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 .
Status of the Claims
Applicant’s election without traverse of Group I in the reply filed on 09/25/25 is acknowledged.
Claims 1-51 are pending.
Claims 37-51 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected Group, there being no allowable generic or linking claim.
The species requirement as required in the action filed 06/03/2025 is hereby withdrawn.
Claims 1-5, 16, 17, 22, and 27-36 are amended,
Claims 1-36 have been examined on the merits.
Claim Rejections - 35 USC § 112(a)
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 1-31 and 33 and 35 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 claims 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.
Scope of the claimed invention
Regarding claims 1-5: The claims recite “pathway agonist” for multiple signaling pathways (SHH, IGF1, CREB, PKA, GDNF, mTOR, BDNF) and “pathway antagonist” for multiple signaling pathways (BMP, MEK, WNT, AKT, PKC, TAK1, TGF-beta, TRK, Notch).
The instant specification defines “agonist" of a cellular signaling pathway as “an agent that stimulates (upregulates) the cellular signaling pathway” (p23 ln 3-5). The broadest reasonable interpretation of a pathway agonist, therefore, is any protein/peptide, small molecule, nucleic acid or compound that stimulates the signaling pathway at any point in the signaling pathway. This includes cytokines, antibodies, small molecules, and genetic manipulation of proteins/polypeptides involved in the pathways of interest. Furthermore, an agonist of a cellular signaling pathway can act on any component of said signaling pathway including receptors, ligands, second messengers/signaling intermediates and effectors.
The instant specification defines “antagonist” as “an agent that inhibits (downregulates) the cellular signaling pathway” (p23 ln10-11). The broadest reasonable interpretation of a pathway antagonist, therefore, is any protein/peptide, small molecule, nucleic acid or compound that inhibits the signaling pathway at any point in the signaling pathway. This includes cytokines, antibodies, small molecules, and genetic manipulation of proteins/polypeptides involved in the pathways of interest.
Furthermore, an antagonist of a cellular signaling pathway can act on any component of said signaling pathway including receptors, ligands, second messengers/signaling intermediates and effectors.
The claims recite the functional limitation “pathway agonist” and “pathway antagonist” for specific signaling pathways, but do not describe a structure capable of performing the functions.
MPEP 2163.02 reads “An objective standard for determining compliance with the written description requirement is, ‘does the description clearly allow persons of ordinary skill in the art to recognize that he or she invented what is claimed’”.
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 by their complete structure. To provide adequate written description and evidence of possession of a claimed genus, the specification must provide sufficient distinguishing identifying characteristics of the genus. The factors to be considered include disclosure of complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, methods of making the claimed product, or any combination thereof. The disclosure of a single species is rarely, if ever, sufficient to describe a broad genus, particularly when the specification fails to describe the features of that genus, even in passing. (see In re Shokal 113USPQ283(CCPA1957); Purdue Pharma L.P. vs Faulding Inc. 56 USPQ2nd 1481 (CAFC 2000).
Teachings of the instant specification
The instant specification teaches examples of pathway antagonists and pathway agonists for the signaling pathways required for the claimed invention. The instant specification teaches exemplary inhibitors of BMP, MEK, WNT, AKT, PKC, TAK1, TGFβ, TRK and Notch and exemplary activators of SSH, IGF1, CREB, PKA, GDNF, mTOR and BDNF.
The instant specification recites antagonists for the signaling pathways BMP, MEK, WNT (p23-24). The instant specification also recites known agonists of the SHH pathway p25 ln 1-12). The instant specification recites known antagonists of the AKT pathway (p25 ln13-27), antagonists of the PKC pathway (p25/26 ln 27-31/1-10), antagonists of TAK1 (MAP3K7) pathway (p26 ln 11-20), antagonists of TGF beta (p26/27 ln 21-30/1-2), antagonists of TRK pathway (p27 ln 3-16), antagonists of the NOTCH pathway (p27 ln 14-25), agonists of the IGF1 pathway (p27/28 ln26-31/1-5), agonists of CREB/PKA pathway p28 ln 6-23), agonists GDNF pathway (p29 ln 9-20), agonists of mTOR (p29/30 ln 19-31/1-2), agonists of the BDNF pathway p30 ln 3-15).
The instant specification further teaches different agonists or antagonists that affect the same signaling pathway are used in different steps of the method when an agonist or antagonist is used in more than one step of the method (p31 ln 27-31), and different concentrations can be used (p32 ln 6-9).
The instant specification also teaches the use of specific antagonists/agonists (here after termed “effectors”) for the various pathways at specific concentrations for 5 specific stages of neural cell development. Table 1 teaches 6 validated effectors for stage 1 (forebrain neural stem cells from human pluripotent stem cells). Table 2 teaches 4 validated effectors for stage 2 (ventral forebrain neural stem cells from the cells of the previous stage). Table 3 teaches 6 validated effectors for stage 3 (medial ganglionic eminence neural progenitor cells from the cells of the previous stage). Table 4 teaches 5 validated effectors for stage 4 (to obtain human immature neurons from the cells of the previous stage). Table 5 teaches 7 validated effectors for stage 5 (to obtain mature parvalbumin+ interneurons from the cells of the previous stage).
The validated effectors in the tables are validated experimentally in Example 3 (stage 1, 2 and 3) and Example 6 (stage 4 and 5), in which cells are cultured with media comprising a specific combination of effectors at a specific concentration (p45 and 51). Neural development is assessed by immunohistochemistry for markers associated with neuronal development.
For each of the five stages of differentiation the instant specification teaches a single combination of effectors, each effector at a single concentration, and each effector targets a single distinct pathway for the indicated developmental stage (Tables 1-5). Thus for each developmental stage the instant specification teaches a single species of effector for each pathway.
The instant specification does not experimentally demonstrate validation for expression of FEZF2 for stage 1 (Fig 16A) or ASCL1 for stage 3 (Fig 16C).
As mentioned supra, the instant specification provides a single example of a single agonist/antagonist concentration for the cell culture stages. For example, the instant specification teaches the effector LDN193189 (BMP antagonist) is used at 275 nM for stage 1 (Table 1) and 250 nM for state 2. However the claimed invention of claim 1 recites any BMP antagonist at any concentration. Different effectors would require different concentrations to achieve the desired result and one of ordinary skill in the art would be required to conduct extensive trial and error experimentation to determine the working concentration ranges for different effectors for which the concentration is not taught.
The state of the art
The instant invention is drawn to a method of use of effectors (agonists/antagonists) to manipulate ~20 signaling pathways for the development of specific neural cell populations. For the sake of brevity and clarity, a general discussion of biological signaling pathways and then a discussion of an exemplary pathway will be used to describe the state of the art for the effects of agonists and antagonist on signaling pathways.
As mentioned supra, a pathway antagonist or agonist is a molecule which can interact with any part of a pathway to perform its function, and thus is a very broad category of molecules. Weng et al (Science (1999) 2:284;1-9) teach signaling in biological systems is complex, and the complexity can be described by the number of components, intricacy of the interfaces between components, the number and intricacy of branches, degree of nesting (p1 ¶2). Weng further teach that the simplest description of a signaling pathway is a three component system (Fig 1A) which comprises three elements that signal in a linear fashion and can be thought of as a wire carrying information (p2 ¶1). Weng further teach that for even in the most simplified model of signaling, complexity is vast due to the vast array of signaling molecules and isoforms with redundant signal transfer functions and different kinetic properties (p2 ¶2).
Weng further teach that interactions between distinct pathways adds further to the complexity as the pathways become part of signaling network (p2 ¶3). Weng teach that, for a system consisting of two interactive pathways of n components, n2 possible interactions would occur (if the interactions occurred in pairs) (p2 ¶3, Fig 1B).
Weng teach that compartmentalization of signaling components contribute a further level of complexity to signaling pathways as some components are anchored in the plasma membrane, while others may be restricted to organelles or specific microenvironments (p3 ¶2, Fig 1 C).
Turning to the MEK pathway as an exemplary pathway recited by the instant disclosure, Barbosa et al (Molecular Cancer Research (2020) 19: 361-74) teach MEK is part of the MAPK signaling cascade and is critical for cell proliferation, differentiation and survival (abstract).
Barbosa further teach a visual overview of the MEK pathway which comprises 19 components (p361 col2 visual overview).
As discussed supra, an antagonist of MEK could target MEK, or target an upstream or downstream component of MEK signaling. Upstream components include receptor tyrosine kinases, RAS, RAF and extracellular signals (p361 col1 visual overview). Downstream components include ERK and ERK substrates (transcription factors, nuclear substrates and cytosolic substrates) (p361 col2 ¶1, visual overview). Nuclear and cytosolic targets of ERK (and thus downstream components of MEK signaling) is an extremely large and diverse substrate pool comprising more than 600 direct substrates of ERK (p365 col 2 ¶2).
Thus a “MEK pathway antagonist” as required by the instant claim 1 encompasses any molecule that can reduce the activity or effect of more than 600 direct substrates of ERK, as ERK is a downstream effector of MEK (p365 col2 ¶2). These substrates include transcription factors, cytoskeletal elements, apoptotic regulators, kinases, phosphatases, mRNA and additional signaling proteins (p365 col2 ¶2).
Guo et al (Cell Research (2009) 19;71-88) teach BMP signaling occurs by signal transduction via Smads (p71 col1 ¶1). Guo also teach that MEK can regulate SMAD3, and thus crosstalk occurs between the MEK and BMP pathways. Guo also teach Wnt signaling is known to effect Smad stability, and thus also cross talks with BMP signaling. Figure 2 demonstrates crosstalk between BMP and AKT signaling pathways (p74).
Zhan et al (Nature Communications (2019) 10:2197;1-17) teach MEK inhibitors can activate Wnt signaling (title). For example the MEK inhibitor reametinib increases expression of the WNT target AXIN2 (p2 col2 ¶4).
Pelullo et al (Frontiers in Genetics (2019)10:711;1-16) teach Hedgehog signaling elements interact with cofactors of Wnt, Notch and SSH pathways (title). For example, both Wnt signaling and SHH activate the downstream transcription factor Gli (p6 Fig 2). Furthermore, activation of Akt signaling results in increased SHH expression (p5 col2 ¶3).
Conclusion
The instant specification provides some guidance and specific examples of “pathway antagonists” and “pathway agonists”, as discussed supra.
However, while the instant specification teaches exemplary inhibitors of BMP, MEK, WNT, AKT, PKC, TAK1, TGFβ, TRK and Notch and exemplary activators of SSH, IGF1, CREB, PKA, GDNF, mTOR and BDNF, the instant specification does not provide sufficient guidance or working examples of the pathway antagonists or pathway agonists claimed in the broad genus “pathway antagonists” and “pathway agonists” for the specific pathways recited. The instant specification teach a single specific media formulation for each developmental stage (1-5) and each formulation comprises one example of a pathway antagonist and/or agonist as required by the instant claims, at a single concentration (Tables 1-5).
The instant specification teaches 12 examples of possible MEK pathway antagonists (p24 ln 4-17). The instant specification also teach one working example of a specific media formulation comprising the use of one specific MEK inhibitor (PD0325901) at one specific concentration (110 nM) during stage 1 of differentiation (differentiation of forebrain neural stem cells) (p42 Table 1) and one working example of one specific MEK inhibitor (PD0325901) at one specific concentration (100nM) during stage 2 of differentiation (differentiation of forebrain neural stem cells) (p43 Table 2).
The 12 specific examples of MEK antagonists and 1 specific working example of MEK antagonist taught by the instant specification. Furthermore, the claims recite multiple agonists/antagonists for multiple pathways. However cellular signaling pathways are complex and cross-talk between pathways results in uncertainty as to what agonists are specific to what pathways. As discussed, Weng teach signaling pathway complexity increases with the number of interacting components comprising said pathway. Barbosa, Guo, Zhan and Pellulo as discussed above teach cross-talk among the cell signaling pathways is complex, and thus the effect of a specific inhibitor on cellular processes is uncertain without trial and error experimentation to determine the effect of any pathway inhibitor on the target cell.
One of ordinary skill in the art would understand that trial and error experimentation is required to test how a specific pathway antagonist or agonist effects cell development because the effect of a pathway agonist or antagonist can effect more than the targeted pathway, and such crosstalk cannot be predicted. Thus all structures of pathway agonists and antagonists cannot be predicted based on the examples of pathway agonists and antagonists taught by the instant specification and the art.
MPEP 2163 states “To satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention”.
As described above, in the case of the instant claims 1-5, the claimed invention is broad and includes cytokines, antibodies, small molecules, and genetic manipulation of proteins/polypeptides involved in the pathways of interest at any point in the pathway of interest.
In view of the unpredictability in the effect of pathway antagonists and agonists agents as taught by the art, and the trial and error necessary for their effective use, the specification fails to support the broad genus of “pathway agonist” and pathway antagonist” as claimed such that one skilled in the art can reasonably conclude the inventor had possession of the claimed invention.
The gap between the limited number of structures corresponding to those that exhibit an agonist or antagonist effect as described in the specification and art compared to the breadth any agent that that is a pathway agonist or antagonist of the recited pathways is very large. Given this large gap, the specification fails to support the broad genus of “pathway agonist” and “pathway antagonist” for the recited pathways as claimed
such that one skilled in the art can reasonably conclude the inventor had possession of the claimed invention.
Claims 1-31, 33 and 35 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a specific embodiment of the disclosed invention, however does not reasonably provide enablement for all pathway agonists and antagonists as recited in the claim. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to perform the claimed method commensurate in scope with these claims.
The factors to be considered in determining whether undue experimentation is required are summarized in In re Wands, 858 F.2d 731, 737, 8 USPQd 1400, 1404 (Fed. Cir. 1988) (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. While all of these factors are considered, a sufficient number are discussed below so as to create a prima facie case.
Scope of the claimed invention
Regarding claims 1-5: The claims recite “pathway agonist” for multiple signaling pathways (SHH, IGF1, CREB, PKA, GDNF, mTOR, BDNF) and “pathway antagonist” for multiple signaling pathways (BMP, MEK, WNT, AKT, PKC, TAK1, TGF-beta, TRK, Notch).
The instant specification defines “agonist" of a cellular signaling pathway as “an agent that stimulates (upregulates) the cellular signaling pathway” (p23 ln 3-5). The broadest reasonable interpretation of a pathway agonist, therefore, is any protein/peptide, small molecule, nucleic acid or compound that stimulates the signaling pathway at any point in the signaling pathway. This includes cytokines, antibodies, small molecules, and genetic manipulation of proteins/polypeptides involved in the pathways of interest. Furthermore, an agonist of a cellular signaling pathway can act on any component of said signaling pathway including receptors, ligands, second messengers/signaling intermediates and effectors.
The instant specification defines “antagonist” as “an agent that inhibits (downregulates) the cellular signaling pathway” (p23 ln10-11). The broadest reasonable interpretation of a pathway antagonist, therefore, is any protein/peptide, small molecule, nucleic acid or compound that inhibits the signaling pathway at any point in the signaling pathway. This includes cytokines, antibodies, small molecules, and genetic manipulation of proteins/polypeptides involved in the pathways of interest.
Furthermore, an antagonist of a cellular signaling pathway can act on any component of said signaling pathway including receptors, ligands, second messengers/signaling intermediates and effectors.
The claims recite the functional limitation “pathway agonist” and “pathway antagonist” for specific signaling pathways, but do not describe a structure capable of performing the functions. Thus the claimed genus of agonist/antagonist is broad and there is insufficient guidance from the specification as to how one of ordinary skill in the art can any agonist/antagonist of the recited pathway at any concentration to achieve the claimed method.
Regarding claims 9-30: These claims depend from claims 1-5. While the dependent claims recite a specific molecule, and in some cases a specific concentration of an agonist or antagonist as required by the claim, a recitation of a single molecule for a method requiring multiple antagonists/agonists does not remedy the deficiencies of the parent claims.
The instant specification
The instant specification teaches examples of pathway antagonists and pathway agonists for the signaling pathways required for the claimed invention. The instant specification teaches exemplary inhibitors of BMP, MEK, WNT, AKT, PKC, TAK1, TGFβ, TRK and Notch and exemplary activators of SSH, IGF1, CREB, PKA, GDNF, mTOR and BDNF.
The instant specification recites antagonists for the signaling pathways BMP, MEK, WNT (p23-24). The instant specification also recites known agonists of the SHH pathway p25 ln 1-12). The instant specification recites known antagonists of the AKT pathway (p25 ln13-27), antagonists of the PKC pathway (p25/26 ln 27-31/1-10), antagonists of TAK1 (MAP3K7) pathway (p26 ln 11-20), antagonists of TGF beta (p26/27 ln 21-30/1-2), antagonists of TRK pathway (p27 ln 3-16), antagonists of the NOTCH pathway (p27 ln 14-25), agonists of the IGF1 pathway (p27/28 ln26-31/1-5), agonists of CREB/PKA pathway p28 ln 6-23), agonists GDNF pathway (p29 ln 9-20), agonists of mTOR (p29/30 ln 19-31/1-2), agonists of the BDNF pathway p30 ln 3-15).
The instant specification further teaches different agonists or antagonists that affect the same signaling pathway are used in different steps of the method when an agonist or antagonist is used in more than one step of the method (p31 ln 27-31), and different concentrations can be used (p32 ln 6-9).
The instant specification also teaches the use of specific antagonists/agonists (here after termed “effectors”) for the various pathways at specific concentrations for 5 specific stages of neural cell development. Table 1 teaches 6 validated effectors for stage 1 (forebrain neural stem cells from human pluripotent stem cells). Table 2 teaches 4 validated effectors for stage 2 (ventral forebrain neural stem cells from the cells of the previous stage). Table 3 teaches 6 validated effectors for stage 3 (medial ganglionic eminence neural progenitor cells from the cells of the previous stage). Table 4 teaches 5 validated effectors for stage 4 (to obtain human immatured neurons from the cells of the previous stage). Table 5 teaches 7 validated effectors for stage 5 (to obtain mature parvalbumin+ interneurons from the cells of the previous stage).
The validated effectors in the tables are validated experimentally in Example 3 (stage 1, 2 and 3) and Example 6 (stage 4 and 5), in which cells are cultured with media comprising a specific combination of effectors at a specific concentration (p45 and 51). Neural development is assessed by immunohistochemistry for markers associated with neuronal development.
For each of the five stages of differentiation the instant specification teaches a single combination of effectors, each effector at a single concentration, and each effector targets a single distinct pathway for the indicated developmental stage (Tables 1-5). Thus for each developmental stage the instant specification teaches a single species of effector for each pathway.
The instant specification does not experimentally demonstrate validation for expression of FEZF2 for stage 1 (Fig 16A) or ASCL1 for stage 3 (Fig 16C).
The state of the art
The instant invention is drawn to a method of use of effectors (agonists/antagonists) to manipulate ~20 signaling pathways for the development of specific neural cell populations. For the sake of brevity and clarity, a general discussion of biological signaling pathways and then a discussion of an exemplary pathway will be used to describe the state of the art for the effects of agonists and antagonist on signaling pathways.
As mentioned supra, a pathway antagonist or agonist is a molecule which can interact with any part of a pathway to perform its function, and thus is a very broad category of molecules. Weng et al (Science (1999) 2:284;1-9) teach signaling in biological systems is complex, and the complexity can be described by the number of components, intricacy of the interfaces between components, the number and intricacy of branches, degree of nesting (p1 ¶2). Weng further teach that the most simple description of a signaling pathway is a three component system (Fig 1A) which comprises three elements that signal in a linear fashion and can be thought of as a wire carrying information (p2 ¶1). Weng further teach that for even in the most simplified model of signaling, complexity is vast due to the vast array of signaling molecules and isoforms with redundant signal transfer functions and different kinetic properties (p2 ¶2).
Weng further teach that interactions between distinct pathways adds further to the complexity as the pathways become part of signaling network (p2 ¶3). Weng teach that, for a system consisting of two interactive pathways of n components, n2 possible interactions would occur (if the interactions occurred in pairs) (p2 ¶3, Fig 1B).
Weng teach that compartmentalization of signaling components contribute a further level of complexity to signaling pathways as some components are anchored in the plasma membrane, while others may be restricted to organelles or specific microenvironments (p3 ¶2, Fig 1 C).
Turning to the MEK pathway as an exemplary pathway recited by the instant disclosure, Barbosa et al (Molecular Cancer Research (2020) 19: 361-74) teach MEK is part of the MAPK signaling cascade and is critical for cell proliferation, differentiation and survival (abstract).
Barbosa further teach a visual overview of the MEK pathway which comprises 19 components (p361 col2 visual overview).
As discussed supra, an antagonist of MEK could target MEK, or target an upstream or downstream component of MEK signaling. Upstream components include receptor tyrosine kinases, RAS, RAF and extracellular signals (p361 col1 visual overview). Downstream components include ERK and ERK substrates (transcription factors, nuclear substrates and cytosolic substrates) (p361 col2 ¶1, visual overview). Nuclear and cytosolic targets of ERK (and thus downstream components of MEK signaling) is an extremely large and diverse substrate pool comprising more than 600 direct substrates of ERK (p365 col 2 ¶2).
Thus a “MEK pathway antagonist” as required by the instant claim 1 encompasses any molecule that can reduce the activity or effect of more than 600 direct substrates of ERK, as ERK is a downstream effector of MEK (p365 col2 ¶2). These substrates include transcription factors, cytoskeletal elements, apoptotic regulators, kinases, phosphatases, mRNA and additional signaling proteins (p365 col2 ¶2).
Furthermore, the claims recite multiple agonists/antagonists for multiple pathways. However cellular signaling pathways are complex and cross-talk between pathways results in uncertainty as to what agonists are specific to what pathways.
Guo et al (Cell Research (2009) 19;71-88) teach BMP signaling occurs by signal transduction via Smads (p71 col1 ¶1). Guo also teach that MEK can regulate SMAD3, and thus crosstalk occurs between the MEK and BMP pathways. Guo also teach Wnt signaling is known to effect Smad stability, and thus also cross talks with BMP signaling. Figure 2 demonstrates crosstalk between BMP and AKT signaling pathways (p74).
Zhan et al (Nature Communications (2019) 10:2197;1-17) teach MEK inhibitors can activate Wnt signaling (title). For example the MEK inhibitor reametinib increases expression of the WNT target AXIN2 (p2 col2 ¶4).
Pelullo et al (Frontiers in Genetics (2019)10:711;1-16) teach Hedgehog signaling elements interact with cofactors of Wnt, Notch and SSH pathways (title). For example, both Wnt signaling and SHH activate the downstream transcription factor Gli (p6 Fig 2). Furthermore, activation of Akt signaling results in increased SHH expression (p5 col2 ¶3).
In regards to the concentration of agonist/antagonist of the claimed invention, at most a single concentration is recited for a single pathway agonist/antagonist. Qi(2017) teach use of PD0325901, the claimed MEK pathway antagonist. Qi teach a dose dependent response of 1uM and 8uM PD0325901 (Fig1i). Thus is it unlikely a working concentration of 0.001uM or 1mM would obtain the desired response. However the concentration of pathway antagonists/agonists for the claimed method is undisclosed for more than one of the multiple claimed small molecules for the recited claims. Thus, one of ordinary skill in the art would be required to conduct trial and error experimentation to determine the working concentrations of the recited agonists/antagonists.
Conclusion
The instant specification provides some guidance and specific examples of “pathway antagonists” and “pathway agonists”, as discussed supra.
However, while the instant specification teaches exemplary inhibitors of BMP, MEK, WNT, AKT, PKC, TAK1, TGFβ, TRK and Notch and exemplary activators of SSH, IGF1, CREB, PKA, GDNF, mTOR and BDNF, the instant specification does not provide sufficient guidance or working examples of the pathway antagonists or pathway agonists claimed in the broad genus “pathway antagonists” and “pathway agonists” for the specific pathways recited. The instant specification teach a single specific media formulation for each developmental stage (1-5) and each formulation comprises one example of a pathway antagonist and/or agonist as required by the instant claims, at a single concentration (Tables 1-5).
The instant specification teaches 12 examples of possible MEK pathway antagonists (p24 ln 4-17). The instant specification also teach one working example of a specific media formulation comprising the use of one specific MEK inhibitor (PD0325901) at one specific concentration (110 nM) during stage 1 of differentiation (differentiation of forebrain neural stem cells) (p42 Table 1) and one working example of one specific MEK inhibitor (PD0325901) at one specific concentration (100nM) during stage 2 of differentiation (differentiation of forebrain neural stem cells) (p43 Table 2).
The 12 specific examples of MEK antagonists and 1 specific working example of MEK antagonist taught by the instant specification
As discussed, Weng teach signaling pathway complexity increases with the number of interacting components comprising said pathway. Barbosa, Guo, Zhan and Pellulo teach cross-talk among the cell signaling pathways is complex, and thus the effect of a specific inhibitor on cellular processes is uncertain without trial and error experimentation to determine the effect of any pathway inhibitor on the target cell.
Therefore, while the instant disclosure is enabling for the working examples which disclose pathway agents, concentrations and culture stage, as disclosed by the validated effectors of Tables 1-5 of the instant specification and instant claims 32, 34, 36, the invention is not enabled for the broad genus of all of the recited pathway antagonists/agonists and pathway agents and unspecified concentrations.
One of ordinary skill in the art would understand that trial and error experimentation is required to test how a specific pathway antagonist or agonist effects cell development because the effect of a pathway agonist or antagonist can effect more than the targeted pathway, and such crosstalk cannot be predicted. Furthermore, one of ordinary skill in the art would understand trial and error experimentation is required to determine the working concentration for the claimed agonists/antagonists. Thus the invention is not enabled for all pathway agonists/antagonists for the recited pathways at all concentrations, as claimed by the instant method..
As described above, in the case of the instant claims 1-5, the claimed invention is broad and includes cytokines, antibodies, small molecules, and genetic manipulation of proteins/polypeptides involved in the pathways of interest at any point in the pathway of interest.
In view of the unpredictability in the effect of pathway antagonists and agonists agents as taught by the art, and the trial and error necessary for their effective use, the specification fails to support the broad genus of “pathway agonist” and pathway antagonist” as claimed such that one skilled in the art can reasonably conclude the inventor had possession of the claimed invention.
The gap between the limited number of structures corresponding to those that exhibit an agonist or antagonist effect as described in the specification and art compared to the breadth any agent that that is a pathway agonist or antagonist of the recited pathways is very large. Given this large gap, the specification fails to support the broad genus of “pathway agonist” and “pathway antagonist” for the recited pathways as claimed
such that one skilled in the art can reasonably use the claimed invention without undue trial and error experimentation.
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.
Regarding claims 24 and 36: The claims recite “MHY1458” as a mTOR pathway agonist. However the art refers mainly to MHY1485 as an mTOR pathway agonist with references to “MHY1458” appearing to be a transposition of the last two digits of the compound. Furthermore, no chemical datasheet or information on the molecule is found in the art for MHY1458, while MHY1485 is a well described mTOR activator with a disclosed structure and mechanism.
Thus the claims are interpreted as referring to MHY1485 as the mTOR pathway agonist.
Appropriate correction is required.
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 (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 pre-AIA 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) the invention was known or used by others in this country, or patented or described in a printed publication in this or a foreign country, before the invention thereof by the applicant for a patent.
Claims 1, 6, 7, and 12 are rejected under pre-AIA 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Liu et al (Nature Protocols (2013) 8:9; 1670-1679) and as evidenced by B27 Datasheet (B-27 Supplement [online]. ThermoFisher corporation [retrieved on 01/30/2026]. Retrieved from the Internet: <URL: B-27 Supplement Minus Vitamin A (50X) liquid | Thermo Fisher Scientific - US) , Xing et al (Biochem and Biophys Research Comm (2015)456;605-609), Hidebrandt(Scientific Reports (2018)8:8711;1-9), Odaka et al (Neural regeneration Research (2017)12:7;1-8), Wang et al (Biochem J (2011)426:3;1-26), Guo et al (Archives of Medical Research (2008) 39;434-442), Ward (The Journal of Biological Chemistry (1998) 273:20;12558-12566).
Regarding claims 1, 6, 7 and 12: Liu(2013) teach maturation of parvalbumin positive (PV) neurons from human embryonic stem cells and induced pluripotent stem cells (abstract, Figure 5c). Liu teach the cells are cultured with medium comprising 1.5 mM purmorphamin (an SHH agnonist) and B27 supplement (step 24).
As evidenced by the B27 datasheet (Gibo Ca 12587-010) and discussed below, the B27 supplement contains insulin (a BMP pathway antagonist), putrescine (an MEK antagonist), corticosterone (a WNT pathway antagonist and AKT pathway antagonist), glutathione (PKC pathway antagonist) and linolenic acid (AKT pathway antagonist).
Xing(2015) teach insulin inhibits BMP2 activity and thus supplementation of B27 supplement reads on a BMP pathway antagonist. Hildebrandt(2018) teaches corticosterone reduces expression of Wnt7b and Wnt10, therefore B27 also reads on a WNT antagonist. Odaka(2017) teach corticosterone also inhibits AKT signaling (Fig 2), thus B27 supplement reads on an AKT antagonist. Wang(2011) teach putrescine inhibits MEK mRNA and protein, and thus B27 supplement also reads on an MEK pathway antagonist. Guo(2008) teaches Linoleic acid inhibits the AKT pathway (title). Thus the B27 reads on an AKT pathway antagonist. Ward(1998) teach glutathione inactivates PKC (title), and thus B27 supplement reads on a PKC antagonist.
Liu further teach cells are cultured for 48h, and a media exchange is conducted with the identical media and cells are cultured for an additional 24 hours. This reads on days 0-3 of the claimed invention. Day 16 of the disclosure of Liu is interpreted as corresponding to day 0 of the instant invention.
Because Liu teach the identical steps as the instant claim 1, the cells derived from the disclosed method would be identical to the cells of the claimed method and thus the cells produced by the method of Liu read on human OTX2+ FEZF2+ SIX3+ FB-NSCs .
While Liu is silent on the characterization of the cells as OTX2+ FEZF2+ SIX3+ FB-NSCs, the Liu teach the active steps identical to the active steps of the claimed method, and thus the cells generated by the disclosure of Liu would be identical to those disclosed in the instant claim 1.
Thus the invention as claimed is anticipated by the teachings of Liu.
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.
Claim 2 is rejected under 35 U.S.C. 103 as being unpatentable over Liu et al (Nature Protocols (2013) 8:9; 1670-1679) as applied supra to claims 1, 6, 7, 12, and further in view of Read et al (Cell. Mol. Life Sci. (2009) 66:1-10) and Dominguez-Garcia et al (Cell Death and Disease (2020) 11:262;1-19).
Liu et al anticipate the invention of claims 1, 6-7 and 12 and thus render them obvious (see above).
Regarding claim 2: The teachings of Liu are described supra. Liu disclose further media exchanges with the media disclosed supra over days 3-6 of the cell culture. Liu do not disclose media lacking an AKT pathway antagonist and a PKC pathway antagonist.
Read(2009) teach Akt promotes neuronal differentiation and promotes neuronal survival (p2 col1 ¶2). Dominguez-Garcia(2020) teach PKC signaling regulates neurogenesis through the release of neurogenic growth factors (Abstract).
It would have been obvious to one of ordinary skill in the art to adapt the methods of Liu drawn to generating parvalbumin positive (PV) neurons from human stem cells by using media lacking an AKT antagonist as taught by Read and lacking a PKC antagonist as taught by Dominguez-Garcies.
One of ordinary skill in the art would have been motivated to modify the method of Liu as taught by Read and Dominguez-Garcia to for the purposes of generating ventral forebrain neural stem cells because Reed and Dominguez-Gacia teach these factors promote neuronal differentiation and survival, and thus one of ordinary skill in the art would understand that omitting the factors which inhibit the pathways which promote neurogenesis and differentiation would improve efficiency of a neural differentiation method.
One would have had a reasonable expectation of success because Dominggez-Garcia teaches PKC activation promotes neuroblast differentiation (abstract) and Reed teach AKT is well documented to promote neuronal survival (abstract).
While Liu, Reed and Dominguez-Garcia are silent on the characterization of the cells as NKX2-1 + VFB-NSCs, the combination of Liu, Reed and Dominguez-Garcia teach the active steps identical to the active steps of the claimed method, and thus the cells generated by the combination of Liu, Reed and Dominguez-Garcia would be identical to those disclosed in the instant claim 2.
Claims 3, 16-19 are rejected under 35 U.S.C. 103 as being unpatentable over Read et al (Cell. Mol. Life Sci. (2009) 66:1-10) and Dominguez-Garcia et al (Cell Death and Disease (2020) 11:262;1-19) as applied supra to claim 2; and further in view of Neubert et al (Cell Death and Differentiation (2011)18;1-10), Valizadeh-Arshad et al (Cell J (2018) 19:4;545-551), Qi et al (Nature Biotechnology (2017) 35:2;154-166) and Bilenker et al (US 2021/0023086).
Regarding claims 3, 16-19: The teachings of Liu are described supra. Liu teach culturing the cells as described supra, with a SSH pathway agonist.
Liu does not disclose culturing cells in media that comprises a TAK1 pathway antagonist, a TGF-beta pathway antagonist, a TRK pathway antagonist a Notch pathway antagonist and an IGF1 pathway antagonist on days 22-24 (equivalent to days 6-9 of the instant invention).
While Liu do teach culturing cells with the IGF1 pathway agonist IGF1 at 10 ng/ml (step 30) at day 26 (step 30). Liu do not teach culturing cells with IGF on days 22-24 (corresponding to days 6-9 of the instant invention).
Neubert(2011) teach inhibition of Tak1 protects against neuronal cell death in ischemia models. Neubert further teach the TAK1 inhibitor is 5Z-7-oxozeaenol (5ZO).
Valizadeh-Arshad(2018) teach inhibition of TGFβ, BMP and WNT signaling pathways promotes differentiation of pluripotent stem cells (hESCs) along the neuronal lineage (p545 col2 ¶1). Valizadeh-Arshad further teach A8301 is a selective inhibitor for the TGFβ signaling (p545 col1 ¶1) and use of A8301 promoted neural differentiation effectively (p550 col2 ¶3).
Qi(2017) teach use of the notch pathway antagonist DAPT (also known as GSIXX) at 10 uM and 5uM for the differentiation of functional neurons(p155 col1 ¶2; p164 col2 ¶3). Qi teach DAPT rapidly induces CNS lineage and triggers neuronal differentiation (p155 ¶4).
Thiele et al (Molecular Pathways (2009) teach that Trk was first identified as an oncogene when constitutively active and is capable of transforming immortalized fibroblasts (p1 col1 ¶1). Thiele further teach downstream signaling of TRK can regulate proliferation vs differentiation (p2 col2 ¶1). Bilenker(2021) teach culture of cells with the TRK pathway GNF-5837 antagonist (claim 2).
It would have been obvious to one of ordinary skill in the art to adapt the methods of Liu drawn to a method of differentiation neural cells by using media comprising a TAK1 antagonist, a TGF-beta antagonist, a TRK antagonist, a notch antagonist and and IGF1 antagonist as taught by Neubert, Valizadeh-Arshad, Qi, Thiele and Bilenker.
One of ordinary skill in the art would have been motivated to modify the method of Liu with the teachings of Neubert, Valizadeh-Arshad, Qi, Thiele and Bilenker because Neubert teach a TAK1 inhibitor will protect against neuronal cell death, Valizadeh-Arshad teach TGFβ, BMP and WNT promote neuronal differentiation, Qi teach notch antagonism promotes differentiation of functional neurons and Thiel teach TRK regulates proliferation vs differentiation.
One would have had a reasonable expectation of success because Neubert teach TAK1 inhibition protects neurons in vitro (abstract), Valizadeh-Arshad teach successful neuron differentiation from stem cells, and Qi teach successful differentiation of functional neurons and Bilenker teaches use of the TRK antagonist in cell cultures.
Claims 4 and 20-23 are rejected under 35 U.S.C. 103 as being unpatentable over Neubert et al (Cell Death and Differentiation (2011)18;1-10), Valizadeh-Arshad et al (Cell J (2018) 19:4;545-551), Qi et al (Nature Biotechnology (2017) 35:2;154-166) and Bilenker et al (US 2021/0023086) as applied supra to claims 3, 17 and 19; and further in view of Park et al (J Neurosurg (2007)107;593-599) and Sawada et al (PLOS ONE (2021) April;1-22).
Regarding claims 4 and 20-23: The teachings of Liu are discussed supra. Liu teach that on day 26 cells are cultured with cAMP (a PKA activator), BDNF (an mTOR signaling agonist), GDNF (a GDNF pathway agonist) and (step 27).
Liu do not teach the media comprises Valproic acid or Substance P.
Park(2007) teach Substance P promotes adult neural progenitor cell proliferation in vitro (abstract). Park further teach cells are cultured with 0.1 umol/L (100 nonomole/L) Substance P (p595 col1 ¶3).
Sawada(2021) teach neonatal animals exposed to valproic acid develop significantly greater parvalbumin-positive neuron density (abstract).
It would have been obvious to one of ordinary skill in the art to adapt the methods of Liu drawn to a method of differentiation neural cells by using media comprising Substance and valproic acid as taught by Park and Sawada.
One of ordinary skill in the art would have been motivated to modify methods of Liu as taught by Park and Sawada because Park teaches Subsance P promotes adult neural progenitor cell proliferation and Sawada teach valproic acid promotes development of parvalbumin-positive neuron.
One would have had a reasonable expectation of success because the disclosures are all drawn to neuronal differentiation.
Claims 5, 25-27, 28-30 are rejected under 35 U.S.C. 103 as being unpatentable over Park et al (J Neurosurg (2007)107;593-599) and Sawada et al (PLOS ONE (2021) April;1-22)
as applied supra to claims 4 and 20-23; and further in view of May et al (Subcell Biochem (2012) 56:1-20) and Birgbauer et al (Neuromolecular Med (2021) 23:1;1-32).
Regarding claims 5, 25-26, 28-30: The teachings of Liu are discussed supra. Liu also teach culturing cells in culture media comprising BDNF, GDNF and IGF1 (step 30). BDNF is a BDNF pathway agonist. IGF1 is an IGF1 pathway agonist. Liu also teach the cells are cultured in NDM which comprises neurobasal medium (Gibco 21103), NEAA (1%) and N2 (1%) supplement (sept 30, p1673 col2). Neurobasal medium contains sodium pyruvate (25.0 mg/L) as evidenced by the ThermoFisher Neurobasal medium data sheet.
Liu teach culturing cells in media containing BDNF and IGF1 on days 26- 35, which corresponds to days 10-19 of the instant invention. MPEP 2131.03 reads “"[W]hen, as by a recitation of ranges or otherwise, a claim covers several compositions, the claim is ‘anticipated’ if one of them is in the prior art." Titanium Metals Corp. v. Banner, 778 F.2d 775, 227 USPQ 773 (Fed. Cir. 1985)”
MPEP 2131.03 reads “When the prior art discloses a range which touches or overlaps the claimed range, but no specific examples falling within the claimed range are disclosed, a case by case determination must be made as to anticipation. In order to anticipate the claims, the claimed subject matter must be disclosed in the reference with ‘sufficient specificity to constitute an anticipation under the statute.’”
MPEP 2131.03 further reads “If the prior art disclosure does not disclose a claimed range with "sufficient specificity" to anticipate a claimed invention, any evidence of unexpected results within the narrow range may render the claims nonobvious. See MPEP § 716.02 et seq.”.
In the case of the instant claim, the range disclosed by the prior art clearly overlaps with the claimed range, however in the in the absence of new or unexpected results for values outside the claimed range, the range disclosed by the cited Art is considered to disclose the claimed range with “sufficient specificity” to anticipate the claimed range. Thus the disclosure of Liu reads on days 12-26 of the instant invention, absent absence to the contrary.
Liu do not teach the media comprises ascorbic acid or lysophosphatidic acid.
May(2012) teach neurons in the central nervous system contain some of the highest concentrations of ascorbic acid (Vitamin C) of all mammalian tissues, and ascorbate may protect neurons from oxidant damage (abstract). May also teach ascorbate has a direct role in neuronal maturation and function (p5 ¶3).
Birgbauer(2021) teach Lysophosphatidic acid (LPA) plays a role in neural progenitor proliferation, survival and differentiation (p4 ¶3).
It would have been obvious to one of ordinary skill in the art to adapt the methods of Liu drawn to a method of differentiation neural cells by using media comprising ascorbic acid or lysophosphatidic acid as taught by May and Birgbauer.
One of ordinary skill in the art would have been motivated to modify methods of Liu as taught by May and Birgbauer because May teach ascorbic acid is protective as an antioxicant and has a role in neuronal maturation and Birgbauer teach LPA supports neural progenitor proliferation, survival and differentiation.
One would have had a reasonable expectation of success because the disclosures are drawn to in vitro neuronal differentiation.
Regarding claim 27: The teachings of Liu are discussed supra. Liu teach cells are cultured in , which comprises neurobasal medium, as discussed supra. Neurobasal medium comprises sodium pyruvate at a concentration of 250 mg/L (~227 uM), as evidenced by ThermoFisher Neurobasal fact sheet.
Liu also teach cells are cultured in Neuronal differentiation medium which is 98% neurobasal medium, for a final concentration of ~222uM sodium pyruvate. This reads on the instant claim limitation that sodium pyruvate is present at a concentration of 50-500uM.
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Liu et al (Nature Protocols (2013) 8:9; 1670-1679) as applied to claim 1, and further in view of Parsi et al (Japanese Dental Science Review (2020)56;50-55).
Regarding claim 8: The teachings of Liu are discussed supra. Liu also teach the cells are cultured on laminin-coated plates (step 17). Liu do not teach the stem cells are attached to vitronectin during culturing.
Parsi(2020) teach coating of substrates with fibronecin promotes cell adhesion (p52 col1 ¶2). Parsi also teach cell adhesion is fundamental for proliferation and differentiation, and lack of adhesion can result in apoptosis (p52 col1 ¶1).
It would have been obvious to one of ordinary skill in the art to adapt the methods of Liu drawn to a method of differentiation neural cells by using plates attached to vitronectin as taught by Parsi.
One of ordinary skill in the art would have been motivated to modify methods of Liu as taught by Parsi because Parsi teach fibronectin promotes cell adhesion and cell adhesion is important for proliferation and differentiation.
One would have had a reasonable expectation of success because Liu disclose cells cultured on laminin-coated plates and substituting one plate coating known to promote cell adhesion (laminin) for another plate coating which is known to promote cell adhesion (fibronectin) would have predictable results.
Claims 9 is rejected under 35 U.S.C. 103 as being unpatentable over Liu et al (Nature Protocols (2013) 8:9; 1670-1679) as applied to claim 1, and further in view of Kuwahara et al (US 2017/0313976).
Regarding claim 9: The teachings of Liu are discussed supra. Liu teach the BMP pathway antagonist insulin. Liu do not teach the BMP pathway antagonist is LDN193189.
Kuwahara(2017) teach a method for producing neural tissues and neural cells from pluripotent stem cells (p1 [0017]). Kuwahara further teach culture of pluripotent stem cells with a BMP signal antagonist (p2 [7]). Kuwarara teach culture of the cells with LDN193189 (p2 [8]). While Kuwahara(2017) discloses LDN193189 as a TGFβ pathway inhibitor, it is also known and a BMP signaling inhibitor.
It would have been prima facie obvious to substitute the BMP antagonist taught by Liu with the BMP antagonist taught by Kuwahara into because both inventions are drawn to differentiation of neuronal cells from pluripotent cells. There would have been a reasonable expectation that the LDN193189 of Kuwahara would work equivalently to the insulin in the invention of Liu because of both disclosures teach successful differentiation of neuronal cells, and the results would have been predictable.
Substitution of one element for another known in the field, wherein the result of the substitution would have been predictable, is considered to be obvious. See KSR International Co. v Teleflex Inc 82 USPQ2d 1385 (US 2007) at page 1395.
Claims 10 and 11 are rejected under 35 U.S.C. 103 as being unpatentable over Liu et al (Nature Protocols (2013) 8:9; 1670-1679) as applied to claim 1, and further in view of Qi et al (Nature Biotechnology (2017) 35:2;154-166).
Regarding claim 10: The teachings of Liu are discussed supra. Liu teach the MEK pathway antagonist putrescine. Liu do not teach the MEK pathway antagonist is PD0325901.
Qi(2017) teach culture of cells with the MAPK/ERK (MEK) pathway antagonist PD0325901 at 1uM and 8 uM (p155 col1 ¶5, p155 col2 ¶1). Qi also teach ERK inhibition has been proposed as a strategy to enhance overall neuronal differentiation in hPSCs (p155 col1/2 ¶5/1). Qi also teach PD0325901 accelerates the exit from pluripotentcy to neuroectodermal fates (Fig 6).
It would have been prima facie obvious to substitute the MEK antagonist taught by Liu with the MEK antagonist taught by Qi into because both inventions are drawn to differentiation of neuronal cells from pluripotent cells. There would have been a reasonable expectation that the PD0325901 of Qi would work equivalently to the putrescine in the invention of Liu because of both disclosures teach successful differentiation of neuronal cells, and the results would have been predictable.
Substitution of one element for another known in the field, wherein the result of the substitution would have been predictable, is considered to be obvious. See KSR International Co. v Teleflex Inc 82 USPQ2d 1385 (US 2007) at page 1395.
Regarding claim 11: The teachings of Liu are discussed supra. Liu teach culture of cells with corticosterone, a WNT pathway antagonist. Liu do not teach the WNT pathway antagonist is XAV939.
Qi(2017) teach culture of cells with the WNT pathway antagonist XAV939 at 5uM, 2uM and 1uM (Fig 2, p164 col2 ¶3). Qi teach XAV939 promotes anterior CNS identity (Fig 6).
It would have been prima facie obvious to substitute the WNT antagonist taught by Liu with the WNT antagonist taught by Qi into because both inventions are drawn to differentiation of neuronal cells from pluripotent cells. There would have been a reasonable expectation that the XAV939 of Qi would work equivalently to the XAV939 in the invention of Liu because of both disclosures teach successful differentiation of neuronal cells, and the results would have been predictable.
Substitution of one element for another known in the field, wherein the result of the substitution would have been predictable, is considered to be obvious. See KSR International Co. v Teleflex Inc 82 USPQ2d 1385 (US 2007) at page 1395.
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over Liu et al (Nature Protocols (2013) 8:9; 1670-1679) as applied to claims 1, and further in view of Guo et al (Cell Research (2009) 19;71-88).
Regarding claim 13: The teachings of Liu are discussed supra. Liu teach culture of cells with linolenic acid, an AKT pathway antagonist. Liu do not teach the AKT pathway antagonist is MK2206.
Guo(2020) teach use of MK2206 at 20uM (Fig 7) to as a AKT pathway antagonist (Fig 7, p8 col1/2 ¶3/1).
It would have been prima facie obvious to substitute the AKT antagonist taught by Liu with the AKT antagonist taught by Guo because both inventions are drawn to differentiation of multipotent stem cells. There would have been a reasonable expectation that the MK2206 of Guo would work equivalently to the linolenic acid in the invention of Liu because of both disclosures teach successful differentiation of multipotent stem cells, and the results would have been predictable.
Substitution of one element for another known in the field, wherein the result of the substitution would have been predictable, is considered to be obvious. See KSR International Co. v Teleflex Inc 82 USPQ2d 1385 (US 2007) at page 1395.
Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over Liu et al (Nature Protocols (2013) 8:9; 1670-1679) as applied to claims 1, 6, 7, 12 , and further in view of Yang et al (Molecular Medicine Reports (2020) 22;4763-4771).
Regarding claim 14: The teachings of Liu are discussed supra. Liu teach cells are cultured with glutathione, a PKC pathway antagonist. Liu do not teach the PKC pathway antagonist is Go6983.
Yang(2020) teach culture of cells with Go6983 at 5 uM to direct neural differentiation from human fibroblasts (abstract, p4764 col1 ¶4).
It would have been prima facie obvious to substitute the PKC antagonist taught by Liu with the PKC antagonist taught by Yang because both inventions are drawn to differentiation of neuronal cells. There would have been a reasonable expectation that the Go6983 of Yang would work equivalently to the glutathione in the invention of Liu because of both disclosures teach successful differentiation of neuronal cells, and the results would have been predictable.
Substitution of one element for another known in the field, wherein the result of the substitution would have been predictable, is considered to be obvious. See KSR International Co. v Teleflex Inc 82 USPQ2d 1385 (US 2007) at page 1395.
Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over over Read et al (Cell. Mol. Life Sci. (2009) 66:1-10) and Dominguez-Garcia et al (Cell Death and Disease (2020) 11:262;1-19) as applied supra to claim 2; and further in view of Neubert et al (Cell Death and Differentiation (2011)12:1-10) and Totzke et al (Cell Chemical Biology (2017) 24;1029-1039).
Regarding claim 15: The teachings of Liu and Neubert are discussed supra. Liu and Neubert teach the Tak1 pathway antagonist 5Z-7-oxozeaenol (5ZO). Liu and Neubert do not teach the TAK1 inhibitor is Takinib.
Totzke(2017) teach 5ZO potently inhibits a panel of at least 50 other kinases in addition to Tak1, and is inadequate for therapeutic purposes due to off-target effects (p1031/1032 col2/1 ¶2/1). Totzke further teach Takinib is easily synthesized and exhibits extraordinary selectivity for Tak1 (p1032 col1 ¶2).
It would have been obvious to one of ordinary skill in the art to adapt the methods of Liu and Neubert drawn to a method of differentiation neural cells using media comprising the Tak1 inhibitor 5Zo by using media comprising the Tak1 inhibitor Takinib as taught by Totzke.
One of ordinary skill in the art would have been motivated to modify methods of Liu as taught by Totzke because Totzke teach 5Zo is easily synthesized and more selective than 5ZO.
One would have had a reasonable expectation of success because Totzke discloses Takinib works in a cell culture system (abstract).
Claim 24 is rejected under 35 U.S.C. 103 as being unpatentable over over Park et al (J Neurosurg (2007)107;593-599) and Sawada et al (PLOS ONE (2021) April;1-22)
as applied supra to claim 4; and further in view of Silva et al (Nature Communications (2020)11:3258;1-18).
Regarding claim 24: As discussed supra, the claim is interpreted as requiring MHY1485.
The teachings of Liu are discussed supra. Liu teach culturing cells with BDNF (an mTOR signaling agonist). Liu do not teach the mTor pathway agonist is MHY1485.
Silva(2020) teach culture of cells with MHY1485 (1uM and 2.5 uM) to activate mTor (p9 col2 ¶3). Silva further teach MHY1485 is a potent mTor activator.
It would have been prima facie obvious to substitute the mTor antagonist taught by Liu with the mTor antagonist taught by Siva because both inventions are drawn neuronal cells differentiated from pluripotent cells. There would have been a reasonable expectation that MHY1485 of Silva would work equivalently to the BDNF in the invention of Liu because of both disclosures teach use of the compounds in neuronal cells, and the results would have been predictable.
Substitution of one element for another known in the field, wherein the result of the substitution would have been predictable, is considered to be obvious. See KSR International Co. v Teleflex Inc 82 USPQ2d 1385 (US 2007) at page 1395.
Claim 31 is rejected 35 U.S.C. 103 as being unpatentable in view of Liu et al (Nature Protocols (2013) 8:9; 1670-1679) and as evidenced by B27 Datasheet (B-27 Supplement [online]. ThermoFisher corporation [retrieved on 01/30/2026]. Retrieved from the Internet: <URL: B-27 Supplement Minus Vitamin A (50X) liquid | Thermo Fisher Scientific - US) , Xing et al (Biochem and Biophys Research Comm (2015)456;605-609), Hidebrandt(Scientific Reports (2018)8:8711;1-9), Odaka et al (Neural regeneration Research (2017)12:7;1-8), Wang et al (Biochem J (2011)426:3;1-26), Guo et al (Archives of Medical Research (2008) 39;434-442), Ward (The Journal of Biological Chemistry (1998) 273:20;12558-12566) and further in view of Read et al (Cell. Mol. Life Sci. (2009) 66:1-10) and Dominguez-Garcia et al (Cell Death and Disease (2020) 11:262;1-19).
Regarding claim 31(a): Liu(2013) teach maturation of parvalbumin positive (PV) neurons from human embryonic stem cells and induced pluripotent stem cells (abstract, Figure 5c). Liu teach the cells are cultured with medium comprising 1.5 mM purmorphamin (an SHH agnonist) and B27 supplement (step 24).
As evidenced by the B27 datasheet (Gibo Ca 12587-010) and discussed below, the B27 supplement contains insulin (a BMP pathway antagonist), putrescine (an MEK antagonist), corticosterone (a WNT pathway antagonist and AKT pathway antagonist), glutathione (PKC pathway antagonist) and linolenic acid (AKT pathway antagonist).
Xing(2015) teach insulin inhibits BMP2 activity and thus supplementation of B27 supplement reads on a BMP pathway antagonist. Hildebrandt(2018) teaches corticosterone reduces expression of Wnt7b and Wnt10, therefore B27 also reads on a WNT antagonist. Odaka(2017) teach corticosterone also inhibits AKT signaling (Fig 2), thus B27 supplement reads on an AKT antagonist. Wang(2011) teach putrescine inhibits MEK mRNA and protein, and thus B27 supplement also reads on an MEK pathway antagonist. Guo(2008) teaches Linoleic acid inhibits the AKT pathway (title). Thus the B27 reads on an AKT pathway antagonist. Ward(1998) teach glutathione inactivates PKC (title), and thus B27 supplement reads on a PKC antagonist.
Liu further teach cells are cultured for 48h, and a media exchange is conducted with the identical media and cells are cultured for an additional 24 hours. This reads on days 0-3 of the claimed invention. Day 16 of the disclosure of Liu is interpreted as corresponding to day 0 of the instant invention.
While Liu is silent on the characterization of the cells as OTX2+ FEZF2+ SIX3+ FB-NSCs, the Liu teach the active steps identical to the active steps of the claimed method, and thus the cells generated by the disclosure of Liu would be identical to those disclosed in the instant claim 31a.
Regarding claim 31(b): The teachings of Liu are described supra. Liu disclose further media exchanges with the media disclosed supra over days 3-6 of the cell culture. Liu do not disclose media lacking an AKT pathway antagonist and a PKC pathway antagonist.
Read(2009) teach Akt promotes neuronal differentiation and promotes neuronal survival (p2 col1 ¶2). Dominguez-Garcia(2020) teach PKC signaling regulates neurogenesis through the release of neurogenic growth factors (Abstract).
It would have been obvious to one of ordinary skill in the art to adapt the methods of Liu drawn to generating parvalbumin positive (PV) neurons from human stem cells by doing using media lacking an AKT antagonist as taught by Read and lacking a PKC antagonist as taught by Dominguez-Garcies.
One of ordinary skill in the art would have been motivated to modify the method of Liu as taught by Read and Dominguez-Garcia to for the purposes of generating ventral forebrain neural stem cells because Reed and Dominguez-Gacia teach these factors promote neuronal differentiation and survival, and thus one of ordinary skill in the art would understand that omitting the factors which inhibit the pathways which promote neurogenesis and differentiation would improve efficiency of a neural differentiation method.
One would have had a reasonable expectation of success because Dominggez-Garcia teaches PKC activation promotes neuroblast differentiation (abstract) and Reed teach AKT is well documented to promote neuronal survival (abstract).
While Liu, Reed and Dominguez-Garcia are silent on the characterization of the cells as NKX2-1 + VFB-NSCs, the combination of Liu, Reed and Dominguez-Garcia teach the active steps identical to the active steps of the claimed method, and thus the cells generated by the combination of Liu, Reed and Dominguez-Garcia would be identical to those disclosed in the instant claim 31b.
Claim 32 is rejected 35 U.S.C. 103 as being unpatentable in view the art as applied to claim 31, and further in view of Kuwahara et al (US 2017/0313976), Qi et al (Nature Biotechnology (2017) 35:2;154-166), Liu et al (Nature Protocols (2013) 8:9; 1670-1679), Guo et al (Archives of Medical Research (2008) 39;434-442), Yang et al (Molecular Medicine Reports (2020) 22;4763-4771),
Regarding claim 32: Kuwahara(2017) teach a method for producing neural tissues and neural cells from pluripotent stem cells (p1 [0017]). Kuwahara further teach culture of pluripotent stem cells with a BMP signal antagonist (p2 [7]). Kuwarara teach culture of the cells with LDN193189 (p2 [8]). While Kuwahara(2017) discloses LDN193189 as a TGFβ pathway inhibitor, it is also known and a BMP signaling inhibitor.
Qi(2017) teach culture of cells with the MAPK/ERK (MEK) pathway antagonist PD0325901 at 1uM and 8 uM (p155 col1 ¶5, p155 col2 ¶1). Qi also teach ERK inhibition has been proposed as a strategy to enhance overall neuronal differentiation in hPSCs (p155 col1/2 ¶5/1). Qi also teach PD0325901 accelerates the exit from pluripotentcy to neuroectodermal fates (Fig 6).
Qi(2017) teach culture of cells with the WNT pathway antagonist XAV939 at 5uM, 2uM and 1uM (Fig 2, p164 col2 ¶3). Qi teach XAV939 promotes anterior CNS identity (Fig 6).
Liu teach the cells are cultured with medium comprising 1.5 mM purmorphamine (step 24).
Guo(2020) teach use of MK2206 at 20uM (Fig 7) to as a AKT pathway antagonist (Fig 7, p8 col1/2 ¶3/1).
Yang(2020) teach culture of cells with Go6983 at 5 uM to direct neural differentiation from human fibroblasts (abstract, p4764 col1 ¶4).
It would have been obvious to modify the method as taught by the disclosure of claim 31, as discussed supra for claim 31, with the combined teachings of the specific pathway agents and concentrations as disclosed by the prior art herein. Pathway agents to manipulate cell culture systems, and specifically neuronal differentiation protocols, were known in the art and one of ordinary skill in the art would have had a reasonable expectation of success because the agents and concentrations were shown to be effective for in vitro cell culture, and in most cases in neuronal differentiation from pluripotent stem cells thus one would have had a reasonable expectation of success and the results would have been predictable.
Claim 33 is rejected is rejected under 35 U.S.C. 103 as being unpatentable in view of Liu et al (Nature Protocols (2013) 8:9; 1670-1679) and as evidenced by B27 Datasheet (B-27 Supplement [online]. ThermoFisher corporation [retrieved on 01/30/2026]. Retrieved from the Internet: <URL: B-27 Supplement Minus Vitamin A (50X) liquid | Thermo Fisher Scientific - US) , Xing et al (Biochem and Biophys Research Comm (2015)456;605-609), Hidebrandt(Scientific Reports (2018)8:8711;1-9), Odaka et al (Neural regeneration Research (2017)12:7;1-8), Wang et al (Biochem J (2011)426:3;1-26), Guo et al (Archives of Medical Research (2008) 39;434-442), Ward (The Journal of Biological Chemistry (1998) 273:20;12558-12566) and further in view of Read et al (Cell. Mol. Life Sci. (2009) 66:1-10), Dominguez-Garcia et al (Cell Death and Disease (2020) 11:262;1-19), Neubert et al (Cell Death and Differentiation (2011)18;1-10), Valizadeh-Arshad et al (Cell J (2018) 19:4;545-551), Qi et al (Nature Biotechnology (2017) 35:2;154-166) and Bilenker et al (US 2021/0023086).
Regarding claim 33(a): Liu(2013) teach maturation of parvalbumin positive (PV) neurons from human embryonic stem cells and induced pluripotent stem cells (abstract, Figure 5c). Liu teach the cells are cultured with medium comprising 1.5 mM purmorphamin (an SHH agnonist) and B27 supplement (step 24).
As evidenced by the B27 datasheet (Gibo Ca 12587-010) and discussed below, the B27 supplement contains insulin (a BMP pathway antagonist), putrescine (an MEK antagonist), corticosterone (a WNT pathway antagonist and AKT pathway antagonist), glutathione (PKC pathway antagonist) and linolenic acid (AKT pathway antagonist).
Xing(2015) teach insulin inhibits BMP2 activity and thus supplementation of B27 supplement reads on a BMP pathway antagonist. Hildebrandt(2018) teaches corticosterone reduces expression of Wnt7b and Wnt10, therefore B27 also reads on a WNT antagonist. Odaka(2017) teach corticosterone also inhibits AKT signaling (Fig 2), thus B27 supplement reads on an AKT antagonist. Wang(2011) teach putrescine inhibits MEK mRNA and protein, and thus B27 supplement also reads on an MEK pathway antagonist. Guo(2008) teaches Linoleic acid inhibits the AKT pathway (title). Thus the B27 reads on an AKT pathway antagonist. Ward(1998) teach glutathione inactivates PKC (title), and thus B27 supplement reads on a PKC antagonist.
Liu further teach cells are cultured for 48h, and a media exchange is conducted with the identical media and cells are cultured for an additional 24 hours. This reads on days 0-3 of the claimed invention. Day 16 of the disclosure of Liu is interpreted as corresponding to day 0 of the instant invention.
While Liu is silent on the characterization of the cells as OTX2+ FEZF2+ SIX3+ FB-NSCs, the Liu teach the active steps identical to the active steps of the claimed method, and thus the cells generated by the disclosure of Liu would be identical to those disclosed in the instant claim 31a.
Regarding claim 33(b): The teachings of Liu are described supra. Liu disclose further media exchanges with the media disclosed supra over days 3-6 of the cell culture. Liu do not disclose media lacking an AKT pathway antagonist and a PKC pathway antagonist.
Read(2009) teach Akt promotes neuronal differentiation and promotes neuronal survival (p2 col1 ¶2). Dominguez-Garcia(2020) teach PKC signaling regulates neurogenesis through the release of neurogenic growth factors (Abstract).
It would have been obvious to one of ordinary skill in the art to adapt the methods of Liu drawn to generating parvalbumin positive (PV) neurons from human stem cells by doing using media lacking an AKT antagonist as taught by Read and lacking a PKC antagonist as taught by Dominguez-Garcies.
One of ordinary skill in the art would have been motivated to modify the method of Liu as taught by Read and Dominguez-Garcia to for the purposes of generating ventral forebrain neural stem cells because Reed and Dominguez-Gacia teach these factors promote neuronal differentiation and survival, and thus one of ordinary skill in the art would understand that omitting the factors which inhibit the pathways which promote neurogenesis and differentiation would improve efficiency of a neural differentiation method.
One would have had a reasonable expectation of success because Dominggez-Garcia teaches PKC activation promotes neuroblast differentiation (abstract) and Reed teach AKT is well documented to promote neuronal survival (abstract).
While Liu, Reed and Dominguez-Garcia are silent on the characterization of the cells as NKX2-1 + VFB-NSCs, the combination of Liu, Reed and Dominguez-Garcia teach the active steps identical to the active steps of the claimed method, and thus the cells generated by the combination of Liu, Reed and Dominguez-Garcia would be identical to those disclosed in the instant claim 31b.
Regarding claim 33(c): The teachings of Liu are described supra. Liu teach culturing the cells as described supra, with a SSH pathway agonist.
Liu does not disclose culturing cells in media that comprises a TAK1 pathway antagonist, a TGF-beta pathway antagonist, a TRK pathway antagonist a Notch pathway antagonist and an IGF1 pathway antagonist on days 22-24 (equivalent to days 6-9 of the instant invention).
While Liu do teach culturing cells with the IGF1 pathway agonist IGF1 at 10 ng/ml (step 30) at day 26 (step 30). Liu do not teach culturing cells with IGF on days 22-24 (corresponding to days 6-9 of the instant invention).
Neubert(2011) teach inhibition of Tak1 protects against neuronal cell death in ischemia models. Neubert further teach the TAK1 inhibitor is 5Z-7-oxozeaenol (5ZO).
Valizadeh-Arshad(2018) teach inhibition of TGFβ, BMP and WNT signaling pathways promotes differentiation of pluripotent stem cells (hESCs) along the neuronal lineage (p545 col2 ¶1). Valizadeh-Arshad further teach A8301 is a selective inhibitor for the TGFβ signaling (p545 col1 ¶1) and use of A8301 promoted neural differentiation effectively (p550 col2 ¶3).
Qi(2017) teach use of the notch pathway antagonist DAPT (also known as GSIXX) at 10 uM and 5uM for the differentiation of functional neurons(p155 col1 ¶2; p164 col2 ¶3). Qi teach DAPT rapidly induces CNS lineage and triggers neuronal differentiation (p155 ¶4).
Thiele et al (Molecular Pathways (2009) teach that Trk was first identified as an oncogene when constitutively active and is capable of transforming immortalized fibroblasts (p1 col1 ¶1). Thiele further teach downstream signaling of TRK can regulate proliferation vs differentiation (p2 col2 ¶1). Bilenker(2021) teach culture of cells with the TRK pathway GNF-5837 antagonist (claim 2).
It would have been obvious to one of ordinary skill in the art to adapt the methods of Liu drawn to a method of differentiation neural cells by using media comprising a TAK1 antagonist, a TGF-beta antagonist, a TRK antagonist, a notch antagonist and and IGF1 antagonist as taught by Neubert, Valizadeh-Arshad, Qi, Thiele and Bilenker.
One of ordinary skill in the art would have been motivated to modify the method of Liu with the teachings of Neubert, Valizadeh-Arshad, Qi, Thiele and Bilenker because Neubert teach a TAK1 inhibitor will protect against neuronal cell death, Valizadeh-Arshad teach TGFβ, BMP and WNT promote neuronal differentiation, Qi teach notch antagonism promotes differentiation of functional neurons and Thiel teach TRK regulates proliferation vs differentiation.
One would have had a reasonable expectation of success because Neubert teach TAK1 inhibition protects neurons in vitro (abstract), Valizadeh-Arshad teach successful neuron differentiation from stem cells, and Qi teach successful differentiation of functional neurons and Bilenker teaches use of the TRK antagonist in cell cultures.
Claim 34 is rejected 35 U.S.C. 103 as being unpatentable in view the art as applied to claim 33, and further in view of Kuwahara et al (US 2017/0313976), Qi et al (Nature Biotechnology (2017) 35:2;154-166), Liu et al (Nature Protocols (2013) 8:9; 1670-1679), Guo et al (Archives of Medical Research (2008) 39;434-442), Yang et al (Molecular Medicine Reports (2020) 22;4763-4771), Totzke et al (Cell Chemical Biology (2017) 24;1029-1039), Valizadeh-Arshad et al (Cell J (2018) 19:4;545-551) and Bilenker et al (US 2021/0023086).
Regarding claim 34: Kuwahara(2017) teach a method for producing neural tissues and neural cells from pluripotent stem cells (p1 [0017]). Kuwahara further teach culture of pluripotent stem cells with a BMP signal antagonist (p2 [7]). Kuwarara teach culture of the cells with LDN193189 (p2 [8]). While Kuwahara(2017) discloses LDN193189 as a TGFβ pathway inhibitor, it is also known and a BMP signaling inhibitor.
Qi(2017) teach culture of cells with the MAPK/ERK (MEK) pathway antagonist PD0325901 at 1uM and 8 uM (p155 col1 ¶5, p155 col2 ¶1). Qi also teach ERK inhibition has been proposed as a strategy to enhance overall neuronal differentiation in hPSCs (p155 col1/2 ¶5/1). Qi also teach PD0325901 accelerates the exit from pluripotentcy to neuroectodermal fates (Fig 6).
Qi(2017) teach culture of cells with the WNT pathway antagonist XAV939 at 5uM, 2uM and 1uM (Fig 2, p164 col2 ¶3). Qi teach XAV939 promotes anterior CNS identity (Fig 6).
Liu teach the cells are cultured with medium comprising 1.5 mM purmorphamine (step 24).
Guo(2020) teach use of MK2206 at 20uM (Fig 7) to as a AKT pathway antagonist (Fig 7, p8 col1/2 ¶3/1).
Yang(2020) teach culture of cells with Go6983 at 5 uM to direct neural differentiation from human fibroblasts (abstract, p4764 col1 ¶4).
Totzke teach Takinib is easily synthesized and exhibits extraordinary selectivity for Tak1 (p1032 col1 ¶2). Totzke further teach treatment of cells with Takinib at concentrations including 1uM (Figure 5A).
Valizadeh-Arshad(2018) teach inhibition of TGFβ, BMP and WNT signaling pathways promotes differentiation of pluripotent stem cells (hESCs) along the neuronal lineage (p545 col2 ¶1). Valizadeh-Arshad further teach A8301 is a selective inhibitor for the TGFβ signaling (p545 col1 ¶1) and use of A8301 promoted neural differentiation effectively (p550 col2 ¶3).
Bilenker(2021) teach culture of cells with the TRK pathway GNF-5837 antagonist (claim 2).
Qi(2017) teach use of the notch pathway antagonist DAPT (also known as GSIXX) at 10 uM and 5uM for the differentiation of functional neurons (p155 col1 ¶2; p164 col2 ¶3). Qi teach DAPT rapidly induces CNS lineage and triggers neuronal differentiation (p155 ¶4).
Liu teach culturing cells with IGF1 pathway agonist IGF1 at 10 ng/ml (step 30).
It would have been obvious to modify the method as taught by the disclosure of claim 34, as discussed supra for claim 33, with the combined teachings of the specific pathway agents and concentrations as disclosed by the prior art. Pathway agents to manipulate cell culture systems, and specifically neuronal differentiation protocols, were known in the art and one of ordinary skill in the art would have had a reasonable expectation of success because the agents and concentrations were shown to be effective for in vitro cell culture, and in most cases in neuronal differentiation from pluripotent stem cells thus one would have had a reasonable expectation of success and the results would have been predictable.
Claim 35 is rejected under 35 U.S.C. 103 as being unpatentable over Neubert et al (Cell Death and Differentiation (2011)18;1-10), Valizadeh-Arshad et al (Cell J (2018) 19:4;545-551), Qi et al (Nature Biotechnology (2017) 35:2;154-166) and Bilenker et al (US 2021/0023086) as applied supra to claims 3, 17 and 19; and further in view of Park et al (J Neurosurg (2007)107;593-599), Sawada et al (PLOS ONE (2021) April;1-22), May et al (Subcell Biochem (2012) 56:1-20) and Birgbauer et al (Neuromolecular Med (2021) 23:1;1-32).
Regarding claim 35(a): The teachings of Liu are discussed supra. Liu teach that on day 26 cells are cultured with cAMP (a PKA activator), BDNF (an mTOR signaling agonist), GDNF (a GDNF pathway agonist) and (step 27).
Liu do not teach the media comprises Valproic acid or Substance P.
Park(2007) teach Substance P promotes adult neural progenitor cell proliferation in vitro (abstract). Park further teach cells are cultured with 0.1 umol/L (100 nanomole/L) Substance P (p595 col1 ¶3).
Sawada(2021) teach neonatal animals exposed to valproic acid develop significantly greater parvalbumin-positive neuron density (abstract).
It would have been obvious to one of ordinary skill in the art to adapt the methods of Liu drawn to a method of differentiation neural cells by using media comprising Substance and valproic acid as taught by Park and Sawada.
One of ordinary skill in the art would have been motivated to modify methods of Liu as taught by Park and Sawada because Park teaches Subsance P promotes adult neural progenitor cell proliferation and Sawada teach valproic acid promotes development of parvalbumin-positive neuron.
One would have had a reasonable expectation of success because the disclosures are all drawn to neuronal differentiation.
Regarding claim 35(b): The teachings of Liu are discussed supra. Liu also teach culturing cells in culture media comprising BDNF, GDNF and IGF1 (step 30). BDNF is a BDNF pathway agonist. IGF1 is an IGF1 pathway agonist. Liu also teach the cells are cultured in NDM which comprises neurobasal medium (Gibco 21103), NEAA (1%) and N2 (1%) supplement (sept 30, p1673 col2). Neurobasal medium contains sodium pyruvate (25.0 mg/L) as evidenced by the ThermoFisher Neurobasal medium data sheet.
Liu teach culturing cells in media containing BDNF and IGF1 on days 26- 35, which corresponds to days 10-19 of the instant invention. MPEP 2131.03 reads “"[W]hen, as by a recitation of ranges or otherwise, a claim covers several compositions, the claim is ‘anticipated’ if one of them is in the prior art." Titanium Metals Corp. v. Banner, 778 F.2d 775, 227 USPQ 773 (Fed. Cir. 1985)”
MPEP 2131.03 reads “When the prior art discloses a range which touches or overlaps the claimed range, but no specific examples falling within the claimed range are disclosed, a case by case determination must be made as to anticipation. In order to anticipate the claims, the claimed subject matter must be disclosed in the reference with ‘sufficient specificity to constitute an anticipation under the statute.’”
MPEP 2131.03 further reads “If the prior art disclosure does not disclose a claimed range with "sufficient specificity" to anticipate a claimed invention, any evidence of unexpected results within the narrow range may render the claims nonobvious. See MPEP § 716.02 et seq.”.
In the case of the instant claim, the range disclosed by the prior art clearly overlaps with the claimed range, however in the in the absence of new or unexpected results for values outside the claimed range, the range disclosed by the cited Art is considered to disclose the claimed range with “sufficient specificity” to anticipate the claimed range. Thus the disclosure of Liu reads on days 12-26 of the instant invention, absent absence to the contrary.
Liu do not teach the media comprises ascorbic acid or lysophosphatidic acid.
May(2012) teach neurons in the central nervous system contain some of the highest concentrations of ascorbic acid (Vitamin C) of all mammalian tissues, and ascorbate may protect neurons from oxidant damage (abstract). May also teach ascorbate has a direct role in neuronal maturation and function (p5 ¶3).
Birgbauer(2021) teach Lysophosphatidic acid (LPA) plays a role in neural progenitor proliferation, survival and differentiation (p4 ¶3).
It would have been obvious to one of ordinary skill in the art to adapt the methods of Liu drawn to a method of differentiation neural cells by using media comprising ascorbic acid or lysophosphatidic acid as taught by May and Birgbauer.
One of ordinary skill in the art would have been motivated to modify methods of Liu as taught by May and Birgbauer because May teach ascorbic acid is protective as an antioxicant and has a role in neuronal maturation and Birgbauer teach LPA supports neural progenitor proliferation, survival and differentiation.
One would have had a reasonable expectation of success because the disclosures are drawn to in vitro neuronal differentiation.
Claim 36 is rejected 35 U.S.C. 103 as being unpatentable in view the art as applied to claim 35, and further in view of Liu et al (Nature Protocols (2013) 8:9; 1670-1679), Sawada et al (PLOS ONE (2021) April;1-22), Park et al (J Neurosurg (2007)107;593-599), Silva et al (Nature Communications (2020)11:3258;1-18), May et al (Subcell Biochem (2012) 56:1-20), and Birgbauer et al (Neuromolecular Med (2021) 23:1;1-32).
Regarding claim 36: Liu teach that on day 26 cells are cultured with cAMP (step 27).
Sawada(2021) teach neonatal animals exposed to valproic acid develop significantly greater parvalbumin-positive neuron density (abstract).
Park(2007) teach Substance P promotes adult neural progenitor cell proliferation in vitro (abstract). Park further teach cells are cultured with 0.1 umol/L (100 nanomole/L) Substance P (p595 col1 ¶3).
Liu teach culturing cells in culture media comprising BDNF, GDNF and IGF1 (step 30).
Silva(2020) teach culture of cells with MHY1485 (1uM and 2.5 uM) to activate mTor (p9 col2 ¶3). Silva further teach MHY1485 is a potent mTor activator.
May(2012) teach neurons in the central nervous system contain some of the highest concentrations of ascorbic acid (Vitamin C) of all mammalian tissues, and ascorbate may protect neurons from oxidant damage (abstract). May also teach ascorbate has a direct role in neuronal maturation and function (p5 ¶3). It would be obvious to use a ascorbic acid analog such as 2-phospho-L-ascorbic acid because the analog is known in the art to be more stable in cell culture media (evidenced by R&D systems 2-Phospho-L-ascorbic acid trisodium salt | Compounds for Stem Cell Differentiation: Tocris Bioscience).
Liu also teach the cells are cultured in NDM which comprises neurobasal medium (Gibco 21103), NEAA (1%) and N2 (1%) supplement (sept 30, p1673 col2)Neurobasal medium contains sodium pyruvate (25.0 mg/L) as evidenced by the ThermoFisher Neurobasal medium data sheet.
Birgbauer(2021) teach Lysophosphatidic acid (LPA) plays a role in neural progenitor proliferation, survival and differentiation (p4 ¶3).
It would have been obvious to modify the method as taught by the disclosure of claim 36, as discussed supra for claim 36, with the combined teachings of the specific pathway agents and concentrations as disclosed by the prior art as discussed supra. Pathway agents to manipulate cell culture systems, and specifically neuronal differentiation protocols, were known in the art and one of ordinary skill in the art would have had a reasonable expectation of success because the agents and concentrations were shown to be effective for in vitro cell culture, and in most cases in neuronal differentiation from pluripotent stem cells thus one would have had a reasonable expectation of success and the results would have been predictable.
Conclusion
No claims are allowed
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ANDREA LYNNE MORRIS SPENCER whose telephone number is (571)272-3328. The examiner can normally be reached Monday-Friday 9:30-5:00.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, James (Doug) Schultz can be reached at 571-272-0763. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/ANDREA LYNNE MORRIS SPENCER/Examiner, Art Unit 1631
/JAMES D SCHULTZ/Supervisory Patent Examiner, Art Unit 1631