DETAILED ACTION
Claims 8-13 are currently pending. Claims 1-7 are cancelled.
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 .
Election/Restrictions
Applicant’s election without traverse of Group I, claims 8-12, in the reply filed on 4/15/2026 is acknowledged.
Claim 13 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected subject matter, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 4/15/2026.
Priority
This application claims benefit as a CON of 16/937,530 (filed 7/23/2020; ABN), which claims benefit from U.S. Application No.15/261,683 (filed 9/9/2016; US Patent No. 10,731,135), which claims benefit from provisional U.S. Application No. 62/217,615.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 8/27/2024, 11/5/2024, 2/20/2025 and 7/2/2025 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Claim Objections
Claims 8 and 12 are objected to because of the following informalities: abbreviations.
In claim 8, the terms “GSK3” and “TGF-β1” are abbreviations that should first be spelled out upon their first usage in a claim.
In claim 12, the term “VE-cadherin” recites the abbreviation “VE” which should first be spelled out upon its use in a claim.
Appropriate correction is required.
Specification
The disclosure is objected to because of the following informalities: the specification recites BJRiPS cells, however the abbreviation should be spelled out to provide clarity as to the identity of the recited cells. The referenced cells appear to be a commercial cell line BJ-RiPS 1.1 (accession # CVCL_C922), produced from human foreskin fibroblasts.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 8-12 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. Claim 8 recites the following method:
“A method for differentiating endothelial and perivascular cells from human induced pluripotent stem cells (hiPSCs) comprising:
(a) culturing the hiPSCs in the presence of at least one GSK3 inhibitor;
(b) culturing the hiPSCs in the presence of a complete differentiating medium;
(c) culturing the hiPSCs with the differentiating medium supplemented with a TGF-31 inhibitor; and
(d) separating hiPSC-derived perivascular progenitor cells (hiPSC-PPCs) and hiPSC- derived endothelial cells (hiPSC-ECs).”
Claims 9-12 depend directly from claim.
The claims encompass producing various types of endothelial cells and perivascular cells with unknown cell surface markers by differentiating human induced pluripotent stem cells (hiPSCs) derived from any somatic cells of different tissues or organs by using culture conditions comprising at least one GSK3 inhibitor and a TGF-β1 inhibitor, wherein steps (a)-(c) are conducted for any time period, e.g. 1 minute or 6 months.
It is noted there are numerous types of perivascular cells and endothelial cells, as discussed by Xu et al., 2021 (Stem cells, 39: 1427-1434; IDS 8/27/2024). Xu teaches that “perivascular cells are a heterogeneous population of mesenchymal progenitors. The outermost layer of the vessel wall is a niche with high density and diversity of progenitors, also called adventitial cells. Several antigens including CD10, CD107, CD140a, as well as aldehyde dehydrogenase activity have been identified in adventitial cells showing distinct differentiation potential with implication for bone repair” (e.g. Significant statement, p. 1427).
Conway et al., 2004 (Genome Biology, 5:207, p. 1-5; IDS 8/27/2024) states “vascular endothelia comprises a diverse population of cells that specialize in response to genetic programs and environmental cues to take on distinct roles in different vessels, tissues, and organs, and in response to pathophysiological stress” (e.g. Abstract). “There is mounting evidence that endothelial cells are extraordinarily diverse in their morphology, function and gene-expression profile. Morphologically, they differ in size, shape, thickness, number of microvilli, and position of the nucleus” (e.g. p. 207.2, left column, 1st paragraph).
Upon review of the specification, the specification shows that Applicants have not provided sufficient description of the invention to support they were in possession of differentiating any human induced pluripotent stem cell (hiPSCs) by culturing for any time period in the presence of any amount of at least one GSK3 inhibitor, or culturing for any time period in the presence of any amount of a TGF-B1 inhibitor to result in producing any type of perivascular cell or any type of endothelial cell.
The specification discloses cell differentiation protocols on two-dimensional cell culture that incorporates Wnt activation with CHIR99021 during pre-differentiation, TGF-β inhibition with SB431542 at the end of differentiation and hypoxic culture (4% O2) during entire differentiation. Starting from Day 0, BJRiPS cells were cultured in complete differentiation medium IMDM supplemented with the disclosed ingredients described on page 41, lines 11-15. On Day 1, BJRiPS cells were treated with CHIR99021 in TeSR1 for 24 hours. From Day 4 to Day 6, cells were cultured in complete differentiation medium that was further supplemented with SB431542. On Day 6 of differentiation culture, cells were dissociated to single cells using TrypLE, stained with CD31-BV605 and CD140b-PE antibodies, and separated by fluorescence-activated cell sorting (FACS). BJRiPS-derived endothelial cells (hiPSC-ECs) were defined as CD31+CD140b- population and BJRiPS-derived perivascular progenitor cells (hiPSC-PPCs) were defined as CD31-CD140b+ population. After FACS isolation, hiPSC-ECs and hiPSC-PPCs were cultured in Collagen I-coated flasks using EGM-FBS-SB medium. For differentiation towards smooth muscle cell-like phenotype, hiPSC-PPCs were cultured in Smooth Muscle Growth Medium-2 for 6 days (Example 5, page 40-41). The resulting cell mixture was composed of two main cell types CD31+CD140b- endothelial cells (hiPSC-ECs, 55.1%) and CD31-CD140b+ perivascular progenitor cells (hiPSC-PPCs, 22.1%). hiPSC-ECs homogeneously expressed endothelial markers (CD31, VE-cadherin and KDR) but not the perivascular marker (CD140b). By the end of expansion, hiPSC-ECs maintained homogeneous expression of endothelial markers. Purified hiPSC-PPCs homogeneously expressed CD140b but not endothelial CD31. More than half of hiPSC-PPCs also expressed pericyte marker, NG2 (p. 42).
The specification fails to provide adequate guidance and evidence for the invention as claimed, specifically how to produce any type of endothelial cells and perivascular progenitor cells with unknown cell surface marker identity (i.e., phenotype) by differentiating any human induced pluripotent stem cells (hiPSCs) by using different cell culture conditions comprising at least one GSK3 inhibitor and a TGF-β1 inhibitor, and wherein the culturing time periods encompass as few as 1 minute and as great as 6 months for steps (a)-(c).
Accordingly, the claims are considered to lack sufficient written description and are properly rejected under 35 USC 112, first paragraph.
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 8-12 are 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.
Regarding claim 8, it is noted the phrase “a method for differentiating endothelial and perivascular cells from human induced pluripotent stem cells” in lines 1-2 of claim 8 renders the claim indefinite since it is unclear if it is the “endothelial and perivascular cells” that are intended to be differentiated into what kind of cell. It appears that the claim is intended to produce endothelial and perivascular cells from human induced pluripotent stem cells (differentiation of hiPSCs) rather than differentiating endothelial and perivascular cells into some other type of cells. Claims 9-12 depend from claim 8 but fail to clarify the indefiniteness.
Further regarding claim 8 and steps (a) and (b), it is unclear as to whether the method requires the presence of the at least one GSK inhibitor during the culturing step that requires a complete differentiation medium and is further unclear if the method cultures the hiPSCs for some time period in the complete medium without the presence of a TGF-β1 inhibitor. The claim is unclear as to if step (b) is conducted for some time period without the TGF-β1 inhibitor and thereafter cultured in differentiation medium supplemented with a TGF-β1 inhibitor.
Further regarding claim 8 and the step (d) “…separating hiPSC-derived perivascular progenitor cells (hiPSC-PPCs)…”, this limitation renders the claim indefinite since it is unclear at what step perivascular progenitor cells have been produced.. The claim does note recite an active step of producing perivascular progenitor cells. It is unclear whether the “perivascular cells” and the “perivascular progenitor cells” are intended to be the same type of cells or different types of cells. If they are different types of cells, it is unclear whether the “perivascular cells” are produced by the presently claimed method or not. If they are the same type of cells, it is unclear which type of cell is intended to be produced, perivascular cell or perivascular progenitor cells. Claims 9-12 depend from claim 8 but fail to clarify the indefiniteness.
Further regarding the phrase “perivascular progenitor cells” in claim 8, this limitation renders the claim indefinite. It is unclear as to the metes and bounds of what would be considered “perivascular progenitor cells”. It is unclear what kind of cell is considered “perivascular progenitor cells”. The specification only discloses CD31-CD140b+ perivascular progenitor cells (page 8, lines 1-2, page 41, lines 21-22, page 42, lines 8-9). The specification fails to specifically define “perivascular progenitor cells”. Claims 9-12 depend from claim 8 but fail to clarify the indefiniteness.
Regarding claim 11, the phrase “further comprising maintaining hypoxic culture conditions of 4% or less of O2” renders the claim indefinite. Claim 11 depends from claim 8, which recites three different cell culturing steps. It is unclear which cell culturing step requires the hypoxic condition of 4% or less of O2.
Regarding claim 12, the phrase “further comprising measuring a plateau in an increase… to indicate sufficient vascular and the end of a first phase of culture” renders the claim indefinite. Claim 12 depends from claim 8, which recites four stages of different cell culturing. It is unclear when the measuring step recited in claim 12 occurs. Is it required at any of steps (a)-(d) or only required after step (d).
It is further noted the phrase “sufficient vascular” appears to be an incomplete phrase. It is unclear if this phrase is intended to mean ‘sufficient vascular differentiation’.
Further regarding claim 12 and the phrase “…an increase in endothelial coverage defined by CD31 and VE-cadherin expression…”, it is unclear if this limitation is directed to the percentage of endothelial cells expressing CD31 and VE-cadherin, or if the claim means the confluence of cells covering the culturing vessel.
In the interest of compact prosecution, this limitation is interpreted as measuring the maximum number of endothelial cells having positive expression of CD31 and VE-cadherin. However, despite the above interpretation, such treatment does not relieve Applicant of the responsibility of responding to this rejection. If the actual interpretation of the claims is different than that posited by the Examiner, additional rejections and art may be readily applied in a subsequent final Office action. The rejection to claim 12 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, stands and must be addressed.
Regarding claim 10, claim 10 recites the limitation "the at least one TGFbeta1 inhibitor" in line 1. There is insufficient antecedent basis for this limitation in the claim. Claim 10 depends from claim 8, which recites “…a TGF-beta1 inhibitor”. Claim 8 fails to recite “at least one TGF-beta1 inhibitor”. Thus, there is no antecedent basis for the phrase "the at least one TGF-beta1 inhibitor".
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 8-10 are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Mummery et al., WO 2014/200340, IDS 8/27/2024) (“Mummery”).
Regarding claims 8-10, Mummery is directed to methods for in vitro production of endothelial cells (ECs), pericytes, pericyte-derived smooth muscle cells and vasculature (page 1, lines 4-6). Mummery teaches improved protocols for differentiation of PSCs (pluripotent stem cells), particularly hPSCs (human pluripotent stem cells) toward EC lineages. The protocols result in very efficient generation of ECs from PSCs, preferably human ESCs (embryonic stem cells) and iPSCs (induced pluripotent stem cells) (page 1, line 26 to page 2, line 4).
It is noted that Mummery teaches that pericytes are perivascular progenitor cells (page 8, lines 10 and 32-33).
Mummery teaches a method comprising,
(a) culturing pluripotent stem cells in defined medium containing Activin A, BMP4, VEGF, preferably VEGF165, and a canonical WNT ligand or GSK3 inhibitor (i.e., CHIR99021) (claim 9), to produce a culture comprising differentiated cells,
(b) culturing the cells obtained in step (a) in defined medium containing VEGF, and a TGFbeta signaling inhibitor (i.e., SB431542) (claim 10), to produce endothelial cells, the pluripotent stem cells are preferably iPSC. The method yields a high number of endothelial cells, and the endothelial cells produced can be purified with CD34 coupled magnetic beads (page 12, lines 21-33; page 19, lines 12-20; page 29, lines 29-33 to page 30, line 1). The method further comprises collecting the cells produced in step (b) and obtaining a collection of cells that comprises more than 90% endothelial cells based on the expression of CD31, CD34 or VE-cadherin (page 13, lines 6-9; claims 7-8). The same method can be used to produce pericytes (i.e., perivascular progenitor cells) and further comprises collecting the cells produced in step (b) and obtaining cells comprising more than 90% pericytes based on the expression of CD31. Preferably, the CD31 negative fraction is collected. The cells are typically negative for CD34 and VE-cadherin (page 14, lines 15-28; claims 9-10). hiPSCs differentiation was induced three days after passing colonies by replacing mTeSR medium with the differentiation media based on BPEL with the addition of activin A, BMP4, VEGF165 and small molecule inhibitor CHIR99021. At day 3, and day 7 of differentiation the media was refreshed with BPEL containing VEGF and SB43152 only (page 26, lines 9-19) (For steps (a)-(c) of claim 8).
Thus, claims 8-10 are anticipated by Mummery.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Mummery (set forth above).
The teaching of Mummery is set forth above and anticipates claims 8-10.
Regarding claim 12, it is noted that Mummery does not specifically teach measuring a plateau in an increase of endothelial coverage defined by CD31 and VE-cadherin expression to indicate sufficient vascular and the end of a first phase of culture.
However, as discussed at the rejection under 35 USC 112(b), the claim is interpreted as measuring the maximum number of endothelial cells having positive expression of CD31 and VE-cadherin.
Mummery teaches the cells produced in step (b) and obtaining therefrom a collection of cells that comprises more than 90% endothelial cells, preferably based on the expression of CD31, CD34 or VE-Cadherin. Such collections are typically obtained by separating the endothelial cells from other cells in the culture. Such separation can be done in a number of ways such as FACS cell sorting, cell panning by means of binding molecules that specifically recognize an endothelial cell marker on the surface of the cells (page 13, lines 6-14).
Therefore, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to measure a plateau in an increase of endothelial coverage defined by CD31 and VE-cadherin expression to indicate sufficient vascular and the end of a first phase of culture because Mummery teaches a method of producing endothelial cells and pericytes, and obtaining a collection of cells, including endothelial cells and pericytes, which comprises more than 90% purified cells based on the expression of CD31, CD34 or VE-cadherin. Since Mummery teaches monitoring the expression of CD31, CD34 or VE-cadherin, it would be obvious for one of ordinary skill in the art to measure a plateau in an increase defined by CD31 and VE-cadherin expression in order to determine whether the endothelial cells or the pericytes are obtained by the culturing method taught by Mummery with reasonable expectation of success.
One having ordinary skill in the art before the effective filing date of the claimed invention would have been motivated to do so in order to produce a cell culture comprising endothelial cells or pericytes with more than 90% purity via the differentiation of hiPSCs as taught by Mummery with reasonable expectation of success.
Claim 11 is rejected under 35 U.S.C. 103 as being unpatentable over Mummery, in view of Gerecht et al., (WO 2015/119642; IDS 8/27/2024) (“Gerecht”).
The teaching of Mummery is set forth above.
Regarding claim 11, and the limitation “maintaining hypoxic culture conditions of 4% or less of O2”, it is noted that Mummery does not specifically teach maintaining hypoxic culture conditions of 4% or less of O2. However, Gerecht is directed to differentiation of pluripotent stem cells (PSCs) under hypoxic conditions toward early vascular cells (EVCs) and teaches that low oxygen tension is a critical regulator of the developing or regenerating vasculature (Abstract and [0003]), wherein the cells have positive expression of VE-cadherin ([0012]). Gerecht’s method cultures under hypoxic conditions that are characterized by oxygen concentrations less than 10%, and more specifically ranging from 1% to 10% oxygen (O2) ([0048]).
Thus, Gerecht has established it was known to employ hypoxic conditions to promote differentiation of pluripotent stem cells to endothelial lineage.
Therefore, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to maintain hypoxic culture conditions of 10% or less (claimed range overlaps the prior art range) of O2 during the culturing of hiPSCs toward endothelial cells because Gerecht teaches low oxygen tension, such as 1% O2 condition, enhances endothelial fate of human pluripotent stem cells. It would be obvious for one of ordinary skill in the art to culture the hiPSCs taught by Mummery in hypoxic condition to produce endothelial cells in order to enhance endothelial lineage commitment as taught by Gerecht with reasonable expectation of success. One of ordinary skill in the art would try different hypoxic condition, including 4% or less O2 condition, to optimize and enhance the efficiency of differentiating hiPSCs into endothelial cells with reasonable expectation of success.
One having ordinary skill in the art before the effective filing date of the claimed invention would have been motivated to do so in order to produce a cell culture comprising endothelial cells or pericytes with more than 90% purity via the differentiation of hiPSCs as taught by Mummery or to increase expression of VEcad as taught by Gerecht with reasonable expectation of success.
Conclusion
No claim is allowed. No claim is free of prior art.
Examiner Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to E. YVONNE PYLA whose telephone number is (571)270-7366. The examiner can normally be reached M-F 9am - 6pm.
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E. YVONNE PYLA
Primary Examiner
Art Unit 1633
/EVELYN Y PYLA/Primary Examiner, Art Unit 1633
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