trimethylation is “super”. Xiang discussion of “Super bivalent gene identification” says: “Among all bivalent genes (described above), we sought to search for those with unusually strong H3K4me3 and H3K27me3 by the following criteria: 1) H3K4me3 enrichment (normalized by Z-score) is higher than that of average H3K4me3 at housekeeping genes in each lineage; and 2) H3K27me3 (normalized by Z-score) is also strong (with an arbitrary cutoff, normalized RPKM > 1). We required strong H3K27me3 because bivalent genes can have very low H3K27me3 when they become activated in E6.5 Epi.” (pg 1, col. 2, last full paragraph, of “Methods” of Xiang. The specification, Xiang, and the art at the time of filing do not teach when “bivalency” of the trimethylation of the 4th or 27th lysine on H3 is “super”, so those of skill could not determine when this trimethylation is “super”.
Fig. 3A discusses “H3K4me3 and H3K27me3” but does not teach all H3 histones within all 200 promoters of the “ABLIM2, ADAM22, ADM,… …and ZFP703” genes have a “trimethylation modification of the 4th lysine of histone H3, and H3K27me3 refers to the trimethylation modification of the 27th lysine on histone H3”.
Accordingly, the concept in claim 7 is not enabled.
G) The specification does not enable making/using mammalian pluripotent cells that have “super bivalency of H3K4me3 and H3K27me3 in the promoter region of [ ] UTF1, FGF5, ZFP13, ZSCAN10, ZIC2, and ESRP1, wherein the super bivalency of H3K4me3 and H3K27me3 refers to bivalent modifications that meet both conditions in chromatin immunoprecipitation-sequencing (ChIP- seq) assays: (i) the enrichment signal of H3K4me3 in the promoter region is stronger than that of a house-keeping gene; and (ii) the promoter region has strong H3K27me3 modification with reads per kilobase per million mapped reads (RPKM) of more than 1 after normalization” as required in claim 38 for reasons set forth above for claim 7.
H) The specification does not enable making/using mammalian pluripotent cells that have “super bivalency of H3K4me3 and H3K27me3 in the promoter region of [ ] HOXA1, HOXA11, HOXA2, HOXA3, HOXA5, HOXA6, GATA4, GATA6, HAND1, PAX2, PAX6, FOXF2, T or LHX, wherein the super bivalency of H3K4me3 and H3K27me3 refers to bivalent modifications that meet both conditions in chromatin immunoprecipitation-sequencing (ChIP- seq) assays: (i) the enrichment signal of H3K4me3 in the promoter region is stronger than that of a house-keeping gene; and (ii) the promoter region has strong H3K27me3 modification with reads per kilobase per million mapped reads (RPKM) of more than 1 after normalization” as required in claim 40 for reasons set forth above for claim 7.
Given the lack of guidance in the specification taken with the art at the time of filing, it would have required those of skill undue experimentation to determine how to make/use mammalian pluripotent cells having the features claimed.
Response to arguments
Applicants arguments are repeated and refer to the written description rejection. Applicants’ arguments are not persuasive for reasons set forth above.
Indefiniteness
Claims 1, 7, 13, 18, 22-34, 36, 38 remain and claim 40 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
A) The metes and bounds that define the structures and functions of pluripotent cell in claim 1 cannot be determined. Nor can the metes/bounds of the structures and functions associated with “naïve” pluripotent cells used for the comparison in claim 1 be determined. The specification uses the term “naïve” to describe ES and iPS cells (pg 1, last partial paragraph) and discusses a “formative” state of pluripotency between a “naïve” state and a “primed” state (pg 2, 1st full paragraph). However, the specification and the art do not clearly and explicitly delineate the structures and functions that define when a pluripotent cell is “formative” (or “primed”) as compared to a “naïve” pluripotent cell. The concept is especially perplexing in context of iPS cells because they are somatic cells that have been artificially induced to become pluripotent; they are not “naïve” because they have been manipulated by the hand of man. If there are structures/functions associated with “formative” (or “primed”) pluripotent cells that distinguish them from “naïve” pluripotent cells, then the specification and the art has failed to provide a definition so that those of skill would be able to make such a determination. Without such guidance, there is no way for those of skill to determine when the desired pluripotent cells that express 2X CLDN6, WNT3,… …or ZIC5 as compared to “naïve” ES cells as required in claim 1.
B) The metes and bounds of carrying out three-dimensional culture of a pre-gastrulation pluripotent stem cell in a formation medium [comprising a basal medium supplemented with a serum substitute in an amount of 0.1-20% (v/v), Activin A, bFGF, and a Wnt/R-catenin signal transduction inhibitor] in the presence of a hydrogel in claim 1 cannot be determined. It is unclear how/whether the term “formation” in the phrase “formation medium” indicates the medium must be capable of making “formative” pluripotent cells out of “naïve” pluripotent cells. While the components of the medium are clearly set forth, it is unclear whether the “carrying out three dimensional culture of a pre-gastrulation pluripotent stem cell” must result in “formative” pluripotent cells. The metes and bounds of “formative” pluripotent cells as inferred by the term/step cannot be determined because the phrase is not defined in the specification or the art at the time of filing. If there are structures/functions associated with “formative” pluripotent cells that distinguish them from “naive” or “primed” pluripotent cells, then the specification and the art has failed to provide a definition so that those of skill would be able to make such a determination. Accordingly, there is no way for those of skill to determine how to make/use medium that is “formation” medium as claimed. It is also unclear whether the cells in claim 1 ARE ”formative” pluripotent cells (whatever they are).
Likewise, the metes and bounds of “pre-gastrulation” pluripotent cells cannot be determined because the phrase is not defined in the specification or the art at the time of filing. If there are structures/functions associated with “pre-gastrulation” pluripotent cells that distinguish them from “naive”, “formative”, or “primed” pluripotent cells, then the specification and the art has failed to provide a definition so that those of skill would be able to make such a determination. Without such guidance, there is no way for those of skill to determine how to determine whether they were starting with a “pre-gastrulation” pluripotent cell as claimed.
To further complicate the matter, it is unclear how the process of culturing a pre-gastrulation” pluripotent cell in “formation” medium further limits the structure/function of the pluripotent cells that expresses 2X CLDN6, WNT3,… …and ZIC5 as compared to “naïve” ES cells as required in claim 1. It is wholly unclear how the process claimed alters the structure/function of any pluripotent cell, specifically a “pre-gastrulation” pluripotent cell to arrive at the overexpression claimed.
It is again noted that all tissue culture is 3D, so it is unclear how/why the phrase “tree-dimensional culture” is being used. If there are particular structures that are associated with 3D culture beyond any cell culture technique, then those structures are missing from the claim.
Accordingly, the “carrying out three-dimensional culture” step in claim 1 is indefinite.
C) The concept of deriving a “pre-gastrulation” pluripotent cell from a naive ESC, an iPSC, an epiblast, a blastocyst inner cell mass, a pluripotent stem cell induced from a somatic cell, or an early embryo from post-implantation to pre- gastrulation makes the claim indefinite. It is unclear when/why/how a pluripotent cell is a “pre-gastrulation” pluripotent cell for reasons set forth above. It is unclear how to take a “naive ESC, an iPSC, an epiblast, a blastocyst inner cell mass, a pluripotent stem cell induced from a somatic cell, or an early embryo from post-implantation to pre- gastrulation” and turn it into a “pre-gastrulation” pluripotent cell as claimed. The concept of “naïve” ES cells is indefinite for reasons set forth above. The “pluripotent cell induced from a somatic cell” is equivalent to the iPSC. The concept of an “early embryo from post-implantation to pre- gastrulation” does not make sense - it is unclear when an embryo is “from post-implantation to pre- gastrulation”. It is also unclear when an embryo is “early”. Accordingly, the specification fails to teach those of skill to determine when they had arrived at a “pre-gastrulation” pluripotent cell or how to derive one from a naive ESC, an iPSC, an epiblast, a blastocyst inner cell mass, a pluripotent stem cell induced from a somatic cell, or an early embryo from post-implantation to pre- gastrulation. This makes the claim indefinite.
D) The specification does not enable making/using mammalian pluripotent cells that have “H3K4me3 and H3K27me3 super bivalency in [a] HOXA, GATA4, GATA6,… …LHX5” promoter region as required in claim 7.
Claim 7 requires “wherein the super bivalency of H3K4me3 and H3K27me3 refers to bivalent modifications that meet both conditions in chromatin immunoprecipitation-sequencing (ChIP- seq) assays: (i) the enrichment signal of H3K4me3 in the promoter region is stronger than that of a house-keeping gene; and (ii) the promoter region has strong H3K27me3 modification with reads per kilobase per million mapped reads (RPKM) of more than 1 after normalization.”
The metes and bounds of “both conditions” or any “conditions” in a ChIP-seq assay cannot be determined. The assay name “chromatin immunoprecipitation-sequencing” is jargon and fails to set forth the reagents or active steps in the assay. The assay name also fails to clearly set forth the result, so it cannot be determined what was sequenced or how the sequencing data was process, how sequences of interest were selected, the desired “conditions,” or when “both conditions” of a ChIP-seq assay are/were met.
The metes and bound of a house-keeping gene in context of the genes listed in claim 7 cannot be determined. Therefore, it cannot be determined when “superbivalency” is “stronger than that of a house-keeping gene”.
The metes and bounds of a “strong H3K27me3 modification” cannot be determined. It is unclear when “H3K27me3 modification” are “strong”. It is unclear when super bivalency is “strong.
The metes and bounds of “RPKM” cannot be determined; it is unclear how it relates to the “strong” H3K27me3 modification or when it is “after normalization” or when it is “more than 1 after normalization”.
Pg 8, 2nd full paragraph, says: “H3K4me3 refers to the trimethylation modification of the 4th lysine of histone H3, and H3K27me3 refers to the trimethylation modification of the 27th lysine on histone H3”. However, the meaning cannot be discerned. The structure of modification of the trimethylation is not disclosed. The meaning and location of “the 4th lysine of histone H3” or “the 27th lysine on histone H3” cannot be discerned because the amino acid sequences in each species, subspecies, variants, and individuals are not the same. It is also unclear when a pluripotent cell has “H3K4me3 and H3K27me3 super bivalency” of any nucleic acid sequence because it is unclear how “super bivalency” further limits the “H3K4me3 and H3K27me3” state or how “H3K4me3 and H3K27me3” further limits “super bivalency”. It is unclear whether the phrase is attempting to say one H3 histone within a promoter has a modification of a trimethylation at the 4th and 27th lysine or if it is attempting to say ALL H3 histones within the promoter must have a modification of a trimethylation at the 4th and 27th lysine. The specification fails to teach any reasonable number of representative structures within the genus. Fig. 3A discusses “H3K4me3 and H3K27me3” but does not teach all H3 histones within a “HOXA, GATA4, GATA6,… …LHX5” promoter have a “trimethylation modification of the 4th lysine of histone H3, and H3K27me3 refers to the trimethylation modification of the 27th lysine on histone H3”. Pg 8 teaches: “It is known in the art that when used to describe epigenetics, "covalent" (also referred to as bivalent/bivalency) refers to an epigenetic modification with both a transcriptional activation marker H3K4me3 and a transcriptional repression marker H3K27me3, wherein, H3K4me3 refers to the trimethylation modification on the 4th lysine of histone H3, and H3K27me3 refers to the trimethylation modification of the 27th lysine on histone H3. A gene in which the above-mentioned covalent modifications exist in the promoter region is called a bivalent gene. "Super bivalent/super bivalency" refers to a bivalent modification that meets the following conditions in ChIP-seq assays: 1) the enrichment signal of H3K4me3 in the promoter region is stronger than that of the house-keeping gene; 2) the promoter region has strong H3K2 7me3 modification (RPKLVl> l after normalization). Gene with the above-mentioned super bivalent modifications in the promoter region is called a super bivalent gene. Detailed description of super bivalent modification can be found in Xiang Y, et al. Nat Genet. 2020 Jan;52(1):95-l05, which is incorporated herein by reference. Herein, when used to describe epigenetics, "bivalent" and "covalent" are two translations of bi valency, both have the same meaning and can be used interchangeably.” However, the only way the promoter is “bivalent” for anything is the trimethylation modification on the 4th lysine of histone H3 and trimethylation modification of the 27th lysine on histone H3 which is missing from the claim. The specification lacks written description for when this “bivalent” trimethylation is “super” as claimed. Xiang (Nature Genetics, 2020, Vol. 52, No. 1, pg 95-105) relies on this ChIP-seq assay without defining when the desired trimethylation is “super”. Xiang discussion of “Super bivalent gene identification” says: “Among all bivalent genes (described above), we sought to search for those with unusually strong H3K4me3 and H3K27me3 by the following criteria: 1) H3K4me3 enrichment (normalized by Z-score) is higher than that of average H3K4me3 at housekeeping genes in each lineage; and 2) H3K27me3 (normalized by Z-score) is also strong (with an arbitrary cutoff, normalized RPKM > 1). We required strong H3K27me3 because bivalent genes can have very low H3K27me3 when they become activated in E6.5 Epi.” (pg 1, col. 2, last full paragraph, of “Methods” of Xiang. The specification, Xiang, and the art at the time of filing do not teach when “bivalency” of the trimethylation of the 4th or 27th lysine on H3 is “super”, so those of skill could not determine when this trimethylation is “super”.
Fig. 3A discusses “H3K4me3 and H3K27me3” but does not teach all H3 histones within all 200 promoters of the “ABLIM2, ADAM22, ADM,… …and ZFP703” genes have a “trimethylation modification of the 4th lysine of histone H3, and H3K27me3 refers to the trimethylation modification of the 27th lysine on histone H3”.
Accordingly, the concept in claim 7 is not indefinite.
E) The metes and bounds of when mammalian pluripotent cells have “super bivalency of H3K4me3 and H3K27me3 in the promoter region of [ ] UTF1, FGF5, ZFP13, ZSCAN10, ZIC2, and ESRP1, wherein the super bivalency of H3K4me3 and H3K27me3 refers to bivalent modifications that meet both conditions in chromatin immunoprecipitation-sequencing (ChIP- seq) assays: (i) the enrichment signal of H3K4me3 in the promoter region is stronger than that of a house-keeping gene; and (ii) the promoter region has strong H3K27me3 modification with reads per kilobase per million mapped reads (RPKM) of more than 1 after normalization” in claim 38 cannot be determined for reasons set forth above for claim 7.
F) The metes and bounds of when mammalian pluripotent cells that have “super bivalency of H3K4me3 and H3K27me3 in the promoter region of [ ] HOXA1, HOXA11, HOXA2, HOXA3, HOXA5, HOXA6, GATA4, GATA6, HAND1, PAX2, PAX6, FOXF2, T or LHX, wherein the super bivalency of H3K4me3 and H3K27me3 refers to bivalent modifications that meet both conditions in chromatin immunoprecipitation-sequencing (ChIP- seq) assays: (i) the enrichment signal of H3K4me3 in the promoter region is stronger than that of a house-keeping gene; and (ii) the promoter region has strong H3K27me3 modification with reads per kilobase per million mapped reads (RPKM) of more than 1 after normalization” in claim 40 cannot be determined for reasons set forth above for claim 7.
Response to arguments
Applicants arguments are repeated and refer to the written description rejection. Applicants’ arguments are not persuasive for reasons set forth above.
Claim Rejections - 35 USC § 102
Claims 1, 7, 13, 18, 32, 35, 38 remain and claim 40 is rejected under 35 U.S.C. 102a1 as being anticipated by Smith (WO 2018/138281).
There is nothing in the specification or the art at the time of filing that says pluripotent cells having 2X increased expression of “CLDN6, WNT3,… …and ZIC5” as compared to ES cells and having “bivalent” “H3K4me3 and H3K27me3” promoters listed in claim 1 MUST be made by any particular method.
There is nothing in the specification that says the pluripotent cells having 2X increased expression of “CLDN6, WNT3,… …and ZIC5” as compared to ES cells in claim 1 have any function that distinguishes them from any well-known pluripotent cell.
Smith isolated pluripotent cells using 3D culture comprising basal medium, KO serum replacement, Activin A, FGF, and XAV (a Wnt/β-catenin signal transduction inhibitor) (pg 5-8, “production of FS cell lines using FS cell culture media”; pg 8, “Culture conditions”).
The pluripotent cells were cultured in the “formation medium” as required in claim 1 (pg 22, Example 1, “Establishing FS cells from epiblast”). For example, Smith taught the media may contain KSR or serum to obtain a “mixed or metastable” population of “predominantly primed” pluripotent cells (pg 5, lines 8-15; pg 6, lines 18-27); KSR is a “serum substitute” described by applicants as being part of the invention (claim 24). Smith is not limited to the absence of KSR; it is optional (pg 8, lines 12-13). They were obtained using the exact same method in lines 15-17 and 22-23 as required in claim 1 and described by applicants as being part of the invention; therefore, the pluripotent cells obtained by Smith after culture inherently MUST have the marker pattern claimed as compared to “naïve” pluripotent cells.
The cells pluripotent because they are capable of germline induction and somatic differentiation (pg 25, Example 6; pg 31, Table 4), “morphological characteristics” (pg 24, Example 3; pg 31, Table 4), genomic characteristics (pg 24, Example 3; pg 31, Table 4), and epigenomic characteristics, e.g. DNA methylation (pg 31, Table 4).
The cells can be passages 5 times (pg 12, line 8) as required in claim 1.
The cells obtained by Smith inherently MUST have “super bivalency in the promoter region of [an] ABLIM2, ANKRD33B, BARX1, DMRT2… …[or] VLDLR [gene]” as required in claim 7 because they were made using the exact same means in claim 1 and described by applicants as being part of the invention for reasons set forth above.
The cells obtained by Smith inherently MUST have “at least 200 super bivalency genes” as required in claim 7 because they were made using the exact same means in claim 1 and described by applicants as being part of the invention for reasons set forth above and because the metes and bounds of “super bivalent genes” cannot be determined (see 112/2nd).
The HAND1, T, FOXA2, NKX2-5,… …or ESRP1 promoter of the cells described by Smith inherently MUST have “lower DNA methylation” as compared to a “naïve” ES cell as required in claim 13 because they were made using the exact same means in claim 1 and described by applicants as being part of the invention for reasons set forth above.
The cells obtained by Smith are mammalian (pg 22-24) as required in claim 18.
The cells obtained by Smith are in pH balanced culture media which is a “pharmaceutical composition” in a “pharmaceutically acceptable carrier” as required in claim 32.
The OTX2, ZIC5, UTF1,… …or ESRP1 promoter of the cells described by Smith inherently MUST have “increased H3K4me3 level and/or H3K27me3 level” as compared to an ES cell as required in claim 38 because they were made using the exact same means in claim 1 and described by applicants as being part of the invention for reasons set forth above.
The cells obtained by Smith inherently MUST have “super bivalency in the promoter region of [an] HOXA1… …LHX5 [gene]” as required in claim 40 because they were made using the exact same means in claim 1 and described by applicants as being part of the invention for reasons set forth above.
Response to arguments
Applicants argue Smith did not teach the same technique for making cells as those described by applicants as being part of the invention because Smith is limited to culturing pluripotent cells in the absence of serum, KSR, or serum replacement. Therefore, applicants conclude Smith could not have taught pluripotent cells having the expression pattern and super bivalency claimed because claim 24 says the method requires using serum replacement. Applicants’ argument is not persuasive.
There is nothing in the specification or the art at the time of filing that says pluripotent cells having 2X increased expression of “CLDN6, WNT3,… …and ZIC5” as compared to “naïve” ES cells as required in claim 1 MUST be made by any particular method. There is nothing in the specification that says the pluripotent cells having 2X increased expression of “CLDN6, WNT3,… …and ZIC5” as compared to “naïve” ES cells as required in claim 1 have any function that distinguishes them from any well-known pluripotent cell. There is nothing in the specification that says “formative” pluripotent cells or pluripotent cells having the expression pattern claimed MUST be made using culture medium in the absence of serum, KSR, or serum replacement.
The cells obtained by Smith inherently MUST express 2X the expression level of CLDN6, WNT3, MIXL1, SOX4,… …FOXH1, GRHL2, or ZIC5 as compared to ES cells as required in claim 1 because Smith taught the media may contain KSR or serum to obtain a “mixed or metastable” population of “predominantly primed” pluripotent cells (pg 5, lines 8-15; pg 6, lines 18-27); KSR is a “serum substitute” described by applicants as being part of the invention in claim 24. The cells made with KSR described by Smith were made using the exact same means described by applicants as being part of the invention in claim 24.
Conclusion
No claim is allowed.
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Michael C. Wilson
/MICHAEL C WILSON/
Primary Examiner, Art Unit 1638
Prosecution Insights