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 .
Claim Status
Claims 1-15 are currently pending in this application.
Election/Restriction
Applicant’s election with traverse of Group I, claims 1-8, in the reply filed on Dec. 3, 2025 is acknowledged. The traversal is on the ground(s) that there would not be a serious or undue burden. This is not found persuasive because burden is immaterial to a finding of lack of unity of invention pursuant to Patent Cooperation Treaty (PCT) Rule 13.1. See MPEP 1893.03(d). The requirement is still deemed proper and is therefore made final. Claims 9-15 have been withdrawn for being drawn to non-elected subject matter, and claims 1-8 have been considered on the merits.
Specification Objections
The disclosure is objected to because of the following informalities:
The disclosure is objected to because it contains references to colors within the drawings; however, the drawings lack any color. For example, pg. 2-3 refers to “green” and “red” in FIGs 1-3.
The disclosure also has numerous typographical errors regarding the term “flask” or “flasks” misspelled as “flaks.”
Appropriate correction is required.
Claim Interpretation
In the claims, the term “neurosphere” is interpreted as a free-floating 3D cellular aggregate comprising neural precursor cells and being mostly spherical in shape, such as, e.g., wherein the standard deviation of diameter measurements at different axes is less than 5%. See instant pg. 5, lines 24-25; pg. 7, lines 6-9.
In the claims, the term “brain organoid” is interpreted as a mostly spherical 3D cellular aggregate comprising brain cell types and cytoarchitecture resembling human brain tissue(s). Brain organoids may also be referred to in the prior art as “neural organoids,” “cerebral organoids” or “mini-brains.” See instant pg. 5, line 25, to pg. 6, line 4; pg. 7, lines 6-9.
In the claims, the term “homogeneous” is defined in the instant application as meaning essentially all the neurospheres or brain organoids of a given plurality of such all have a similar diameter, shape, anatomy, cytoarchitecture, functionality and cell diversity. See instant pg. 6, lines 19-22; pg. 7, lines 1-25; pg. 2, lines 21-22.
In the claims, the term “compartment” is interpreted as encompassing any division of microwell sets within a single multi-well culture plate, e.g., a 256-well plate may comprise 2 to 128 compartments each having a plurality of wells.
In claims 1 and 2, the term “seeding” is defined to mean “applying a cell containing solution to a cell culture device in a manner that the cells are evenly distributed over the whole surface of the cell culture device.” See instant pg. 8, lines 12-14. In view of dependent claims 5 and 7, evenly distributed does not guarantee each compartment of the device receives the “same” number of cells, such as wherein the compartments have different dimensions.
In claims 1 and 2, the term cell “culture device” “comprising a plurality of compartments comprising a plurality of microwells” is interpreted as excluding 3D culture dishes lacking two or more compartments each comprising at least two microwells.
In claims 1 and 2, the phrase culturing said pluripotent cells is interpreted to mean any condition known in the art to develop neurospheres from pluripotent cells, such as known to induce differentiation of neuroectoderm and the formation of neurospheres. See instant pg. 10, lines 23-26.
In claims 1 and 2, the phrase “wherein the pluripotent cells do not form embryoid bodies” is interpreted to mean during none of the steps, or the method as a whole, is any embryoid body formed from a pluripotent cell.
In claim 2, the neurospheres formed in the first culturing step are considered “homogeneous” as the same exact steps are performed as in claim 1, which may be critical to ensure the resulting brain organoids are homogeneous.
In claim 2, the phrase culturing said neurospheres is interpreted to mean any condition known in the art to form brain organoids from neurospheres in a 3D culture dish, such as in a medium compatible for use with spinner flasks, bioreactors, orbital shakers, clinostats, and/or at 37° C. See instant pg. 11, lines 9-28.
In claim 7, the phrase “receives the same number of pluripotent cells” is interpreted as the “same” only statistically or approximately instead of meaning identical in absolute cell count, e.g., ~10,000 cells each. See instant pg. 8, lines 22-24; Example 5.
Claim Rejections - 35 USC § 112(a) - Written Description
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-8 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The claimed invention as a whole is not adequately described if the claims require essential or critical elements that are not adequately described in the specification and that is not conventional in the art as of applicant’s effective filing date. Possession may be shown by actual reduction to practice, clear depiction of the invention in a detailed drawing, or by describing the invention with sufficient relevant identifying characteristics such that a person skilled in the art would recognize that the inventor had possession of the claimed invention. Pfaff v. Wells Electronics, Inc., 48 USPQ2d 1641,1646 (1998).
In making a determination of whether the application complies with the written description requirement under 35 U.S.C. 112(a) or 35 U.S.C. 112, first paragraph, it is necessary to understand what Applicant is claiming and what Applicant has possession of.
The claims are directed to methods of producing homogeneous neurospheres or neurospheres that develop into homogeneous brain organoids via active step(s) of culturing. The claims are broad in that the culturing steps have no required conditions beyond those functionally defined as “allow the cells to form neurospheres” or “allow said neurosphere to develop into brain organoids.” The claims are also broad in that the culturing duration has no minimum and, thus, encompasses 1 second or less.
In view of dependent claims 5 and 7, claim 1 encompasses wherein the pluripotent cells are seeded in different numbers and/or volumes per microwell. Thus, the claims are also broad in that homogeneous neurospheres are forming despite each microwell being a different size or shape and/or seeded with a different number of cells and/or volume of cell solutions.
In analyzing whether the written description requirement is met for genus claims, it is first determined whether a representative number of species have been described. In the instant case, the specification fails to provide any other representative species for conditions and durations other than:
(1-a) direct differentiation of pluripotent cells for about 4-7 days at 37° C 5% CO2 in a serum-free medium composition comprising 5 pM SB431542 and 0.5 pM dorsomorphin (pg. 10, line 24, to pg. 11, line 4; pg. 12, lines 11-14) or
(1-b) culturing pluripotent cells for 5 days at 37° C, 5% CO2, in serum-free Neural induction medium (NIM) (Stemcell Technologies) supplemented with 10 pM of an unnamed ROCK inhibitor (Example 5; Fig. 1B-C); and
(2) forming brain organoids over about 30-50 days at 37° C 5% CO2 in a serum-free medium composition comprising 5 pM SB431542 and 0.5 pM dorsomorphin (pg. 11, lines 27-31; Example 2; FIG. 1B-D), as shown by mature neural markers, such as TLIJ1, DCX, Map2, PCP4, synapsin-1, and/or S100B (FIG. 2A, 2C, 3-5; pg. 3).
Thus, the instant application describes only one representative species of claim 2 and two representative species for claim 1, and in all these method species the neurosphere formation takes at least 4-5 days in a medium that is serum free comprising a small molecule inhibitor at least at a picomolar concentration.
The claims fail to recite, and the specification fails to disclose, the necessary structure/function/action-taking step(s) nexus between the combination of each of: culturing conditions and durations that predictably ensures the formation of neurospheres or brain organoids. The claims also fail to recite, and the specification also fails to disclose, the necessary structure/function/action-taking step(s) nexus between the combination of each of: the seeding step and varying the number of pluripotent cells seeded per microwell and/or varying the dimensions of each microwell that predictably ensures the formation of homogeneous neurospheres or brain organoids.
The prior art provides additional guidance as to specific methods validated to work under specific conditions. The prior art teaches methods of developing neurospheres from pluripotent stem cells (embryonic stem cells) without forming any embryoid body via suspension culturing for 4-7 days in a serum-free medium often comprising TGF-β and BMP receptors inhibitors (SB431542 and dorsomorphin) and/or a ROCK inhibitor (e.g., Y-27632) (Li et al., BMC Neurosci 10: 122 (2009) at Fig. 1; pg. 2, right col., 2nd para.; Birenboim et al., J Neurosci Methods 214: 9-14 (2013), IDS ref., at Fig. 3A-B, Abstract; pg. 10, left col., 2nd para.; Gabriel et al., J Vis Exp 122: 55372 (2017) at pg. 2-3, Protocol, Initiation of neuroectoderm, steps 3-6).
The prior art also teaches culturing neuronal stem cells into neurospheres in a 400-microwell dish wherein the microwell dimensions exert size constraints that homogenize neurosphere size and the cells were evenly distributed into identically sized microwells and any outside the wells were remoived (Yang et al., Journal of Industrial and Engineering Chemistry 74:148-57 (2019) at pg. 149, left col., last para, to right col., 1st para.; Fig. 1).
Regarding brain organoids, the prior art teaches methods of differentiating a plurality of homogeneous neurospheres from human induced pluripotent stem cells into brain organoids in 23 days without forming any embryoid bodies in a medium comprising 0.5 µM dorsomorphin and 5 µM SB431542, such as to model neurodevelopmental disorders (Gabriel et al., at Fig. 1; pg. 3, Protocol, Organoids in a Rotary Suspension Culture, steps 1-6; pg. 1).
Therefore, in view of the prior art, brain organoids can be formed in as short as a total of 23 days using a 14-day organoid culturing step in the presence of the TGF-β receptors and BMP receptors inhibitors dorsomorphin and SB431542. However the prior art is silent as to attaining homogeneity when microwell dimensions are varied in a single culture device or when the cell number seeded is not held constant.
In view of the instant specification, the current claims were not sufficiently described over the entire scope of the claims regarding culturing conditions, culturing durations, microwell shape and/or volume variability, or cell seeding variability. None of dependent claims 3-6 and 8 remedy all these deficiencies.
Because it is unpredictable based on the art that short culturing durations, e.g., less than 4 days or 1 day at 37° C, would generate neurospheres under any conditions, the applicant has not demonstrated possession of such methods. Similarly, it is unpredictable that neurospheres can be generated without a serum-free medium and TGF-β receptors and BMP receptors inhibitors, at least at a high femtomolar or low picomolar concentration.
Because it is unpredictable based on the art that short culturing durations, e.g., less than 14 days, would generate brain organoids under any conditions, the applicant has not demonstrated possession of such methods. Further, performing such a 14 day culturing duration was only achieved using picomolar amounts of TGF-β receptors and BMP receptors inhibitors, whereas the instant working embodiments did not validate brain organoid formation until at least day 30.
Finally, both the prior art and the instant working examples all relied on using a constant microwell size/shape and approximately the same number of cells seeded per microwell. Achieving homogeneity when deviating from these constants is unpredictable, especially when one microwell may have a single cell and a neighboring microwell may have 50,000 cells, or when one microwell has a flat bottom and a neighboring microwell has a conical bottom physically constraining maximum neurosphere size.
The skilled artisan could not rely upon the disclosure in the specification such that the specification would sufficiently describe that Applicant was in possession of the entire scope of culturing conditions and durations to ensure the predictable production of homogeneous neurospheres or brain organoids by the claimed methods.
35 USC § 112(a), Scope of Enablement
Claims 1-8 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because while enabling the method of claim 1 that further comprises seeding approximately the same number of cells per microwell, suspension culturing the cells for a sufficient duration (e.g., at least 4 days) under suitable cell physiological conditions (37° C, 5% CO2) in a serum-free medium comprising at least 0.5 picomolar amounts of SB431542 and dorsomorphin and wherein the microwells are at least wide enough to accommodate a single neurosphere (e.g, > 50 µm), wherein all the plurality of microwells have the same sized and shaped bottoms, and wherein the microwells lack any cell adherent coating or are nonadherent; the specification does not enable any person, skilled in the art to which it pertains, or with which it is most nearly connected to, to produce a plurality of homogeneous neurospheres by merely culturing the cells under any culture conditions for any duration.
Enablement is considered in view of the Wands factors (MPEP 2164.01 (a)). The court in Wands states that "Enablement is not precluded by the necessity for some experimentation such as routine screening. However, experimentation needed to practice the invention must not be undue experimentation. The key word is 'undue.' Not 'experimentation;" (Wands, 8 USPQ2d 104). Clearly, enablement of a claimed invention cannot be predicated on the basis of quantity of experimentation required to make or use the invention. "Whether undue experimentation is needed is not a single, simple factual determination, but rather is a conclusion reached by weighting many factual considerations." (Wands, 8 USPQ2d 1404).
The factors to be considered when determining whether there is sufficient evidence to support a determination that a disclosure does not satisfy the enablement requirement and whether any necessary experimentation required is “undue” include, but are not limited to:
(A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
Furthermore, the USPTO does not have laboratory facilities to test if an invention will function as claimed when working examples are not disclosed in the specification. Therefore, enablement issues are raised and discussed based on the state of knowledge pertinent to an art at the time of the invention. And thus, skepticism raised in the enablement rejections are those raised in the art by artisans of expertise.
All of the Wands factors have been considered with regard to the instant claims, with the most relevant factors discussed below.
Breadth of claims
The claims are directed to methods of producing homogeneous neurospheres via an active step of culturing. Claim 2 is directed methods of producing homogeneous brain organoids via two active culturing steps, one for pluripotent cells to form neurospheres and the other for neurospheres to form brain organoids. The claims are broad in that the culturing steps have no required conditions beyond those functionally defined as “allow the cells to form neurospheres” or “allow said neurosphere to develop into brain organoids.” The claims are also broad in that the culturing duration has no minimum.
In view of dependent claims 5 and 7, claim 1 encompasses wherein the pluripotent cells are seeded in different numbers and/or volumes per microwell. Thus, the claims are also broad in that homogeneous neurospheres are forming despite each microwell being a different size or shape and/or seeded with a different number of cells and/or volume of cell solutions.
The state of the art:
The prior art teaches methods of developing neurospheres from pluripotent stem cells (embryonic stem cells) without forming any embryoid body via suspension culturing for 4-7 days in a serum-free medium, typically comprising TGF-β receptors and BMP receptors inhibitors (10 µM SB431542 and 2 µM dorsomorphin) and/or a ROCK inhibitor (10 µM Y-27632) (Li et al., BMC Neurosci 10: 122 (2009) at Fig. 1; pg. 2, right col., 2nd para.; Birenboim et al., J Neurosci Methods 214: 9-14 (2013), IDS ref., at Fig. 3A-B, Abstract; pg. 10, left col., 2nd para.; Gabriel et al., J Vis Exp 122: 55372 (2017) at pg. 2-3, Protocol, Initiation of neuroectoderm, steps 3-6). Regarding brain organoids, the prior art teaches methods of differentiating a plurality of homogeneous neurospheres from human induced pluripotent stem cells into brain organoids in 23 days without forming any embryoid bodies in a medium comprising TGF-β receptors and BMP receptors inhibitors (0.5 µM dorsomorphin and 5 µM SB431542) (Gabriel et al., at Fig. 1; pg. 3, Protocol, Organoids in a Rotary Suspension Culture, steps 1-6; pg. 1).
Thus, the prior art is silent as to methods of culturing pluripotent stem cells into neurospheres in less than 4 days at 37 °C or into brain organoids in less than 23 days at 37 °C. The prior art is silent as to methods of culturing pluripotent stem cells into neurospheres without using serum free media.
The prior art is also silent as to methods of achieving neurosphere homogeneity without using a constant microwell size/dimension and without seeding an approximately constant number of stem cells per microwell. This can be challenge even when using a constant cell or cellular aggregate number (Yang et al., ACS Omega 4: 18136-46 (2019) at pg. 18136, right col., 1st para.).
Thus, these aspects of using must be shown to a reasonable extent so that one of the ordinary skills in the art would be able to practice the invention without any undue or unreasonable burden being on such artisan. However applicant is invited to furnish evidence to the contrary.
The amount of direction and guidance as well as any working examples provided:
The instant specification provides a single working example of methods of differentiating human iPSCs into neurospheres via culturing for 5 days at 37° C in Neural induction medium (NIM) (Stemcell Technologies) supplemented with 10 pM ROCK inhibitor (Example 5; Fig. 1B-C).
The instant specification provides a single working example of methods of forming brain organoids from neurospheres via culturing for 30-50 days at 37° C and 5% CO2 in a serum-free medium composition comprising 1:1 DMEM/F12 and Neural Basal medium, supplemented with N2 supplement (1:200), B-27 supplement w/o vitamin A (1:100), 0.05 mM MEM non-essential amino acids, L-glutamine (1:100), Pen-strep (100 pg/mL each), insulin (1.6 g/L), 0.05 mM β-mercaptoethanol, 5 pM SB431542 and 0.5 pM Dorsomorphin (Example 2; FIG. 1B-D).
Nowhere does the instant specification show how to perform the method of claim 1 in less than 4 days at 37 C 5% CO2 to produce neurospheres or without either a ROCK inhibitor or TGFβ/BMP receptors inhibitors. Nowhere does the instant specification show how to perform the method of claim 2 in less than 30 days at 37 C 5% CO2 to produce brain organoids or without TGFβ/BMP receptors inhibitors. Thus, there is no evidence in the instant application or the prior art that the full scope of the claims would predictably result in neurospheres or brain organoids as recited in the claims.
Nowhere does the instant specification show how to perform the method of claim 1 or claim 2 using varied microwell dimensions or varied numbers of cells per microwell. Thus, there is no evidence in the instant application or the prior art that the full scope of the claims would predictably result in homogeneous neurospheres or brain organoids as recited in the claims. To the contrary, the instant specification notes that the bigger the surface of each compartment, the more difficult it is to evenly distribute the cells so as to ensure an equal/essentially the same number of cells per microwell (pg. 9, line 25, to pg. 10, line 2).
Undue experimentation would be required to fill these gaps and unpredictability. Undue experimentation is required to shorten culturing times to hours or use the inhibitors at low femtomolar concentrations. Undue experimentation is required to ensure the requisite homogeneity when microwell dimensions are not constant and/or the amount of cells seeded are not constant. None of dependent claims 3-8 remedy all these deficiencies.
In summary, the claims are rejected under 35 U.S.C. 112(a) because the specification does not reasonably provide enablement to a person skilled in the art to produce homogeneous neurospheres from any culture duration and conditions. Additionally, as defined by the application the neurospheres must be free-floating but the claims do not ensure this aspect, such as by noting the surface of the microwell are non-adherent or the culturing is in suspension or the cells are not embedded in a hydrogel.
Further, claims 2-3 are rejected under 35 U.S.C. 112(a) because the specification does not reasonably provide enablement to a person skilled in the art to produce brain organoids from any culture durations and conditions. Moreover the specification does not reasonably provide enablement to a person skilled in the art to produce neurospheres or brain organoids in a way that ensures homogeneity over the full scope of the claims.
Given the lack of working examples, the limited guidance provided in the specification, the lack of guidance in the prior art, and the broad scope of the claims, undue and/or unreasonable experimentation would have been required for one skilled in the art to produce the recited results over the full scope of the claimed methods using these broadly and sparsely recited method steps.
Claim Rejections - 35 USC § 112(b)
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 1-8 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.
The claims are interpreted as set forth in a previous section.
In the specification, the term “neurospheres” is defined as meaning free-floating (pg. 5, lines 24-25), which is contrary to its ordinary meaning (e.g., spontaneously or cultured on certain types of polystyrene) and as described in the instant specification (pg. 11, lines 16-24, “to prevent adhesion of the neurospheres” or “detaching the neurospheres from the microwells”). If the applicant acts as her own lexicographer to specifically define a term of a claim contrary to its ordinary meaning, the written description must clearly redefine the claim term and set forth the uncommon definition so as to put one reasonably skilled in the art on notice that the applicant intended to so redefine that claim term. Process Control Corp. v. HydReclaim Corp., 190 F.3d 1350, 1357, 52 USPQ2d 1029, 1033 (Fed. Cir. 1999). In the instant case, there is no such redefinition, and, thus, the term “neurospheres” as used in the claims is indefinite.
Claim 1 recites the term “said pluripotent stem cells”, which lacks sufficient antecedent basis in the claim. Claims 4-8 are included in this rejection for depending from indefinite claim 1.
Claims 1 and 2 each recites the term “homogeneous” regarding either a plurality of neurospheres or brain organoids, respectively. As the term “homogeneous” is defined in the instant specification using the relative terms “essentially” and “similar”, the term homogeneous is indefinite because the claims, specification and prior art all fail to provide a standard for ascertaining the requisite degree for “essentially all” and “similarity” of diameter, shape, anatomy, cytoarchitecture, functionality, and cell diversity.” “When a term of degree is used in the claim, the examiner should determine whether the specification provides some standard for measuring that degree” and if the specification does not provide some standard for measuring that degree, “a determination must be made as to whether one of ordinary skill in the art could nevertheless ascertain the scope of the claim (e.g., a standard that is recognized in the art for measuring the meaning of the term of degree). See MPEP 2173.05(b) (I). As “essentially” or “similar” and functionality or diversity can mean a variety of deviations from identical, a person of ordinary skill in the art would not be appraised of the metes and bounds of the scope of the claim term.
Claims 1 and 2 each recites the term “seeding” whereby the cells are evenly distributed over the whole surface of the cell culture device as defined in the instant application; however a person of ordinary skill in the art reading the claim would not understand the metes and bounds of the whole surface of the device “comprising a plurality of compartments comprising a plurality of microwells.” It is ambiguous as to whether the cells are evenly distributed over or within each microwell and/or over or within each compartment or merely over the entire device “surface” regardless of the features termed “compartments” and/or “microwells.” Claims 3-8 are included in this rejection for depending from indefinite claim 1 or 2.
Claim 4 recites the relative term “essentially free” regarding flat surfaces. A person of ordinary skill in the art reading the claim would not understand the metes and bounds of the relative term “essentially” because neither the claim nor the instant application provides any definition or standard. See MPEP 2173.05(b). Thus, one of ordinary skill in the art would not be reasonably appraised of the scope of this claim limitation. Furthermore, even wherein the side walls of the microwells have rounded edges, there is still an infinitesimally small flat region (Euclidean plane) at the top of each microwell wall as part of the compartment bottom regardless of geometry, which could have a surface area of at least as wide as a pluripotent cell depending on the wall size.
Claim 6 recites a shape that is both pyramidal and has “a rounded tip and rounded edges,” which is incoherent and ambiguous as pyramid shapes are not ordinarily rounded and the descriptive language is closer to describing an inverted cone shape instead of a pyramid shape (see FIG. 1A). If the applicant acts as her own lexicographer to specifically define a term of a claim contrary to its ordinary meaning, the written description must clearly redefine the claim term and set forth the uncommon definition so as to put one reasonably skilled in the art on notice that the applicant intended to so redefine that claim term.
Claim 7 recites the “same number” which is determined statistically as noted in a previous section. Claim 7 is indefinite because neither the claim nor the instant specification explains how the “same number” is achieved by statistics beyond mentioning preferred embodiments using a figure-8 movement during seeding as the seeded cells are settling into the microwells (pg. 8, lines 14-17; Example 5). Furthermore, the specification seems to mention that prior art methods cannot achieve this due to human pipetting errors (id.)
Claim 8 recites the phrase “preferably human induced pluripotent stem cells,” which is ambiguous and unclear as to whether the language after preferably is a limitation or optional feature.
Claim Rejections - 35 USC § 112(d)
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 3 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 3 attempts to further limit the scope of claim 2 to wherein the brain organoids develop within less than 50 days; however there is no recitation of any limitation to the active steps of claim 2. As nothing about the characteristic recited in claim 3 implies any additional feature to the method of claim 2, then claim 3 appears to merely recite a natural result or an inherent property of the organoids formed by the performance of all active method steps of claim 2. Thus, absent evidence to the contrary, the result of performing the method of claim 2 inherently produces brain organoids with such capability. If it does not, then claim 3 is indefinite for failing to adequately describe all essential feature(s) to ensure production of the brain organoids recited therein.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1, 5, and 7-8 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Birenboim (Birenboim et al., J Neurosci Methods 214: 9-14 (2013), IDS ref.).
Regarding claim 1, Birenboim discloses culturing pluripotent stem cells (hESC) for 4 days in a medium (Medium I comprising GMEM, 10 µM SB431542, 2 µM dorsomorphin, and 10 µM ROCK inhibitor) in a cell culture device (micro multiwell petri dishes made using agarose and rubber micromolds) comprising compartments with microwells to produce hundreds of neurospheres of homogeneous size without forming any embryoid bodies (Fig. 3A-B; Abstract; pg. 10, left col., 2nd para.; pg. 10, right col., para. 2, to pg. 12, left col., 1st para.; pg. 12, right col., last para.).
Regarding claim 5, Birenboim discloses the device comprises 256 similarly sized microwells due to the shape of the mold used from MicroTissues Inc. (pg. 10, left col., last para.).
Regarding claim 7, Birenboim discloses seeding equivalent numbers of cells on average per micro well over 256 microwells total (~ 1,172 cells per well distributed from a 300,000 cell stock solution) (pg. 10, right col., 2nd para.).
Regarding claim 8, Birenboim discloses wherein the pluripotent cells (hESC) are human cells (Abstract).
Thus, Birenboim anticipates the claimed invention.
Claims 1-3, 5, and 7-8 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Gabriel (Gabriel et al., J Vis Exp 122: 55372 (2017)).
Regarding claim 1, Gabriel discloses a method of producing a plurality of homogeneous neurospheres from pluripotent cells via seeding and culturing in media (neural induction medium comprising Y-27632) for 5 days in a cell culture device comprising compartments comprising microwells without forming any embryoid bodies (non-adherent, v-bottom, 96-microwell plate) (pg. 2-3, Protocol, Initiation of neuroectoderm, steps 3-6). Regarding claims 5 and 7, Gabriel discloses wherein each microwell contains 100 µL comprising 45,000 pluripotent cells (id.). Regarding claim 8, Gabriel discloses wherein the pluripotent cells are human iPSC (id.).
Regarding claim 2, Gabriel discloses a method of producing a plurality of homogeneous brain organoids in 23 days from neurospheres derived from pluripotent cells without forming any embryoid bodies as described above and wherein the neurospheres are transferred into 3D culture dishes (spinner flasks) and cultured for 14 days into brain organoids in a medium comprising 0.5 µM dorsomorphin and 5 µM SB431542 (Fig. 1; pg. 3, Protocol, Organoids in a Rotary Suspension Culture, steps 1-6). Regarding claim 3, Gabriel discloses brain organoid formation at day 14, e.g., comprising p-Vim and Arl13b staining ventricular zones (VZ) with palisade-like nuclei and primitive cortical plates with TUJ1+ neurons (Fig. 1E-F). Thus, Gabriel anticipates the claimed invention.
Claims 1-3, 5, and 7-8 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Goranci (Goranci-Buzhala et al., Cell Rep 31: 107738 (2020), published 2020 Jun 9).
Regarding claim 1, Goranci discloses a method of producing a plurality of homogeneous neurospheres from pluripotent cells via seeding and culturing in media (neural induction medium (NIM) comprising Y-27632 and glioma stem cells) for 5 days in a cell culture device comprising compartments comprising microwells without forming any embryoid bodies (CellCarrier Spheroid ULA 96-well microplates) (Fig. 1A; Fig. 2A; pg. e3, 3rd para.). Regarding claims 5 and 7, Goranci discloses wherein each microwell contains 100 µL comprising 35,000 pluripotent cells (id.). Regarding claim 8, Goranci discloses wherein the pluripotent cells are human iPSC (pg. e3, para. 1 and 3).
Regarding claim 2, Goranci discloses a method of producing a plurality of homogeneous brain organoids from neurospheres derived from pluripotent cells without forming any embryoid bodies as described above and wherein the neurospheres are transferred into 3D culture dishes (spinner flasks) and cultured for 10-60 days into brain organoids in a medium comprising 2.5 µM dorsomorphin and 25 µM SB-431542 (Fig. 1A; Fig. 2A; pg. e3, para. 3-6). Regarding claim 3, Goranci discloses brain organoid formation at day 30 or less, e.g., 20-day old or 40-day old organoids expressing Synapsin 1, MAP2, and NeuN and comprising ventricular zones and cortical plates (Fig. 3; pg. 2, right col., 1st para.; pg. 4 left col., last para., to right col., 1st para.). Thus, Goranci anticipates the claimed invention.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1-3, 5, and 7-8 are rejected under 35 U.S.C. 103 as being unpatentable over Birenboim (Birenboim et al., J Neurosci Methods 214: 9-14 (2013), IDS ref.) in view of Durens (Durens et al., J Neurosci Methods 335:108627 (2020) Epub 2020 Feb 4; IDS ref.).
As set forth fully above, Birenboim anticipates claims 1, 5, and 7-8 and thus, Birenboim renders obvious the subject matter of claims 1, 5, and 7-8.
Birenboim teaches transferring the smooth homogeneous neurospheres to 3D culture dishes (3 cm plastic dishes) for suspension culture in a neurosphere medium (NSPM comprising DMEM:F12 and FGF2) (Fig. 3B) and then after, e.g., 20 days, plated for adherent culture in NSPM further comprising the neurotrophins NGF, BDNF and NT3 to drive terminal neural differentiation for at least 7 days without forming embryoid bodies (Fig. 3C-G; pg. 10, right col., last para., to pg. 11, left col., 2nd para.). The resulting cellular aggregates comprised mostly neurons, as shown by NeuN and neurofilament-H staining and at greater efficiency than methods generating embryoid bodies, 92% vs 5 % (pg. 11, right col., last para., to pg. 12, left col., 1st para.).
Regarding claim 2, Birenboim does not expressly teach culturing the neurospheres in 3D culture dishes into brain organoids.
However Durens teaches a multi-step protocol for 3D culturing brain organoids developed from human pluripotent cells via an embryoid body intermediate (SFEB) taking 54 days or at least 45-60 cell doublings (DIV45-DIV60) using 3D culture dishes comprising inserts and the factors BDNF, NT3, β-NGF, and FGF, and later DAPT, such as to obtain brain organoids for use in cell based assays to investigate development, diseases, and for drug screening of artificial brain tissues in vitro (Fig. 1, pg. 3, left col., para. 2-3; Abstract; pg. 2).
It would have been prima facie obvious to one of ordinary skill in the art at the effective time of filing to modify the method of Durens of making brain organoids using the neurospheres generated by the method of Birenboim in lieu of the SFEBs in order to obtain human brain organoids more quickly and reduce variability introduced by using SFEBs. One of ordinary skill in the art would be motivated to make brain organoids because Duren teaches methods of using human brain organoids for high throughput screening for disease modeling and drug discovery and the method of Birenboim provides a method of making more homogeneous neurospheres in a shorter timeframe (4-7 days instead of 14 days to early embryoid bodies) with many more neural progenitor cells than a method dependent on an embryoid body intermediate.
Regarding claim 3, modifying Durens protocol to start with neurospheres of Birenboim shortens the entire protocol from 54 days to less than 50 days, i.e., at least 7-10 days shorter.
Thus, the claimed invention as a whole is prima facie obvious in the absence of evidence to the contrary.
Claims 1-8 are rejected under 35 U.S.C. 103 as being unpatentable over Birenboim in view of Durens as applied above, and further in view of Kugelmeier (US20120149051A1, IDS ref.).
Regarding claim 4, the combination of Birenboim and Durens does not teach wherein each compartment bottom is free of flat surfaces because it is completely covered with microwell features.
However Kugelmeier teaches a cell culture device specially designed for homogeneous stem cell aggregation comprising compartments comprising inverted pyramidal (or conical) microwells (e.g., 4 compartments each comprising cavities of about 100 µm by 350 µm) with regular dimensions wherein the bottom of the compartments are essentially free of flat surfaces to avoid capturing cells outside of the wells by packing the microwells as close together as possible in each compartment and because no side walls are oriented at 90°, instead being angled (FIG. 1C-D, 2C-D, 5C-D) ([0034]; [0044]; [0050], Abstract; [0010]; [0016], [0041], [0046]).
It would have been prima facie obvious to one of ordinary skill in the art at the effective time of filing to perform the method of Birenboim and Durens using a predesigned cell culture device taught by Kugelmeier for more homogeneous stem cell aggregation instead of using the molded agarose setup taught by Birenboim. One of ordinary skill in the art would be motivated to use a culture device of Kugelmeier shaped to slide cells down to the bottom of the wells and more naturally aggregate stem cells that promotes homogeneity by avoiding single cells resting on surfaces outside of the microwells upon seeding ([0010]; [0016], [0035], [0041], [0046]).
Regarding claim 6, the combination of Birenboim and Durens does not teach wherein each microwell has a pyramid shape with a rounded tip and rounded edges between microwell side walls.
However Kugelmeier teaches a cell culture device for stem cell aggregation comprising compartments comprising inverted pyramidal microwells (quadrangular or triangular) with rounded bottoms and no side walls oriented at 90°, instead being angled with only rounded edges ( FIG. 1D-E, 2D-E, 5D-E) ([0034]; [0036], [0044]; [0050], Abstract).
It would have been prima facie obvious to one of ordinary skill in the art at the effective time of filing to perform the method of Birenboim and Durens using a cell culture device taught by Kugelmeier with microwell dimensions designed for more homogeneous stem cell aggregation. One of ordinary skill in the art would be motivated to use a culture device of Kugelmeier shaped to slide cells down to the bottom of the wells and more naturally aggregate stem cells in the rounded microwell bottoms in a dimensionally controlled manner ([0035]-[0036], [0041]). Thus, the claimed invention as a whole is prima facie obvious in the absence of evidence to the contrary.
Claims 1-8 are rejected under 35 U.S.C. 103 as being unpatentable over Gabriel (Gabriel et al., J Vis Exp 122: 55372 (2017)) in view of Kugelmeier (US20120149051A1, IDS ref.).
As set forth fully above, Gabriel anticipates claims 1-3, 5 and 7-8, and thus, Gabriel renders obvious the subject matter of claims 1-3, 5, and 7-8.
Regarding claim 4, the Gabriel does not teach wherein each compartment bottom is free of flat surfaces because it is completely covered with microwell features.
However Kugelmeier teaches a cell culture device specially designed for homogeneous stem cell aggregation comprising compartments comprising inverted pyramidal (or conical) microwells (e.g., 4 compartments each comprising cavities of about 100 µm by 350 µm) with regular dimensions wherein the bottom of the compartments are essentially free of flat surfaces to avoid capturing cells outside of the wells by packing the microwells as close together as possible in each compartment and because no side walls are oriented at 90°, instead being angled (FIG. 1C-D, 2C-D, 5C-D) ([0034]; [0044]; [0050], Abstract; [0010]; [0016], [0041], [0046]).
It would have been prima facie obvious to one of ordinary skill in the art at the effective time of filing to perform the method of Gabriel using a predesigned cell culture device taught by Kugelmeier for more homogeneous stem cell aggregation. One of ordinary skill in the art would be motivated to use a culture device of Kugelmeier shaped to slide cells down to the bottom of the wells and more naturally aggregate stem cells that promotes homogeneity by avoiding single cells resting on surfaces outside of the microwells upon seeding ([0010]; [0016], [0035], [0041], [0046]).
Regarding claim 6, Gabriel does not teach wherein each microwell has a pyramid shape with a rounded tip and rounded edges between microwell side walls.
However Kugelmeier teaches a cell culture device for stem cell aggregation comprising compartments comprising inverted pyramidal microwells (quadrangular or triangular) with rounded bottoms and no side walls oriented at 90°, instead being angled with only rounded edges ( FIG. 1D-E, 2D-E, 5D-E) ([0034]; [0036], [0044]; [0050], Abstract).
It would have been prima facie obvious to one of ordinary skill in the art at the effective time of filing to perform the method of Gabriel using a cell culture device taught by Kugelmeier with microwell dimensions designed for more homogeneous stem cell aggregation. One of ordinary skill in the art would be motivated to use a culture device of Kugelmeier shaped to slide cells down to the bottom of the wells and more naturally aggregate stem cells in the rounded microwell bottoms in a dimensionally controlled manner ([0035]-[0036], [0041]). Thus, the claimed invention as a whole is prima facie obvious in the absence of evidence to the contrary.
Conclusion
No claim is allowed.
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/ERIC J ROGERS/Examiner, Art Unit 1638
/KEVIN K HILL/Primary Examiner, Art Unit 1638