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 .
DETAILED ACTION
Election/Restriction
Applicant’s election of Group I, claims 1-13, 21-23 and 26-27, drawn to a method of generating human cochlear hair cells, in the reply filed on 07/31/2026 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 § 818.03(a)).
The requirement is still deemed proper and is therefore made FINAL.
Claims 30-31 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claim Status
Claims 1-13, 21-23, 26-27 and 30-31 are pending.
Claims 30-31 are withdrawn.
Claims 1-13, 21-23 and 26-27 are considered on the merits.
Priority
This application is a 371 of PCT/US2022/081292 (filed on 12/09/2022), which claims benefit from provisional application 63/287,761 (filed on 12/09/2021). The priority claim of the instant application has been granted and the earliest benefit date is 12/09/2021 from the application 63/287,761.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 01/07/2026 and 05/13/2026 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner. The corresponding signed and initialed PTO forms 1449 have been mailed with this action.
Claim Objections
Claims 1, 8-10, 21-22 and 26 are objected to because of the following informalities:
Claim 1, step (b) does not recite a punctuation mark at the end. It is recommended to add “; and” at the end of step (b).
Claim 8 recites “a sufficient amount of time”. It is recommended to change to “the sufficient amount of time” since base claim 1 has recited a sufficient amount of time.
Claim 9 recites “a sufficient amount of time”. It is recommended to change to “the sufficient amount of time” since base claim 1 has recited a sufficient amount of time.
Claim 10 recites “two or markers” in line 1. It is recommended to change to “two or more markers”.
Claim 21, claim 22, and claim 26 do not recite a conjunction between the steps. It is recommended to add “and” at the end of claim 21 step (c), at the end of claim 22 step (f), and at the end of claim 26 step (g).
Claim 22 recites “for about at least about 78 days” in step (g). It is recommended to change to “for at least about 78 days”.
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 1-7 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 recites the phrase “a sufficient amount of time”. A claim may be rendered indefinite by reference to term of an object that is variable (see MPEP 2173.05(b), II). Specifically, the term “sufficient” is a relative term which renders the claim indefinite. The phrase "a sufficient amount of time" 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. Claims 2-7 are rejected as being dependent on claim 1 but not resolving the ambiguity.
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 1-13, 21-23 and 26-27 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
Specifically, independent claim 1 encompasses a genus of method of generating human cochlear hair cells comprising (a) culturing PAX2b+ otic progenitor cells derived from human pluripotent stem cells in a medium comprising a genus of activator of sonic hedgehog, in any concentration with any other activators and inhibitors, for about 5 days; and (b) subsequently culturing the cells of step (a) in a medium comprising a genus of activator of sonic hedgehog and a genus of Wnt inhibitor, in any concentrations with any other activators and inhibitors, for about 4 days after step (a); and (c) further culturing the cells of step (b) for any amount of time in a medium with any activators and inhibitors to differentiate the cells into human cochlear hair cells. Independent claims 21 and 26 (and dependent claim 22) encompass a genus of method comprising the steps of culturing pluripotent stem cells in media comprising claimed supplements, in any concentration with any other activators and inhibitors, for the claimed durations to generate PAX2b+ progenitor cells and human cochlear hair cells.
However, the specification only discloses a single species of “optimized” method of generating human cochlear hair cells comprising (a) culturing pluripotent stem cells in a medium (in 100 µL) consisting essentially of 4 ng/mL FGF-2, optional at least 100 pg/mL BMP-4, and 10 µM SB431542 for about 3 days on coated plates; (b) further culturing the cells of step (a) in a medium (now 125 µL after adding 25 µL) consisting essentially of about 13 ng/mL FGF-2 (added 25 µL of 50 ng/mL FGF-2), about 8 µM SB431542 and about 40 nM LDN193189 (added 25 µL of 200 nM LDN) for about 4 days; (c) further culturing the cells of step (b) in a medium consisting essentially of 3 µM CHIR99021, 200 nM LDN193189, and 50 ng/mL FGF-2 for about 4 days; (d) further culturing the cells of step (c) in a medium consisting essentially of 3 µM CHIR99021 on coated plates for about 2 days; (e) further culturing the cells of step (d) in a medium consisting essentially of 3 µM CHIR99021 and 1 µM purmorphamine for about 5 days; (f) further culturing the cells of step (e) in a medium consisting essentially of 3 µM CHIR99021, 1 µM purmorphamine, and 1 µM IWP-2 for about 4 days; (g) further culturing the cells of step (f) in a medium without supplement for about 28 days; (h) further culturing the cells of step (g) in a medium consisting essentially of 250 ng/mL thyroxine for about 50 days to generate human cochlear hair cells (see e.g., Fig 2a “Schematic comparison of the inventors' original vs. optimized protocol”, see p. 37 “Organoid culture” for the supplements and concentrations and see Fig 7 for treatment of 250 ng/mL thyroxine. It is noted that the above steps (e)-(h) are equivalent to claim 1 steps (a)-(c)).
Under the written description guidelines (see MPEP 2163) the Examiner is directed to determine whether one skilled in the art would recognize that the Applicant was in possession of the claimed invention as a whole at the time of filing. The following considerations are critical to this determination.
To satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention. See, e.g., Moba, B.V. v. Diamond Automation, Inc., 325 F.3d 1306, 1319, 66 USPQ2d 1429, 1438 (Fed. Cir. 2003); Vas-Cath, Inc. v. Mahurkar, 935 F.2d at 1563, 19 USPQ2d at 1116. An original claim may lack written description support when (1) the claim defines the invention in functional language specifying a desired result but the disclosure fails to sufficiently identify how the function is performed or the result is achieved or (2) a broad genus claim is presented but the disclosure only describes a narrow species with no evidence that the genus is contemplated. See Ariad Pharms., Inc. v. Eli Lilly & Co., 598 F.3d 1336, 1349-50 (Fed. Cir. 2010) (en banc). The written description requirement is not necessarily met when the claim language appears in ipsis verbis in the specification. Enzo Biochem, Inc. v. Gen-Probe, Inc., 323 F.3d 956, 968, 63 USPQ2d 1609, 1616 (Fed. Cir. 2002).
Accordingly, to satisfy the written description requirement, the specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention. Vas-Cath, Inc. v. Mahurkar, 935 F.2d 1555, 1562-63, 19 USPQ2d 1111 (Fed. Cir. 1991). See also MPEP 2163.
SCOPE OF THE INVENTION
As stated supra, the breadth of the claims encompasses a genus of method of generating PAX2b+ progenitor cells and human cochlear hair cells comprising culturing pluripotent stem cells in media comprising supplements in any concentrations with any other activators and inhibitors, or comprising a genus of activator of sonic hedgehog and a genus of Wnt inhibitor in any concentrations with any other activators and inhibitors. However, the specification only discloses and provides guidance for a single species of such method with defined supplements in the media, including defined Shh activator purmorphamine and Wnt inhibitor IWP-2, in defined concentrations (see above). Thus, the specification fails to describe the genus of methods.
ACTUAL REDUCTION TO PRACTICE
Accordingly, Applicant did not demonstrate a reduction to practice of a genus of method of generating PAX2b+ progenitor cells and human cochlear hair cells, nor did Applicant adequately set forth in terms of distinguishing identifying characteristics as evidenced by other descriptions of the invention that are sufficiently detailed to show that Applicant was in possession of the claimed genus of method.
DISCLOSURE OF STRUCTURE
The Applicant claims a genus of method of generating PAX2b+ progenitor cells and human cochlear hair cells, but has provided no other methods than the single species of “optimized” method of generating PAX2b+ progenitor cells and human cochlear hair cells. Certainly, a skilled artisan could use the claimed method comprising culturing cells in media with supplements in any concentrations with addition of any other activators or inhibitors. However, the prior art is silent on the genus of method would result in predictably generating PAX2b+ progenitor cells and human cochlear hair cells. Furthermore, neither the specification nor the art indicates a relationship between the structure of the claimed genus of supplements or concentrations and the capability of generating PAX2b+ progenitor cells and human cochlear hair cells.
SUFFICIENT RELEVANT IDENTIFYING CHARACTERISTICS
As mentioned above, the activators and inhibitors of signaling pathways were known in the art field, and the skilled artisan could use the claimed method comprising culturing cells in media with supplements in any concentrations with addition of any other activators or inhibitors. However, the prior art is silent on the capability of generating PAX2b+ progenitor cells and human cochlear hair cells.
The breadth of the claims encompasses a genus of method of generating PAX2b+ progenitor cells and human cochlear hair cells comprising culturing pluripotent stem cells in media comprising supplements in any concentrations with any other activators and inhibitors, or comprising a genus of activator of sonic hedgehog and a genus of Wnt inhibitor in any concentrations with any other activators and inhibitors, yet the present specification provides no guidance nor description what concentrations of the supplements, what specific Shh activator or Wnt inhibitor, or what other activators or inhibitors may be added, other than the defined supplements in the media, including defined Shh activator purmorphamine and Wnt inhibitor IWP-2, in defined concentrations to generate PAX2b+ progenitor cells and human cochlear hair cells. Therefore, the skilled artisan would not know what rational approach to take to use the genus of method, with any predictable outcome on generating PAX2b+ progenitor cells and human cochlear hair cells. Therefore, it is incumbent on the applicant to provide this nexus between structure and function, in order to be given credit for possession of the claimed genus of method.
An applicant may show that an invention is complete by disclosure of sufficiently detailed, relevant identifying characteristics which provide evidence that applicant was in possession of the claimed invention, i.e., complete or partial structure, other physical and/or chemical properties, functional characteristics when coupled with a known or disclosed correlation between function and structure, or some combination of such characteristics. Enzo Biochem, 323 F.3d at 964, 63 USPQ2d at 1613.
STATE OF THE ART & QUANTITY OF EXPERIMENTATION
The method of using the claimed invention is not well established. Although generating PAX2b+ progenitor cells and human cochlear hair cells was known in the state of the art, one of skill in the art would neither expect nor predict the appropriate outcome of PAX2b+ progenitor cells and human cochlear hair cells produced according to the claimed genus of method.
In fact, the instant specification discloses that “while all otic lineage differentiation requires BMP signalling to establish the non-neural ectoderm, the inventors found that the inventors' cochlear organoid cultures were especially sensitive to this parameter for efficient otic induction and subsequent hair cell generation (FIG. 10). Following optimization, the number of hair cell-producing aggregates more than doubled vs. the inventors' previous protocol, and the number of hair cells per aggregate increased 20-fold.” (specification, p. 28, underlined by examiner). The specification also discloses that “the inventors examined effects of the small molecule H89, a cell permeable PKA inhibitor, alone or in combination with PUR and IWP2, but none of these treatments were efficacious in deriving hair cells (FIG. 12).” (specification, p. 30). Thus, the instant application discloses that culturing pluripotent stem cells in media comprising supplements in any concentration with addition of any other activators or inhibitors to generate PAX2b+ progenitor cells and human cochlear hair cells, was not well established.
Applicant has claimed a genus of method of generating PAX2b+ progenitor cells and human cochlear hair cells comprising culturing pluripotent stem cells in media comprising supplements in any concentrations with any other activators and inhibitors, or comprising a genus of activator of sonic hedgehog and a genus of Wnt inhibitor in any concentrations with any other activators and inhibitors, yet the present specification has not disclosed such methods other than the single species of “optimized” method of generating PAX2b+ progenitor cells and human cochlear hair cells by culturing pluripotent stem cells with defined supplements in defined concentrations including defined Shh activator purmorphamine and Wnt inhibitor IWP-2, has not set forth in terms of distinguishing identifying characteristics as evidenced by other descriptions of the invention that are sufficiently detailed to show that Applicant was in possession of the claimed genus of method. Furthermore, the state of the art indicated that using the claimed genus of method to generate PAX2b+ progenitor cells and human cochlear hair cells is not well established and would require undue experimentation, and one of skill in the art would neither expect nor predict the claimed function of generating PAX2b+ progenitor cells and human cochlear hair cells produced according to the claimed genus of method.
CONCLUSION
The Examiner concludes that there is insufficient written description of the instantly claimed genus of method of generating PAX2b+ progenitor cells and human cochlear hair cells comprising culturing pluripotent stem cells in media comprising supplements in any concentrations with any other activators and inhibitors, or comprising a genus of activator of sonic hedgehog and a genus of Wnt inhibitor in any concentrations with any other activators and inhibitors. Specifically, Applicant has only provided one single species of method comprising defined supplements in defined concentrations including defined Shh activator purmorphamine and Wnt inhibitor IWP-2, thus does not provide sufficient number of species to represent the entire scope of the claimed extremely broad genus of method. Therefore, the Examiner concludes that there is insufficient written description to show that Applicant was in possession of the claimed genus of method of generating PAX2b+ progenitor cells and human cochlear hair cells comprising culturing pluripotent stem cells in media comprising supplements in any concentrations with any other activators and inhibitors, or comprising a genus of activator of sonic hedgehog and a genus of Wnt inhibitor in any concentrations with any other activators and inhibitors.
(Scope of Enablement)
Claims 1-13, 21-23 and 26-27 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 generating human cochlear hair cells comprising (a) culturing pluripotent stem cells in a medium (in 100 µL) consisting essentially of 4 ng/mL FGF-2, optional at least 100 pg/mL BMP-4, and 10 µM SB431542 for about 3 days on coated plates; (b) further culturing the cells of step (a) in a medium (now 125 µL after adding 25 µL) consisting essentially of about 13 ng/mL FGF-2 (added 25 µL of 50 ng/mL FGF-2), about 8 µM SB431542 and about 40 nM LDN193189 (added 25 µL of 200 nM LDN) for about 4 days; (c) further culturing the cells of step (b) in a medium consisting essentially of 3 µM CHIR99021, 200 nM LDN193189, and 50 ng/mL FGF-2 for about 4 days; (d) further culturing the cells of step (c) in a medium consisting essentially of 3 µM CHIR99021 on coated plates for about 2 days; (e) further culturing the cells of step (d) in a medium consisting essentially of 3 µM CHIR99021 and 1 µM purmorphamine for about 5 days; (f) further culturing the cells of step (e) in a medium consisting essentially of 3 µM CHIR99021, 1 µM purmorphamine, and 1 µM IWP-2 for about 4 days; (g) further culturing the cells of step (f) in a medium without supplement for about 28 days; (h) further culturing the cells of step (g) in a medium consisting essentially of 250 ng/mL thyroxine for about 50 days to generate human cochlear hair cells (see e.g., Fig 2a “Schematic comparison of the inventors' original vs. optimized protocol”, see p. 37 “Organoid culture” for the supplements and concentrations and see Fig 7 for treatment of 250 ng/mL thyroxine. It is noted that the above steps (e)-(h) are equivalent to claim 1 steps (a)-(c)), does not reasonably provide enablement for a genus of method of generating human cochlear hair cells comprising (a) culturing PAX2b+ otic progenitor cells derived from human pluripotent stem cells in a medium comprising a genus of activator of sonic hedgehog, in any concentration with any other activators and inhibitors, for about 5 days; and (b) subsequently culturing the cells of step (a) in a medium comprising a genus of activator of sonic hedgehog and a genus of Wnt inhibitor, in any concentrations with any other activators and inhibitors, for about 4 days after step (a); and (c) further culturing the cells of step (b) for any amount of time in a medium with any activators and inhibitors to differentiate the cells into human cochlear hair cells, or for a genus of method comprising the steps of culturing pluripotent stem cells in media comprising claimed supplements, in any concentration with any other activators and inhibitors, for the claimed durations to generate PAX2b+ progenitor cells and human cochlear hair cells. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
The factors to be considered in determining whether undue experimentation is required are summarized In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988). The Court in Wands states: “Enablement is not precluded by the necessity for some 'experimentation.'” 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: (1) the quantity of experimentation necessary, (2) the amount or direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims. While all of these factors are considered, a sufficient amount for a prima facie case is discussed below.
The office has analyzed the specification in direct accordance to the factors outlined in In re Wands. MPEP 2164.04 states: "[W]hile the analysis and conclusion of a lack of enablement are based on factors discussed in MPEP 2164.01(a) and the evidence as whole, it is not necessary to discuss each factor in written enablement rejection." These factors will be analyzed, in turn, to demonstrate that one of ordinary skill in the art would have had to perform "undue experimentation" to make and/or use the invention and therefore, Applicant's claims are not enabled commensurate with the scope of the invention.
SCOPE OF THE INVENTION
As stated supra, the breadth of the claims encompasses a genus of method of generating PAX2b+ progenitor cells and human cochlear hair cells comprising culturing pluripotent stem cells in media comprising supplements in any concentrations with any other activators and inhibitors, or comprising a genus of activator of sonic hedgehog and a genus of Wnt inhibitor in any concentrations with any other activators and inhibitors. However, the specification only discloses and provides guidance for a single species of such method with defined supplements in the media, including defined Shh activator purmorphamine and Wnt inhibitor IWP-2, in defined concentrations (see above). Thus, the specification fails to describe the genus of methods.
ACTUAL REDUCTION TO PRACTICE
Accordingly, Applicant did not demonstrate a reduction to practice of a genus of method of generating PAX2b+ progenitor cells and human cochlear hair cells. Since the specification only discloses and provides guidance for a single species of method comprising culturing pluripotent stem cells with defined supplements in defined concentrations including defined Shh activator purmorphamine and Wnt inhibitor IWP-2 to generate PAX2b+ progenitor cells and human cochlear hair cells, the absence of working examples necessitates further experimentation. Therefore, the specification does not provide sufficient guidance on how to use the claimed genus of methods.
STATE OF THE ART & QUANTITY OF EXPERIMENTATION
The state of the art teaches that a method of generating PAX2b+ progenitor cells and human cochlear hair cells is not a highly successful technique or has highly variable results.
In fact, the instant specification discloses that “while all otic lineage differentiation requires BMP signalling to establish the non-neural ectoderm, the inventors found that the inventors' cochlear organoid cultures were especially sensitive to this parameter for efficient otic induction and subsequent hair cell generation (FIG. 10). Following optimization, the number of hair cell-producing aggregates more than doubled vs. the inventors' previous protocol, and the number of hair cells per aggregate increased 20-fold.” (specification, p. 28, underlined by examiner). The specification also discloses that “the inventors examined effects of the small molecule H89, a cell permeable PKA inhibitor, alone or in combination with PUR and IWP2, but none of these treatments were efficacious in deriving hair cells (FIG. 12).” (specification, p. 30). Thus, the instant application discloses that culturing pluripotent stem cells in media comprising supplements in any concentration with addition of any other activators or inhibitors to generate PAX2b+ progenitor cells and human cochlear hair cells, was not well established.
Since the state of the art did not provide guidance for generating PAX2b+ progenitor cells and human cochlear hair cells from culturing pluripotent stem cells encompassed by the instant invention, it is incumbent upon the instant specification to do so. The physiological art is recognized as unpredictable (MPEP 2164.03). As set forth in In re Fisher, 166 USPQ 18 (CCPA 1970), compliance with 35 USC 112, first paragraph requires: “That scope of claims must bear a reasonable correlation to scope of enablement provided by specification to persons of ordinary skill in the art; … in cases involving unpredictable factors, such as most chemical reactions and physiological activity, scope of enablement varies inversely with degree of unpredictability of factors involved.” Moreover, the courts have also stated that reasonable correlation must exist between scope of exclusive right to patent application and scope of enablement set forth in the patent application (27 USPQ2d 1662 Ex parte Maize!.). In view of the foregoing, due to the lack of sufficient guidance provided by the specification regarding the issues set forth above, the state of the relevant art, and the breadth of the claims, it would have required undue experimentation for one skilled in the art to use the instant broadly claimed invention.
CONCLUSION
In conclusion, since the art teaches that the method of generating PAX2b+ progenitor cells and human cochlear hair cells by culturing pluripotent stem cells is prone to influence by multiple factors (e.g., concentrations or what activators/inhibitors used), and is highly unpredictable, and the specification does not provide ample guidance, one would be burdened with undue experimentation to use the claimed invention for generating PAX2b+ progenitor cells and human cochlear hair cells.
In conclusion, given the breadth of the claims and the limited scope of the specification, an undue quantity of experimentation is required to use the invention beyond the scope of a method of generating human cochlear hair cells comprising (a) culturing pluripotent stem cells in a medium consisting essentially of 4 ng/mL FGF-2, optional at least 100 pg/mL BMP-4, and 10 µM SB431542 for about 3 days on coated plates; (b) further culturing the cells of step (a) in a medium consisting essentially of about 13 ng/mL FGF-2 (added 25 µL of 50 ng/mL FGF-2 to original 100 µL), about 8 µM SB431542 and about 40 nM LDN193189 (added 25 µL of 200 nM LDN) for about 4 days; (c) further culturing the cells of step (b) in a medium consisting essentially of 3 µM CHIR99021, 200 nM LDN193189, and 50 ng/mL FGF-2 for about 4 days; (d) further culturing the cells of step (c) in a medium consisting essentially of 3 µM CHIR99021 on coated plates for about 2 days; (e) further culturing the cells of step (d) in a medium consisting essentially of 3 µM CHIR99021 and 1 µM purmorphamine for about 5 days; (f) further culturing the cells of step (e) in a medium consisting essentially of 3 µM CHIR99021, 1 µM purmorphamine, and 1 µM IWP-2 for about 4 days; (g) further culturing the cells of step (f) in a medium without supplement for about 28 days; (h) further culturing the cells of step (g) in a medium consisting essentially of 250 ng/mL thyroxine for about 50 days to generate human cochlear hair cells.
Examiner’s comment
Based on the limited genus of method disclosed by Applicant’s specification to result in generating PAX2b+ progenitor cells and human cochlear hair cells, the prior art of Longworth-Mills and others have been applied to make obvious this limited genus of method.
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 1-13, 21-23 and 26-27 are rejected under 35 U.S.C. 103 as being unpatentable over Koehler et al., (Nat Biotechnol. 2017;35(6):583-589. Cited in IDS 01/07/2026) in view of Longworth-Mills (thesis abstract, publicly available in August 2019, downloaded from https://scholarworks.indianapolis.iu.edu/items/7576b175-38f5-4a3e-a8fc-9db825fc456e, downloaded on 09/16/2026, p. 1-2), Zhao et al., (NeuroReport. 2006; 17:121-124), Karp et al., (US 2017/0071937 A1) and Weber et al., (Proc Natl Acad Sci U S A. 2002; 99(5): 2901-2906. Cited in IDS 01/07/2026).
With respect to independent claims 21, 26 and 1, Koehler teaches a method of generating otic progenitor cells and functional hair cells from human pluripotent stem cells (e.g., title and abstract).
Regarding claim 21 step (a) and claim 26 step (a), Koehler teaches the hPSCs are dissociated and “the aggregates were transferred to low-adhesion 96-well U-bottom plates in 100 μl of chemically defined medium (CDM) containing 4 ng/mL FGF-2, 10 μM SB-431542, and, for some experiments, 2.5 ng/mL BMP4, and 2% growth factor reduced (GFR) Matrigel” and after 4 days of incubation, additional medium is added (Online Method, p. 1, left col, para 2 “hPSC differentiation”), thus teaches (a) culturing pluripotent stem cells in a medium comprising FGF-2, BMP-4, and SB431542 for about 3 days on Matrigel coated plates in claims 21 and 26.
Regarding claim 21 step (b) and claim 26 step (b), Koehler teaches “After 4 d of incubation, 25 μl of CDM containing a 250 ng/mL FGF-2 (50 ng/ml final concentration) and 1 μM LDN-193189 (200 nM final concentration) was added to the pre-existing 100 μl of media in each well” and after an additional 4 d (8 d total), additional medium is added (Online Method, p. 1, left col, para 2 “hPSC differentiation”), thus teaches (b) further culturing the cells of step (a) in a medium comprising FGF-2, SB431542 (from the pre-existing medium) and LDN193189 for about 4 days in claims 21 and 26.
Regarding claim 21 step (c) and claim 26 step (c), Koehler teaches “After an additional 4 d (8 d total), 25 μl of CDM was added to the media. For some experiments, CDM containing a 18 μM CHIR99021 (3 μM final concentration) was added to the pre-existing 125 μl of media in each well on day 8” and on differentiation day 12, the aggregates are pooled (Online Method, p. 1, left col, para 2 “hPSC differentiation”), thus teaches (c) further culturing the cells of step (b) in a medium comprising CHIR99021, LDN193189 (from the pre-existing medium), and FGF-2 (from the pre-existing medium) for about 4 days in claims 21 and 26.
Regarding claim 21 step (d) and claim 26 step (d), Koehler teaches on differentiation day 12, “For non-droplet otic induction, the aggregates were washed and plated individually into each well of a 24-well low-cell-adhesion plate in OMM containing 3 μM CHIR and 1% GFR Matrigel” and the medium was changed completely on day 15 (Online Method, p. 1, left col, para 2 “hPSC differentiation”), thus teaches (d) further culturing the cells of step (c) in a medium comprising CHIR99021 on Matrigel coated plates for about 2 days in claims 21 and 26.
In regard to PAX2b+ otic progenitor cells in claim 21 (d) and claim 1, Koehler teaches “On day 15 or 16, a subset of at least three aggregates were fixed, cryosectioned, and immunostained using PAX2/PAX8 antibodies to positively identify otic-pit-like structures” (Online Method, p. 1, left col, para 2 “hPSC differentiation”), and teaches “aggregates were treated with CHIR99021 (CHIR), a GSK3β inhibitor that activates the WNT pathway, 90.9 ± 5.2% (± s.d.) of the aggregates on days 12–16 (n = 84, seven experiments) showed epithelial protrusions reminiscent of the otic pits that precede vesicle development in vivo (Fig. 2c). These structures were PAX2+ PAX8+ SOX2+ SOX10+ JAG1+, confirming their otic identity (Fig. 2d–h and Supplementary Fig. 10)” (p. 584, right col, para 1). Thus, Koehler teaches the above method steps result in generating PAX2+ otic progenitor cells. Although Koehler does not specifically teach the otic progenitor cells are positive for PAX2b (i.e., a specific isoform of PAX2, see p. 35 of specification), one of ordinary skill in the art would have immediately expected that the PAX2+ otic progenitor cells of Koehler would likely have been PAX2b+ as claimed in claim 21 (d) and claim 1.
However, Koehler is silent on the otic progenitor cells being further cultured with a sonic hedgehog activator such as purmorphamine in claims 1, 2, 22 and 26.
Longworth-Mills, from the same lab as Koehler, teaches in the thesis abstract that “Our laboratory has established a three-dimensional culture system for deriving functional sensory hair cells from human pluripotent stem cells. A major limitation of this approach is that derived hair cells exhibit a morphological and gene expression phenotype reflective of native vestibular hair cells” (see p. 1), likely referring to the method of Koehler (who teaches “the hPSC-derived organoids appear to form vestibular sensory epithelia by default; thus, additional signaling manipulation will be needed to initiate cochlear organogenesis”, see Koehler, p. 589, left col, last para. before Methods). Longworth-Mills teaches “Sonic hedgehog (SHH) signaling has been shown to play a key role in specification of the ventral otic vesicle and subsequent cochlear development. Here, SHH treatment was pursued as a potential strategy for inducing a patterning phenotype permissive to cochlear induction in vitro” and teaches treatment with the SHH pathway agonist Purmorphamine reduces expression of markers for the vestibular-yielding dorsal otic vesicle and upregulates expression of ventral otic marker genes (i.e., cochlear induction, see p. 2). Thus, Longworth-Mills suggests further culturing the cells of step (d) (i.e., the otic progenitor cells) in a medium comprising CHIR99021 (from the pre-existing medium) and purmorphamine in claims 22 and 26 (e)-(f), and suggests culturing PAX2b+ otic progenitor cells derived from human pluripotent stem cells in a medium comprising an activator of sonic hedgehog (e.g., purmorphamine) in claim 1 (a)-(b) and claim 2.
Furthermore, prior art Zhao teaches sonic hedgehog promotes inner ear progenitor cell proliferation and hair cell generation in vitro (see e.g., abstract).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of generating otic progenitor cells and functional human hair cells disclosed by Koehler, by combining a step of culturing the otic progenitor cells in a medium comprising Shh activator such as purmorphamine as suggested by Longworth-Mills and Zhao with a reasonable expectation of success. Since Koehler aims to provide a source of cells for cell-based therapies of the inner ear (e.g., abstract) and acknowledges and suggests that “the hPSC-derived organoids appear to form vestibular sensory epithelia by default; thus, additional signaling manipulation will be needed to initiate cochlear organogenesis” (see e.g., p. 589, left col, last para. before Methods), and since Longworth-Mills, from the same lab as Koehler, teaches “SHH treatment was pursued as a potential strategy for inducing a patterning phenotype permissive to cochlear induction in vitro” and teaches treatment with the SHH pathway agonist Purmorphamine reduces expression of markers for the vestibular-yielding dorsal otic vesicle and upregulates expression of ventral otic marker genes (p. 2) and since Zhao teaches sonic hedgehog promotes inner ear progenitor cell proliferation and hair cell generation in vitro (see e.g., abstract), one of ordinary skill in the art would have had a reason to combine a step of culturing the otic progenitor cells in a medium comprising Shh activator such as purmorphamine as suggested by Longworth-Mills and Zhao in order to obtain human cochlear hair cells of the inner ear for cell-based therapies for deafness (see Koehler abstract and Longworth-Mills p. 1).
However, Koehler is silent on the otic progenitor cells being further cultured with a Wnt inhibitor such as IWP-2 in claims 1, 4-5, 22 and 26.
As stated supra, Koehler teaches that the hPSC-derived organoids appear to form vestibular sensory epithelia (i.e., the dorsal structure) by default; thus, additional signaling manipulation will be needed to initiate cochlear organogenesis (i.e., the ventral structure) (see e.g., p. 589, left col, last para. before Methods), and Longworth-Mills, from the same lab as Koehler, teaches localized domains of gene expression along the dorso-ventral axis of the developing otic vesicle is necessary for proper morphogenesis of both auditory and vestibular inner ear structures (p. 1).
Zhao teaches that Shh is essential for the development of ventral inner ear structures. Conversely, Wnt signaling is critical for dorsal patterning of the otic vesicle and influences the expression of the genes that are necessary for vestibular morphogenesis. Shh also restricts Wnt target genes, and the balance between Wnt and Shh signaling activities plays a major role in distinguishing between vestibular and auditory cell types (p. 121, Introduction), thus suggests Shh activation and Wnt inhibition play an additive role in inducing ventral inner ear structures such as cochlea, related to claims 1 (b), 22 (f) and 26 (f). Zhao reduces to practice Shh treatment to promote inner ear progenitor cell proliferation and hair cell generation in vitro (see e.g., abstract).
In regard to a Wnt inhibitor IWP-2, Karp teaches a method for activating Wnt pathway such as CHIR99021 in a cochlear supporting cell population (LGR5+) to increase self-renewal and differentiation into hair cells (e.g., [0018] and [0065]). Karp reduces to practice IWP-2 (2 µM) as a Wnt inhibitor in this context (e.g., [0082], [0979]), related to claims 4-5, 22 (f) and 26 (f).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of generating human cochlear hair cells comprising culturing otic progenitor cells with an activator of Shh suggested by Koehler in view of Longworth-Mills and Zhao, by combining a Wnt inhibitor with the Shh activator to additively promote induction of ventral inner ear structures, i.e., cochlear induction, as suggested by Zhao with a reasonable expectation of success. Since Zhao teaches Shh and Wnt play opposing patterning roles and Shh restricts Wnt target genes (see above) and since Longworth-Mills teaches “SHH treatment was pursued as a potential strategy for inducing a patterning phenotype permissive to cochlear induction in vitro” and treatment with the SHH pathway agonist Purmorphamine reduces expression of markers for the vestibular-yielding dorsal otic vesicle and upregulates expression of ventral otic marker genes (p. 2), one of ordinary skill in the art would have had a reason to combine a Wnt inhibitor in combination with the Shh activator of Longworth-Mills, as suggested by Zhao, in order to further additively restrict the dorsal Wnt signal to promote induction of ventral structures, i.e., cochlear hair cells. Furthermore, one of ordinary skill in the art would have had a reason to choose IWP-2 as a Wnt inhibitor since Karp reduces to practice IWP-2 (at a concentration of 2 µM) as a Wnt inhibitor in the context of differentiating otic progenitor cells into hair cells. Applicant is reminded that claim 1 steps (a)-(b) and claims 22 and 26 steps (e)-(f) recite the transitional phrase “comprising”, thus encompass using the same medium comprising both purmorphamine and IWP-2 together with CHIR99021 in those steps, as suggested by Koehler in view of Longworth-Mills, Zhao and Karp.
In regard to the concentrations and durations recited in claims 1, 3, 5, 22 and 26, MPEP states “generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical” and “where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation”. See MPEP 2144.05(II)(A). In the instant case, Longworth-Mills, Zhao and Karp make obvious culturing otic progenitor cells in a medium comprising Shh activator purmorphamine and Wnt inhibitor IWP-2 for induction of ventral inner ear structures such as cochlea, and Karp reduces to practice the concentration of IWP-2 within the claimed range in this context. Therefore, it would have been obvious for one ordinary skill in the art before the effective filing date of the claimed invention to apply the claimed concentration range of purmorphamine and IWP-2, and to apply the claimed culture durations because they are the result of “routine optimization”.
Regarding claim 6 and claim 26 (h) directed to culturing with thyroxine, Weber teaches thyroid hormone is a critical determinant for the regulation of the cochlear motor protein Prestin which has a thyroid hormone (TH) response element in coding region (see title and abstract). Weber teaches a severe reduction of Prestin mRNA and protein was noted in cochleae of same-aged animals in the absence of thyroid hormone (TH) at P6, P8, and P16, and when hypothyroid rats were injected with T4 (i.e., thyroxine) on P3 and killed 12 h later, the suppression of Prestin mRNA in outer hair cells (OHC) in cochlea, typically noted in the absence of TH (Fig. 5C, Hypo P3) in comparison to the control (Fig. 5C, Control P3), was overcome (Fig. 5C, Hypo P3+T4) (e.g., p. 2905, left col, also see Fig 6C for the restored structure of outer hair cells). Thus, Weber suggests that administration of thyroxine increases expression of cochlear motor protein Prestin and the structure of outer hair cells.
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of generating human cochlear hair cells suggested by Koehler in view of Longworth-Mills, Zhao and Karp, by combining a step of further culturing the cells in a medium comprising thyroxine as suggested by Weber with a reasonable expectation of success. Since Weber teaches thyroid hormone is a critical determinant for the regulation of the cochlear motor protein Prestin and thyroid hormone receptor β (TRβ) mutant mice are deaf (abstract and p. 2906, right col) and treatment of thyroxine increases Prestin mRNA and protein and restores structure of outer hair cells (see e.g., Fig 5C and 6C), one of ordinary skill in the art would have had a reason to combine thyroxine in the culture medium in order to increase expression of cochlear motor protein Prestin that relates to the function of cochlear outer hair cells.
In regard to the start time/duration and concentration of thyroxine recited in claims 6-7 and claim 26 (g)-(h), as stated supra, Weber suggests culturing the otic progenitor cells with thyroxine. Therefore, it would have been obvious for one ordinary skill in the art before the effective filing date of the claimed invention to apply the claimed start time/duration and concentration of thyroxine because they are the result of “routine optimization”. See MPEP 2144.05(II)(A).
Regarding claim 1 (c), claim 22 (g) and claim 26 (g)-(h) directed to differentiating the cells into human cochlear hair cells, as stated supra, Longworth-Mills teaches treatment with the SHH pathway agonist Purmorphamine reduces expression of markers for the vestibular-yielding dorsal otic vesicle and upregulates expression of ventral otic marker genes (i.e., cochlear induction, see p. 2), Zhao suggests Shh activation and Wnt inhibition play an additive role in inducing ventral inner ear structures such as cochlea, and Weber teaches thyroid hormone is a critical determinant for the regulation of the cochlear motor protein Prestin and thyroid hormone receptor β (TRβ) mutant mice are deaf (abstract and p. 2906, right col) and treatment of thyroxine increases Prestin mRNA and protein and restores structure of outer hair cells (see e.g., Fig 5C and 6C). Therefore, one of ordinary skill in the art would have had a reasonable expectation of success in differentiating the otic progenitor cells into human cochlear hair cells by culturing the progenitor cells in media comprising Shh activator such as purmorphamine and Wnt inhibitor such as IWP-2, as well as thyroxine.
In regard to the culture durations to generate human cochlear hair cells recited in claim 1 (c), claims 8-13, claim 22 (g) and claim 26 (g)-(h), as stated supra, it would have been obvious for one ordinary skill in the art before the effective filing date of the claimed invention to apply the claimed culture durations to generate human cochlear hair cells because they are the result of “routine optimization”. See MPEP 2144.05(II)(A).
In regard to the cochlear hair cells expressing markers such as Prestin recited in claim 1 (c) and claims 10-13, it must be noted that these wherein clauses do not recite an active step in the claimed method, but only the results of the culturing steps as suggested by Koehler in view of Longworth-Mills, Zhao, Karp, and Weber. MPEP 2111.04 I states a whereby clause (or a wherein clause) “in a method claim is not given weight when it simply expresses the intended result of a process step positively recited.” Therefore, these wherein clauses do not provide any patentable weight in determining patentability of the claimed method. Nevertheless, as stated supra, Weber suggests that culturing the otic cells with thyroxine increases expression of Prestin.
Regarding the medium about 11 days after start of claim 1 step (a) does not contain additional agonists or inhibitors in claims 8-9 and the medium in step (g) does not comprise CHIR99021, purmorphamine, or IWP-2 in claims 23 and 27, Koehler teaches “On day 18 of differentiation, the CHIR was removed from the medium by washing” and the aggregates are maintained in stir-plates or orbital shaker for up to 140 days (Online Method, p. 1, left col, para 2 “hPSC differentiation” – right col, para 1). It is noted that day 12 of Koehler is equivalent to the start of step (a) in claim 1 and to the start of step (d) in claims 21 and 26 (see discussion above regarding steps (a)-(c) in claims 21 and 26). Thus, the day 18 of Koehler, after which time point the medium does not contain CHIR99021 or other agonists or inhibitors such as purmorphamine or IWP-2, is equivalent to about 9 days after the start of step (a) in claim 1 (i.e., after steps (a) and (b)), and is equivalent to the step (g) in claims 22 and 26. Therefore, Koehler makes obvious the cells, after culturing in a medium comprising CHIR99021, are maintained in a medium without CHIR99021 (or other additional agonists or inhibitors), related to claims 8-9 and claims 23 and 27.
Hence, the claimed invention as a whole was prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention in the absence of evidence to the contrary.
Conclusion
No claims are allowed.
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/JIANJIAN ZHU/Examiner, Art Unit 1631
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