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 .
Priority
The instant application is a national stage entry under 35 U.S.C. § 371 of PCT/US2021/026659 (filed 04/09/2021). Acknowledgement is made of Applicants’ claim for benefit of U.S. Provisional Application No. 63/007,698 (filed 04/09/2020).
Response to Amendment
The amendment filed on 01/21/2026 has been received and entered into the application file.
Status of Prior Rejections/Response to Arguments
RE: Objection to claims 1 and 16:
The amendments to claims 1 and 16 to remedy the typographical errors are sufficient to overcome the objections thereto.
The objections are withdrawn.
RE: Rejection of claim 17 under 35 U.S.C. 101:
The cancellation of claim 17 renders the rejection thereof moot.
RE: Rejection of claim 17 under 35 U.S.C. 102(a)(1) over Lacolley:
The cancellation of claim 17 renders the rejection thereof moot.
RE: Rejection of claims 1-17 under 35 U.S.C. 103 over Kwong in view of Patsch, Maldonado, Schreier, and Koutsouki; as evidenced by Yang, Deumens, and Zhi:
The cancellation of claims 2-3, 6, and 17 renders the rejections thereof moot.
Applicants have traversed the instant rejection.
Applicants assert Koutsouki does not disclose or suggest purifying the induced vascular smooth muscle cells (VSMCs) by selecting cells that express cadherin-2, and Zhi only teaches use of a cadherin-2 antibody to identify leukemic cells. Respectfully, this argument is unpersuasive.
As set forth in the non-final Office action dated 09/22/2025, Koutsouki teaches N-cadherin (i.e., cadherin-2) modulates cell survival via cell-cell contact in VSMCs (pg. 986; col. 1, par. 1); thus, it would have been prima facie obvious to a person having ordinary skill in the art to have purified the VSMC population by selecting cells positive for cadherin-2 expression for the increased cell survival taught by Koutsouki. That is to say, the Koutsouki disclosure provides the necessary motivation for a skilled artisan; Zhi is merely relied upon as an evidentiary reference illustrating the selection of live cells through FACS sorting is a well-known technique in the art, providing a reasonable expectation of success in the instant modification.
Applicants further assert: the differences in growth factor concentration are not result effective variables that would have been routinely optimized by a person having ordinary skill in the art; a skilled artisan would not have a reasonable expectation of success as the invention of the instant application yielded surprising and unexpected results in the generation of VSMCs in maintaining a contractile phenotype evaluated through expression of contractile VSMC markers such (e.g., ACTA2, CNN1, SMTN, MYH11), as well as a commercially useful proliferation capacity; and, the differences in concentration are critical.
Respectfully, a showing of unexpected results must be based on evidence, not argument or speculation. In re Mayne, 104 F.3d 1339, 1343-44, 41 USPQ2d 1451, 1455-56 (Fed Cir. 1997) and MPEP 716, especially 716.02(a)-(b). Kwong teaches the expression of ACTA2, MYH11, SMTN, and CNN1 in the VSMCs of the disclosure (Fig. 5), and the SMCs are highly proliferative (pg. 510; col. 1, par. 1). Therefore, lacking any comparative data evidencing unexpected results, and as the primary references do teach expression of contractile VSMC markers as well as proliferation capacity, the argument of unexpected results is found unpersuasive. Likewise, Applicants’ argument the differences in concentration is critical is also unpersuasive.
Additionally, it is noted that the features upon which applicant relies (i.e., a contractile phenotype and a commercially useful proliferation capacity) are not recited in the rejected claims. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). Further, absent any guidance in the specification of the instant application as to what growth rate constitutes a “commercially useful” proliferation capacity, the proliferative capacities of the VSMCs taught by Kwong would read on the instant unclaimed feature under broadest reasonable interpretation. Therefore, based on the teachings of the Kwong disclosure regarding VSMC proliferation as well as expression of contractile markers, a skilled artisan would have a reasonable expectation of success in the generation of VSMCs with a contractile phenotype and “commercially useful” proliferation capacity. Thus, respectfully, this argument is unpersuasive.
The remaining rejections of record are modified and set forth below.
RE: Rejection of claims 1-15 under 35 U.S.C. 103 over Cheung in view of Patsch, Maldonado, Schreier, and Koutsouki; as evidenced by Zhi:
The cancellation of claims 2-3 and 6 renders the rejections thereof moot.
Applicants’ traversal of the instant rejection is the same as the traversal regarding the Kwong disclosure. Respectfully, as Cheung teaches the expression of CN11 and MYH11 (Fig. 1), and the HPSC-derived SMCs demonstrate functional properties including proliferation (pg. 930; col. 2, par. 1), the arguments are found unpersuasive for the same reasons as set forth above.
The remaining rejections of record are modified and set forth below.
RE: Rejection of claims 18-19 under 35 U.S.C. 103 over Dahl, et al. in view of Cheung, et al., further in view of Patsch, Maldonado, Schreier, and Koutsouki; as evidenced by Zhi and Zhou:
Applicants’ traversal of the instant rejection hinges on the same traversal as set forth above for the base claims from which the instant claims depend. Therefore, respectfully, the arguments are likewise found unpersuasive and for the same reasons.
The remaining rejections of record are modified and set forth below.
RE: Rejection of claims 16-19 under 35 U.S.C. 103 over Qyang in view of Koutsouki:
The cancellation of claim 17 renders the rejection thereof moot.
Claim 16 has been amended to depend from claim 1 as an active step in the method of claim 1, and is effective to render the product-by-process limitation of previous claim 16 moot.
Accordingly, the remaining rejections of record are withdrawn.
Claim Interpretation
The following comments are made to establish broadest reasonable interpretation for the record.
Regarding claims 1, 11-15: These claims recite limitations regarding replicating or replicated vascular smooth muscle-like cells. The terms replicating or replicated are interpreted as meaning the duplication of a vascular smooth muscle-like cell population; this encompasses both passaging (i.e., subculturing cells to maintain growth and/or prevent altered culture kinetics, cell signaling, etc.) as well as growth (i.e., expansion or proliferation) of the vascular smooth muscle-like cell population(s).
Additionally, claim 1 recites the limitation cadherin-2, which is interpreted as Neural cadherin, N-cadherin, CDH2 gene products.
Regarding claim 16: This claim recites, “…wherein the vascular smooth muscle like cells further express vascular smoothelin and vascular smooth muscle myosin heavy chain.”. Vascular smoothelin is interpreted as smoothelin, smoothelin-B, SMTN gene products; vascular smooth muscle myosin heavy chain is interpreted as smooth muscle myosin heavy chain, SM-1 myosin heavy chain, MYH11 gene products; cadherin-2 is interpreted as Neural cadherin, N-cadherin, CDH2 gene products.
Modified Prior Art Rejections
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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.
Claims 1, 4-5, and 7-16 are rejected under 35 U.S.C. 103 as being unpatentable over Kwong, et al. (Stem Cell Reports. 2019), in view of Patsch, et al. (Nat Cell Biol. 2015), Maldonado, et al. (Stem Cell Res. 2016), Schreier, et al. (Biochim Biophys Acta. 2016), Koutsouki, et al. (Aterioscler Thromb Vasc Biol. 2005), and Davis-Dusenbery, et al. (Arterioscler Thromb Vasc Biol. 2011); as evidenced by Yang and Xiong (Biomed Tissue Cult. 2012), Deumens, et al. (Prog Neurobiol. 2010), and Zhi, et al. (Cancer Lett. 2010).
Kwong, et al. teaches the generation of a purified iPSC-derived smooth muscle-like population for cell sheet engineering (Title).
Patsch, et al. teaches GSK3 inhibition and BMP4 treatment rapidly commit pluripotent cells to a mesodermal fate and subsequent exposure to PDGF-BB results in the differentiation of vascular smooth muscle cells (Abstract).
Maldonado, et al. teaches ROCK inhibitor Y-27632 primes human induced pluripotent stem cells to selectively differentiate towards a mesendodermal lineage via epithelial-mesenchymal transition-like modulation (Title, Abstract).
Schreier, et al. teaches ligands involved in vascular dysfunction and remodeling can transactivate the epidermal growth factor receptor (EGFR) of vascular smooth muscle cells (VSMCs), and characterizes the importance of VSMC-EGFR for proliferation, migration and marker gene expression for inflammation, fibrosis and reactive oxygen species homeostasis (Abstract).
Koutsouki, et al. teaches vascular smooth muscle cell survival is dependent on N-cadherin-mediated cell-cell contacts (Abstract).
Davis-Dusenbery, et al. teaches the dynamic regulation of vascular smooth muscle cell phenotype in response to various stimuli (Abstract).
Regarding claims 1, 4-5, 7-8: Kwong, et al. teaches human iPSC clones were differentiated into KDR+ mesoderm using a serum-free medium supplemented with BMP4, vascular endothelial growth factor (VEGF), FGF2, and activin A for 24h, followed by 72h with BMP4, VEGF, and FGF2 (pg. 506; col. 1, par. 3); as FGF2 reads on basic fibroblast growth factor, this reads on the contacting pluripotent stem cells with a mesoderm induction growth medium for a day or more, wherein the pluripotent stem cells are induced pluripotent stem cells, and the mesoderm induction growth medium comprises basic fibroblast growth factor, under conditions such that the pluripotent stem cells formed induced mesodermal-like cells limitation recited in step a) of claim 1, the wherein contacting pluripotent stem cells with a mesoderm induction growth medium for a day or more is for four days limitation recited in claim 7, as well as the wherein contacting pluripotent stem cells with a mesoderm induction growth medium for a day or more is for not more than five days limitation recited in claim 8.
Kwong, et al. does not teach the mesoderm induction medium as comprising a rho-associated protein kinase (ROCK) inhibitor or a glycogen synthase kinase-3 (GSK3) inhibitor, as required by the remaining limitations recited in step a) of claim 1.
Regarding the ROCK inhibitor:
Maldonado, et al. teaches ROCK inhibitor Y-27632 primes hiPSCs to selectively differentiate towards mesendodermal lineage; by effectively regulating the cell cytoskeleton and cell-cell junction proteins, Y-27632 induces hiPSCs to undergo EMT-like changes which predispose the cells to differentiate towards mesendodermal lineage (“Conclusion”; pg. 226). Simultaneously, such a disruption of actin and E-cadherin organization results in an inhibition of ectodermal differentiation; thus, Y-27632 enhances directed differentiation of human PSCs towards mesendodermal target cell types (“Conclusion”; pg. 226).
It would have been prima facie obvious to a person having ordinary skill in the art to have modified the mesoderm induction medium used in the method taught by Kwong, et al. to have included ROCK inhibitor Y-27632, as taught by Maldonado, et al. This conclusion of obviousness is based on the ‘teaching, suggestion, or motivation rationale’; one would be motivated to do so for the enhanced directed differentiation of hiPSCs towards mesendodermal cell types, as taught by Maldonado, et al. Further, as Maldonado, et al. teaches the use of Y-27632 in human PSCs, one skilled in the art would have more than a reasonable expectation of success in using it with the human iPSCs of the method taught by Kwong, et al.
Regarding the GSK3 inhibitor:
Patsch, et al. teaches Wnt signaling directs differentiation of hPSCs into mesoderm and GSK3β inhibition activates this pathway; a panel of GSK3 inhibitors was evaluated for their selectivity and potential to inhibit GSK3 and to activate Wnt signaling, including CHIR99021 (pg. 995; col. 1, pars. 2-3). As BMP4 is also a potent inducer of mesoderm, Patsch, et al. tested BMP4 alone or combined with Wnt activation by GSK3 inhibitors CP21R7 and CHIR99021, wherein gene expression analyses revealed BMP4 treatment with Wnt activation led to upregulation of genes associated with mesoderm (pg. 995; col. 1, par. 5). Patsch, et al. teaches activation of Wnt signaling by GSK3 inhibition with CHIR99021 combined with BMP4 induced commitment of hPSCs towards mesoderm (pg. 995; col. 2, par. 1).
It would have been prima facie obvious to a person having ordinary skill in the art to have modified the mesoderm induction medium used in the method taught by Kwong, et al. to have included GSK3 inhibitor CHIR99021, as taught by Patsch, et al. This conclusion of obviousness is based on the ‘teaching, suggestion, or motivation rationale’; one would be motivated to do so for the directed differentiation of hiPSCs towards mesoderm lineage and upregulation of mesoderm-associated genes, as taught by Patsch, et al. Further, as Patsch, et al. teaches the use of CHIR99021 in hPSCs in a similar protocol to the one taught by Kwong, et al. (i.e., induction of iPSCs to a mesoderm fate in media comprising BMP4 and subsequent differentiation of the induced mesoderm-like cells to VSMCs), one skilled in the art would have more than a reasonable expectation of success in using it with the iPSCs of the method taught by Kwong, et al.
Therefore, the limitations of step a) of claim 1 are thus rendered obvious. Further, the modifications outlined above read on the wherein the rho-associated protein kinase inhibitor is trans-4- [(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide (Y-27632) limitation recited in claim 4, as well as the wherein the glycogen synthase kinase-3 inhibitor is 6-[[2-[[4-(2, 4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3- pyridine-carbonitrile (CHIR-99021) limitation recited in claim 5.
Kwong, et al. teaches cells were then dissociated with Gentle Cell Dissociation Reagent at 37°C for 3m and passaged into Matrigel™-coated plates in serum-free differentiation medium comprising PDGF-BB and TGF-β, with cells dissociated and sorted for further analysis between days 20 and 30 (“Experimental Procedures: Directed Differentiation of Human iPSCs into ACTA2eGFP+ Cells”; pgs. 511-512); this reads on the contacting the induced mesodermal-like cells with a first vascular smooth muscle cell growth medium for a day or more, wherein the first vascular smooth muscle cell growth medium comprises transforming growth factor-beta (TGF-β) and platelet-derived growth factor (PDGF), under conditions such that the mesodermal-like cells form induced vascular smooth muscle like cells limitation recited in step b) of claim 1.
Kwong, et al. does not teach the vascular smooth muscle cell growth medium as comprising epidermal growth factor, as required by the remaining limitation in step b) of claim 1.
Briefly, Schreier, et al. teaches mice containing floxed EGFR alleles were crossed with SM22-cre transgenic mice, in which the cyclization recombination recombinase is under the control of the smooth muscle cell–specific SM22 promoter, as evidenced by a previous disclosure from Schreier, et al. (“Methods: Generation of EGFRΔ/Δ VSMC Mice”; Aterioscler Thromb Vasc Biol. 2011). Primary culture of VSMC from 4- to 5-month-old mice was performed, with EGFRflox/flox × SM22-Cre+/− → VSMCΔ/ΔEGFR = knockout (KO) VSMC cells, and EGFRflox/flox × SM22- Cre−/− → VSMC+/+EGFR = wild-type (WT) VSMC cells (pg. 1520; section 2.1, par. 2). After 24h starvation, the addition of epidermal growth factor (EGF) increased VSMC proliferation of WT cells but not KO cells (Fig. 2A; pg. 1521; section 3.2, par. 1). Further, in WT VSMC, but not in KO VSMC, EGF enhanced gap closure, an effect detectable already within the first 4h and even more pronounced after 48h (Fig. 2B, C, E, and F); since an effect of EGF on proliferation within 4h is highly unlikely, these data already indicate that EGF stimulates chemokinesis via the EGF receptor (pg. 1521; section 3.2, par. 2). To distinguish between accelerated motion and directed movement in single cells Boyden chamber assays (directed movement) were performed over 24h and single cell tracking experiments (accelerated motion) over 6h; EGF increased the directed movement of WT cells in the Boyden chamber (Fig. 2D); additionally, EGF increased gross velocity in WT VSMC (pg. 1522; section 3.2, par. 2). Schreier, et al. further teaches EGF-activated EGFR induces VSMC proliferation, single cell migration, chemotaxis, as well as collective migration, and that EGF increases gap closure, as the gap closure effect is abolished by EGFR inhibition using AG-1478 (pg. 1529; section 4.1, par. 7).
It would have been prima facie obvious to a person having ordinary skill in the art to have modified the VSMC induction medium used in the method taught by Kwong, et al. to have included EGF, as taught by Schreier, et al. This conclusion of obviousness is based on the ‘teaching, suggestion, or motivation rationale’; one would be motivated to do so for the induction of VSMC proliferation, migration, and chemotaxis, as well as the increased gap closure, as taught by Schreier, et al. Further, as Schreier, et al. teaches the use of EGF in VSMCs, one skilled in the art would have more than a reasonable expectation of success in using it in the method taught by Kwong, et al.
Therefore, the limitations of step b) of claim 1 are thus rendered obvious.
Regarding steps c), d), e) of claim 1:
Kwong, et al. does not explicitly teach the limitations recited in steps c) and d) of claim 1; namely, passaging VSMCs by using a protease or collagenase to detach the cells, i.e., step c), and subculturing the passaged cell population, i.e., steps d) and e).
However, it would have been prima facie obvious to a person having ordinary skill in the art to have modified the method of Kwong, et al. by further incorporating a step wherein the iPSC-SMCs were passaged for expansion and plated onto Matrigel™-coated plates in the VSMC induction medium. This conclusion of obviousness is based on the ‘teaching, suggestion, or motivation rationale’; one would be motivated to do so for the increased cell yield with an expansion step. An increased cell yield would be desirable to one skilled in the art for reasons including assays requiring a high cell number, or increased technical replicates for statistical analysis. Further, as evidenced by Yang and Xiong (pgs. 15-16; par. 5.2.1), passaging cells using digestion media such as trypsin is a basic cell culture technique; thus, a person skilled in the art would have more than a reasonable expectation of success.
As evidenced by Yang and Xiong, trypsin is a protease called endopeptidase (pgs. 15-16; par. 5.2.1); as evidenced by Deumens, et al., Matrigel™ is an extracellular matrix solution comprising type IV collagen (pg. 258; par. 3.3.3). Therefore, the modifications set forth above read on the contacting the induced vascular smooth muscle like cells with a protease under conditions such that induced vascular smooth muscle like cells detach from each other providing detached induced vascular smooth muscle like cells limitation recited in part c) of claim 1, the replicating the detached induced vascular smooth muscle like cells by exposure to collagen and the first vascular smooth muscle cell growth medium for a day or more providing replicated vascular smooth muscle like cells limitation recited in part d) of claim 1, as well as the contacting replicated vascular smooth muscle like cells with a second vascular smooth muscle cell growth medium for a day or more, wherein the second vascular smooth muscle cell growth medium comprises transforming growth factor-beta, epidermal growth factor, and platelet-derived growth factor, under conditions such that the replicated vascular smooth muscle like cells form a second batch of induced vascular smooth muscle like cells limitation recited in part e) of claim 1.
Kwong, et al. does not teach wherein the concentration of transforming growth factor-beta in the second vascular smooth muscle cell growth medium is increased compared to the concentration of transforming growth factor-beta in the first vascular smooth muscle cell growth media, and wherein the concentration of platelet-derived growth factor in the second vascular smooth muscle cell growth medium is decreased compared to the concentration of platelet-derived growth factor in the first vascular smooth muscle cell growth media limitations, as required by the remaining limitations recited in part e) of claim 1.
However, Davis-Dusenbery, et al. teaches the natural cycle of VSMC plasticity, wherein the dedifferentiated VSMC phenotype is characterized by increased rates of proliferation and migrations, while the differentiated VSMC phenotype is characterized by high levels of contractile gene expression (pg. 1; par. 1). Davis-Dusenbery, et al. further teaches VSMC phenotype is determined through environmental cues; e.g., cytokines, cell-cell contact, cell adhesions, extracellular matrix interactions (pg. 2; par. 1). In particular, PDGF promotes increased proliferation and migration but reduced expression of contractile genes, while TGF-β reduces VSMC proliferation and migration but increases expression of contractile gene (pg. 2; par. 1).
Therefore, it would have been prima facie obvious to a person having ordinary skill in the art to have further modified the method of Kwong, et al. by reducing the concentration of PDGF-BB and increasing the concentration of TGF-β in the cell culture medium after passaging the VSMCs for expansion. This conclusion of obviousness is based on the ‘teaching, suggestion, or motivation rationale’. One would have been motivated to do so to mimic the natural differentiation process of VSMCS taught by Davis-Dusenbery, et al.; i.e., a state characterized by increased proliferation, followed by a state characterized by increased expression of contractile genes. Further, as the same disclosure teaches PDGF for increasing proliferation of migration of VSMCs and TGF-β for increasing expression of contractile genes, a skilled artisan would have a reasonable expectation of success in doing so.
This renders obvious the remaining limitations recited in step e) of claim 1.
Regarding step f) of claim 1:
Kwong, et al. does not teach purifying the induced vascular smooth muscle-like cells by selecting cells that express cadherin-2, as required by the limitation recited in step f) of claim 1.
However, Koutsouki, et al. teaches in VSMCs, N-cadherin modulates cell survival via cell-cell contacts (pg. 986; col. 1, par. 1), and cell aggregation mediated by N-cadherin and subsequent activation of Akt promotes cell survival (pg. 987; col. 2, par. 2).
Therefore, it would have been prima facie obvious to a person having ordinary skill in the art to have modified the method of Kwong, et al. by further incorporating a step wherein VSMCs are purified by selecting cells that express N-cadherin, in light of the Koutsouki, et al. disclosure. This conclusion of obviousness is based on the ‘teaching, suggestion, or motivation rationale’; one would be motivated to do so as Koutsouki, et al. teaches N-cadherin promotes cell survival (pgs. 986-987), and it would have been obvious to one skilled in the art that purifying the VSMC population by selecting cells that already express N-cadherin (i.e., cadherin-2) would increase cell survival in said VSMC population. Further, as evidenced by Zhi, et al., the use of anti-human CD325 (N-Cadherin) antibody to sort cells is a well-known technique in the art (“Materials and methods: 2.2. FACS Sorting”; pg. 66. Cancer Lett. 2010); thus, a person of ordinary skill in the art would have more than a reasonable expectation of success in selecting VSMCs that express cadherin-2.
Thus, the limitations of claims 1, 4-5, and 7-8 are rendered obvious.
Regarding claims 9-14: These instant claims are drawn to limitations regarding timepoints of the method of claim 1.
Kwong, et al. does not teach: contacting the induced mesodermal-like cells with a first VSMC growth medium for twenty days (claim 9), or not more than twenty-one days (claim 10); replicating the detached induced vascular smooth muscle like cells by exposure to collagen and the first vascular smooth muscle cell growth medium for fifteen days (claim 11), or not more than sixteen days (claim 12); contacting replicated vascular smooth muscle like cells with a second vascular smooth muscle cell growth medium for twenty-five days or more (claim 13), or not more than twenty-six days (claim 14).
However, the timing of the method steps would have been routinely optimized by one having ordinary skill in the art based on the other culture conditions. Kwong, et al. clearly teaches the disclosed method is provided in a timeframe effective to achieve VSMC differentiation. That means the timeframe necessary to achieve VSMC differentiation was a result effective variable. Result effective variables would be optimized by routine experimentation by one having ordinary skill in the art. Furthermore, differences in timepoints will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such differences are critical. See MPEP 2144.05(II)(A).
This renders obvious the limitations of claims 9-14.
Regarding claim 15: Following the above discussion, Kwong, et al. does not teach replicating the purified cadherin-2-expressing vascular smooth muscle cells by exposing said cells to collagen and the second VSMC growth medium, as required by the limitation recited in the instant claim.
However, it would have been prima facie obvious to a person having ordinary skill in the art to have included an additional step in the modified method of Kwong, et al. wherein the cadherin-2 expressing VSMCs were replicated by passaging cells and plating them onto Matrigel™-coated plates in the VSMC growth medium. This conclusion of obviousness is based on the ‘teaching, suggestion, or motivation rationale’. One would be motivated to do so in order to expand the population of VSMCs wherein cadherin-2 modulates the survival thereof via cell-cell contact, as taught by Koutsouki, et al. (pg. 986; col. 1, par. 1); reasons for expansion of a cell population are well-known in the art, e.g., expansion of a cell population for sufficient cell numbers for an assay. Further, as evidenced by Yang and Xiong (pgs. 15-16; par. 5.2.1), passaging cells using digestion media such as trypsin is a basic cell culture technique; thus, a person skilled in the art would have more than a reasonable expectation of success.
Thus, the limitations of claim 15 are rendered obvious.
Regarding claim 16: Kwong, et al. teaches the human iPSC-derived ACTA2eGFP+ cells (i.e., the VSMCs) express smooth muscle cell markers, including SMTN and MYH11 (Fig. 5E); this reads on the wherein the vascular smooth muscle cells further express vascular smoothelin and vascular smooth muscle myosin heavy chain limitation recited in claim 16.
Claims 1-15 are rejected under 35 U.S.C. 103 as being unpatentable over Cheung, et al. (Nat Protoc. 2014), in view of Patsch, et al. (Nat Cell Biol. 2015), Maldonado, et al. (Stem Cell Res. 2016), Schreier, et al. (Biochim Biophys Acta. 2016), Koutsouki, et al. (Aterioscler Thromb Vasc Biol. 2005), and Davis-Dusenbery, et al. (Arterioscler Thromb Vasc Biol. 2011); as evidenced by Zhi, et al. (Cancer Lett. 2010).
Cheung, et al. teaches directed differentiation of embryonic origin-specific vascular smooth muscle subtypes from human pluripotent stem cells (Title).
The teachings of Patsch, et al., Maldonado, et al., Schreier, et al., Koutsouki, et al., and Davis-Dusenbery, et al. are set forth above.
Regarding claims 1, 4-5, 7-8:
Cheung, et al. teaches a method wherein human pluripotent stem cells (hPSCs) are differentiated to vascular smooth muscle cells (SMCs); on Days 0-5, hPSCs are differentiated to an early mesoderm lineage in media comprising FGF2 and BMP4, followed by subsequent differentiation to either lateral plate mesoderm or paraxial mesoderm lineage in media comprising FGF2 as well as BMP4 or PI3K inhibitor LY294002, respectively (Fig. 1; pg. 930). As FGF2 reads on basic fibroblast growth factor (bFGF), this reads on the contacting pluripotent stem cells with a mesoderm induction growth medium for a day or more, wherein the pluripotent stem cells are induced pluripotent stem cells, and wherein the mesoderm induction growth medium comprises basic fibroblast growth factor, under conditions such that the pluripotent stem cells form induced mesodermal-like cells limitation recited in step a) of claim 1, the wherein contacting pluripotent stem cells with a mesoderm induction growth medium for a day or more is for four days limitation recited in claim 7, as well as the wherein contacting pluripotent stem cells with a mesoderm induction growth medium for a day or more is for not more than five days limitation recited in claim 8.
Cheung, et al. does not teach the mesoderm induction medium as comprising a rho-associated protein kinase (ROCK) inhibitor or a glycogen synthase kinase-3 (GSK3) inhibitor, as required by the remaining limitations recited in step a) of claim 1.
Regarding the ROCK inhibitor:
Maldonado, et al. teaches ROCK inhibitor Y-27632 primes hiPSCs to selectively differentiate towards mesendodermal lineage; by effectively regulating the cell cytoskeleton and cell-cell junction proteins, Y-27632 induces hiPSCs to undergo EMT-like changes which predispose the cells to differentiate towards mesendodermal lineage (“Conclusion”; pg. 226). Simultaneously, such a disruption of actin and E-cadherin organization results in an inhibition of ectodermal differentiation; thus, Y-27632 enhances directed differentiation of human PSCs towards mesendodermal target cell types (“Conclusion”; pg. 226).
It would have been prima facie obvious to a person having ordinary skill in the art to have modified the mesoderm induction medium used in the method taught by Cheung, et al. to have included ROCK inhibitor Y-27632, as taught by Maldonado, et al. This conclusion of obviousness is based on the ‘teaching, suggestion, or motivation rationale’; one would be motivated to do so for the enhanced directed differentiation of hiPSCs towards mesendodermal cell types, as taught by Maldonado, et al. Further, as Maldonado, et al. teaches the use of Y-27632 in human PSCs, one skilled in the art would have more than a reasonable expectation of success in using it with the hiPSCs of the method taught by Cheung, et al.
Regarding the GSK3 inhibitor:
Patsch, et al. teaches Wnt signaling directs differentiation of hPSCs into mesoderm and GSK3β inhibition activates this pathway; a panel of GSK3 inhibitors was evaluated for their selectivity and potential to inhibit GSK3 and to activate Wnt signaling, including CHIR99021 (pg. 995; col. 1, pars. 2-3). As BMP4 is also a potent inducer of mesoderm, Patsch, et al. tested BMP4 alone or combined with Wnt activation by GSK3 inhibitors CP21R7 and CHIR99021, wherein gene expression analyses revealed BMP4 treatment with Wnt activation led to upregulation of genes associated with mesoderm (pg. 995; col. 1, par. 5). Patsch, et al. teaches activation of Wnt signaling by GSK3 inhibition with CHIR99021 combined with BMP4 induced commitment of hPSCs towards mesoderm (pg. 995; col. 2, par. 1).
It would have been prima facie obvious to a person having ordinary skill in the art to have modified the mesoderm induction medium used in the method taught by Cheung, et al. to have included GSK3 inhibitor CHIR99021, as taught by Patsch, et al. This conclusion of obviousness is based on the ‘teaching, suggestion, or motivation rationale’; one would be motivated to do so for the directed differentiation of hiPSCs towards mesoderm lineage and upregulation of mesoderm-associated genes, as taught by Patsch, et al. Further, as Patsch, et al. teaches the use of CHIR99021 in hPSCs in a similar protocol to the one taught by Cheung, et al. (i.e., induction of hPSCs to a mesoderm fate in media comprising BMP4 and subsequent differentiation of the induced mesoderm-like cells to VSMCs), one skilled in the art would have more than a reasonable expectation of success in using it with the hiPSCs of the method taught by Cheung, et al.
Therefore, the limitations of step a) of claim 1 are thus rendered obvious. Further, the modifications outlined above read on the wherein the rho-associated protein kinase inhibitor is trans-4- [(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide (Y-27632) limitation recited in claim 4, as well as the wherein the glycogen synthase kinase-3 inhibitor is 6-[[2-[[4-(2, 4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3- pyridine-carbonitrile (CHIR-99021) limitation recited in claim 5.
Cheung, et al. teaches the subsequent differentiation of the cells to SMCs in media comprising PDGF-BB and TGF-β1 for at least 12 days, starting on Day 5 (Fig. 1; pg. 930); this reads on the contacting the induced mesodermal-like cells with a first vascular smooth muscle cell growth medium for a day or more, wherein the first vascular smooth muscle cell growth medium comprises transforming growth factor-beta (TGF-β) and platelet-derived growth factor (PDGF), under conditions such that the mesodermal-like cells form induced vascular smooth muscle like cells limitation recited in step b) of claim 1.
Cheung, et al. does not teach the vascular smooth muscle cell growth medium as comprising epidermal growth factor, as required by the remaining limitation in step b) of claim 1.
Briefly, Schreier, et al. teaches mice containing floxed EGFR alleles were crossed with SM22-cre transgenic mice, in which the cyclization recombination recombinase is under the control of the smooth muscle cell–specific SM22 promoter, as evidenced by a previous disclosure from Schreier, et al. (“Methods: Generation of EGFRΔ/Δ VSMC Mice”; Aterioscler Thromb Vasc Biol. 2011). Primary culture of VSMC from 4- to 5-month-old mice was performed, with EGFRflox/flox × SM22-Cre+/− → VSMCΔ/ΔEGFR = knockout (KO) VSMC cells, and EGFRflox/flox × SM22- Cre−/− → VSMC+/+EGFR = wild-type (WT) VSMC cells (pg. 1520; section 2.1, par. 2). After 24h starvation, the addition of epidermal growth factor (EGF) increased VSMC proliferation of WT cells but not KO cells (Fig. 2A; pg. 1521; section 3.2, par. 1). Further, in WT VSMC, but not in KO VSMC, EGF enhanced gap closure, an effect detectable already within the first 4h and even more pronounced after 48h (Fig. 2B, C, E, and F); since an effect of EGF on proliferation within 4h is highly unlikely, these data already indicate that EGF stimulates chemokinesis via the EGF receptor (pg. 1521; section 3.2, par. 2). To distinguish between accelerated motion and directed movement in single cells Boyden chamber assays (directed movement) were performed over 24h and single cell tracking experiments (accelerated motion) over 6h; EGF increased the directed movement of WT cells in the Boyden chamber (Fig. 2D); additionally, EGF increased gross velocity in WT VSMC (pg. 1522; section 3.2, par. 2). Schreier, et al. further teaches EGF-activated EGFR induces VSMC proliferation, single cell migration, chemotaxis, as well as collective migration, and that EGF increases gap closure, as the gap closure effect is abolished by EGFR inhibition using AG-1478 (pg. 1529; section 4.1, par. 7).
It would have been prima facie obvious to a person having ordinary skill in the art to have modified the VSMC induction medium used in the method taught by Cheung, et al. to have included EGF, as taught by Schreier, et al. This conclusion of obviousness is based on the ‘teaching, suggestion, or motivation rationale’; one would be motivated to do so for the induction of VSMC proliferation, migration, and chemotaxis, as well as the increased gap closure, as taught by Schreier, et al. Further, as Schreier, et al. teaches the use of EGF in VSMCs, one skilled in the art would have more than a reasonable expectation of success in using it in the method taught by Cheung, et al.
Therefore, the limitations of step b) of claim 1 are thus rendered obvious.
Regarding steps c), d), e) of claim 1:
Cheung, et al. does not explicitly teach the limitations recited in steps c) and d) of claim 1; namely, passaging VSMCs by using a protease or collagenase to detach the cells, i.e., step c), and subculturing the passaged cell population, i.e., steps d) and e).
However, Cheung, et al. teaches that during the at least 12 days of differentiation in VSMC media, cells that become confluent may be passaged at any point by dissociating cells using TrypLE Express and then plating them onto gelatin-coated plates at a ratio of 1:2, using the VSMC media comprising PDGF-BB and TGF-β1 (pg. 935, step 16; pgs. 934-935, steps 10-14). As TrypLE Express is a protease, this reads on the contacting the induced vascular smooth muscle like cells with a protease under conditions such that induced vascular smooth muscle like cells detach from each other providing detached induced vascular smooth muscle like cells limitation recited in part c) of claim 1; additionally, as gelatin is denatured collagen, the modified method of Cheung, et al. reads on the replicating the detached induced vascular smooth muscle like cells by exposure to collagen and the first vascular smooth muscle cell growth medium for a day or more providing replicated vascular smooth muscle like cells limitation recited in part d) of claim 1, as well as the contacting replicated vascular smooth muscle like cells with a second vascular smooth muscle cell growth medium for a day or more, wherein the second vascular smooth muscle cell growth medium comprises transforming growth factor-beta, epidermal growth factor, and platelet-derived growth factor, under conditions such that the replicated vascular smooth muscle like cells form a second batch of induced vascular smooth muscle like cells limitation recited in part e) of claim 1.
Cheung, et al. does not teach the wherein the concentration of transforming growth factor-beta in the second vascular smooth muscle cell growth medium is increased compared to the concentration of transforming growth factor-beta in the first vascular smooth muscle cell growth media, and wherein the concentration of platelet-derived growth factor in the second vascular smooth muscle cell growth medium is decreased compared to the concentration of platelet-derived growth factor in the first vascular smooth muscle cell growth media limitations, as required by the remaining limitations recited in part e) of claim 1.
However, Davis-Dusenbery, et al. teaches the natural cycle of VSMC plasticity, wherein the dedifferentiated VSMC phenotype is characterized by increased rates of proliferation and migrations, while the differentiated VSMC phenotype is characterized by high levels of contractile gene expression (pg. 1; par. 1). Davis-Dusenbery, et al. further teaches VSMC phenotype is determined through environmental cues; e.g., cytokines, cell-cell contact, cell adhesions, extracellular matrix interactions (pg. 2; par. 1). In particular, PDGF promotes increased proliferation and migration but reduced expression of contractile genes, while TGF-β reduces VSMC proliferation and migration but increases expression of contractile gene (pg. 2; par. 1).
Therefore, it would have been prima facie obvious to a person having ordinary skill in the art to have further modified the method of Cheung, et al. by reducing the concentration of PDGF-BB and increasing the concentration of TGF-β in the cell culture medium after passaging the VSMCs. This conclusion of obviousness is based on the ‘teaching, suggestion, or motivation rationale’. One would have been motivated to do so to mimic the natural differentiation process of VSMCS taught by Davis-Dusenbery, et al.; i.e., a state characterized by increased proliferation, followed by a state characterized by increased expression of contractile genes. Further, as the same disclosure teaches PDGF for increasing proliferation of migration of VSMCs and TGF-β for increasing expression of contractile genes, a skilled artisan would have a reasonable expectation of success in doing so.
This renders obvious the remaining limitations recited in step e) of claim 1.
Regarding step f) of claim 1:
Cheung, et al. does not teach purifying the induced vascular smooth muscle-like cells by selecting cells that express cadherin-2, as required by the limitation recited in step f) of claim 1.
However, Koutsouki, et al. teaches in VSMCs, N-cadherin modulates cell survival via cell-cell contacts (pg. 986; col. 1, par. 1), and cell aggregation is mediated by N-cadherin and subsequent activation of Akt promotes cell survival (pg. 987; col. 2, par. 2).
It would have been prima facie obvious to a person having ordinary skill in the art to have modified the method of Cheung, et al. by incorporating a step wherein VSMCs are purified by selecting cells that express N-cadherin, in light of the Koutsouki, et al. disclosure. This conclusion of obviousness is based on the ‘teaching, suggestion, or motivation rationale’; one would be motivated to do so as Koutsouki, et al. teaches N-cadherin modulates cell survival via cell-cell contact (pg. 986; col. 1, par. 1), and it would have been obvious to one skilled in the art that purifying the VSMC population by selecting cells that already express N-cadherin (i.e., cadherin-2) would increase cell survival in said VSMC population. Further, as evidenced by Zhi, et al., the use of anti-human CD325 (N-Cadherin) antibody to sort cells is a well-known technique in the art (“Materials and methods: 2.2. FACS Sorting”; pg. 66. Cancer Lett. 2010); thus, a person of ordinary skill in the art would have more than a reasonable expectation of success in selecting VSMCs that express cadherin-2.
Thus, the limitations of claim 1 are rendered obvious.
Regarding claims 9-14: These instant claims are drawn to limitations regarding timepoints of the method of claim 1.
Cheung, et al. does not teach: contacting the induced mesodermal-like cells with a first VSMC growth medium for twenty days (claim 9), or not more than twenty-one days (claim 10); replicating the detached induced vascular smooth muscle like cells by exposure to collagen and the first vascular smooth muscle cell growth medium for fifteen days (claim 11), or not more than sixteen days (claim 12); contacting replicated vascular smooth muscle like cells with a second vascular smooth muscle cell growth medium for twenty-five days or more (claim 13), or not more than twenty-six days (claim 14).
However, the timing of the method steps would have been routinely optimized by one having ordinary skill in the art based on the other culture conditions. Cheung, et al. clearly teaches the designated timepoints of the disclosed method are provided in a timeframe effective to achieve VSMC differentiation. That means the conditions to achieve the VSMC differentiation necessarily were result effective variables. Result effective variables would be optimized by routine experimentation by one having ordinary skill in the art. Furthermore, differences in timepoints will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such differences are critical. See MPEP 2144.05(II)(A).
This renders obvious the limitations of claims 9-14.
Regarding claim 15: Following the above discussion, Cheung, et al. does not teach replicating the purified cadherin-2-expressing vascular smooth muscle cells by exposing said cells to collagen and the second VSMC growth medium, as required by the limitation recited in the instant claim.
However, Cheung, et al. teaches cells that become confluent may be passaged at any point by dissociating cells using TrypLE Express and then plating them onto gelatin-coated plates at a ratio of 1:2, using the VSMC media comprising PDGF-BB and TGF-β1 (pg. 935, step 16; pgs. 934-935, steps 10-14).
It would have been prima facie obvious to a person having ordinary skill in the art to have included an additional step in the modified method of Cheung, et al. wherein the cadherin-2 expressing VSMCs were replicated by passaging the cells using TrypLE Express and plating them onto gelatin-coated plates, as taught by Cheung, et al. This conclusion of obviousness is based on the ‘teaching, suggestion, or motivation rationale’. One would be motivated to do so in order to expand the population of VSMCs; reasons for expansion of a cell population are well-known in the art, e.g., expansion of a cell population for sufficient cell numbers for an assay. Further, as Cheung, et al. teaches passaging the cells if confluency is reaches, a person of ordinary skill in the art would have more than a reasonable expectation of success in passaging the cells in order to split and expand the existing cell population.
Thus, the limitations of claim 15 are rendered obvious.
Regarding claim 16: Following the above discussion, Cheung, et al. teaches VSMCs express MYH11 by day 19 (Fig. 1). Additionally, as the modified method of Cheung, et al. carries out the same active steps of claim 1, the effect of expression of vascular smoothelin is necessarily achieved; see MPEP 2112.02(I). This renders obvious the wherein the vascular smooth muscle cells further express vascular smoothelin and vascular smooth muscle myosin heavy chain limitation recited in the instant claims.
Claims 18-19 are rejected under 35 U.S.C. 103 as being unpatentable over Dahl, et al. (Sci Transl Med. 2011) in view of Cheung, et al. (Nat Protoc. 2014), further in view of Patsch, et al. (Nat Cell Biol. 2015), Maldonado, et al. (Stem Cell Res. 2016), Schreier, et al. (Biochim Biophys Acta. 2016), and Koutsouki, et al. (Aterioscler Thromb Vasc Biol. 2005); as evidenced by Zhi, et al. (Cancer Lett. 2010) and Zhou, et al. (Stem Cell Rev Rep. 2015).
The teachings of Cheung, et al., Patsch, et al., Maldonado, et al., Schreier, et al., and Koutsouki, et al. are set forth above.
Dahl, et al. teaches engineered vascular grafts grown on a tubular polyglycolic acid scaffold (Abstract).
Regarding claim 18: Briefly, Dahl, et al. teaches a method wherein allogeneic smooth muscle cells (SMCs) obtained from cadaveric donors were cultured on rapidly degradable polyglycolic acid (PGA) tubular scaffolds (Fig. 1A) in a bioreactor, where the SMCs secrete extracellular matrix proteins to form biosynthetic vascular tissue (Fig. 1B), and the PGA degrades (“Results: Generation of TEVGs from allogeneic cells and decellularization”; pgs. 1-2). At the end of the culture period, the resultant tissue is decellularized, leaving only the secreted collagenous matrix (Fig. 1C); the TEVGs (Fig. 1C) can then be used for placement as an arteriovenous graft (Fig. 1D), or seeded with autologous endothelial cells to reduce the risk of thrombosis associated with small-diameter vascular grafting in peripheral or coronary arteries (Fig. 1E) (“Results: Generation of TEVGs from allogeneic cells and decellularization”; pg. 2). To assess the function of TEVGs, nine TEVGs grown from human cells (6 mm in diameter, 12.5 ± 1.1 cm in length) were implanted into baboons as arteriovenous conduits (Fig. 2A) and observed for 1 to 6 months (“Results: Decellularized human TEVGs in an arteriovenous model”; pg. 2).
As Dahl, et al. further teaches the vascular grafts are functional as arteriovenous conduits and as small-caliber arterial bypasses in the peripheral (carotid) and coronary circulations, supporting a use for decellularized human TEVGs in a range of vascular applications for patients (pg. 8; col. 1, par. 2), the TEVGs comprising SMCs reads on the treating a cardiovascular disease or condition comprising administering an effective amount of cells to a subject in need thereof limitation recited in claim 18. Dahl, et al. does not teach the cells as made by the method of claim 1, as required by the remaining limitation recited in the instant claim.
However, it is set forth above the modified method of Cheung, et al. renders obvious the method of claim 1.
It would have been prima facie obvious to a person having ordinary skill in the art to have modified the method of Dahl, et al. by using the VSMCs rendered obvious by the modified method of Cheung, et al. This conclusion of obviousness is based on the ‘substitution rationale’; the use of the hPSC-derived VSMCs taught by Cheung, et al. in place of the allogeneic SMCs taught by Dahl, et al. is a predictable use of prior art elements according to their established functions, leading to the predictable result of decellularized human TEVGs functional as arteriovenous conduits and as small-caliber arterial bypasses. This rationale aligns with the principle of simple substitution of one known element for another to obtain predictable results; see MPEP 2141.
Thus, the modification as set forth above results in VSMCs generated by the modified method of Cheung, et al. (i.e., the method of claim 1) subsequently used in the generation of vascular grafts for arteriovenous conduits and as small-caliber arterial bypasses in the peripheral (carotid) and coronary circulations, rendering the limitations of claim 18 obvious.
Regarding claim 19: Following the above discussion, the modified method of Cheung, et al. does not teach the pluripotent cells used to generate the hPSC-derived VSMCs as derived from the subject, as required by the instant claim.
However, it would have been prima facie obvious to a person having ordinary skill in the art to have used pluripotent cells derived from the subject. This conclusion of obviousness is based on the ‘teaching, suggestion, or motivation rationale’; one would be motivated to do so because an autologous source for the therapeutic administration of cells reduces the risk of complications such as rejection or graft-versus-host disease. Further, as evidenced by Zhou, et al. (Fig. 1; Stem Cell Rev Rep. 2015), the derivation of human iPSCs from human peripheral blood samples is well-known in the art; thus, a person having ordinary skill in the art would have more than a reasonable expectation of success.
Thus, the limitations of claim 19 are rendered obvious.
Conclusion
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/GINA PRONZATI/Examiner, Art Unit 1633
/ALLISON M FOX/Primary Examiner, Art Unit 1633