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 .
Election/Restrictions
Applicant’s election without traverse of Group I (Claims 1-14; drawn to a method of culturing umbilical cord derived mesenchymal stem cells) in the reply filed on June 24, 2026, is acknowledged.
Claims 15-20 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention (Groups II and III), there being no allowable generic or linking claim.
DETAILED ACTION
The claims filed on January 8, 2024, have been acknowledged. In light of the Applicant’s elected invention, claims 15-20 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Claims 1-14 are pending and examined on the merits.
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.
Claim 6 is 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.
Claim 6 contains the trademark/trade name FACS. 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 is used to identify/describe fluorescence-assisted cell sorting and, accordingly, the identification/description is indefinite.
Applicant is recommended to remove the acronym FACS.
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.
Claims 1-2 and 5 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Musial-Wysocka et al. (Int. J. Mol. Sci. 20: 1-14. 2019).
Musial-Wysocka teaches a method of isolating and culturing mesenchymal stem cells under hypoxic conditions comprising:
umbilical cords were collected from three patients after Caesarean sections after parents’ consent. After washing with phosphate-buffered saline (PBS) solution supplemented with antibiotic-antimycotic solution, umbilical cords were cut into small (5 mm) pieces. Around 50 small slices of umbilical cord were put onto the plastic flask. Explants were cultured with growth medium for mesenchymal stem cells (DMEM Low Glucose, Biowest, USA), supplemented with platelet lysate under hypoxic condition with 5% of O2 and 5% of CO2 at 37°C. After 7 days, the explants were removed, and the cells were passaged using Accutase cell detachment solution. WJ-MSCs were then cultured under hypoxic conditions (5% oxygen) in the Quantum Cell Expansion Bioreactor.
Musial-Wysocka teaches that cytometric analysis confirmed the presence of WJ-MSCs specific markers characteristic for MSCs—namely CD73, CD90, and CD105—and a lack of expression for CD3 and CD45 antigens (Figure 1D,E). WJ-MSCs cultured under standard conditions and with low partial pressure of oxygen express the aforementioned markers at similar level. Figure 1E shows that CD73, CD90, and CD105 were expressed in at least 98% of cells (page 3, paragraph 5-page 3, paragraph 1, page 7, paragraph 4, page 8, paragraph 3, and Figure 1).
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.
Claims 1 and 3-4 are rejected under 35 U.S.C. 103 as being unpatentable over Musial-Wysocka et al. (Int. J. Mol. Sci. 20: 1-14. 2019) as applied to claim 1 above, and further in view of Chen et al. (CNS Neurosci Ther. 28:1519–1531. 2022).
Regarding claim 3, the teachings of Musial-Wysocka are as discussed above.
Musial-Wysocka does not teach adding Roxadustat to their mesenchymal stem cells.
However, Chen teaches that BMSCs were cultured in a glucose-free DMEM in an anaerobic incubator and flushed with CO2 (5%), O2 (1%) and N2 (94%) for 24 h at 37°C. FG-4592 (Roxadustat) (10 μmol/L) was given prior to the oxygen–glucose deprivation (OGD) insult. The role of FG-4592 pretreatment in BMSCs under OGD exposure was evaluated. CCK-8 assay showed that the viability of BMSCs, which were pretreated with 10 μmol/L of FG-4592 for 24 h, improved significantly (Figure 1A). Immunofluorescent staining with Ki67 following OGD exposure showed decreased expression of Ki67, while FG-4592 pretreatment increased the quantity of Ki67 positive cells (Figure 1B,C). These results indicated that FG-459 enhanced the survival ability of BMSCs under OGD exposure. They further explored the effect of FG-4592 against OGD-induced apoptosis in BMSCs. Through flow cytometry assay, they found that the apoptosis ratio of BMSCs was distinctly increased in the OGD group, whereas FG-4592 administration decreased the apoptosis rate in BMSCs after OGD (Figure 2A,B). Similarly, pretreatment with FG-4592 notably suppressed the level of Bax and restored the level of Bcl-2 (Figure 2C–E), indicating the anti-apoptosis activity of FG-4592 in OGD-treated BMSCs (page 1521, column 1, paragraph 2, page 1522, column 2, paragraph 5-page 1523, column 1, paragraph 1, and Figures 1-2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the hypoxic WJ-MSC isolation and expansion method of Musial-Wysocka by adding Roxadustat to the media during isolation and expansion under hypoxic conditions to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because Chen teaches that oxygen and glucose deprivation increases the rate of apoptosis in BMSCs whereas Roxadustat (FG-459) administration decreased the apoptosis rate in BMSCs through increased anti-apoptotic activity and enhanced the survival ability of BMSCs under oxygen and glucose deprivation exposure. As oxygen deprivation reduces the survivability of bone marrow derived mesenchymal stem cells, one of ordinary skill in the art would reasonably conclude that this would also occur with mesenchymal stem cells derived from other sources and placed in oxygen deprivation conditions, As Roxadustat increased survivability when added prior to the start of oxygen deprivation, it would have been obvious to add Roxadustat to the WJ-MSCs during explant culture and expansion culture to increase the survivability of the cells under hypoxic conditions, a known cell stressor, as this would increase the overall yield of MSCs. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Regarding claim 4, as stated supra, Chen teaches that they used 10 μmol/L of Roxadustat (FG-4592). This equates to 35.23 μg/mL.
Claims 1 and 6-7 are rejected under 35 U.S.C. 103 as being unpatentable over Musial-Wysocka et al. (Int. J. Mol. Sci. 20: 1-14. 2019) as applied to claim 1 above, and further in view of Ciria et al. (Stem Cells and Development 26: 973-985. 2017) and Ji et al. (J. Cell. Physiol. 221: 54–66. 2009).
Regarding claim 6, the teachings of Musial-Wysocka are as discussed above.
Musial-Wysocka does not teach using fluorescence-assisted cell sorting to derive a subset of UC-MSCs that is positive for HIF-1α.
However, Ciria teaches that they transduced human MSCs with a lentivirus encoding HIF-1α and GFP (pWPI-HIF-1a-GFP) for over-expression of HIF-1α. Ciria teaches that hypoxia-inducible factor 1 alpha (HIF-1a) overexpression improves several MSC functions, including cell adhesion, migration, and proliferation, thereby increasing their therapeutic potential (abstract, page 973, column 1, paragraph 1-page 977, column 1, paragraph 1, and Figure 1).
Ji teaches that they transduced cells with a lentivirus encoding HIF-1α and GFP (pBPLV-HIF-1a). The GFP positive expressing cells were sorted by flow cytometry. Following sorting most cells showed high expression of GFP (page 57, column 1, paragraph 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined the hypoxic WJ-MSC isolation and expansion method of Musial-Wysocka with the method of transducing MSCs with a lentiviral vector encoding HIF-1α and GFP and sorting for the GFP+ cells overexpressing HIF-1α of the combined teachings of Ciria and Ji to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to combine with a reasonable expectation of success because Ciria teaches that hypoxia-inducible factor 1 alpha (HIF-1a) overexpression improves several MSC functions, including cell adhesion, migration, and proliferation, thereby increasing their therapeutic potential. Ciria successfully reduces to practice that a lentivirus encoding HIF-1a and GFP can be used for overexpressing HIF-1a in MSCs with GFP expression to identify cells that have been successfully transduced. Ji successfully reduces to practice that cells transduced with a lentivirus encoding HIF-1α and GFP can be sorted based on GFP expression. Therefore, it would have been obvious that one of ordinary skill would want to transduce the cells of Musial-Wysocka with a lentivirus encoding HIF-1α and GFP as HIF-1α overexpression improves proliferation of MSCs which would increase the yield of MSCs from the expansion method of Musial-Wysocka. One of ordinary skill in the art would want to perform cell sorting based on GFP expression to ensure that they are only expanding successfully transduced cells. Therefore, it would have been obvious to combine the method of Musial-Wysocka with the combined methods of Ciria and Ji. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Regarding claim 7, as stated supra, Musial-Wysocka teaches that greater than 98% of their MSCs expressed CD73, CD90, and CD105. As the combined method of Ciria and Ji would sort for only HIF-1α+; GFP+ cells, this would lead to greater than 90% of MSCs positive for HIF-1α+ and ~98% of MSCs expressing CD73, CD90, and CD105.
Claims 1, 6, and 8-10 are rejected under 35 U.S.C. 103 as being unpatentable over Musial-Wysocka et al. (Int. J. Mol. Sci. 20: 1-14. 2019), Ciria et al. (Stem Cells and Development 26: 973-985. 2017), and Ji et al. (J. Cell. Physiol. 221: 54–66. 2009) as applied to claims 1 and 6 above, and further in view of World Intellectual Property Organization Application No. 2022190091 (Levenberg) and Phelps et al. (Stem Cell Research & Therapy 14: 1-15. 2023).
Regarding claim 8, the teachings of Musial-Wysocka, Ciria, and Ji are as discussed above.
The combined teachings of Musial-Wysocka, Ciria, and Ji do not teach isolating extracellular vesicles produced the umbilical cord MSCs.
However, Levenberg teaches a method of isolating extracellular vesicles from MSCs. Levenberg teaches that the secreted extracellular vesicles can be isolated using differential ultracentrifugation using a centrifugal force of 100,000 g for 70 min (page 45, line 13-page 62, line 6).
Phelps teaches that when MSCs are cultured under physiologically relevant oxygen conditions (2–9% O2), they have been found to secrete higher amounts of survival and angiogenic factors. Culturing MSCs under physioxic conditions increased their expression of angiogenic genes SDF1 and VEGF, and subsequently elevated VEGF-A content in the EV fraction. MSC-EVs demonstrated an ability to induce CMEC angiogenesis by promoting tube formation, with the EV fraction from physioxic cultures having the greatest effect. The physioxic EV fraction further upregulated the expression of CMEC angiogenic genes FGF2, HIF1, VEGF and TGFB1, as well as genes (OCLN and TJP1) involved in BBB maintenance. EVs from physioxic MSC cultures hold promise in the generation of a cell-free therapy to induce angiogenesis. Their positive angiogenic effect on cerebral microvascular endothelial cells demonstrates that they may have utility in treating ischemic cerebral conditions, where the induction of angiogenesis is critical to improving recovery and neurological function (abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined the hypoxic WJ-MSC isolation and expansion method of Musial-Wysocka with the EV isolation method of Lvenberg to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to combine with a reasonable expectation of success because Phelps teaches that culturing MSCs under physioxic conditions increased their expression of angiogenic genes SDF1 and VEGF, and subsequently elevated VEGF-A content in the EV fraction. MSC-EVs demonstrated an ability to induce CMEC angiogenesis by promoting tube formation, with the EV fraction from physioxic cultures having the greatest effect. EVs from physioxic MSC cultures hold promise in the generation of a cell-free therapy to induce angiogenesis. Their positive angiogenic effect on cerebral microvascular endothelial cells demonstrates that they may have utility in treating ischemic cerebral conditions, where the induction of angiogenesis is critical to improving recovery and neurological function. Therefore, extracellular vesicles from MSCs cultured under hypoxic conditions are known to have their own therapeutic applications, such as treating ischemic cerebral conditions. Therefore, it would have been obvious that one could isolate the extracellular vesicles produced by the cells of Musial-Wysocka using the known method of Levenberg to collect therapeutic products for future use in treating diseases. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Claims 1, 6, 8-9, and 11-12 are rejected under 35 U.S.C. 103 as being unpatentable over Musial-Wysocka et al. (Int. J. Mol. Sci. 20: 1-14. 2019), Ciria et al. (Stem Cells and Development 26: 973-985. 2017), Ji et al. (J. Cell. Physiol. 221: 54–66. 2009), World Intellectual Property Organization Application No. 2022190091 (Levenberg), and Phelps et al. (Stem Cell Research & Therapy 14: 1-15. 2023) as applied to claims 1, 6, and 8-9 above, and further in view of Sivanantham et al. (Life 12: 1-19. 2022).
The teachings of Musial-Wysocka, Ciria, Ji, Levenberg, and Phelps are as discussed above. Levenberg teaches that the medium comprising the EV of the present invention may be concentrated using methods known in the art to increase the concentration of its constituents and then lyophilized and reconstituted in saline for injection.
The combined teachings of Musial-Wysocka, Ciria, Ji, Levenberg, and Phelps do not teach storing the dry lyophilized powder comprising the EVs at 4°C.
However, Sivanantham teaches that 4°C is the optimal storage temperature for lyophilized EVs (page 8, paragraph 2).
As Sivanantham teaches that 4°C is the optimal storage temperature for lyophilized EVs, it would have been obvious to store the lyophilized EVs in a vial at this temperature. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Claims 1 and 13-14 are rejected under 35 U.S.C. 103 as being unpatentable over Musial-Wysocka et al. (Int. J. Mol. Sci. 20: 1-14. 2019) as applied to claim 1 above, and further in view of Yang et al. (Bio-Medical Materials and Engineering 28: S217–S228. 2017).
The teachings of Musial-Wysocka are as discussed above.
Musial-Wysocka does not teach further differentiating their cells with chondrogenic media.
However, Yang teaches that they cultured Wharton’s Jelly mesenchymal stem cells (WJ-MSCs) under 2% oxygen and examined their chondrogenic differential potential by culturing the cells in chondrogenic induction media in hypoxic conditions (2% oxygen). Chondrogenic differentiation of hWJ-MSCs produced cells expressing Collagen II (page S219, paragraph 5-page S222, paragraph 4 and Figure 4).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined the hypoxic WJ-MSC isolation and expansion method of Musial-Wysocka with the chondrogenic differentiation method of Yang to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because Yang successfully reduces to practice that WJ-MSCs can undergo chondrogenic differentiation under hypoxic conditions. Furthermore, this is a well understood protocol for differentiation of mesenchymal stem cells and it would have been well understood that the cells of Musial-Wysocka could undergo further differentiation to assess differentiation potential of the cells. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Conclusion
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/KEENAN A BATES/Examiner, Art Unit 1631
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