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 .
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 07-14-2025 has been entered.
Applicant's amendments to the claims and arguments filed on 07-14-2025 have been received and entered. Claims 1, 5, 6, 9 have been amended. Claims 12, 16-34 have been canceled. Claim 35-38 have been added. Claims 1-11, 13-15, 35-38 are pending.
Election/Restrictions
Applicant’s election of Group I (claims 1-15) in the reply filed on 05-24-2024 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claims 1-11, 13-15, 35-38 are pending and under consideration.
Priority
This application is a 371 of PCT/US2020/014572 filed 01/22/2020, which claims
priority from US provisional application 62/795,520 filed on 01/22/2019.
New - Claim Rejections - 35 USC § 112- necessitated by amendments
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 1-11, 13-15, 35-38 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 1 recites the phrase “a human brain endothelial cell (BEC) vessel of about 5 to 10 microns in diameter and 5 to 50 microns in length” which render the claim vague and indefinite because it is unclear the phrase “5 to 50 microns in length” is diameter length or width length or diagonal length or what length. It is unclear if the how the term “length” is defined or just arbitrary cutoff of any random portion of the vessel. The specification of the claimed invention does not define/provide guidance for the term “length” other than general information for the term “diameter”: the instant disclosure teaches that “The vessel has a size on the order of a capillary. A capillary is an extremely small blood vessel located within the tissues of the body that transports blood. Capillaries measure in size from about 5 to 10 microns in diameter. Capillary walls are thin and are composed of endothelium. The iBBB is on the order of approximately 5 to 50 microns in length ……” (see instant disclosure page 17, lines 19-22). Also, instant claim 15 also requires BEC vessel is a capillary size. Additionally, brain blood vessel (blood brain barrier in capillary size) is interconnected branching system, a skilled person in the art would not know how to measure something from end to end without knowing where to start and stop or just arbitrary cutoff portion of the brain blood vessel.
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Furthermore, the specification of the claimed invention provides Example 1 to teach reconstruction of anatomical and physiological properties of the human blood-brain barrier in vitro that specifies “ after two weeks in the hydrogel matrix, BECs assembled into large (> 5 mm2 ) networks of interconnected CD144-positive cells resembling blood vessels (Fig. 1b; FIG. 7a)” (see instant disclosure on page 39, lines 25-27). Thus, the BECs network is large (> 5 mm2 or > 5,000,000 µm2), a skilled person in the art would not know which portion of the network is being measured and why, without guidance.
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According to Félétou M. (The Endothelium: Part 1: Multiple Functions of the Endothelial Cells—Focus on Endothelium Derived Vasoactive Mediators. San Rafael (CA): Morgan & Claypool Life Sciences; 2011.) who teaches that “the shape of the endothelial cells varies across the vascular tree, but they are generally thin and slightly elongated, their dimension being roughly 50–70 μm long, 10–30 μm wide and 0.1–10 μm thick” (Chapter 1-Introduction) . Thus, the size of single endothelial cells is already ranging from 50–70 μm long, 10–30 μm wide it is unclear how many endothelial cells the “vessel” is composed of to have the length of approximately 5 to 50 microns.
For the sake of compact prosecution, the phrase “5 to 50 microns in length” is interpreted as in diameter length (see page 17, lines 19-22 of the instant disclosure).
Claim 8 depends from the product iBBB of claim 1; however, there’s no culturing step for the claimed product (iBBB structure), so it is unclear how a culturing step fits into the scope of the claimed product.
Claim 36 recites the phrase “wherein the 3D matrix is a hydrogel formed of Matrigel, Myogel and Cartigel, or a combination of Matrigel, Myogel and Cartigel and a naturally derived biomaterial or biomaterials or hydrophilic polymers that are linear or branched wherein the polymers are synthetic.”. It is unclear which 3D matrix is required for the claim.
Claims 2-7, 9-11, 13-15, 35, 37-38 are included in the rejection because they directly or indirectly depend from the rejected base claim. Appropriate correction is required.
Claim Rejections - 35 USC § 112
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 37 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 37 does not satisfy the requirements of 112(d), as they contain a reference to a previous claim but do not specify a further limitation of the subject matter claimed. Claim 37 depend from claim 11 which recites “wherein the iBBB is generated using about 1 million endothelial cells per ml, about 200,000 astrocytes per ml and about 200,000 pericytes per ml”. A simple calculation would show that 1 million endothelial cells: 200,000 astrocytes: 200,000 pericytes is 5 parts: 1 part: 1 part which is required by claim 37. Thus, Claim 37 does not specify a further limitation of the subject matter claimed.
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 Objections
Claim 38 is objected to under 37 CFR 1.75 as being a substantial duplicate of claim 11. When two claims in an application are duplicates or else are so close in content that they both cover the same thing, despite a slight difference in wording, it is proper after allowing one claim to object to the other as being a substantial duplicate of the allowed claim. See MPEP § 608.01(m).
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Maintained in modified form - Claim Rejections - 35 USC § 101 - necessitated by amendments
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1-11, 13-15, 35-38 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a judicial exception (i.e., a law of nature, a natural phenomenon, or an abstract idea) without significantly more. Based upon an analysis with respect to the claim as a whole, claims do not recite something significantly different than a judicial exception. The rationale for this determination is explained below.
Step 1: Is the claim to a process, machine, manufacture or composition of matter?
Under the broadest reasonable interpretation, the terms of the claim are presumed to have their plain meaning consistent with the specification as it would be interpreted by one of ordinary skill in the art. See MPEP 2111.
Claims 1-2 are directed to blood brain barrier (iBBB) comprising a 3-dimensional (3D) matrix comprising a human brain endothelial cell (BEC) vessel of about 5 to 10 microns in diameter and 5 to 50 microns in length comprised of a large interconnected network of human induced pluripotentderived positive stem cell (iPSC) endothelial cells encapsulated in a 3D matrix, human induced pluripotent-derived stem cell (iPSC) pericytes proximal to the BEC vessel on an apical surface, and human induced pluripotent-derived stem cell (iPSC) astrocytes dispersed throughout the 3D matrix, wherein a plurality of the astrocytes are proximal to the BEC vessel and have glial fibrillary acidic protein (GFAP)- positive projections into the perivascular space, wherein the astrocytes have glial fibrillary acidic protein (GFAP)- positive projections into the perivascular space and express AQP4. Claims 3-6 are directed to the 3D matrix comprises (LAMA4), and the BEC express at least any one of PgP, LRP1, and RAGE, wherein PgP and ABCG2 are expressed on the apical surface and are 2-3 times greater than levels of PgP and ABCG2 expressed on BEC cultured alone or co-cultured with astrocytes. Claim 7 is directed to the iBBB that has a TEER that exceeds 5,500 Ohm x cm2, exhibits reduced molecular permeability and polarization of efflux pumps relative to BEC cultured alone or co-cultured with astrocytes. Claim 8 is directed to the iBBB that is not cultured with retinoic acid. Claim 9 is directed to the human pluripotent are iPSC-derived CD144 cells. Claims 10-11, 37-38 are directed to ratio and concentration of endothelial cells and astrocytes and pericytes. Claims 13-15 are directed to the length of iBBB.
Thus, the broadest reasonable interpretation of the claimed in vitro blood brain barrier (iBBB) is a product comprising endothelial cell vessel, pericytes and 3-dimensional matrix, human brain endothelial cell vessel, a human pericytes and a human astrocytes that are composition of matter. Thus, the claims are directed to at least one statutory category of invention (Step 1: YES).
Step 2(A), Prong 1: Does the claim recite an abstract idea, law of nature or natural phenomenon?
The claims are then analyzed to determine whether the in-vitro blood brain barrier is directed to a judicial exception. First, the phrases and “in-vitro” and “pluripotent-derived” in claim 1 are interpreted as product by process as directed to endothelial cells, pericytes, astrocytes. See MPEP 2113. Even though product-by-process claims are limited by and defined by the process, determination of patentability is based on the product itself. The patentability of a product does not depend on its method of production. If the product in the product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process." In re Thorpe, 777 F.2d 695, 698, 227 USPQ 964, 966 (Fed. Cir. 1985) (citations omitted). Second, the markedly different characteristics analysis is performed by comparing the nature-based product limitation in the claim to its naturally occurring counterpart to determine if it has markedly different characteristics from the counterpart. MPEP 2106.04(c) (II).
Regarding to claim 1, a neurovascular unit comprising endothelial cells, pericytes, and astrocytes can be assembled into a blood brain barrier: Yan et al (Theranostics 2021, Vol. 11, Issue 20, 10148-10170. doi: 10.7150/thno.63195) teaches that Primary cell lines from brain tissues such as primary brain microvascular endothelial cells (BMECs), pericytes, and astrocytes are widely used to construct the neurovascular unit (NVU) and investigate the blood-brain barrier (BBB) function in vitro (Page 10149, left column 2nd para.) …… hPSCs derived from healthy and diseased patients can be an ideal alternative to primary cell or animal models for human diseases in vitro …… can differentiate into various NVU cell types such as BMECs, pericytes and astrocytes, which can be assembled into an in vitro BBB model (Page 10149, left column 3rd para.). To characterize the hPSC-derived astrocytes, the presence of glial fibrillar acidic protein (GFAP) has been considered as the gold standard for identifying astrocytes (Page 10156, left column, 1st para.).
Lippmann et al ( herein after Lippmann-2013 Fluids Barriers CNS 10, 2 (2013). Doi: 10.1186/2045-8118-10-2) teaches that during development, the BBB arises as a result of complex multicellular interactions between immature endothelial cells and neural progenitors, neurons, radial glia, and pericytes. As the brain develops, astrocytes and pericytes further contribute to BBB induction and maintenance of the BBB phenotype (Abstract).
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Figure 1 Schematic representation of the developmental and adult BBB.
While it is obvious that cell cultures do not naturally occur, the combination of the constituent culture parts do in fact occur in nature and as taught above in Lippmann-2013 that all the claimed combination of cells are known to play a critical role to form complex structure of BBB (see figure 1). It is further disclosed that “Unlike the early stages of embryonic BBB development when astrocytes are absent, astrocytes play important roles in BBB maturation and maintenance. As a result of this adult brain microenvironment and in contrast to the developmental BBB, the adult BBB boasts an elevated TEER, measured at average values between 1000–2000 Ωxcm2 (and maximum values up to 6000 Ωxcm2) and a correspondingly lower passive permeability to molecular tracers. These mature brain endothelial cells also express a broad array of large and small molecule transport systems including nutrient influx transporters and efflux transporters such as p-glycoprotein (p-gp), multi-drug resistance-associated proteins (MRP), and breast cancer resistance protein (BCRP). While the mechanisms driving the further induction and maintenance of the adult BBB are unresolved, several growth factors and signaling molecules such as angiopoietin-1, cyclic adenosine monophosphate, basic fibroblast growth factor, glial-derived neurotrophic factor, glucocorticoids, retinoic acid, and Wnt3a (see Lippmann et al 2013, page 2, col. 2).
Additionally, the specification of the claimed invention discloses that the matrix may be formed of naturally derived biomaterials such as polysaccharides, gelatinous proteins, or ECM components comprising the following or functional variants thereof: agarose; alginate; chitosan; dextran; gelatin; laminins; collagens; hyaluronan; fibrin, and mixtures thereof. …… (Page 18, lines 19-22). The 3D matrix may be generated using an optimal mixture of endothelial cells, pericytes, and astrocytes (Page 19, lines 1-2). Nakamura et al (J. Cell Biol. 2019 Vol. 218 No. 10 3506–3525, doi: 10.1083/jcb.201807178) teaches Laminin produced by astrocytes regulates BBB integrity and pericyte differentiation. Fibronectin, which is produced by endothelial cells, pericytes, and astrocytes, stimulates angiogenesis and fibrosis after stroke suggesting an involvement of perlecan in BBB repair under pathological conditions (i.e., following ischemic stroke) (page 3511, left column, last para.). Campisi et al (Biomaterials 180 (2018) 117e129, Doi: 10.1016/j.biomaterials.2018.07.014) teaches 3D self-organized microvascular model of the human blood-brain barrier with endothelial cells, pericytes and astrocytes (title) and lateral and transverse vessel diameter distributions are ranging between about 0-55 µm and 0-50 µm (see Figure 3F on page 123 and bridging last para on left column to right column on page 121 ). DeStefano et al. (Fluids Barriers CNS (2018) 15:32; doi: 10.1186/s12987-018-0117-2, published: 04 December 2018) teach “Benchmarks for blood–brain barrier models: Ultrastructure: ……. Capillaries in the human brain are 8–10 µm in diameter, with 50–100 µm long segments between bifurcations (Fig. 1A, B) …… In capillaries, BMECs wrap around to form junctions with themselves and their upstream and downstream neighbors (Fig. 1C, D) such as pericytes and astrocytes (Page 2 left column, last para. and Figure 1). Thus, a human brain endothelial cell vessel of about 5 to 10 microns in diameter are also natural occurring, and the claimed human blood-brain barrier is not markedly different from product of nature.
Regarding to claims 2-11, 13-15, 35-38, the expression of GFAP (claim 1), AQP4 (claim 2), LAMA4 (claim 3), PgP, LRP1, RAGE, ABCG2 (Claims 4-6), iBBB having a TEER, reduced molecular permeability and polarization of efflux pumps (claim 7), cell having CD144 (Claim 9) are natural occurring events that happen in natural occurring products (see the USC 103 rejection below). Additionally, claim 8 specifies that the iBBB is not cultured with retinoic acid. Claims 10-11, 13-15, 35-38 only specify the cell ratio, cell concentration and the length of iBBB, 3D matrix container. Thus, the limitations in claim 8, 10-11, 13-15, 35-38 would not be subject to the markedly different characteristics analysis as they are not nature-based products, but would be evaluated as additional elements in Prong Two (and Step 2B). See, e.g., Funk Bros. Seed Co. v. Kalo Inoculant Co., 333 U.S. 127, 130, 76 USPQ 280, 281 (1948).
Therefore, there is no difference between the claimed blood in-vitro brain barrier and naturally occurring blood brain barrier, the claimed blood brain barrier does not have markedly different characteristics, and thus are a "product of nature" exception. In re Ros/in Institute (Edinburgh), 750 F.3d 1333, 1338-39 (Fed. Cir. 2014). Accordingly, the answer to step 2A prong one of the 101 Subject Matter Eligibility Test is "Yes". The claims are directed to “Judicial Exception”.
Step 2(A), Prong 2: Do the claims recite additional elements that integrate the judicial exception into a practical application?
In view of foregoing analysis, claim 1 and its dependent claims 2-11, 13-15, 35-38 recite a judicial exception of a naturally occurring blood brain barrier they would still be patent-eligible if the claim as a whole integrates the recited judicial exception (blood brain barrier) into a practical application of the exception. Here the claims recite the expression of GFAP (claim 1), AQP4 (claim 2), LAMA4 (claim 3), PgP, LRP1, RAGE, ABCG2 (Claims 4-6), iBBB having a TEER, reduced molecular permeability and polarization of efflux pumps (claim 7), cell having CD144 (Claim 9) are natural occurring events that specify the gene expression in natural occurring products. Thus, the judicial exception of blood brain barrier is not integrated into a practical application because the claims do not recite any additional elements that integrate the exception into a practical application of the exception. Additionally, claim 8 specifies that the iBBB is not cultured with retinoic acid, and claims 10-11, 13-15, 37-38 specify the cell ratio, cell concentration and the length of iBBB. Claims 35-36 specify the 3D matrix is contained in a glass bottom culture dish, and the 3D matrix is a hydrogel. However, holding natural occurring products together with a hydrogel such as Matrigel does not integrate the judicial exception into a practical application because keeping natural occurring products together with a hydrogel is a generic concept of containing a human brain endothelial cell, pericytes, astrocytes as evidenced by Urich et al (SCIENTIFIC REPORTS | 3 : 1500 | DOI: 10.1038/srep01500, 20 March 2013) who teach that in a matrigel setup the human primary brain endothelial cells (hpBECs), primary
pericytes (hpPs) and primary astrocytes (hpAs) undergo self-arrangement to form endothelial tube-like structures tightly covered by hpPs and loosely attached hpAs mainly at the junctions (see abstract).
Thus, these additional elements do not apply or use the judicial exception in a manner that imposes a meaningful limit on the judicial exception such as providing improvements to any technology or technical field, or applying or using a judicial exception to affect a particular treatment or prophylaxis for a disease or medical condition. Thus, the answer to step 2A prong two is NO. The claims do NOT recite additional elements that integrate the judicial exception into a practical application. Therefore, the answer to step 2A of the 101 Subject Matter Eligibility Test is "Yes". The claims are directed to “Judicial Exception”.
Step 2(B): Does the claim recite additional elements that amount to significantly more than the judicial exception?
The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception. The only elements other than the naturally occurring blood brain barrier includes the iBBB is not cultured with retinoic acid in claim 8, and the cell ratio, cell concentration and the length of iBBB (claims 1, 10-11, 13-15, 37-38). These limitations are well-understood, routine, and conventional activity in the art because measuring cells concentration, cells numbers, cells length is routinely performed in the art. Thus, they do not, individually or in combination, provide sufficient inventive concept to render the claims patent eligible, and the claimed blood brain barrier is not integrated to amount to significantly more than the judicial exception. Further, only a blood brain barrier is examined as a product not a method with respect to their status as judicial exceptions. The phrase "in vitro” is considered; however, the claims are interpreted as a product-by-process that lack any structural feature(s) recited in the claims that would distinguish a blood brain barrier derived in vitro from pluripotent stem cells versus any other blood brain barrier (e.g., a naturally blood brain barrier).
In summary, the blood brain barrier as claimed is indistinguishable from those that exist in nature and there are no limitations that add any additional elements to the claimed blood brain barrier. The blood brain barrier has the same function as they do in nature and the fact that they may exist in an isolated (e.g., in vitro) form does not change the blood brain barrier in a significant or meaningful way to amount to more than the judicial exception. The claim as a whole does not amount to significantly more than the "product of nature" itself. Therefore, the answer to step 2B of the 101 Subject Matter Eligibility Test is "No". Thus, the claim does not qualify as patent eligible subject matter.
Response to Arguments
Applicant's arguments filed 07-14-2025 have been fully considered but they are not persuasive. The declaration of Dr. Joel Blanchard under 37 C.F.R. § 1.132 filed on 07-14-2025 is discussed below.
Applicant argue that the claimed populations of cells are not found in nature. In order to clarify the distinction, Applicant presents declaratory evidence establishing that the claimed cells are not naturally occurring and that the differences between the naturally occurring cells and the claimed cells is significant. The claim recites three cell types, human induced pluripotent-derived stem cell (iPSC) BEC, pericytes and astrocytes. Each of the cells is distinct from naturally occurring BEC, pericytes and astrocytes. A publication by two of the inventors provides a transcriptional analysis of iPSCs versus naturally occurring cells and the data presented in Extended Figure 1q highlights the differences between the cell types. (Blanchard, J.W., Bula, M., Davila-Velderrain, J. et al. Reconstruction of the human blood-brain barrier in vitro reveals a pathogenic mechanism of APOE4 in pericytes. Nat Med 26, 952-963 (2020). https://doi.org/10.1038/s41591-020-0886-4).
Response to Arguments:
Applicants argue that the three cell types, human induced pluripotent-derived stem cell (iPSC) BEC, pericytes and astrocytes are distinct from naturally occurring BEC, pericytes and astrocytes. However, while “human induced pluripotent-derived stem cell (iPSC)” is not naturally occurring cells, the claims recite iPSC-derived BEC, pericytes and astrocytes which are interpreted as product by process. The BEC cells, pericytes, astrocytes recited in the claims are structurally, functionally, physiologically similar to naturally occurring endothelial cells, pericytes, astrocytes. The claims do not specify how these cells are different from the naturally occurring cells. See MPEP 2113: Even though product-by-process claims are limited by and defined by the process, determination of patentability is based on the product itself. The patentability of a product does not depend on its method of production. If the product in the product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process." In re Thorpe, 777 F.2d 695, 698, 227 USPQ 964, 966 (Fed. Cir. 1985) (citations omitted). Second, the markedly different characteristics analysis is performed by comparing the nature-based product limitation in the claim to its naturally occurring counterpart to determine if it has markedly different characteristics from the counterpart. MPEP 2106.04(c) (II).
The Blanchard’s declaration and the remarks (page 11) cited Blanchard, et al. (Nat Med 26, 952-963 (2020). https://doi.org/10.1038/s41591-020-0886-4) to argue that in Extended Figure 1q highlights the differences between the cell types; however, Extended Figure 1q (see below) only has in-vivo human pericyte to compare with primary human pericytes not iPSC-derived pericytes (see Extended Figure 1q below). Primary human pericytes are not iPSC-derived pericytes : Blanchard et al teach “Isolation of mouse primary brain pericytes: Primary brain pericytes were isolated from 6–8-week-old APOE4KO mice as previously described” (see the method of Blanchard et al, second page). There is no comparison between in-vivo human BEC cells, pericytes, astrocytes with iPSC-derived BEC cells, pericytes, astrocytes under the same environmental conditions (for gene expression comparison using RNAseq). Additionally, Extended Figure 1q shows heatmap of global hierarchical clustering of transcriptomes (13,338 genes) which is RNAseq analysis. It is noted that gene expression with RNAseq data for exact same cell/clone under different environmental conditions (temperature, pH, medium condition) may also be different due to epigenetic regulations let alone comparison between in-vivo human pericytes with primary brain pericytes, iPSC-derived astrocyte, and there is no comparison for BEC cells as described in Figure 1q. Thus, the use of Figure 1q as evidence for iPSC-derived BEC cells, pericytes, astrocytes to be distinct from product of nature is not persuasive.
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Nevertheless, even if Applicant can provide evidences for the iPSC-derived BEC cells, pericytes, astrocytes are distinct from naturally occurring (in-vivo) BEC, pericytes and astrocytes, the claims should be amended to include those limitations to reflect the differences because the claims, as currently written, can encompass / be interpreted that the above cells with exact natural occurring characteristics (product of nature) can be produced from iPSC via another methods (Product by Process).
Maintained & modified & new-Claim Rejections - 35 USC § 102 - necessitated by amendments
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 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.
Claims 1-3 and 8 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Gastfriend et al (Curr Opin Biomed Eng. 2018 March ; 5: 6–12. doi:10.1016/j.cobme.2017.11.002.) as evidenced by Kataoka (Int. J. Mol. Sci. 2016, 17, 1306; doi:10.3390/ijms17081306) and Thomsen et al (J. Neurochem. (2017) 140, 741-754, doi: 10.1111/jnc.13747) and Campisi et al (Biomaterials 180 (2018) 117e129, Doi: 10.1016/j.biomaterials.2018.07.014.
Regarding to claim 1, Gastfriend et al teach “in vitro models of the BBB based on brain endothelial cells have been developed to facilitate screening drugs for BBB permeability …… they work in concert with other brain-resident cells such as neural progenitor cells, pericytes, astrocytes, ……” (Abstract), and “in vitro models of the NVU, including those derived from patient-specific induced pluripotent stem cells (iPSCs)” (Page 2, 2nd para.)
Gastfriend et al teach “Pericytes are also required for the maintenance of the BBB in adulthood, as demonstrated by pericyte-dependent endothelial gene expression, reduction in endothelial transcytosis, and astrocyte end-foot polarization” (Page 3, 1st para.) and “Recently developed multicellular BBB models have incorporated neural progenitor cells, pericytes, astrocytes, and neurons” (Page 3, 2nd para.), and “Transwell-based BBB models typically consist of endothelial cells cultured on an extracellular matrix-coated permeable membrane of a cell culture insert……Additionally, for permeability screening, molecules or cells can be added to the culture medium in the top (apical or “blood-side”) chamber” (Page 3, 3rd para.), and “Endothelial cells could be cultured on the bottom of the insert to facilitate a comparison of monolayer (two-dimensional, 2D) and collagen hydrogel (three-dimensional, 3D) astrocyte culture in the top chamber” (Page 4, 1st para.).
Regarding to claim 1-2, the expression of GFAP and AQP4 are inherently expressed in the end-feet (perivascular) of astrocytes as evidenced by Kataoka (Abstract, Page 2-8th para., Page 6-6th para., Page 7-1st para.) (For claims 1-2).
Regarding to claim 3, Likewise, the expression of basement membrane proteins such as Laminin subunit alpha-4 (Lama4) is inherent to brain capillary endothelial cell as evidenced by Thomsen et al (Page 750, Table 2) (For claim 3).
Further, as evidenced by Campisi et al, endothelial cells, pericytes and astrocytes can form 3D self-organized microvascular model of the human blood-brain barrier with microvessels (Title and abstract) that have lateral and transverse vessel diameter distributions are ranging between about 0-55 µm and 0-50 µm (see Figure 3F on page 123 and bridging last para on left column to right column on page 121 ) .
Regarding to claim 8, Gastfriend et al are silent about any step of culturing their iBBB in the presence of retinoic acid. Given that there’s no apparent evidence that Gastfriend et al performed the step that is precluded by the language of claim 8, it is reasonable to conclude that Gastfriend et al anticipates claim 8.
Thus, claims 1-3 and 8 are anticipated by Gastfriend et al.
Response to Arguments
Applicant's arguments filed 07-14-2025 have been fully considered but they are not persuasive.
1. Applicant argue that The BEC of Gastfriend et al are plated on an ECM coated membrane of a transwell device. The layer of cells disclosed therein does not form a vessel. Thus, the claimed element of the BEC vessel is missing from Gastfriend et al. (Remarks, page 6).
Response to Arguments:
Gastfriend et al teach Figure 1: The neurovascular unit (NVU) and multicellular BBB models. (a) Cross-section of a brain capillary, showing the organization of brain microvascular endothelial
cells (BMECs), pericytes, astrocytes, and neurons (see page 12). Capillary is blood vessel (BBB) made of brain microvascular endothelial cells (BMECs). It is noted that the instant claim 15 specifies the BEC vessel is a capillary size. Thus, Gastfriend et al teach BEC vessel.
2. Applicant argue that the claimed invention discloses pericytes positioned on an apical surface of the BEC vessel. The pericytes of Gastfriend et al are not positioned on an apical surface of the BEC vessel. Rather, the pericytes are positioned on a membrane surface and are not in physical contact with the BEC. A physical membrane separates the two cell types ….. Thus, Gastfriend et al does not disclose pericytes positioned on an apical membrane of the BEC (Remarks, page 6-7).
Response to Arguments:
It is noted that base claim 1 recites “human induced pluripotent-derived stem cell (iPSC) pericytes proximal to the BEC vessel on an apical surface ” (see instant claim 1). There is no requirement for pericytes to be in physical contact with the BEC but only to be proximal. Additionally, there is no definition for the term “proximal” in the instant disclosure.
Specifically, Gastfriend et al teach the BEC vessel on an apical surface: “transwell-based BBB models typically consist of endothelial cells cultured on an extracellular matrix-coated permeable membrane of a cell culture insert, …… molecules or cells can be added to the culture medium in the top (apical or “blood-side”) chamber …. The Transwell system can be readily adapted to multicellular BBB models, and offers flexibility in the arrangement of different cell types depending on the intended application of the model. NVU cell types can be cultured on the bottom of the well, allowing the exchange of soluble factors with BMECs cultured on the insert. For example, human pluripotent stem cell (hPSC)-derived BMECs have been co-cultured sequentially with primary human pericytes and human neural progenitor cell-derived neurons and astrocytes in this manner” (see page 3, last 2 para.). Gastfriend et al teach “ “Contact” co-culture of pericytes and BMECs or astrocytes and BMECs increased expression of the gene encoding the tight junction protein claudin-5, an effect not observed in analogous “non-contact” co-cultures” (Page 4, 1st para.). Thus, endothelial cells can be added on apical side and can be co-cultured with primary human pericytes (proximal).
3. Applicant argue that astrocytes dispersed throughout the 3D matrix, but rather on the bottom surface of the well with endothelial cells on the top surface (page 3, last 4 lines) or the top surface of the well with endothelial cells on the bottom surface (page 4 last two sentences of the first paragraph) (Remarks, page 7).
Response to Arguments:
There is no requirement that astrocytes cannot be dispersed on the bottom surface of the well with endothelial cells on the top surface (page 3, last 4 lines) or the top surface of the well with endothelial cells on the bottom surface (page 4 last two sentences of the first paragraph. The instant disclosure does not provide guidance for how astrocytes dispersed throughout the 3D matrix.
Moreover, Gastfriend et al teach the transwell system can be readily adapted to multicellular BBB models, and offers flexibility in the arrangement of different cell types depending on the intended application of the …...” (see page 3, 3rd para.). Gastfriend et al teach “ “Contact” co-culture of pericytes and BMECs or astrocytes and BMECs increased expression of the gene encoding the tight junction protein claudin-5, an effect not observed in analogous “non-contact” co-cultures” (Page 4, 1st para.). Gastfriend et al also teach Figure 1 showing different ways of cell arrangement :
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Thus, Gastfriend et al teach astrocytes dispersed throughout the 3D matrix.
4. Applicant argue that the claimed invention recites astrocytes dispersed throughout the 3D matrix. Gastfriend et al teaches that the astrocytes are cultured on the bottom of the cell. The Office refers to description of the microfluidic models in Gastfriend et al to support this element. However, the transwell and microfluidic models are separate models having completely different structure. One aspect of the microfluidic model should not be read into the transwell model. The element of astrocytes being dispersed throughout the 3D matrix in the context of the other claim elements is not disclosed in Gastfriend (Remarks, page 7).
Response to Arguments:
As explained above, Gastfriend et al teach the transwell system can be readily adapted to multicellular BBB models, and offers flexibility in the arrangement of different cell types depending on the intended application of the …...” (see page 3, 3rd para.). Gastfriend et al teach in transwell BBB model : “Contact” co-culture of pericytes and BMECs or astrocytes and BMECs increased expression of the gene encoding the tight junction protein claudin-5, an effect not observed in analogous “non-contact” co-cultures. Transwells are also amenable to alternative arrangements of cells.” (Page 4, 1st para)
Nevertheless, this is product claims which are interpreted as product by process. Applicant’s arguments regarding the process are not persuasive. Furthermore, the claims do not specify using transwell or microfluidic models, and Gastfriend et al teach both. Applicant provides opinion without evidence that one aspect of the microfluidic model should not be read into the transwell model. According to MPEP 716.01(c) (II), Arguments presented by the applicant cannot take the place of evidence in the record. In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965) and In re De Blauwe, 736 F.2d 699, 705, 222 USPQ 191, 196 (Fed. Cir. 1984). Examples of statements which are not evidence and which must be supported by an appropriate affidavit or declaration include statements regarding unexpected results, commercial success, solution of a long-felt need, inoperability of the prior art, invention before the date of the reference, and allegations that the author(s) of the prior art derived the disclosed subject matter from the inventor or at least one joint inventor.
New-Claim Rejections - 35 USC § 103 - necessitated by amendments
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.
Claims 1, 4, 5, 6, 8-9 13, 14, 15 are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al (Mol. Pharmaceutics 2016, 13, 895−906, DOI: 10.1021/acs.molpharmaceut.5b00805) in view of Campisi et al (Biomaterials 180 (2018) 117e129, Doi: 10.1016/j.biomaterials.2018.07.014) and as evidenced by Burkhart et al (Fluids Barriers CNS (2015) 12:19, DOI 10.1186/s12987-015-0015-9 ) and as evidenced by Urich et al (SCIENTIFIC REPORTS | 3 : 1500 | DOI: 10.1038/srep01500, 20 March 2013).
Regarding to claim 1, Wang et al teach “organization of endothelial cells, pericytes, and astrocytes into a 3d microfluidic in vitro model of the blood-brain barrier” (title). Wang et al teach “The endothelial cells lining the capillaries that supply the brain with oxygen and nutrients present a highly regulated barrier known as the blood-brain barrier (BBB)…….” (Page 895, left column). Wang et al teach coculturing mouse brain endothelial cells (b.End3) (proximal) with pericytes and astrocytes in layered microfluidic devices following the same organization of the neurovascular unit observed in vivo to develop a three-dimensional (3D) in vitro BBB model that successfully mimics the restrictive transport properties observed in vivo (Page 897 left column, last para.).
Wang et al teach Figure 1 (Page 896) and Figure 4A (page 901) showing “pericytes proximal to the BEC vessel on an apical surface” and “astrocytes dispersed throughout the 3D matrix, wherein a plurality of the astrocytes is proximal to the BEC vessel” see below:
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Wang et al do not specifically teach human iPSC brain endothelial cell, pericytes and astrocytes. Campisi et al cure the deficiency.
Campisi et al teach “3D self-organized microvascular model of the human blood-brain
barrier with endothelial cells, pericytes and astrocytes” (Title) and “This microfluidic system includes human induced pluripotent stem cell-derived endothelial cells, brain pericytes, and astrocytes as self-assembled vascular networks in fibrin gel” (Abstract).
Campisi et al teach “direct physical contacts were observed between AC end feet (Glial Fibrillary Acidic Protein, (GFAP), violet) and the abluminal surface of the brain vessels (CD31, green, Fig. 2f; Supplementary Figs. 6b and c).” (Page 121, left column, 4th para) (For the claimed: “have glial fibrillary acidic protein (GFAP)- positive projections into the perivascular space”).
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the rejected claims to combine the teachings of prior art to modify the method of Wang et al to use human iPSC brain endothelial cell, pericytes and astrocytes as taught by Campisi et al as instantly claimed, with a reasonable expectation of success. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to do so because Campisi et al stated that “these results indicate that the networks formed with all three cell types – iPSC-ECs + PCs + ACs – contained more stable and shorter vessel branches, with more circular cross-sections and smaller vessel diameters compared to the other conditions. These networks also exhibited more random interconnections and improved 3D structural orientation into the gel region: such structure is more similar to in vivo vessel morphology” (Page 121, right column, 4th para.). One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Campisi et al were successful in generation of 3D self-organized microvascular model of the human blood-brain barrier with endothelial cells, pericytes and astrocytes with detailed instructions and data.
Regarding to claim 1 the claimed : a human brain endothelial cell (BEC) vessel of about 5 to 10 microns in diameter and 5 to 50 microns in length, and claims 13, 14, 15: Campisi et al teach “The overall transverse diameter distributions were similar for all three conditions, ranging between 10 and 40 µm, and centered around 30 µm (Fig. 3def (ii))” (Page 121, left column, last para). Campisi et al also stated that “Our 3D BBB mVN model incorporating three cell-types (Figs. 1b and 7a) expressed both functional and morphological characteristics present in the human BBB, with stable and perfusable mVNs, comprising small lumens with circular cross-section comparable with in vivo human microcirculation (arterioles and venules 10-90 µm; capillaries 7-10 µm)” (Page 124, right column). It is noted that the instant disclosure of the claimed invention teaches that “The vessel has a size on the order of a capillary.” (see the instant specification on page 17 line 19-20), and Campisi et al teach their 3D BBB model “comparable with in vivo human microcirculation” such as capillaries; therefore, the claimed in vitro blood brain barrier (iBBB) and the 3D BBB model are similar.
Regarding to claim 4, Wang et al teach both the bi- and triculture models exhibited functional expression of the P-glycoprotein efflux pump, which increased with the increase in the number of days in culture. These results collectively indicate that the triculture model is a robust in vitro model of the BBB (Abstract).
Regarding to claims 5, 6, the above references teach triple culture conditions (culturing BCECs together with astrocytes and pericytes). Triple cell cultures can result in levels of PgP and ABCG2 expressed on the apical surface of the BEC that are 2-3 times greater than levels of PgP and ABCG2 expressed on BEC cultured alone or co-cultured with astrocytes as evidenced by Burkhart et al who teach gene expression analyses performed on brain capillary endothelial cells (BCECs) cultured in mono-, co- and triple culture conditions (culturing BCECs together with astrocytes and pericytes as co- or triple cell cultures), and to assess the expression profile of BCECs specific proteins, quantitative RT-PCR was performed with primers specific for ATP-binding cassette, sub-family G, member 2 (ABCG2) and ATP-binding cassette, sub-family B, member 1 (ABCB1), also known as p-glycoprotein (PgP) (Page 4, right column, 1st para.). Burkhart et al teach Fig. 2: Gene expression analysis of the hall mark proteins related to brain capillary endothelial cells (BCECs) showing levels of PgP and ABCG2 expressed on the BEC are 2-3 times greater than levels of PgP and ABCG2 expressed on BEC cultured alone
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Regarding to claim 8, Wang et al or Campisi et al do not teach culturing with retinoic acid. Thus, a person of ordinary skill in the art would not use retinoic acid for culturing.
Regarding to claim 9, the human pluripotent-derived positive endothelial cells are iPSC-derived CD144 cells as evidenced by Urich et al who teach the use of cell-specific markers such as CD31 and CD144 for human primary brain endothelial cells (hpBECs) (Fig. 1A) (Page 2, left column, 2nd para).
Claims 2-3, 7, 35-36 are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al (Mol. Pharmaceutics 2016, 13, 895−906, DOI: 10.1021/acs.molpharmaceut.5b00805) in view of Campisi et al (Biomaterials 180 (2018) 117e129, Doi: 10.1016/j.biomaterials.2018.07.014) and as evidenced by Burkhart et al (Fluids Barriers CNS (2015) 12:19, DOI 10.1186/s12987-015-0015-9 ) and as evidenced by Urich et al (SCIENTIFIC REPORTS | 3 : 1500 | DOI: 10.1038/srep01500, 20 March 2013) as applied to claims 1, 4, 5, 6, 8-9, 13, 14, 15 above, and further in view of DeStefano et al (Fluids Barriers CNS (2018) 15:32 , Doi:10.1186/s12987-018-0117-2).
The teachings of Wang et al and Campisi et al and Burkhart et al and Urich et al above are incorporated herein in their entirety.
The above references do not specifically teach astrocytes express Aquaporin 4 (AQP4) ; 3D matrix comprises laminin α4 (LAMA4) and wherein the 3D matrix is a hydrogel formed of Matrigel, Myogel and Cartigel, or a combination. DeStefano et al cure the deficiency.
Regarding to claim 2, DeStefano et al teach “Benchmarking in vitro tissue-engineered blood–brain barrier models” (title), and “Suggested benchmarks for incorporation of astrocytes into BBB models: …… High expression of astrocyte markers KiR4.1 and AQP4” (Page 8, left column, 3rd para).
Regarding to claim 3, DeStefano et al teach “…. the basement membrane in capillaries is typically characterized by the presence of laminin α4 …. ” (Page 4, left column, 1st para.).
Regarding to claim 7, DeS