Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Detailed Action
This action is in response to the papers filed on December 12th 2023. Claims 1-2, 5, 6, 7, 10-12, 16, 18, 20, 22, 24-26 and 28-32 are currently pending. It is noted that claims 1 and 28 are independent claims.
Therefore, claims 1-2, 5, 6, 7, 10-12, 16, 18, 20, 22, 24-26 and 28-32 are under examination to which the following grounds of rejection are applicable.
Priority
The present application is a 35 U.S.C. 371 national stage filing of International Application NoPCT/CA2022/050943, filed on June 14th, 2022.
Applicant’s claim for the benefit of a prior-filed parent provisional applications 63/286,173 filed December 6, 2021, and 63/210,248, filed June 14th 2021 under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, or 365(c) is acknowledged.
Thus, the earliest possible priority for the instant application is June 14th, 2021 since the scope of provisional application encompasses the claims of the instant application.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on March 11th, 2024 and June 24th, 2025 were filed. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Claim Objections
Claims 1, 11, 12, and 28 are objected to because of the following informalities:
Claims 1, 11, 12, and 28 recite the acronym “TGF” but it is not defined in the claim. An acronym should be defined the first time it appears in an independent claim or in the group of claims under the independent claim. For the purposes of compact prosecution, “TGF” will be interpreted as transforming growth factor.
Claims 22, 24 and 25 are objected to because of the following informalities: : abbreviations such as FOXJ1, TUBB4B, TJP1, ACE2 and MUC5AC should be spelled out at the first encounter in the claims. Appropriate correction is required
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-2, 5, 6, 7, 10-12, 16, 18, 20, 22, 24-26 and 28-32 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.
Regarding claim 1 and 28, they are indefinite in its recitation of the phrase “notch signaling and/or an inhibitor of signaling” in line 3. The use of the term “and/or” is improper because it creates ambiguity as to the claim scope (e.g. does it mean notch signaling, an inhibitor of signaling through a TGF or both?). For the sake of compact prosecution, the claim will be interpreted as “comprising an inhibitor of notch signaling”.
Claims 1 and 28 are indefinite in its recitation of “apical -out organoids in culture” . The term " apical -out organoids " is not defined by the claim. ”. The specification does not provide any closed definition as to what is meant by “apical -out organoids”. Although it is acknowledged the specification discloses some epithelial organoids that are considered by applicant to grow with the apical surface facing a central lumen of the organoid and to require polarity reversal to access the apical side via direct external environment (paragraph [0004]) , these are merely exemplary and non-limiting. The metes and bounds of the claims are unclear particularly since “apical -out organoids in culture” would vary depending on type of cell, orientation of basal membrane and apical surface relative to a central lumen of the organoid and culture conditions. As such, the metes and bounds of the claims cannot be determined.
In view of the above, a person of ordinary skill in the art cannot unequivocally interpret the metes and bounds of the claim so as to understand how to avoid infringement. Applicant is reminded that any amendment must point to a basis in the specification so as not to add New Matter. See MPEP 714.02 and 2163.06.
Claim 1 and by dependence, claim 18, are indefinite for recitation of the phrase “at least a portion” since it is unclear how the phrase “at least” is defined, what its metes and bounds are, or to what the term is directed towards. It is not clear in reference to the organoid apical surface which portion or portions contain the faces away.
Clam 1 is indefinite in its recitation of “faces away from a core thereof” because it is unclear if the quoted phrase refers to the core of the organoid or any other core. As such the metes and bounds are indefinite.
Regarding claim 1 and 30, it is indefinite in its recitation of the phrase “in the absence of an added extracellular matrix or extracellular matrix protein” and “the organoid medium does not contain or come into contact with an added extracellular matrix or extracellular matrix protein”. It is unclear if extracellular matrix protein is added into the medium or removed.
Regarding claim 2, it is indefinite in its recitation of the phrase “preferably bronchial or nasal epithelial cells”. Use of a narrow numerical range that falls within a broader range in the same claim renders the claim indefinite when the boundaries of the claim are not discernible. Description of examples and preferences is properly set forth in the specification rather than in a single claim. As such, the metes and bounds of this claim are indefinite.
Claim 24 uses parentheses to comments on or qualify part of the sentences. It is unclear whether the limitations in parentheses are meant to be limitations in the claims or whether they are only suggestions/examples. As such, the metes and bounds of the claims cannot be determined.
Regarding claims 20, 22, and 24-26, it is indefinite in its recitation of the phrase “the organoid” as it lacks proper antecedent basis. Claim 1 recites only an “apical-out organoid”.
Claims 2, 5-7, 10-12, 16, and 18 are indefinite insofar as they depend on claim 1. Claims 29, 31 and 32 are indefinite insofar as they depend on claim 28 ,
For the sake of compact prosecution, the phrase “apical -out organoids in culture” is interpreted as a three-dimensional tissue model with reversed polarity, such that the luminal surface or inner cavity of an organoid containing the epithelial layer of cells is exposed to an environment (the medium).
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.
Claims 1-2, 5, 6, 7, 10-12, 16, 18, 20, 22, 24-26 and 28-32 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 apical-out organoids comprising pulmonary lineage epithelial cells , does not reasonably provide enablement for apical-out organoids with different cell types . 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.
The criteria for enablement set out In re Wans, MPEP 2162.01(a), considers the following factors:
Breadth of the Claims
The instant claims are directed to a method for forming apical-out organoids in a culture and an organoid medium for generating apical-out organoids. Thus, the claims encompass all apical-out organoids comprising any populations of cells. The population of cells includes cells of any species or origin. As such, the breadth of the claims is great.
State of Prior Art
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The prior art demonstrates that apical out organoids is a tissue-specific and varies depend on the culture conditions and cell types used. Co et al. (Published: 2019. Cell reports, 26(9), 2509–2520.e4) teaches “a method to reverse the epithelial polarity of the enteroids such that the apical surface faces outward” (page 2510 col 1 para 2). Thus, Co teaches a model for gastrointestinal epithelium (e.g, epithelial cells) where upon removing ECM scaffold proteins, basal out enteroids evert to apical-out polarity
in a beta1 integrin-dependent manner (In Brief) and where the apical surface of epithelial cells reverts outward from the interior of the spheroid facing the lumen (page 2510; col. 2) .Furthermore, Co also teaches that “this model can be used to recapitulate and advance our understanding of intestinal pathogens and host-microbe interactions” (page 2510 col 1 para 2). Therefore, the Co’s disclosure of the apical-out enteroids from gastrointestinal cells does not enable the pulmonary apical out organoids claimed in the instant application. The art fails to teach or guide one skilled in the art that the polarity inverse methodology for apical-out enteroids could translate into pulmonary apical-out organoids discussed in the instant application, which are biologically and functionally distinct.
Amount of Direction Provided by the Inventor
The specification provides insufficient direction and guidance to enable a person of ordinary skill in the art to practice the full scope of the claimed invention. Although the generic claims encompass apical-out organoids by contacting ANY cell population, the specification only provides working examples for primary bronchial epithelial cells (Specification, para [00077], “A population of pulmonary lineage cells may be expanded and passaged using a commercially available kit, such as PneumaCultTM Ex or PneumaCultTM Ex Plus (STEMCELL Technologies).”; para [00083], “Primary normal human bronchial epithelial cells (hBECs) were commercially sourced, such as from LONZA or EPITHELIX SARL. In the experiments described herein, all cells were collected from non-smoking, healthy donors.”). Furthermore, the specification fails to disclose any step-by-step protocols, cell culture conditions, organoid medium formulations, or ranges that would enable a skilled artisan to adapt the methods to other organoid cell types encompassed by the claims. As a result, the guidance provided is not commensurate with the breadth of the claims, and a skilled artisan would require undue experimentation to practice the embodiments outside the apical-out organoid that uses pulmonary lineage cells.
The Presence or Absence of Working Examples
The specification provides working examples for apical-out organoids using primary normal human bronchial epithelial cells (hBECs), and does not include any examples demonstrating the successful preparations or performance of apical-out organoids contacting different cell populations, as encompassed by the claims. The absence of working examples for all species of cell populations is highly significant because the prior art establishes that apical-out polarity systems can be achieved in other models in response to different culture conditions and factors, indicating that polarity outcomes can vary across tissue types and experimental setups. As such, the limited working examples do not encompass the full scope of the claims and the specification does not fully enable the entire genus of polymers.
The Quantity of Experimentation Necessary
Given the absence of working examples or specific guidance on non-pulmonary lineages, a person with ordinary skill in the art would need to engage in undue experimentation to determine whether and how apical-out polarity can be done in each claimed tissue/cell type. Therefore, the claims are not enabled for all types of apical-out polarity utilizing different cell lineages.
Conclusion
In light of the unpredictability surrounding the claimed subject matter and the lack of adequate guidance, one wishing to practice the presently claimed invention would be unable to do so without engaging undue experimentation. The specification provides working examples limited to pulmonary lineage epithelial cells, offers no meaningful guidance or data for other epithelial lineages encompassed by the claims. One wishing to practice the presently claimed invention would have to produce additional data and experimentation to determine if non-pulmonary lineage cells can achieve the same apical-out fate described in the specification. Thus, the claims are not enabled across their full scope and rejected under 35 U.S.C. 112(a) for failing to be enabling for the full scope of the claims.
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.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1, 2, 5, 6, 10, 11-12, 16, 18, 24, 26, 28, 29, 30-32 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Salahudeen (Cited in IDS Filed: 3/11/2024. Published: Nov 25 2020. Nature, 588(7839):670-675).
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Regarding claim 1, 28, and 30, Salahudeen teaches a method for forming apical-out organoids in a culture (Abstract, page 670, "We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population."), contacting a population of cells within an organoid medium comprising an inhibitor of TGF (page 676 col 1 para 2, “To isolate distal airway cells, lung parenchyma from 1 cm from the visceral pleura was mechanically dissociated with Castro scissors…resuspended in two tissue volumes of lung organoid medium comprising advanced DMEM/F12…and TGF-β inhibitor A83-01 (100 nM, Tocris). This lung organoid medium was used for all experiments except for those shown in Extended Data Fig. 4f”), and culturing the organoid medium in the absence of an extracellular matrix protein to obtain apical-out organoids (Figure 10b caption: Diagram of ECM removal and suspension culture leading to apical-out polarity of lung organoids), wherein an apical surface of at least a portion of the apical-organoids faces away from a core thereof (Abstract, page 670, “We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface”).
Regarding claim 2, Salahudeen teaches the population of cells are pulmonary lineage cells, preferably bronchial or nasal epithelial cells (Abstract, page 370, “Here we describe the development of a long-term feeder-free, chemically defined culture system for distal lung progenitors as organoids
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derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells.”).
Regarding claim 5, Salahudeen teaches aggregating the population of cells (Figure 2g caption: “The survival of single cells in suspension could be prolonged by promoting the aggregation of LS174T single cells”).
Regarding claim 6, Salahudeen teaches that the aggregation of the populations of cells is within the organoid medium (page 676, col 1 para 2, “To isolate distal airway cells, lung parenchyma from 1 cm from the visceral pleura was mechanically dissociated with Castro scissors, washed and incubated with 5 units per ml porcine elastase (Worthington), 100 Kunitz units per ml DNase I (Worthington), and Normocin (InvivoGen), and resuspended in two tissue volumes of lung organoid medium…Cells in matrix were then plated in 24-well plates in 50-μl droplets, and warm medium was added after the droplets had solidified for 10 min at room temperature Medium was changed every 3–4 days and organoids were passaged every 3–4 weeks by dissociation with TrypLE”).
Regarding claims 10 and 29, Salahudeen teaches that the organoid medium is serum-free (Chemically defined EGF/NOGGIN medium was sufficient for baseline clonal proliferation of AT2 organoids, which was attenuated by blocking global endogenous WNT biosynthesis consistent with the requirement for autocrine WNT signalling in mouse AT2 cells16. Growth was enhanced by adding fibroblast conditioned medium containing serum and WNT agonists.). Note that serum was later added to the medium to improve growth.
Regarding claim 11,12, 31, 32, Salahudeen teaches the organoid medium comprises an inhibitor of signaling through a TGFβ (page 676 col 1 para 2, “To isolate distal airway cells, lung parenchyma from 1 cm from the visceral pleura was mechanically dissociated with Castro scissors…resuspended in two tissue volumes of lung organoid medium comprising advanced DMEM/F12…and TGF-β inhibitor A83-01 (100 nM, Tocris)).
Regarding claim 16, Salahudeen teaches that the culturing the population of cells is under non-adherent conditions (page 678 col 2 para 4, “The pellet was resuspended in growth medium in ultra-low attachment six-well tissue culture plates (Corning Costar 3471).
Regarding claim 18, Salahudeen teaches that 25% of cells of the portion of the organoids are ciliated (page 671 col 2 para 2 bridging into page 672, "However, by one month of culture, about 50% of
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organoids had developed single or occasionally multiple lumens (Fig. 2a, b, Extended Data Fig. 5a–c) with club (SCGB1A1+KRT5–) and ciliated (AcTUB+KRT5–) cells lining the interior surface (Fig. 2a, b). Basal cultures purified by density sedimentation exhibited serial clonal outgrowth, dependence on EGF and NOGGIN, and cavitation lined by luminal SCGB1A1+ club and AcTUB+ ciliated cells (Fig. 2c, d, Extended Data Fig. 5d–h, Supplementary Videos 1, 2).").
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Regarding claim 24, Salahudeen teaches that the organoids express ZO-1 and ACE2 (Figure 10a and 10d: a) scRNA-seq plots of ACE2 and TMPRSS2 gene expression in ECM-embedded mixed distal lung organoids as in Fig. 1a–h, d) By day 2 in suspension (d2) ZO-1 (white) forms junctional rings in the apical periphery of each cell facing the external side of the organoids and the actin cytoskeleton forms microvilli (green) facing outward (apical-out polarity)).
Regarding claim 26, Salahudeen teaches that the apical-out pulmonary organoids support the replication of viruses (page 673 col 1 para 3, “Influenza virus H1N1 avidly infected distal lung organoids, which also expressed influenza receptors (Extended Data Fig. 9a–d), similar to proximal airway organoids24,25. Infection of organoids with influenza”; page 673 col 2 para 3, “In addition, infected organoids showed replication-specific SARS-CoV-2 spliced subgenomic RNA (sgRNA; Fig. 4c, right) and production of infectious virions with VeroE6 cell plaque formation (35 PFU ml−1 from organoid lysates and 65 PFU ml−1 from organoid supernatants).”).
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.
Claim(s) 1, 5, 7 and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Salahudeen (Cited in IDS Filed: 3/11/2024. Published: Nov 25 2020. Nature, 588(7839):670-675).
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Regarding claim 1, Salahudeen teaches a method for forming apical-out organoids in a culture (Abstract, page 670, "We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population."), contacting a population of cells within an organoid medium comprising an inhibitor of TGF (page 676 col 1 para 2, “To isolate distal airway cells, lung parenchyma from 1 cm from the visceral pleura was mechanically dissociated with Castro scissors…resuspended in two tissue volumes of lung organoid medium comprising advanced DMEM/F12…and TGF-β inhibitor A83-01 (100 nM, Tocris). This lung organoid medium was used for all experiments except for those shown in Extended Data Fig. 4f”), and culturing the organoid medium in the absence of an extracellular matrix protein to obtain apical-out organoids (Figure 10b caption: Diagram of ECM removal and suspension culture leading to apical-out polarity of lung organoids), wherein an apical surface of at least a portion of the apical-organoids faces away from a core thereof (Abstract, page 670, “We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface”).
With regard to instant claims 1 and 5, the teachings of the apical-out organoid of Salahudeen render obvious the claimed method, as iterated above in the 102 rejection the content of which is incorporated herein, in its entirety. Moreover, Salahudeen teaches that the population of cells were aggregated between 1 and 7 days in a microwell device of claim 7 (page 676 col 1 para 3, “The cell pellet was then washed and resuspended in 10 volumes of reduced growth factor Basement Membrane Extract II (Trevigen). Cells in matrix were then plated in 24-well plates in 50-μl droplets, and warm medium was added after the droplets had solidified for 10 min at room temperature Medium was changed every 3–4 days and organoids were passaged every 3–4 weeks by dissociation with TrypLE.”).
However, regarding claim 7, Salahudeen fails to teach that that the aggregation of the population of cells is between 1 and 7 days and the aggregation of the cells comprises depositing between 10 and 2000 single cells comprised in clumps into a microwell device.
It would have been obvious to optimize the number of aggregated single cells into a microwell device based on influential considerations in the design of the apical-out organoid, such as confluency of cells, number of cells per organoid, cell culture medium conditions, number of passages etc. to result at the utilization of 10-2000 single cells into a microwell device to develop the apical-out organoids.
The Court has stated that generally such differences amount to mere optimization and will not support patentability unless there is evidence indicating the claimed feature 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). (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.); see also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382 (“The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages.”); In re Hoeschele, 406 F.2d 1403, 160 USPQ 809 (CCPA 1969) (Claimed elastomeric polyurethanes which fell within the broad scope of the references were held to be unpatentable thereover because, among other reasons, there was no evidence of the criticality of the claimed ranges of molecular weight or molar proportions.). For more recent cases applying this principle, see Merck & Co. Inc. v. Biocraft Laboratories Inc., 874 F.2d 804, 10 USPQ2d 1843 (Fed. Cir.), cert. denied, 493 U.S. 975 (1989); In re Kulling, 897 F.2d 1147, 14 USPQ2d 1056 (Fed. Cir. 1990); and In re Geisler, 116 F.3d 1465, 43 USPQ2d 1362 (Fed. Cir. 1997). In KSR International Co. v. Teleflex Inc., 550 U.S. 398 (2007), the Supreme Court held that "obvious to try" was a valid rationale for an obviousness finding, for example, when there is a "design need" or "market demand" and there are a "finite number" of solutions. 550 U.S. at 421.
MPEP 2144 sets forth Applicant' s burden for rebuttal of a prima facie case of obviousness based upon routine optimization. Applicant must provide either a showing that the particular amount or range recited within the claims is critical; and/or a showing that the prior art reference teaches away from the claimed amount.
Regarding claim 20, Saluhadeen teaches the method of claim 1, wherein among at least 60% of the organoids the apical surface thereof is in contact with the organoid medium (page 678, col 2 para 1, "Organoids were then plated in suspension in lung organoid medium (apical-out organoids)").
It would have been obvious to optimize the number of apical-out cells design, such as confluency of cells, number of cells per organoid, concentrations of EDTA or dissociate the matrix to achieve apical out polarity, such that 60% of the organoids with an apical surface contact the organoid medium.
***
Claim(s) 1, 22 and 25 are rejected under 35 U.S.C. 103 as being unpatentable over Salahudeen (Cited in IDS Filed: 3/11/2024. Published: Nov 25 2020. Nature, 588(7839):670-675) and in further view of Dye et al. (Published: 2015. In vitro generation of human pluripotent stem cell derived lung organoids. eLife, 4, e05098).
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Regarding claim 1, Salahudeen teaches a method for forming apical-out organoids in a culture (Abstract, page 670, "We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population."), contacting a population of cells within an organoid medium comprising an inhibitor of TGF (page 676 col 1 para 2, “To isolate distal airway cells, lung parenchyma from 1 cm from the visceral pleura was mechanically dissociated with Castro scissors…resuspended in two tissue volumes of lung organoid medium comprising advanced DMEM/F12…and TGF-β inhibitor A83-01 (100 nM, Tocris). This lung organoid medium was used for all experiments except for those shown in Extended Data Fig. 4f”), and culturing the organoid medium in the absence of an extracellular matrix protein to obtain apical-out organoids (Figure 10b caption: Diagram of ECM removal and suspension culture leading to apical-out polarity of lung organoids), wherein an apical surface of at least a portion of the apical-organoids faces away from a core thereof (Abstract, page 670, “We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface”).
However, Salahudeen does not teach wherein the organoids express one or more markers of ciliogenesis and the one or more markers of ciliogenesis comprise FOXJ1 and/or TUBB4B, as required in claim 22.
Dye teaches that the organoids express markers of ciliogenesis comprising FOXJ1 (page 15 para 2, “Here, we show that HLOs possess both mesenchymal and lung epithelial (∼60% NKX2.1+) cells with proximal airway-like structures that possess P63+ (∼40%) and FOXJ1+ cells (∼3%) along with distal airway-like structures that possess SFTPC+ (∼5%) and HOPX+ (∼4%) cells.”). Furthermore, Dye teaches that the expression of FOXJ1 cells is vital to understand basal cell differentiation in organoids (page 15 para 3, “. For example, the location of P63+ cells adjacent to FOXJ1+ cells in the HLOs will be necessary to study basal cell differentiation into different proximal airway cell types during homeostasis or after injury”).
It would have been obvious for someone with ordinary skill in the art to implement the expression of FOXJ1 in organoids taught by Dye into the apical-out organoids taught by Salahudeen in order to study basal cell differentiation as well as potential airway dynamics in lung tissue. Such incorporation of FOXJ1 would have been expected to achieve predictable results in resulting in apical-out lung organoids that would express FOXJ1, a marker of ciliogenesis. Additionally, a skilled artisan would have been motivated to incorporated ciliated cells to encourage the resemblance naturally occurring mesenchymal and lung epithelial cells of proximal airways.
Regarding claim 25, the teachings of Saluhadeen render obvious the claimed methodology of claim 1. Moreover, Dye teaches the organoids express markers of ciliogenesis comprising FOXJ1 (page 15 para 2) and that the organoids do not express MUC5AC (page 9 para 1, “Although the goblet cell marker MUC5AC mRNA expression was detected, protein expression was not detected by immunofluorescence (Figure 4A and data not shown).”).
It would have been obvious for someone with ordinary skill in the art to implement the teachings of Dye into the apical-out distal lung organoids of Saluhadeen with apical-out polarity in order to detect lack of expression of goblet cells within the apical-out organoid structure by lack of MUC5AC expression with a reasonable expectation of success.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Katriel B Kasayan whose telephone number is (571)272-1402. The examiner can normally be reached 10-4p.
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/KATRIEL BARCELLANO KASAYAN/Examiner, Art Unit 1634
/MARIA G LEAVITT/Supervisory Patent Examiner, Art Unit 1634