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 .
Priority
This application 17/914,323 filed on 09/24/2022 is a national phase application under 35 U.S.C. § 371 that claims priority to International Application No. PCT/JP2021/011891 field on 03/23/2021, and claims priority of foreign application JP 2020-052748 filed on 03/24/2020.
A certified copy of foreign application JP 2020-052748 filed on 03/24/2020 has been submitted of the record by Applicants on 09/24/2022. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
No English translation of foreign application JP 2020-052748 filed on 03/24/2020 has been provided. The priority date of claim set filed on 04/25/2023 is determined to be 03/23/2021, the filing date of PCT/JP2021/011891.
Should applicant desire to obtain the benefit of foreign priority under 35 U.S.C. 119(a)-(d) prior to declaration of an interference, a certified English translation of the foreign application must be submitted in reply to this action. 37 CFR 41.154(b) and 41.202(e).
Failure to provide a certified translation may result in no benefit being accorded for the non-English application.
Restriction/Election
Applicant’s election of Group III, claims 15-19, in the reply filed on 10/30/2025 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claims 1, 3-4, 7-9, 11, 13-27 are pending.
Claims 1, 3-4, 7-9, 11, 13-14, and 20-27 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected inventions, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 10/30/2025.
Claims 15-19 are currently under examination.
Claim Objections
Claim 15 is objected to because of the following informalities: the limitation “except for a for a primordial germ cell” recited in line 3 of claim 15 should read “except for a primordial germ cell”. 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 15-19 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.
(i) Claim 15 filed on 04/25/2023 reads as follows: An embryogenesis arrest inhibitory method that reduces damage caused by manipulation of an in-vitro-culture including any
or any combination of a germ cell, except for a primordial germ cell, a fertilized egg, and an embryo, and suppresses embryonic arrest, comprising the step of: treating with a temporary treatment medium including an intracellular skeleton regulator and/or an apoptosis inhibitor for a specific period of time before and/or after a damaging manipulation; wherein the intracellular skeleton regulator and/or the apoptosis inhibitor is a Rho kinase inhibitor; wherein the manipulation excepts freezing and thawing and is a treatment involving damage to the in-vitro-culture; and wherein the damage is accompanied by damage or alteration to various structures necessary for survival and normal differentiation of a cell and by double strand break of DNA in the nucleus.
The limitation “including any or any combination of a germ cell, except for a primordial germ cell, a fertilized egg, and an embryo” is unclear regarding whether the limitation “a fertilized egg, and an embryo” is intended to be part of limitation “except for a primordial germ cell” or the limitation “a fertilized egg, and an embryo” is intended to be part of limitation “including any or any combination of a germ cell”.
The metes and bounds of limitation “wherein the damage is accompanied by damage or alteration to various structures necessary for survival and normal differentiation of a cell” cannot be determined in the context of limitation “any or any combination of a germ cell, except for a primordial germ cell, a fertilized egg, and an embryo”.
Claims 16-19 depend from claim 15.
(ii) Claim 17 filed on 04/25/2023 reads as follows: The embryogenesis arrest inhibitory method according to claim 15, wherein the specific period is within one hour when by using an animal species, a strain, and/or a frozen egg that is sensitive to the manipulation.
Claim 15 recites the limitation “treating with a temporary treatment medium including an intracellular skeleton regulator and/or an apoptosis inhibitor for a specific period of time before and/or after a damaging manipulation”. Claim 17 depends from claim 15 and recites “wherein the specific period is within one hour when by using an animal species, a strain, and/or a frozen egg that is sensitive to the manipulation”.
The limitation “a frozen egg that is sensitive to the manipulation” recited in claim 17 renders the metes and bounds of limitation “excepts freezing and thawing” recited in claim 15 indefinite.
Furthermore, claim 15 recites the limitation “treating with a temporary treatment medium including an intracellular skeleton regulator and/or an apoptosis inhibitor for a specific period of time before and/or after a damaging manipulation”. Claim 17 depends from claim 15 and recites “wherein the specific period is within one hour when by using an animal species, a strain, and/or a frozen egg that is sensitive to the manipulation”. It is unclear whether the limitation “within one hour” recited in claim 17 is intended to be “within one hour before and/or after a damaging manipulation” or the limitation “within one hour” recited in claim 17 is intended to be the length of time for “treating with a temporary treatment medium including an intracellular skeleton regulator and/or an apoptosis inhibitor”.
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 15-18 are rejected under 35 U.S.C. 102 (a)(1) as being anticipated by Emre et al. (2010), (Emre et al., The ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-activated cell sorting with multiple cell surface markers, PLoS One 2010 Aug 13;5(8):e12148. doi: 10.1371/journal.pone.0012148. cited in PTO-892 on 05/30/2025).
Claim interpretations: (i) The limitation “except for a primordial germ cell” is interpreted as directed to limiting “a germ cell”. In other words, the limitation “including any or any combination of a germ cell, except for a primordial germ cell, a fertilized egg, and an embryo” is interpreted as “a fertilized egg, and an embryo” being part of limitation “including any or any combination of a germ cell”. (ii) The limitation “within one hour” recited in claim 17 which depends from claim 15 is interpreted as “within one hour before and/or after a damaging manipulation”
Regarding claims 15-18, Emre et al. (2010) teaches that “Due to the inherent sensitivity of human embryonic stem cells (hESCs) [which reads on originated from Primates recited in claim 16, and derived from a mammal recited in claim 18] to manipulations, the recovery and survival of hESCs after fluorescence-activated cell sorting (FACS) can be low. Additionally, a well characterized and robust methodology for performing FACS on hESCs using multiple-cell surface markers has not been described. The p160-Rho-associated coiled kinase (ROCK) inhibitor, Y-27632, previously has been identified as enhancing survival of hESCs upon single-cell dissociation, as well as enhancing recovery from cryopreservation. Here we examined the application of Y-27632 to hESCs after FACS to improve survival in both feeder-dependent and feeder-independent growth conditions.” (See Background).
Emre et al. (2010) teaches that “HESCs were sorted using markers for SSEA-3, TRA-1-81, and SSEA-1. Cells were plated after sorting [which reads on within one hour recited in claim 17] for 24 hours in either the presence or the absence of Y-27632. In both feeder-dependent and feeder-independent conditions, cell survival was greater when Y-27632 was applied to the hESCs after sort. Specifically, treatment of cells with Y-27632 improved post-sort recovery up to four-fold. To determine the long-term effects of sorting with and without the application of Y-27632, hESCs were further analyzed. Specifically, hESCs sorted with and without the addition of Y-27632 retained normal morphology, expressed hESC-specific markers as measured by immunocytochemistry and flow cytometry, and maintained a stable karyotype. In addition, the hESCs could differentiate into three germ layers in vitro and in vivo in both feeder-dependent and feeder-independent growth conditions.” (See Methodology/principal findings).
Emre et al. (2010) teaches that “The application of Y-27632 to hESCs after cell sorting improves cell recovery with no observed effect on pluripotency, and enables the consistent recovery of hESCs by FACS using multiple surface markers.” (See Conclusions/significance).
PNG
media_image1.png
406
1128
media_image1.png
Greyscale
Figure 5. In vitro differentiative capacity of hESCs upon extended passaging after sorting with Y-27632. Bright field images of A) day 5 embryoid bodies from hESCs on feeders at passage 13 post-sort and E) day 6 embryoid bodies from hESCs feeder-free at passage 12 post-sort. Immunofluorescence of differentiated hESCs shows labeling for mesoderm (B, F) (GATA4 and cTnI and Desmin), ectoderm (C, G) (Nestin and Sox1), and endoderm (D, H) (FoxA2, Sox17). For feeder conditions (B–D), hESCs are at passage 14 post-sort (B); passage 13 post-sort (C); and passage 16 post-sort (D). For feeder-free conditions (F–H), hESCs are at passage 12 post-sort (F); passage 12 post-sort (G); and passage 13 post-sort (H). Scale bars are 100 mm.
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.
Claims 15, 17 and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Emre et al. (2010), (Emre et al., The ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-activated cell sorting with multiple cell surface markers, PLoS One 2010 Aug 13;5(8): e12148. doi: 10.1371/journal.pone.0012148. Cited in PTO-892 on 05/30/2025) in view of Mellott et al. (2014) (Mellott et al., Improving viability and transfection efficiency with human umbilical cord wharton's jelly cells through use of a ROCK inhibitor, Cell Reprogram., 2014 Apr;16(2):91-7. doi: 10.1089/cell.2013.0069. Epub 2014 Feb 19. Cited as NPL # 5 in the IDS filed by Applicants on 10/17/2022).
Claim interpretations: (i) The limitation “except for a primordial germ cell is interpreted as directed to limiting “a germ cell”. In other words, the limitation “including any or any combination of a germ cell, except for a primordial germ cell, a fertilized egg, and an embryo” is interpreted as “a fertilized egg, and an embryo” being part of limitation “including any or any combination of a germ cell”. (ii) The limitation “within one hour” recited in claim 17 is interpreted as the length of time for “treating with a temporary treatment medium including an intracellular skeleton regulator and/or an apoptosis inhibitor”.
Regarding claims 15 and 19, Emre et al. (2010) teaches that “Due to the inherent sensitivity of human embryonic stem cells (hESCs) to manipulations, the recovery and survival of hESCs after fluorescence-activated cell sorting (FACS) can be low. Additionally, a well characterized and robust methodology for performing FACS on hESCs using multiple-cell surface markers has not been described. The p160-Rho-associated coiled kinase (ROCK) inhibitor, Y-27632, previously has been identified as enhancing survival of hESCs upon single-cell dissociation, as well as enhancing recovery from cryopreservation. Here we examined the application of Y-27632 to hESCs after FACS to improve survival in both feeder-dependent and feeder-independent growth conditions.” (See Background).
Emre et al. (2010) teaches that “HESCs were sorted using markers for SSEA-3, TRA-1-81, and SSEA-1. Cells were plated after sorting for 24 hours in either the presence or the absence of Y-27632. In both feeder-dependent and feeder-independent conditions, cell survival was greater when Y-27632 was applied to the hESCs after sort. Specifically, treatment of cells with Y-27632 improved post-sort recovery up to four-fold. To determine the long-term effects of sorting with and without the application of Y-27632, hESCs were further analyzed. Specifically, hESCs sorted with and without the addition of Y-27632 retained normal morphology, expressed hESC-specific markers as measured by immunocytochemistry and flow cytometry, and maintained a stable karyotype. In addition, the hESCs could differentiate into three germ layers in vitro and in vivo in both feeder-dependent and feeder-independent growth conditions.” (See Methodology/principal findings).
Emre et al. (2010) teaches that “The application of Y-27632 to hESCs after cell sorting improves cell recovery with no observed effect on pluripotency, and enables the consistent recovery of hESCs by FACS using multiple surface markers.” (See Conclusions/significance).
PNG
media_image1.png
406
1128
media_image1.png
Greyscale
Figure 5. In vitro differentiative capacity of hESCs upon extended passaging after sorting with Y-27632. Bright field images of A) day 5 embryoid bodies from hESCs on feeders at passage 13 post-sort and E) day 6 embryoid bodies from hESCs feeder-free at passage 12 post-sort. Immunofluorescence of differentiated hESCs shows labeling for mesoderm (B, F) (GATA4 and cTnI and Desmin), ectoderm (C, G) (Nestin and Sox1), and endoderm (D, H) (FoxA2, Sox17). For feeder conditions (B–D), hESCs are at passage 14 post-sort (B); passage 13 post-sort (C); and passage 16 post-sort (D). For feeder-free conditions (F–H), hESCs are at passage 12 post-sort (F); passage 12 post-sort (G); and passage 13 post-sort (H). Scale bars are 100 mm.
Emre et al. (2010) does not explicitly teach (i) The limitation “within one hour” recited in claim 17; and (ii) The limitation “wherein the manipulation is a multiple electroporation method, and the mammal is a hybrid, an inbred, a disease model, or heterogenous” recited in claim 19.
(i) Regarding claim 17, Emre et al. (2010) teaches that “HESCs were sorted using markers for SSEA-3, TRA-1-81, and SSEA-1. Cells were plated after sorting [which reads on within one hour recited in claim 17] for 24 hours in either the presence or the absence of Y-27632. Moreover, Mellott et al. (2014) teaches that “On the day of transfection, medium from all wells was removed, and cells were washed twice with phosphate Buffered saline (PBS). Afterward, cells were incubated for 1 h at 370C in traditional hUCMSC medium (10% FBS-MSC Qualified/1% penicillin-streptomycin/low-glucose DMEM) or traditional hUCMSC medium with 10 mM of Y-27632-RI (Reagents Direct, Encinitas, CA, USA). After 1 h, hUCMSCs were washed twice with PBS, trypsinized, and then resuspended in 95 mL of 4D NucleofectorTM P1 Primary Solution (P1PS) (Lonza) at a density of 500,000 cells per 95 mL in a 50-mL conical tube (Phenix Research Products, Candler, NC, USA) (See right column, page 92).
(ii) Regarding claim 19, Mellott et al. (2014) teaches that “Differentiating stem cells using gene delivery is a key strategy in tissue engineering and regenerative medicine applications. Nonviral gene delivery bypasses several safety concerns associated with viral gene delivery; however, leading nonviral techniques, such as electroporation, subject cells to high stress and can result in poor cell viabilities. Inhibition of Rho-associated coiled-coil kinase (ROCK) has been shown to mitigate apoptotic mechanisms associated with detachment and freezing of induced pluripotent stem cells and embryonic stem cells; however, inhibiting ROCK in mesenchymal stromal cells (MSCs) for improving gene delivery applications has not been reported previously. In this study, we hypothesized that ROCK Inhibitor (RI) would improve cell viability and gene expression in primary human umbilical cord mesenchymal stromal cells (hUCMSCs) when transfected via NucleofectionTM. As hypothesized, the pre-treatment and post-treatment of hUCMSCs transfected via nucleofection with Y-27632-RI significantly improved survival rates of hUCMSCs and gene expression as measured by green fluorescent protein intensity. This study provides the first comparative look at the effect of Y-27632-RI on hUCMSCs that underwent transfection via nucleofection and shows that using Y-27632-RI in concert with nucleofection could greatly enhance the utility of differentiating and reprogramming hUCMSCs for tissue engineering applications” (See Abstract).
It would have been prima facie obvious for a skilled artisan to incorporate the disclosure of “improving viability and transfection efficiency with human umbilical cord Wharton's jelly cells through use of a ROCK inhibitor” taught in the title of reference by Mellott et al. (2014) into the teachings of Emre et al. (2010) regarding “the ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-activated cell sorting with multiple cell surface markers” as stated in the title, to reach the claimed methods recited in instant claims 17 and 19 regarding the length of time and a damaging manipulation in the context of limitation “treating with a temporary treatment medium including an intracellular skeleton regulator and/or an apoptosis inhibitor for a specific period of time before and/ or after a damaging manipulation” recited in instant claim 15.
A skilled artisan would be motivated to combine the teachings Emre et al. (2010) with the teachings of Mellott et al. (2014) with reasonable expectation of success because the two references are analogous arts in the same field of endeavor by using the same ROCK inhibitor Y-27632 and focusing on improving the recovery of human embryonic stem cells after FACS analysis taught by Emre et al. (2010), and improving viability of human umbilical cord Wharton’s Jelly Cell after electroporation taught by Mellott et al. (2014).
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Wu-Cheng Winston Shen whose telephone number is (571)272-3157. The examiner can normally be reached Mon.-Fri. 8:00 AM-5:00 PM.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
Prosecution Insights