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 .
Claims 2-6, 8-12, 14, 15, 17, 20, 21, 37 have been canceled. Claim 39 has been added. Claims 1, 7, 13, 16, 18, 19, 22-36, 38, 39 are pending.
Election/Restrictions
Applicant's election with traverse of Group I, claims 1, 3, 4, 6, 7, 13, 16, 18, 19, 35, 38 in the reply filed on 8-11-25 is acknowledged. The traversal is on the ground(s) that Smith (WO 2018/138281) did not teach the claims as newly amended. This is not found persuasive because Smith taught isolating pluripotent cells using 3D culture comprising basal medium, KO serum replacement, Activin A, FGF, and XAV (a Wnt/β-catenin signal transduction inhibitor) (pg 5-6, “production of FS cell lines using FS cell culture media”). The cells obtained by Smith inherently MUST express 2X the expression level of CLDN6, WNT3, MIXL1, LIN28B, EPHA1, MMP25, MOXD1, MYC, NECTIN1, SLC39A8, SLC7A8, TMEM59L, GRHL2, ZSCAN10, ETV4, ETV5, FOXH3, or ZIC5 as compared to ES cells as required in claim 1 because they were made using the means described by applicants as being part of the invention in example 1 (pg 30) and in claims 22-24. Claim 32, drawn to a pharmaceutical composition comprising the pluripotent cell of claim 1, was combined with Group I in the office action sent 9-30-25.
Claims 22-31, 33, 34, 36 remain withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 8-11-25.
Claims 1, 7, 13, 16, 18, 19, 32, 35, 38, 39 are under consideration.
Applicant's arguments filed 3-2-26 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 Rejections - 35 USC § 112
Written Description
Claims 1, 3, 4, 6, 7, 13, 16, 18, 19, 32, 35, 38 remain and claim 39 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
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A) The specification lacks written description for pluripotent cells that have “the pluripotency of a mammalian embryonic epiblast cell from post-implantation to pre-gastrulation” as broadly encompassed by claim 1 other than ---pluripotency---. The metes and bounds of the phrase cannot be determined because pluripotency is pluripotency. It is unclear how the pluripotency of an “embryonic epiblast cell” differs from any other pluripotent cell. It is unclear how the pluripotency of an “embryonic epiblast cell from post-implantation” differs from an “embryonic epiblast cell from pre-gastrulation” or any other pluripotent cell. The metes and bounds of when an epiblast is “post-implantation” cannot be determined when the pluripotent cell is in a laboratory and not in a uterus. The metes and bounds of when an epiblast is “early post-implantation” cannot be determined when the pluripotent cell is in a laboratory and not in a uterus and because the line at which a “post-implantation” epiblast is “early” is not defined in the specification or the art at the time of filing.
B) The specification lacks written description for pluripotent cells that have a desired “formative differentiation function” or a desired “morphological characteristics, genomic characteristics, or epigenomic characteristics” as required in lines 6-8 of claim 1 other than pluripotent cells. The metes and bounds of when cells have a “formative differentiation function” in line 6-7 of claim 1 cannot be determined because the phrase is not defined in the specification or the art at the time of filing. The metes and bounds of when the cells have desired “morphological characteristics”, genomic characteristics, or epigenomic characteristics in lines 7-8 of claim 1 cannot be determined because the specification does not teach these characteristics that define the cells of interest. Accordingly, the concept in lines 6-8 of claim 1 lack written description other than for pluripotent cells – the phrases do nothing to further limit the cells other than to say they are pluripotent.
C) The specification lacks written description for mammalian pluripotent cells in which CLDN6, WNT3, MIXL1, LIN28B, EPHA1, MMP25, MOXD1, MYC, NECTIN1, SLC39A8, SLC7A8, TMEM59L, GRHL2, ZSCAN10, ETV4, ETV5, FOXH3, or ZIC5 expression levels are “at least 2 times of that in an embryonic stem cell (ESC)” in lines 11-15 as required in claim 1. Applicants point 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.
Smith (WO 2018/138281) 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 cells have “the pluripotency of a mammalian embryonic epiblast cell from post-implantation to pre-gastrulation (early post-implantation epiblast)” as required in claim 1 because Smith isolated the cells from epiblast (pg 22, Example 1, “Establishing FS cells from epiblast”), because the cells obtained by Smith were pluripotent, and because the metes and bounds of the phrase cannot be determined.
The cells have a “differentiation function” 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 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 they were made using the exact same means described by applicants as being part of the invention in claims 22-24. 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).
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 1 because they were made using the exact same means described by applicants as being part of the invention in claims 22-24.
The specification does not teach any specific mammalian pluripotent cells that have the expression pattern claim in its narrowest embodiments (2X expression of all of the markers as compared to ESC), nor in its broadest embodiment (2X expression of one of the markers as compared to ESC). Pg 6 lists a number of markers, but the specification does not contemplate excluding certain markers, i.e. SOX4, JPH4, ZIC2, EOMES, TCFL5, FOXH1 as newly required in claim 1. Furthermore, the comparison to any embryonic stem cell of any species is meaningless because ES cells are also pluripotent and have “formative differentiation function” as well as “morphological”, “genomic”, and “epigenomic characteristics” as required in lines 5-7 of claim 1.
D) 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 lines 16-18 of claim 1. 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”. Accordingly, the concept lacks written description.
E) The specification lacks written description for mammalian pluripotent cells that have “H3K4me3 and H3K27me3 super bivalency in [the promoter of] at least 200 genes selected from the group ABLIM2, ADAM22, ADM,… …ZFP703” as required in claim 7. The meaning of “H3K4me3 and H3K27me3 super bivalency” and “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” on pg 6 cannot be determined for reasons set forth above. 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 lacks written description.
F) The specification lacks written description for mammalian pluripotent cells that have “H3K4me3 and H3K27me3 super bivalency in [a] HOXA, GATA4, GATA6,… …LHX5” and a promoter region of a OTX2, ZIC5, UTF1, FGF5, ZFP13, ZSCAN10, ZIC2, or an ESRP1 gene that “has increased H3K4me3 level and/or decreased H3K27me3 level compared to the promoter region of the corresponding gene in an ESC” as required in claim 38.. 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 “increased H3K4me3 level and/or decreased H3K27me3 level”. It is unclear whether the phrase is attempting to say one H3 histone within the 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 “OTX2, ZIC5,… …ESRP1” 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 is recited above. 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”. If applicants are simply attempting to further limit the list of promoters in claim 1, then much clarification will be required. Accordingly, the concept lacks written description.
Response to arguments
Applicants argue support is found on pg 6, Examples 3 and 6; pg 8-10, Examples 4 and 7 (pg 11 of the response). Applicants’ argument is not persuasive for reasons set forth above.
Applicants argue support for the expression levels in claim 1 are found on pg 3 and Example 5 (pg 12). Applicants’ argument is not persuasive for reasons set forth above.
Applicants argue support for “formative differentiation function” in claim 1 is found on pg 3 (pg 13). Applicants’ argument is not persuasive for reasons set forth above. The phrase means nothing more than “pluripotent”. Pg 3 says nothing that indicates otherwise. If pg 3 further limits a specific type of “pluripotency”, then it is unclear how.
Applicants argue support for “morphological” “genomic” and “epigenomics characteristic” of pluripotent cells in claim 1 is found in Example 2 and pg 8-11 (pg 14-16). Applicants’ argument is not persuasive for reasons set forth above. The phrases mean nothing more than the cells are “pluripotent”. Example 2 says nothing other than the cells are pluripotent. If Example 2 says the cells have a specific type of “pluripotency”, then it is unclear how.
Applicants argue a super bivalent promoter has support on pg 8 and Example 3 (pg 18-20). Applicants’ argument is not persuasive for reasons set forth above.
Applicants argue decreased H3K27me3 in the promoters listed in claim 38 has support (pg 20-21). Applicants’ argument is not persuasive. The concept cannot be found in the specification. The specification does not teach any pluripotent cells with a “super bivalent” promoter in claim 1 and a promoter with increased H3K4me3 or increased H3K27me3 as required in claim 38.
Enablement
Claims 1, 3, 4, 6, 7, 13, 16, 18, 19, 32, 35, 38 remain and claim 39 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 have “the pluripotency of a mammalian embryonic epiblast cell from post-implantation to pre-gastrulation” as broadly encompassed by claim 1 other than ---pluripotency---. The metes and bounds of the phrase cannot be determined because pluripotency is pluripotency. It is unclear how the pluripotency of an “embryonic epiblast cell” differs from any other pluripotent cell. It is unclear how the pluripotency of an “embryonic epiblast cell from post-implantation” differs from an “embryonic epiblast cell from pre-gastrulation” or any other pluripotent cell. The metes and bounds of when an epiblast is “post-implantation” cannot be determined when the pluripotent cell is in a laboratory and not in a uterus. The metes and bounds of when an epiblast is “early post-implantation” cannot be determined when the pluripotent cell is in a laboratory and not in a uterus and because the line at which a “post-implantation” epiblast is “early” is not defined in the specification or the art at the time of filing.
B) The specification does not enable making/using pluripotent cells that have a desired “formative differentiation function” or a desired “morphological characteristics, genomic characteristics, or epigenomic characteristics” as required in lines 6-8 of claim 1 other than pluripotent cells. The metes and bounds of when cells have a “formative differentiation function” in line 6-7 of claim 1 cannot be determined because the phrase is not defined in the specification or the art at the time of filing. The metes and bounds of when the cells have desired “morphological characteristics”, genomic characteristics, or epigenomic characteristics in lines 7-8 of claim 1 cannot be determined because the specification does not teach these characteristics that define the cells of interest. Accordingly, the concept in lines 6-8 of claim 1 is not enabled other than for pluripotent cells – the phrases do nothing to further limit the cells other than to say they are pluripotent.
C) The specification does not enable making/using mammalian pluripotent cells in which CLDN6, WNT3, MIXL1, LIN28B, EPHA1, MMP25, MOXD1, MYC, NECTIN1, SLC39A8, SLC7A8, TMEM59L, GRHL2, ZSCAN10, ETV4, ETV5, FOXH3, or ZIC5 expression levels are “at least 2 times of that in an embryonic stem cell (ESC)” in lines 11-15 as required in claim 1. Applicants point 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.
Smith (WO 2018/138281) 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 cells have “the pluripotency of a mammalian embryonic epiblast cell from post-implantation to pre-gastrulation (early post-implantation epiblast)” as required in claim 1 because Smith isolated the cells from epiblast (pg 22, Example 1, “Establishing FS cells from epiblast”), because the cells obtained by Smith were pluripotent, and because the metes and bounds of the phrase cannot be determined.
The cells have a “differentiation function” 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 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 they were made using the exact same means described by applicants as being part of the invention in claims 22-24. 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).
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 1 because they were made using the exact same means described by applicants as being part of the invention in claims 22-24.
The specification does not teach any specific mammalian pluripotent cells that have the expression pattern claim in its narrowest embodiments (2X expression of all of the markers as compared to ESC), nor in its broadest embodiment (2X expression of one of the markers as compared to ESC). Pg 6 lists a number of markers, but the specification does not contemplate excluding certain markers, i.e. SOX4, JPH4, ZIC2, EOMES, TCFL5, FOXH1 as newly required in claim 1. Furthermore, the comparison to any embryonic stem cell of any species is meaningless because ES cells are also pluripotent and have “formative differentiation function” as well as “morphological”, “genomic”, and “epigenomic characteristics” as required in lines 5-7 of claim 1. 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 other than those described by Smith.
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 lines 16-18 of claim 1. 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”. Accordingly, the concept is not enabled.
E) The specification does not enable making/using mammalian pluripotent cells that have “H3K4me3 and H3K27me3 super bivalency in [the promoter of] at least 200 genes selected from the group ABLIM2, ADAM22, ADM,… …ZFP703” as required in claim 7. The meaning of “H3K4me3 and H3K27me3 super bivalency” and “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” on pg 6 cannot be determined for reasons set forth above. 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 is not enabled.
F) The specification does not enable making/using mammalian pluripotent cells that have “H3K4me3 and H3K27me3 super bivalency in [a] HOXA, GATA4, GATA6,… …LHX5” and a promoter region of a OTX2, ZIC5, UTF1, FGF5, ZFP13, ZSCAN10, ZIC2, or an ESRP1 gene that “has increased H3K4me3 level and/or decreased H3K27me3 level compared to the promoter region of the corresponding gene in an ESC” as required in claim 38.. 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 “increased H3K4me3 level and/or decreased H3K27me3 level”. It is unclear whether the phrase is attempting to say one H3 histone within the 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 “OTX2, ZIC5,… …ESRP1” 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 is recited above. 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”. If applicants are simply attempting to further limit the list of promoters in claim 1, then much clarification will be required. Accordingly, the concept is not enabled.
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’ argument are not persuasive for reasons set forth above.
Indefiniteness
Claims 1, 3, 4, 6, 7, 13, 16, 18, 19, 32, 35, 38 remain and claim 39 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.
Withdrawn rejections
The rejection regarding the term “stable” in the phrase “capable of stable passage” in item iii) of claim 1 has been withdrawn because the term “stable” has been deleted.
The rejection regarding the Markush group in lines 8-10 of claim 1 has been withdrawn in view of the amendment.
The rejection regarding the phrase “mRNA and/or protein” in the phrase “mRNA and/or protein expression levels” being redundant has been withdrawn in view of the amendment.
The rejection regarding the phrase “the differentiation efficiency” in claims 3, 4 lacking antecedent basis has been withdrawn because the claims have been deleted.
The rejection regarding the phrase “epiblast-like embryoid” in claim 6 has been withdrawn because the claim has been deleted.
The rejection regarding the phrase “(EpiBlastoid)” in claim 6 has been withdrawn because the claim has been deleted.
The rejection regarding the term “EpiBlastoid” in claim 6 has been withdrawn because the claim has been deleted.
Pending rejections
A) The phrase “the pluripotency of a mammalian embryonic epiblast cell from post-implantation to pre-gastrulation” in claim 1 makes the claim indefinite. The metes and bounds of the phrase cannot be determined because pluripotency is pluripotency; it is unclear how the pluripotency of an “embryonic epiblast cell” differs from any other pluripotent cell. It is unclear how the pluripotency of an “embryonic epiblast cell from post-implantation” differs from an “embryonic epiblast cell from pre-gastrulation” or any other pluripotent cell. The metes and bounds of when an epiblast is “post-implantation” cannot be determined when the pluripotent cell is in a laboratory and not in a uterus. The metes and bounds of when cells have a “formative differentiation function” cannot be determined because the phrase is not defined in the specification or the art at the time of filing. The metes and bounds of when the cells have desired “morphological characteristics”, genomic characteristics, or epigenomic characteristics cannot be determined because the specification does not teach these characteristics that define the cells of interest. Accordingly, these phrases in lines 3-8 of claim 1 make the claim indefinite because it is unclear how they mean anything other than “pluripotent”. If the phrase have meaning beyond “pluripotent”, then it is unclear how the phrases further limit the cells other than to say they are pluripotent.
Response to arguments
Applicants arguments on pg 26-28 have been reviewed but do not fully address this rejection. Pg 26, last para, says the phrases are “sufficiently clear in view of the as-filed specification, and their metes and bounds can be easily determined”. Applicants’ argument is not persuasive because those metes and bounds cannot be found in the specification or the art at the time of filing. And the metes and bounds are not disclosed in applicants’ arguments.
B) The Markush group in lines 16-18 of claim 1 lists proteins, but the beginning of the phrase says they contain “promoter regions”. The list should contain “genes” not proteins.
Response to arguments
Applicants argue the amendment overcomes the rejection. Applicants’ argument is not persuasive because promoters are only found in genes, but applicants’ Markush group only lists proteins.
C) The metes and bounds of “H3K4me3 and H3K27me3 super bivalency in the promoter regions of HOXA cluster, GATA4, GATA6, HAND1, PAX2, PAX6, FOXF2, T, and LHX5” in lines 16-18 of claim 1 cannot be determined.
HOXA cluster is a plurality of genes, each of which has their own promoter; the genes in the HOXA cluster should be listed separately.
If applicants are attempting to say an “H3K4me3 and H3K27me3” promoter has a trimethylation modification on the 4th lysine of histone H3 and trimethylation modification of the 27th lysine on histone H3, then term “bivalent” is irrelevant. The term “bivalent” simply means “associated in pairs”, but the phrase “super bivalency” fails to capture the structure/function of what is bivalent, i.e. the trimethylation modification on the 4th lysine of histone H3 and trimethylation modification of the 27th lysine on histone H3.
The term “super” in the phrase “super bivalent” makes claim 1 even more indefinite because it is unclear why/when bivalency is “super”.
If applicants are attempting to say an “H3K4me3 and H3K27me3” promoter has a trimethylation modification on the 4th lysine of histone H3 and trimethylation modification of the 27th lysine on histone H3, that makes claim 1 indefinite. 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”. Accordingly, the concept is not enabled.
Response to arguments
Applicants argue the amendment overcomes the rejection. Applicants’ argument is not persuasive because it is not clear when
D) The phrase “wherein the pluripotent cell has the H3K3me3 and H3K27me3 super bivalency in the promoter regions of at least 200 genes selected from the group consisting of: ABLIM2… …and ZFP703” in claim 7 makes the claim indefinite for reasons set forth above regarding “super bivalency” above. The phrase is also indefinite because it is broader than the scope of claim 1. It does not appear to further limit the bivalency of the promoters in claim 1. Nor does it appear to say at least 200 additional promoters selected from the group consisting of ABLIM2… …and ZFP703” are “bivalent”.
Response to arguments
Applicants argue the amendment overcomes the rejection (pg 30-31). Applicants’ argument is not persuasive. The amendment has made the claim even more confusing for reasons set forth above.
E) The phrase “wherein when the isolated pluripotent stem cell is a female isolated pluripotent stem cell, it comprises two X chromosomes, one of which is an activated X chromosome, and the other is a silent X chromosome” in claim 19 makes the claim indefinite. The metes and bounds of when X chromosomes are activated or silent cannot be determined. The metes and bounds of when an X chromosome is “silent” or “activated” are not defined in the specification or the art at the time of filing. Therefore, those of skill would not be able to determine when they were infringing on the claim.
Response to arguments
Applicants argue Fig. 1F and Example 2 discuss inactivated chromosomes. Applicants point to means of detecting the chromosome state on pg 33 including Plath who discussed inactivated chromosomes. Applicants’ argument is not persuasive because the claim does not require inactivating chromosomes. There is nothing that says in the specification or the art that an “activated” is just a normal, “non-activated” chromosome. There is nothing in the specification or art that says a “silent” chromosome must be “inactivated”.
F) It is unclear how the phrase “disease model or drug screening model” in claim 35 further limits the pluripotent cells of claim 1 other than them being isolated pluripotent cells. As written, the phrase appears as just an intended use as a means for using the cells as a model of disease or to screen drugs. If applicants intend the phrase to mean something more than isolated cells for use as a disease model or for screening drugs in some sort of apparatus, then much clarification will be required. And possibly further restriction.
Response to arguments
Applicants argue the phrase “appears as just an intended use as a means for using the cells as a model of disease or to screen drugs”. If that’s the case, then the phrase adds NOTHING to the structure or function of the cells. ANY cell can be used “as a model of disease or to screen drugs”.
G) The concept of a pluripotent cell that has “super bivalency in the promoter” of an ABLIM2, ANKRD33,… …or VLDLR gene and increased H3K4me3 or decreased H3K27me3 level in the promoter of an OTX2, ZIC5,… …or ESRP1 gene in claim 38 makes the claim indefinite. The claim lists proteins but the Markush group says they are supposed to be genes. Xiang taught trimethylation was strong in both H3K4me3 and H3K27me3 level (pg 1 of Methods). It is unclear if the promoters listed in claim 38 are “bivalent” or “super bivalent”. It is unclear whether the promoters listed in claim 38 are “bivalent” in addition to the promoters listed in claim 1. Accordingly, the metes and bounds of claim 38 are unclear.
Response to arguments
Applicants argue proteins listed are selected from a separate category than the genes listed in claim 1. Applicants’ argument is not persuasive. There is nothing in claim 38 that say a promoter of a OTX2, ZIC5, UTF1, FGF56, ZFP13, ZSCAN10, ZIC2, or ESRP1 gene is also “bivalent” or “H3K4me3 and H3K27me3”. The language used in claim 38 conflates and confounds the “bivalent” or “H3K4me3 and H3K27me3” language in claim 1.
New rejection
H) The concept of a pluripotent cell that has “super bivalency in the promoter” of a “HOXA cluster, HOXB cluster, HOXC cluster,… …or LHX5” in claim 39 makes the claim indefinite. The claim lists proteins but the Markush group says they have “promoter regions” which are limited to genes. Xiang taught trimethylation was strong in both H3K4me3 and H3K27me3 level (pg 1 of Methods). It is unclear if the promoters listed in claim 39 are “bivalent” or “super bivalent”. It is unclear whether the promoters listed in claim 38 are “bivalent” in addition to the promoters listed in claim 1. Accordingly, the metes and bounds of claim 38 are unclear.
Claim Rejections - 35 USC § 102
Claims 1, 7, 13, 16, 18, 19, 32, 35, 38 remain and claim 39 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 and having “bivalent” “H3K4me3 and H3K27me3” promoters listed 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 cells have “the pluripotency of a mammalian embryonic epiblast cell from post-implantation to pre-gastrulation (early post-implantation epiblast)” as required in claim 1 because Smith isolated the cells from epiblast (pg 22, Example 1, “Establishing FS cells from epiblast”), because the cells obtained by Smith were pluripotent, and because the metes and bounds of the phrase cannot be determined.
The cells have a “differentiation function” 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 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 they were made using the exact same means described by applicants as being part of the invention in claims 22-24. 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).
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 1 because they were made using the exact same means described by applicants as being part of the invention in claims 22-24.
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 described by applicants as being part of the invention in claims 22-24 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 an ES cell as required in claim 13 because they were made using the exact same means described by applicants as being part of the invention in claims 22-24.
The cells obtained by Smith are derived from ESCs or epiblast, blastocyst inner cell mass, early embryo (pg 22-24) as required in claim 16.
The cells obtained by Smith are mammalian (pg 22-24) as required in claim 18.
The cells obtained by Smith are female (pg 2, line 28; pg 15, line 3; pg 20, line 31) one of which is silenced (pg 2, line 28; pg 15, line 3) as required in claim 19.
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 described by applicants as being part of the invention in claims 22-24.
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 39 because they were made using the exact same means described by applicants as being part of the invention in claims 22-24.
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. Applicants argue 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 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 and having “bivalent” “H3K4me3 and H3K27me3” promoters listed in claim 1 have any function that distinguishes them from any well-known pluripotent cell.
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.
Applicants argue a later study by Wang (2021) compared “fPSCs” (formative pluripotent stem cells) (pg 2, 2nd para) of applicants’ invention with “FS” (formative pluripotent stem cells) (pg 23, line 2) of Smith and conclude “that fPSCs are [sic] FS cells are different” (pg 35 of the response filed 3-2-26). Applicants’ argument is not persuasive. The teachings of Smith are not limited to “FS” cells– the cells of Smith also encompass pluripotent cells cultured with KSR (pg 5-6) as described by applicants in claim 24.
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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Michael C. Wilson
/MICHAEL C WILSON/
Primary Examiner, Art Unit 1638