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
Election/Restriction
Applicant’s election, without traverse, of Group I, claims 1-9, drawn to a method for producing human PSC-derived cardiac organoids, in the reply filed on 11/06/2025 is acknowledged.
Claim 10 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claim Status
Claims 1-10 are pending.
Claim 10 is withdrawn.
Claims 1-9 are considered on the merits.
Priority
This application is a 371 of PCT/KR2022/004329 (filed on 03/28/2022), which claims benefit from foreign application KR10-2021-0060265 (filed on 05/10/2021). The priority claim of the instant application has been granted and the earliest benefit date is 05/10/2021 from the application KR10-2021-0060265.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 01/13/2023 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner. The corresponding signed and initialed PTO form 1449 has been mailed with this action.
Claim Objections
Claims 1, 3 and 6 are objected to because of the following informalities:
Claim 1, the wherein clause recites the phrase “self-organized” cells. Since these self-organized cells have been recited in step (D), it is recommended to change the phrase in the wherein clause to “the self-organized” cells.
Claim 3, line 2 recites the phrase “plating human pluripotent stem cells”. Since base claims 1 and 2 have recited human pluripotent stem cells, it is recommended to change the phrase in claim 3 to “plating the human pluripotent stem cells”.
Claim 6 does not end with a period. MPEP 608.01(m) states, “Each claim begins with a capital letter and ends with a period.” See Fressola v.Manbeck, 36 USPQ2d 1211 (D.D.C. 1995)”.
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 1-9 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.
Claims 1 and 3-7 recite the phrase “embryonic bodies” that seems to mean embryoid bodies (see specification, [0024] reciting embryoid bodies in Figs 2 and 3, and [0042] reciting embryonic bodies “As shown in Figure 2”). The phrase is indefinite because the specification does not clearly define the phrase “embryonic bodies”. Where applicant acts as his or her own lexicographer to specifically define a term of a claim contrary to its ordinary meaning, the written description must clearly redefine the claim term and set forth the uncommon definition so as to put one reasonably skilled in the art on notice that the applicant intended to so redefine that claim term. Process Control Corp. v. HydReclaim Corp., 190 F.3d 1350, 1357, 52 USPQ2d 1029, 1033 (Fed. Cir. 1999). Claims 2 and 8-9 are rejected as being directly or indirectly dependent from claim 1 but not resolving the ambiguity. The phrase is examined as “embryoid bodies”.
Claims 3 and 4 contain the trademark/trade name “mTeSR”. Claims 5-9 contain the trademark/trade name “B-27”. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name mTeSR is used to identify/describe the cell culture medium for growing stem cells, and the trademark/trade name B-27 is used to identify/describe the nutritive supplements for the growth and differentiation of cells. Accordingly, the identification/description is indefinite.
Claims 3 and 4 recite the phrase ROCK inhibitor “(Y-27632)”. The use of parentheses renders the claims indefinite because there are multiple ROCK inhibitors, thus it is unclear whether the limitation “Y-27632” between the parentheses is part of the claimed invention.
Claim 5 steps (C-1) and (C-2), claim 6, claim 7 and claim 8 step (D-1), recite the phrase “B-27 supplement (minus insulin)”. Claim 8 steps (D-3) and (D-4) and claim 9, recite the phrase “B-27 supplement (minus vitamin A)”. The use of parentheses renders the claims indefinite because it is unclear whether the limitation “minus insulin” or “minus vitamin A” between the parentheses is part of the claimed invention.
Claim Rejections - 35 USC § 112(a)
(Written Description)
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 1-9 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
Under the written description guidelines (see MPEP 2163) the Examiner is directed to determine whether one skilled in the art would recognize that the Applicant was in possession of the claimed invention as a whole at the time of filing. The following considerations are critical to this determination.
To satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention. See, e.g., Moba, B.V. v. Diamond Automation, Inc., 325 F.3d 1306, 1319, 66 USPQ2d 1429, 1438 (Fed. Cir. 2003); Vas-Cath, Inc. v. Mahurkar, 935 F.2d at 1563, 19 USPQ2d at 1116. An original claim may lack written description support when (1) the claim defines the invention in functional language specifying a desired result but the disclosure fails to sufficiently identify how the function is performed or the result is achieved or (2) a broad genus claim is presented but the disclosure only describes a narrow species with no evidence that the genus is contemplated. See Ariad Pharms., Inc. v. Eli Lilly & Co., 598 F.3d 1336, 1349-50 (Fed. Cir. 2010) (en banc). The written description requirement is not necessarily met when the claim language appears in ipsis verbis in the specification. "Even if a claim is supported by the specification, the language of the specification, to the extent possible, must describe the claimed invention so that one skilled in the art can recognize what is claimed. The appearance of mere indistinct words in a specification or a claim, even an original claim, does not necessarily satisfy that requirement." Enzo Biochem, Inc. v. Gen-Probe, Inc., 323 F.3d 956, 968, 63 USPQ2d 1609, 1616 (Fed. Cir. 2002).
Accordingly, to satisfy the written description requirement, the specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention. Vas-Cath, Inc. v. Mahurkar, 935 F.2d 1555, 1562-63, 19 USPQ2d 1111 (Fed. Cir. 1991). See also MPEP 2163.
SCOPE OF THE INVENTION
Independent claim 1 encompasses a genus of method for producing human PSC-derived cardiac organoids. It is noted that the instant claim 1 does not provide any specific culturing condition in regard to culture medium, supplements, culture duration, culture container etc. The only two limitations required by claim 1 are (1) undergoing an embryoid body stage, and (2) the final product of cardiac organoids having a specific cell number ratio of three types of cells. Thus, the independent claim 1 encompasses a genus of method comprising culturing hPSCs into embryoid bodies and differentiating the embryoid bodies into cardiac organoids comprising self-organized cardiomyocytes, fibroblasts and vascular endothelial cells in a specific cell number ratio, the genus of method comprising culturing hPSCs in any culture medium, with any supplements, for any culture duration, on any culture container.
However, the specification only discloses a single species of method for culturing and differentiating hPSCs into cardiac organoids with the claimed cell composition by sequentially culturing the hPSCs in specific media and supplements (e.g., mTeSR1 with or without supplemented ROCK inhibitor) for a specific duration (e.g., 1 day or 2-3 days in each step) on a specific container (e.g., an uncoated 6-well plate (Matrigel not used) or a low-attachment plate) (see [0040], [0041], [0047] for merely differentiating hPSCs into embryoid bodies as an example, and see p. 12-24 for the method for producing hPSC-derived cardiac organoids with the claimed cell composition).
Dependent claims 2-9 encompass type of hPSCs, steps of differentiation of embryoid bodies, steps of differentiation of structures comprising mesoderm and endoderm cells, steps of differentiation of cardiomyocytes, fibroblasts and vascular endothelial cells, and step of maturation of cardiac organoids containing the self-organized three types of cells. However, the specification only discloses a single species of method for differentiating hPSCs into cardiac organoids with the claimed cell composition by sequentially culturing the hPSCs in specific media and supplements for a specific duration on a specific container. In other words, the specification only discloses a single method comprising all the above steps sequentially.
ACTUAL REDUCTION TO PRACTICE
As stated supra, the specification only discloses a single species of method comprising all the above steps sequentially, for producing hPSC-derived cardiac organoids with the claimed cell composition. Accordingly, Applicant did not demonstrate a reduction to practice a genus of method for producing hPSC-derived cardiac organoids with the claimed cell composition, nor did Applicant adequately set forth in terms of distinguishing identifying characteristics as evidenced by other descriptions of the invention that are sufficiently detailed to show that Applicant was in possession of the claimed genus of method.
DISCLOSURE OF STRUCTURE
The Applicant has provided a single species of method in the working examples to produce hPSC-derived cardiac organoids with the claimed cell composition. Although producing hPSC-derived cardiac organoids is known in the field, the prior art is silent on a method for producing hPSC-derived cardiac organoids with the claimed cell composition, nor indicate a relationship between the structure of the method, e.g., in regard to culture medium, supplements, duration, container, and the ability to produce a cardiac organoid with the claimed cell composition.
SUFFICIENT RELEVANT IDENTIFYING CHARACTERISTICS
As mentioned above, producing hPSC-derived cardiac organoids is known in the field, and a skilled artisan could differentiate hPSCs into cardiac organoids.
The breadth of the claims encompasses a genus of method for differentiating hPSCs, in any culture medium, with any supplements, for any duration, on any container, into cardiac organoids having a specific cell composition, yet the present specification merely provides one single species of method, but does not provide any guidance or description on how to adjust the above conditions to result in the cardiac organoids with the claimed cell composition. Thus, the skilled artisan would not know what rational approach to take to use the claimed method to obtain any predictable outcome on producing the cardiac organoids with the claimed cell compositions. Therefore, it is incumbent on the applicant to provide this nexus between structure and function, in order to be given credit for possession of the claimed genus of method.
An applicant may show that an invention is complete by disclosure of sufficiently detailed, relevant identifying characteristics which provide evidence that applicant was in possession of the claimed invention, i.e., complete or partial structure, other physical and/or chemical properties, functional characteristics when coupled with a known or disclosed correlation between function and structure, or some combination of such characteristics. Enzo Biochem, 323 F.3d at 964, 63 USPQ2d at 1613.
STATE OF THE ART & QUANTITY OF EXPERIMENTATION
The method of making the claimed invention is not well established. Although the method for producing hPSC-derived cardiac organoids was known in the state of the art, one of skill in the art would neither expect nor predict the method may produce a cardiac organoids with the claimed cell composition.
With respect to a method for producing hPSC-derived cardiac organoids, the instant specification provides a side-by-side comparison on culturing conditions (Experimental Groups 1 and 2), with the only difference being the Group 2 has more supplements from Day 4 of culture (see Figs 8-10 and p. 25-29 of specification). Only Group 2 results in cardiac organoids with the claimed cell composition (see Fig 10). Thus, the instant specification clearly demonstrates that the method for producing hPSC-derived cardiac organoids with the claimed cell composition is highly unpredictable, and is not well established.
Applicant has claimed a genus of method for producing hPSC-derived cardiac organoids with the claimed cell composition, yet the specification has only disclosed a single method, has not set forth in terms of distinguishing identifying characteristics as evidenced by other descriptions of the invention that are sufficiently detailed to show that Applicant was in possession of the claimed genus of method. Furthermore, the instant specification indicated that a method for producing hPSC-derived cardiac organoids with the claimed cell composition is not well established and would require undue experimentation, and one of skill in the art would neither expect nor predict the claimed outcome of cardiac organoids with the claimed cell composition obtained according to the claimed genus of method of differentiating hPSCs.
CONCLUSION
In summary, the Examiner concludes that there is insufficient written description of the instantly claimed genus of method for producing hPSC-derived cardiac organoids with the claimed cell composition. Specifically, there is one single species of the method, and limited description of the structure-function relationship between the claimed genus of method and their ability to produce cardiac organoids having the claimed cell composition, and the Examiner further concludes a skilled artisan would find the specification inadequately describes the claimed genus of method.
Therefore, the specification fails to provide sufficient written description to inform a skilled artisan that inventors were in possession of the entire scope of the claimed invention.
(Scope of Enablement)
Claims 1-9 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 comprising differentiating hPSCs sequentially in the conditions recited in the dependent claims 3-9 to obtain cardiac organoids with the claimed cell composition (see specification, p. 12-24 for the method for producing hPSC-derived cardiac organoids) or a method comprising differentiating hPSCs in a first and second CHIR99021 to obtain cardiac organoids, does not reasonably provide enablement for a method for differentiating hPSCs, in any culture medium, with any supplements, for any duration, on any container, into cardiac organoids having the claimed cell composition. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
The factors to be considered in determining whether undue experimentation is required are summarized In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988). The Court in Wands states: “Enablement is not precluded by the necessity for some 'experimentation.'” 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: (1) the quantity of experimentation necessary, (2) the amount or direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims. While all of these factors are considered, a sufficient amount for a prima facie case is discussed below.
The office has analyzed the specification in direct accordance to the factors outlined in In re Wands. MPEP 2164.04 states: "[W]hile the analysis and conclusion of a lack of enablement are based on factors discussed in MPEP 2164.01(a) and the evidence as whole, it is not necessary to discuss each factor in written enablement rejection." These factors will be analyzed, in turn, to demonstrate that one of ordinary skill in the art would have had to perform "undue experimentation" to make and/or use the invention and therefore, Applicant's claims are not enabled commensurate with the scope of the invention.
SCOPE OF THE INVENTION
The breadth of the claims encompasses a genus of method for differentiating hPSCs, in any culture medium, with any supplements, for any duration, on any container, into cardiac organoids having a specific cell composition.
However, the specification fails to describe the genus of methods. The present specification merely provides one single species of method for culturing and differentiating hPSCs into cardiac organoids with the claimed cell composition by sequentially culturing the hPSCs in specific media and supplements for a specific duration on a specific container.
Independent claim 1 encompasses a genus of method comprising culturing hPSCs into embryoid bodies and differentiating the embryoid bodies into cardiac organoids comprising self-organized cardiomyocytes, fibroblasts and vascular endothelial cells in a specific cell number ratio, the genus of method comprising culturing hPSCs in any culture medium, with any supplements, for any culture duration, on any culture container, while the specification only discloses a single species of method comprising culturing hPSCs sequentially in the conditions recited in dependent claims 3-9 (see p. 12-24 for the method for producing hPSC-derived cardiac organoids with the claimed cell composition).
Dependent claims 2-9 encompass type of hPSCs, steps of differentiation of embryoid bodies, steps of differentiation of structures comprising mesoderm and endoderm cells, steps of differentiation of cardiomyocytes, fibroblasts and vascular endothelial cells, and step of maturation of cardiac organoids. However, the specification only discloses a single species of method for producing hPSC-derived cardiac organoids with the claimed cell composition by culturing the hPSCs in specific media and supplements for a specific duration on a specific container. In other words, the specification only discloses a method comprising all the above steps sequentially.
ACTUAL REDUCTION TO PRACTICE
As stated supra, the specification only discloses a single species of method. The specification does not provide guidance for or a working example for a method for differentiating hPSCs, in any culture medium, with any supplements, for any duration, on any container, into cardiac organoids having a specific cell composition. The absence of working examples directed to a method for differentiating hPSCs, in any culture medium, with any supplements, for any duration, on any container, into cardiac organoids, necessitates further experimentation. Therefore, the specification does not provide sufficient guidance on how to use the claimed genus of methods for producing hPSC-derived cardiac organoids.
STATE OF THE ART & QUANTITY OF EXPERIMENTATION
The method of making the claimed invention is not well established, and one of skill in the art would neither expect nor predict any method may produce a cardiac organoids with the claimed cell composition.
With respect to a method for producing hPSC-derived cardiac organoids, the instant specification provides a side-by-side comparison on culturing conditions (Experimental Groups 1 and 2), with the only difference being the Group 2 has more supplements from Day 4 of culture (see Figs 8-10 and p. 25-29 of specification). Only Group 2 results in cardiac organoids with the claimed cell composition (see Fig 10). Thus, the instant specification clearly demonstrates that the method for producing hPSC-derived cardiac organoids with the claimed cell composition is highly unpredictable, and is not well established.
Since the prior art did not provide guidance for producing hPSC-derived cardiac organoids with the claimed cell composition by culturing hPSC in any medium with any supplement for any duration on any container, encompassed by the instant invention, it is incumbent upon the instant specification to do so. The physiological art is recognized as unpredictable (MPEP 2164.03). As set forth in In re Fisher, 166 USPQ 18 (CCPA 1970), compliance with 35 USC 112, first paragraph requires: “That scope of claims must bear a reasonable correlation to scope of enablement provided by specification to persons of ordinary skill in the art; …; in cases involving unpredictable factors, such as most chemical reactions and physiological activity, scope of enablement varies inversely with degree of unpredictability of factors involved.” Moreover, the courts have also stated that reasonable correlation must exist between scope of exclusive right to patent application and scope of enablement set forth in the patent application (27 USPQ2d 1662 Ex parte Maize!.). In view of the foregoing, due to the lack of sufficient guidance provided by the specification regarding the issues set forth above, the state of the relevant art, and the breadth of the claims, it would have required undue experimentation for one skilled in the art to use the instant broadly claimed invention.
CONCLUSION
In conclusion, since the instant specification teaches that the method for producing hPSC-derived cardiac organoids is prone to influence by multiple factors, and is highly unpredictable, and the specification does not provide ample guidance with respect to obtaining cardiac organoids with the claimed cell composition by a genus of method of differentiating hPSCs, one would be burdened with undue experimentation to use the claimed invention for producing hPSC-derived cardiac organoids with the claimed cell composition by differentiating hPSCs in any medium with any supplement for any duration on any container encompassed by the instant claims.
Therefore, given the breadth of the claims and the limited scope of the specification, an undue quantity of experimentation is required to use the invention beyond the scope of a method comprising differentiating hPSCs sequentially in the conditions recited in the dependent claims 3-9 to obtain cardiac organoids with the claimed cell composition, or a method comprising differentiating hPSCs in a first and second CHIR99021 to obtain cardiac organoids.
Examiner’s comment
Based on the limited scope of a method comprising differentiating hPSCs sequentially in the conditions recited in the dependent claims 3-9 to obtain cardiac organoids with the claimed cell composition, or a method comprising differentiating hPSCs in a first and second CHIR99021 to obtain cardiac organoids, enabled by Applicant’s specification and prior art, the prior art Israeli et al have been applied to make obvious this limited scope of method.
Claim Rejections - 35 USC § 103
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.
Claims 1-4 are rejected under 35 U.S.C. 103 as being unpatentable over Israeli et al., (bioRxiv preprint, doi: https://doi.org/10.1101/2020.06.25.171611; posted June 26, 2020, p. 1-25. Cited in IDS 01/13/2023) in view of Hoang et al., (Nat Protoc. 2018;13(4):723-737. Cited in IDS 01/13/2023).
With respect to claim 1, Israeli teaches a method for producing heart organoids (i.e., cardiac organoids) by culturing human pluripotent stem cells (hPSCs) (see e.g., abstract), thus teaches a method for producing hPSC-derived cardiac organoids.
In regard to claim 1 step (A) and claim 3, and claim 1 step (B) and claim 4, Israeli teaches “We started by assembling hPSCs into spherical aggregates by centrifugation in ultra-low attachment 96-well plates followed by a 48-hour incubation at 37 °C and 95% O2 prior to induction” (p. 4, last para, also see Fig 1a and Fig 2a schematic diagrams showing EB formation in the beginning of the culture). It is noted that these “spherical aggregates” are equivalent to the claimed embryoid bodies (EB) as shown in Fig 1a and Fig 2a, and figure legend (see p. 13). Thus, Israeli teaches steps (A) and (B) preparing and culturing embryoid bodies in claim 1.
However, Israeli is silent on the diameters of the embryoid bodies recited in claim 1 (A) and (B), nor teach the media and supplements recited in claims 3 and 4.
Hoang teaches a method for generation of cardiac organoids using human pluripotent stem cells (see e.g. abstract). Hoang teaches the method starts with patterning of hiPSCs (see p. 730, section “Patterning of hiPSCs”, and see Figure 2 (i) for the schematic diagram of the method that starts from cell seeding at -72 h and culturing to spherical aggregates (i.e., embryoid bodies) at Day 0 before CHIR99021 treatment (image #3 from left in Fig 2i). Thus, Hoang teaches a method for producing hPSC-derived cardiac organoids comprising steps of preparing and culturing embryoid bodies in claim 1 (A) and (B).
Regarding the culture media and duration, Hoang teaches the PSCs are plated in an mTeSR1 medium containing Y-27632 (see p. 730, step 32) at -72 h and culture to -48 h (see Fig 2i diagram), thus teaches claim 3 plating hPSCs in an mTeSR1 medium supplemented with a ROCK inhibitor Y-27632 and culturing the plated cells for 1 day.
Hoang teaches twenty-four hours after seeding, aspirate the mTeSR1 containing Y-27632 and add fresh mTeSR1 without Y-27632, and change the mTeSR1 (without Y-27632) daily (about 2 additional days) to begin cardiac differentiation (p. 731, step 33, and see Fig 2i from -48h to Day 0), thus teaches claim 4 culturing the embryoid bodies in mTeSR medium free of a ROCK inhibitor for 2 to 3 days.
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method for producing hPSC-derived cardiac organoids disclosed by Israeli, by substituting with the steps of preparing and culturing embryoid bodies as taught by Hoang with a reasonable expectation of success. Since Hoang reduces to practice the method comprising the steps of preparing and culturing embryoid bodies and teaches the protocol successfully produces spatial-patterned early-developing cardiac organoids using human PSCs (see e.g., abstract), one of ordinary skill in the art would have had a reason to substitute with Hoang’s steps of preparing and culturing embryoid bodies in the method of Israeli in order to successfully prepare and culture embryoid bodies from hPSCs for the cardiac differentiation. Furthermore, since Israeli’s steps and Hoang’s steps are for the same purpose (i.e., to prepare and culture embryoid bodies for cardiac differentiation), these steps are art-recognized obvious equivalents to each other. Therefore, it would have been obvious for one of ordinary skill in the art to have substituted Hoang’s steps for Israeli’s steps. See MPEP 2144.06.
Finally, regarding the diameters of the embryoid bodies recited in claim 1 (A) and (B), it is noted that these limitations are directed to the results of culturing human PSCs under the conditions recited in claims 3 and 4. Since Israeli, in view of Hoang, suggest a method comprising the same steps (A) and (B) recited in claims 3 and 4 (i.e., the same starting material human PSCs in the same culture medium with the same supplement for the same culture duration), these method steps would have inherently produced the identical results as claimed in claims 1 (A) and (B). See MPEP 2112. II.
In regard to claim 1 steps (C), (D) and (E) directed to differentiating the embryoid bodies into cardiac organoids wherein the cardiac organoids contain cardiomyocytes, fibroblasts and vascular endothelial cells at a cell number ratio of 55 to 65 : 15 to 30 : 15, Israeli teaches the embryoid bodies are cultured in various medium and supplements, specifically undergone two rounds of CHIR99021 culturing (see Fig 2a schematic diagram), to differentiate into cardiac organoids that contain multicell type cardiac lineages (see p. 6, last para “A second Wnt activation leads to the formation of multicell type cardiac lineages”). Israeli teaches “A 4 μM CHIR99021 exposure resulted in the highest cardiomyocyte content with 64±5% TNNT2+ cells at day 15” (see p. 4, lines 4-5 from the bottom), and teaches “the presence of cardiac fibroblasts positive for THY1 and VIM (Fig. 4d), which made up 12±2% of the tissues in the hHOs (Fig4 c,e),” (p. 6, line 5-6 from bottom) and “Confocal imaging revealed a robust interconnected network of endothelial cells (PECAM1+), and vessel-like formation throughout the organoid (Fig. 4a). Higher magnification uncovered a complex web of endothelial cells adjacent or embedded into myocardial tissue (Fig. 4b)” (p. 6, last para). Israeli teaches an exemplary heart organoid has cell compositions shown in Fig 4e in which cardiomyocytes are about 58%, fibroblasts are about 12% and endothelial cells are about 1.3%. Thus, Israeli teaches the cardiac organoids contain cardiomyocytes in the claimed ratio range (55% to 65%), fibroblasts that is close to the claimed ratio 15% to 30%, and vascular endothelial cells that although less than claimed ratio, functionally form blood vessels throughout the hHOs. Israeli further teaches treatment of BMP4 and Activin A increases the PECAM+ endothelial cell percentage by 400%, indicating a significant effect on organoid vascularization (see p. 7, para “BMP4 and activin A improve heart organoid chamber formation and vascularization” and Fig 6f).
Accordingly, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method for producing hPSC-derived cardiac organoids having cardiomyocytes, fibroblasts and vascular endothelial cells within or close to the claimed cell number ratio as suggested by Israeli in view of Hoang, by combining treating the cardiac organoid with BMP4 and activin A as suggested by Israeli to increase endothelial cells to arrive at the claimed ratio with a reasonable expectation of success. Since Israeli teaches treatment of BMP4 and Activin A increases the PECAM+ endothelial cell percentage, indicating a significant effect on organoid vascularization which accounts for the increase in organoid size by facilitating gas diffusion and transport of nutrients (see p. 7, para “BMP4 and activin A improve heart organoid chamber formation and vascularization” and Fig 6f), one of ordinary skill in the art would have had a reason to increase the endothelial cell percentage to arrive at the claimed ratio range in order to facilitate gas diffusion and transport of nutrients to the cardiac organoids.
With respect to claim 2, Israeli (p. 10, para “Stem cell culture”) and Hoang (p. 725, left col, para “Cell type”) teach both human ESCs and human iPSCs.
Hence, the claimed invention as a whole was prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention in the absence of evidence to the contrary.
Conclusion
No claims are allowed.
Examiner Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Jianjian Zhu whose telephone number is (571)272-0956. The examiner can normally be reached M - F 8:30AM - 4PM (EST).
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/JIANJIAN ZHU/Examiner, Art Unit 1631