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
Claim Status
This action is in response to the amended claim set filed on 10/28/2024.
Claims 19-38 are pending and are considered on the merits.
Priority
This application is a DIV of 17/025,953 (filed on 09/18/2020), which claims benefit from provisional application 62/902,561 (filed on 09/19/2019). The priority claim of the instant application has been granted and the earliest benefit date is 09/19/2019 from the application 62/902,561.
Information Disclosure Statement
Applicant has not disclosed all information material to patentability. See MPEP §2001.04 and 37 C.F.R. § 1.56. Applicant is respectfully reminded the individuals covered by 37 C.F.R. § 1.56 have a duty to bring to the attention of the examiner, or other Office official involved with the examination of a particular application, information within their knowledge as to other copending United States applications which are "material to patentability" of the application in question. As set forth by the court in Armour & Co. v. Swift & Co., 466 F.2d 767, 779, 175 USPQ 70, 79 (7th Cir. 1972). See MPEP §2001.06(b).
Furthermore, Applicant is reminded that the listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Claim Objections
Claims 20-22, 25, 31 and 36 are objected to because of the following informalities:
Claims 20-22 recite “a chemically defined liquid cell culture medium” in line 2. It is recommended to change the phrase to “the chemically defined liquid cell culture medium”. Additionally, claims 20-22 recite the phrase “at least 2 days” in line 3. It is recommended to change the phrase to “at least 2 days each time”.
Claims 25 recites “wherein culturing the cell population on a surface contacting a chemically defined liquid cell culture medium” in lines 1-2. It is recommended to change to “wherein the culturing the cell population on the surface contacting the chemically defined liquid cell culture medium”.
Claim 31 recites the term “37C”. It is recommended to change to “37 °C”.
Claim 36 recites “wherein culturing the cell population” in line 1. It is recommended to change to “wherein the culturing the cell population”. Additionally, claim 36 recites “at least one” in lines 2-3. It is recommended to change to “at least once”.
Appropriate correction is required.
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.
Claims 25 and 36-37 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 25 and 36 recite “wherein culturing the cell population … comprises…”. There is insufficient antecedent basis for this limitation in the claims because the base claim 19 recites culturing the cell population “a first time” and “a second time”. Thus, it is not clear the limitation in claims 25 and 36 is referring to the culturing a first time or the culturing a second time. It is examined as the culturing either time. Claim 37 is rejected as being dependent from claim 36 but not resolving the ambiguity.
Claim Rejections - 35 USC § 102
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.
Claims 19-38 are rejected under 35 U.S.C. 102 (a)(1) as being anticipated by Kuo et al., (bioRxiv preprint doi: https://doi.org/10.1101/685503; posted September 6, 2019, p. 1-44).
Kuo et al appears to be Applicant’s own work. Applicant may rely on the exception under 35 U.S.C. 102(b)(1)(A) to overcome this rejection under 35 U.S.C. 102(a)(1) by a showing under 37 CFR 1.130(a) that the subject matter disclosed in the reference was obtained directly or indirectly from the inventor or a joint inventor of this application, and is therefore not prior art under 35 U.S.C. 102(a)(1) . Alternatively, applicant may rely on the exception under 35 U.S.C. 102(b)(1)(B) by providing evidence of a prior public disclosure via an affidavit or declaration under 37 CFR 1.130(b).
In regard to claims 19-38, Kuo discloses culturing 34 hiPSC lines and maintenance of pluripotency long-term (over 100 passages) in a novel hiPSC culture medium (B8), which is specifically optimized for fast growth and robustness at low seeding densities (p. 2, “Summary”), thus teaches a method of expanding a pluripotent cell population, comprising culturing the cell population on a surface (“2 ml medium per 9.6 cm2 of surface area”, e.g., p. 16) contacting a chemically defined liquid cell culture medium (i.e., the B8 medium) a first time and a second time for at least 2 days (“passaging cells every 3.5 days”, e.g., p. 11, last para) (claim 24), and subjecting the cell population to a monolayer differentiation protocol (“We used our existing cardiac, endothelial, and epithelial differentiation protocols”, e.g., p. 11, last line - p. 12, para 1 and Fig 5F-5H) (claim 26). Kuo teaches the components of the B8 medium (e.g., p. 40, Fig S1B far right column) that comprises a cell culture base medium DMEM/F12 (claim 33), thermostable FGF2-G3 40 ng/mL (also in p. 19), TGFb3 0.1 ng/mL (also in p. 19) (claims 19 and 29-31), insulin, ascorbic acid 2-phosphate, transferrin, a source of selenium, NRG1 0.1 ng/mL (also in p. 20), and sodium bicarbonate (e.g., p. 7) (claims 32, 34, 35, 37 and 38). Kuo teaches the pluripotent cells are maintained over 100 passages and maintain the expression of markers of undifferentiated status such as SSEA4 and TRA-1-60 (e.g., p. 10, last para), thus teaches claims 20-22 and 27-28. Kuo teaches the population doubling numbers being more than 10 after 3 passages (Fig 5D), thus teaches claim 23. Kuo teaches low seeding density of 10,000 cells per well of a 12-well plate (e.g., Fig 1B), thus teaches claim 25. Kuo teaches the medium is changed after the first 24 h (e.g., p. 1, last para), thus teaches claim 36.
Accordingly, Kuo anticipates instant claims.
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 19-29 and 36 are rejected under 35 U.S.C. 103 as being unpatentable over Wu et al., (US 9,234,176 B2).
With respect to claim 19, Wu teaches a method of growing and passaging hESC/hPSC as a monolayer by culturing hPSCs in a T25 flask (a flask with a surface area of 25 cm2) with E8 medium and passaging the cells on a 4 day cycle (e.g., col. 31, last full para), thus teaches a method of expanding a pluripotent cell population, the method comprising culturing the cell population on a surface (i.e., the T25 flask) contacting a chemically defined liquid cell culture medium (i.e., E8 medium) a first time for at least 2 days (e.g., 4 days), passaging the cell population, culturing the cell population on the surface contacting the chemically defined liquid cell culture medium a second time for at least 2 days. Wu teaches the hPSCs are subjected to directed cardiac differentiation procedure in a T25 flask (e.g., col 31, past para), thus teaches subjecting the cell population to a monolayer differentiation protocol. Wu teaches the E8 medium comprises a basal medium DMEM/F12 supplemented with 7 components, including about 2 ng/ml recombinant human TGFB1 and about 100 ng/ml recombinant human FGF2 (see col. 19, last para and col. 20, Table 1), thus teaches the E8 medium being a chemically defined liquid cell culture medium comprising a nonzero amount of TGF beta about 2 ng/mL and a nonzero amount of FGF2 about 100 ng/mL.
Although Wu does not specifically teach the amount of TGF beta being less than 2 ng/mL and the amount of FGF2 being less than 100 ng/mL, notably, in the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990). It is routine procedure to optimize component amounts to arrive at an optimal product that is superior for its intended use, since it has been held where the general conditions of a claim are disclosed in the prior art, discovering the optimum or workable ranges involves only routine skill in the art. See M.P.E.P. §2144.05. In the instant case, since Wu teaches the amount of TGF beta being about 2 ng/mL, encompassing a range of TGF beta being from less than 2 ng/mL to more than 2 ng/mL, and similarly a range of FGF2 being from less than 100 ng/mL to more than 100 ng/mL, the claimed ranges of TGF beta being less than 2 ng/mL and FGF2 being less than 100 ng/mL overlap the ranges disclosed by the prior art, thus are prima facie obvious over Wu.
With respect to claim 20 directed to repeating the culturing for at least 5 times, claim 21 directed to repeating the culturing for at least 10 times, and claim 22 directed to repeating the culturing for at least 50 times, Wu teaches “This method of growing hESC/hPSC as a monolayer has been demonstrated to be successful for the growth of hESC for over 40 passages without karyotypic abnormality. We commonly grow cells for up to 12 passages in this manner” (col. 31, lines 18-21) and teaches “The eight Sendai virus-derived lines were maintained using the E8/EDTA culture method for more than 1 year and overall all lines repeatedly differentiated efficiently from passage 25 to >80” (col. 40, lines 52-55). Wu teaches passaging the cells on a 4 day cycle (e.g., col. 31, last full para). Thus, Wu teaches repeating the culturing for at least 5 times, at least 10 times, and at least 50 times (e.g., >80) for at least 2 days each time/passage.
With respect to claim 23 directed to the cell population exhibiting at least 3 population doublings per cell passage, it must be noted that this wherein clause does not recite an active step in the claimed method, but only the results of the culturing the cell population as taught by Wu. MPEP 2111.04 I states a whereby clause (or a wherein clause) “in a method claim is not given weight when it simply expresses the intended result of a process step positively recited.” Therefore, this wherein clause does not provide any patentable weight in determining patentability of the claimed method. Nevertheless, Wu teaches the cumulative population doubling in Fig 8d in which the cumulative population doubling number of each passage (4 days in culture) is about 1/3 of 10, i.e., about 3.3. Wu also teaches the cell number increases 10 times in 4 days of culture (e.g., col 31, last full para). Thus, Wu teaches the cell population exhibits at least 3 population doublings per cell passage.
With respect to claim 24 directed to the cell population being passaged no more than twice per week, as stated supra, Wu teaches passaging the cells on a 4 day cycle (e.g., col. 31, last full para), thus teaches the cell population is passaged no more than twice per week.
With respect to claim 25, Wu teaches optimizing pluripotent cell seeding density from 0.6 – 1.6 x 104/cm2 (see Fig 10f), and teaches 3-4 x 106 cells in a T25 flask (25 cm2) is approximately 85% confluent (col. 31, last full para and col. 32, lines 51-52, i.e., 12-16 x 104/cm2 being 85% confluent). Thus, the seeding density of 0.6 – 1.6 x 104/cm2 is equivalent to 3.2% - 11% confluency (0.6/16 x 85% - 1.6/12 x 85%), overlapping the claimed range of less than 5% confluency, thus makes obvious the claimed range. See M.P.E.P. §2144.05, and In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990).
In regard to observing monolayer growth of at least one doubling per day, it must be noted that this wherein clause does not recite an active step in the claimed method, but only the results of the culturing the cell population as taught by Wu, thus does not provide any patentable weight in determining patentability of the claimed method. See MPEP 2111.04 I. Nevertheless, as stated supra, Wu teaches the cumulative population doubling in Fig 8d in which the cumulative population doubling number of each passage (4 days in culture) is about 1/3 of 10, i.e., about 3.3, and the cell number increases 10 times in 4 days of culture (e.g., col 31, last full para). Thus, Wu suggests monolayer growth of about one doubling per day.
With respect to claim 26 directed to observing at least 70% of the cell population to differentiate, this is examined as at least 70% of the cell population are undergoing differentiation. It is noted that this limitation is not an active step but only the result of the subjecting to a differentiation protocol, thus does not provide any patentable weight in determining patentability of the claimed method. See MPEP 2111.04 I. Nevertheless, Wu teaches the subjecting to a differentiation protocol results in up to 95% TNNT2+ cardiomyocytes (e.g., col. 34, para 1), thus teaches at least 70% of the cell population are undergoing differentiation.
With respect to claim 27 directed to the cell population exhibiting at least 80% maintenance of a pluripotency marker prior to inducing the cell population to differentiate, and claim 28 directed to flow cytometry detection of SSEA4 and flow cytometry detection of TRA1-60, Wu teaches checking the pluripotency of the hiPSCs using flow cytometry assay of pluripotency markers, and teaches 99.5% cells are double positive for SSEA4 and POU5F1 and 99.9% cells are double positive for TRA-1-60 and NANOG (see e.g., Fig 7d), thus teaches the cell population exhibits at least 80% maintenance of a pluripotency marker prior to differentiation and the markers include flow cytometry detection of SSEA4 and TRA1-60.
With respect to claim 29 directed to TGF beta 1, as stated supra, Wu teaches the E8 medium comprises TGFB1 (see col. 19, last para and col. 20, Table 1).
With respect to claim 36 directed to changing the medium at least once during the at least 2 days, Wu teaches “Every day aspirate media and replace with fresh E8 media” (e.g., col. 31, last full para), thus teaches changing the medium at least once during the at least 2 days.
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.
Claims 30-31 are rejected under 35 U.S.C. 103 as being unpatentable over Wu et al., (US 9,234,176 B2) in view of Dvorak et al., (Biotechnology and Bioengineering. 2018;115:850-862).
Claim 30 is directed to the FGF2 comprising FGF2-G3 and claim 31 is directed to the FGF2 being thermostable at 37 °C for at least 2 days in culturing condition.
However, Wu is silent on the FGF2 being FGF2-G3 or thermostable.
Dvorak teaches the wild-type FGF2 has low stability and thus creates a unique modified FGF2 with nine mutations displaying unprecedented stability and uncompromised biological function (e.g., abstract). Dvorak teaches the modified FGF2 with nine mutations being FGF2-G3 (see e.g., Figure 2, related to claim 30) and the G3 mutant retains most of their activity for the full length of the study period (20 days) at the physiological temperature (e.g., p. 859, right col, para 1 and Figure 5, related to claim 31). Dvorak teaches the stabilized FGF2-G3 maintains pluripotency marker expression of hESCs (e.g., see Figure 7).
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 substituted Dvorak’s thermostable FGF2-G3 for the FGF2 of Wu with a reasonable expectation of success. Since Dvorak teaches the wild-type FGF2 has low stability and FGF2-G3 retains activity at 37 °C for 20 days, displaying unprecedented stability and uncompromised biological function (e.g., abstract and Figs 5 and 7), one of ordinary skill in the art would have had a reason to substitute Dvorak’s thermostable FGF2-G3 for the FGF2 of Wu in order to take advantage of the stability and uncompromised biological function of FGF2-G3 to maintain pluripotent cells without the need of daily media change as used by Wu.
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.
Claims 32-35 and 38 are rejected under 35 U.S.C. 103 as being unpatentable over Wu et al., (US 9,234,176 B2) in view of Wang et al., (Blood. 2007;110(12):4111-9).
With respect to claims 32-34, Wu teaches the E8 medium comprises a basal medium DMEM/F12 (related to claim 33) with L-Glutamine and HEPES, supplemented with 7 components including recombinant human Insulin, L-ascorbic acid 2-phosphate, recombinant human transferrin, sodium selenite and sodium bicarbonate (see col. 19, last para and col. 20, Table 1, related to claim 32). One of ordinary skill in the art would have immediately expected that the basal medium DMEM/F12 would have comprised salts, amino acids and vitamins. Since Wu teaches the E8 medium is a defined medium and does not teach Activin A in the list of components, one of ordinary skill in the art would have immediately expected that the chemically defined liquid cell culture medium (i.e., E8 medium) of Wu does not comprise Activin A at a concentration sufficient to influence cell growth, differentiation or health in claim 34.
However, Wu is silent on the medium comprising NRG in claim 32 or NRG1 in claim 35.
Wang teaches a cell culture medium for the growth and maintenance of pluripotent stem cells (e.g., abstract). Wang teaches “HRG (aka NRG1) played the major roles in self-renewal and proliferation in the context of this defined medium (Figure 3B,C), and cultures could not be maintained without HRG (NRG1) beyond approximately 3 weeks” (p. 4115, right col, line 5, also see Fig 3B and 3C to compare HAIF vs. AIF, and HAF vs. AF for the HRG/NRG1 effect wherein H stands for HRG/NRG1), related to claims 32 and 35. Thus, Wang teaches NRG1 in culture medium enhances long-term self-renewal and proliferation of pluripotent stem cells.
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 combined an NRG1 as suggested by Wang in the chemically defined medium of Wu with a reasonable expectation of success. Since Wu teaches the cell culture medium maintains pluripotency of cultured pluripotent cells (col 42, line 45), and since Wang teaches addition of NRG1 in culture medium enhances long-term self-renewal and proliferation of pluripotent stem cells (p. 4115, right col, line 5, also see Fig 3B and 3C), one of ordinary skill in the art would have had a reason to combine NRG1 taught by Wang to the cell culture medium of Wu to improve long-term self-renewal and proliferation of the cultured pluripotent stem cells. Furthermore, since both Wu and Wang teach a chemically defined medium (see Wu, col. 19, last para and Wang, p. 4112, left col, para “Culture of hESCs in defined conditions”), one of ordinary skill in the art would have immediately expected that the chemically defined medium as suggested by Wu in view of Wang comprises no other cell growth factors at a concentration sufficient to influence cell growth, differentiation or health in claim 38.
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.
Claim 37 is rejected under 35 U.S.C. 103 as being unpatentable over Wu et al., (US 9,234,176 B2) in view of Frank et al., (PLoS ONE. 2012; 7(7): e41958, p. 1-13).
Claim 37 is directed to the medium comprising less than 1 ng/mL TGF beta and less than 1 ng/mL NRG1. It is noted that the scope of the limitation “less than 1 ng/mL NRG1” encompasses 0 ng/mL NRG1, i.e., the medium does not comprise NRG1.
As a first matter, as stated supra, Wu teaches the E8 medium is a defined medium and does not teach NRG1 in the list of components (see col. 19, last para and col. 20, Table 1). Thus, one of ordinary skill in the art would have immediately expected that the chemically defined liquid cell culture medium (i.e., E8 medium) of Wu does not comprise NRG1, and thus is encompassed by instant claim 37.
However, Wu teaches the E8 medium comprises about 2 ng/mL TGFB1 (see col. 19, last para), but is silent on the medium comprising less than 1 ng/mL TGF beta.
Frank systematically evaluates over 20 potential media ingredients for culturing hPSCs and ultimately combines components that reproducibly gave beneficial effects to yield a simple and cost-effective formulation termed FTDA including FGF2 and TGF beta 1 (e.g., abstract). Frank titrates the concentration of TGFB1 and reveals that near-saturating levels are already obtained at a rather moderate dosage of 0.5 ng/ml (Figure 2D) (p. 2, end of left col. to beginning of right col.), related to claim 37.
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 substituted the dosage of less than 1 ng/mL TGF beta as suggested by Frank for the dosage of about 2 ng/mL TGFB1 of Wu with a reasonable expectation of success. Since Frank titrates the concentration of TGFB1 in a defined medium for culturing pluripotent cells and reveals that near-saturating levels are already obtained at a dosage of 0.5 ng/ml (p. 2, end of left col. to beginning of right col. and Figure 2D), one of ordinary skill in the art would have had a reason to substitute this lower dosage of less than 1 ng/mL TGF beta as suggested by Frank for the high dosage of Wu in order to take advantage of the lower concentration to reduce cost of agent.
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.
Double Patenting Rejections
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/process/file/efs/guidance/eTD-info-I.jsp.
Claims 19-38 are rejected on the ground of nonstatutory double patenting as being unpatentable over patent claims 1, 9-11 and 14 of US Patent No. 9,234,176 (‘176) in view of Wu et al., (US 9,234,176 B2), Dvorak et al., (Biotechnology and Bioengineering. 2018;115:850-862), Wang et al., (Blood. 2007;110(12):4111-9) and Frank et al., (PLoS ONE. 2012; 7(7): e41958, p. 1-13). Although the claims at issue are not identical, they are not patentably distinct from each other.
Patent claims 1, 9-11 and 14 of ‘176 recite a method of producing a cardiomyocyte population from a human pluripotent stem cell population (reference claim 1) and prior to contact with the Wnt signaling agonist, the mammalian pluripotent stem cell population is cultured in maintenance media (reference claim 9). Reference claim 10 recites a ROCK inhibitor being supplemented for a period of about 24 hours each time the human pluripotent stem cell population is passaged and reference claim 11 recites the human pluripotent stem cell population is cultured as a monolayer on a matrix. Reference claim 14 recites at least 80% of the cells are determined to be cardiomyocytes (related to instant claim 26). Thus, patent claims recite a method of expanding a pluripotent cell population comprising culturing the cell population on a surface contacting a maintenance media and passaging the cell population (by culturing and passaging recited in reference claims 9 and 10, on a matrix in reference claim 11), and subjecting the cell population to a monolayer differentiation protocol (by the method recited in reference claim 1), related to instant claim 19.
However, patent claims are silent on the duration of culturing between passaging or the component of the maintenance media recited in instant claims 19-25 and 27-38.
Wu teaches a method of growing and passaging hESC/hPSC as a monolayer with a chemically defined E8 medium and passaging the cells on a 4 day cycle (e.g., col. 31, last full para). Wu teaches the E8 medium comprises a basal medium DMEM/F12 (related to instant claim 33) with L-Glutamine and HEPES, supplemented with 7 components including recombinant human Insulin, L-ascorbic acid 2-phosphate, recombinant human transferrin, sodium selenite and sodium bicarbonate, and about 2 ng/ml recombinant human TGFB1 and about 100 ng/ml recombinant human FGF2 (see col. 19, last para and col. 20, Table 1, related to instant claims 19, 29 and 32). Since the claimed ranges of TGF beta being less than 2 ng/mL and FGF2 being less than 100 ng/mL overlap the ranges disclosed by Wu, the claimed ranges in instant claim 19 are prima facie obvious over Wu. One of ordinary skill in the art would have immediately expected that the basal medium DMEM/F12 would have comprised salts, amino acids and vitamins. Since Wu teaches the E8 medium is a defined medium and does not teach Activin A in the list of components, one of ordinary skill in the art would have immediately expected that the chemically defined liquid cell culture medium (i.e., E8 medium) of Wu does not comprise Activin A at a concentration sufficient to influence cell growth, differentiation or health in instant claim 34. Wu teaches “The eight Sendai virus-derived lines were maintained using the E8/EDTA culture method for more than 1 year and overall all lines repeatedly differentiated efficiently from passage 25 to >80” (col. 40, lines 52-55), related to instant claims 20-22. Wu teaches the cumulative population doubling in Fig 8d in which the cumulative population doubling number of each passage, related to instant claims 23 and 25. Wu teaches passaging the cells on a 4 day cycle (e.g., col. 31, last full para), related to instant claim 24. Wu teaches optimizing pluripotent cell seeding density from 0.6 – 1.6 x 104/cm2 (see Fig 10f), related to instant claim 25. Wu teaches 99.5% cells are double positive for SSEA4 and POU5F1 and 99.9% cells are double positive for TRA-1-60 and NANOG by flow cytometry assay (see e.g., Fig 7d), related to instant claims 27 and 28. Wu teaches changing media every day (col. 31, last full para), related to instant claim 36.
Dvorak teaches the wild-type FGF2 has low stability and thus creates a unique modified FGF2 with nine mutations (e.g., abstract), being FGF2-G3 (see e.g., Figure 2, related to instant claim 30) and the G3 mutant retains most of their activity for the full length of the study period (20 days) at the physiological temperature (e.g., p. 859, right col, para 1 and Figure 5, related to instant claim 31).
Wang teaches a cell culture medium for the growth and maintenance of pluripotent stem cells (e.g., abstract). Wang teaches “HRG (aka NRG1) played the major roles in self-renewal and proliferation in the context of this defined medium (Figure 3B,C), and cultures could not be maintained without HRG (NRG1) beyond approximately 3 weeks” (p. 4115, right col, line 5, also see Fig 3B and 3C to compare HAIF vs. AIF, and HAF vs. AF for the HRG/NRG1 effect wherein H stands for HRG/NRG1), related to instant claims 32, 35 and 37. It would have been obvious for one of ordinary skill in the art to titer the concentration of NRG1 to arrive at the claimed range in instant claim 37 with a reasonable expectation of success. One of ordinary skill in the art would have had a reason to do so since It is routine procedure to optimize component amounts to arrive at an optimal product that is superior for its intended use. See M.P.E.P. §2144.05.
Frank systematically evaluates over 20 potential media ingredients for culturing hPSCs and ultimately combines components that reproducibly gave beneficial effects to yield a simple and cost-effective formulation termed FTDA including FGF2 and TGF beta 1 (e.g., abstract). Frank titrates the concentration of TGFB1 and reveals that near-saturating levels are already obtained at a rather moderate dosage of 0.5 ng/ml (Figure 2D) (p. 2, end of left col. to beginning of right col.), related to instant claim 37.
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 claimed method of culturing and passaging pluripotent cells in the cited patent claims, by choosing the chemically defined medium and the passaging protocol suggested by Wu, by substituting a thermostable FGF2-G3 as suggested by Dvorak, by combining an NRG1 in the medium as suggested by Wang, and by substituting a lower dosage of TGFB1 as suggested by Frank with a reasonable expectation of success. Since Wu has reduced to practice a method of culturing and passaging pluripotent cells in a defined medium with a verified protocol, since Dvorak teaches the mutant FGF2-G3 is more stable than the wildtype FGF2, since Wang teaches addition of NRG1 promotes self-renewal and proliferation of pluripotent stem cells, and since Frank titrates the concentration of TGFB1 and reveals near-saturating levels obtained at a lower dosage of less than 1 ng/ml, one of ordinary skill in the art would have had a reason to modify the claimed method as suggested by Wu, Dvorak, Wang, and Frank in order to maintain the pluripotent stem cells for induction of cardiomyocytes. Furthermore, since both Wu and Wang teach a chemically defined medium (see Wu, col. 19, last para and Wang, p. 4112, left col,), one of ordinary skill in the art would have immediately expected that the chemically defined medium comprises no other cell growth factors at a concentration sufficient to influence cell growth, differentiation or health in instant claim 38.
Since the instant application claims are obvious over cited patent claims, in view of Wu, Dvorak, Wang, and Frank, said claims are not patentably distinct.
Provisional Double Patenting Rejections
Claims 19-38 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over copending claims 1, 20-22 and 24-25 of copending Application No. 19/109,550 (‘550) in view of Wu et al., (US 9,234,176 B2). Although the claims at issue are not identical, they are not patentably distinct from each other.
Copending claim 22 of ‘550 recites a method for culturing iPSCs comprising seeding the iPSCs in the media of reference claim 1 (equivalent to the basal medium in instant claims 32 and 33), and culturing the iPSCs under suitable conditions wherein the media is not changed during culturing of the iPSCs (interpreted as using the same type of maintenance medium during culturing). Copending claim 24 recites the iPSCs express makers such as SSEA (related to instant claims 27-28). Copending claim 25 recite the iPSCs are further differentiated into cardiomyocytes, endothelial, vascular smooth muscle, neural and hepatocyte-like cells (related to instant claims 19 and 26). Copending claims 21-22 recite the media comprises supplements of insulin, L-ascorbic acid, transferrin, selenite, 30-50 ng/mL FGF2-G3, 0.05-0.5 ng/mL TGFb3, and 0.05-0.5 ng/mL NRG1, and copending claim 1 recites the medium comprising a salt, a vitamin and an amino acid, including sodium bicarbonate. Thus, copending claims 1 and 21-22 teach instant claims 19, 29-35 and 37-38.
However, the copending claims are silent on the culture duration between passaging and passage numbers recited in instant claims 19-25 and 36.
Wu teaches a method of growing and passaging hESC/hPSC as a monolayer with a chemically defined medium and passaging the cells on a 4 day cycle (e.g., col. 31, last full para), related to instant claims 19 and 24. Wu teaches the iPSCs are maintained for more than 1 year and overall all lines repeatedly differentiated efficiently from passage 25 to >80 (col. 40, lines 52-55), related to instant claims 20-22. Wu teaches the cumulative population doubling in Fig 8d in which the cumulative population doubling number of each passage, related to instant claims 23 and 25. Wu teaches optimizing pluripotent cell seeding density from 0.6 – 1.6 x 104/cm2 (see Fig 10f), related to instant claim 25. Wu teaches changing media every day (col. 31, last full para), related to instant claim 36.
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 claimed method of culturing and passaging pluripotent cells in the cited copending claims, by choosing the passaging protocol suggested by Wu with a reasonable expectation of success. Since Wu has reduced to practice a method of culturing and passaging pluripotent cells in a defined medium with a verified protocol, one of ordinary skill in the art would have had a reason to choose the protocol of Wu in order to successfully culture and passage the pluripotent stem cells.
Since the instant application claims are obvious over cited application claims, in view of Wu, said claims are not patentably distinct.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims in the copending application have not in fact been patented.
Conclusion
No claims are allowed.
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/JIANJIAN ZHU/Examiner, Art Unit 1631
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