DETAILED ACTION
Claim Status
As of the Non-Final Office Action mailed 4/7/2026, claims 1-4 and 6-24 were pending.
In Applicant's Response filed on 7/72026, claim 15 was amended and claim 20 was canceled.
As such, claims 1-4, 6-19, 21-24 are pending and have been examined herein.
Withdrawn Objections/Rejections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 Any objections or rejections not specifically reiterated are hereby withdrawn.
The rejection of record of claims 15-16 under 35 USC § 112(a) have been withdrawn in view of Applicant’s amendment to claim 15.
Claim Rejections - 35 USC § 103 - Modified
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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.
Claim(s) 1-4, 6-14, 17-19, and 21-24 are rejected under 35 U.S.C. 103 as being unpatentable over Ilmarinen et al (WO2018037161A1, 24 August 2017; published 1 March 2018; Ref. 6 of Foreign Patent Documents in IDS filed 30 Sept 2021; of record) in view of Deng et al (US20160376555A1, 28 June 2016; published 29 Dec 2016; Ref. 2 of US Patent Documents in IDS filed 30 September 2021; of record), Iwata et al (WO2017188378A1; 27 April 2017; published 2 Nov 2017; of record), and as evidenced by Stasi et al (Invest Ophthalmol Vis Sci. 2014 Jan 20;55(1):375-86; of record).
Regarding claim 6 in-part, Ilmarinen teaches a method for producing eye cells selected from the group consisting of corneal epithelial precursor cells comprising a) culturing pluripotent stem cells in the absence of feeder cells, b) culturing said cells in a cell culture medium comprising a TGF-beta inhibitor and a fibroblast growth factor (FGF) followed by culturing said cells in a cell culture medium comprising bone morphogenetic protein 4 (BMP-4) thereby producing eye precursor cells, and c) culturing said eye precursor cells in a cell culture medium comprising one or more supplements selected from the group consisting of epidermal growth factor (EGF), hydrocortisone, insulin, isoproterenol, and tri-iodo- thyronine in the absence of a TGF-beta inhibitor, FGF, or BMP-4, thereby producing corneal epithelial precursor cells (see claim 1 of Ilmarinen).
Ilmarinen continues to teach that the corneal epithelial precursor cells express markers such as p63 and ABCG2 (“Differentiation phase” para 2) (i.e., corneal limbal stem cells, which are p63 and ABCG2 positive cells, see instant specification p.2, Fig. 2E and 3A).
Regarding claim 10, Ilmarinen teaches that the stem cells are selected from induced pluripotent stem (iPS) cells and embryonic stem (ES) cells, with the proviso that if human embryonic stem (hESC) cells are used, the method does not include the destruction of human embryos (see claim 9 of Ilmarinen).
Regarding claim 12, Ilmarinen teaches that step (a) comprises forming embryoid bodies from the pluripotent stem cells (see claim 10 of Ilmarinen) and the formation of embryoid bodies is carried out by physical or chemical method, such as culturing cells in the presence of attachment-preventing agents, culturing cells in hanging drops, microfabrication techniques, forced aggregation e.g. by centrifugation, and culturing cells in the presence of one or more aggregation-promoting agents such as macromolecular crowders, blebbistatin and ROCK inhibitors (see claim 11 of Ilmarinen). The reference also teaches that the culturing in step (c) is carried out on a substrate coated with at least collagen IV and Laminin (see claim 12 of Ilmarinen).
Regarding claim 13, Ilmarinen teaches that the addition of laminins and collagens allow for the culturing of cells without the use of feeder cells (see p. 6 lines 30-36 and p. 7 lines 1-22) such that one of ordinary skill would readily use coated surfaces with laminin and collagen to avoid the use of feeder cells.
Regarding claims 1 in-part and 6 in-part, Ilmarinen teaches obtaining precursor cells requires culturing the cells for about 10 to about 35 days and that the expression of CK12 and CK3 is present in only matured corneal epithelium cells that result from maturing the corneal epithelial precursor cells (p. 17 lines 22-30, 34-35).
Ilmarinen does not teach:
ABCG2 expression in the corneal limbal epithelial cells are maintained by culturing in a maintenance medium comprising EGF and at least one Wnt activator.
Regarding (i), Deng teaches a method for maintaining human limbal stem cells of the corneal epithelium in an undifferentiated limbal stem cell phenotype by culturing the cells in a culture media for human limbal stem cells and that the human limbal stem cell phenotype is characterized by observing expression of ATP-binding cassette subfamily G member 2 (ABCG2) in the human limbal stem cells (see claims 1 and 2 of Deng). The corneal limbal stem cells can be characterized by the presence, absence, and/or expression biomarkers such as cytokeratin (K) 1, K3, K10, K12, K14, or K15 (para 0041).
Also regarding (i), Iwata teaches a method of culturing pancreatic progenitor cells wherein the cells are cultured in 1) EGF and/or FGF and 2) a Wnt agonist (see claim 1 of Iwata translation) (“maintenance cell culture medium comprising EGF and at least one Wnt activator” as in instant claim 6 in-part). The reference teaches that culturing the cells refers to maintaining the cells (see translation, p. 4 “Detailed Description of the Invention”). The reference also teaches that the Wnt agonist is a protein belonging to the R-spondin family and / or a GSK inhibitor (see claim 2 of Iwata) (“wherein the Wnt activator is selected from the group consisting of GSK3 inhibitors and proteins of R-spondin family” as in instant claim 7). The reference teaches that substances that maintain undifferentiation and promotes proliferation can be added to the medium such as GSK-3 inhibitors like CHIR99021 and TGF-b inhibitors like LDN193189 (p. 7 of translation, para 16) (“wherein the Wnt activator is CHIR99021” as in instant claim 8 and 23; “wherein the culture medium further comprises . . . LDN-193189” as in instant claim 9). The reference also teaches that noggin, a BMP signal inhibitor, can be used in place of the TGF-b inhibitor for the same purpose (p. 6 of translation, para 3) (“wherein the culture medium further comprises noggin” as in instant claim 9). This shows that EGF and Wnt agonists can be added to mediums to maintain the undifferentiated state of stem cells.
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to produce ABCG2-expressing corneal limbal stem cells from pluripotent stem cells as taught by Ilmarinen, where the corneal limbal stem cells are maintained in a culture medium containing EGF and a Wnt agonist as taught by Deng and Iwata in combination, to arrive at the instantly claimed invention. Deng shows that ABCG2-positive corneal limbal stem cells can be maintained in a culture medium and Iwata shows EGF and a Wnt agonist like CHIR99021 can be put in a culture medium to maintain stem cells. One of ordinary skill would have been motivated to combine the method of producing corneal epithelial stem cells that express ABCG2 (i.e., corneal limbal stem cells) with the method of maintaining corneal limbal stem cells in a medium containing at least EGF and Wnt agonist as taught by Deng and Iwata in combination according to known methods to yield the predictable and advantageous result of obtaining ABCG-2 positive corneal limbal stem cells that maintain their undifferentiated status (e.g., maintain expression of corneal limbal stem cell markers) and are able to proliferate.
Regarding claim 21, Ilmarinen teaches the medium is free of BSA and HSA (see entirety of pg. 21).
Regarding claim 22, Ilmarinen teaches culturing eye precursor cells in a cell culture medium comprising one or more supplements selected from the group consisting of epidermal growth factor (EGF), hydrocortisone, insulin, isoproterenol, and tri-iodo- thyronine in the absence of a TGF-beta inhibitor, FGF, or BMP-4 (see claim 1).
Regarding 24, Ilmarinen teaches use of laminin-521 and collagen IV (see claim 12).
Response to Arguments
Applicant’s arguments and the Skottman Declaration have been fully considered but they are not persuasive.
On p. 5-7 of Remarks, Applicant argues that the Skottman declaration shows unexpected results over the Ilmarinen reference in that when the cells of Ilmarinen are continually cultured in differentiation medium as described in said reference, the ABCG2 expression is lost. Applicant argues that the switch to a maintenance medium containing EGF and CHIR99021 maintains ABCG2+ phenotype and is purportedly unexpected. Applicant further argues that the Lee reference discussed in the Skottman declaration shows that EGF and Wnt activator does not have the same properties depending on the culture conditions because CHIR99021 decreased stemness expression (ABCG2) and proliferation when utilized in limbal biopsy explant cultures while increasing ABCG2 expression when utilized in cultures of isolated limbal stem cells.
While Applicant’s arguments have been considered, they are not persuasive. First, the Ilmarinen reference was cited to describe the creation of the limbal stem cells from pluripotent stem cells as required by claim 6 and not to render obvious the maintenance in EGF and CHIR99021. ). Deng shows that ABCG2 phenotype of limbal stem cells can be maintained in culture, and Iwata shows that maintenance of undifferentiation and promotion of proliferation can be stimulated by adding GSK-3 inhibitors like CHIR99021 and TGF-b inhibitors like LDN193189 to mediums. The combination of Deng and Iwata shows that 1) it was known prior to the effective filing date of the instant invention to maintain the stemness of limbal stem cells via culturing, and 2) that the EGF and Wnt activators are known components to drive stemness maintenance generally. While the provided Lee reference shows that CHIR99021 decreases stemness of limbal explants (not within the scope of the instant claims), it does show that isolated cell cultures (within the scope of the instant claims) do have increased stemness (increased ABCG2 and p63) when cultured in the presence of CHIR99021. Based on the teachings of Deng, Iwata, and Lee, one of ordinary skill in the art would have recognized that CHIR99021 increases stemness of isolated limbal stem cells such that one of ordinary skill would have used such medium components to maintain limbal stem cell stemness. Thus, Applicant’s arguments are not persuasive.
Claim(s) 15-16 is/are rejected under 35 U.S.C. 103 as being unpatentable over Moore et al (US 11,987,809 B2, 5/11/2018; published 3/21/2019) in view of Lee et al (Sci Rep. 2017 Nov 10;7(1):15241.; provided by Applicant). Please note that this is a new ground of rejection necessitated by amendment.
Regarding claim 15, Moore teaches method of treating corneal dystrophies comprising manipulating a nucleic acid mutation in a corneal dystrophy target nucleic acid in a limbal epithelial stem cell to correct the mutation and transplanting the manipulated limbal stem cell into the subject (see claim 1 of Moore).
Moore continues to teach that the plurality of stem cells is positive for expression of p63, ABCG2 or combinations thereof and that the stem cells are negative for expression of CK3, CK12, connexin 43, involucrin, or combinations thereof (“Stem cells” para 2).
Moore continues to teach that the isolated cells are cultured in a medium containing EGF (col. 32, line 67).
Moore does not teach that the ABCG2-positive, CK3-negative, and CK-12 negative phenotype is maintained by culturing in the presence of a WNT activator.
However, Lee teaches the divergent effects of Wnt signaling on the growth and differentiation of limbal epithelial progenitor cells when cultured as isolated cells seeded in low calcium versus as biopsy explants (abstract).
Lee continues to teach that CHIR99021 reduced the growth rate, stem/progenitor cell marker content and clonogenic capacity in the explants but also had the opposite effect on the isolated cells. These results show that the outcome of Wnt/β-catenin signaling modification is dependent on the culture systems.
Notably, Fig. 3A of Lee shows that isolated cells had statistically significant increase of ABCG2, p63, and beta-actin expression, which are associated with limbal epithelial cells stemness and proliferation potential, when cultured in the presence of CHIR99021 (Effect of Wnt signaling, para 1).
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to transplant ABCG2-positve, CK-3-, CK-12-negative limbal stem cells as taught by Moore, where the cells’ stemness is maintained by medium containing CHIR99021 as taught by Lee, to arrive at the instantly claimed invention. Lee shows that isolated limbal stem cells can be successfully cultured in CHIR99021. One of ordinary skill would have been motivated to modify the method of Moore to include culturing in CHIR99021 as taught by Lee with a reasonable expectation of advantageously increasing ABCG2 expression and improving epithelial cells stemness and proliferation potential as taught by the prior art.
Conclusion
No claim is allowed.
THIS ACTION IS MADE FINAL. 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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/G.R./Examiner, Art Unit 1632
/MARCIA S NOBLE/Primary Examiner, Art Unit 1632
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