Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant has elected group I, drawn to a method of producing neural organoids which does not comprise embedding of cells into a gel and the organoids obtained by the method and multiwell plates comprising said organoids (claims 1-6).
Claims 7-15 are pending but withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a non-elected invention, there being no allowable generic or linking claim.
Election of Applicant’s invention(s) was made with traverse in the reply filed
on July 1, 2024.
DETAILED ACTION
The amended claims filed on December 3, 2025, have been acknowledged. Claims 3 and 11 were cancelled. Claims 2 and 4-5 were amended. In light of the Applicant’s elected invention, claims 7-10 and 12-15 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Claims 1-2 and 4-6 are pending and examined on the merits.
Priority
Acknowledgment is made of Applicant’s claim for foreign priority under 35 U.S.C. 119(a)-(d).The applicant claims foreign priority from EP18193698.0 filed on September 11, 2018. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55, received March 10, 2021. Claims 1-2 and 4-6 find support in foreign application EP18193698.0 filed on September 11, 2018.
Declaration under 37 CFR 1.132
The declaration under 37 CFR 1.132 filed by Dr. Jan Markus Bruder on December 3, 2025, has been considered and is persuasive regarding the prior rejections of record for the instant claims based upon 35 U.S.C 102/103 as set forth in the previous Office action mailed on September 4, 2025 for the following reasons:
Dr. Bruder’s Declaration is convincing regarding how one of ordinary skill in the art would not be considered neuronal tissue-specific (Items 5-6 and 13 of the Declaration).
As such, these rejections have been withdrawn.
Regarding Dr. Bruder’s assertions about the size distribution of the spheroids of Sloan and Pasca, these arguments are considered unpersuasive. As identified by Dr. Bruder, Sloan specifically references the spheroids of Pasca when discussing what results can be expected using the protocol disclosed therein and that spheroids should be uniform in distribution across the same hPSC line (item 8). In item 9, Dr. Bruder cites to a part of Pacsa discussing the size distribution of the spheroids of Pasca. From Pasca: The hCSs grew in size to more than 300 um in diameter by 2 weeks of culture and reached up to 4 mm in diameter by 2.5 months (4.2 ±0.3 mm, mean± s.e.m., n = 16 hCSs from 4 differentiated hiPSC lines). It is worth noting that Pasca measured and combined the results of 16 hCSs from 4 different hiPSC lines whereas Sloan disclosed that there should be uniform distribution across the same hiPSC line. As such, the calculated standard deviation from the Declaration is not representative of what would be found if only comparing spheroids from the same cell line. As an example, Figure 1d of Pasca provides an image of a collection of 15 spheroids at day 26 of culture. Based on an analysis of the image (see below), the organoids have a diameter ranging from ~1300 μm to ~1800 μm and a calculated standard deviation from the mean of ~10%. This is also in line with the analysis performed in the previous Non-Final Office action mailed on September 4, 2025, of Figure 3 of Sloan that showed two organoids have a diameter of ~1500 μm and ~1750 μm that equates to a standard deviation from the mean of ~8%.
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Annotated Figure 1D of Pasca
Therefore, the method of Sloan is considered to generate spheroids that fall within the homogenous size distribution requirements of a standard deviation of less than 20% of the mean.
Dr. Bruder further argues that their method results in unexpected results. First, instead of random, mitotic neural rosettes like those of Sloan and Jo, the claimed organoids self-organize into a reproducible, globally radially symmetric structure with concentric zones. This ensures a predictable biological state across samples. Second, the claimed methods achieve unprecedented homogeneity (Item 15).
As identified above, the organoids of Pasca are able to generate a homogenous size between organoids with a ~10% standard deviation and Pasca and Jo show that their organoids consist of concentric VZ, Deep layers, and superficial zones in Pasca in concentric VZ, IZ, and MZ zones in Jo. Therefore, this would not be unexpected.
Regarding the new rejections discussed below, when all of the evidence is considered, the totality of the rebuttal evidence of nonobviousness fails to outweigh the evidence of obviousness regarding the newly recited rejections of record.
Claim Objections
Claims 1-2 and 4 are objected to because of the following informalities:
Claims 1-2 and 4 use the terms homogeneous and homogenous interchangeably. Choose one spelling for the terms for each claim.
Appropriate correction is required.
Withdrawn Claim Rejections - 35 USC § 112
The prior rejection of claim 2 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 is withdrawn in light of Applicant’s amendments to claim 2 to remove the term preferably and fix the antecedent basis issue.
New Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 4-5 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 term “comprised mostly of” in claim 4 is a relative term which renders the claim indefinite. The term “comprised mostly of” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. It is not clear what amount of neural precursor cells are required to read on primarily comprising. It is unclear whether 50%, 75%, or 90% of the core comprising neural precursor cells would reach primarily comprising. Applicant is recommended to further define the term (such as by stating wherein comprised mostly of refers to greater than 50%) or to identify a specific percentage of the core that is made up of neural precursors.
Claims 5 is also rejected because of their dependence on claim 4.
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 4 recites the broad recitation one neural organoid, and the claim also recites more than one neural organoids which is the narrower statement of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims. Subclaims a-b identify features that can be found in a single neural organoid or more than one neural organoid. However, subclaim c requires that the neural organoids are homogenous in terms of size. In order for there to be homogeneity, there would necessarily need to be more than one neural organoid. Therefore, it is unclear whether this subclaim is a limitation for a single neural organoid or only a plurality of neural organoids.
Claims 5 is also rejected because of their dependence on claim 4.
Furthermore, claim 5 has a similar issue as claim 5 requires a multiwell plate with a plurality of wells with each well comprising a single neural organoid selected from the one or more neural organoids of claim 4. However, if there is only a single neural organoid, it is not clear how each well can comprise a single neural organoid as the single neural organoid of claim 4 can only reside in one well. If there is only one neural organoid, even in a 2 well plate, there would be an empty well which would not fulfill the requirements of claim 5. Therefore, claim 5 is also considered indefinite.
Withdrawn Claim Rejections - 35 USC § 102
The prior rejection of claim 1 under 35 U.S.C. 102(a)(1) as being anticipated by Sloan et al. (Nature Protocols 13: 2062-2085. 2018. Published September 10, 2018; referenced in IDS) is withdrawn in light of Applicant’s persuasive arguments against considering pluripotent stem cells as a neuronal tissue-specific precursor cell.
The prior rejection of claim 4 under 35 U.S.C. 102(a)(1) as being anticipated by Jo et al. (Cell Stem Cell 19: 248-257. 2016; referenced in IDS) is withdrawn in light of Applicant’s persuasive arguments against considering pluripotent stem cells as a neuronal tissue-specific precursor cell.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1-2 and 6 are rejected under 35 U.S.C. 103 as being unpatentable over Pasca et al. (Nature Methods 12: 671-678. 2015; referenced in IDS) and further in view of Reinhardt et al. (PLoS ONE 8: 1-18. 2013; cited in IDS) and Monzel et al. (Stem Cell Reports 8: 1144-1154. 2017; referenced in IDS). This a new rejection made in response to Applicant’s compelling arguments against the prior rejection based on Sloan. Any aspect of Applicant’s traversal that is relevant to the rejection as newly written is addressed below.
Regarding claims 1 and 6, Pasca teaches a method of generating human cortical spheroids (hCSs) (i.e. neural organoids) (Figure 1) from iPSCs, comprising:
a) To generate suspended cellular aggregates of pluripotent cells, they cultured seven hiPSC lines derived from five subjects in a dish (i.e. seeded iPSCs in a container) and successfully generated neural organoids from each clone.
b-i) Rather than using single-cell suspensions, they enzymatically detached intact hiPSC colonies. Suspended colonies were subsequently transferred into low-attachment plates in a KnockOut Serum (Invitrogen)-based medium without fibroblast growth factor 2 (FGF2) and the floating hiPSC colonies folded into spherical structures (as aggregation occurred in this media, it is considered aggregation media).
(b-ii) To achieve rapid and efficient neural induction, both the BMP and TGF-β signaling pathways were inhibited with small molecules: dorsomorphin (also known as compound C) and SB-431542. On the sixth day in suspension, the floating spheroids were moved to serum-free Neurobasal with B-27 medium containing FGF2 and epidermal growth factor (EGF). By day 18, over 85% of cells expressed PAX6, and more than 80% of these progenitors expressed FOXG1 (Fig. 1b; Supplementary Fig. 1). To promote differentiation of neural progenitors into neurons, FGF2 and EGF were replaced with brain-derived neurotrophic factor (BDNF) and neurotrophic factor 3 (NT3) starting at day 25 (i.e. organoids were cultured neural maturation media) (page 672, column 1, paragraph 2-column 2, paragraph 2, Methods page 1, column 1, paragraphs 1-2, and Figure 1).
Pasca specifically teaches that these neural structures, which they named human cortical spheroids (hCSs), were generated from intact hiPSC colonies that were cultured and minimally patterned in exclusively nonadherent conditions and in the absence of extracellular scaffolding (page 671, column 2, paragraph 3).
Regarding the diameter and homogenous size limitation, as an example, Figure 1d of Pasca provides an image of a collection of 15 spheroids at day 26 of culture. Based on an analysis of the image (see below), the organoids have a diameter ranging from ~1300 μm to ~1800 μm and a calculated standard deviation from the mean of ~10%.
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Annotated Figure 1D of Pasca
Therefore, the organoids developed by the method of Pasca fall within 20% standard deviation and have homogeneous size.
Pasca teaches that hCSs represent a convenient and physiologically relevant platform for studying neuropsychiatric disorders, including synaptopathies and epilepsies and a versatile platform for designing large-scale drug screening in vitro (page 677, column 2, paragraph 2).
Pasca does not teach wherein they used neuronal tissue-specific precursor cells.
However, Reinhardt teaches that they generated small molecule neural precursor cells from iPSCs from two patients with Parkinson’s disease to model a neurological disease and determined smNPCs are suitable for modeling neurological diseases (page 10, column 1, paragraphs 2-3 and Figures 7-8). Reinhardt teaches that smNPCs are robust, exhibit immortal expansion, and do not require cumbersome manual culture and selection steps (abstract).
Monzel teaches that they used the neuroepithelial stem cells (referred to as small molecule neural precursor cells in Reinhardt) of Reinhardt to generate human midbrain organoids. The utilization of neural stem cells as the starting population has the advantage that already patterned cells might differentiate into the desired structures more efficiently (cheaper, faster cell doublings, ease of handling, and so forth) (page 1144, column 2, paragraph 1-page 1150, column 2, paragraph 1 and Figure 1).
As can be seen in Monzel Figure 1B, the hMOs generated from the same cell line showed homogenous size distributions.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have substituted the patient derived hiPSCs of Pasca with the patient derived smNPCs of Reinhardt to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to substitute with a reasonable expectation of success because accessing patient derived cells are not easily accessible at all times, especially for rare genetic disorders and increase variability if multiple collections are required to generate the neural organoids. On the contrary, Reinhardt teaches that patient derived smNPCs are robust, exhibit immortal expansion, and do not require cumbersome manual culture and selection steps. Therefore, smNPCs are readily accessible and easier to use. Furthermore, Monzel showed that smNPCs can be used to generate human brain organoids with homogenous sizes and that using these cells as the starting population has the advantage that already patterned cells might differentiate into the desired structures more efficiently (cheaper, faster cell doublings, and ease of handling). Pasca teaches that hCSs represent a convenient and physiologically relevant platform for studying neuropsychiatric disorders and Reinhardt showed that smNPCs differentiated from patient-derived iPSCs were suitable for modeling patient diseases. Therefore, it would have been obvious to one of ordinary skill to use smNPCs as they are easier to obtain and maintain than patient derived hiPSCs for repeat experimentation and can recapitulate patient disease, similar to hiPSCs, leading to a method that is cheaper and simpler. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
As Pasca and Monzel show that their organoids were homogeneous in size, it would naturally flow that the combined method of Pasca, Reinhardt, and Monzel would have a homogeneous size.
Regarding claim 2, Pasca is silent as to the number of iPSCs they seeded to start their method.
However, Monzel teaches that 9000 NESCs can be seeded an ultra-low-attachment 96-well round-bottomed plate and cultured in N2B27 medium (DMEM-F12/Neurobasal 50:50 with 1:200 N2 supplement to start the human brain organoid development process.
As Monzel teaches that a starting population of 9000 NESCs is sufficient to generate a brain organoid, it would have been obvious that the combined method of Pasca, Reinhardt, and Monzel could use a similar starting population size.
Response to Arguments
Applicant's arguments filed December 3, 2025, are acknowledged.
Applicant argues that Pasca addresses size distribution in their document when Psaca states that the hCSs grew in size to more than 300 μm in diameter by 2 weeks of culture and reached up to 4 mm in diameter by 2.5 months (4.2 ±0.3 mm, mean± s.e.m., n = 16 hCSs from 4 differentiated hiPSC lines) (Fig. Id). This would equate a standard deviation of 1.2 mm for the spheroids of Pasca and would fall outside the limitations of claim 1 (page 12, paragraph 2-page 13, paragraph 2).
Applicant's arguments and the cited Declaration have been fully considered but they are not persuasive.
Regarding Dr. Bruder’s assertions about the size distribution of the spheroids of Sloan and Pasca, these arguments are considered unpersuasive. As identified by Dr. Bruder, Sloan specifically references the spheroids of Pasca when discussing what results can be expected using the protocol disclosed therein and that spheroids should be uniform in distribution across the same hPSC line (item 8). Dr. Bruder cites to a part of Pacsa discussing the size distribution of the spheroids of Pasca. It is worth noting that Pasca measured and combined the results of 16 hCSs from 4 different hiPSC lines whereas Sloan disclosed that there should be uniform distribution across the same hiPSC line. As such, the calculated standard deviation from the Declaration is not representative of what would be found if only comparing spheroids from the same cell line. As shown above, Figure 1d of Pasca provides an image of a collection of 15 spheroids at day 26 of culture with a calculated standard deviation from the mean of ~10%. This is also in line with the analysis performed in the previous Non-Final Office action mailed on September 4, 2025, of Figure 3 of Sloan that showed two organoids have a diameter of ~1500 μm and ~1750 μm that equates to a standard deviation from the mean of ~8%.
Therefore, the method of Pasca is considered to generate spheroids that fall within the homogenous size distribution requirements of a standard deviation of less than 20% of the mean.
Applicant further argues that their method results in unexpected results. First, instead of random, mitotic neural rosettes like those of Sloan and Jo, the claimed organoids self-organize into a reproducible, globally radially symmetric structure with concentric zones. This ensures a predictable biological state across samples. Second, the claimed methods achieve unprecedented homogeneity (page 19, paragraphs 2-3).
As identified above, the organoids of Pasca are able to generate a homogenous size between organoids with a ~10% standard deviation and Pasca show that their organoids consist of concentric VZ, SVZ, and a lumen at day 52 and VZ, Deep layers, and superficial zones at day 137 (Figure 2). Therefore, this would not be unexpected.
Claims 4-5 are rejected under 35 U.S.C. 103 as being unpatentable over Jo et al. (Cell Stem Cell 19: 248-257. 2016; referenced in IDS) and further in view of Pasca et al. (Nature Methods 12: 671-678. 2015), Reinhardt et al. (PLoS ONE 8: 1-18. 2013), and Monzel et al. (Stem Cell Reports 8: 1144-1154. 2017). This is a new rejection made in response to Applicant’s amendments to claim 4. Applicant’s traversal has been fully considered but is moot in response to the new rejection.
Regarding claim 4, as an initial matter, as claim 4 recites that there can be one or more neural organoids, the de minimis construction is a single neural organoid. Therefore, as identified in the 112b rejection above, step c is not considered to be required when there is a single organoid. As such, homogeneity of structure and/or size is not required when there is a single neural organoid.
Furthermore, the limitation reciting that the one or more neural organoids are obtained by the method of claim 2 is considered a product-by-process limitation. As such, only the positively recited structures of the neural organoid are considered germane to the patentability of the product. The recited structures of the product are considered:
A. Midbrain organoid
B. At least three concentric zones
C. A core comprising neural precursor cells (as stated in the 112b above, it is unclear what is meant by mostly composed of. Therefore, anything above 50% is considered to fall under primarily)
D. Electrical activity in neurons
E. Not embedded in Matrigel
F. Derived from neuron tissue-specific precursor cells
G. Spherical diameter of > 500 μM
Jo teaches that they generated a midbrain organoid with three concentric zones (ventral zone, intermediate zone, and mantle zone). The vental zone is considered to be the core as it is the closest to the center of the organoid. The ventral zone predominantly (over 50%) comprises cells that express OTX2 and MASH1, markers of progenitor cells (i. e. neural precursor cells). Jo teaches that their organoid comprises midbrain dopaminergic neurons and that these neurons show electrical activity (Figures 1 and 4 and page 248, column 2, paragraph 3-page 254, column 1, paragraph 1). Figure 1A of Jo shows that their organoid has a spherical diameter of > 500 μM
The recitation of a process limitation in claim 4 is not viewed as positively limiting the claimed product absent a showing that the process of making recited in claim 2 imparts a novel or unexpected property to the claimed product, as it is assumed that equivalent products are obtainable by multiple routes. The burden is placed upon the applicants to establish a patentable distinction between the claimed and referenced products. The method in which theorganoid were produced is immaterial to their patentability.
"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 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, 227 USPQ 964, 966 (Fed. Cir. 1985). See also MPEP §2113.
Furthermore, Jo teaches that their organoid could be derived from Parkinson’s disease (PD) patients to shed light onto the underlying pathophysiological mechanisms of PD (page 254, column 2, paragraph 2).
Jo does not teach wherein their organoid was not embedded in Matrigel.
However, Pasca teaches a method of generating human brain spheroids (i.e. neural organoids) (Figure 1) from iPSCs, comprising:
a) To generate suspended cellular aggregates of pluripotent cells, they cultured seven hiPSC lines derived from five subjects in a dish (i.e. seeded iPSCs in a container) and successfully generated neural organoids from each clone.
b-i) Rather than using single-cell suspensions, they enzymatically detached intact hiPSC colonies. Suspended colonies were subsequently transferred into low-attachment plates in a KnockOut Serum (Invitrogen)-based medium without fibroblast growth factor 2 (FGF2) and the floating hiPSC colonies folded into spherical structures (as aggregation occurred in this media, it is considered aggregation media).
(b-ii) To achieve rapid and efficient neural induction, both the BMP and TGF-β signaling pathways were inhibited with small molecules: dorsomorphin (also known as compound C) and SB-431542. On the sixth day in suspension, the floating spheroids were moved to serum-free Neurobasal with B-27 medium containing FGF2 and epidermal growth factor (EGF). By day 18, over 85% of cells expressed PAX6, and more than 80% of these progenitors expressed FOXG1 (Fig. 1b; Supplementary Fig. 1). To promote differentiation of neural progenitors into neurons, FGF2 and EGF were replaced with brain-derived neurotrophic factor (BDNF) and neurotrophic factor 3 (NT3) starting at day 25 (i.e. organoids were cultured neural maturation media). Pasca teaches that their neural organoids reached a size of ~1-4 mm depending on the length of culture (page 672, column 1, paragraph 2-column 2, paragraph 2, Methods page 1, column 1, paragraphs 1-2, and Figure 1).
Pasca specifically teaches that these neural organoids were generated from intact hiPSC colonies that were cultured and minimally patterned in exclusively nonadherent conditions and in the absence of extracellular scaffolding and that this method addresses many of the difficulties previously identified in the art for generating neural organoids, including reproducibility between hiPSC clones within and across differentiations (page 671, column 2, paragraph 3).
Pasca shows that their neural organoids comprised three concentric zones (lumen, VZ, and SVZ at day 52 and VZ, deep layers, and superficial layers at day 137) and electrical activity of their neurons (Figures 2 and 5)
The ventral zone predominantly (over 50%) comprises cells that express Pax6, a marker of progenitor cells (i. e. neural precursor cells) (Figure 2 and page 674, column 1, paragraph 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have generated the neural organoids of Jo without embedding the cells into Matrigel, as identified by Pasca, to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because Pasca has successfully reduced to practice that neural organoids can be produced without embedding the organoids in Matrigel and that this may help improve reproducibility between hiPSC clones within and across differentiations. Furthermore, even without embedding the organoids in Matrigel, Pasca generated similar structural and functional components as the organoids of Jo, namely development of three concentric zones, electrical activity in neurons, organoid size (greater than 1 mm in diameter), and a core of neural precursor cells. Therefore, it would have been obvious that one could generate the organoid of Jo without embedding the organoid in Matrigel.
The combined teachings of Jo and Pasca do not teach wherein they used neuronal tissue-specific precursor cells as the starting cell population.
However, Reinhardt teaches that they generated small molecule neural precursor cells from iPSCs from two patients with Parkinson’s disease to model a neurological disease and determined smNPCs are suitable for modeling neurological diseases (page 10, column 1, paragraphs 2-3 and Figures 7-8). Reinhardt teaches that smNPCs are robust, exhibit immortal expansion, and do not require cumbersome manual culture and selection steps (abstract).
Monzel teaches that they used the neuroepithelial stem cells (referred to as small molecule neural precursor cells in Reinhardt) of Reinhardt to generate human midbrain organoids. The utilization of neural stem cells as the starting population has the advantage that already patterned cells might differentiate into the desired structures more efficiently (cheaper, faster cell doublings, ease of handling, and so forth) (page 1144, column 2, paragraph 1-page 1150, column 2, paragraph 1 and Figure 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have substituted the patient derived hiPSCs of Jo with the patient derived smNPCs of Reinhardt to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to substitute with a reasonable expectation of success because accessing patient derived cells are not easily accessible at all times, especially for rare genetic disorders and increase variability if multiple collections are required to generate the neural organoids. On the contrary, Reinhardt teaches that patient derived smNPCs are robust, exhibit immortal expansion, and do not require cumbersome manual culture and selection steps. Therefore, smNPCs are readily accessible and easier to use. Furthermore, Monzel showed that smNPCs can be used to generate human brain organoids (the same type of organoids made by Monzel) and that using these cells as the starting population has the advantage that already patterned cells might differentiate into the desired structures more efficiently (cheaper, faster cell doublings, and ease of handling). Jo teaches that using PD patient-derived cell lines may shed light onto the underlying pathophysiological mechanisms of PD and Reinhardt showed that smNPCs differentiated from patient-derived iPSCs were suitable for modeling patient diseases. Therefore, it would have been obvious to one of ordinary skill to use smNPCs as they are easier to obtain and maintain than patient derived hiPSCs for repeat experimentation and can recapitulate patient disease, similar to hiPSCs, leading to a method that is cheaper and simpler. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Regarding claim 5, due to the 112b issue described above, only one well of a multiwell plate is considered to require a neural organoid.
Jo teaches that during the long-term culture of the hMLO, they observed sparse, black/brown-colored deposits in hMLOs by light microscopy after approximately 2 months corresponding to neuromelanin deposits. Neuromelanin is a byproduct of DA synthesis in midbrain dopaminergic neurons. Jo assessed the level of dopamine in their organoids using high performance liquid chromatography (HPLC) (page 251, column 1, paragraph 3-page 243, column 1, paragraph 1 and Figures 3-4). Jo teaches that for HPLC measurements of dopamine, they took a single midbrain organoid and homogenized it in perchloric acid and that this procedure can be done in a well of a 6 well dish (page 14 of the Supplemental Information (page 25 of the PDF), paragraph 3).
Jo is silent as to whether they dissolved the single organoid in the well of a multiwell plate.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have examined the organoid of Jo for neuromelanin expression under light microscopy and then used a multiwell plate for homogenizing an individual organoid in a well for HPLC analysis to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to use a multiwell plate for homogenizing an organoidsfor HPLC analysis with a reasonable expectation of success because Jo already indicates that homogenization with 2D-dopaminergic neurons can occur in one well of a 6-well dish and that they want to homogenize a single organoid for analysis, and compare the results to other organoids and other cultured cells (human cerebral organoid and 2D-DA neurons, respectively) (Figure 4). Therefore, it would be obvious to examine the organoid for neuromelanin expression under light microscopy to determine when it expresses neuromelanin (a sign of dopamine production) and to then place the individual midbrain organoid that expresses neuromelanin into an individual well of a 6 well plate along with other types of organoids and cells to homogenize each in separate wells for HPLC analysis and comparison. This would simplify the need for multiple dishes as all samples could fit in a single 6 well dish for homogenization while reducing the potential variability if each was done individually.
Conclusion
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/KEENAN A BATES/Examiner, Art Unit 1631
/JAMES D SCHULTZ/Supervisory Patent Examiner, Art Unit 1631