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 .
Election/Restrictions
Applicant’s election without traverse of Group 1 (claims 1-2, 4, 6-7, 10-12, 15-16 and 30-31) in the reply filed on 7/1/2026 is acknowledged.
Claims 17-19, 21-26, and 36 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 7/1/2026.
Accordingly, claims 1-2, 4, 6-7, 10-12, 15-19, 21-26, 30-31 and 36 are pending and claims 1-2, 4, 6-7, 10-12, 15-16 and 30-31 have been examined herein.
Priority
The instant claims herein are examined utilizing the accepted effective filing date of 9/29/2020 for the basis of any prior art rejections. The instant application claims benefit to U.S. provisional application 63/084,980.
I don’t like the “as opposed to” language in claim 12 as it is not necessary. Enriching for PECAM1+ is sufficient. You could object and just say it is not necessary to recite “as opposed to”.
Claim Objections
Claim 12 is objected to because of the following informalities:
Claim 12 recites “enriching for PECAM-1+ endothelial cells as opposed to PECAM-1- smooth muscle-like cells”. The recitation of “as opposed to PECAM-1- smooth muscle-like cells” is not necessary and the examiner suggests removing this language from the claim.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
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.
Claim 6 and 15 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.
Claim 6 recites “wherein the cells are cultured on a surface comprising a permeable support within a tissue culture system.” It is unclear which cells (either in steps (a), (b), or (c)) are cultured on the permeable support surface as claimed. Thus, the claim is indefinite.
Claim 15 recites the limitation "wherein the culturing of the C31+ EECM-BMECs with at least one pro-inflammatory cytokine" in line 2. There is insufficient antecedent basis for this limitation in the claim as there is no previous recitation of any culturing with one pro-inflammatory cytokine recited in the claim nor in claim 1, of which claim 15 depends. Thus, the claim is indefinite.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 6 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 6 recites “wherein the cells are cultured on a surface comprising a permeable support”. As set forth in the above 112(b) rejection, it is unclear which cell is cultured on the surface comprising a permeable support. Accordingly, claim 6 does not further limit claim 1 because this recitation is broader than the “culturing . . . on collagen coated surface” in steps (a) and (b) as recited in claim 1. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
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 (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claim(s) 1-2, 6, 10-12, 15-16, and 30-31 is/are rejected under 35 U.S.C. 103 as being unpatentable over Qian et al (US20170283772A1, 4/4/2017; published 10/5/2017; in IDS filed 12/26/2024) in view of Shusta et al (US9902940B2, 6/8/2011; published 2/27/2018; in IDS filed 12/26/2024) as evidenced by Hillyer et al (Clin Exp Immunol, 2003. 134: 431-441; in IDS filed 12/26/2024).
Regarding claim 1, Qian teaches a method for generating a population of human brain microvascular endothelial cells (BMECs) comprising culturing the cells expressing mesodermal markers for about 5 days in the presence of a chemically defined, serum-free culture medium comprising B27 supplement, whereby cells that express endothelial progenitor marker Flk-1 are obtained; and (c) culturing the Flk-1+ cells of (b) for about two days in the presence of a chemically defined, serum-free endothelial medium comprising B27 supplement, bFGF/FGF2, and retinoic acid (RA), whereby a cell population comprising human BMECs is obtained (claim 1 of Qian).
Qian continues to teach that at least 95% of cells of the cell population of (c) are BMECs cells positive for expression of one or more of CD31 (claim 3 of Qian).
Qian continues to teach that the endothelial progenitor cells express CD31 and CD34 (para 18-19).
Regarding claims 1 and 6, Qian also teaches that during differentiation, cells were cultured on Matrigel coated wells (para 15) and replated on human placenta-derived collagen IV surfaces (para 86).
Qian continues to teach growing the BMECs of step (c) as a monolayer to confluence (claim 4 of Qian).
Regarding claims 1, 10, 11, Table 3 of Qian describes gene expression similarity between human BMECs, hPSC-derived BMECs differentiated under defined condition, and hPSC-derived BMECs differentiated in unconditioned-medium including CAV-1, VCAM-1, MCAM, and PECAM-1.
Regarding claim 1, 2, and 10 in-part, Qian continues to teach that the resultant day 10 BMEC-like cells were a pure population expressing endothelial markers (CD31, VE-Cadherin), BBB glucose transporter (GLUT-1), tight junction proteins (ZO-1, claudin-5, occludin) and efflux transporters (BCRP, MRP1, Pgp) (FIGS. 2A-2I; para 84). BMECs exhibited endothelial cell properties, including expression of von Willebrand factor (vWF) (FIG. 3A), formation of tube-like structures on Matrigel® in the presence of VEGF (FIG. 3B), uptake of acetylated low-density lipoprotein (LDL) (FIG. 3C), and upregulation of ICAM-1 (para 88).
Regarding claim 2, Qian teaches that the BMECs are positive for occludin and claudin 5 (claim 3 of Qian) as well as ZO-1 (para 19 of Qian).
Regarding claim 15, Qian teaches ICAM-1 induction in hPSC-derived BMECs (para 15). hPSC-derived BMECs at day 10 were treated with 10 ng/mL of TNF-α for 16 hours (same para). Cells were stained for ICAM-1 before and after TNF-α treatment (Fig. 3D-E). The cells experienced 62% increase in ICAM-1 after TNF-alpha stimulation
Regarding claim 16, Qian teaches that the endothelial stem cells are differentiated from human pluripotent stem cells (claim 1 of Qian).
Regarding claim 30 and 31, Qian teaches chemically defined, serum-free culture medium comprising an activator of Wnt/β-catenin signaling, the Wnt signaling is a GSK3 inhibitor, and the GSK3 inhibitor is a small molecule selected from the group consisting of CHIR99021, CHIR98014, BIO-acetoxime, BIO, LiCl, SB216763, SB415286, AR A014418, 1-Azakenpaullone, and Bis-7-indolylmaleimide (claims 2 and 6-7 of Qian).
Qian does not teach:
selectively passaging the endothelial cells on collagen surface for at least two passages, and
the BMECs express ICAM-2, E-selectin, P-selectin, and CD99.
(i) However, regarding claim 1 and selective passaging, Shusta teaches broadly a method of producing brain specific endothelial cells (EC) (see Summary of Invention). Shusta continues to teach the selective culturing of cells to confluence after 1-2 days, their cells were contact-inhibited, exhibited characteristic EC morphology (FIG. 4b), and were capable of acetylated low-density lipoprotein uptake (FIG. 4c). Approximately 100% of the cells in the cultures expressed the requisite BBB markers, indicating effective and facile purification using this extracellular matrix-based selective passaging method.
Shusta continues to teach that in EC medium, the cells grew to confluence after 1-2 days, were contact-inhibited, exhibited characteristic EC morphology (FIG. 4b ), and were capable of acetylated low-density lipoprotein uptake (FIG. 4c ). Approximately 100% of the cells in the cultures expressed the requisite BBB markers, indicating effective and facile purification using this extracellular matrix-based selective passaging method (FIGS. 4d and 4e ). Importantly, the cells also acquired vWF and VE-cadherin expression during subculture, as demonstrated both by RT-PCR and immunocytochemistry, indicating further EC maturation (FIGS. 4a and 4e ).
Regarding claim 12, Shusta teaches expanding the EC regions by culturing the cells in EC medium, wherein the expanded cells are PECAM-1 (claim 4 of Shusta).
(ii) Regarding ICAM-2, E- and P-selectin expression, the teachings of Hillyer are relied upon as providing evidence that BMECs express markers including ICAM-1, ICAM-2 VCAM, PECAM, and E/P selectin regardless of cytokine stimulation (p. 434 para 1; p. 435, Fig. 2). This evidences that the BMECs as taught by Qian would also express ICAM-2, E- and P-selectin.
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to create BMECs from endothelial cells as taught by Qian, where the endothelial cells are passaged as taught by Shusta, to arrive at the instantly claimed invention.
Importantly, Shusta shows that endothelial cells can be selectively passaged successfully to produce BMEC and one of ordinary skill would have been motivated to use the known technique of selective passaging as taught by Shusta in the method as taught by Qian with a reasonable expectation of advantageously having effective and facile purification of cells expressing blood-brain barrier markers to produce BMECs as taught by the prior art. One of ordinary skill in the art would have been capable of applying this known method of enhancement to a "base" method in the prior art and the results would have been predictable to one of ordinary skill in the art.
Claim(s) 4 and 7 is/are rejected under 35 U.S.C. 103 as being unpatentable over Qian and Shusta as applied to claims 1-2, 6, 10-12, 16, and 30-31 above, and further in view of Cader et al (WO2019058140A1, 9/24/2018; published 3/28/2019).
The teachings of Qian and Shusta in combination were recited in the above 35 U.S.C. 103 rejection as applied to claim 1 of which claim 4 depends. The teachings will not be repeated here.
Neither Qian nor Shusta teaches:
step (a) comprises culturing the endothelial progenitor cells in the presence of a ROCK inhibitor,
removing the ROCK inhibitor at subsequent culturing steps, and
the serum-free endothelial medium of steps (b)-(c) comprises conditioned medium from smooth muscle- like cells (SMLCs); or the cells are co-cultured with smooth muscle-like cells (SMLCs).
Regarding (i), (ii), and (iii), Cader teaches a method for producing brain microvascular endothelial cells (claim 1 of Cader). The medium of the endothelial cell induction step can be an endothelial cell growth medium. The medium can comprise a ROCK-pathway inhibitor such as Y-27632. Such an inhibitor may prevent loss of tight junctions and adherens junctions.
Cader continues to teach that providing a ROCK-pathway inhibitor can improve cell viability post-thaw and also it has been reported that Rho-associated protein kinase increases actomyosin contractility, which facilitates breakdown of intercellular junctions.
Regarding claim 7, Cader teaches that the conditioned media from one or more of neuronal cell culture, astrocyte cell culture, and mural cell culture is added onto the brain microvascular endothelial cells (claim 25 of Cader). It also describes co-culturing the BMECs with neurons, astrocytes, or mural cells (claim 27 of Cader).
Cader continues to teach that astrocytes and mural cells, such as pericytes and smooth muscle cells participate in maintaining the integrity of the BBB (p. 1, Lines 22-23).
Cader continues to teach that the barrier properties of the brain microvascular endothelial cells may be enhanced in co-culture with mural cells (smooth muscle cells and/or pericytes), enhance the distribution of adherens and tight junctions, and increase the tightness of the barrier formed.
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to produce BMECs from endothelial cells as taught by Qian and Shusta in combination, where the cells are cultured in the presence of ROCK inhibitor as taught by Cader, to arrive at the instantly claimed invention.
Cader shows that BMECs can be successfully produced from endothelial cells when initially cultured in the presence of ROCK inhibitors. One of ordinary skill would have been motivated to modify the culture method of Qian and Shusta to include ROCK inhibitor with a reasonable expectation of advantageously improving cell viability post-thaw and prevent the loss of tight junctions as taught by the prior art. One of ordinary skill in the art would recognize that the improvements caused by the addition of ROCK inhibitors would improve similar cultures in the same way.
It also would have been obvious prior to the effective filing date of the instantly claimed invention to produce BMECs from endothelial cells as taught by Qian and Shusta in combination, where the cells are co-cultured with smooth muscle cells or smooth muscle cell-conditioned media as taught by Cader, to arrive at the instantly claimed invention. Cader shows that BMECs can be successfully co-cultured with mural cells such as smooth muscle cells. One of ordinary skill would have been motivated to modify the culture method of Qian and Shusta to include co-culture with smooth muscle cells or smooth muscle cell conditioned media with a reasonable expectation of advantageously enhancing barrier properties of the cells, enhance the distribution of adherens and tight junctions, and increase the tightness of the barrier formed as taught by the prior art. One of ordinary skill in the art would recognize that the improvements caused by the addition of smooth muscle cell co-culture or smooth muscle cell conditioned media would improve similar cultures in the same way.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-2, 6-7, 10-12, 15-16, and 30-31 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 3-4, and 6-7 of U.S. Patent No. 10,214,724 B2 in view of Shusta et al (US9902940B2, 6/8/2011; published 2/27/2018; in IDS filed 12/26/2024) and Cader et al (WO2019058140A1, 9/24/2018; published 3/28/2019) as evidenced by Hillyer et al (Clin Exp Immunol, 2003. 134: 431-441; in IDS filed 12/26/2024).
Regarding claim 1-2, 10 in-part, 16 and 30-31, patented claim 1 of ‘724 recites “A method for generating a population of human brain microvascular endothelial cells (BMECs) from human pluripotent stem cells, wherein the method comprises, in order, (a) culturing human pluripotent stem cells for about 24 hours in a chemically defined, serum-free culture medium that comprises an activator of Wnt/β-catenin signaling, whereby cells that express mesodermal markers are obtained; (b) culturing the cells expressing mesodermal markers for about 5 days in the presence of a chemically defined, serum-free culture medium comprising a neuronal cell culture supplement, whereby cells that express endothelial progenitor marker Flk-1 are obtained; and (c) culturing the Flk-1+ cells of (b) for about two days in the presence of a chemically defined, serum-free endothelial medium comprising a neuronal cell culture supplement, bFGF/FGF2, and retinoic acid (RA), whereby a cell population comprising human BMECs is obtained.” Claim 4 of ‘724 recites “further comprising growing the human BMECs of step (c) as a monolayer to confluence.”
Claim 3 of ‘724 recites “at least 95% of cells of the cell population comprising human BMECs are BMECs positive for expression of one or more of CD31, p-glycoprotein (Pgp), occludin, and claudin-5”
Claim 6 of ‘724 recites “wherein the activator of Wnt/β-catenin signaling is a Gsk3 inhibitor.”
Claim 7 of ‘724 recites “wherein the Gsk3 inhibitor is a small molecule selected from the group consisting of CHIR99021, CHIR98014, BIO-acetoxime, BIO, LiCl, SB216763, SB415286, AR A014418, 1-Azakenpaullone, and Bis-7-indolylmaleimide.”
‘724 patent does not disclose:
selective passaging of the endothelial cells, and
BMECs express VE-cadherin, ICAM-2, PECAM-1, ICAM-1, VCAM-1, E-selectin, P-selectin, and/or CD99.
However, Shusta teaches that the cells grew to confluence after 1-2 days, were contact-inhibited, exhibited characteristic EC morphology (FIG. 4b), and were capable of acetylated low-density lipoprotein uptake (FIG. 4c ). Approximately 100% of the cells in the cultures expressed the requisite BBB markers, indicating effective and facile purification using this extracellular matrix-based selective passaging method.
Shusta continues to teach that in EC medium, the cells grew to confluence after 1-2 days, were contact-inhibited, exhibited characteristic EC morphology (FIG. 4b ), and were capable of acetylated low-density lipoprotein uptake (FIG. 4c ). Approximately 100% of the cells in the cultures expressed the requisite BBB markers, indicating effective and facile purification using this extracellular matrix-based selective passaging method (FIGS. 4d and 4e ). Importantly, the cells also acquired vWF and VE-cadherin expression during subculture, as demonstrated both by RT-PCR and immunocytochemistry, indicating further EC maturation (FIGS. 4a and 4e ).
Regarding claim 12, Shusta teaches expanding the EC regions by culturing the cells in EC medium, wherein the expanded cells are PECAM-1 (claim 4 of Shusta).
Cader teaches Cader teaches a method for producing brain microvascular endothelial cells (claim 1 of Cader). The medium of the endothelial cell induction step can be an endothelial cell growth medium. The medium can comprise a ROCK-pathway inhibitor such as Y-27632. Such an inhibitor may prevent loss of tight junctions and adherens junctions.
Cader continues to teach that providing a ROCK-pathway inhibitor can improve cell viability post-thaw and also it has been reported that Rho-associated protein kinase increases actomyosin contractility, which facilitates breakdown of intercellular junctions.
Regarding claim 7, Cader teaches that the conditioned media from one or more of neuronal cell culture, astrocyte cell culture, and mural cell culture is added onto the brain microvascular endothelial cells (claim 25 of Cader). It also describes co-culturing the BMECs with neurons, astrocytes, or mural cells (claim 27 of Cader).
Cader continues to teach that astrocytes and mural cells, such as pericytes and smooth muscle cells participate in maintaining the integrity of the BBB (p. 1, Lines 22-23).
Cader continues to teach that the barrier properties of the brain microvascular endothelial cells may be enhanced in co-culture with mural cells (smooth muscle cells and/or pericytes), enhance the distribution of adherens and tight junctions, and increase the tightness of the barrier formed.
Regarding ICAM-2, E- and P-selectin expression, the teachings of Hillyer are relied upon as providing evidence that BMECs express markers including ICAM-1, ICAM-2 VCAM, PECAM, and E/P selectin regardless of cytokine stimulation (p. 434 para 1; p. 435, Fig. 2). This evidences that the BMECs as taught by Qian would also express ICAM-2, E- and P-selectin.
Thus, the instant claims are obvious variants of the method claim of the ‘724 patent in view of Shusta, Cader, and as evidenced by Hillyer given their teachings.
Claims 1-2, 6-7, 10-12, 15-16, and 30-31 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 and 4of U.S. Patent No. 9,902,940 B2 in view of Qian et al (US20170283772A1, 4/4/2017; published 10/5/2017; in IDS filed 12/26/2024) and Cader et al (WO2019058140A1, 9/24/2018; published 3/28/2019) as evidenced by Hillyer et al (Clin Exp Immunol, 2003. 134: 431-441; in IDS filed 12/26/2024).
Regarding claim 1-2 and 16, claim 1 and 4 of ‘940 patent recites “method of producing a blood brain barrier model from endothelial cells, comprising the steps of: (a) growing human pluripotent stem cells (hPSCs), wherein all the hPSCs are grown adherently on an extracellular matrix; (b) inducing differentiation of the hPSCs by culturing the hPSCs in unconditioned medium on the extracellular matrix for a sufficient amount of time until the endothelial cells form from the hPSCs and endothelial cell (EC) regions are observed, wherein no fibroblast growth factor (FGF) is present in the unconditioned medium, and (c) expanding the EC regions by culturing the endothelial cells in EC medium, wherein the expanded endothelial cells are glucose transporter+(GLUT-1+), platelet endothelial cell adhesion molecule+(PECAM-1+), claudin-5+, occludin+, zonula occludens-1+(ZO-1+) and p-glycoprotein+, wherein the EC regions are purified after step (b), and the method additionally comprising the step of growing the endothelial cells of step (c) to confluent monolayers of the endothelial cells, wherein the blood brain barrier model is obtained, wherein the blood brain barrier model is evinced by expression of various blood-brain barrier transporters.”
Claim 7 of ‘940 patent recites “wherein the expanded cells are von Willebrand factor+ and vascular endothelial (VE)-cadherin+.”
However, Qian teaches a method for generating a population of human brain microvascular endothelial cells (BMECs) comprising culturing the cells expressing mesodermal markers for about 5 days in the presence of a chemically defined, serum-free culture medium comprising B27 supplement, whereby cells that express endothelial progenitor marker Flk-1 are obtained; and (c) culturing the Flk-1+ cells of (b) for about two days in the presence of a chemically defined, serum-free endothelial medium comprising B27 supplement, bFGF/FGF2, and retinoic acid (RA), whereby a cell population comprising human BMECs is obtained (claim 1 of Qian).
Qian continues to teach that at least 95% of cells of the cell population of (c) are BMECs cells positive for expression of one or more of CD31 (claim 3 of Qian).
Qian continues to teach that the endothelial progenitor cells express CD31 and CD34 (para 18-19).
Regarding claims 1 and 6, Qian also teaches that during differentiation, cells were cultured on Matrigel coated wells (para 15) and replated on human placenta-derived collagen IV surfaces (para 86).
Qian continues to teach growing the BMECs of step (c) as a monolayer to confluence (claim 4 of Qian).
Regarding claim 1, 10, 11, Table 3 of Qian describes gene expression similarity between human BMECs, hPSC-derived BMECs differentiated under defined condition, and hPSC-derived BMECs differentiated in unconditioned-medium including CAV-1, VCAM-1, MCAM, and PECAM-1.
Regarding claim 1, 2, and 10 in-part, Qian continues to teach that the resultant day 10 BMEC-like cells were a pure population expressing endothelial markers (CD31, VE-Cadherin), BBB glucose transporter (GLUT-1), tight junction proteins (ZO-1, claudin-5, occludin) and efflux transporters (BCRP, MRP1, Pgp) (FIGS. 2A-2I; para 84). BMECs exhibited endothelial cell properties, including expression of von Willebrand factor (vWF) (FIG. 3A), formation of tube-like structures on Matrigel® in the presence of VEGF (FIG. 3B), uptake of acetylated low-density lipoprotein (LDL) (FIG. 3C), and upregulation of ICAM-1 (para 88).
Regarding claim 2, Qian teaches that the BMECs are positive for occludin and claudin 5 (claim 3 of Qian) as well as ZO-1 (para 19 of Qian).
Regarding claim 15, Qian teaches ICAM-1 induction in hPSC-derived BMECs (para 15). hPSC-derived BMECs at day 10 were treated with 10 ng/mL of TNF-α for 16 hours (same para). Cells were stained for ICAM-1 before and after TNF-α treatment (Fig. 3D-E). The cells experienced 62% increase in ICAM-1 after TNF-alpha stimulation
Regarding claim 16, Qian teaches that the endothelial stem cells are differentiated from human pluripotent stem cells (claim 1 of Qian).
Regarding claim 30 and 31, Qian teaches chemically defined, serum-free culture medium comprising an activator of Wnt/β-catenin signaling, the Wnt signaling is a GSK3 inhibitor, and the GSK3 inhibitor is a small molecule selected from the group consisting of CHIR99021, CHIR98014, BIO-acetoxime, BIO, LiCl, SB216763, SB415286, AR A014418, 1-Azakenpaullone, and Bis-7-indolylmaleimide (claims 2 and 6-7 of Qian).
Cader teaches Cader teaches a method for producing brain microvascular endothelial cells (claim 1 of Cader). The medium of the endothelial cell induction step can be an endothelial cell growth medium. The medium can comprise a ROCK-pathway inhibitor such as Y-27632. Such an inhibitor may prevent loss of tight junctions and adherens junctions.
Cader continues to teach that providing a ROCK-pathway inhibitor can improve cell viability post-thaw and also it has been reported that Rho-associated protein kinase increases actomyosin contractility, which facilitates breakdown of intercellular junctions.
Regarding claim 7, Cader teaches that the conditioned media from one or more of neuronal cell culture, astrocyte cell culture, and mural cell culture is added onto the brain microvascular endothelial cells (claim 25 of Cader). It also describes co-culturing the BMECs with neurons, astrocytes, or mural cells (claim 27 of Cader).
Cader continues to teach that astrocytes and mural cells, such as pericytes and smooth muscle cells participate in maintaining the integrity of the BBB (p. 1, Lines 22-23).
Cader continues to teach that the barrier properties of the brain microvascular endothelial cells may be enhanced in co-culture with mural cells (smooth muscle cells and/or pericytes), enhance the distribution of adherens and tight junctions, and increase the tightness of the barrier formed.
Regarding ICAM-2, E- and P-selectin expression, the teachings of Hillyer are relied upon as providing evidence that BMECs express markers including ICAM-1, ICAM-2 VCAM, PECAM, and E/P selectin regardless of cytokine stimulation (p. 434 para 1; p. 435, Fig. 2). This evidences that the BMECs would also express ICAM-2, E- and P-selectin.
Thus, the instant claims are obvious variants of the method claim of the ‘940 patent in view of Qian, Cader, and as evidenced by Hillyer given their teachings.
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to GILLIAN C REGLAS whose telephone number is (571)270-0320. The examiner can normally be reached M-F 9-5.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Peter Paras Jr can be reached at (571) 272-4517. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/G.R./Examiner, Art Unit 1632 /DAVID A MONTANARI/ Examiner, Art Unit 1632
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