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 .
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 03-17-2026 has been entered.
Applicant's amendments and arguments filed on 03-17-2026 have been received and entered. Claims 1 has been amended. Claims 1-27 are pending.
Election/Restrictions
Applicant’s election without traverse of Group I, claims 1-19 and 26, in the reply filed on 01-27-2025 is acknowledged.
Claims 20-25, 27 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected subject matter, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 01-27-2025.
Claims 1-19 and 26 are under consideration.
Priority
This application is a CON of PCT/US2020/048733 filed on 08/31/2020 which claims
priority from US provisional application 62/893,674 filed on 08/29/2019.
Information Disclosure Statement
The information disclosure statements (IDSs) submitted on 11-11-2025 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement has been considered by the examiner.
New-Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-19 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 the phrase “at least about” which render the claim indefinite because “About” encompasses values above and below a reference point whereas “at least” encompasses values above the reference point. Therefore, the combination of both terms (at least about) is unclear because one term (about) is including values below the reference point whereas “at least” appears to be intended to limit the recited range to values above the reference point, which is inconsistent with the term “about”.
Similarly, claims 2, 11 and 14 also have this same issue regarding the metes and bounds of the term “at least about” as used in those claims: claim 2 recites the phrase “…at least about 1 days…”; claim 11 recites the phrase “…at least about 80% identical…”; claim 14 recites the phrase “…at least about 10 days …” Appropriate correction of these claims is also required
Claims 3-10, 12-13, 15-19 are included in the rejection because they directly or indirectly depend from the rejected claims.
New-Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 3 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 3 does not further limit claim 1. Claim 3, which depends on claim 1, requires the initial contact of the cells with the at least one activator of FGF signaling is about 10 days or 12 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling. Claim 1 requires the at least one activator of FGF signaling is contacted with cells ‘at least about 12 days’ from the initial contact of the cells with the at least one inhibitor of SMAD signaling. As noted above in the 112(b) section of this action, the metes and bounds of the term “at least about 12 days” are unclear and the limitations recited in claim 3 does not further limit claim 1 because:
One alternative embodiment of claim 3 is “about 10 days” from the initial contact. This alternative embodiment doesn’t appear to further limit claim 1 because one could reasonably interpret the phrase “about 10 days” as encompassing embodiments where the cells are contacted with the at least on activator of FGF signaling prior to the time frame encompassed by “at least about 12 days” (e.g., embodiments that fall outside of the base claim’s scope). For example, the definition provided in the instant disclosure for the subjective term “about” is open ended and doesn’t refer to a range of days (e.g., see definition of the term “about” in the instant disclosure on page 12, lines 21-28).
For the other alternative embodiment recited in claim 3, where the contacting takes place “12 days” from the initial contact of the cells with the at least one inhibitor of SMAD signaling, it is not clear how this embodiment necessarily further limits claim 1 because the time variance encompassed by “at least about 12” days from claim 1 is unclear.
Therefore, it does not appear that either of the two alternatives recited in claim 3 necessarily further limit the scope of claim 1.
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.
Maintained and New - 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.
Claims 1-19 and 26 are rejected under 35 U.S.C. 103 as being unpatentable over Studer et al (Pub .No.: US 2018 /0094242 A1, Pub Date : Apr 5, 2018) (Applicant’s own work) in view of Schoeller et al (Pub. No.: US 2015/0010515 A1, Pub. Date: Jan. 8, 2015) and Chambers et al (Pub. No.: US 2017/0130199 A1, Pub. Date: May 11, 2017 ) (Applicant’s own work) and Liu et al (STEM CELLS AND DEVELOPMENT Volume 27, Number 5, January 17, 2018, DOI: 10.1089/scd.2017.0282).
Regarding to claim 1, Studer et al disclosed subject matter relates to the discovery that midbrain dopamine (DA) neurons, and precursors thereof, can be differentiated from human stem cells by dual inhibition of SMAD signaling (for example, by inhibition of TGF-β/Activin -Nodal signaling and BMP signaling), along with the activation of Sonic Hedgehog (SHH) signaling, and activation of wingless (Wnt) signaling ([0006], page 1) (For the claimed: 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)
Studer et al teach the cells can be further contacted with DA neuron lineage activators and inhibitors ([0007], page 1). Studer et al also teach the use of signal transduction protein include fibroblast growth factors (FGF) ([0047], page 4), and E8/E6 cell culture medium comprising FGF2 (e.g., activator of FGF signaling) ([0099], page 8) (For the claimed: contacting the cells with at least one activator of fibroblast growth factor (FGF) signaling).
Studer et al teach cells were then tested for expression of early midbrain DA marker combinations: 1) OTX2/EN / LMX1A and 2) PAX6/FOXA2/EN/ and NKX2.2 or NKX6.1; or late midbrain DA marker combinations: 1) TH /EN /FOXA2 and 2) LMX1A /OTX2/NURR1 (For the claimed: to obtain a population of differentiated cells expressing at least one marker indicating a midbrain dopamine neuron (mDA) or a precursor thereof).
Although Studer et al teach signal transduction protein include fibroblast growth factors (FGF) ([0047], page 4) and FGF2 ([0099], page 8), Studer et al do not specifically teach FGF18, FGF17 and combination thereof. Schoeller et al cure the deficiency.
Schoeller et al teach method of differentiating the neural plate border stem cells (NPBSCs) into midbrain dopaminergic neurons ([0160], page 13). Schoeller et al teach “culturing the NPBSCs obtained in step (d) in a neural medium comprising an activator of the FGF signaling pathway ……... thereby differentiating the NPBSCs into midbrain dopaminergic neurons” (see [0174]-[0185], page 14). Preferably, the activator of the FGF signaling pathway is selected from the group consisting of FGF2, FGF1, FGF4, FGF8, FGF17 and FGF18 ([0188], page 14).
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the rejected claims to combine the teachings of prior art to modify the method of Studer et al by using FGF17 and FGF18 as activator of the FGF signaling pathway as taught by Schoeller et al as instantly claimed, with a reasonable expectation of success. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to do so because Schoeller et al stated that “the overall efficiency of differentiation of mDA(midbrain dopaminergic) neurons was consistent between 3 different NPBSC lines (FIG. 11D). Taken together, these results demonstrate the formation of mDA neurons by NPBSCs using developmentally appropriate patterning signals.” (see [0307], right column, page 22). Also, Schoeller et al teach the use of activator of the FGF signaling pathway such as FGF17 and FGF18 ([0188], page 14), and the term “activator” is defined as a compound enhancing the activity of a target molecule, preferably by performing one or more of the following effects: (i) the transcription of the gene encoding the protein to be activated is enhanced, (ii) the translation of the mRNA encoding the protein to be activated is enhanced, (iii) the protein performs its biochemical function with enhanced efficiency in the presence of the activator, and (iv) the protein performs its cellular function with enhanced efficiency in the presence of the activator ([0086], page 7). One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Schoeller et al were successful in differentiation of neural plate border stem cells (NPBSCs) into midbrain dopaminergic neurons (mDA) with working examples and data.
Studer et al does not specifically disclose wherein the initial contact of the cells 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. However, Chambers et al cure the deficiency.
Chambers et al teach methods including induction of neural plate development in hESCs for obtaining midbrain dopamine (DA) neurons (Abstract). Chambers et al teach “dual SMAD
inhibition (SB431542 and Noggin)”([0023], page 3) and “FIG. 7 showing exemplary neuralization of hESC by dual SMAD inhibition permits a pre-rosette …….(m) showing dopaminergic neuronal patterning was initiated with the addition of supersonic on day 5-9, followed by the addition of BDNF, ascorbic acid, sonic hedgehog, and FGF8 on day 9-12” ([0029], page 4) (For the claimed: the initial contact of the cells 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 ).
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Therefore, it would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the rejected claims to combine the teachings of prior art to modify the method of Studer et al by adding activator of FGF signaling on day 9-12 after dual SMAD inhibition as taught by Chambers et al, with a reasonable expectation of success. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to do so because Chambers et al teach “FIG. 1 showing exemplary dual SMAD inhibition that allowed for a highly efficient feeder-free neural induction in adherent cultures within seven days ….. The dual SMAD inhibition greatly improves neural differentiation” ([0023], page 3) and Chambers et al stated that “this method was adapted to culture systems which may enhance the ease, yield efficiency, speed at which neural cells are derived” ([0161], page 13). Chambers et al also describe a modified N-SB protocol that allows the efficient derivation of highly enriched populations of placodal precursors and enable the derivation of unlimited numbers of placode derivates such as cranial sensory neurons for the study of sensory function and pain ([0180], page 16). One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Chambers et al were successful in reliably generating a broad repertoire of hiPSC derived neural cell types including midbrain dopamine (DA) neurons populations with detailed instructions and working example with data.
Regarding to claim 2-3, as described above, Chambers et al teach the use of activator of
fibroblast growth factor (FGF) signaling such as FGF8 on day 9-12 after dual SMAD inhibition that lead to Dopaminergic cells were maturated on days 12-19 ([0029], page 4).
Regarding to claim 4, Studer et al teach the one or more inhibitor of TGF-β/Activin -Nodal signaling is contacted to the cells for at least about 4 , 5 , 6 , 7 , 8 , 9 , or 10 or more days ([0101], page 8), and the one or more inhibitor of BMP/SMAD signaling is contacted to the cells for at least about 4 , 5 , 6 , 7 , 8 , 9 , or 10 or more days ([0103], page 9).
Regarding to claim 5, Studer et al teach the at least one activator of Sonic hedgehog (SHH) signaling are contacted to the plurality of pluripotent cells for between about 4 and 10 days (see claim 3, page 21).
Regarding to claim 6, Studer et al teach the cells are contacted with the increased concentration of the Wnt activator for at least about 4, 5, 6, 7, 8, 9, or 10 days or more ([0008], page 1).
Regarding to claim 7, Studer et al teach the concentration of the activator of Wnt signaling is increased at least about 2, 3, 4, 5 or 6 days after the cells are initially contacted with the Wnt activator ([0008], page 1).
Regarding to claim 8, Studer et al teach one or more activator of Wnt signaling lowers GSK3β for activation of Wnt signaling. Thus, the activator of Wnt signaling can be a GSK3β inhibitor. A GSK3P inhibitor is capable of activating a WNT signaling pathway ([0091], page 7).
Regarding to claims 8-9, Studer et al teach one or more inhibitor of TGF-β /Activin -Nodal signaling is a small molecule selected from the group consisting of SB431542, derivatives thereof ([0018], page 2).
Regarding to claim 10, the above references do not teach derivative of SB431542 is A83-01. Liu et al cure the deficiency.
Liu et al teach small molecules for neural stem cell induction (title). Liu et al teach the use of both A83-01 and SB431542 to generate NSCs/NPCs from different cell types (see table 2, page 305). SB431542 could be a substitution for A83- 01 (Page 306, right column, 3rd para.). Since Studer et al teach derivatives of SB431542 can be used as inhibitor of TGF-β/Activin -Nodal signaling, a person of ordinary skill in the art would be able to use A83-01 as a derivative of SB431542 to inhibit TGF-β/Activin -Nodal signaling.
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the rejected claims to combine the teachings of prior art to modify the method of the above references by using A83-01 as a derivative of SB431542 to inhibit TGF-β/Activin-Nodal signaling as taught by Liu et al, as instantly claimed, with a reasonable expectation of success. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to do so because Liu et al teaches that TGF-β inhibitor, A83-01, could switch the cell fate from iPSCs into NPCs in TF (OSKM)-induced HUC reprogramming, indicating the importance of TGF-β inhibitors for NSC/NPC induction (Page 306, 3rd para). One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Liu et al demonstrated examples with successful generation of NSCs/NPCs from different cell types with the use of A83-01.
Regarding to claim 11, Studer et al teach the activator of SHH signaling is selected from the group consisting of recombinant SHH, purified SHH, C25II ([0018], page 2)
Regarding to claim 12, Studer et al teach one activator of Sonic hedgehog (SHH) signaling comprises purmorphamine, recombinant SHH, purified SHH, and/or Smoothened agonists (e.g., claim 20, page 21).
Regarding to claim 13, Studer et al teach the cells are contacted with the foregoing agents in amounts effective to increase detectable levels of expression of one or more markers of midbrain DA neurons, or precursors thereof, for example, but not limited to, engrailed-1 (EN-1), orthodenticle homeobox 2 (OTX2), tyrosine hydroxylase (TH), nuclear receptor related - 1 protein (NURR1), forkhead box protein A2 (FOXA2), and LIM homeobox transcription factor 1 alpha (LMX1A) ([0009], page 1).
Regarding to claim 14, Studer et al teach FIG.6A - B shows (A) that hESCs maintained under E8/matrigel conditions and then differentiated for 25 days in NB/N2 media according to the GMP V2A and GMP V2B culture methods (with the at least one inhibitor of SMAD signaling) described by Example 1, induced differentiation of midbrain DA cells expressing high levels of EN-1 and TH. Midbrain DA neurons were fixed and stained for TH and EN-1 in vitro (upper panels) and NURR1 and LMX1A (lower panel) ([0030], page 3).
Regarding to claim 15, Studer et al teach the cells do not express detectable levels of PAX6 and/or Ki67 ([0012], page 1).
Regarding to claim 16, Studer et al teach the method further comprises subjecting the population of differentiated cells to conditions favoring maturation of the cells into dopamine neurons (see claim 15, page 21).
Regarding to claim 17, Studer et al teach the conditions favoring maturation of the cells into dopamine neurons comprise contacting the cells with brain -derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), Cyclic adenosinemonophosphate (cAMP), Transforming growth factor beta 3 (TGF-β3), ascorbic acid (AA), and/or DAPT (see claim 16, page 21).
Regarding to claim 18-19, Studer et al teach the pluripotent cells are selected from the group consisting of human, nonhuman primate or rodent nonembryonic stem cells; human, nonhuman primate or rodent embryonic stem cells; human, nonhuman primate or rodent induced pluripotent stem cells; and human, nonhuman primate or rodent recombinant pluripotent cells (see claim 17, page 21).
Regarding to claim 26, Studer et al disclosed subject matter provides for kits for inducing differentiation of stem cells. In certain embodiments, the kit comprises (a) one or more inhibitor of transforming growth factor beta (TGF-β)/Activin -Nodal signaling, (b) one or more inhibitor of BMP/SMAD signaling, (c) one or more activator of Wnt signaling, (d) one or more activator of SHH signaling. Studer et al also teach the use of signal transduction protein include fibroblast growth factors (FGF) ([0047], page 4), and E8/E6 cell culture medium comprising FGF2 (activator of FGF signaling) ([0099], page 8). Schoeller et al teach the use of activator of the FGF signaling pathway which is selected from the group consisting of FGF2, FGF1, FGF4, FGF8, FGF17 and FGF18 ([0188], page 14). Thus, one of ordinary skill in the art would also include at least one activator of FGF signaling to prepare a kit for inducing differentiation of stem cells to midbrain dopamine neurons.
Response to Arguments
Applicant's arguments filed on 03-17-2026 have been fully considered but they are not persuasive.
1. Applicants use Figure 6 and argue that contacting cells specifically with FGF18 and/or FGFI 7 at the recited timing provides unexpected advantages, including: 1) increased expression of EN1, a marker of midbrain dopamine neurons or precursors, relative to other FGF pathway activators, and 2) reduced expression of SMA1, a marker of contaminating cells which are not midbrain dopamine neurons or precursors, relative to other FGF pathway activators. FGF18 and FGF17 exhibit advantageous marker expression profiles relative to the use of other FGF activators, in particular, FGF8A or FGF8B (Remarks, page 8-9).
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Response to Arguments: As per 716.02, Allegations of Unexpected Results, Any differences between the claimed invention and the prior art may be expected to result in some differences in properties. The issue is whether the properties differ to such an extent that the difference is really unexpected. In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). In the instant case:
It is note that the recited reference Schoeller et al teach method of differentiating the neural plate border stem cells (NPBSCs) into midbrain dopaminergic neurons ([0160], page 13) using a neural medium comprising an activator of the FGF signaling pathway ( [0174]-[0185], page 14), preferably, the activator of the FGF signaling pathway is selected from the group consisting of FGF2, FGF1, FGF4, FGF8, FGF17 and FGF18 ([0188], page 14). Thus, since the prior art reference specifically teach the use of FGF17 and FGF18, a person of ordinary skill in the art would expect that FGF17 and FGF18 function to differentiate the stem cells into midbrain dopaminergic neurons as same as the claimed invention.
The expression data of Figure 6 is without statistical analysis, and it is not unexpected to have variable gene expression data. For example, according to figure 6, the expression of EN1 (a marker of midbrain dopamine neurons or precursors) in presence of FGF17 or FGF18 is not stronger upregulated as compared with FGF8b, or the expression of SMA1 (a marker of contaminating cells which are not midbrain dopamine neurons or precursors) in presence of FGF17 or FGF18 is not significantly downregulated as compared to FGF8A.
2. Applicants argue that the cited art cannot not support the Examiner's position that the claimed subject matter could be achieved by simply "combining prior art elements according to known methods to yield predictable results." (See page 4 and page 6 of the instant Office Action; emphasis added). For example, nowhere does Schoeller provide results supporting the alleged predictability of contacting cells with FGF18 or FGFl 7, let alone that contacting the cells at day 12, rather than specifically between days 9-12 as in Chambers, would lead to the production of the claimed neurons (Remarks, page 10).
Response to Arguments:
It appears that Applicant is arguing that the cited references do not expressly suggest the claimed invention. However, it is well established in case law that a reference must be considered not only for what it expressly teaches, but also for what it fairly suggests. In re Burkel, 201 USPQ 67 (CCPA 1979). Furthermore, in the determination of obviousness, the state of the art as well as the level of skill of those in the art are important factors to be considered. The teaching of the cited references must be viewed in light of these factors. It also appears that applicant is attempting to attack each reference individually. However, in a 103 rejection the references must be considered as a whole. In the instant case, Studer et al (Applicant’s own work) teach the use of signal transduction protein include fibroblast growth factors (FGF) ([0047], page 4). Schoeller et al teach method of differentiating the neural plate border stem cells (NPBSCs) into midbrain dopaminergic neurons ([0160], page 13), using a neural medium comprising an activator of the FGF signaling pathway ( [0174]-[0185], page 14). Preferably, the activator of the FGF signaling pathway is selected from the group consisting of FGF2, FGF1, FGF4, FGF8, FGF17 and FGF18 ([0188], page 14). One of ordinary skill in the art would have been motivated to combine the references because Schoeller et al stated that “the overall efficiency of differentiation of mDA(midbrain dopaminergic) neurons was consistent between 3 different NPBSC lines (FIG. 11D) ( [0307], right column, page 22). Also, Schoeller et al teach the use of activator of the FGF signaling pathway such as FGF17 and FGF18 ([0188], page 14), and the term “activator” is defined as a compound enhancing the activity of a target molecule, preferably by performing one or more of the following effects: (i) the transcription of the gene encoding the protein to be activated is enhanced, (ii) the translation of the mRNA encoding the protein to be activated is enhanced, (iii) the protein performs its biochemical function with enhanced efficiency in the presence of the activator, and (iv) the protein performs its cellular function with enhanced efficiency in the presence of the activator ([0086], page 7).
Chambers et al (Applicant’s own work) teach methods including induction of neural plate development in hESCs for obtaining midbrain dopamine (DA) neurons (Abstract) using “dual SMAD inhibition (SB431542 and Noggin)”([0023], page 3) and “addition of BDNF, ascorbic acid, sonic hedgehog, and FGF8 on day 9-12” ([0029], page 4). One of ordinary skill in the art would have been motivated to combine the references because Chambers et al teach “FIG. 1 showing exemplary dual SMAD inhibition that allowed for a highly efficient feeder-free neural induction in adherent cultures within seven days ….. The dual SMAD inhibition greatly improves neural differentiation” ([0023], page 3) and Chambers et al stated that “this method was adapted to culture systems which may enhance the ease, yield efficiency, speed at which neural cells are derived” ([0161], page 13).
Claims 1-19 and 26 are rejected under 35 U.S.C. 103 as being unpatentable over Studer et al (Pub .No.: US 2018 /0094242 A1, Pub Date : Apr 5, 2018) (Applicant’s own work) in view of Schoeller et al (Pub. No.: US 2015/0010515 A1, Pub. Date: Jan. 8, 2015) and Xi et al (Stem Cells . 2012 Aug;30(8):1655-63. doi: 10.1002/stem.1152.) and Liu et al (STEM CELLS AND DEVELOPMENT Volume 27, Number 5, January 17, 2018, DOI: 10.1089/scd.2017.0282).
Regarding to claim 1, Studer et al disclosed subject matter relates to the discovery that midbrain dopamine (DA) neurons, and precursors thereof, can be differentiated from human stem cells by dual inhibition of SMAD signaling (for example, by inhibition of TGF-β/Activin -Nodal signaling and BMP signaling), along with the activation of Sonic Hedgehog (SHH) signaling, and activation of wingless (Wnt) signaling ([0006], page 1).
Studer et al teach the cells can be further contacted with DA neuron lineage activators and inhibitors ([0007], page 1). Studer et al also teach the use of signal transduction protein include fibroblast growth factors (FGF) ([0047], page 4), and E8/E6 cell culture medium comprising FGF2 (e.g., activator of FGF signaling) ([0099], page 8) (For the claimed: contacting the cells with at least one activator of fibroblast growth factor (FGF) signaling).
Studer et al teach cells were then tested for expression of early midbrain DA marker combinations: 1) OTX2/EN / LMX1A and 2) PAX6/FOXA2/EN/ and NKX2.2 or NKX6.1; or late midbrain DA marker combinations: 1) TH /EN /FOXA2 and 2) LMX1A /OTX2/NURR1 (For the claimed: to obtain a population of differentiated cells expressing at least one marker indicating a midbrain dopamine neuron (mDA) or a precursor thereof).
Although Studer et al teach signal transduction protein include fibroblast growth factors (FGF) ([0047], page 4) and FGF2 ([0099], page 8), Studer et al do not specifically teach FGF18, FGF17 and combination thereof. Schoeller et al cure the deficiency.
Schoeller et al teach method of differentiating the neural plate border stem cells (NPBSCs) into midbrain dopaminergic neurons ([0160], page 13). Schoeller et al teach “culturing the NPBSCs obtained in step (d) in a neural medium comprising an activator of the FGF signaling pathway ……... thereby differentiating the NPBSCs into midbrain dopaminergic neurons” (see [0174]-[0185], page 14). Preferably, the activator of the FGF signaling pathway is selected from the group consisting of FGF2, FGF1, FGF4, FGF8, FGF17 and FGF18 ([0188], page 14).
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the rejected claims to combine the teachings of prior art to modify the method of Studer et al by using FGF17 and FGF18 as activator of the FGF signaling pathway as taught by Schoeller et al as instantly claimed, with a reasonable expectation of success. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to do so because Schoeller et al stated that “the overall efficiency of differentiation of mDA(midbrain dopaminergic) neurons was consistent between 3 different NPBSC lines (FIG. 11D). Taken together, these results demonstrate the formation of mDA neurons by NPBSCs using developmentally appropriate patterning signals.” (see [0307], right column, page 22). Also, Schoeller et al teach the use of activator of the FGF signaling pathway such as FGF17 and FGF18 ([0188], page 14), and the term “activator” is defined as a compound enhancing the activity of a target molecule, preferably by performing one or more of the following effects: (i) the transcription of the gene encoding the protein to be activated is enhanced, (ii) the translation of the mRNA encoding the protein to be activated is enhanced, (iii) the protein performs its biochemical function with enhanced efficiency in the presence of the activator, and (iv) the protein performs its cellular function with enhanced efficiency in the presence of the activator ([0086], page 7). One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Schoeller et al were successful in differentiation of neural plate border stem cells (NPBSCs) into midbrain dopaminergic neurons (mDA) with working examples and data.
Studer et al does not specifically disclose wherein the initial contact of the cells 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. However, Xi et al cure the deficiency.
Xi et al teach “specification of midbrain dopamine neurons from primate pluripotent stem cells” (Title) and “For DA neuron differentiation, PSCs at approximately 10% confluence (1 day after passaging) on mouse embryonic fibroblast feeder layer were cultured in the neural induction medium …. in the presence of SB431542 (10 µM) and LDN193189 (200 nM, both from Stemgent), similar to that reported by Studer and coworkers [25]. … On day 12, some of the FP progenitor clusters were plated onto laminin-coated coverslips overnight for immunocytochemical analysis ……. and FGF8b (100 ng/ml) was added to the culture to expand the progenitors in suspension for 16 days (D12–28).” (Page 1656, left column, 2nd para, Materials And Methods). Thus, Xi et al teach method of Studer and coworkers (authors of primary reference) with step of adding activator of FGF signaling such as FGF8b about 12 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling such as SB431542 and LDN193189. One of ordinary skill in the art would have been motivated to do so because Xi et al stated that “rhesus iPSCs were efficiently patterned to the midbrain FP progenitors that expressed FoxA2, Corin, Otx2, En1, and Lmx1a (Supporting Information Fig. S5B, S5D)” (Page 1160, right column, 1st para.) and “the strategy developed in this study can efficiently induce human and monkey iPSCs to midbrain DA neurons” (Page 1661, left column, 1st para.)
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the rejected claims to combine the teachings of prior art to modify the method of Studer et al and by adding activator of FGF signaling on day 12 after dual SMAD inhibition (SB431542 and LDN193189) as taught by Xi et al, with a reasonable expectation of success. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to do so because Xi et al teach DA neuron differentiation with PSCs on mouse embryonic fibroblast feeder layer in the presence of SB431542 and LDN193189, using the from Studer and coworkers with adding activator of FGF signaling such as FGF8b from D12–28 (Page 1656, left column, 2nd para, Materials And Methods). Xi et al also stated that “rhesus iPSCs were efficiently patterned to the midbrain FP progenitors that expressed FoxA2, Corin, Otx2, En1, and Lmx1a (Supporting Information Fig. S5B, S5D)” (Page 1160, right column, 1st para.) and “the strategy developed in this study can efficiently induce human and monkey iPSCs to midbrain DA neurons” (Page 1661, left column, 1st para.). One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Xi et al were successful in specification of midbrain dopamine neurons from pluripotent stem cells with detailed instructions and working example with data.
Regarding to claim 2-3, Xi et al teach DA neuron differentiation in the neural induction medium in the presence of SB431542 and LDN193189 (both are inhibitors of SMAD signaling) and “On day 12, some of the FP progenitor clusters were plated onto laminin-coated coverslips overnight for immunocytochemical analysis…… FGF8b (100 ng/ml) was added to the culture to expand the progenitors in suspension for 16 days (D12–28)” (Page 1656, left column, materials and methods, 2nd para.). Thus, Xi et al teach cells are contacted with the at least one activator of FGF signaling for at least about 1 days and initial contact of the cells with the at least one activator of FGF signaling is 12 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling.
Regarding to claim 4, Studer et al teach the one or more inhibitor of TGF-β/Activin -Nodal signaling is contacted to the cells for at least about 4 , 5 , 6 , 7 , 8 , 9 , or 10 or more days ([0101], page 8), and the one or more inhibitor of BMP/SMAD signaling is contacted to the cells for at least about 4 , 5 , 6 , 7 , 8 , 9 , or 10 or more days ([0103], page 9).
Regarding to claim 5, Studer et al teach the at least one activator of Sonic hedgehog (SHH) signaling are contacted to the plurality of pluripotent cells for between about 4 and 10 days (see claim 3, page 21).
Regarding to claim 6, Studer et al teach the cells are contacted with the increased concentration of the Wnt activator for at least about 4, 5, 6, 7, 8, 9, or 10 days or more ([0008], page 1).
Regarding to claim 7, Studer et al teach the concentration of the activator of Wnt signaling is increased at least about 2, 3, 4, 5 or 6 days after the cells are initially contacted with the Wnt activator ([0008], page 1).
Regarding to claim 8, Studer et al teach one or more activator of Wnt signaling lowers GSK3β for activation of Wnt signaling. Thus, the activator of Wnt signaling can be a GSK3β inhibitor. A GSK3P inhibitor is capable of activating a WNT signaling pathway ([0091], page 7).
Regarding to claims 8-9, Studer et al teach one or more inhibitor of TGF-β /Activin -Nodal signaling is a small molecule selected from the group consisting of SB431542, derivatives thereof ([0018], page 2).
Regarding to claim 10, the above references do not teach derivative of SB431542 is A83-01. Liu et al cure the deficiency.
Liu et al teach small molecules for neural stem cell induction (title). Liu et al teach the use of both A83-01 and SB431542 to generate NSCs/NPCs from different cell types (see table 2, page 305). SB431542 could be a substitution for A83- 01 (Page 306, right column, 3rd para.). Since Studer et al teach derivatives of SB431542 can be used as inhibitor of TGF-β/Activin -Nodal signaling, a person of ordinary skill in the art would be able to use A83-01 as a derivative of SB431542 to inhibit TGF-β/Activin -Nodal signaling.
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the rejected claims to combine the teachings of prior art to modify the method of the above references by using A83-01 as a derivative of SB431542 to inhibit TGF-β/Activin-Nodal signaling as taught by Liu et al, as instantly claimed, with a reasonable expectation of success. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to do so because Liu et al teaches that TGF-β inhibitor, A83-01, could switch the cell fate from iPSCs into NPCs in TF (OSKM)-induced HUC reprogramming, indicating the importance of TGF-β inhibitors for NSC/NPC induction (Page 306, 3rd para). One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Liu et al demonstrated examples with successful generation of NSCs/NPCs from different cell types with the use of A83-01.
Regarding to claim 11, Studer et al teach the activator of SHH signaling is selected from the group consisting of recombinant SHH, purified SHH, C25II ([0018], page 2)
Regarding to claim 12, Studer et al teach one activator of Sonic hedgehog (SHH) signaling comprises purmorphamine, recombinant SHH, purified SHH, and/or Smoothened agonists (e.g., claim 20, page 21).
Regarding to claim 13, Studer et al teach the cells are contacted with the foregoing agents in amounts effective to increase detectable levels of expression of one or more markers of midbrain DA neurons, or precursors thereof, for example, but not limited to, engrailed-1 (EN-1), orthodenticle homeobox 2 (OTX2), tyrosine hydroxylase (TH), nuclear receptor related - 1 protein (NURR1), forkhead box protein A2 (FOXA2), and LIM homeobox transcription factor 1 alpha (LMX1A) ([0009], page 1).
Regarding to claim 14, Studer et al teach FIG.6A - B shows (A) that hESCs maintained under E8/matrigel conditions and then differentiated for 25 days in NB/N2 media according to the GMP V2A and GMP V2B culture methods (with the at least one inhibitor of SMAD signaling) described by Example 1, induced differentiation of midbrain DA cells expressing high levels of EN-1 and TH. Midbrain DA neurons were fixed and stained for TH and EN-1 in vitro (upper panels) and NURR1 and LMX1A (lower panel) ([0030], page 3).
Regarding to claim 15, Studer et al teach the cells do not express detectable levels of PAX6 and/or Ki67 ([0012], page 1).
Regarding to claim 16, Studer et al teach the method further comprises subjecting the population of differentiated cells to conditions favoring maturation of the cells into dopamine neurons (see claim 15, page 21).
Regarding to claim 17, Studer et al teach the conditions favoring maturation of the cells into dopamine neurons comprise contacting the cells with brain -derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), Cyclic adenosinemonophosphate (cAMP), Transforming growth factor beta 3 (TGF-β3), ascorbic acid (AA), and/or DAPT (see claim 16, page 21).
Regarding to claim 18-19, Studer et al teach the pluripotent cells are selected from the group consisting of human, nonhuman primate or rodent nonembryonic stem cells; human, nonhuman primate or rodent embryonic stem cells; human, nonhuman primate or rodent induced pluripotent stem cells; and human, nonhuman primate or rodent recombinant pluripotent cells (see claim 17, page 21).
Regarding to claim 26, Studer et al disclosed subject matter provides for kits for inducing differentiation of stem cells. In certain embodiments, the kit comprises (a) one or more inhibitor of transforming growth factor beta (TGF-β)/Activin -Nodal signaling, (b) one or more inhibitor of BMP/SMAD signaling, (c) one or more activator of Wnt signaling, (d) one or more activator of SHH signaling. Studer et al also teach the use of signal transduction protein include fibroblast growth factors (FGF) ([0047], page 4), and E8/E6 cell culture medium comprising FGF2 (activator of FGF signaling) ([0099], page 8). Schoeller et al teach the use of activator of the FGF signaling pathway which is selected from the group consisting of FGF2, FGF1, FGF4, FGF8, FGF17 and FGF18 ([0188], page 14). Thus, one of ordinary skill in the art would also include at least one activator of FGF signaling to prepare a kit for inducing differentiation of stem cells to midbrain dopamine neurons.
Conclusion
No claim is allowed.
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/KHOA NHAT TRAN/Examiner, Art Unit 1632
/PETER PARAS JR/Supervisory Patent Examiner, Art Unit 1632
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