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 .
1. Claims 1 – 24 are pending.
Election/Restrictions
2. Applicant’s election without traverse of Group I (claims 1 – 18) in the reply filed on 08/07/2025 is acknowledged.
3. Claims 19 – 24 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.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 08/07/2025.
4. Claims 1 – 18 are under consideration.
Priority
5. This application is a national stage application under 35 U.S.C. 371 and claims the benefit of PCT application PCT/US2021/025414 filed 04/01/2021 which claims priority to U.S. provisional application 63/003/661 and 63/003/670 both filed on 04/01/2020.
Information Disclosure Statement
6. The information disclosure statement (IDS) submitted on 12/30/2022 and 09/29/2022 are acknowledged. The submissions are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Drawings
7. The drawings filed on 09/29/2022 are acknowledged.
Specification
8. The use of the term NutriStem, AlexaFluor, HistoChoice, QuantStudio Chromium, TaqMan, FlowJo, mTeSR1, iMatrix, Transwell, GlutaMax, AggreWell, CryoStem, Dynabeads, Knockout, Nanodrop, B27, which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
9. Claim 1 is objected to because of the following informalities: in line 2 “a.” should read “(a)” and in line 3, “b.” should read “(b)”. MPEP 608.01(m) states “Each claim begins with a capital letter and ends with a period. Periods may not be used elsewhere in the claims except for abbreviations”. Appropriate correction is required.
10. Claim 3 is objected to because of the following informalities: in line 2, “(iPSCs)comprises:” should read “(iPSCs) comprises:”. Appropriate correction is required.
11. Claim 3 is objected to because of the following informalities: in line 3, “a.” should read “(a)”; in line 4, “b.” should read “(b)”; in line 5, “c.” should read “(c)”; in line 6, “d.” should read “(d)”. MPEP 608.01(m) states “Each claim begins with a capital letter and ends with a period. Periods may not be used elsewhere in the claims except for abbreviations”. Appropriate correction is required.
12. Claim 4 is objected to under 37 CFR 1.75(c) as being in improper form because a multiple dependent claim should refer to other claims in the alternative only. See MPEP § 608.01(n).
13. Claim 4 is objected to because of the following informalities: in line 3, “a.” should read “(a)”; in line 5, “b.” should read “(b)”; in line 6, “c.” should read “(c)”. MPEP 608.01(m) states “Each claim begins with a capital letter and ends with a period. Periods may not be used elsewhere in the claims except for abbreviations”. Appropriate correction is required.
14. Claim 4 is objected to because of the following informalities: in line 7 – 8, “wherein the culture conditions comprise Duel-SMAD” should read “wherein culturing comprises Dual-SMAD”. Appropriate correction is required.
15. Claim 8 is objected to because of the following informalities: in line 2, “antibody-bases” should read “antibody-based”. Appropriate correction is required.
16. Claim 10 is objected to under 37 CFR 1.75(c) as being in improper form because a multiple dependent claim should refer to other claims in the alternative only. See MPEP § 608.01(n).
17. Claim 11 is objected to under 37 CFR 1.75(c) as being in improper form because a multiple dependent claim should refer to other claims in the alternative only. See MPEP § 608.01(n).
18. Claim 12 is objected to under 37 CFR 1.75(c) as being in improper form because a multiple dependent claim should refer to other claims in the alternative only. See MPEP § 608.01(n).
19. Claim 12 is objected to because of the following informalities: in line 2, “by taking a tissue or cell sample” should read “from a tissue or cell sample”. Appropriate correction is required.
20. Claim 14 is objected to under 37 CFR 1.75(c) as being in improper form because a multiple dependent claim should refer to other claims in the alternative only. See MPEP § 608.01(n).
21. Claim 16 is objected to under 37 CFR 1.75(c) as being in improper form because a multiple dependent claim should refer to other claims in the alternative only. See MPEP § 608.01(n).
22. Claim 17 is objected to under 37 CFR 1.75(c) as being in improper form because a multiple dependent claim should refer to other claims in the alternative only. See MPEP § 608.01(n).
23. Claim 18 is objected to under 37 CFR 1.75(c) as being in improper form because a multiple dependent claim should refer to other claims in the alternative only. See MPEP § 608.01(n).
Claim Interpretation
24. Claim 3 is interpreted as depending from claim 1.
25. Claim 4 is interpreted as depending from claim 1.
26. Claim 6 is interpreted as depending from claim 4.
27. Claim 10 is interpreted as depending from claim 6.
28. Claim 11 is interpreted as depending from claim 6.
29. Claim 12 is interpreted as depending from claim 3.
30. Claim 14 is interpreted as depending from claim 3.
31. Claim 16 is interpreted as depending from claim 1.
32. Claim 17 is interpreted as depending from claim 1.
33. Claim 18 is interpreted as depending from claim 1.
34. Induced basal cells (iBCs) of claim 1 are interpreted as cells expressing at least one basal cell stem markers from the group consisting of KRT5, TP63, and NKX2.1 (see Applicant’s specification at page 7, lines 10 – 12).
35. Claim 3 step c is interpreted as RNA-based reprogramming factors comprise Oct4, Sox2, Klf4, cMyc, Nanog, and Lin28 (see Applicant’s specification at page 8, lines 25 – 28 and page 26, lines 5 – 6).
36. Claim 3 step d is interpreted as identifying iPSCs that express Oct3/4 and purifying Oct3/4+ iPSC colonies (see Applicant’s specification at page 9, lines 20 – 26 and page 26, lines 16 – 19).
37. It is noted in claim 4, step c that culture conditions can optionally comprise a ROCK inhibitor. The term “optionally” does not require that the ROCK inhibitor be present in the culture conditions.
38. Claim 5 is interpreted as steps a and b of claim 4 comprises 3D organoid culture but step c does not comprise 3D organoid culture (see Applicant’s specification at page 29, lines 8 – 26).
39. Claims 7 and 8 are interpreted as lung progenitor cells are enriched by cell sorting (see Applicant’s specification at page 11, lines 3 – 7).
Claim Rejections - 35 USC § 112
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.
40. Claims 1 – 18 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 producing induced basal cells (iBCs) comprising (a) obtaining induced pluripotent stem cells (iPSCs); (b) differentiating iPSCs by culturing iPSCs in media comprising a WNT pathway activator and a BMP pathway activator to obtain into lung progenitors; (c) differentiating the lung progenitors in media comprising a Notch pathway inhibitor to obtain airway epithelial organoids; and (iii) dissociating the airway epithelial organoids into single cells and culturing the single cells in media comprising dual-SMAD inhibitors to obtain iBCs, wherein the directing lacks genetic manipulation.
does not reasonably provide enablement for any other method of generating iBCs that lacks genetic manipulation and produces iBCs where iBCs are interpreted as cells that express at least one basal cell stem marker selected from the group consisting of KRT5, TP63, and NKX2.1 (page7, lines 10 – 12 of Applicant’s specification). The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
It is noted that the instant rejection is based on absence of an enabling disclosure for the breadth of claimed method for generating iBCs from iPSCs without genetic manipulation. The claim encompasses any method of culturing iPSCs under any conditions and with any agent including conditions that may not result in the formation of basal cells that express at least one of KRT5, TP63, and NKX2.1 or may result in the formation of cells that express at least one of KRT5, TP63, and NKX2.1 but are not airway basal cells.
Enablement is considered in view of the Wands factors (MPEP 2164.01(a)). The court in Wands states: "Enablement is not precluded by the necessity for some experimentation such as routine screening. However, experimentation needed to practice the invention must not be undue experimentation. The key word is 'undue,' not 'experimentation.' " (Wands, 8 USPQ2d 1404). 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: (a) the breadth of the claims, (b) the nature of the invention, (c) the state of the prior art, (d) the level or ordinary skill in the art, (e) the level of predictability in the art, (f) the amount of direction provided by the inventor, (g) the existence of working examples, and (h) the quantity of experimentation needed to make use of the invention based on the content of the disclosure. While all of these factors are considered, a sufficient amount for a prima facie case are discussed below.
(a) The breadth of the claims:
The claim is broad, encompassing many different methods for differentiation of iPSCs with any differentiation factors for any amount of time. Thus, the claim encompasses differentiation methods that do not produce cells expressing at least one of KRT5, TP63, and NKX2.1 and methods that produce cells expressing at least one of KRT5, TP63, and NKX2.1 but that are not airway basal cells.
(b) The nature of the invention: The nature of the invention is a method of generating airway basal cells from iPSCs where the airway basal cells produce normal mucociliary epithelium (page 1, lines 20 – 28).
(c) The state of the prior art:
Regarding the issue of absence of an enabling disclosure for any method of directing generation of iBCs from iPSCs, the state of the art teaches protocols for differentiating iPSCs to lung progenitors includes differentiation of iPSCs to definitive endoderm (DE) and then to anterior foregut endoderm (AFE) (Yiangou, Loukia, et al. Cell Stem Cell 22.4 (2018): 485-499, page 488, left col. para. 2). The state of the art teaches that AFE can be specified into lung or thyroid lineages and WNT and BMP signaling are essential to drive lung progenitor formation and WNT signaling is essential to block thyroid cell specification expressing NKX2.1/PAX8 (Yiangou, page 488, left col. para. 3). The state of the art teaches WNT controls the proximodistal patterning of lung endoderm (NKX2.1+/PAX8-) (Yiangou, page 488, left col. para. 3). The state of the art teaches multiple active signaling pathways in NKX2.1 endodermal progenitors including BMP, WNT, EGF, and FGF (Kurmann, Anita A., et al. Cell stem cell 17.5 (2015): 527-542; Figure 1A; page 530, right col. para. 1) The state of the art teaches that BMP4 and FGF2 induces thyroid competent NKX2.1 progenitors (Kurmann, page 532, left col. para. 2). The state of the art fails to teach that any combination of signaling pathway activators/inhibitors will produce iBCs from iPSC-derived AFE.
(d) The level of one of ordinary skill:
The level of skill in the art of iPSC differentiation is high, as an artisan in this art needs specialized knowledge such as a postgraduate degree (Ph.D.) given the complex nature of the signaling pathways that when activated/inhibited can produce diverse cell types from iPSCs.
(e) The level of predictability in the art:
Regarding the issue of absence of an enabling disclosure for any method of directing generation of iBCs from iPSCs, the protocols for differentiation of iPSCs to DE and AFE are well-known in the art. However, differentiation of AFE to lung progenitors is not predictable. For instance, multiple signaling pathways are active in NKX2.1+ progenitors and differentiation protocols of AFE to lung or thyroid comprise culturing AFE with BMP but different factors are used to specify each cell type (Yiangyou, page 488, left col. para. 3 and right col. para. 3; Kurmann, page 530, right col. para. 1; page 532, left col. para. 2). Thus, it is unpredictable whether any agent used for any amount of time for directing differentiation of iPSCs to one cell type can be used to specify another cell type. Additionally, when the same agent is used (BMP), both lung and thyroid cells are produced and both express NKX2.1, which is a marker of iBCs (Yiangyou, page 488, left col. para. 3; Kurmann, Figure 1A). Thus, it is unpredictable that iBCs will be produced with a given agent because protocols using BMP produce different cell types both expressing an iBC marker. The predictability of applying any agent for any amount of time to differentiate iPSCs to iBCs encompassed by the instant claim would be low given that there does not appear to be a link between any agent and iBC production.
(f) The amount of direction provided by the inventor:
The specification teaches differentiating iPSCs to DE with STEMDIFF DE kit, differentiating DE to AFE with dorsomorphin and SB431542, differentiating AFE to lung progenitors with CHIR99021, BMP4, and retinoic acid, differentiating lung progenitors to airway epithelial organoids with bFGF, FGF10, dexamethasone, ROCK inhibitor, and DAPT, and differentiation of airway epithelial cells to iBCs with dual-SMAD inhibitors (page 28 – 29, E, F, G; Figures 6 – 7). Since the agents used are well known in the differentiation of iPSCs to lung basal cells, there is a nexus between in vitro results and the producing iBCs, but not to the full scope of the method claimed. The specification does not provide guidance on how to use any other agents to produce iBCs.
(g) The existence of working examples:
The applicants have provided one example of generating lung progenitor cells from iPSCs by differentiating iPSCs to definitive endoderm, differentiation of the definitive endoderm into anterior foregut and lung progenitors, differentiation of lung progenitors into airway epithelial cells (page 28, E and F and page 29, G; page 34 – 35, Example 3 and 4). The applicants have not provided any other working examples of any other method for forming airway basal cells. Thus, the specification provides sufficient teachings only for the enabled method of producing iBCs.
(h) The quantity of experimentation needed to make use of the invention based on the content of the disclosure:
The amount of experimentation would be undue because it would require determining which activators/inhibitors would form iBCs from AFE and would require determining the timing of addition/withdrawal of the activators/inhibitors. Since, as discussed above, it is not routine to treat AFE with any agent for any amount of time to produce iBCs, knowing only that an agent activates or inhibits a given signaling pathway would mean that significant experimentation would be required to determine which other agents and timing of addition/withdrawal of the agents would produce iBCs. This is because one cannot extrapolate between the activity of an agent (activation/inhibition of a signaling pathway) and differentiation of AFE to iBCs since there is little guidance (in both the state of the art and the specification) with respect to any other method for producing iBCs.
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.
41. Claims 5 and 17 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.
42. Regarding claim 5, it is unclear if step c requires 3D organoid culture because steps a and b recite organoids but the organoids are dissociated in step c and then cultured to form iBCs.
43. Claim 17 recites the limitation "the genome" in line 1. There is insufficient antecedent basis for this limitation in the claim.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
44. Claim(s) 1 and 16 – 18 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by McCauley (McCauley, Katherine B., et al. Cell stem cell 20.6 (2017): 844-857), hereinafter McCauley which is cited on the IDS filed 12/30/2022.
Claim 1 is drawn to A method of producing induced basal cells (iBCs ), comprising:
a. obtaining induced pluripotent stem cells (iPSCs); and
b. directing generation of iBCs from the iPSCs, wherein the directing lacks genetic manipulation.
Claim 18 is drawn to an induced basal cell (iBC) prepared using the method of any one of claims 1 – 17.
Regarding claim 1 and 18, McCauley teaches a method of differentiation of iPSCs obtained from a normal donor (step a) to basal cells that express NKX2.1, KRT5, and TP63 (step b; claim 18) (page 860, last para; page 861, para. 1 and 3; Figure 1A; Figure 2B, D, E – I; page 853, right col. para. 3; page 854, left col. para. 2; Figure 5G and J). McCauley’s method lacks genetic manipulation (Figure 1A).
Regarding claim 16, McCauley teaches the iPSCs are human (page 860, Table, “Experimental Models: Cell Lines and last para.; page 854, left col. para. 2).
Regarding claim 17, McCauley teaches the human iPSCs have been engineered to carry one corrected CFTR allele (page 860, Table, “Experimental Models: Cell Lines; page 861, para. 1; page 854, left col. last para.).
Therefore, McCauley anticipates claims 1 and 16 – 18.
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.
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.
45. Claim(s) 1 and 2 is/are rejected under 35 U.S.C. 103 as being unpatentable over McCauley (McCauley, Katherine B., et al. Cell stem cell 20.6 (2017): 844-857), hereinafter McCauley which is cited on the IDS filed 12/30/2022 in view of Rock (Rock, Jason R., et al. Proceedings of the National Academy of Sciences 106.31 (2009): 12771-12775.), hereinafter Rock.
McCauley anticipates claim 1 as set forth above. McCauley teaches the method produces iBCs that express KRT5, TP63, and NKX2.1 (page 853, right col. para. 3; Figure 2I; Figure 5G) but does not teach a homogenous population of iBCs in which at least 80% iBCs express KRT5, TP63, and NKX2.1. However, McCauley teaches withdrawal of CHIR during differentiation results in increased frequencies of cells co-expressing NKX2.1 and TP63 and a subset of cells co-expressing NKX2.1, TP63, and KRT5 which is a triad unique to airway basal cells (page 849, right col. para. 2; Figure 2I). McCauley teaches a method of purification of NKX2.1 cells by cell sorting (page 861, para. 5). McCauley teaches recent cell sorting methods have enabled the derivation of more homogeneous populations of lung epithelial progenitor cells or their differentiated progeny from human PSCs (page 845, right col. para. 1). McCauley teaches a proposed pathway for the generation of basal cells from iPSCs in Figure 5J and that airway spheres generated in low-Wnt conditions resemble airway epithelium and provide a mechanistic roadmap by which hPSC-derived lung progenitors can be differentiated to diverse lineages of distinct clinical interest (page 853, right col. last para.; page 854, left col. para. 1).
Rock teaches human basal cell isolation from human bronchi by FACS that resulted in a population of 96% p63+ basal cells and basal cells express TP63 and KRT5(page 12771, left col. para. 1; page 12774, right col. para. 1; page 12775, left col. last para. and right col. para. 1). Rock teaches the isolated human basal cells form broncospheres suggesting human basal cells are capable of both self-renewal and differentiation (page 12774, right col. para. 1). Rock teaches sphere formation of basal cells will greatly facilitate studies on the self -renewal and differentiation of basal cells (page 12771, left col. last para.). Rock teaches tracheal basal cells function as stem cells for repair of tracheal cell injury (page 12771, left col. para. 2). Rock teaches despite evidence that basal cells are stem cells, relatively little is known about their biology and mechanisms regulating their behavior (page 12771, left col. last para.).
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 McCauley regarding a method of generating basal cells expressing KRT5, TP63, and NKX2.1 and FACS sorting for the purification of NKX2.1 cells with the teachings of Rock regarding FACS sorting of basal cells that result in a population of basal cells greater that 80% to arrive at the claimed method where a homogenous population of iBCs in which at least 80% of the iBCs are KRT5+, TP63+, and NKX2.1+. One would have been motivated to combine the teachings of McCauley and Rock in a method of producing airway basal cells to understand the stem cell behavior of these cells as Rock teaches despite evidence that basal cells are stem cells, relatively little is known about their biology and mechanisms regulating their behavior. One would have a reasonable expectation of success in combining the teachings as McCauley teaches the method produces KRT5+, TP63+, and NKX2.1+ airway basal cells, methods for purification of NKX2.1 cells by cell sorting, and recent cell sorting methods have enabled the derivation of more homogeneous populations of lung epithelial progenitor cells or their differentiated progeny from human PSCs, and Rock teaches isolation of basal cells by cell sorting and one of ordinary skill in the art would be able to isolate a homogenous population of KRT5+, TP63+, and NKX2.1+ iBCs based on the teachings of McCauley and Rock and because cell sorting is routine in the art.
46. Claim(s) 1, 3, and 12 – 15 is/are rejected under 35 U.S.C. 103 as being unpatentable over McCauley (McCauley, Katherine B., et al. Cell stem cell 20.6 (2017): 844-857), hereinafter McCauley which is cited on the IDS filed 12/30/2022 in view of Somers (Somers, Aba, et al. Stem cells 28.10 (2010): 1728-1740.), hereinafter Somers in view of Ji (Ji, Hong, et al. Journal of Allergy and Clinical Immunology 135.1 (2015): 236-244.), hereinafter Ji.
McCauley anticipates claim 1 as set forth above.
McCauley teaches the iPSCs were generated from donors (page 860, Table, “Experimental Models: Cell Lines; page 861, para. 1; page 854, left col. last para.) but does not teach the method of making these iPSCs as outlined in claim 3. However, McCauley teaches the cells the CFTR mutant iPSC lines were obtained by the method of Somers. McCauley does not teach how the primary cells are obtained of claim 12 and 13 or the PCs are AECs that are nasal epithelial cells of claims 14 and 15. McCauley teaches directed differentiation of functional lung epithelial cell types from human PSCs holds promise for in vitro modeling of complex respiratory diseases and for future cell-based regenerative therapies (page 845, left col. para. 1). McCauley teaches the method of producing basal cells is used with cystic fibrosis patient-specific iPSC lines both before and after gene editing to correct the CFTR mutation responsible for the disease thus allowing precise interrogation of mutant versus corrected CFTR function (page 846, left col. para. 3; page 854, left col. para. 2 – 3). McCauley teaches iPSC-derived lung organoids contain functional epithelial cells with the potential for in vitro lung-specific disease modeling and gene correction of cystic fibrosis (page 854, right col. para. 1).
Regarding claims 3 step a, 12, and 13, Somers teaches a method of generating iPSCs by obtaining a full thickness skin punch biopsy from patients with cystic fibrosis (step a of claim 3; “taking tissue or cell sample from an individual” of claim 12; “surgical excision” of claim 13) (page 1730, right col. last para.; page 1731, left col. para. 2).
Regarding claim 3 step b and step c, Somers teaches culturing the fibroblasts from the skin biopsy overnight (step b) and transfecting the fibroblasts with RNA encoding Oct4, Sox2, Klf4, and c-Myc (step c) (page 1730, left col. para. 3; page 1731, left col. para. 2; Figure 1A).
Regarding claim 3 step d, Somers teaches iPSC clones were characterized based on staining for SSEA-4 (“identifying”) and were mechanically isolated (“purifying”) (page 1731, left col. para. 2 – 3 and right col. last para.).
Somers does not teach the iPSCs were derived from PCs that are airway epithelial cells that are nasal epithelial cells of claims 14 and 15. However, Somers teaches a sufficiently efficient reprogramming system is still needed to achieve the widespread derivation of disease-specific iPSCs from humans with inherited or degenerative diseases (Abstract, left col. ). Somers teaches the method of reprogramming primary cells produces iPSCs free of reprogramming genes (Abstract, right col.). Somers teaches the ability of reprogramming postnatal cells by defined transcription factors to iPSCs suggests generating autologous iPSCs from easily accessible human tissues (page 1728; page 1729, left col. para. 1). Somers teaches the method generates iPSC lines from individuals with diseases affecting the epithelial, endothelial, and interstitial compartments of the lung (page 1730, left col. para. 3). Somers teaches primary cells from individuals with cystic fibrosis are difficult to expand in culture for studies of disease pathogenesis, drug therapy, or targeted correction of their diseased loci and thus the prospect of deriving an inexhaustible supply of disease-specific lung epithelia from iPSCs is attractive for modeling or treating the disease (page 1740, left col. para. 2).
Regarding nasal epithelial primary cells of claims 14 and 15, Ji teaches derivation of iPSCs from nasal epithelial cells isolated from the nasal mucosa of children with asthma using a CytoSoft Brush (page 237, left col. para. 2 – 3; Figure 1). Ji teaches culturing the nasal epithelial cells until confluence and lentiviral-mediated delivery of Oct4, Sox2, Klf4, and c-Myc (page 237, left col. last para.; Figure 1). Ji teaches the iPSCs express Oct4, readily formed embryonic bodies in vitro and differentiated into cardiomyocytes (Figure 2; page 238, left col. last para. and right col. para. 1). Ji teaches the iPSCs are functionally similar to hESCs and comparable to iPSCs generated from airway epithelial cells from a healthy donor (page 240, left col. para. 2). Ji teaches nasal epithelial cells are an easily accessible tissue for the pediatric population (page 240, 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 combine the teachings of McCauley regarding a method of generating basal cells from iPSCs where the iPSCs were produced by the method of Somers with the teachings of Somers regarding a method of reprogramming primary cells into iPSCs that results in reprogramming factor-free iPSCs with the teachings of Ji regarding a method of deriving iPSCs from nasal epithelial cells of children to arrive at the claimed method where iPSCs are obtained by culturing and expanding a sample of primary cells, transfecting the primary cells with RNA-based reprogramming factors and identifying and purifying the iPSCs. One would have been motivated to combine the teachings of McCauley, Somers, and Ji in a method of producing iBCs from autologous iPSCs to study lung disease as McCauley teaches directed differentiation of functional lung epithelial cell types from human PSCs holds promise for in vitro modeling of complex respiratory diseases and for future cell-based regenerative therapies and Somers teaches a sufficiently efficient reprogramming system is still needed to achieve the widespread derivation of disease-specific iPSCs from humans with inherited or degenerative diseases and Ji teaches nasal epithelial cells are an easily accessible tissue for the pediatric population. One would have a reasonable expectation of success in combining the teachings because the iPSCs used in the method of McCauley were obtained by the method of Somers and Ji teaches the iPSCs obtained from nasal epithelial cells are capable of differentiation.
47. Claim(s) 1 and 4 – 11 is/are rejected under 35 U.S.C. 103 as being unpatentable over McCauley (McCauley, Katherine B., et al. Cell stem cell 20.6 (2017): 844-857), hereinafter McCauley which is cited on the IDS filed 12/30/2022 in view of Butler (Butler, Colin R., et al. American journal of respiratory and critical care medicine 194.2 (2016): 156-168.), hereinafter Butler in view of McCauley-2018 (McCauley, Katherine B., et. al. Current protocols in stem cell biology 45.1 (2018): e51), hereinafter McCauley-2018 which is cited on the IDS filed 12/30/2022.
McCauley anticipates claim 1 as set forth above.
Regarding claim 4 step a, McCauley teaches culturing iPSCs to form airway progenitor organoids from the iPSCs where the method is shown in Figure 1A and 5J (step a) (page 853, left col. last para. and right col. para. 1 – 2; page 861, para. 3).
Regarding claim 4 step b, McCauley teaches differentiating the lung organoids to form airway epithelial spheroids where the method is shown in Figure 5A and 5J (page 853, right col. para. 2 – 4; page 861, last para.; page 862, para. 1, 5, and 6). McCauley teaches the method uses a low-Wnt protocol for the reproducible and efficient generation and maturation of functional proximal airway organoids from patient-specific lines for the purposes of disease modeling and the testing of gene therapies (page 853, left col. last para.).
Regarding claim 4 step c, McCauley teaches proximalized cells were trypsinized and replated in two-dimensional culture on TransWell inserts and expansion was performed with dual SMAD inhibition by Dorsomorphin and SB431542 for cell expansion (page 853, right col. para. 3; page 862, para. 8). McCauley teaches dorsomorphin blocked CHIR-induced distal patterning (page 846, right col. para. 4). McCauley teaches the spheres produced were basal in nature and multiciliated epithelial cells could be generated after transfer into 2D conditions in air liquid interface culture (Figure 5I; page 853, right col. para. 4). McCauley does not teach culturing on gamma-irradiated fibroblasts.
Regarding claim 5, McCauley teaches 3D organoid culture as shown in Figure 5J (page 861, para. 5; page 862, para. 5 – 6; page 853, left col. last para.; page 853, left col. last para. and right col. para. 2).
Regarding claim 6, McCauley teaches lung organoids are formed by inducing definitive endoderm (“form definitive endoderm”), patterning DE to anterior foregut endoderm (“differentiation of the DE into anterior foregut endoderm”), and culturing the AFE to product lung epithelial progenitors (“differentiation of AFE into lung progenitor cells”) where lung organoids are formed (page 861, para. 3; page 862, para. 5 – 6).
Regarding claim 7 and 8, McCauley teaches enriching NKX2.1+ lung progenitors by antibody-based cell sorting prior to the formation of lung organoids to (page 861, para. 5; page 862, para. 5 – 6; page 859, Antibodies).
Regarding claim 10, McCauley teaches Matrigel in the formation of AFE from DE (page 861, para. 3).
Regarding claim 11, McCauley teaches culturing DE in SB431542 (inhibitor of BMP) and dorsomorphin (inhibitor of TGF-b/Activin/NODAL) to pattern cells to AFE and culturing AFE with CHIR99021 (inhibitor of GSK3) to induce lung epithelial progenitor fate (page 861, para. 3).
McCauley does not teach culturing on gamma-irradiated fibroblasts of claim 4 step c or enriching lung progenitor cells by antibody-based cell sorting wherein the antibody is an anti-carboxypeptidase antibody of claim 9. However, McCauley teaches 2D culture with dual SMAD inhibition for cell expansion and formation of multiciliated cells (page 853, right col. para. 3; page 862, para. 8). McCauley teaches iPSCs carrying a GFP reporter targeted to NKX2.1 for following differentiation because NKX2.1 is the first gene locus activated in developing endoderm at the time of lung lineage specification (page 846, right col. para. 1; page 856, left col. para. 2). cCauley teaches to ensure that airway organoids originated from NKX2.1+ progenitors population, sorted NCK2.1 cells were replated and cultured in 3D (page 853, right col. para. 2). McCauley teaches the method produces a potentially inexhaustible source of human proximal airway organoids that contain multiple airway epithelial lineages and express airway markers at levels comparable to the adult lung (page 856, left col. para. 3). McCauley teaches the method allows the production of patient-specific airway epithelial organoids both before and after gene editing to correct the CFTR genetic lesion responsible for cystic fibrosis (page 856, left col. last para.). McCauley teaches the produced organoids allow for assay to analyze CFTR function (page 856, left col. last para.). McCauley teaches the iPSC airway organoid system may also be readily adaptable for in vitro modeling, drug development and regenerative medicine (page 856, right col. para. 1). McCauley teaches effective derivation of functional airway organoids from iPSCs would provide valuable models of lung disease and facilitate cell-based regenerative therapies for airway disorders (Summary; page 845, left col.). M
Regarding culturing on gamma-irradiated fibroblasts of claim 4 step c, Butler teaches a method for expansion of larger numbers of functional human airway basal epithelial cells comprising culturing the basal cells on mitotically inactivated fibroblasts with the ROCK inhibitor Y-27632 (Abstract; page 159, middle col.; page 160, right col. para. 2; page 166, right col. last para.). Butler teaches generation of mitotically inactivated fibroblasts using irradiation treatment (page E1, last para.; page E2, para. 1). Butler does not teach enriching lung progenitor cells by antibody-based cell sorting wherein the antibody is an anti-carboxypeptidase antibody of claim 9. However, Butler teaches the cultured cells were capable of generating both ciliated and goblet cell lineages and cilial were functional (Abstract). Butler teaches high cell-seeding densities are required for bioengineering applications and obtaining sufficient numbers of autologous epithelial cells is a challenge (page 159, left col. para. 2). Butler teaches epithelial cells obtained from endobronchial biopsies and cultured for multiple passages is useful to generate basal cells for in vitro investigations but inefficient for regenerative applications as many cultures fail and those that grow cannot provide sufficient cell numbers (page 159, left col. para. 2). Butler teaches a method to generate sufficient numbers of airway epithelial cells for use in tissue-engineered tracheal transplants is a significant and unmet need (page 159, left col. para. 2). Butler teaches successful long-term expansion of human epidermal stem cells is achieved by coculture with mitotically inactive mouse embryonic fibroblast feeder cells and inhibition of ROCK increases cell proliferation (page 159, left col. para. 3). Butler teaches the method rapidly and efficiently expands cells expressing airway basal stem cell markers suggesting the technique may generate quantities of functional cells demanded by future tissue-engineered constructs (page 159, left col. last para.). One would have been motivated to combine the teachings of McCauley regarding a method of generating basal cells from iPSCs with the teachings of Butler regarding a method of rapid and efficient expansion of basal cells on gamma irradiated fibroblasts with a ROCK inhibitor to arrive at the claimed method where the airway epithelial spheroids produced by the method of McCauley are dissociated and cultured on gamma-irradiated fibroblasts with dual-SMAD inhibition and ROCK inhibition to generate large numbers of iBCs to study airway disease and for potential regenerative therapies because Butler teaches a method to generate sufficient numbers of airway epithelial cells for use in tissue-engineered tracheal transplants is a significant and unmet need.
Regarding enriching lung progenitor cells by antibody-based cell sorting wherein the antibody is an anti-carboxypeptidase antibody of claim 9, McCauley-2018 teaches the same method of McCauley regarding differentiation of iPSCs to NKX2.1+ lung progenitors (page 2, para. 3). McCauley-2018 teaches as directed differentiation can be inefficient and heterogeneous, the engineering of reporter cell lines allows for live-cell sorting of populations of interest (page 24, last para.). McCauley-2018 teaches in the absence of reporter lines, cell surface markers can be used as a surrogate to enrich for the population of interest and to identify PSC-derived NKX2.1+ lung epithelial progenitors using two reliable cell surface markers, CPM and CD47 (page 25, para. 2). Applicant’s specification uses “CPM” as an abbreviation for an anti-carboxypeptidase M antibody at page 11, line 7 – 8, page 28, line 25, and page 34, line 26.
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 McCauley regarding a method of generating basal cells from iPSCs where the method includes enriching NKX2.1+ progenitors by antibody-based cell sorting with the teachings of Butler regarding a method of rapid and efficient expansion of basal cells on gamma irradiated fibroblasts with a ROCK inhibitor with the teachings of McCauley-2018 regarding enriching NKX2.1 lung progenitors using CPM antibody cell sorting to arrive at the claimed method where the airway epithelial spheroids produced by the method of McCauley are dissociated and cultured on gamma-irradiated fibroblasts with dual-SMAD inhibition and ROCK inhibition and where NKX2.1 lung progenitors are enriched by anti-carboxypeptidase antibody cell sorting. One would have been motivated to combine the teachings of McCauley, Butler, and McCauley-2018 in a method of producing iBCs to study airway disease and for potential regenerative therapies as McCauley teaches effective derivation of functional airway organoids from iPSCs would provide valuable models of lung disease and facilitate cell-based regenerative therapies for airway disorders and Butler teaches epithelial cells obtained from endobronchial biopsies is inefficient for regenerative applications as many cultures fail and those that grow cannot provide sufficient cell numbers. One would have a reasonable expectation of success in combining the teachings as Butler teaches the method rapidly and efficiently expands cells expressing airway basal stem cell markers suggesting the technique may generate quantities of functional cells demanded by future tissue-engineered constructs and McCauley-2018 teaches CPM is a reliable marker to identify PSC-derived NKX2.1+ lung progenitors.
Conclusion
No claims allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ZANNA M BEHARRY whose telephone number is (571)270-0411. The examiner can normally be reached Monday - Friday 8:45 am - 5:45 pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Peter Paras can be reached at (571)272-4517. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/Z.M.B./Examiner, Art Unit 1632
/ANOOP K SINGH/Primary Examiner, Art Unit 1632