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 .
Response to Amendment
The amendment filed 3/25/2026, amending claim(s) 1, 3-23, 25-32, 35, 37, 39, 41-45, 48, 50, 52-53, 55-56, 58-65, 71, 74-77, 80-82 and cancelling claim(s) 54 is acknowledged.
Claims 1-53 and 55-82 are pending. Claims 7 and 66-82 were previously withdrawn due to being drawn to a nonelected invention.
Claims 1, 5, 6, 8, 9, 13-25, 28-37, 39-50, 52, 53, and 55-65 are pending and under examination.
Applicant’s amendments to the claims have overcome the objections previously set forth in the Non-Final Office Action mailed 11/26/2025.
Withdrawn Rejections
The rejection of claim 1 under 35 U.S.C. 102(a)(1) as being anticipated by Song is withdrawn. Applicant added the new limitation of “wherein the cells are contacted with the at least one inhibitor of Wnt signaling at least about 12 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling; wherein the cells are contacted with the at least one activator of FGF signaling at least about 12 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling; and wherein the at least one marker indicating a midbrain dopamine neuron or a precursor thereof is selected from the group consisting of EN1, OTX2, TH, NURR1, FOXA2, PITX3, LMX1A, LMO3, SNCA, ADCAP1, CHRNA4, SOX6, DAT, VMAT2, WNT1, GIRK2, and combinations thereof.”, which is not disclosed by Song.
Regarding the previous 112(b) rejection related to the presence of activators and inhibitors in the solutions, the Applicant clarified that “activators and inhibitors can be present in the same solutions and in contact with the cells at the same time”. As such, the previous 112(b) rejection is withdrawn.
Priority
This application is a continuation of PCT application PCT/US2021/025596, filed on April 2, 2021. Applicant’s claim for the benefit of a prior-filed application provisional application 63/004,138 filed on April 2, 2020 under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, or 365(c) is acknowledged.
Drawings
The amended drawings received on 03/25/2026 are accepted.
Claim Objections
Claims 49 and 55 are objected to because of the following informalities:
Claim 49: recitations of “a un-modified” should be replaced with “an un-modified”.
Claim 55: line 2 recites “do no express” instead of “do not express”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(a) – Written Description
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1, 5, 6, 8, 9, 13-25, 28-37, 39-50, 52, 53, and 55-65 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The claims recite an in vitro method that comprises a) contacting stem cells with at least one SMAD signaling inhibitor, at least one SHH signaling activator, and at least one Wnt signaling activator; b) contacting the cells with at least one FGF signaling activator, and at least one Wnt signaling inhibitor.
The claims recite the cells being contacted with the at least one inhibitor of Wnt signaling at least about 12 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling, and the cells being contacted with the at least one activator of FGF signaling at least about 12 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling.
The cells may be contacted with the Wnt inhibitor anywhere from about 1 day to about 20 days (claims 6, 8, 9).
Contact with the FGF activator may be initiated 10-13 days after a SMAD inhibitor (claim 13).
The cells may be contacted with the FGF activator anywhere from about 1 day to about 20 days (claims 14-16).
The cells may be contacted with the SMAD inhibitor for about 5-7 days (claims 17, 18).
The cells may be contacted with the SHH activator for about 5-7 days (claims 19, 20).
The cells may be contacted with the Wnt activator for about 15-17 days (claims 21, 22).
The concentration of the Wnt activator may increase (claims 23-25).
The method may further comprise isolating the cells based on surface markers (claims 56-61).
Claim 1 recites the differentiated stem cells “expressing at least one marker indicating a midbrain dopamine neuron or a precursor thereof selected from the group consisting of EN1, OTX2, TH, NURR1, FOXA2, PITX3, LMX1A, LMO3, SNCA, ADCAP1, CHRNA4, SOX6, DAT, VMAT2, WNT1, GIRK2, and combinations thereof.”
Claim 28 recites “the at least one inhibitor of Wnt signaling is capable of inhibiting non-canonical Wnt signaling and canonical Wnt signaling.”
Claim 32 recites “the at least one activator of FGF signaling is capable of causing expansion of the midbrain and upregulating midbrain gene expression.
Claim 52 recites “at least about 80% of the differentiated cells express FOXA2 and EN1 about 15 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling.”
Claim 53 recites “wherein greater than about 80% or greater than about 90% of the differentiated cells express FOXA2 and EN1 16 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling.
Claim 55 recites “wherein the differentiated cells do no express at least one marker selected from the group consisting of PAX6, EMX2, LHX2, SMA, SIX1, PITX2, SIM1, POU4F1, PHOX2A, BARHL1, BARHL2, GBX2, HOXA1, HOXA2, HOXB1, HOXB2, POU5F1, NANOG, and combinations thereof.”
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).
The court explained that “reading a claim in light of the specification, to thereby interpret limitations explicitly recited in the claim, is a quite different thing from ‘reading limitations of the specification into a claim,’ to thereby narrow the scope of the claim by implicitly adding disclosed limitations which have no express basis in the claim.” The court found that applicant was advocating the latter, i.e., the impermissible importation of subject matter from the specification into the claim.). See also In re Morris, 127 F.3d 1048, 1054-55, 44 USPQ2d 1023, 1027-28 (Fed. Cir. 1997).
Either these are inherent properties of (that naturally flows from) the method steps of claim 1, or they are not. The claims denote that not all of the structures/method steps of the independent claim are able to achieve the recited property(ies) recited in the dependent claim(s).
To the extent it is not an inherent property (that naturally flows) from the product/method of the independent claim, then something must change. The claim is considered to lack adequate written description for failing to recite the structure that is necessary and sufficient to cause the resulting cells of the claimed method to possess these characteristics/marker profiles. For example, the concentrations of the activators and inhibitors are recited at a high level of generality, e.g., no positively recited concentration(s) in any claims. The claim limitations recited above merely state functional characteristics without providing any indication about how the characteristic is provided. If the characteristic does not follow from (is not an inherent property of) the structure/active method steps recited in the claim, it is unclear whether the claim requires some other structure to be added to the composition to provide the characteristic.
The recitation of the method steps/activators and inhibitors recited in claim 1 is broader in scope than the working examples of the specification. For example, pg. 51, lines 4-6 of the specification note that the concentration of CHIR (a Wnt activator) to use can vary depending on hPSC/hiPSCs lines. Pgs. 52-54 also recite 6 different mediums, all which vary in in composition and concentrations. Examples 1 and 2 provide different exemplary midbrain DA neuron differentiation protocols, each of which was used in Example 3. Example 3 also recites a “Wnt-boost” protocol, which may be combined with the protocols of Examples 1 and 2 (pg. 54, lines 21-25). As pointed out by the specification, exposure to the different differentiation mediums, which vary in their composition and concentrations, for different lengths of time produces varied results (e.g., resulting marker expression) (e.g., Figure 1, 2A). Further, Example 4 discusses optimizing WNT inhibition, but provides no details on how to do so (i.e., what/how to change the method steps and differentiation medium(s)). Example 9 details increasing exposure to Wnt inhibitors, which follows a different protocol than previously described in the other examples. Given all the differing protocols in the working examples alone, specification fails to disclose what structural changes to the method steps of claim 1 is necessary and sufficient to produce differentiated stem cells sharing the same functional properties (e.g., expressing at least one marker indicating a midbrain dopamine neuron or a precursor thereof selected from the group consisting of EN1, OTX2, TH, NURR1, FOXA2, PITX3, LMX1A, LMO3, SNCA, ADCAP1, CHRNA4, SOX6, DAT, VMAT2, WNT1, GIRK2, and combinations thereof), and thus the ordinary artisan would not know what modification(s) must be made in order to fulfill the instant recitation.
A search of the prior art showed the timing of exposure to the activators and inhibitors in relation to one another (e.g., contacting with FGF activator at least 12 days from initiation of contact with SMAD inhibitor) is different from the standard in the art. For example, previously cited Song teaches beginning exposure to a FGF activator 1 day after the start of exposure to a SMAD inhibitor:
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A review of pluripotent stem cell therapies for Parkinson disease by the Applicant (Kim, Tae Wan, So Yeon Koo, and Lorenz Studer. "Pluripotent stem cell therapies for Parkinson disease: present challenges and future opportunities." Frontiers in cell and developmental biology 8 (2020): 729.) after the effective filing date of the instant application also demonstrates the variability in timing in the art. Figure 1 compares published differentiation protocols for dopamine neuron derivation from human pluripotent stem cells. Only one of the seven compared protocols teach exposing the cells to a FGF activator 10 days after the initiation of SMAD inhibitor exposure, which is still less than the claimed method.
The claim fails to recite, and the specification fails to disclose, a nexus between the required method steps (and differentiation medium used) and the corresponding functional property(ies) of inducing differentiation of stem cells (i.e., resulting in the same type of differentiated cell).
Additionally, claim 1 recites “at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, at least one activator of Sonic hedgehog (SHH) signaling, and at least one activator of wingless (Wnt) signaling”, and “at least one activator of fibroblast growth factor (FGF) signaling and at least one inhibitor of Wnt signaling”. Further, claim 1 also recites “at least one marker indicating a midbrain dopamine neuron or a precursor thereof”.
The claim is broad for reciting a genus of SMAD inhibitors, SHH activators, Wnt activators, FGF activators, and Wnt inhibitors. As written, the claim encompasses any molecule/compound of any structure and function that acts directly or indirectly as an activator or inhibitor for their corresponding signaling pathway, resulting in an enormously vast genus of possible activators and inhibitors.
The specification offers little guidance on these genera. For examples, on Wnt inhibitors, the specification discloses:
“In certain embodiments, the at least one inhibitor of Wnt signaling is capable of inhibiting canonical Wnt signaling. In certain embodiments, the at least one inhibitor of Wnt signaling is capable of inhibiting non-canonical Wnt signaling and canonical Wnt signaling. In certain embodiments, the at least one inhibitor of Wnt signaling is selected from the group consisting of IWP2, IWR1-endo, XAV939, IWP-01,IWP12, Wnt-C59, IWP-L6, ICG-001, LGK-974, IWR-1, ETC-159, iCRT3, IWP-4, Salinomycin, Pyrvinium Pamoate, iCRT14, FH535, CCT251545, KYA1797K, Wogonin, NCB-0846, Hexachrorophene, PNU-74654, KY02111, 503031 (KYO1-I),502031 (KY02-I), Triptonide, BC2059, PKF115-584, Quercetin, NSC668036, G007-LK, MSAB, LF3, JW55, Isoquercitrin, WIKI4, derivatives thereof, and combinations thereof” (pg. 4-5).
“The present disclosure is based on the discovery that treatment with a Wnt inhibitor can improve mDA neuron derivation. In addition, such treatment with the Wnt inhibitor does not negatively impact the expression of EN1 and other mDA neuron markers, e.g., the differentiation methods disclosed herein including a Wnt inhibitor lead to sustained expression of EN1 and other mDA neuron markers. Furthermore, the treatment with the Wnt inhibitor does not increase the emergence of contaminating markers (non- mDA neuron markers). The present disclosure is also based on the discovery that the Wnt inhibitor treatment leads to better segregation of A9 subtype neurons and A10 subtype neurons. In certain embodiments, the Wnt inhibitor treatment impacts (e.g., increases) the mRNA expression of markers indicating A9 subtype mDAs (e.g., ALDH1A1). Non-limiting examples of markers indicating A9 subtype neurons include LMO3, ALDH1A1, SOX6, VGLUT2, and NDNF. In certain embodiments, the Wnt inhibitor treatment increases number of ALDH1AI+ cells in vitro and in vivo (among the EN1+) cells. ALDH1A1 expression can be high without EN1 co-expression, and the ALDH1AI'EN1- cells are not necessarily A9 subtype neurons and are not clearly defined cells.
The differentiation methods disclosed herein including the Wnt inhibitor treatment result in high generation of cells expressing both ALDH1A1 and EN1 in vitro and in vivo after graft. In certain embodiments, the Wnt inhibitor treatment further impacts (e.g., increases) the mRNA expression of markers indicating A10 subtype mDAs. Non-limiting examples of markers indicating A10 subtype neurons include CALB1, CALB2, OTX2, CCK, VGAT (Slc32al), and VIP. Increased mRNA expressions of A9 and A10 subtype markers support for proper specification of A9 and A10 subtype neurons. For example, certain A10 subtype neuron markers (e.g., CALB1 and CALB2) can only be seen once the cells are properly specified to exhibit A9 or A10 identity. Furthermore, the Wnt inhibitor treatment can reduce proliferation and increase expressions of mDA neuron maturity markers. Non-limiting examples of mDA neuron maturity markers include DAT, VMAT2, PITX3, CHRNA6, and CHRNB3. In addition, the Wnt inhibitor treatment can improve differentiation and reduce remaining Ki67* proliferating cells, which can lead to improved safety profile of DA neurons” (pg. 12).
Additionally, the specification defines “inhibitor” as:
"Inhibitor" as used herein, refers to a compound or molecule (e.g., small molecule, peptide, peptidomimetic, natural compound, siRNA, anti-sense nucleic acid, aptamer, or antibody) that interferes with (e.g., reduces, decreases, suppresses, eliminates, or blocks) the signaling function of the molecule or pathway (e.g., Wnt signaling pathway, and SMAD signaling). An inhibitor can be any compound or molecule that changes any activity of a named protein (signaling molecule, any molecule involved with the named signaling molecule, a named associated molecule, such as a glycogen synthase kinase 3p (GSK33)). (e.g., including, but not limited to, the signaling molecules described herein). For example, an inhibitor of SMAD signaling can function, for example, via directly contacting SMAD, contacting SMAD mRNA, causing conformational changes of SMAD, decreasing SMAD protein levels, or interfering with SMAD interactions with signaling partners, and affecting the expression of SMAD target genes.
Inhibitors also include molecules that indirectly regulate biological activity, for example, SMAD biological activity, by intercepting upstream signaling molecules (e.g., within the extracellular domain, examples of a signaling molecule and an effect include: Noggin which
sequesters bone morphogenic proteins, inhibiting activation of ALK receptors 1,2,3, and 6, thus preventing downstream SMAD activation” (pg. 14-15).
The specification defines “activator” as:
"Activators," as used herein, refer to compounds that increase, induce, stimulate, activate,
facilitate, or enhance activation the signaling function of the molecule or pathway, e.g., Wnt
signaling, SHH signaling, etc.” (pg. 15).
Given the breadth of the claim language, the definitions of “inhibitors” and “activators”, a reiteration of the claim limitations, and a list of non-limiting examples does not represent an adequate disclosure of the complete structure of the broad genus.
In addition, claim 56 recites the claimed method “further comprising isolating cells that express at least one positive surface marker and do not express at least one negative surface marker”. Claims 57-61 further limit the positive markers to CD171 and CD184 and the negative markers to CD49e, CD99, and CD340. However, there is no further guidance on what is considered a positive or negative marker, and what cells are being isolated (e.g., the differentiated cells expressing one of the recited markers indicating a midbrain dopamine neuron or a precursor thereof? the differentiated cells before these markers are expressed?). A search of the specification provided minimal guidance, reciting “In certain embodiments, the differentiation methods disclosed herein further comprise isolating mDA neurons and precursors thereof based on at least one or at least two surface markers.” No parameters for positive or negative markers were provided. Further, no guidance is provided for when this isolation step is to take place in the method.
The prior art does not inform the ordinary artisan of the structure/function nexus, let alone which activators and inhibitors will retain the claimed function(s) (e.g., differentiated cells expressing at least one marker indicating a midbrain dopamine neuron or a precursor thereof), as opposed to which modifications will not retain the claimed function(s).
Arenas, Ernest. "Wnt signaling in midbrain dopaminergic neuron development and regenerative medicine for Parkinson's disease." Journal of molecular cell biology 6.1 (2014): 42-53. is considered relevant prior art for reviewing Wnt signaling in midbrain dopaminergic neuron development.
Arenas teaches that Wnts are known to regulate three main different pathways (CTNNB1 [canonical], PCP [non-canonical], and Ca2+ [non-canonical] pathways), and notes that the capacity of Wnts to activate distinct signaling pathways and its branches is cell type- or context-dependent (pg. 42-43, “Wnt signaling pathways”). Additionally, Arenas outlines the complexity of the Wnt/PCP pathway, which has multiple co-receptors and signaling branches, and teaches that these Wnt pathways interact with one another at multiple levels, not only because they share ligands and the Fz-Dvl module, but also because there are multiple mechanisms to regulate each other. (pg. 43, last para col 1- first para col 2; col 2, para 3).
Further, Arenas discusses how mDA neurons are mainly classified into two anatomically, molecularly, and functionally distinct subtypes, A9 substantia nigra pars compacta (SNc) and A10 ventral tegmental area (VTA). Additionally, genetic fate-mapping experiments have indicated that both SNc and VTA DA neurons are generated by proliferating progenitors in the ventricular zone (VZ)of the mFP (pg. 44, “Wnt signaling in mDA neuron development”).
In summary, Arenas teaches that there are multiple Wnt genes (e.g., Wnt1, Wnt2, Wnt5a) and multiple signaling pathways that may be targeted. Additionally, there are two structurally and functionally distinct mDA neurons. In addition, Arenas notes the variability in protocols and results of differentiating stem cells into mDA neurons, such as Kirkeby et al. (2012) finding that low and high concentrations of GSK3b inhibitors induced diencephalic and hindbrain cells, respectively, while intermediate concentrations resulted in cells with midbrain identity, and that while GSK3b inhibitors can improve the mDA neuron differentiation of pluripotent cells in vitro, they have off target effects and can modulate different pathways (pg. 48, col 2, para 3-pg. 49, col 1).
Thus, for the reasons outlined above, it is concluded that the claims do not meet the requirements for written description under 35 U.S.C. 112, first paragraph, and the Artisan would not have understood Applicant to have been in possession of the invention as claimed.
Claim Rejections - 35 USC § 112(a) – Scope of Enablement
Claims 1, 5, 6, 8, 9, 13-25, 28-37, 39-50, 52, 53, and 55-65 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:
an in vitro method for inducing differentiation of stem cells, comprising: contacting the stem cells with 10 microM SB (TFGbeta inhibitor), 250 nM LDN (BMP inhibitor), 500 ng/ml SHH C25II (SHH activator), 1 or 6 microM CHIR (Wnt activator), 1 microM IWP2 (Wnt inhibitor), and 100 ng/ml FGF18 (FGF activator) (as described in Examples 1-3 of specification).
does not reasonably provide enablement for an in vitro method for inducing differentiation of stem cells, comprising: contacting the stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, at least one activator of Sonic hedgehog (SHH) signaling, and at least one activator of wingless (Wnt) signaling; and contacting the cells with at least one activator of fibroblast growth factor (FGF) signaling and at least one inhibitor of Wnt signaling to obtain a population of differentiated cells expressing at least one of the recited markers indicating a midbrain dopamine neuron or a precursor thereof. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
While determining whether a specification is enabling, one considers whether the claimed invention provides sufficient guidance to make and use the claimed invention. If not, whether an artisan would have required undue experimentation to make and use the claimed invention and whether working examples have been provided. When determining whether a specification meets the enablement requirements, some of the factors that need to be analyzed are: the breadth of the claims, the nature of the invention, the state of the prior art, the level of one of ordinary skill, the level of predictability in the art, the amount of direction provided by the inventor, the existence of working examples, and whether the quantity of any necessary experimentation to make or use the invention based on the content of the disclosure is “undue” (In re Wands, 858 F.2d 731, 737, 8 USPQ2ds 1400, 1404 (Fed. Cir. 1988)). Furthermore, USPTO does not have laboratory facilities to test if an invention will function as claimed when working examples are not disclosed in the specification. Therefore, enablement issues are raised and discussed based on the state of knowledge pertinent to an art at the time of the invention. And thus, skepticism raised in the enablement rejections are those raised in the art by artisans of expertise.
The Examiner incorporates herein the analysis discussed above in the 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, written description rejection.
The Breadth of the Claims and The Nature of the Invention
The claims are directed to an in vitro method for inducing differentiation of stem cells.
The claims are broad for reasonably encompassing a large genus of activators and inhibitors.
The State of the Prior Art, The Level of One of Ordinary Skill and The Level of Predictability in the Art
The art teaches many different protocols to differentiate stem cells into midbrain dopaminergic neurons.
Tofoli, Fabiano Araújo, et al. "Midbrain dopaminergic neurons differentiated from human-induced pluripotent stem cells." Neural Stem Cells: Methods and Protocols. New York, NY: Springer New York, 2019. 97-118. is considered relevant prior art for teaching “there are about 40 different published protocols for mDAN differentiation, which are eventually modified according to the respective laboratory. In many cases, protocols are not fully described, failing to provide essential tips for researchers starting in the field. Considering that commercial kits produce low mDAN percentages (20–50%), we chose to follow a mix of four main protocols based on Kriks and colleagues’ protocol, from which the resulting mDAN were engrafted with success in three different animal models of Parkinson’s disease” (Abstract). Figure 1 of Tofoli outlines inhibitors used in their own differentiation protocol, including DAPT which is not required in the instant method. Tofoli also teaches that “midbrain neuron specification requires the expression of FOXA2 and LMX1A transcription factors… One of the gold standard
markers for identification of mDAN is tyrosine hydroxylase (TH)” (pg. 99, para 3). Table 1 of Tofoli also details the final concentrations of reagents and medium supplements used in their protocol, with some of the reagents not being required in the instant method such as cAMP, and ROCK inhibitor, or the same reagents being used at a different concentration, such as 3 microM of CHIR and 100nM of LDN.
Hegarty, Shane V., Aideen M. Sullivan, and Gerard W. O'keeffe. "Midbrain dopaminergic neurons: a review of the molecular circuitry that regulates their development." Developmental biology 379.2 (2013): 123-138. is considered relevant prior art for teaching the Shh, FGF8, and Wnt1 are key in the induction of VM DA neurogenesis, and that Lmx1a, Lmx1b, and FoxA2 expression is essential to VM DA NPs (see “Induction of a dopaminergic phenotype in ventral midbrain neural precursors”, beginning on pg. 126). Additionally, Hegarty teaches 3 ventral midbrain dopaminergic neuron subtypes- A8, A9, and A10, each of which having differences in development. In addition, Hegarty teaches A9 cells undergoing progressive degeneration in Parkinson’s disease (Abstract; pg. 133, “Concluding remarks and future perspectives”; pg. 123, col 2, para 2). Further, Hegarty teaches differences in midbrain dopaminergic neurons from the VTA vs. SNc, such as Pitx3 may be critical for the induction of TH expression in SNc DA neurons, but not those of the VTA, as the absence of Pitx3 results in a failure of SNc DA neurons to express TH, while VTA neurons do so. Also, lateral VM DA neurons express Pitx3 prior to TH, while the medial VM DA neurons express Pitx3 coincidently with TH (pg. 130, col 2, para 1). In summary, Hegarty teaches structural and functional differences between subtypes of midbrain dopaminergic neurons and where they originate from, along with key factors that must be present for the development of VM DA neurons.
Tabar, Viviane, and Lorenz Studer. "Pluripotent stem cells in regenerative medicine: challenges and recent progress." Nature Reviews Genetics 15.2 (2014): 82-92. (NPL cited in IDS; authored by Applicant) is considered relevant prior art for reviewing directed differentiation of hPSCs. Figure 1a of Tabar teaches a protocol for creating hESC-derived mDA neurons by exposing hESCs or hiPSCs to Pur (SHH), SHIR (WNT), SB (TGFbeta), and LDN (BMP) on days 0-13, followed by exposure to a “cocktail” of growth factors that promote mDA neuron fate (PDGF-AA, BDNF, GDNF, cAMP, and TGF-beta3) on days 13-25. Tabar also notes that a key feature of this protocol is the transition of the cells through a floor plate intermediate stage instead of the neuroepithelial intermediate, a key step of which was the activation of canonical WNT signaling using a small-molecule inhibitor of glycogen synthase kinase (GSK) (pg. 83, col 2, para 1). The teachings of Tabar differ from the instant method as the instant method does not require the presence of any growth factors in order to obtain a population of differentiated cells expressing at least one marker for midbrain dopaminergic neurons or a precursor thereof.
The Existence of Working Examples and The Amount of Direction Provided by the Inventor
As discussed in the 112(a) written description rejection above, the specification provides little guidance for one of ordinary skill to carry out the claimed method.
The specification fails to make up for the deficiencies of the global scientific community.
The Quantity of Any Necessary Experimentation to Make or Use the Invention
Thus, the quantity of necessary experimentation to make or use the invention as claimed, based upon what is known in the art and what has been disclosed in the specification, will create an undue burden for a person of ordinary skill in the art to necessarily and predictably use the claimed method comprising the broadly claimed genus of activators and inhibitors that is capable of differentiating into a population of differentiated cells expressing at least one of the recited markers for midbrain dopaminergic neurons or a precursor thereof.
In conclusion, the specification fails to provide any guidance as to how an artisan would have dealt with the art-recognized limitations of the claimed cells and therefore, limiting the claimed invention to an in vitro method for inducing differentiation of stem cells, comprising: contacting the stem cells with 10 microM SB (TFGbeta inhibitor), 250 nM LDN (BMP inhibitor), 500 ng/ml SHH C25II (SHH activator), 1 or 6 microM CHIR (Wnt activator), 1 microM IWP2 (Wnt inhibitor), and 100 ng/ml FGF18 (FGF activator).
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 5, 6, 8, 9, 13-25, 28-37, 39-50, 52, 53, and 55-65 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 recites “An in vitro method for inducing differentiation of stem cells, comprising:
contacting the stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, at least one activator of Sonic hedgehog (SHH) signaling, and at least one activator of wingless (Wnt) signaling; and
contacting the cells with at least one activator of fibroblast growth factor (FGF) signaling and at least one inhibitor of Wnt signaling to obtain a population of differentiated cells expressing at least one marker indicating a midbrain dopamine neuron or a precursor thereof.”
As written, it is unclear whether a) and b) designate 2 distinct active method steps, e.g., cells must first be contacted with a SMAD inhibitor, SHH activator, and Wnt activator, then the cells can be exposed to a FGF activator and Wnt inhibitor. The claim clarifies that FGF activator and Wnt inhibitor exposure follows the start of SMAD inhibitor exposure. However, does this timing also apply to the SHH activator and Wnt activator since they are also recited in step a) with the SMAD inhibitor?
It would be remedial to clarify the timing of exposure of the SHH activator and Wnt inhibitor in relation to the other activators and inhibitors, e.g., if contact with the FGF signaling activator(s) and Wnt signaling inhibitor(s) is supposed to occur after contact with the other recited activator(s)/inhibitor(s), add “followed by contacting the cells…”.
Claims 1, 6, 14, 48, 52, and 53 recite “at least about”. The term “at least about” is a relative term which renders the claim indefinite. The term “at least about” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. “At least” is a recitation that describes a definitive cut-off of a range. In contrast, “about” is a term that is intended to provide wiggle room and a small range. In addition, the specification does not define nor offer further guidance of what “about” encompasses. As such, the two terms together are conflicting. “About” renders the definite cut-off, “at least” indefinite because the overalls metes and bounds of the range are not apparent. Does the recited range have a definite cut-off or not?
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 14 recites the broad recitation “about 1 day and/or for up to about 20 days”, and the claim also recites, e.g., “at least 4 days and/or for up to 7 days” which is the narrower statement of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims.
In kind, claim 53 is rejected (e.g., broadly recites greater than about 80%, while also reciting the narrower range of greater than about 90%).
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 5 and 13 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Base claim 1 recites “cells are contacted with the at least one inhibitor of Wnt signaling at least about 12 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling”.
Claim 5 recites “the contact of the cells with the at least one inhibitor of Wnt signaling is initiated 10 days, 11 days, 12 days, or 13 days from the initial contact of the stem cells with the at least one inhibitor of SMAD signaling.” Claim 5 recites a broader range (i.e., 10 or 11 days) than the base claim. Therefore, claim 5 fails to include all limitation of claim 1.
In kind, claim 13 is similarly rejected.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Double Patenting
The Examiner notes that the instant claims are similar to the claimed inventions of U.S. Patent Nos. US10858625B2, US10711243B2, US11970712B2, and US12084679B2, and copending Application No. 18591734 (Notice of Allowance mailed 6/1/2026) but are not presently considered nonstatutory double patenting. For example, as currently written, the instant method does not require the cells to be exposed to the at least one inhibitor of GSK3β signaling (e.g., Wnt activator) three (3) days from the initial exposure of the cells to the at least one inhibitor of TGFβ/Activin-Nodal signaling and the at least one inhibitor of BMP signaling (e.g., SMAD inhibitors) as recited in base claim 1 of U.S. Patent No. US11970712B2. However, depending on future claim amendments, a nonstatutory double patenting rejection may be appropriate.
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 33 and 34 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of copending Application No. 17681113 (reference application). Although the claims at issue are not identical, they are not patentably distinct from each other because the instant application is a species of the reference case application.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 33 and 34: Reference claim 1 is drawn to in vitro method for inducing differentiation of stem cells. The method comprises contacting the stem cells with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, at least one activator of Sonic hedgehog (SHH) signaling, and at least one activator of wingless (Wnt) signaling; and contacting the cells with at least one activator of fibroblast growth factor (FGF) signaling to obtain a population of differentiated cells expressing at least one marker indicating a midbrain dopamine neuron (mDA) or a precursor thereof. Reference claim 1 requires the FGF activator to be FGF18, and for the cells to be contacted with the at least one activator of FGF signaling is at least about 12 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling. Reference claim 1 does not require an Wnt inhibitor.
Response to Arguments
Applicant's arguments filed 3/25/2026 have been fully considered but they are not persuasive.
Regarding the 112a rejections, while adding limitations reciting midbrain dopamine markers and timing of administration of certain activators and inhibitors do address some concerns in the rejections, these amendments still do not address all concerns. Regarding the genus of activators and inhibitors, the Applicant argues that the instant specification “provides a number of compounds that have been previously shown to function in connection with each genus. Thus, the instant specification describes several approaches for each genus including specific species falling within each genus, which demonstrates both an actual reduction to practice and a constructive reduction to practice”. This argument is not persuasive because defining a species functionally does not further limit or clarify the structure of the species. Additionally, it is unclear how this demonstrates both an actual and constructive reduction to practice.
Regarding the arguments related to concentrations and timing, the Applicant references paragraph numbers. The instant specification does not recite any paragraph numbers, so it is unclear what part of the specification the Applicant is referring to. Additionally, the Applicant fails to provide evidence that “a skilled person would be capable of utilizing the specific concentration ranges of species falling within each genus” or how “a skilled person would be capable of utilizing the timing associated with both actual reductions to practice and constructive reductions to arrive at strategies effective for achieving inhibition/activation of designated pathways” based on the instant specification. The Applicant has failed to fully address the teachings of cited references such as Arenas in the 112a rejections.
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ALLISON M JOHNSON whose telephone number is (703)756-1396. The examiner can normally be reached Monday-Friday 9am-5pm.
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ALLISON M. JOHNSON
Examiner
Art Unit 1638
/ALLISON MARIE JOHNSON/ Examiner, Art Unit 1638
/ROBERT M KELLY/ Primary Examiner, Art Unit 1638
Prosecution Insights