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 .
Priority
The instant application is a national stage entry under 35 U.S.C. § 371 of PCT/TR2023/050203 (filed 03/02/2023). Acknowledgement is made of Applicants’ claim for priority to foreign application TR 2022/003171 (filed 03/03/2022).
Specification
The disclosure is objected to because of the following informalities:
Line 1 of pg. 21 recites the term “bEGF” as part of step c) of the method of the instant invention. As Table 1 on pg. 19 recites use of bFGF in the culture medium, “bEGF” is presumed to be a typographical error.
Appropriate correction is required.
Claim Objections
Claim 1 is objected to because of the following informalities:
In step c) of claim 1, the term “bEGF” is recited twice; for the same reason set forth above in the objection to the specification, this is presumed to be a typographical error.
Additionally, claim 1 recites the terms “bFGF”, “RA”, and “SAG”; the description for Figs. 2A-2B on pg. 16 of the specification indicates these terms are abbreviations for basic fibroblast growth factor, retinoic acid, and the small molecule Smoothened Agonist, respectively. While these abbreviations are known in the art, it is proper for the initial introduction of a term to be spelled out in full, with the abbreviation subsequently denoted in enclosed parentheses.
Appropriate correction is required.
Claim Interpretation
The following comments are made to establish broadest reasonable interpretation for the record.
Regarding claim 1: Following the above discussion, the “bEGF” limitation is interpreted as bFGF, or basic fibroblast growth factor; “RA” is interpreted as retinoic acid; and “SAG” is interpreted as Smoothened Agonist, the small molecule which activates the Hedgehog signaling pathway.
Step c) of claim 1 recites, “…adding, respectively, RA and SAG; RA, Shh, and bEGF [sic] to the suspended serum free N2B27 culture medium…”; this is interpreted as adding RA and SAG to the culture media as a first sub step of step c), and adding RA, Shh, and bFGF as a second sub step of step c). This interpretation is supported by the stepwise addition of the growth factors and timing requirements therefor as set forth in Table 1 in the specification (Table 1; pg. 19).
Regarding claims 1, 5-6: These claims recite the term “suspended serum-free N2B27 culture medium”. The term N2B27 culture medium is known in the art; as evidenced by Mulas, et al. (Development. 2019), N2B27 culture medium comprises a 1:1 mixture of DMEM/F12 media: Neurobasal® media, supplemented with N2 and B27® (Table S1). Thus, the suspended serum-free N2B27 culture medium limitation is interpreted as a serum-free culture media comprising DMEM/F12 media and Neurobasal® media at a 1:1 ratio and wherein the media further comprises N2 and B27® supplements. The “suspended” portion of this limitation is not given any patentable weight; it appears to be used throughout the claims as a descriptive term for said culture media.
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 1-6 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.
Regarding claims 1, 5-6: Claim 1 is directed to a method for culturing stem cells by using a culture medium to differentiate human pluripotent stem cells into motor neurons through neuromesodermal stem cells (NMSCs), the method comprising the steps set forth in a), b), and c) of claim 1. Claims 5 and 6 are directed to limitations which further define the method recited in claim 1.
This rejection will focus on three points of indefiniteness: (1) the suspended serum-free N2B27 culture medium limitation, as recited in claims 1, 5, and 6; (2) the adding, respectively, RA and SAG; RA, Shh, and bEGF limitation, as recited in claim 1; and (3) trademarks/trade names recited in claims 1, 5, and 6.
Regarding (1):
Steps a) – c) of claim 1 and claims 5-6 recite the limitation suspended serum-free N2B27 culture medium. The interpretation for this limitation is set forth above; however, as it pertains to the instant rejection, its recitation results in a lack of clarity. Is the suspended portion of this term used as a modifier for the serum-free N2B27 culture medium? Or is its recitation meant to further clarify the steps of the claimed method; i.e., culturing the populations of human pluripotent stems cells (hPSCs) and neuromesodermal stem cells (NMSCs) in suspension for the duration of the method?
For instance, part b) of claim 1 recites, “…attaching the NMSCs formed to a surface in the suspended serum-free N2B27 culture medium…”. As evidenced by Valamehr, et al. (PNAS. 2008), stem cells can be differentiated into specialized cell types, wherein the formation of three-dimensional (3D) cell aggregates known as embryoid bodies (EBs) is a common and critical intermediate to the induction of lineage-specific differentiation (pg. 14459; col. 1, par. 1); this method comprises the use of low-attachment tissue culture plates, which typically use neutral, hydrophilic hydrogels to prevent protein adsorption and subsequent cell attachment when differentiating stem cells into lineage-specific, specialized cell types (pg. 14459; col. 1, par. 2). Is step b) of claim 1 referring to switching the NMSCs formed in step a) from suspension culture to two-dimensional culture? Or is step b) re-affirming the claimed method comprises culturing the cells in suspension; i.e., dissociate any cells which have spontaneously attached due to protein adsorption from the use of non-low attachment tissue culture plates?
As per the specification, “…cells forming the embryoid bodies in suspended culture until the third day were transferred to the surface coated with matrigel-gelatin mixture on the third day, thereby attaching thereto, and the culturing was then carried out in two-dimensional culture” (pg. 19; par. 6); thus, it appears step b) refers to switching from suspension culture to two-dimensional culture. However, this would not be immediately understood by a person having ordinary skill in the art upon reading claim 1; as currently written the metes and bounds are not clearly or precisely defined, rendering claims 1, 5, and 6 indefinite.
Claims 2-4 depend from claim 1, inherit its deficiencies, and are likewise included in this first part of the indefiniteness rejection.
Regarding (2):
Step c) of claim 1 likewise suffers from a lack of clarity. This step recites, “…adding, respectively, RA and SAG; RA, Shh, and bEGF [sic] to the suspended serum-free N2B27 culture medium…”. While the interpretation for this limitation is set forth above, the metes and bounds of the method of the instant claims as currently written is unclear. Does this limitation refer to a complete media exchange? Or does it refer to supplementation only? And, if the latter, how much time is required to pass after adding RA and SAG to the media before it is appropriate to add RA, Shh, and bFGF? As this step explicitly states it is the bFGF which ensures the NMSCs differentiate into functional motor neurons, it is important the metes and bounds are clearly and precisely defined; as currently written, they are not, rendering claim 1 indefinite.
Claims 2-5 depend from claim 1, inherit its deficiencies, and are likewise included in this second part of the indefiniteness rejection. It is noted for the record the limitations recited in claim 6 cure the deficiencies related to the adding, respectively, RA and SAG; RA, Shh, and bEGF limitation.
Regarding (3):
Claims 1, 5, and 6 recite limitations which contain the trademarks/trade names N2B27 culture medium (claims 1, 5, and 6), B27® supplement (claim 5), and Neurobasal® medium (claim 5). Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name.
In the present case, the trademark/trade name “N2B27 culture medium” is used to identify/describe the culture medium of the claimed method; specifically, “N2B27 culture medium” is used to describe a culture medium which comprises a 1:1 mixture of DMEM/F12 media: Neurobasal® media, supplemented with N2 and B27®. However, as B27® supplement is a registered trademark, this limitation is necessarily directed to a particular trademarked product.
The trademark/trade name “B27® supplement” is used to identify/describe a proprietary formulation of serum-free neuronal culture supplement.
The trademark/trade name “Neurobasal® medium” is used to identify/describe a basal medium for the culture of neuronal cells.
Accordingly, the identification/description of these terms is indefinite.
Allowable Subject Matter
In the interest of compact prosecution, there is allowable subject matter, and the following is a statement of reasons for the indication of allowable subject matter:
It appears the method of the instant application is non-obvious in view of the prior art. This is based on the Examiner’s interpretation according to the particulars of the disclosure; e.g., the time sensitive and stepwise differentiation steps recited in Table 1, Working Example 1, pars. 2-5 on pg. 21 of the specification, and Fig. 2A. The method comprises culturing hPSCs in suspension in a culture medium comprising 12 ng/mL bFGF and 3 µM CHIR-99021 from day 0 to day 3 to obtain a population of NMSCs; culturing the obtained NMSCs in a monolayer in a culture medium comprising 0.1 µM RA and 0.5 µM SAG from day 3 to day 5; culturing the NMSCs in a culture medium comprising 0.1 µM RA and 50 ng/mL Shh from day 5 to day 7; culturing the NMSCs in a culture medium comprising 0.1 µM RA, 50 ng/mL Shh, and 10 ng/mL bFGF from day 7 to day 8; and culturing the NMSCs in a culture medium comprising 10 ng/mL bFGF from day 8 onwards.
Jordan, et al. (J Neurosci Res. 2009) teaches the role of bFGF in the differentiation of motor neurons from neural stem and/or progenitor cells; i.e., from NMSCs (Abstract). Jordan, et al. teaches bFGF stimulates the proliferation of neural stem cells (NSCs) in vitro and induces or inhibits neuronal differentiation from cultured NSCs in a dose- and time-dependent manner (pg. 318; col. 2, par. 1). Although 4-day priming with bFGF induces ventralization and expression of transcription factors for the spinal motor neuron lineage, these bFGF-primed NSCs are still dividing progenitors and NOT neurons; the acquisition of a final motor neuron phenotype requires the removal of bFGF and further differentiation (pg. 330; col. 1, par. 2).
Jha, et al. (Stem Cell Rev and Rep. 2015) teaches a four stage protocol to generate spinal motor neurons from human embryonic stem cells (Abstract). The four stages include a pluripotent stem cell (PSC) stage, a neural stem cell (NSC) stage, a motor neuron precursor (MNP) stage, and a mature motor neuron stage (pg. 195; col. 2, par. 2). Once cells have differentiated from NSCs to MNPs, bFGF is withdrawn from the culture medium for further differentiation to mature motor neurons (pg. 195; Fig. 1).
The withdrawal of bFGF is standard in the art, as further evidenced by Lippmann, et al. (US 2017/0283774), which teaches a method for generating motor neuron progenitor cells from hPSCs comprising transiently exposing Sox2+/T+ neuromesodermal progenitors (NMPs) cultured in a neural differentiation medium comprising FGF and a first concentration of a Wnt/β-catenin agonist (e.g., CHIR 99021) to a second higher concentration of the agonist until NKX6.1+ NMPs are obtained; then, NKX6.1+ NMPs are cultured in the neural differentiation medium comprising a retinoid and at least one Shh agonist (in the absence of a Wnt agonist) until OLIG2+NKX6.1+PAX6+ motor neuron progenitors cells are obtained (pars. 0068, 0073, 0077). The OLIG2+NKX6.1+PAX6+ motor neuron progenitors cells are further differentiated to post-mitotic motor neurons by culturing them in a neural differentiation medium comprising retinoid and at least one SHH agonist (par. 0076). Lippmann, et al. teaches OLIG2 expression, required for differentiation to motor neuron progenitors cells, is only achieved in the absence of FGF (Figs. 5A, 5E; pars. 0035, 0039).
The Jordan, et al., Jha, et al., and Lippmann, et al. disclosures follow the same generic protocol: a first step of differentiating pluripotent stem cells to NMSCs in a medium comprising an FGF (e.g., bFGF) and/or CHIR-99021; a second step of differentiating the NMSCs to motor neuron progenitor cells; and a third step of differentiating the motor neuron progenitor cells to post-mitotic, mature motor neurons. While the application of RA and/or the activation of Hedgehog signaling (e.g., SAG, Shh) varies slightly in the art for the second and third differentiation steps, the withdrawal of bFGF is clearly standard.
Applicants, however, have indeed demonstrated the disclosed method is sufficient to differentiate NMSCs to functional motor neurons. As seen in Figs. 5 and 6, axonal elongation and synaptic transmission were observed in the obtained neurons. Further, the obtained neurons had an active neuron phenotype by day 15 (pg. 19). As seen in Fig. 3, these neurons expressed active neuron markers β-tubulin III, Isl-1, Isl-2, MNX-1, and Neun. As evidenced by Sagner, et al. (PLoS Biol. 2018), Isl-1/2, MNX-1, and β-tubulin III are postmitotic motor neuron markers (pg. 5; Fig. 1); as evidenced by Gusel’nikova and Korzhevskiy (Acta Naturae. 2015), NeuN is also a postmitotic neuron marker (pg. 42; col. 1, par. 1).
Thus, the invention of the instant application is considered non-obvious in view of the prior art.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to GINA PRONZATI whose telephone number is (571)270-5725. The examiner can normally be reached Monday - Friday 9:00a - 5:00p ET.
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/GINA PRONZATI/Examiner, Art Unit 1633
/ALLISON M FOX/Primary Examiner, Art Unit 1633
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