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 .
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Information Disclosure Statement
The information disclosure statement (IDS) submitted on 11/18/2022 was filed before the
mailing date of the non-final first action on the merits. The submission is in compliance with the
provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being
considered by the examiner.
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 text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
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.
Claims 16-33 are rejected under 35 U.S.C. 103 as being unpatentable over Chal et al ( Nature biotechnology, 2015), in view of Hudson et al (WO 2020/028957 A1), as evidenced by (Boulting et al,2011).
Regarding claims 1 and 32, Chal et al teach a method of differentiating pluripotent stem cells (PCSs) into skeletal muscle. The method of Chal et al comprises of four steps, that involves the sequential culturing of PSCs in a serum-free medium containing a mixture of selective small molecules. The first step in Chal et al’s method involves culturing human induced PSCs (iPSCs) in a serum-free medium supplemented with 3 μM CHIR99021 ,a Wnt pathway inhibitor, and 0.5 μM LDN193189 ,a BMP pathway inhibitor. Chal et al teach that cells are maintained in this medium for 6 days, this reads on claim 16 step (a). The second step involves switching the cells to a culture medium comprising 10 ng/ml HGF, 2 ng/ml IGF-1, 20 ng/ml FGF-2 , and 0.5 μM LDN193189. Chal et al teach that the cells are maintained in this media for 2 days, this reads on claim 16 step (b). The method of Chal et al further involves a third step, wherein the cells are switched to a medium supple-mented with 2 ng/ml IGF1 but lacking HGF and a BMP pathway inhibitor. Chal et al teach that the cells are maintained in this medium for 4 days, this reads on claim 16 step (c). The fourth step in Chal et al’s method teaches that the differentiated cells can be maintained in a medium supplemented with both 10 ng/ml HGF and 2 ng/ml IGF-1. ( See Methods “ Serum-free myogenic differentiation of the human iPS cells” on page 3297, and Fig.6a). Chal et al disclose that the differentiated fibers exhibited spontaneous in vitro contractions, indicating that the differentiated muscle fiber are functional.
Chal et al teach that the cells can be maintained in a serum-free medium comprising IGF-1. However, Chal et al do not teach step (d), which involves culturing cells in a medium comprising IGF1, and γ-secretase/Notch pathway inhibitor
Hudson et al teach a method of differentiating myoblasts into functional myotubes. The method of Hudson et al involves culturing myoblast in a serum-free medium comprising MEM supplemented with ITS , B-27, DAPT (γ-secretase and Notch pathway inhibitor ), and Dabrafenib (Raf inhibitor). Hudson et al refer to this method as the D&D protocol. Hudson et al demonstrate that culturing myoblasts in the serum-free protocol based on Notch and Raf signaling inhibition enables rapid production of functional skeletal muscle and within 7 days. Hudson et al teach that the optimized D&D protocol enhances myoblast differentiation and drives rapid formation of myofibers. (See the Results “ Iterative Screening for Optimal Serum-free Differentiation” on pages 27-28). Hudson et al further state that “Notch signaling is known to play a critical role in the development and regeneration of skeletal muscle, with Notch activation driving self-renewal of undifferentiated PAX7 myoblasts and inhibiting myogenic differentiation”. (See Discussion on page 33 lines 1-11). It is noted that the method of Hudson et al involves differentiating myoblasts into functional myotubes, whereas the method of Chal et al involves differentiating iPSCs into skeletal muscle. However, Chal et al demonstrate that at day 20, a subset of the differentiated iPSCs give rise to myogenic precursor cells (e.g. myocytes and undifferentiated myoblasts), as demonstrated by the presence of cells expressing Myogenin+ (Fig. 6c) and PAX7+ (Fig. 6d).
Therefore, claim 16 would have been obvious to one with ordinary skill in the art at the time the invention was filed, as there was some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention. Chal et al teach a method of differentiating PSCs into skeletal muscle comprising of four steps. Chal et al, further demonstrate that their method of differentiation produce a subset of cells that are undifferentiated myoblasts. Hudson et al describe a method for rapidly producing of functional skeletal muscle fiber from myoblasts by culturing them in serum-free medium comprising γ secretase and Notch pathway inhibitor. Hudson et al further teach that Notch activation inhibits myogenic differentiation. Thus, one would be motivated to consider adding step (d) to the method of Chal et al, wherein the cells are cultured in serum-free medium comprising γ secretase and Notch inhibitor for 7 days, as taught by Hudson et al. There would be a reasonable expectation of success because doing so because would increase the likelihood of producing functional muscle fibers innervated by motoneurons.
Furthermore, it is noted that Chal et al do not teach a functional skeletal muscle that is innervated with motoneurons. However, upon combining the teachings of Chal and Hudson the presence of functional skeletal muscle that are innervated with neuronal cells would be an inherent outcome of performing the method.
Regarding claims 17-19, the method of Chal et al involves using medium supplemented with CHIR99021 to inhibit the Wnt pathway. ( See Methods “ Serum-free myogenic differentiation of the human iPS cells” on page 3297).
Regarding claims 20-21, the method of Chal et al also teach culturing the PSCs in culture medium supplemented with ROCK pathway inhibitor, wherein the ROCK inhibitor is Y-27632. (See Methods section “ Serum-free myogenic differentiation of the human iPS cells”).
Regarding claims22-24, the method of Chal et al also involves using medium supplemented with LDN193189 to inhibit the BMP pathway, wherein the LDN193189 act as inhibitor of both ALK2 and ALK2 receptors. (See Methods “ Serum-free myogenic differentiation of the human iPS cells” on page 3297).
Regarding claims 25-26, following the discussion of claim 16 above, the combined teaching of Chal and Hudson render obvious using γ-secretase and Notch pathway inhibitor to drive a rapid and directed differentiation of myoblasts into functional skeletal muscle. Hudson et al teach using DATP as an inhibitor of the γ-secretase/Notch pathway. (See Hudson et al, Material and methods on page 23 lines 1-5).
Regarding claim 27, following the discussion of claim 16 above. Chal et al render obvious steps (a-c) of claim 16. Hudson et al teach that culturing myoblast in serum free medium comprising γ secretase and Notch inhibitor enables rapid production of functional skeletal muscle within 7 days. Therefore, the recited timeframe for the sequential culturing of PSCs in serum-free medium comprising the selective small molecules would have been obvious to one with ordinary skill in the art, as there was some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention. The method of Chal et al involves culturing cells for 6 days in step (a), 2 days in step (b), 4 days in step (c). Thus one would be motivated to add step (d) to the method of Chal et al and culture the cells for 1-7 days in a serum-free medium comprising Notch inhibitor, as suggested by Hudson et al. There would be a reasonable expectation of success because doing so would promote rapid differentiation and production of functional skeletal muscle.
Regarding claims 28-31, the method of Chal et al involves utilizing hiPSCs 11a cell lines (See the Method section “Human PS cell culture and differentiation”), which are iPSCs derived from fibroblast, as evidence by Boulting et al (See Table.1 on page 16). Chal et al, do not use iPSCs that is derived from a subject with muscular disease. However, Chal et al demonstrate how to differentiate embryonic stem cells derived from the blastocysts of a mdx mouse, a mouse model of DMD syndrome, into skeletal muscle fiber. Chal et al state that “ Fibers derived from ES cells of mdx mice exhibit an abnormal branched phenotype resembling that described in vivo, thus providing an attractive model to study the origin of the pathological defects associated with DMD”. ( See abstract). Therefore, claims 30-31 are combining prior art elements according to known methods to yield predictable results, namely the predictable result being the use of iPSCs derived from a subject with muscular disorder, such as DMD, to drive myogenic differentiation. Chal et al demonstrate that the myogenic differentiation method (as described in claim 16) may be applied to iPSCs derived from normal fibroblast to drive myogenic differentiation. Chal et al also demonstrate that the method may be applied on stem cells derived from a subject with muscular disease, and Chal et al strongly suggest such method can also be employed on stem cells from a subject with muscular disease to study the origin of the pathological defects. However, Chal et al fails to mention iPSCs derived from a subject with muscular disease. An ordinary skill in the art who had reviewed Chal et al, could have immediately noticed the strong possibility that using iPSCs derived from a subject with muscular disease, instead of the mdx-ES of Chal et al, would have the predictable result of being used as an attractive model to study the pathological defects associated with muscular diseases.
Regarding 33, Chal et al teach culturing the cells in 24 and 12-well plate coated with Matrigel, which is a basement membrane matrix composed of laminin, type IV collagen and proteoglycans.
Conclusion
No claim is allowed.
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/FATIMAH KHALAF MATALKAH/Examiner, Art Unit 1638
/Tracy Vivlemore/Supervisory Primary Examiner, Art Unit 1638