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 .
This action is in response to the papers filed 03/07/2023.
Claims 52-71 are pending in the application and examined on the merits. Claims 52, 64, 65, 66, 68, 70 and 71 are independent.
Priority
The present application is a 35 U.S.C. 371 national stage filing of International Application No. PCT/CA2021/051239 filed September 08, 2021.
Applicant’s claim for the benefit of a prior-filed parent provisional applications 63/075,575 filed September 08, 2020 under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, or 365(c) is acknowledged.
Thus, the earliest possible priority for the instant application is September 08, 2020.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper."
For example, there are over 50 references listed on the last pages of the specification (p. 61-68) which do not have a corresponding IDS.
Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Claim Objections
Claims 52 is objected to because of the following informalities:
The first time an acronym is utilized in a claim-set, said acronym should be spelled out in its entirety followed by said acronym in parenthesis (e.g. neural progenitor cell (NPC)).
This applies to terms such as “NPC” and “RAR” (Claim 52), “NIM” (Claim 56), and “RA” (Claim 59).
Appropriate correction is required.
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 52-71 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 53, 59 and 66, the phrase "such as" renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Claim 52 is in improper Markush form; a Markush group should be in the format “expressing reduced levels of at least one marker selected from the group consisting of A, B, and C.”
Claim 53 is in improper Markush form; a Markush group should be in the form “lower levels of at least one marker selected from the group consisting of A, B, and C.”
Claim 64 is in improper Markush form; a Markush group should be in the form “expressing reduced levels of at least one marker selected from the group consisting of A, B, and C.”
Claim 66 is in improper Markush form; a Markush group should be in the form “a Wnt agonist selected from the group consisting of A, B, and C.”
Claim 68 is in improper Markush form; a Markush group should be in the form “one or more detectable markers selected from the group consisting of A, B, and C.”
Currently, it is not clear which species are included in the Markush group and which are not.
Claims 53-56 are rejected as being indefinite as the metes and bounds of the term “unpatterned NPCs” is unclear. Neither the claim or the specification provides a definition of the term “unpatterned NPCs.” Though the Specification discloses at paragraph [0244] that unpatterned NPCs express Nestin, Sox2, and Pax6 this is just this is merely exemplary and non-limiting. The metes and bounds of the claims are unclear particularly since unpatterned NPCs would vary depending on the process of culturing progenitor cells and culture conditions
Claim 66 recites the limitation “essentially serum free media” in step c. This is a relative a relative term which renders the claim indefinite. The term “essentially serum free” 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. It makes the limitations unclear to whether or not there is or isn’t a presence of serum and what amount contained in the media therein would be “essentially free.” Claim 66 additionally recites the term “high concentration” when describing FGF2 + FGF8 in step f. 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. The specification in regard to this limitation states “(for example greater than 20 ng/ml and up to about 400 ng/mL for FGF2 or FGF8)” on page 31. However this is exemplary and not a definition. Furthermore, it is unclear if this is FGF2 and FGF8 combined or separately as their own concentrations. For instance whether FGF2 is 400 ng/mL and FGF8 is 400 ng/ml added into the same media (a total of 800 ng/mL), or if the combination of FGF2 and FGF8 amounts to 400 ng/mL within the media.
Claim 69 contains reference to Table 1 of the specification.
According to MPEP 2173.05(s), “Where possible, claims are to be complete in themselves. Incorporation by reference to a specific figure or table "is permitted only in exceptional circumstances where there is no practical way to define the invention in words and where it is more concise to incorporate by reference than duplicating a drawing or table into the claim. Incorporation by reference is a necessity doctrine, not for applicant’s convenience." Ex parte Fressola, 27 USPQ2d 1608, 1609 (Bd. Pat. App. & Inter. 1993) (citations omitted).”
Claim 53 recites the limitation " the primed unpatterned NPCs" in lines 2-3. There is insufficient antecedent basis for this limitation in the claim as there is no mention of primed unpatterned NPCs.
As the independent claims are rendered indefinite, the claims which depend from the independent claims are also considered indefinite.
Therefore, claims 52-71 are rejected as being indefinite.
The examiner notes that the terms “posteriorized NPCs” and “caudalized NPCs” are defined at paragraphs [0094] and [0111] of the published application.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claim 52, 58-59, and 62-65 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Fehlings (WO2019/023793; published February 2019; Applicant’s own work).
Regarding claim 52, Fehlings teaches a method of making neural progenitor cells (spNPCs) having spinal identity comprising producing first caudalized NPCs which includes steps of passaging NPCs and incubating the NPCs in culture media supplemented with a RAR agonist such as Retinoic Acid (RA) to produce caudalized NPCs (Fig 1A and 10A, para. 0015, 0046).
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As seen in Figure 10D below, the caudalized cells express reduced levels of Otx2 levels compared to that of unpatterned NPCs, although posteriorized NPCs are not explicitly stated, these are viewed as an intermediary while the caudalized NPCs are produced from unpatterned NPCs exposed to retinoic acid to eventually produce caudalized NPCs in the same population (para. 0046).
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As retinoic acid is utilized in both the claimed invention and caudalization of Fehlings, the same method steps yield the same results. Therefore, the caudalized cells would express reduced levels of Otx2 levels compared to the posteriorized cells. Fehlings additionally teaches passaging the caudalized NPCs in suitable culture media supplemented with retinoic acid and an EGFR agonist (para. 0015, step g). Fehlings teaches ventralization after caudalization utilizing a suitable media and then culturing in the presence of a FGF agonist such as FGF2, EGFR, PDGFR agonist (para. 0015, steps h-I, para. 0064,). Fehlings discloses “the term "PDGFR agonist" as used herein means any protein or small molecule that can activate the PDGF receptor A and/or PDGF receptor B (e.g. molecules that bind to PDFGR, induce the dimerization of the receptor and activate the signaling PI3K pathway and STAT1/3 pathways) including any members of the PDGF family such as PDGF- A, -B, -C and -D, and either homo- or heterodimers (e.g. PDGF-AA, -AB, -BB, -CC, -DD). In addition to PDGF, PDGF analogues are known and include for example 740 Y-P (PDGFR 740Y-P)” (para. 0070).
While Fehlings does not teach explicitly that the method is for producing spinal identity neural progenitor cells (spNPCs), the progenitor cells are made through the same methods steps as the invention, therefore, having the same properties.
Regarding claim 58, Fehlings teaches a ROCK inhibitor added after each passage in the first 24 hours (para. 0033).
Regarding claim 59, Fehlings teaches that retinoic acid (RA) is used (para. 0015).
Regarding claim 62 and 63, Fehlings teaches the FGF2 agonist is FGF2 and EGF receptor agonist is EGF (para. 0015, 0065).
Regarding claim 64, the isolated cell population is made by the method of claim 52 which is anticipated by Fehlings, therefore, the isolated cell population claimed is also anticipated by Fehlings. Moreover, Fehlings teaches that the population of cells made can be isolated, purified or diluted (para. 00143).
Regarding claim 65, Fehlings teaches the isolated cell population of claim 64 as discussed above. Morever, Fehlings teaches spinal cord injury treatment or demyelination disease (i.e. neurodegenerative) as uses for the isolated cells which would be administered to a subject (para. 0027, 00150-151).
Therefore, by teaching all of the above claimed limitations, the invention is anticipated by Fehlings.
Claim Rejections - 35 USC § 103
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 53-55 are rejected under 35 U.S.C. 103 as being unpatentable over Fehlings (WO2019/023793; published February 2019; Applicant’s own work) in view of Bicker (2017, Nat. Commun. 8, 15922).
As discussed above in the 102 rejection and incorporated herein in its entirety, Fehlings teaches a method of making spNPCs via making caudalized NPCs from iPSC derived unpatterned NPCs. Moreover, Fehlings teaches that unpatterned NPCs are subjected to culture with one or more of FGFR agonists such as FGF2 and FGF8 (para. 0064, 0078) and anticipates claims 52, 58-59, and 62-65.
However, regarding claims 53 and 54, Fehlings does not teach priming unpatterned NPCs through the method step of adding EGF-L7 agonist to culture media to achieve a primed unpatterned NPCs and required in claim 54, step b. or supplementing the primed unpatterned NPCs in culture media with FGF2 and FGF8 as required in claim 53.
Bicker teaches that the addition of EGFL7 pushes activated NSCs (neural stem cells) towards quiescence and neuronal progeny such as NPCs and activated NSCs towards differentiation through promoting the Notch signaling system (Abstract, p. 7, 2nd column).
It would have been obvious to the ordinary artisan, that the EGF-L7 of Bicker could have been applied to the method of Fehlings, to produce primed unpatterned NPCs from the unpatterned NPCs with a reasonable expectation of success. As Fehlings teaches a process of differentiation, an artisan would have been motivated to utilize a protein such as EGF-L7 known to induce differentiation and promote the Notch signaling system. As discussed above, as each and every method step is the same, the same results will occur such as posteriorized NPCs expressing higher levels of at least one Hox gene, and lower levels of at least one of the brain markers such as Gbx2, Otx2 and FoxG1 compared to unpatterned NPCs.
Regarding claim 55, as discussed above, Fehlings and Bicker make obvious claims 53 and 54. Moreover, Fehlings discloses culturing cells differentiating from hiPSCs in the presence of DLL4 (i.e. notch signaling activator) with EGF and FGF2 (para. 000167).
Therefore, the invention would have been obvious to one of ordinary skill in the art at the time of the effective filing date.
Claims 56 and 57 are rejected under 35 U.S.C. 103 as being unpatentable over Fehlings (supra) in view of Bicker (supra) as applied to claim 53 above, and in further view of Plaistead (PLoS ONE 11(6): e0157620), Hung (“ Formation of Embryonic Bodies to Produce Neural Stem Cells from iPSCs Using the Corning® Spheroid Microplate” 2018) and Ding (J. Cell. Physiol. 225: 417–428, 2010)
As discussed above in the 103 rejection and incorporated here in its entirety, Fehlings and Bicker make obvious a method of making spNPCs via making caudalized NPCs from iPSC derived unpatterned NPCs which are primed via EGF7 and a notch signaling receptor.
Regarding claim 56, Fehlings teaches that the iPSCs are passaged and incubated in an iPSC media for at least about 2 days (para. 0015, step a). Regarding step b, BMP inhibitors and dual SMAD inhibitors are added on day 2 (Fig 3). Regarding step d, rosettes are produced through the method of Fehlings after culturing the cells in NIM with a FGF2 agonist wherein dual SMAD and BMP inhibitors are removed on day 9 (cultured for 7 days) (para. 00160, Step 9; Figure 3). Regarding the limitation wherein the BMP inhibitor or dual SMAD inhibitors are removed from the media on about day 2, to produce unpatterned NPCs, it is interpreted in light of the specification that because day 2 is when the BMP inhibitor is placed, the claim is referring to day 2 of the inhibitors presence not day 2 of the culture (see protocol on p. 37), day 4 overall.
However, Fehlings and Bicker do not teach steps b and c wherein the iPSC culture media lacks a FGF2 agonist and wherein the NIM lacks the FGF2 agonist. The references also do not teach the production of embryoid bodies which are cultured to produce neural rosettes in the presence of BMP inhibitors for 2 days.
Plaistead teaches a method wherein hiPSC form embryoid bodies (EBs) on the fifth day without FGF supplementation in the media which form rosette structures when supplemented with bmp inhibitors then cultured in the presence of FGF to obtain NPCs (p. 3, Derivation and maintenance).
It would have been obvious to one of ordinary skill in the art at the time of the effective filing date to modify the teachings of Fehlings and Bicker with the teachings of Plaistead to arrive at the claimed method involving steps lacking FGF to achieve embryoid bodies with a reasonable expectation of success (current claim 56, steps b. and c, in part.). An artisan would be utilizing a known technique to modify the method of producing NPCs from hiPSCs.
However, the above references do not teach EB formation in 2 days.
Hung teaches EB formation in 2 days from iPS utilizing an essential 8 medium that does not contain FGF (Protocol 1).
Therefore, it would have been obvious to one of ordinary skill in the art at the time of the effective filing date to modify the teachings of Fehlings, Bicker, Plaistead with the teachings of Hung to arrive at the claimed method involving steps able to achieve embryoid bodies in 2 days of culture with a reasonable expectation of success. An artisan would be utilizing a known technique to modify the method of producing NPCs from hiPSCs. As the EB formation is a smaller 2 day time period, it would be obvious to one of ordinary skill in the art to remove bmp inhibitors at a shorter time period such as 2 days instead of the 7 days of Fehlings.
Regarding claim 57, Fehlings, Bicker, Plaistead and Hung make obvious the method of claim 56 as discussed above. Moreover, Fehlings show a BMP inhibitor, TGFbeta inhibitor, FGF2 agonist combined in their induction media.
However, these inhibitors are not present in the iPSC media, nor is a Wnt inhibitor utilized.
Ding teaches FGF-2 directly regulates activity to maintain ESC and iPS cells in an undifferentiated state (p. 422, 1st paragraph). Both Wnt inhibitor (DKK-1) and FGF-2 were cultured together and no significant change was found from regulating activity of TCF/LEF (p. 427, 1st paragraph).
It would have been obvious to one of ordinary skill in the art at the time of the effective filing date to put a BMP inhibitor, TGFP inhibitor, FGF2 agonist, and Wnt inhibitor in the media of step a) as some are known to be part of neural differentiation in figure 3 of Fehlings (See FGF, TGFbi, BMPi, Noggin) in addition to a Wnt inhibitor as taught by Ding with a reasonable expectation of success. An artisan would be motivated to do so as all of Fehlings inhibitors and FGF are applied to the media after the iPSC media in the method, therefore it would be obvious to one of ordinary skill in the art to apply the same inhibitors to the step previous in culturing iPSCs to the same end result of NPCs. Additionally, Ding teaches both Wnt inhibitor (DKK-1) and FGF-2 were cultured together regulating activity of TCF/LEF. Therefore, Wnt inhibitors and FGF-2 are known to work together to maintain iPSCs.
Therefore, the invention would have been obvious to one of ordinary skill in the art.
Claims 60 and 61 are rejected under 35 U.S.C. 103 as being unpatentable over Fehlings (supra) in view of Bicker (supra) as applied to claim 52 above, and in further view of Bejoy (Organogenesis, 12:1–15, 2016)
As discussed above in the 103 rejection and incorporated here in its entirety, Fehlings and Bicker make obvious a method of making spNPCs via making caudalized NPCs from iPSC derived unpatterned NPCs which are primed via EGF7 and a notch signaling receptor.
However, regarding 60 and 61, these references do not teach wherein the posteriorized NPCs are incubated in culture media supplemented with a Wnt signaling activator in addition to the RAR agonist, wherein the Wnt signaling activator is Wnt3a, AZD2858, Wnt agonist 1, CP21R7 (CP21), Wnt or BML-284 hydrochloride.
Bejoy teaches due to the neural patterning effect, Wnt signaling can efficiently promote motor neuron differentiation from hPSCs, in combination with caudalization factor retinoic acid (RA) and ventralization factor SHH. Wnt signaling may elevate the threshold level of SHH signaling to enrich motor neural progenitors. (p. 4, 1st column). Wnt activation induces a caudal fate of neural progenitors that more easily differentiate into motor neurons (p. 4, 1st column). Known wnt activators/agonists are wnt3a (Table 2).
It would have been obvious to one of ordinary skill in the art at the time of the effective filing date to add wnt3a as taught by Bejoy at the time of introducing RA in Fehlings and Bicker with a reasonable expectation of success. An artisan would have been motivated to do so as the RA is being utilized to caudalize the NPCs in Fehlings and Bejoy teaches wnt activation induces caudal fate.
Therefore, the invention would have been obvious to one of ordinary skill in the art.
Claims 66-71 are rejected under 35 U.S.C. 103 as being unpatentable over Fehlings (WO2019/023793; published February 2019; Applicant’s own work) in view of Bicker (2017, Nat. Commun. 8, 15922), Plaistead (PLoS ONE 11(6): e0157620), Ding (J. Cell. Physiol. 225: 417–428, 2010) and Bejoy (Organogenesis, 12:1–15, 2016)
Regarding independent claim 66 steps h and i, Fehlings teaches a method of making neural progenitor cells (spNPCs) having spinal identity comprising producing first caudalized NPCs which includes steps of passaging NPCs and incubating the NPCs in culture media supplemented with a RAR agonist such as Retinoic Acid (RA) to produce caudalized NPCs (Fig 1A and 10A, para. 0015, 0046).
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As seen in Figure 10D below, the caudalized cells express reduced levels of Otx2 levels compared to that of unpatterned NPCs, although posteriorized NPCs are not explicitly stated, these are viewed as an intermediary while the caudalized NPCs are produced from unpatterned NPCs exposed to retinoic acid to eventually produce caudalized NPCs in the same population (para. 0046).
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As retinoic acid (RA) is utilized in both the claimed invention and caudalization of Fehlings, the same method steps yield the same results. Therefore, the caudalized cells would express reduced levels of Otx2 levels compared to the posteriorized cells. Fehlings additionally teaches passaging the caudalized NPCs in suitable culture media supplemented with retinoic acid and an EGFR agonist (para. 0015, step g). Fehlings teaches ventralization after caudalization utilizing a suitable media and then culturing in the presence of a FGF agonist such as FGF2, EGFR, PDGFR agonist (para. 0015, steps h-I, para. 0064,). Fehlings discloses the use of 740 Y-P (PDGFR 740Y-P)” (para. 0070). The unpatterned NPCs are obtained from rosettes.
While Fehlings does not teach explicitly that the method is for producing spinal identity neural progenitor cells (spNPCs), the progenitor cells are made through the same methods steps as the invention, therefore, having the same properties.
Moreover, regarding step j, Fehlings teaches the addition of heparin and FGF after the NPCs are produced (Fig. 7). This reads on dual activation of PI 3-kinase-Akt pathway and FGF pathway, therefore, increasing the proliferation with a reasonable expectation of success.
Thus, Fehlings reads on step a (in part), step d (in part), step g (in part), step h, step i, and step j.
However, Fehlings does not teach in step a of culturing iPSCs (pluripotent stem cells), that TGFP inhibitor, FGF2 agonist, Wnt inhibitor, and BMP inhibitor are present in the media. TGFP inhibitor, FGF2 agonist, and BMP inhibitors are only in their induction media.
Ding teaches FGF-2 directly regulates activity to maintain ESC and iPS cells in an undifferentiated state (p. 422, 1st paragraph). Both Wnt inhibitor (DKK-1) and FGF-2 were cultured together and no significant change was found from regulating activity of TCF/LEF (p. 427, 1st paragraph).
It would have been obvious to one of ordinary skill in the art at the time of the effective filing date to put a BMP inhibitor, TGFP inhibitor, FGF2 agonist, and Wnt inhibitor in the media of step a) as some are known to be part of neural differentiation in figure 3 of Fehlings (See FGF, TGFbi, BMPi, Noggin) in addition to a Wnt inhibitor as taught by Ding with a reasonable expectation of success. An artisan would be motivated to do so as all of Fehlings inhibitors and FGF are applied to the media after the iPSC media in the method, therefore it would be obvious to one of ordinary skill in the art to apply the same inhibitors to the step previous in culturing iPSCs to the same end result of NPCs. Additionally, Ding teaches both Wnt inhibitor (DKK-1) and FGF-2 were cultured together regulating activity of TCF/LEF. Therefore, Wnt inhibitors and FGF-2 are known to work together to maintain iPSCs.
Regarding step b, as the combination of Fehlings and Ding teach iPSCs in a TGFP inhibitor, FGF2 agonist, Wnt inhibitor, and BMP inhibitor, this reads on a culture media containing Wnt inhibitor, and BMP inhibitor.
Regarding step c, Fehlings and Ding do not teach the formation of embryoid bodies (EBs) in serum free media.
Plaistead teaches a method wherein hiPSC form embryoid bodies (EBs) on the fifth day without FGF supplementation in the media which form rosette structures when supplemented with bmp inhibitors then cultured in the presence of FGF to obtain NPCs (p. 3, Derivation and maintenance). As seen in the method detailed, the human ESC medium is serum free (p. 3).
It would have been obvious to one of ordinary skill in the art at the time of the effective filing date to modify the teachings of Fehlings and Ding with the teachings of Plaistead to arrive at the claimed method involving steps in serum free media to achieve embryoid bodies with a reasonable expectation of success. An artisan would be utilizing a known technique to modify the method of producing NPCs from hiPSCs.
Regarding step d, the combination of Fehlings, Ding and Plaistead make obvious the formation of rosettes. Fehlings teaches rosettes are neural tube-like rosettes formed during the course of neural induction (p. 28, step 10). Therefore, neuroectodermal cells are present.
Regarding step e, the combination of Fehlings, Ding and Plaistead make obvious the formation of neuroectodermal cells from tube-like rosettes. However these references do not teach priming them to stay in the ectodermal cell fate by using EGF-L7 or its agonist.
Bicker teaches that the addition of EGFL7 pushes activated NSCs (neural stem cells) towards quiescence and neuronal progeny such as NPCs and activated NSCs towards differentiation through promoting the Notch signaling system (Abstract, p. 7, 2nd column).
It would have been obvious to the ordinary artisan, that the EGF-L7 of Bicker could have been applied to the method of Fehlings, to produce primed unpatterned NPCs from the unpatterned NPCs with a reasonable expectation of success. As Fehlings teaches a process of differentiation, an artisan would have been motivated to utilize a protein such as EGF-L7 known to induce differentiation and promote the Notch signaling system. As discussed above, as each and every method step is the same, the same results will occur such as posteriorized NPCs expressing higher levels of at least one Hox gene, and lower levels of at least one of the brain markers such as Gbx2, Otx2 and FoxG1 compared to unpatterned NPCs.
Regarding step f, the combination of Fehlings, Ding, Plaistead, and Bicker make obvious the primed cells of e). Moreover, Fehlings teaches that unpatterned NPCs are subjected to culture with one or more of FGFR agonists such as FGF2 and FGF8 (para. 0064, 0078). There is no definition for “high” as it is a relative term, therefore, it is interpreted that the exemplary concentrations of FGF agonists at 40ng/mL (p. 28, step 12). It would have been obvious to one of ordinary skill in the art at the time of the effective filing date to utilize both FGF2 and FGF8 in combination at those concentrations as they are both known FGF agonists utilized for the same purpose.
Regarding step g, the combination of Fehlings, Ding, Plaistead, and Bicker make obvious the cells of f). As discussed above, Fehlings teaches caudalization with RA. However, these references do not additionally teach a wnt agonist such as AZD2858, Wnt agonist 1, CP21R7 (CP21) or Wnt.
Bejoy teaches due to the neural patterning effect, Wnt signaling can efficiently promote motor neuron differentiation from hPSCs, in combination with caudalization factor retinoic acid (RA) and ventralization factor SHH. Wnt signaling may elevate the threshold level of SHH signaling to enrich motor neural progenitors. (p. 4, 1st column). Wnt activation induces a caudal fate of neural progenitors that more easily differentiate into motor neurons (p. 4, 1st column). Known wnt activators/agonists are canonical ligands such as wnt3a and gsk inhibitors such as CHIR99021 (Table 2). As evidenced by Fehlings, other known wnt agonists are wnt agonist 1.
It would have been obvious to one of ordinary skill in the art at the time of the effective filing date to add wnt agonists such as wnt agonist 1 as taught by Bejoy and Fehlings at the time of introducing RA in Fehlings and Bicker with a reasonable expectation of success. An artisan would have been motivated to do so as the RA is being utilized to caudalize the NPCs in Fehlings and Bejoy teaches wnt activation induces caudal fate.
Regarding results such as marker expression, cell type induction and others, as each and every method step is made obvious, the same results would also occur with a reasonable expectation of success.
Regarding claim 67, Fehlings, Bicker, Plaistead, Ding and Bejoy make obvious the method of claim 66. Moreover, claim 67 is merely stating an inherent characteristic or intended use which does not provide further structure to 740Y-P or an active method step of claim 66.
Regarding claim 68, Fehlings, Bicker, Plaistead, Ding and Bejoy make obvious the method of claim 66. Thus, the culture composition utilized within 66 is also rendered obvious.
Regarding claim 69, Fehlings, Bicker, Plaistead, Ding and Bejoy make obvious the culture composition based on claim 68. Moreover, Fehlings teaches components of the compositions of Table 1 in the present application in their table on p. 39.
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It is interpreted that “base media” is the DMEM/F12 medium and DMEM/F12 medium supplemented with sodium pyruvate and Neurobasal Media components. As seen above DMEM/F12 medium supplemented with sodium pyruvate is taught by Fehlings. Moreover, for neuronal differentiation medium from EBs, Bicker utilizes Neurobasal medium (p. 3, last paragraph). Therefore, it would be obvious to combine the two different media which are utilized for the same purpose of neuronal differentiation and neural induction.
Regarding claim 70, Fehlings, Bicker, Plaistead, Ding and Bejoy make obvious the method of claim 66. Thus, the isolated population of spNPC of 66 is also rendered obvious..
Regarding claim 71, Fehlings, Bicker, Plaistead, Hung and Ding make obvious the method of claim 66, Fehlings teaches spinal cord injury treatment or demyelination disease (i.e. neurodegenerative) as uses for the isolated cells which would be administered to a subject (para. 0027, 00150-151).
Therefore, the invention would have been obvious to one of ordinary skill in the art.
Conclusion
No claims are allowed.
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/ALEXANDRA F CONNORS/Examiner, Art Unit 1634
/MARIA G LEAVITT/Supervisory Patent Examiner, Art Unit 1634