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 .
Claim Status
1. The amendment filed 03/16/2026 has been entered. Claims 1 – 20 remain pending. Claims 1 – 15 are under consideration.
Election/Restrictions
2. Applicant’s election without traverse of Group I (claims 1 – 15) in the reply filed on 10/30/2025 is acknowledged.
3. Claims 16 – 20 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 10/30/2025.
Priority
4. This application is the U.S. National Stage application of PCT International Patent Application No. PCT/AU2021/051204 filed 10/14/2021 which claims priority to Australian Patent Application No. 2020903734 filed 10/14/2020.
Withdrawn Claim Objections
5. The objections to claim 1 are withdrawn in view of Applicant’s amendment to claim 1.
6. The objection to claim 2 are withdrawn in view of Applicant’s amendment to claim 2.
7. The objection to claim 13 are withdrawn in view of Applicant’s amendment to claim 13.
8. The objection to claim 14 are withdrawn in view of Applicant’s amendment to claim 14.
Withdrawn Claim Rejections
9. The rejection of claim 15 under 35 U.S.C. 112(d) is withdrawn in view of Applicant’s amendment to the claim.
10. The rejection of claims 1 – 4, and 7 – 15 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 1.
11. The rejection of claims 5 and 6 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 1.
Maintained and Necessitated by Amendment 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.
12. Claims 13 and 14 remain rejected and claim 15 is 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. Although maintained, the rejection is revised in light of Applicant’s amendment to the claims.
13. Regarding claim 13, the recited number of days lack antecedent basis because claim 1 does not recite any method steps for the formation of otic prosensory vesicles and instead merely recites culturing otic prosensory vesicles. Likewise, the recited number of days in steps (b) and (c) appear to be based on otic prosensory vesicle formation and not steps A, B, or C of claim 1 and claim 1 does not recite maturation. Further, it is unclear if steps (b) is further limiting step A of claim 1 to require culturing with a SHH inhibitor for about 15 days and if step (c) is further limiting step C of claim 1 to require culturing for about 68 days in total or 68 days starting from step A. Therefore, the metes and bounds of the recited number of days is unclear relative to the limitations of claim 1.
14. Regarding claim 14, the recited number of days lack antecedent basis because step A of claim 1 does not recite any method steps for the formation of sensory epithelium formation or maturation. Further, it is unclear if step (d) is further limiting step A of claim 1 to require culturing with a SHH inhibitor for 18 to 32 days and if step (e) is further limiting step C of claim 1 to require culturing for 33 days. Therefore, the metes and bounds of the recited number of days is unclear relative to the limitations of claim 1. Claim 15 is also rejected as it depends from claim 14 and does not clarify the grounds of rejection.
Claim Interpretation
15. For the purpose of applying prior art, “that originate from human induced pluripotent stem cells” of claim 1 is interpreted as a product-by-process limitation. MPEP 2113(I) states that “product-by-process claims are not limited to the manipulation of the recited steps, only the structure implied by the steps”. As such, the structure implied by that originate from human induced pluripotent stem cells” is that of otic prosensory vesicles. MPEP 2113 (II) states that “once a product appearing to be substantially identical is found and a prior art rejection is made, the burden shifts to the applicant to show an nonobvious difference”.
16. For the purpose of applying prior art, “gelatinous protein mixture secreted by EHS mouse sarcoma cells” of claim 1 is interpreted as Matrigel because Applicant’s specification discloses at page 15, para. 0091 this protein mixture can be Matrigel.
17. For the purpose of applying prior art, “with neural innervation” is interpreted as the inner ear hair cells express myosin VIIa and Tuj1 based on Applicant’s specification at page 21, para. 00123.
18. For the purpose of applying prior art, claims 2 and 8 – 15 are not given patentable weight because they recite product-by-process limitations of the “otic prosensory vesicles that originate from human induced pluripotent stem cells” of claim 1. MPEP 2113(I) states that “product-by-process claims are not limited to the manipulation of the recited steps, only the structure implied by the steps”. As such, the structure implied by claims 2 and 8 – 15 is that of otic prosensory vesicles.
19. For the purpose of applying prior art, “inner hair cells” of claim 7 is interpreted as inner hair cells that are Tuj1 positive.
Objections and Rejections Necessitated by Amendment
Claim Objections
20. Claim 1 is objected to because of the following informalities: in line 4, “pluripotent stem-cells” should read “pluripotent stem cells” without the dash between “stem” and “cells”. Appropriate correction is required.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
21. Claim(s) 1 – 4, and 7 – 15 is/are rejected under 35 U.S.C. 103 as being unpatentable over Yangling (Zhao, Yanling, et al. Neuroreport 17.2 (2006): 121-124; previously cited), hereinafter Yangling in view of Panek (Panek, M. et. al. Cytotechnology 70.3 (2018): 1085-1095; previously cited), hereinafter Panek as evidenced by Roccio (Roccio, M. et. al. Development 146.17 (2019): dev177188; previously cited), hereinafter Roccio in view of Koehler (US-12077778-B2; Filed 08/10/2020; Published 09/03/2024), hereinafter Koehler.
Regarding step (A) of claim 1 and “otic prosensory vesicles” of claims 1, 2, and 8 – 15, Yangling teaches a method of producing hair cells by culturing an aggregate of otic vesicle cells (“otic prosensory vesicles” of claims 1, 2, and 8 – 15; “3D shaped inner ear organoid” of claim 1) in hanging drop in the presence of in a culture medium comprising 2.5 µM cyclopamine (“SHH inhibitor” of claim 1) (page 122, left col. para. 3; Figure 1 and 2). Yangling teaches when otic vesicles where cultured in monolayers, only very few hair cells were observed but culturing otic vesicles in aggregates resulted in robust and reproducible hair cells (page 122, right col. last para.). Yangling teaches it has been shown that in vitro differentiating hair cells in otic vesicle cell aggregates organize into specific patterns and are surrounded by supporting cells in a manner similar to the in vivo pattern (page 122, right col. last para.). Yangling teaches that intimate signaling among the aggregated otic vesicle cells was required for generating hair cells because hair cells failed to develop when the otic vesicles were cultured in dispersed monolayers (page 123, left col. para. 2 and right col. para. 1). Yangling does not teach the culture medium comprises 5 – 10% Matrigel.
Regarding step (B) of claim 1, Yangling teaches culturing the aggregate of otic vesicle cells in culture medium with Shh and this promoted cell growth, while cyclopamine inhibited cell growth (page 121, left col. para. 3 and right col. para. 2). Yangling does not teach removing cyclopamine from the culture medium.
Regarding step (C) of claim 1, Yangling teaches culturing the aggregate of otic vesicle cells in culture medium with Shh promoted cell growth and increased the number of myosin VIIA-positive cells in the aggregates, while culturing with cyclopamine decreased the number of myosin VIIA-positive cells in the aggregates (page 121, left col. para. 3 and right col. para. 2 – 3; Figure 2; page 123, left col. para. 2). Yangling teaches when Shh signaling was blocked with cyclopamine, myosin VIIA-positive cell generation was remarkably reduced (page 122, right col. para. 3). Yangling does not teach the myosin VIIA-positive cells are also positive for Tuj1 (“with neural innervation”).
Yangling teaches the number of hair cells produced with Shh was significantly higher than that in control cultures and when Shh signaling was blocked with cyclopamine, hair cell generation was largely inhibited suggesting Shh may be a regulator of inner ear progenitor cell growth and hair cell generation (Abstract; page 123, right col. para. 2). Yangling teaches Shh is expressed in the developing ear and in Shh-/- knockout mice, the auditory compartment of the inner ear fails to form which may be due to inhibition of auditory progenitor cell proliferation or massive cell death of the progenitor cells (page 123, left col. para. 2). Yangling teaches Wnt signaling is critical for dorsal patterning of the otic vesicle and influences the expression of the genes that are necessary for vestibular morphogenesis (page 121, left col.). Yangling teaches the balance between Wnt and Shh signaling activities plays a major role in distinguishing between vestibular and auditory cell types (page 121, left col.). Thus, Yangling teaches culturing a 3D aggregate of otic prosensory vesicles in a culture medium with a SHH inhibitor produces hair cells that express myosin VIIA. Yangling provides motivation to remove the SHH inhibitor from the culture (step (B)) to increase the number of myosin VIIa-positive hair cells produced because Yangling teaches that the number of myosin VIIa-positive hair cells produced by culturing the aggregate of otic vesicles was reduced in the presence of the SHH inhibitor.
Regarding claim 3, Yangling teaches cyclopamine (page 122, left col. para. 3).
Regarding claim 4, Yangling teaches culturing with 2.5 µM cyclopamine (page 122, left col. para. 3) which is close to the recited concentration of 2 µM. Because the concentration of cyclopamine taught by Yangling is very close to the claimed concentration, one skilled in the art would have expected 2 µM and 2.5 µM to have the same effect of inhibiting SHH (see MPEP 2144.05(I)). Further, Benito-Gonzalez teaches that treating cochlear explants with 5 µM cyclopamine increases inner hair cell differentiation compared to untreated control cultures or cultures treated with SHH after 24 or 48 hours (page 12782, right col.; Figure 5A – I). Benito-Gonzalez teaches deletion of Shh causes prosensory cells to drop out of the cell cycle prematurely and to differentiate precociously (page 12782, left col.). Benito-Gonzalez teaches that modulation of Hedgehog signaling does not globally alter progenitor-specific gene expression (page 12873, left col. para. 1; Figure 5K, L). Benito-Gonzalez teaches auditory hair cells derive from a common pool of postmitotic prosensory cells within the developing cochlear duct (page 12865, left col.).
Yangling does not teach “a culture media comprising 5 – 10% of the gelatinous protein mixture secreted by EHS mouse sarcoma” or “with neural innervation” of claim 1 or “the inner ear hair cells are inner hair cells” of claim 7. However, Yangling teaches that otic vesicles yielded a very low number of hair cells in a monolayer culture but the number of hair cells was substantially increased when cultured in aggregates or in the presence of Shh (page 122, right col. para. 3). Yangling teaches otic vesicles cultured with cyclopamine yielded myosin VIIA-positive hair cells (page 122, left col. para. 3 and right col. para. 3). Yangling teaches Shh promotes inner ear progenitor cell proliferation and hair cell generation (page 123, right col. para. 2). Yangling teaches the specific effects of Shh on inner ear progenitor cell proliferation and differentiation are largely unclear (page 121, right col. para. 1).
Regarding “5 – 10% of the gelatinous protein mixture secreted by EHS mouse sarcoma” of claim 1, Panek teaches the formation of intestinal organoids in hanging drop with 5% Matrigel (1086, right col. last para.; page 1087, left col. para. 1; page 1090, right col. para. 1; page 1094, left col. para. 1). Panek teaches if the Matrigel was excluded from the medium cells were not able to aggregate and connect to one another and contained less proliferating cells (page 1093, left col. last para. and right col.). Panek teaches organoids have become widely used in vitro models of many tissues and organs and the advantage of 3D in vitro models involves their ability to partially reconstruct the dynamics typical for the tissue and therefore provide researchers a more physiological context for experiments (Abstract; page 1086, left col. para. 3). The gut organoid generation methods can be applied to generation of inner ear sensory epithelial cells as evidenced by Roccio (page 4, right col. last para.; Figure 2). Panek does not teach “with neural innervation” of claim 1 and “inner hair cells” of claim 7. One would have been motivated to combine the teachings of Yangling and Panek because Yangling teaches the formation of hair cells from aggregates of otic vesicles formed by hanging drop and Panek teaches the addition of 5% Matrigel in the hanging drop method increases cell proliferation and promotes aggregation into organoids.
Regarding “with neural innervation” of claim 1 and “inner hair cells” of claim 7, Koehler teaches a method of producing inner ear hair cells with neural innervation that are Tuj1 positive (“with neural innervation” of claim 1 and “inner hair cells” of claim 7) from inner ear organoids comprising culturing otic vesicles in Matrigel where the otic vesicles are produced from human pluripotent stem cells (col. 1, lines 26 – 33; Figure 4A and 17; col. 3, lines 35 – 39; col. 23, lines 59 – 67; col. 24, lines 7 – 67; col. 25, lines 9 – 30). Koehler teaches the hair cells are myosin 7A positive (col. 3, line 54; col. 25, lines 24 – 30). Koehler teaches aggregates plated in Matrigel produced neurite outgrowths that are Tuj1 positive (col. 5, lines 31 – 33; col. 6, lines 34 – 44; col. 14, lines 1 – 2; col. 23, lines 50 – 58 and 65 – 66). Koehler teaches the method of producing the inner ear organoids comprises forming aggregates of human pluripotent stem cells in media containing the ROCK inhibitor Y-27632 for 2 days; transferring the aggregates to media containing 2% growth factor reduced Matrigel and FGF2 (4 ng/mL) and SB-431542 (10 µM) (col. 16, lines 41 – 51; col. 24, lines 7 – 50; col. 27 – 30). Koehler teaches after 4 days, FGF2 (50 ng/mL) and LDN-193189 (200 nM) was added (col. 16, lines 59 – 63; col. 24, lines 7 – 50; col. 27 – 30). Koehler teaches at 8 days, CHIR99021 (3 µM) was added (col. 16, lines 63 – 67; col. 24, lines 51 – 67; col. 27 – 30). Koehler teaches at day 12 the aggregates were resuspended in undiluted growth factor reduced Matrigel and cultured with CHIR99021 (3 µM) and after 18 days of differentiation the CHIR was removed and the droplets transferred to spinner flasks and maintained in the spinner flasks for up to 180 days of differentiation or maintained in plates for up to 140 days (col. 17, lines 32 – 50; col. 24, lines 51 – 67; col. 25, lines 9 – 30 col. 27 – 30). Koehler teaches growth factor reduced Matrigel was chosen because it has been shown to be a reliable inducer of self-organizing epithelia from pluripotent stem cells in 3D culture (col. 17, lines 61 – 63). Koehler teaches growth factor reduced Matrigel is a more defined alternative to Matrigel because the concentrations of growth factors have been minimized to levels that should have a negligible effect on cell fate specification (col. 17, lines 64 – 67). Koehler teaches a low concentration of Matrigel does not seem to be supportive of otic vesicle formation and otic vesicle formation was observed in >95% of the aggregates that go on to differentiate into sensory neurons (col. 24, lines 52 – 54 and 63 – 67).
Koehler teaches nearly half a billion people have hearing loss world-wide, yet there are no pharmacological, genetic, or cell therapies that can treat hearing loss (col. 1, lines 46 – 47). Koehler teaches there remains a need for efficient, reproducible, and xenogeneic material-free methods for differentiating human pluripotent stem cells into inner ear sensory tissue suitable for clinical cell therapies (col. 1, lines 52 – 55). Koehler teaches the organoid model may recapitulate assembly of the sensorineural circuit between hair cells and sensory neurons (col. 24, lines 1 – 2).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Yangling regarding a method of culturing otic vesicle cell aggregates in cyclopamine to form myosin VIIa-positive hair cells with the teachings of Panek regarding a method of forming organoids with 5% Matrigel in the hanging drop method increases the formation of organoids with the teachings of Koehler regarding a method of culturing otic vesicles in Matrigel to form Tuj1-positive and myosin VIIa-positive hair cells to arrive at the claimed method for producing inner ear hair cells, comprising the steps of: (A) culturing otic prosensory vesicles that originate from human induced pluripotent stem-cells in the presence of ill a SHH inhibitor in an amount sufficient to partially inhibit the SHH pathway and (ii) a culture medium comprising 5-10% of the a gelatinous protein mixture secreted by EHS mouse sarcoma cells to form a 3D shaped inner ear organoid; (B) removing the SHH inhibitor from the culture in step A, medium while the inner ear organoid remains in the culture; and (C) culturing the inner ear organoid from step B in a culture medium comprising 5-10% of the a gelatinous protein mixture secreted by EHS mouse sarcoma cells to form inner ear hair cells with neural innervation. One would have been motivated to combine the teachings of Yangling, , Panek, and Koehler in a method of producing inner ear hair cells as Koehler teaches nearly half a billion people have hearing loss world-wide, yet there are no pharmacological, genetic, or cell therapies that can treat hearing loss and Koehler teaches there remains a need for efficient, reproducible, and xenogeneic material-free methods for differentiating human pluripotent stem cells into inner ear sensory tissue suitable for clinical cell therapies. One would have a reasonable expectation of success in combining the teachings as Yangling teaches culturing cells with cyclopamine resulted in myosin VIIA-positive hair cells and Yangling teaches both Wnt and Shh are required for development of functional inner ear but Shh inhibition reduces hair cell formation from otic vesicles in vitro and Koehler teaches Wnt inhibition by CHIR99021 promotes sensory neurons from otic vesicles and Matrigel promotes neurite outgrowth and Yangling teaches when otic vesicles where cultured in monolayers, only very few hair cells were observed but culturing otic vesicles in aggregates resulted in robust and reproducible hair cells and Yangling teaches it has been shown that in vitro differentiating hair cells in otic vesicle cell aggregates organize into specific patterns and are surrounded by supporting cells in a manner similar to the in vivo pattern and Panek teaches if the Matrigel was excluded from the medium cells were not able to aggregate and connect to one another and contained less proliferating cells.
22. Claim(s) 5 and 6 is/are rejected under 35 U.S.C. 103 as being unpatentable over Yangling (Zhao, Yanling, et al. Neuroreport 17.2 (2006): 121-124; previously cited), hereinafter Yangling in view of Panek (Panek, M. et. al. Cytotechnology 70.3 (2018): 1085-1095; previously cited), hereinafter Panek as evidenced by Roccio (Roccio, M. et. al. Development 146.17 (2019): dev177188; previously cited), hereinafter Roccio in view of Koehler (US-12077778-B2; Filed 08/10/2020; Published 09/03/2024), hereinafter Koehler as applied to claims 1 – 4, and 7 – 15 above, and further in view of Lauth (Lauth M, et. al. Proc Natl Acad Sci U S A. 2007 May 15;104(20):8455-60; previously cited), hereinafter Lauth.
Yangling in view of Panek and Koeher make obvious the limitations of claim 1 as set forth above. Yangling teaches cyclopamine as the SHH inhibitor and does not teach GANT61 of claim 5 or GANT58 of claim 6. However, Yangling teaches Gli is a target of Shh and both are expressed in the otocyst of the developing inner ear (page 123, left col. para. 2). Yangling teaches the specific effects of Shh on inner ear progenitor cell proliferation and differentiation are largely unclear (page 121, right col. para. 1).
Lauth teaches GANT61 (claim 5) and GANT58 (claim 6) are Gli antagonists and inhibit endogenous Hedgehog (Hh) signaling in Shh-L2 cells (Figure 2; page 8456, left col. para. 2). Lauth teaches the IC50 was ~5 µM for both compounds for inhibiting Hh signaling, which is lower than that of cyclopamine estimated from Figure 2A (page 8456, left col. para. 2). Lauth teaches no toxicity or reduction in cell viability could be observed for several cell lines tested (page 8456, left col. para. 2). Lauth teaches both GANTs significantly reduced Gli1 mRNA levels at 10 µM which is indicative of strong pathway inhibition (page 8456, right col. para. 1; Figure 2B). Lauth teaches similar findings using a readout of endogenous Ptch1 expression where both GANTs and cyclopamine showed similar results at 5 µM (page 8456, right col. para. 1; Figure 2C). Lauth teaches Smoothened is targeted for the development of Hh pathway signaling inhibitors but these types of inhibitors would not address the scenario in which pathway activation occurs downstream of Smoothened (Abstract). Lauth teaches GANT 61 and 58 target GLI-mediated transcription which is the final step of the Hh pathway (Abstract). Lauth teaches cyclopamine is ineffective in blocking Hh signaling downstream of Smoothened (Abstract). Lauth teaches GANTs are selective for inhibiting the Hh pathway (page 8457, left col. para. 2). Lauth teaches GANTs will be valuable research tools in elucidating the involvement of Gli in normal embryonic development (page 8460, left col. para. 1).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to substitute the cyclopamine of Yangling with GANT61 or GANT58 at 2 µM to arrive at the claimed method where the SHH inhibitor is GANT61 or GANT58. One would have been motivated to make such a substitution to understand the role of Gli in inner ear organoid development as Yangling teaches Gli is a target of Shh and both are expressed in the otocyst of the developing inner ear and Yangling teaches the specific effects of Shh on inner ear progenitor cell proliferation and differentiation are largely unclear and Lauth teaches GANTs will be valuable research tools in elucidating the involvement of Gli in normal embryonic development. One would have a reasonable expectation of success in carrying out the substitution as Lauth teaches no toxicity or reduction in cell viability could be observed for several cell lines tested and the GANTs show lower IC50 values for Hh inhibition relative to cyclopamine.
Applicant’s Arguments/ Response to Arguments
23. Applicant Argues: On page 5, para. 6, Applicant asserts that the amendment to claims 13 and 14 render the rejections moot.
Response to Argument: The rejections of claims 13 and 14 are maintained and revised as set forth above in light of the amendments to these claims. Claim 13 lacks antecedent basis for otic prosensory vesicles formation because claim 1 recites culturing otic prosensory vesicles and does not recite method steps for forming otic prosensory vesicles. Further, the metes and bounds of the recited number of days are unclear because based on Figure 1 of Applicant’s disclosure, the number of days refer to starting with the formation of non-neural ectoderm from iPSCs. However, claim 1 does not recite any method steps for forming non-neural ectoderm or otic prosensory vesicles. Similarly, claim 14 lacks antecedent basis for the recited days for formation of sensory epithelium and maturation. Should Applicant amend claim 1 to recite active method steps for the formation of otic prosensory vesicles and hair cell maturation, the rejection may be overcome upon further consideration.
Applicant Argues: On page 6, para. 3, Applicant asserts that the hindsight reconstruction.
Response to Argument: In response to applicant’s argument that the examiner’s conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant’s disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
Applicant Argues: On page 6, last para., Applicant asserts that none of the cited references alone or in combination teach amended claim 1. On page 8, Applicant asserts the rationale for motivation noted in the Office Action is flawed and the cited references do not render obvious the claimed invention recited in amended claim 1. On page 8, Applicant asserts that pending claims 5 and 6 depend from claim 1 and are not obvious because the rationale for motivation to combine the references is flawed and the references do not teach or suggest amended claim 1.
Response to Argument: The previous rejection of the claims over prior art has been withdrawn in view of Applicant’s amendment to the claim to require an inner ear organoid and hair cells with neural innervation. In the necessitated rejection set forth above, Yangling teaches a method of producing hair cells by culturing an aggregate of otic vesicle cells in hanging drop in the presence of in a culture medium comprising 2.5 µM cyclopamine (page 122, left col. para. 3; Figure 1 and 2). Yangling teaches when otic vesicles where cultured in monolayers, only very few hair cells were observed but culturing otic vesicles in aggregates resulted in robust and reproducible hair cells (page 122, right col. last para.). Yangling teaches the number of hair cells produced with Shh was significantly higher than that in control cultures and when Shh signaling was blocked with cyclopamine, hair cell generation was largely inhibited suggesting Shh may be a regulator of inner ear progenitor cell growth and hair cell generation (Abstract; page 123, right col. para. 2). Thus, Yangling teaches culturing a 3D aggregate of otic prosensory vesicles in a culture medium with a SHH inhibitor produces hair cells that express myosin VIIA but not as many as when the aggregates are cultured without cyclopamine. Yangling provides motivation to remove the SHH inhibitor from the culture to increase the number of myosin VIIa-positive hair cells produced because Yangling teaches that the number of myosin VIIa-positive hair cells produced by culturing the aggregate of otic vesicles was reduced in the presence of the SHH inhibitor. Yangling teaches the balance between Wnt and Shh signaling activities plays a major role in distinguishing between vestibular and auditory cell types (page 121, left col.). Yangling does not teach “a culture media comprising 5 – 10% of the gelatinous protein mixture secreted by EHS mouse sarcoma” or “with neural innervation” of claim 1.
Regarding “a culture media comprising 5 – 10% of the gelatinous protein mixture secreted by EHS mouse sarcoma” of claim 1, Panek teaches the formation of intestinal organoids in hanging drop with 5% Matrigel (1086, right col. last para.; page 1087, left col. para. 1; page 1090, right col. para. 1; page 1094, left col. para. 1). Panek teaches if the Matrigel was excluded from the medium cells were not able to aggregate and connect to one another and contained less proliferating cells (page 1093, left col. last para. and right col.). The gut organoid generation methods can be applied to generation of inner ear sensory epithelial cells as evidenced by Roccio (page 4, right col. last para.; Figure 2). One would have been motivated to combine the teachings of Yangling and Panek because Yangling teaches the formation of hair cells from aggregates of otic vesicles formed by hanging drop and Panek teaches the addition of 5% Matrigel in the hanging drop method increases cell proliferation and promotes aggregation into organoids.
Regarding “with neural innervation” of claim 1, Koehler teaches a method of producing inner ear hair cells with neural innervation that are Tuj1 positive from inner ear organoids comprising culturing otic vesicles in Matrigel where the otic vesicles are produced from human pluripotent stem cells (col. 1, lines 26 – 33; Figure 4A and 17; col. 3, lines 35 – 39; col. 23, lines 59 – 67; col. 24, lines 7 – 67; col. 25, lines 9 – 30). Koehler teaches the hair cells are myosin 7A positive (col. 3, line 54; col. 25, lines 24 – 30). Koehler teaches aggregates plated in Matrigel produced neurite outgrowths that are Tuj1 positive (col. 5, lines 31 – 33; col. 6, lines 34 – 44; col. 14, lines 1 – 2; col. 23, lines 50 – 58 and 65 – 66). Koehler teaches at day 12 the aggregates were resuspended in undiluted growth factor reduced Matrigel and cultured with CHIR99021 (3 µM) and after 18 days of differentiation the CHIR was removed and the droplets transferred to spinner flasks and maintained in the spinner flasks for up to 180 days of differentiation or maintained in plates for up to 140 days (col. 17, lines 32 – 50; col. 24, lines 51 – 67; col. 25, lines 9 – 30 col. 27 – 30). Koehler teaches growth factor reduced Matrigel was chosen because it has been shown to be a reliable inducer of self-organizing epithelia from pluripotent stem cells in 3D culture (col. 17, lines 61 – 63).
One would have been motivated to combine the teachings of Yangling, , Panek, and Koehler to arrive at the method of amended claim 1 as Koehler teaches nearly half a billion people have hearing loss world-wide, yet there are no pharmacological, genetic, or cell therapies that can treat hearing loss and Koehler teaches there remains a need for efficient, reproducible, and xenogeneic material-free methods for differentiating human pluripotent stem cells into inner ear sensory tissue suitable for clinical cell therapies. One would have a reasonable expectation of success in combining the teachings as Yangling teaches culturing cells with cyclopamine resulted in myosin VIIA-positive hair cells and Yangling teaches both Wnt and Shh are required for development of functional inner ear but Shh inhibition reduces hair cell formation from otic vesicles in vitro and Koehler teaches Wnt inhibition by CHIR99021 promotes sensory neurons from otic vesicles and Matrigel promotes neurite outgrowth and Yangling teaches when otic vesicles where cultured in monolayers, only very few hair cells were observed but culturing otic vesicles in aggregates resulted in robust and reproducible hair cells and Yangling teaches it has been shown that in vitro differentiating hair cells in otic vesicle cell aggregates organize into specific patterns and are surrounded by supporting cells in a manner similar to the in vivo pattern and Panek teaches if the Matrigel was excluded from the medium cells were not able to aggregate and connect to one another and contained less proliferating cells.
Conclusion
No claims allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/Z.M.B./Examiner, Art Unit 1632
/MARCIA S NOBLE/Primary Examiner, Art Unit 1632
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