DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election of Group I, drawn to claims 1-16, in the reply filed on 05/28/2026 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)). Claims 17-18 are withdrawn from further consideration as being drawn to a non-elected invention.
Priority
The instant application is a CON of PCT/IL2022/050474 filed 05/05/2022, which claims benefit of US Provisional 63/283,242 filed 11/25/2021.
Acknowledgment is made of applicant's claim for foreign priority based on an application filed in Israel on 05/06/2021. It is noted, however, that applicant has not filed a certified copy of the IL283011 application as required by 37 CFR 1.55.
Claim Objections
Claims 6, 12, and 16 are objected to because of the following informalities:
Claim 6 recites the phrase “Induced Pluripotent Stem cells” in lines 4-5, which should be corrected to “induced pluripotent stem cells” for the sake of consistency.
Claim 12 recites the limitation, “wherein said proliferation medium is devoid of molecules selected from the group consisting of” (lines 1-2), which amounts to a Markush grouping and is interpreted as such for the purpose of examination. This limitation should be corrected to “wherein said proliferation medium is devoid of at least one molecule[[s]] selected from the group consisting of” or the like.
Claim 16 comprises limitations (d) and (e), which are identical.
Appropriate correction is required.
Claim Interpretation
The term “farmed animal” is defined in the specification as “any animals which are grown (cultivated) for agricultural purposes, and, in particular, for provision of meat for consumption” (p 38, lines 7-8).
The term “myogenic precursor cell” is defined in the specification as “any cell which can differentiate into a muscle cell” (p 21, lines 9-10).
The term “multinucleated myotube” is defined in the specification as “fused myogenic precursors (e.g. fused myoblasts) having 3 or more nuclei” (p 21, lines 21-22). The specification further recites that “Mono- or bi-nucleated myogenic precursors, even if expressing myogenic differentiation markers, are not considered “multinucleated myotubes”” (p 21, lines 22-24).
Claim 1 is drawn to “A method of inducing multinucleated myotube formation, the method comprising contacting myogenic precursor cells from a farmed animal with at least one molecule.” Claim 1 does not recite a causative relationship between the step of contacting myogenic precursor cells with a molecule and the effect of inducing multinucleated myotube formation. For the sake of compact prosecution, claim 1 is interpreted as “A method of inducing multinucleated myotube formation, the method comprising contacting myogenic precursor cells from a farmed animal with at least one molecule, wherein said contacting induces multinucleated myotube formation.”
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 1-16 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 in the list of molecules: a Retinoid-X Receptor (RXR) agonist, a Retinoid-X Receptor (RXR) activator, a Retinoic Acid Receptor (RAR) agonist, a Retinoic Acid Receptor (RAR) activator, a Ryanodine Receptor (RYRl, RYR3) agonist, a Ryanodine Receptor (RYRl, RYR3) activator, a Calmodulin-dependent Protein Kinase II (CaMKII) agonist, and a Calmodulin-dependent Protein Kinase II (CaMKII) activator. The specification does not define the terms “agonist” or “activator,” which are used interchangeably in the art. Therefore, the metes and bounds of the limitations “agonist” and “activator” are unclear.
Furthermore, claim 1 recites the limitation “Extracellular Regulated Signaling Kinase (ERK1/2) inhibitor” (line 3), which is indefinite. “ERK” is an art-recognized abbreviation for extracellular signal-regulated kinase. However, ERK comprises ERK3, ERK4, ERK5, and ERK7 in addition to ERK1 and ERK2 (see Lu and Malemud, Int J Mol Sci, 2019, 20(15): 3792, Abstract). It is unclear if the entire genus of ERK inhibitor is claimed, or whether only inhibitors of ERK1 and ERK2 are claimed.
Furthermore, claim 1 recites the limitation “a Ryanodine Receptor (RYRl, RYR3) agonist, a Ryanodine Receptor (RYRl, RYR3) activator” (lines 7-8), which is indefinite. Ryanodine receptors include RyR1, RyR2, and RyR3, but the parenthetical includes only RyR1 and RyR3. It is unclear if the entire genus of a Ryanodine Receptor agonist is claimed, or whether only agonists of RyR1 and RyR3 are claimed.
Claims 2-16 are included in the rejection because they depend from claim 1.
Claim 2 recites the limitation “Extracellular Regulated Signaling Kinase (ERK1/2) inhibitor” (lines 1-2), which is indefinite for the same reason as set forth above for claim 1.
Claim 3 recites the limitation “wherein said ERK1/2 inhibitor is an ERK1/2 specific inhibitor” (lines 1-2). The limitation “ERK1/2 specific inhibitor” is not defined in the specification, and therefore, the metes and bounds of said limitation, or how it differs from “ERK1/2 inhibitor,” are unclear. For the purpose of examination, “ERK1/2 specific inhibitor” is interpreted as a “direct inhibitor of ERK1/2.”
Claim 4 recites various compounds followed by their alias(es) in parentheticals (e.g., “MK-8353 (SCH900353)” in line 3). While parentheticals can be used to recite abbreviations, the use of parentheticals adjacent to terms which recite alternate names or isoforms renders the claim indefinite because it is not clear whether the items within the parentheticals are required or optional.
Furthermore, claim 4 recites the phrase “said RXR/RAR agonist” in limitation (c). RXR agonist and RAR agonist are separate genera. Thus, it is unclear which of the species listed in limitation (c) are RXR agonists and which are RAR agonists.
Claim 7 recites the limitation "the corresponding orthologs thereof " in line 2. There is insufficient antecedent basis for this limitation in the claim.
Claim 12 recites the limitations “an Extracellular Regulated Signaling Kinase (ERK1/2) inhibitor,” “a Ryanodine Receptor (RYRl, RYR3) agonist,” and “a Ryanodine Receptor (RYRl, RYR3) activator,” which are indefinite for the same reason as set forth above for claim 1.
Claim 15 is drawn to “The method of claim 1, effected in the presence of serum or serum replacement at an amount which allows cell proliferation and/or under normoxic conditions” (emphasis added). It is unclear whether the italicized limitation of the claim means (1) “effected in the presence of serum or serum replacement (a) at an amount which allows cell proliferation and/or (b) under normoxic conditions,” or (2) “effected (a) in the presence of serum or serum replacement at an amount which allows cell proliferation and/or (b) under normoxic conditions.”
Claim 16 recites the limitation “large multinucleated fibers” in line 2 of limitation (f). The term “large” is a relative term which renders the claim indefinite. The term “large” 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.
Furthermore, claim 16 recites the limitation “(a) said multinucleated myotubes comprise at least three nuclei.” The term “multinucleated myotube” is defined in the specification as “fused myogenic precursors (e.g. fused myoblasts) having 3 or more nuclei” (p 21, lines 21-22). Thus, it is unclear how limitation (a) of claim 16 differs from, or narrows the scope of, the multinucleated myotube of claim 1.
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.
Claim 4 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 4 is drawn to “The method of claim 1, wherein: (b) said MEK1 inhibitor is selected from the group consisting of… MEK inhibitor” (line 5-6 of limitation (b) in claim 4). A MEK1 inhibitor, as recited in claim 1, is a species of MEK inhibitor; thus, the limitation of claim 4 broadens 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.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-16 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 a method of inducing multinucleated myotube formation, the method comprising contacting myoblasts from a farmed animal with at least one molecule selected from the group consisting of an Extracellular Regulated Signaling Kinase (ERK l/2) inhibitor, Mitogen-Activated Protein Kinase Kinase 1 (MEK1) inhibitor, a CaMKII activator/agonist, a Retinoid-X Receptor (RXR) agonist/activator, and a Transforming Growth Factor-Beta (TGF-Beta) inhibitor, wherein the cells are grown on a basement membrane matrix, does not reasonably provide enablement for a method of inducing multinucleated myotube formation, the method comprising contacting any myogenic precursor cells from a farmed animal with a Fibroblast Growth Factor (FGF) inhibitor, a Retinoic Acid Receptor (RAR) agonist/activator, a Ryanodine Receptor (RYRl, RYR3) agonist/activator, an upregulator of intracellular Ca 2+, or a calcium ionophore, in the absence of a basement membrane matrix. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to carry out the invention commensurate in scope with these claims.
Enablement is considered in view of the Wands factors (MPEP 2164.01(a)). The court in Wands states: "Enablement is not precluded by the necessity for some experimentation such as routine screening. However, experimentation needed to practice the invention must not be undue experimentation. The key word is 'undue,' not 'experimentation.' " (Wands, 8 USPQ2d 1404). Clearly, enablement of a claimed invention cannot be predicated on the basis of quantity of experimentation required to make or use the invention. "Whether undue experimentation is needed is not a single, simple factual determination, but rather is a conclusion reached by weighing many factual considerations." (Wands, 8 USPQ2d 1404). The factors to be considered in determining whether undue experimentation is required include:
(A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
While all of these factors are considered, a sufficient amount for a prima facie case are discussed below.
The nature of the invention: The nature of the invention is a method of inducing multinucleated myotube formation, the method comprising contacting myogenic precursor cells from a farmed animal with a Mitogen-Activated Protein Kinase Kinase 1 (MEKl) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a Transforming Growth Factor-Beta (TGF-Beta) inhibitor, a Retinoic Acid Receptor (RAR) agonist/activator, a Ryanodine Receptor (RYRl, RYR3) agonist/activator, an upregulator of intracellular Ca 2+, a Calmodulin-dependent Protein Kinase II (CaMKII) agonist/activator, and/or a calcium ionophore.
The breadth of the claims: Claims 1 and 6-16 encompass a method of inducing multinucleated myotube formation, the method comprising contacting any myogenic precursor cells from a farmed animal with a Mitogen-Activated Protein Kinase Kinase 1 (MEKl) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a Transforming Growth Factor-Beta (TGF-Beta) inhibitor, a Retinoic Acid Receptor (RAR) agonist/activator, a Ryanodine Receptor (RYRl, RYR3) agonist/activator, an upregulator of intracellular Ca 2+, a Calmodulin-dependent Protein Kinase II (CaMKII) agonist/activator, or a calcium ionophore, wherein the cells are cultured on any substrate.
Claim 2 narrows the molecule to an ERK l/2 inhibitor and/or an upregulator of intracellular Ca 2+.
Claim 3 narrows the molecule to an ERK l/2 inhibitor. Claim 5 narrows the ERK l/2 inhibitor to SCH772984.
Claim 4 narrows the scope of claim 1 to require the combination of an ERK l/2 inhibitor, a MEK1 inhibitor, an RXR/RAR agonist, an RYR1/RYR3 agonist, an upregulator of intracellular Ca2+, and a CaMKII agonist.
Claim 8 narrows the myogenic precursor cell type to myoblasts.
The state of the prior art:
Genovese (US 2016/0227830 A1) teaches an in vitro method comprising contacting myogenic precursor cells with a medium comprising A 83-01, a TGF-Beta inhibitor, to produce multinucleated myotubes (para 80). The starting cells used in an embodiment of Genovese are porcine induced pluripotent stem cells (para 7, 75). However, the iPSCs were differentiated into a myogenic lineage prior to contacting with the TGF-Beta inhibitor A83-01 to induce multinucleated myotube formation (para 78-80). Genovese teaches culturing the cells on Matrigel, a basement membrane matrix (para 78-80).
AlSudais (The Journal of Biological Chemistry, 2016, 291(6): 3090-3099) teaches that bexarotene, a retinoid X receptor (RXR) activator, promotes myoblast differentiation and fusion through the activation of RXR and the regulation of Akt/PKB isoform-specific expression (Abstract). AlSudais teaches a method for inducing multinucleated myotube formation by culturing mouse primary myoblasts in the presence of bexarotene (p 3091, col 1, para 3; p 3092, col 1, para 2; Fig 1B, 1C). AlSudais teaches culturing the cells on Matrigel, a basement membrane matrix (p 3091, col 1, para 3).
Barron (US 2022/0047552A1) teaches that GW5074, a cRaf1 kinase inhibitor, which Hanna (US 2017/0275593 A1) shows is an ERK1/2 inhibitor and a MEK1/2 inhibitor (para 305-306), facilitates myogenic differentiation of human primary myoblasts into multinucleated myotubes (para 23-26; Fig 2; para 134, 138-141).
The level of one of ordinary skill: One of ordinary skill in the art is a research scientist holding a postgraduate degree or equivalent experience.
The level of predictability in the art:
The prior art, as set forth above in Genovese, AlSudais, and Barron, teaches that a TGF-beta inhibitor, an RXR activator, and an ERK1/2 inhibitor and MEK1/2 inhibitor promotes myoblast fusion and myotube formation.
However, there was a high level of unpredictability regarding the use of a Fibroblast Growth Factor (FGF) inhibitor, a Retinoic Acid Receptor (RAR) agonist/activator, a Ryanodine Receptor (RYRl, RYR3) agonist/activator, an upregulator of intracellular Ca 2+, a Calmodulin-dependent Protein Kinase II (CaMKII) agonist/activator, or a calcium ionophore, wherein each of the molecule is used alone by itself, to induce myotube formation from myogenic precursor cells. Furthermore, there was a high level of unpredictability regarding 1) the induction of multinucleated myotubes in the absence of a basement membrane matrix, and 2) contacting cells other than myoblasts with a TGF-beta inhibitor, an RXR activator, or an ERK1/2 inhibitor and MEK1/2 inhibitor, to induce myotube formation.
Working examples and the amount of guidance:
The instant specification discloses the following relevant examples:
Contacting first passage mouse-derived primary myoblasts in growth medium with the ERK1/2 inhibitor SCH772984 to induce formation of multinucleated myotubes (Example 1);
Contacting primary isolated bovine myoblasts with ERK1/2 inhibitors SCH772984, AZD0364, BVD523, DEL22379, FR180204, GDC0994, KO947, and LY3214996 to induce formation of multinucleated myotubes (Example 10);
Contacting primary chicken myoblasts with the ERK1/2 inhibitor SCH772984 or the MEK inhibitor U0126 to induce myoblast differentiation and fusion (Example 14);
Contacting primary chicken myoblasts with calcium ionophores AND an ERK inhibitor to increase the fusion index of myoblasts (Example 11);
Contacting primary chicken myoblasts with RXR or RAR agonists AND an ERK inhibitor to increase the fusion index of myoblasts (Example 12);
Contacting primary chicken myoblasts with RYR agonists AND an ERK inhibitor to increase the fusion index of myoblasts (Example 13); and
Transducing primary mouse myoblasts in growth medium with an adenoviral vector encoding wildtype CAMK2δ to induce formation of polynucleated myotubes (Example 3).
The specification does not provide guidance on a method of inducing multinucleated myotubes by contacting myoblasts with a FGF inhibitor, a TGF-beta inhibitor, or a Ca2+ upregulator other than a calcium ionophore.
In all of the examples, primary myoblasts are grown on plates coated with a basement membrane matrix (p 60, para 2; p 75, para 4-5).
The specification does not provide guidance on how to carry out the method of claim 1, wherein myogenic precursor cells other than myoblasts are contacted with an ERK1/2 inhibitor, MEK inhibitor, or CaMKII activator/agonist to induce formation of multinucleated myotubes, or wherein myotubes are grown in the absence of a basement membrane matrix.
The quantity of experimentation necessary:
Based on the content of the disclosure and the state of the prior art, undue experimentation is required to carry out the invention as claimed. As discussed above, the experimental examples disclosed in the specification are limited to embodiments of the invention wherein myoblasts cultured on a basement membrane matrix are contacted with an ERK1/2 inhibitor, MEK inhibitor, or CaMKII activator/agonist to induce formation of multinucleated myotubes.
Given the content of the disclosure and the state of the prior art, additional experimentation is required to carry out the method of induce formation of multinucleated myotubes from myogenic precursor cells, wherein cells other than myoblasts are used; wherein the cells are grown in the absence of a basement membrane matrix; and wherein a molecule other than an ERK1/2 inhibitor, MEK inhibitor, CaMKII activator/agonist, RXR agonist/activator, or TGF-Beta inhibitor is sufficient to induce myotube formation.
Therefore, in light of the breadth of the claims, the limited guidance in the specification with respect to the breadth, and the state of the art, undue experimentation is required to carry out the invention as broadly claimed.
In conclusion, the evidence provided in the disclosure, in light of the teachings available in the art, does not enable one skilled in the art to make the claimed invention without undue or reasonable experimentation. Therefore, the method recited in claims 1-16 are not enabled in its full breadth. Claims 2-16 are included in the rejection because they depend from claim 1.
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.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1, 6-9, and 11-16 are rejected under 35 U.S.C. 102(a)(1) and 35 U.S.C. 102(a)(2) as being anticipated by Genovese (US 2016/0227830 A1; cited in IDS 12/07/2023), as evidenced by Almeida (Stem Cells Int, 2016: 1078686).
Genovese teaches an in vitro method for skeletal muscle cultivation that utilizes cell lines with the potential to differentiate as skeletal muscle, wherein the cell lines are from livestock, poultry, game species, aquatic species, or semi-aquatic species (para 5) (reads on myogenic precursor cells from a farmed animal of claim 1). The method comprises contacting myogenic precursor cells with a medium comprising A 83-01(reads on TGF-Beta inhibitor of claim 1) to produce multinucleated myotubes (para 80).
Genovese teaches modifying an O2K porcine stem cell line (claims 1, 6) with an inducible MyoD transcription factor to produce a myogenic-transcription-factor-modified O2KM cell line (para 7, 75). O2KM cells were seeded onto culture dishes coated with poly-D lysine, murine laminin and Matrigel at a density of 4.1×103 cells/cm2 and cultured under 5% O2 in self-renewal medium, which does not comprise the TGF-Beta inhibitor A83-01, for 3 days (para 78) (claims 11-12). To facilitate myogenic differentiation, cultures were transferred to a 20% O2 basal differentiation milieu, designated by withdrawal of PD032591, PD173074, DOX, hLIF and β-mercaptoethanol (para 78) (claim 13). To conditionally induce the expressed MyoDER protein, 10 μM E2 was added to the medium (para 78). E2-directed myogenic lineage specification following 2 days of induction culture was confirmed by (1) adoption of spindle-like morphology characteristic of skeletal myocytes in treated cultures (2) expression of endogenous the MYOG skeletal muscle transcription factor, and (3) uniform expression of the skeletal myocyte cell surface marker, NCAM (para 78; Fig 6-8).
Following 2 days of E2 induction, cultures were either terminally differentiated in situ (claim 14), or passaged to Matrigel-coated culture dished in terminal differentiation medium (TDM) at 1.56×105 cells/cm2 for terminal differentiation (para 80). TDM was formulated from the same components as myogenic induction medium (MIM), except for the following modifications: withdrawal of E2, addition of 4 μM A 83-01(reads on TGF-Beta inhibitor of claim 1), and 100 nM IGF-1 (para 80). N-2 and B-27 supplements were used exclusively as serum replacements, and cultures were differentiated for up to 6 days under 20% O2 (para 80) (claim 15). Myotube polyploidy was observed during terminal differentiation (para 80, Fig 20D) (claims 1, 16(a)). The myotubes exhibited increasing expression of myogenin (MYOG) over 8 days of terminal differentiation (para 80; Fig 20E) (claims 1, 16(b)).
Genovese teaches that primary adult skeletal muscle progenitor cells, which are harvested from a biopsy (claim 9) and include myoblasts (claims 6, 8) can be used for the method taught therein (para 58). Almeida shows that myoblasts express MyoD, Pax3, and Pax7 (Fig 1) (claim 7). Genovese further teaches an embodiment of the method, wherein the method comprises the following steps: i) modifying a selected self-renewing cell line with a myogenic transcription factor, including MYOD1, PAX3, PAX7, or orthologs thereof, to produce a myogenic-transcription-factor-modified cell line, and ii) inducing such modified cell line by exogenous regulation to maintain in self-renewal process or advance to differentiation process (para 64) (claim 7).
Claim(s) 1-2, 6-8, 11-14, and 16 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Barron (US 2022/0047552A1), as evidenced by Hanna (US 2017/0275593 A1; cited in IDS 12/07/2023) and Almeida (Stem Cells Int, 2016: 1078686).
Barron teaches that GW5074, a cRaf1 kinase inhibitor, facilitates myogenic differentiation of human primary myoblasts into multinucleated myotubes (para 23-26; Fig 2; para 134, 138-141) (claims 1, 16(a)).
Hanna shows that “inhibition of ERK1/2 activity can be also achieved by inhibition of a protein(s) which activity thereof directly affects the activity of ERK1/2. Such a protein(s) is considered to be “upstream” of the MEK/ERK pathway, i.e., a protein (e.g., A-RAF, B-RAF and C-RAF), which inhibition thereof results in a subsequent inhibition of the ERK1/2 protein activity” (para 306). Therefore, GW5074, which inhibits cRaf1, also inhibits ERK1/2 (claims 1-2). Hanna further shows that ERK1/2 inhibitors are also known as MEK1/2 inhibitors (para 305) (claim 1).
Barron teaches that cells from avian, bovine, ovine, or porcine animals can be used in the method taught therein (para 121) (claim 1).
Barron teaches the use of primary myoblasts in the method taught therein (para 134) (claims 6, 8). Barron teaches that primary myoblasts express Pax7 and MyoD (para 135-137) (claim 7). Almeida shows that myoblasts express MyoD, Pax3, and Pax7 (Fig 1) (claim 7).
Barron teaches that human primary myoblasts were cultured in skeletal muscle growth medium, which does not comprise GW5074, prior the addition of GW5074 in the same culture plate to induce myotube formation (para 141) (claims 11-14).
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.
Claim(s) 1 and 6-16 are rejected under 35 U.S.C. 103 as being unpatentable over AlSudais (The Journal of Biological Chemistry, 2016, 291(6): 3090-3099), in view of Genovese (US 2016/0227830 A1), as evidenced by Almeida (Stem Cells Int, 2016: 1078686).
AlSudais teaches that bexarotene, a retinoid X receptor (RXR) activator, promotes myoblast differentiation and fusion through the activation of RXR and the regulation of Akt/PKB isoform-specific expression (Abstract).
Regarding claim 1: AlSudais teaches isolating mouse primary myoblasts (reads on myogenic precursor cells) from lower hind limb muscles, then inducing differentiation in the presence or absence of bexarotene (p 3091, col 1, para 3). The addition of bexarotene resulted in multinucleated myotubes after 24 and 48 hours (p 3092, col 1, para 2; Fig 1B, 1C).
AlSudais does not teach the use of myogenic precursor cells from a farmed animal.
Genovese teaches an in vitro method for skeletal muscle cultivation that utilizes cell lines with the potential to differentiate as skeletal muscle, including myoblasts, wherein the cell lines are from livestock, poultry, game species, aquatic species, or semi-aquatic species (para 4-5, 54-55, 58). Genovese teaches that the production of animal-autonomous meat, or cultured meat, by in vitro cell culture has benefits including production efficiency, reduced environmental impacts, expanded culinary application utility, enhanced nutritional value, cruelty-free production and improved food safety relative to conventionally produced meats (para 4).
It would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of AlSudais by using myoblasts from farmed animals instead of murine myoblasts. One of ordinary skill in the art would have been motivated to make this modification to produce cultured meat products to reduce environmental impacts, expand culinary application utility, enhanced nutritional value, promote animal welfare, and improve food safety relative to conventionally produced meats, as taught in Genovese. One of ordinary skill in the art would have had a reasonable expectation of successfully making this modification because Genovese teaches that myoblasts can be isolated from farmed animals for in vitro myogenesis (para 58).
Regarding claims 6-15: Following the discussion of claim 1, Genovese teaches that primary adult skeletal muscle progenitor cells, which are harvested from a biopsy (claim 9) and include myoblasts (claims 6, 8) can be used for the method taught therein (para 58). Almeida shows that myoblasts express MyoD, Pax3, and Pax7 (Fig 1) (claim 7).
Following the discussion of claim 1, AlSudais teaches isolating mouse primary myoblasts by dissecting lower hind limb muscles from mice (claim 9), then digesting the tissue with dispase and collagenase (claim 10) (p 3091, col 1, para 3). AlSudais teaches growing the isolated cells on Matrigel-coated dishes in DMEM supplemented with 20% FBS, 10% horse serum in the presence of 10 ng/ml basic FGF and 2 ng/ml HGF at 37 °C with 5% CO2 (p 3091, col 1, para 3) (claims 11, 15), which does not comprise the RXR-activator bexarotene (claim 12). To induce differentiation, the medium of 70% confluent cell cultures was changed to DMEM containing 2% FBS and 10% horse serum (p 3091, col 1, para 3) (claims 13, 15). The method of AlSudais implies that the same Matrigel-coated dish is used for the duration of culture, with changes of media therein (claim 14).
Regarding claim 16: AlSudais teaches that the addition of bexarotene resulted in multinucleated myotubes with at least 3 nuclei (limitation (a)) (p 3092, col 1, para 2; Fig 1B, 1C), with increased fraction of MyoD-positive and Myog-positive cells compared to cells cultured in the absence of bexarotene (limitation (b) and (c)) (p 3092, col 1, para 2; Fig 1D-E), and fusion of primary myoblasts and expansion of multinucleated myotubes into large multinucleated fibers (limitation (f)) (p 3092, col 1, para 2; Fig 1B, 1C).
Claim(s) 1, 3, and 5 are rejected under 35 U.S.C. 103 as being unpatentable over Barron (US 2016/0227830 A1), in view of Yohe (Sci Transl Med. 2018, 10(448):eaan4470), as evidenced by Hanna (US 2017/0275593A1).
The teachings of Barron and evidence from Hanna are set forth above.
Barron anticipates claim 1.
Regarding claims 3 and 5: Barron teaches that GW5074, which is an ERK1/2 inhibitor as evidenced by Hanna, facilitates myogenic differentiation of human primary myoblasts into multinucleated myotubes (para 23-26; Fig 2; para 134, 138-141).
Barron does not teach the method of claim 1, wherein the ERK1/2 inhibitor is SCH772984.
Yohe teaches that SCH772984 (claim 5), which functions both as a direct ERK1/2 kinase inhibitor (claim 3) and as an inhibitor of ERK phosphorylation by MEK, was the most potent ERK inhibitor in inducing differentiation in RD cells (a PAX3/7 fusion-negative rhabdomyosarcoma cell line with an activating mutation in NRAS) and SMS-CTR cells (p 5, col 1, para 3; Fig 2F).
It would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Barron by using SCH772984 instead of GW5074 as the ERK1/2 inhibitor for myogenic differentiation. One of ordinary skill in the art would have been motivated to make this modification because Barron teaches that inhibition of ERK1/2 facilitates myogenic differentiation of primary myoblasts into multinucleated myotubes, and Yohe teaches that SCH772984 is an especially potent ERK1/2 inhibitor that induces differentiation.
Claim(s) 1 and 4 are rejected under 35 U.S.C. 103 as being unpatentable over AlSudais (The Journal of Biological Chemistry, 2016, 291(6): 3090-3099), in view of Genovese (US 2016/0227830 A1), Barron (US 2016/0227830 A1), Yohe (Sci Transl Med. 2018, 10(448):eaan4470), Hindi (Science Signaling, 2013, 6(272): re2), Alvarellos (Pharmacogenetics and Genomics, 2016, 26:138–144), Morgan (Biochemical Journal, 1994, 300(3): 665-672), and Moradi (Int J Mol Sci, 2020, 21(3):1016).
The teachings of AlSudais, Genovese, Barron, and Yohe are set forth above.
AlSudais, in view of Genovese, renders obvious claim 1.
Regarding claim 4: AlSudais teaches the use of bexarotene ((c) RXR agonist), which promotes myoblast differentiation and fusion through the activation of RXR and the regulation of Akt/PKB isoform-specific expression (Abstract).
AlSudais, in view of Genovese, does not teach the method of claim 1, wherein the myogenic precursor cells are further contacted with an ERK1/2 inhibitor, MEK1 inhibitor, RYR1/3 agonist, upregulator of intracellular Ca2+, and CaMKII agonist.
Regarding (a) the ERK1/2 inhibitor: Barron, in view of Yohe, renders obvious the method of claim 1, wherein the ERK1/2 inhibitor is SCH772984, as set forth above.
Regarding (b) the MEK1 inhibitor: Barron teaches the use of GW5074, which is a MEK inhibitor, as set forth above.
Regarding (d) the RYR1/3 agonist: Hindi teaches that intracellular Ca2+ influx promotes myoblast fusion (Fig 2; p 4, col 1, para 2).
Alaverellos teaches that RYR is a homotetrameric calcium channel found on the terminal cisternae of the sarcoplasmic reticulum of skeletal muscle, cardiac muscle, smooth muscle cells, and that the activation of RYR1 releases calcium stores from the sarcoplasmic reticulum (Abstract; p 138, col 1, para 1). Alaverellos teaches that caffeine activates RYR1 (p 139, col 1, para 3).
Regarding (e) the upregulator of intracellular Ca2+: Hindi teaches that calcium-dependent signaling regulates the fusion of myoblasts into multinucleated myotubes (p 4, col 1, para 2), and that intracellular Ca2+ influx promotes myoblast fusion (Fig 2).
Morgan teaches that ionomycin upregulates intracellular Ca2+ influx (e.g., Title, Abstract).
Regarding (f) the CaMKII agonist: Moradi teaches that Calmodulin (CaM) is an important Ca2+-sensing protein with numerous downstream targets that are either CaM-dependent or CaM-regulated (Abstract). Moradi teaches that Calmodulin upregulates CaMKII, which in turn results in increased myoblast fusion (Fig 1A).
It would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of AlSudais, in view of Genovese, by further contacting the myoblast with a) the ERK1/2 inhibitor SCH772984, as taught by Barron and Yohe; b) the MEK1 inhibitor (MEK inhibitor) GW5074, as taught by Barron; d) the RYR1/3 agonist caffeine, as taught by Hindi and Alaverellos; e) the intracellular Ca2+ upregulator ionomycin, as taught by Hindi and Morgan; and the CaMKII agonist Calmodulin, as taught by Moradi. One of ordinary skill in the art would have been motivated to make this modification to induce myotube formation from myoblasts, as taught by Barron, Yohe, Hindi, Alvarellos, Morgan, and Moradi. One of ordinary skill in the art would have had a reasonable expectation of successfully making this modification because Barron, Yohe, and Hindi teaches that each of these molecules promotes myoblast fusion and myotube formation.
Conclusion
No claims are allowed.
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/RISA TAKENAKA/Examiner, Art Unit 1632
/KARA D JOHNSON/Primary Examiner, Art Unit 1632
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