DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election without traverse of Group II, claims 3-6, in the reply filed on 01/21/2026 is acknowledged.
Claims 1-4 and 7-45 withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected Groups, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 01/21/2026.
Status of the Claims
Claims 1-45 are currently pending.
Claims 1-4, 31-33, and 40-43 are amended.
Claims 1-2 and 7-45 have been withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected Invention, there being no allowable generic or linking claim.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 3-6 have been considered 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 3-6 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph.
Regarding independent claim 3, the specification is enabling for:
A method of generating human TH+ KCNJ6+ mature dopaminergic neurons comprising:
(a) culturing human OTX2+ FOXA2+ LMX1A+ midbrain neural progenitor cells (MB NPCs) in a culture media comprising a Wingless-related integration site (WNT) pathway agonist, a Mammalian Target of Rapamycin (mTOR) pathway antagonist, a retinoic acid receptor (RAR) pathway antagonist, a MEK pathway antagonist, a Notch pathway antagonist, and a Bone Morphogenetic Protein (BMP) pathway agonist for three days (day 0-day 3) to obtain human EOXA2+ LMX1A+ MSX1+ PITX3+ DCX+ immature midbrain neurons (MB-immature neurons);
wherein in step (a) the WNT pathway agonist is CHIR99021 at a concentration of 1 µM, the mTOR pathway antagonist is AZD3147 at a concentration of 15 nM, the RAR antagonist is AGN193109 at a concentration of 100 nM, the MEK pathway antagonist is PD0325901 at a concentration of 100 nM, the Notch pathway antagonist is DBZ at a concentration of 100 nM, and the BMP pathway agonist is BMP7 at a concentration of 15 ng/ml; and
(b) culturing the MB-immature neurons in a culture media comprising a Brain-Derived Neurotrophic (BDNF) pathway agonist, a Glial cell line-Derived Neurotrophic Factor (GDNF) pathway agonist, a Peroxisome Proliferator-Activated Receptor Alpha (PPAR-a) pathway agonist, heparin or heparin mimetic, a Notch pathway antagonist, and a dopamine agonist for at least fourteen days (day 3-day 17) to obtain TH+ KCNJ6+ mature dopaminergic neurons;
wherein in step (b) the BDNF pathway agonist is BDNF at a concentration of 10 ng/ml, the GDNF pathway agonist is GDNF at a concentration of 10 ng/ml, the PPAR-a pathway agonist is GW7647 at a concentration of 250 nM, the heparin or heparin mimetic is heparin art a concentration of 5 µg/ml, the notch pathway antagonist is DBZ at a concentration of 100 nM, and the dopamine agonist is dopamine at a concentration of 10 µM.
However, the specification does not reasonably provide enablement for the method of claim 3 employing (i) any agonist or antagonist of the claimed pathways provided at (ii) any concentration.
Regarding independent claim 4, the specification is enabling for:
A method of generating human TH+ KCNJ6+ mature dopaminergic neurons comprising:
(a) culturing human pluripotent stem cells in a culture media comprising a Wingless-related integration site (WNT) pathway agonist, a Sonic Hedgehog (SHH) pathway agonist, a Bone Morphogenetic Protein (BMP) pathway antagonist, an AKT pathway antagonist, and a MEK pathway antagonist on days 0-3 to obtain committed midbrain neural stem cells (MB NSCs);
wherein in step (a) the WNT pathway agonist is CHIR99021 at a concentration of 1.1 µm, the SHH pathway agonist is purmorphamine at a concentration of 550 nM, the BMP pathway antagonist is LDN193189 at a concentration of 275 nM, the AKT pathway antagonist is MK2206 at a concentration of 138 nM, and the MEK pathway antagonist is PD0325901; and
(b) culturing the MB NSCs in a culture media comprising a BMP pathway agonist, a retinoic acid (RA) pathway agonist, a Liver X Receptor (LXR) pathway agonist, an AKT pathway antagonist, a Mammalian Target of Rapamycin (mTOR) pathway antagonist, and a Transforming Growth Factor Beta (TGFb) pathway antagonist on days 4-6 to obtain human OTX2+ FOXA2+ LMX1A+ midbrain neural progenitor cells (MB NPCs);
wherein in step (b) the BMP pathway agonist is BMP7 at a concentration of 15 ng/ml, the RA pathway agonist is TTNPB at a concentration of 50 nM, the LXR pathway agonist is GW3965, the AKT pathway antagonist is MK2206 at a concentration of 50 nM, the mTOR pathway antagonist is AXD3147 at a concentration of 15 nM, the TGFb pathway antagonist is A83-01 at a concentration of 300 nM; and
(c) culturing the MB NPCs in a culture media comprising a WNT pathway agonist, an mTOR pathway antagonist, a retinoic acid receptor (RAR) pathway antagonist, a MEK pathway antagonist, a Notch pathway antagonist, and a BMP pathway agonist on days 6-9 to obtain human FOXA2+ LMX1A+ MSX1+ PITX3+ DCX+ immature midbrain neurons (MB-immature neurons); and
wherein in step (c) the WNT pathway agonist is CHIR99021 at a concentration of 1 µM, the mTOR pathway antagonist is AZD3147 at a concentration of 15 nM, the RAR antagonist is AGN193109 at a concentration of 100 nM, the MEK pathway antagonist is PD0325901 at a concentration of 100 nM, the Notch pathway antagonist is DBZ at a concentration of 100 nM, and the BMP pathway agonist is BMP7 at a concentration of 15 ng/ml; and
(d) culturing the MB-immature neurons in a culture media comprising a Brain-Derived Neurotrophic Factor (BDNF) pathway agonist, a Glial cell line-Derived Neurotrophic Factor (GDNF) pathway agonist, a Peroxisome Proliferator-Activated Receptor Alpha (PPAR-a) pathway agonist, heparin or heparin mimetic, a Notch pathway antagonist, and a dopamine agonist on days 9-23 to obtain TH+ KCNJ6+ mature dopaminergic neurons;
wherein in step (b) the BDNF pathway agonist is BDNF at a concentration of 10 ng/ml, the GDNF pathway agonist is GDNF at a concentration of 10 ng/ml, the PPAR-a pathway agonist is GW7647 at a concentration of 250 nM, the heparin or heparin mimetic is heparin art a concentration of 5 µg/ml, the notch pathway antagonist is DBZ at a concentration of 100 nM, and the dopamine agonist is dopamine at a concentration of 10 µM.
However, the specification does not reasonably provide enablement for the method of claim 4 employing (i) any agonist or antagonist of the claimed pathways provided at (ii) any concentration.
Thus, the specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Enablement is considered in view of the Wands factors (MPEP 2164.01(a)). The court in Wands states: "Enablement is not precluded by the necessity for some experimentation such as routine screening. However, experimentation needed to practice the invention must not be undue experimentation. The key word is 'undue,' not 'experimentation.' " (Wands, 8 USPQ2d 1404). Clearly, enablement of a claimed invention cannot be predicated on the basis of quantity of experimentation required to make or use the invention. "Whether undue experimentation is needed is not a single, simple factual determination, but rather is a conclusion reached by weighing many factual considerations." (Wands, 8 USPQ2d 1404). The factors to be considered in determining whether undue experimentation is required include: (1) the quantity of experimentation necessary, (2) the amount of direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims. While all of these factors are considered, a sufficient amount for a prima facie case are discussed below.
(1) The nature of the invention
The specification describes the invention as a method of making immature and mature dopaminergic neurons from midbrain neuronal progenitor cells which are derived from human pluripotent stem cell.
(2) the breadth of the claims:
Independent claims 3 and 4, broadly encompass a method of generating human TH+ KCNJ6+ mature dopaminergic neurons beginning from the starting material of midbrain neural progenitor cells (claim 3) or human pluripotent stem cells (claim 4). Dependent claims 5 and 6, which depend from claim 4, further require that the pluripotent stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells, respectively. None of claims 3-6 provide specific pathway agonists/antagonists or concentrations necessary to ultimately result in the production of human TH+ KCNJ6+ mature dopaminergic neurons.
Thus, the claims taken together with the specification imply that the method is able to generate human TH+ KCNJ6+ mature dopaminergic neurons employing (i) any compounds which act as the claimed pathway agonists/antagonists in (ii) any concentration, which is unpredictable.
(3) The state of the prior art
The prior art shows the application of various pathway agonist/antagonist combinations at specified concentrations to generate human dopaminergic neurons. The art shows that different pathway agonists/antagonists having varying levels of effectiveness which are concentration dependent. However does not appear to demonstrate that any pathway agonist/antagonist at any concentration is capable of producing human dopaminergic neurons.
(4) the predictability or unpredictability of the art:
The claims embody a method of generating human TH+ KCNJ6+ mature dopaminergic neurons employing (i) any compounds which act as the claimed pathway agonists/antagonists in (ii) any concentration, which is unpredictable.
The method is found unpredictable with regards to the ability of the method to produce the claimed cells without limitation to the specific pathway agonists/antagonists used.
Zheng et al (Mol Cell Pharmacol., “mTOR inhibitors at a Glance”, 2015) teaches that mTOR is comprised of two catalytic subunits, mTORC1 and mTORC2, and that various mTOR inhibitors act on mTORC1 or mTORC2 or both (introduction, pg. 1 last para, spanning pg. 2 para 2). Zheng teaches that “Because the sequence similarity of mTOR with PI3K, many ATP competitive PI3K inhibitors were found to display various degrees of mTOR inhibitory activity” (pg. 3, para 3). Zheng teaches that rapamycin only inhibits mTORC1 activity (Introduction, pg. 2, para 2), AZD8055 is a selective mTOR inhibitor with high potency and high selectivity toward both mTORC1 and mTORC2 (pg. 4, para 1-2), and “PI-103 inhibits several isoforms of PI3K with an IC50 of 2–3 nM but is less selective for mTORC1 and mTORC2 with IC50 of 20 nM and 83 nM, respectively (pg. 3, para 3). The specification states “the mTOR pathway antagonist is selected from the group consisting of AZD3147, rapamycin, sirolimus, temsirolimus, everolimus, ridaforolimus, umirolimus, zotarolimus, torin-1, torin-2, vistusertib, AZD8055, dactolisib, PI-103, NU7441, BC-LI-0186, eCF 309, ETP 45658, niclosamide, omipalisib, PF 04691502, PF 05212384, WYE 687, XL 388, STK16-IN-1, PP 242, torkinib, sapanisertib, voxtalisib, and combinations thereof”([0106]). Although the specification states that the mTOR antagonist can be chosen from the list provided in [0106], the specification only provides detailed information on how to employ the method using AZD3147. Based on the teachings of Zheng that mTOR antagonists vary significantly in which catalytic unit of mTOR is affected and vary significantly in the level of inhibition, one of ordinary skill in the art would be required to complete undue experimentation to practice the method employing any other mTOR antagonist than the demonstrated AZD3147. Additionally, the methods of the examples, specifically example 1 required a screening of 12 compounds in 96 various combinations to produce the desired gene regulation and expression profiles. Therefore, one of ordinary skill in the art would need to complete a screening of an undue amount of compounds in an undue amount of combinations to arrive at a working method.
Shu et al (Cells, “Insights into Bone Morphogenetic Protein—(BMP-) Signaling in Ocular Lens Biology and Pathology”, 2021) teaches about BMP signaling and the classification of BMPs. Shu teaches that BMPs are broken down into different subgroups based on the amino acid sequences and functional differences which are: BMP-2/4, BMP-5/6/7/8, BMP-14/13/12 (GDF5/6/7), GDF8/11, BMP-9 (GDF2)/BMP-10, GDF1/3 and GDF10/BMP-3 (pg. 2, para 5). Shu also states “[a]lthough their monikers imply that all BMP members are inducers of bone, some can act as inhibitors of bone formation [10]. For instance, BMP-3 is a negative regulator of bone density [22], and BMP-13 strongly inhibits bone formation” (pg. 2, para 5). The specification states “ the BMP pathway agonist is selected from the group consisting of BMPs, sb4, ventromorphins… or combinations thereof” ([0104]). Although the specification states that the BMP agonist can be chosen from any BMP or additional BMP agonists in [0104], the specification only provides detailed information on how to employ the method using BMP7. Based on the teachings of Shu that BMP agonists vary significantly and some BMPs, BMP3 and BMP13, are actually antagonists of their pathway, one of ordinary skill in the art would be required to complete undue experimentation to practice the method employing any other BMP agonist than the demonstrated BMP7. In fact, the specification includes BMP3 and BMP13 as possible BMP agonists and the art supports that if one of ordinary skill in the art were to choose to employ BMP3 or BMP13 based on the specification at [0104], they would not be able to reproduce the claimed results of the method. Additionally, the methods of the examples, specifically example 1 required a screening of 12 compounds in 96 various combinations to produce the desired gene regulation and expression profiles. Therefore, one of ordinary skill in the art would need to complete a screening of an undue amount of compounds in an undue amount of combinations to arrive at a working method.
Misiorek et al (Cells, “Context Matters: NOTCH Signatures and Pathway in Cancer Progression and Metastasis, 2021) teaches about Notch ligands. Misiorek teaches that “The type and ratio of Notch receptors to ligands; the exact nature of physical contact; the quality, duration, and precise topology of interactions: all of this matters. In addition, different Notch ligands can trigger very different responses, probably due to differential activation rates and variable strength of the corresponding receptors.” (pg. 3, para 1). Misiorek also teaches that Notch singalling is connected to SHH signaling and that Notch inhibition alone with GSI results in SHH and WNT signaling activity, whereas “Targeting both Notch (with GSI) and Hh (by cyclopamine) induced cell death by apoptosis and blocked colony formation in comparison to either inhibitor alone, thus confirming the nature of compensatory signaling cascades” (pg. 11, para 1). Misiorek concludes that “Altogether, the balance and synergies between [notch and SHH] signaling pathways, and many others such as EGFR and BMP, determine the outcome of cell fate instructions by Notch signaling (pg. 11, para 1). Misiorek provides strong support for the synergism between Notch signalling, SHH signaling, and even EGFR and BMP signaling. The methods of the examples, specifically example 1 required a screening of 12 compounds in 96 various combinations to produce the desired gene regulation and expression profiles. Therefore, one of ordinary skill in the art would need to complete a screening of an undue amount of compounds in an undue amount of combinations to arrive at a working method. Additionally, Misiorek demonstrates that without explicit evidence of a working combination of agonists/antagonists there would be an undue experimental burden on a person of ordinary skill in the art to arrive at the combination of pathway agonist/antagonists which might result in the claimed human TH+ KCNJ6+ mature dopaminergic neurons.
The method is also found to be unpredictable with regards to the ability of the claimed method to produce the claimed cells without limitation to the concentrations of specific agonists/antagonists employed.
Delepine et al (PLOS ONE, “GSK3ß inhibitor CHIR 99021 modulates cerebral organoid development through dose-dependent regulation of apoptosis, proliferation, differentiation and migration”, 2021) teaches about the importance of specific concentrations of CHIR99021, which is a GSK3ß inhibitor and a WNT activator/agonist. Delepine “demonstrate[s] that while low dose of GSK3β inhibitor CHIR 99021 increases organoid size, higher dose actually reduces organoid size; with the highest dose arresting organoid growth” in cerebral organoids (abstract). The claims do not require a specific concertation of the claimed WNT agonist, and the specification only provides support for CHIR99021 as the WNT agonist at concentrations of 1.1 for step (a) of the methods and 1.1 µm for step (c) of the methods. Delepine provides strong support that without explicit guidance as to the concentrations of the various components there would be an undue experimental burden on a person of ordinary skill in the art to arrive at the concentration of pathway agonist/antagonists which might result in the claimed human TH+ KCNJ6+ mature dopaminergic neurons.
The specification also provides evidence as to the importance of concentration with regards to the desired results of the method. The specification provides Fig. 4 which is a set of graphs depicting the expression change of FOXA2 and other genes of interest, with purmorphamine treatment as well as other components. Specifically, the figure provides 13 parameters and only two of which are the same compound, purmorphamine, at varying concentrations, 200 and 500 nM. Example 1 describes that Fig. 4 is an optimization of maximum FOXA2 expression and “Since Purmorphamine did not have a positive impact on expression of OTX2, DMBX1 and LMX1A in previous setting, we expected a drop in the expression level of the genes, however, it was observed that their expression level stayed in the same vicinity as the previous condition with 3000, 450 and 11000 for DMBX1, LMX1A and OTX2, respectively (FIG. 4 )”. Fig. 4 clearly shows that at 200 nM of purmorphamine, the FOXA1 expression remains relatively constant, whereas at 500 nM of purmorphamine, FOXA1 expression is increased more than any other parameter. If the inventors only included purmorphamine at the 200 nM concentration, they would not have chosen purmorphamine so as to optimize for FOXA1 expression since the 200 nM concentration had virtually no effect. This demonstrates that without guidance from the instant disclosure, the currently claimed method has a great deal of variability in outcome tied to specific concentrations. Thus, the instant specification supports that there would be an undue experimental burden on a person of ordinary skill in the art to arrive at the concentration of pathway agonist/antagonists which might result in the claimed human TH+ KCNJ6+ mature dopaminergic neurons.
Therefore, this method is found to be unpredictable with regards to the method’s ability to generate human TH+ KCNJ6+ mature dopaminergic neurons through employing (i) any compounds as the claimed pathway agonist/antagonists and at (ii) any concentration.
(5) The relative skill of those in the art:
The relative skill of those in the art is high.
(6) The amount of direction or guidance presented
The specification details examples 1-7. Example 1 details the process of culture protocol development for the generation of stem cell-derived midbrain neural progenitors expressing FOXA2 and LMX1A. Example 2 details factor criticality analysis of stem cell derived midbrain neural progenitor-inducing culture conditions. Example 3 details immunochemistry validation of stem cell-derived midbrain neural progenitors expressing FOXA2 and LMX1A. Example 4 details RNA-seq validation of stem cell-derived midbrain neural progenitors expressing LMX1A and FOXA2. Example 5 details culture protocol development for the generation of dopaminergic neurons expressing TH and KCNJ6. Example 6 details factor criticality analysis of dopaminergic neuron-inducing culture conditions. Example 7 details immunocytochemistry validation of midbrain dopaminergic neurons expressing TH and KCNJ6.
(7) the presence or absence of working examples:
With specific regards to working examples, the specification teaches Examples 1 and 5 . The examples detail the validation of specific factors which are the claimed pathway agonist/antagonists at specific concentrations. The method of claim 3 requires stages 3 and 4 of the protocol described in example 5 and the method of claim 4 requires stages 1-4 of the protocol described in examples 1 and 5. The specific factors and concentrations for stages 1-4 are validated through many rounds of experimentation which included various combinations of conditions. Example 1 states that a 12-factor experiment which 96 conditions of combinations were tested, the subsequent examples do not provide the specific number of conditions of combinations tested.
(8) The quantity of experimentation necessary:
Considering the state of the art as discussed above and the high unpredictability and the lack of guidance provided in the specification, one of ordinary skill in the art would be burdened with undue experimentation to use the claimed invention within the broad scope as instantly claimed.
It is the examiner’s position that one skilled in the art could not practice the invention commensurate in the breadth of the claims without undue experimentation.
Therefore, claims 3-6 are rejected under 35 U.S.C. 112, first paragraph, for a lack of scope of enablement.
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.
Claim 3 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 3 contains the phrase “for three days (day 0-day 3)” in line 7, and “for at least 14 days (day 3- day 17)” in lines 12-13, which are indefinite. It is not clear what limitation is intended by including the days in parentheses due to the recitation of the number of days prior to the claimed phrases in parentheses. Appropriate correction is required.
Conclusion
No claims are allowed.
Examiner Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CONSTANTINA E STAVROU whose telephone number is (571)272-9899. The examiner can normally be reached M-F 8:00-5:00.
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CONSTANTINA E. STAVROU
Examiner
Art Unit 1632
/ANOOP K SINGH/Primary Examiner, Art Unit 1632