DETAILED ACTION
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 6/18/2026 has been entered.
Withdrawn Objections/Rejections
The objections and rejections presented herein represent the full set of objections and rejections currently pending in this application. Any objections or rejections not specifically reiterated are hereby withdrawn.
Claim Rejections - 35 USC § 103 – New Grounds of Rejection
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.
Claim(s) 1-4, 6-13, and 15 is/are rejected under 35 U.S.C. 103 as being unpatentable over Huang et al (Sci Rep 6, 19727 (2016); published 27 January 2016) in view of Ikeya et al (WO2019107485A1, 11/29/2018; published 6/6/2019; refer to provided translation) and as evidenced by Chen et al (Exp Ther Med. 2015 Feb;9(2):351-360. Epub 2014 Dec 3.; of record).
Regarding claim 1 and 12, Huang teaches differentiation of induced pluripotent stem cells and embryonic stem cells into neural crest cells. Differentiation of human PSC to trunk NCC was performed using the modified Dalton protocol with Retinoic Acid (1 uM) added starting at Day 3 and maintained throughout the duration of the protocol (Day 11). The modified Dalton protocol consists of SB431542 and CHIR99021 (Methods para 3 and 4).
Regarding claim 2, Huang teaches the use of SB431542 as the TGF-beta inhibitor.
Regarding claim 3 and 4, Huang teaches Wnt activator is GSK3 inhibitor and the GSK3 inhibitor is CHIR99021.
Regarding claim 6, the culture period of Dalton is 11 days, which is close to “3 to 10 days” as in instantly claimed (see MPEP 2144.05(I), which states that a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close. Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 783, 227 USPQ 773, 779 (Fed. Cir. 1985)).
Regarding claim 8 and 9, Huang teaches concentration of SB431542 is used at 10 uM and the CHIR99021 is used at 3 uM (see MPEP 2144.05(I) which states “In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990)”).
Regarding claim 10, while Huang does not explicitly teach the use of 10 uM of retinoic acid to produce neural crest cells, as per MPEP 2144.05(II), “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. ‘[W]here 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.’ In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).”
Huang does not teach:
culturing the resultant cells (neural crest cells) in TGF-beta inhibitor and Wnt activator to produce ZO-1-positive CEC-like cells, and
that the CEC-like cells have hexagonal morphology and express Na/K-ATPase.
(i) Regarding culturing the neural crest cells in TGF-beta inhibitor and Wnt activator to produce CEC-like cells, Ikeya teaches that neural crest cells derived from pluripotent stem cells can be differentiated into corneal cells (Obtaining neural crest cells para 3).
Ikeya continues to teach that neural crest cells are multipotent cells able to differentiate into many types of cells such as corneal cells (Background of the invention, para 1). Neural crest cells are seeded on a plate, and cultured in CDM medium containing 10 μM SB431542 and 1 μM CHIR99021 (i.e., in the absence of retinoic acid). The medium is replaced one day later with a conditioned medium. The medium is changed once every two days, and the expression of ZO-1, which is a marker molecule of corneal cells, is confirmed via immunostaining. COL4A1 and COL8A1 expression (expressed in corneal endothelial cells) was also confirmed.
Regarding claim 7, Ikeya teaches that the neural crest cells are cultured for at least 1 day to produce CEC cells.
Regarding claim 11, Ikeya does not explicitly teach gradually decreasing retinoic acid, however, it would have been a matter of routine optimization using standard laboratory techniques available at the time of filing to determine the appropriate time to remove or reduce the amount of differentiation regulators such as retinoic acid. In re Williams, 36 F.2d 436, 438, 4 USPQ 237 (CCPA 1929) ("It is a settled principle of law that a mere carrying forward of an original patented conception involving only change of form, proportions, or degree, or the substitution of equivalents doing the same thing as the original invention, by substantially the same means, is not such an invention as will sustain a patent, even though the changes of the kind may produce better results than prior inventions."). See also KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 416, 82 USPQ2d 1385, 1395 (2007) (identifying "the need for caution in granting a patent based on the combination of elements found in the prior art.").
Regarding claim 13, Ikeya teaches that the plate is coated with fibronectin.
(ii) Regarding hexagonal morphology and Na/K-ATPase expression, the teachings of Chen are relied upon as providing evidence that CECs had positive immunostaining of ZO-1 and Na+-K+-ATPase (Fig. 1B–G).
Regarding claim 15, Chen teaches that the CECs also had positive immunostaining of AQP1, VE-cadherin, N-cadherin and vimentin, which have been used as general markers for CECs, and was used to identify the cultured cells as CECs (Fig. 1B–G).
Chen continues to evidence that the cells displayed hexagonal or polygonal morphology (Results, p. 355; Fig. 1A). Thus, one of ordinary skill would reasonably expect the ZO-1 positive cells of Huang and Ikeya in combination to also express at least NA-K-ATPase, AQP1, VE-cadherin, N-cadherin and vimentin because, as per MPEP 2112.01(II), "Products of identical chemical composition cannot have mutually exclusive properties." In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990). A chemical composition and its properties are inseparable. Therefore, if the prior art teaches the identical chemical structure, the properties applicant discloses and/or claims are necessarily present. Id. (Applicant argued that the claimed composition was a pressure sensitive adhesive containing a tacky polymer while the product of the reference was hard and abrasion resistant. ‘The Board correctly found that the virtual identity of monomers and procedures sufficed to support a prima facie case of unpatentability of Spada’s polymer latexes for lack of novelty.’).
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to create neural crest cells from pluripotent stem cells as taught by Huang, where the neural crest cells are further cultured in SB431542 and CHIR99021 to produce CECs as taught by Ikeya, to arrive at the instantly claimed invention. As Ikeya teaches that neural crest cells can be successfully transformed into corneal cells with CEC marker expression via culture in SB431542 and CHIR99021, one of ordinary skill would have been motivated to modify the method of Huang to include the culture steps of Ikeya with a reasonable expectation of producing cells that can be used cell preparation for regenerative medicine as taught by the prior art.
Claim(s) 5 is/are rejected under 35 U.S.C. 103 as being unpatentable over Huang et al in view of Ikeya et al (WO2019107485A1, ) as applied to claims 1-4, 6-13, and 15 above, and further in view of Chen et al (Exp Ther Med. 2015 Feb;9(2):351-360. Epub 2014 Dec 3.; of record).
The teachings of Huang and Ikeya in combination were recited in the above 35 U.S.C. 103 rejection as applied to claim 1 of which claim 5 depends. The teachings will not be repeated here.
Neither Huang nor Ikeya teach:
that the retinoic acid is all-trans retinoic acid.
Regarding (i), Chen teaches the in vitro directed the differentiation of pluripotent stem cells towards corneal endothelial cell-like cells (title). Mouse ESCs and iPSCs were induced to differentiate into CECs using CEC embryonic development events as a guide (Materials and Methods).
Chen continues to teach that all-trans retinoic acid (RA) treatment during the embryoid body (EB) differentiation step was used to promote neural crest (NC) cell differentiation as first step and was followed by a second induction in CEC- or lens epithelial cell (LEC)-conditioned medium (CM) to ultimately generate CEC-like cells. The data indicated that 4 days of treatment with 1 μM RA starting on day 4 of EB formation favored NC cell differentiation and that plating on gelatin-coated plates led to cell migration out of the EBs (abstract). The addition of RA accelerated the differen-tiation process, as shown by the severe reduction in the mRNA expression of Nanog, a marker of the undifferentiated state, at EBd4+4 after 1 μM RA treatment (Fig. 3H). These data indicate that EB differentiation for 4 days followed by 1 μM RA treatment for another 4 days (EBd4+4, 1 μM RA) could be used to induce NC differentiation as the first step of CEC differentiation (p. 357, col 1 para 2).
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to produce CEC-like cells from pluripotent cells by culturing on ECM substrate as taught by Huang and Ikeya in combination, where the retinoic acid is all-trans retinoic acid as taught by Chen, to arrive at the instantly claimed invention. Chen shows that neural crest cells can be induced using all-trans retinoic acid. One of ordinary skill would have been motivated to simply substitute one known element [retinoic acid of Huang and Ikeya in combination] for another [all-trans retinoic acid as taught by Chen] to advantageously obtain the predictable result of accelerating differentiation of pluripotent stem cells into neural crest cells during the first step of CEC differentiation as taught by the prior art.
Response to Arguments
Applicant’s arguments and the Skottman Declaration have been fully considered but are not persuasive. The response to arguments will focus on previously cited Chen reference and are also applicable to the rejection as described below for instant claim 5. Arguments related to the Zhao reference are moot.
On p. 9-10 of Remarks, Applicant argues that Chen demonstrates a two-step EB-based induction of mouse ESCs and iPSCs into CEC-like cells and that RA is applied only on day 4. The Skottman Declaration argues that mouse pluripotent stem cells are distinct from human pluripotent stem cells and once of ordinary skill would not think that differentiation protocols developed for mouse pluripotent stem cells would be successfully applied to human pluripotent stem cells. Applicant further argues that Chen only applies RA after cells progress beyond pluripotent stage and that Chen teaches that RA is insufficient to drive differentiation into CEC-like cells.
These arguments have not been found persuasive. First, newly cited reference Huang shows that neural crest cells can be induced from human pluripotent stem cells by the combination of RA, SB431542, and CHIR99021 and newly cited Ikeya shows that neural crest cells derived from pluripotent stem cells can be differentiated into ZO-1-positive corneal cells via culturing with SB431542 and CHIR99021. Second, the claims merely require “pluripotent stem cells,” not human pluripotent stem cells. The claims necessarily encompass protocols utilizing pluripotent stem cells from any species. One of ordinary skill in the art would find Huang as sufficient teachings that RA can be used to induce NCCs from human pluripotent stem cells. Third, Applicant is not claiming any particular time frames in which the RA is added to the culture. Finally, Chen states that “the addition of RA (all-trans retinoic acid) accelerated the differen-tiation process [of the neural crest cells] . . . and could be used to induce NC differentiation as the first step of CEC differentiation” as is instantly claimed. Thus, Applicant’s arguments are not persuasive.
Claim(s) 14 is/are rejected under 35 U.S.C. 103 as being unpatentable over Huang et al and Ikeya et al as applied to claims 1-4, 6-13, and 15 above, and further in view of Okumura et al (Invest Ophthalmol Vis Sci. 2015 May; 56(5):2933-42; previously cited).
The teachings of Huang and Ikeya in combination were recited in the above 35 U.S.C. 103 rejection as applied to claim 1 of which claim 14 depends.
Regarding claim 14 in-part, Ikeya teaches that culture vessels such as dishes, flasks, microplates and cell culture sheets such as OptiCell can be used. The culture vessel may be surface-treated to improve adhesion (hydrophilicity) with cells, with, for example, collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, and Matrigel.
However, Huang and Ikeya do not teach that the ECM protein is laminin-521.
Okumura teaches the usefulness of laminin isoforms as substrates for culturing human corneal endothelial cells (abstract). Specific laminin isoforms expressed in Descemet's membrane may transduce biological signals to CECs, and that these signals may trigger the in vitro expansion of CECs for clinical use (Introduction, para 4). Primary HCECs seeded on laminin-511 and -521 formed a monolayer of cells with a hexagonal phenotype after 48 hours of culture (“Effect of Laminin-511 and -521” para 1). In contrast, control HCECs seeded on laminin-211 coated or uncoated culture plates formed patchy colonies rather than a confluent monolayer (same para; Fig. 2A). The reference concludes that laminin-511 and -521 were the laminin isoforms present in the Descemet's membrane, and that these laminins modulate the adhesion and proliferation of CECs (Discussion, last para).
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to produce CEC-like cells from pluripotent cells by culturing on ECM substrate as taught by Huang and Ikeya in combination, where the ECM is laminin-521 as taught by Okumura, to arrive at the instantly claimed invention. As Okumura shows that CECs can be cultured on laminin-521, one of ordinary skill would have been motivated to simply substitute one known element [fibronectin substrate of Huang and Ikeya in combination] for another [laminin-521 as taught by Okumura] to advantageously obtain the predictable result of having a substrate that modulates the adhesion and proliferation of the CEC-like cells and have the cells obtain their hexagonal phenotype during culture as taught by the prior art.
Response to Arguments
On p. 10-11, Applicant argues that the Okumura teaches that laminin521 supporting adhesion and proliferation of already differentiated CECs but does not function as a differentiation cue. Applicant argues that Okumura does not provide neither a motivation to alter the differentiation substrate nor a reasonable expectation of success to include laminin 521 in a differentiation protocol to induce CEC production.
This argument is also not found to be persuasive. Okumura describes that specific laminin isoforms such as laminin-521 expressed in Descemet's membrane may transduce biological signals to CECs, and that these signals may trigger the in vitro expansion of CECs for clinical use. Moreover, it allows CECs to form confluent monolayers. Newly cited reference Ikeya also states that culture vessels coated in, for example, laminin, fibronectin, and Matrigel improve adhesion of cells in culture (i.e., interchangeable for the same purpose) and can be used for the induction of differentiation of neural crest cells. Thus, Applicant’s argument is not persuasive.
Conclusion
No claim is allowed.
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/G.R./Examiner, Art Unit 1632
/MARCIA S NOBLE/Primary Examiner, Art Unit 1632
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