DETAILED ACTION
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 .
Current Action Summary and Claim Status
This action is in response to the papers filed on March 2, 2026.
Currently, claims 1, 2, and 9-15 are pending.
Claims 13-15 were previously withdrawn as directed to a non-elected invention.
Claims 3-8 were canceled by applicant.
Claims 1, 2, and 9-12 are under examination.
Any objections and rejections not reiterated below are hereby withdrawn.
The rejections of record under U.S.C. 112(b) are withdrawn in view of the amendments to the claims.
The rejection of record under U.S.C. 112(d) is moot in view of the cancellation of rejected claims.
The provisional double patenting rejection over 17/910,206 is moot because the ‘206 application was abandoned on January 28, 2026.
Effective Filing Date
The present application was filed on December 21, 2021 and is a 371 of PCT/JP2019/025952, filed June 28, 2019.
Election/Restrictions
Applicant’s election without traverse of the invention of Group I, claims 1-12, drawn to a method for assessing a differentiation state of a cell, comprising detecting Pdk1 mRNA in the response filed March 6, 2025 is acknowledged.
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.
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 1-2, 9-10, and 12 are/remain rejected under 35 U.S.C. 103 as being unpatentable over Prigione et al., 2014 in view of Wiraja et al., “Real-Time Imaging of Dynamic Cell Reprogramming with Nanosensors”, Small 2018, 14, 1703440, April 3, 2018, Zheng et al., “Rationally designed molecular beacons for bioanalytical and biomedical applications”, Chem Soc Rev, 2015. 44, 3036 (2015), Genbank. Bethesda (MD):National Library of Medicine (US), National Center for Biotechnology Information; 2004-[cited April 9, 2025], Hirayama et al., US 2018/0022789 A1, published January 25, 2018, Thakor et al., “Subcutaneous Peripheral Injection of Cationized Gelatin/DNA Polyplexes As A Platform for Non-viral Gene Transfer to Sensory Neurons”, Molecular Therapy vol. 15 no. 12, pp 2124-2131, published December 2007, and Obata et al., “HSP47 siRNA conjugated with cationized gelatin microspheres suppresses peritoneal fibrosis in mice”, Acta Biomateralia 8(2012) 2688-2696, published April 6, 2012.
This rejection has been updated as necessitated by the amendments to the claims (cancellation of claims 6 and 7).
Regarding claims 1-2 and 9, Prigione et al. teach detecting Pdk1 expression by quantitative real-time PCR during dedifferentiation of somatic cells into induced pluripotent stem cells (Prigione et al., Figure 4A) and human embryonic stem cells in the undifferentiated state and assessing glycolytic shift (i.e. whether the cell is in a state in which metabolism by glycolysis is predominant or whether metabolism in mitochondria is activated) (Prigione et al., Abstract).
Prigione et al. do not teach detecting Pdk1 expression by introducing a probe capable of detecting Pdk1 into a cell wherein the introducing includes bringing a cationized gelatin nanoparticle that supports the probe into contact with the cell.
However, Wiraja et al. teach a method of detecting the pluripotent stem cell marker OCT4 in live cells during reprogramming (i.e. a switch in a differentiation state between a state in which glycolysis is predominant and one in which metabolism in mitochondria is activated; detecting expression over time) (Wiraja et al., Figure 3) comprising introducing a molecular beacon (i.e. a probe with a sequence complementary to a target nucleic acid) specific to OCT4 mRNA (Wiraja et al., page 5, column 1, paragraph 6). Wiraja et al. teach coupling molecular beacons to gold nanoparticles or poly(lactic-co-glycolic acid) (PLGA) nanoparticles to allow uptake and sustained release of molecular beacons directly into the cytoplasm of cells (Wiraja, page 2, column 1, paragraphs 2-3).
Zheng et al. teach rational principles for designing molecular beacon probes for both gene-detection assays such as real-time PCR (Zheng et al., page 3044, column 2, paragraph 3), in vivo bioimaging (Zheng et al., page 3048, column 2, paragraph 6- page 3049, column 1, paragraph 1), and intracellular imaging of mRNAs in live cells using molecular beacons supported on gold nanoparticles (Zheng et al., Figure 12).
Furthermore, Genbank teaches the complete nucleotide sequence of Pdk1 (Genbank: NM_002610).
Hirayama et al. teach “easy-uptake gelatin particles” that contain an auxiliary component (i.e. cargo) used for applications such as the measurement of substances (Hirayama et al., paragraph 0042), and may contain nucleic acids (Hirayama et al., paragraph 0044). Hirayama et al. teach that gelatin nanoparticles are taken up through the cells’ own activity, and are readily biodegradable (Hirayama et al., paragraph 0009) and that this property is desirable because it minimizes the loss of the cells’ activity following nanoparticle uptake and allows for sustained release of auxiliary components into cells (Hirayama et al., paragraph 0009-0010). Furthermore, Hirayama et al. teach that the amount of a cargo component inside the gelatin particle is greater than an amount of the cargo on the surface of the gelatin particle (Hirayama et al., Abstract).
Hirayama et al. do not teach how the nucleic acid cargo is attached to the gelatin nanoparticles.
However, Thakor et al. and Obata et al. each teach methods comprising introducing DNA transgenes that produce functional fluorescent reporter gene in live animal models (Thakor et al., abstract) or delivering shRNA capable of binding to and reducing the expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (Thakor et al., abstract) using cationic gelatin nanoparticles (Thakor et al., abstract and Figure 6). Obata et al. similarly teach using cationized gelatin nanoparticles comprising siRNAs to silence expression of target genes in live model animals (Obata et al., abstract).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method of detecting Pdk1 expression in a cell by qPCR, taught by Prigione et al., with the teachings of Wiraja et al., Zheng et al., Genbank, Hirayama et al., and either of Thakor et al. or Obata et al. to arrive at the presently claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to modify the method of Pdk1 detection in induced pluripotent stem cells comprising qRT-PCR, taught by Prigione et al. by detecting the Pdk1 mRNA with the molecular beacon nanosensors taught by Wiraja because of the teaching of Wiraja that PCR-based techniques are disruptive (i.e. not compatible with live-cell imaging) and that in situ, live cell detection of differentiation biomarkers allows for high-throughput assessment, optimization, and biomarker-specific cell enrichment during low-efficiency reprogramming events (Wiraja, Abstract). The ordinary artisan would have used the principles of molecular beacon design taught by Zheng et al., in addition to the complete nucleotide sequence of Pdk1, taught by Genbank, to construct a molecular beacon probe capable of detecting Pdk1. The ordinary artisan would have been motivated to further modify the nanoparticle-probes with the teachings of the teachings of Hirayama et al. that gelatin nanoparticles are readily absorbed by cells’ own activities, are highly biocompatible, allow for non-destructive imaging of live cells (Hirayama et al., paragraphs 008-0010), and can deliver nucleic acids (i.e. molecular beacons) into living cells (Hirayama et al., paragraphs 0042-0044). The ordinary artisan would have been reasonably confident that the “highly biocompatible” gelatin nanoparticles taught by Hirayama would have allowed for easy uptake of a molecular beacon capable of detecting Pdk1 into living cells by their own activities. Finally, the ordinary artisan would have been motivated to modify the gelatin nanoparticles for delivering molecular beacons into living cells, taught by Hirayama et al., with the teachings of Thakor et al., that cationic polymers and peptides have been widely developed for gene delivery (i.e. nucleic acid probes) because they can condense negatively charged nucleic acids through electrostatic interactions to form protective nanoscale polyplexes (Thakor et al., page 2124, column 2, paragraph 3) and that cationized gelatin nanoparticles are less toxic to living cells than alternative polymer carriers (Thakor et al., page 2125, column 2, paragraph 2 and page 2126, column 2, paragraph 1, and page 2127, column 2, paragraph 1). The ordinary artisan would have been similarly motivated to modify the gelatin nanoparticles for delivering molecular beacons into living cells, taught by Hirayama et al., with the teachings of Obata et al., that cationized gelatin nanoparticles successfully deliver functional siRNA in living animal models (Obata et al., abstract). Obata et al. further teach that the cationized gelatin nanoparticles have the advantages of low toxicity (Obata et al., page 2695 and 2688, column 2) and sustained, tunable release of cargo molecules (Obata et al., page 2689, column 1).
Therefore, to summarize, the ordinary artisan would have been motivated to detect Pdk1 expression (as taught by Prigione et al.) in live cells using molecular beacon probes (as taught by Wiraja et al., Zheng et al., and Genbank) coupled to cationized gelatin nanoparticles (as taught by Hirayama et al., Thakor et al., and/or Obata et al.) because of the teachings of Wiraja at al. that nanoparticle-labeled molecular beacons allow for live-cell imaging of differentiation markers (such as Pdk1, as taught by Prigione) and by the teachings of Hirayama, Thakor, and Obata that cationized gelatin nanoparticles provide the predictable, known advantages of reduced toxicity in live cells and allow for sustained, controlled release of cargo molecules (specifically nucleic acids).
Regarding claim 10, Prigione et al. teach the cell is a stem cell (Prigione et al., abstract).
Regarding claim 12, Wiraja et al. teaches detecting the expression levels of multiple genes in live cancer cells using molecular beacon probes (Wiraja et al., page 3048, column 1, paragraphs 2-3).
Claim 11 is rejected under 35 U.S.C. 103 as being unpatentable over Prigione et al. in view of Wiraja et al., Zheng et al., Genbank, Hirayama et al., Thakor et al., and Obata et al. as applied to claims 1, 2, 9-10, and 12 above, and further in view of Tan et al., “Pyruvate Dehydrogenase Kinase Participates in Macrophage Polarization via Regulating Glucose Metabolism” J Immunol (2015) 194(12):6082-6089.
Regarding claim 11, Prigione et al. in view of Wiraja et al., Zheng et al., Genbank, Hirayama et al., Thakor et al., and Obata et al. teach methods comprising detecting Pdk1 expression by contacting live stem cells or cancer cells with cationized gelatin nanoparticles coupled to molecular beacons capable of detecting Pdk1 expression as documented in the preceding updated 103 rejection.
Prigione et al. in view of Wiraja et al., Zheng et al., Genbank, Hirayama et al., Thakor et al., and Obata et al. do not teach detecting Pdk1 expression in immune cells.
However, Tan et al. teach methods comprising detecting PDK1 expression in macrophages at multiple timepoints following various treatments (i.e. immune cells) by qPCR or Western blot (Tan et al., figure 4 B and C) and reducing Pdk1 expression using siRNA in immune cells.
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the methods comprising contacting live cells with cationized gelatin nanoparticles coupled to molecular beacons (i.e. probes) capable of detecting Pdk1, taught by Prigione et al. in view of Wiraja et al., Zheng et al., Genbank, Hirayama et al., Thakor et al., and Obata et al. to detect Pdk1 expression in immune cells, as taught by Tan et al.
The ordinary artisan would have been motivated to modify the method of Pdk1 detection in cancer or stem cells taught by Prigione et al. in view of Wiraja et al., Zheng et al., Genbank, Hirayama et al., Thakor et al., and Obata et al. to measure Pdk1 expression in immune cells because of the teaching of: Tan et al. that Pdk1 expression is required for macrophage activation (i.e. immune cell differentiation) (Tan et al., page 6084, column 2-page 6085, column 1) and Wiraja et al. that PCR-based techniques are disruptive (i.e. not compatible with live-cell imaging) and that in situ, live cell detection of differentiation biomarkers allows for high-throughput assessment, optimization, and biomarker-specific cell enrichment during low-efficiency reprogramming events (Wiraja, Abstract). The ordinary artisan would therefore have had a reasonable expectation that the cationized gelatin nanoparticles for detection of Pdk1 taught by Prigione et al. in view of Wiraja et al., Zheng et al., Genbank, Hirayama et al., Thakor et al., and Obata et al. would have successfully detected the expression of Pdk1 in immune cells with similar predictable advantages of in situ, live cell detection of Pdk1 expression with reduced toxicity relative to endpoint (i.e. qPCR or western blot) detection or other methods for transfecting detection reagents (i.e. probes) into live cells.
Response to arguments
The response makes the following arguments against the prima facie case of obviousness presented in the rejections of record under 35 U.S.C. 103.
The response cites applicant’s post-filing date publication, Murata et al., “Intracellular Controlled Release of Molecular Beacon Prolongs the Time Period of mRNA Visualization” (Tissue Eng Part A. 2019 Nov; 25(21-22):1527-1537 as “evidence that it would not have been obvious to replace Wiraja’s particles with Hirayama’s gelatin particles” because Murata compares the duration of fluorescence signals observed when using (i) a simple cationized gelatin-molecular beacon complex with (ii) cationized gelatin nanospheres incorporating molecular beacon and reports that fluorescence from (i) disappears within about 5 days, while the fluorescence from (ii) is observed for over 14 days. The response asserts “in light of Murata’s disclosures, successful long-term, time-course visualization in live cells could not have been expected to be achieved by simply combining molecular beacons and gelatin.”
This argument has been thoroughly reviewed and is not persuasive.
First, applicant’s post-filing date publication is not prior art and is not part of the disclosure of the present application.
MPEP 2142 (emphasis added) states: "To reach a proper determination under 35 U.S.C. 103, the examiner must step backward in time and into the shoes worn by the hypothetical "person of ordinary skill in the art". That time is "before the effective filing date of the claimed invention" for 35 U.S.C. 103 or "at the time the invention was made" for pre-AIA 35 U.S.C. 103. In view of all factual information, the examiner must then make a determination whether the claimed invention "as a whole" would have been obvious at that time to a hypothetical person of ordinary skill in the art."
As such, the findings presented in the Murata reference do not inform the determination of obviousness because they were not available to one of ordinary skill in the art before the effective filing date of the claimed invention.
Second, the combination of references in the rejection of record does not teach “simply combining molecular beacons and gelatin”. Rather, as described in the rejection, Prigione et al., Wiraja et al., Zheng et al., and Genbank teach methods for detecting a differentiation state of live cells over time comprising introducing molecular beacons (i.e. a nucleic acid probe) into cells using nanoparticles that allow uptake and sustained release of molecular beacons into the cytoplasm of cells. Hirayama et al. teach “easy-uptake gelatin particles” that contain cargo components for sustained release over time that may expressly be nucleic acids. Finally, Thakor et al. and Obata et al. teach cationized gelatin nanoparticles advantageously improve uptake of functional nucleic acids (e.g. siRNAs) into live cells, including into live model animals. The combination of references described in the rejection of record results in delivering the molecular beacons for measuring Pdk1 expression in live cells, taught by Prigione et al., Wiraja et al., Zheng et al., and Genbank using the cationized gelatin nanoparticles taught by Hirayama et al., Thakor et al., and Obata et al. as motivated by the previously described advantageous properties of “highly biocompatible” gelatin nanoparticles taught by Hirayama et al., the advantages of live cell imaging taught by Wiraja et al., and the advantageous properties of cationized gelatin nanoparticles taught by Thakor et al. and Obata et al.
Third, the opinions expressed regarding the post filing date publication are taken as applicant’s arguments which cannot take the place of evidence on the record (See MPEP 716.01(c).
Fourth, arguments regarding the comparison between “simple cationized gelatin-molecular beacon complex[es]” and “cationized gelatin nanospheres incorporating molecular beacon” in the post-filing date Murata reference disclosing differences in the duration of fluorescence observed in cells contacted with the two types of cationized gelatin compositions appear to rely on a requirement of “long-term, time-course visualization”. It is noted that, in addition to the other reasons the argument is not persuasive, this feature is not recited in the rejected claims. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
The response argues that “it would not have been straightforward or obvious… to replace Wiraja’s PLGA nanoparticles with gelatin particles and achieve a long-term, time-course visualization in live cells because “Hirayama (paras. 0009-0010 and 0031-0032) indicates difficulties in cellular uptake with conventional gelatin particles and proposes quantitative, engineered distribution design conditions”.
This argument has been thoroughly reviewed and is not persuasive.
Paragraphs 0009-0010 of Hirayama describes the need for their invention in the context of “findings by [Hirayama that] gelatin particles described in [prior art relative to Hirayama et al.] are unlikely to be taken up into cells through the cells’ own activity.
Paragraphs 0031-0032 of Hirayama summarize their invention of “gelatin particles that are easily taken up into cells by the cells themselves”… “where an auxiliary component, which is likely to be recognized as a foreign substance by cells and unlikely to be taken up”… “is mostly present in the inner part of the particles and is not or hardly present in the surface part… because there is no auxiliary component exposed on the particle surface, or… an extremely small amount [on the surface]… the gelatin particles are unlikely to be recognized as a foreign substance by cells and are easily taken up into cells through the cells’ own activity”.
Hirayama’s disclosure describes methods for producing gelatin nanoparticles and introducing said nanoparticles into cells wherein auxiliary components such as nucleic acids are mostly or are entirely present within the interior of the nanoparticle in contrast to the referenced “conventional” (i.e. known before Hirayama’s disclosure) gelatin particles.
Because Hirayama et al. is prior art according to 35 U.S.C. 102(a)(1) and 35 U.S.C. 102(a)(2) with a publication date of January 25, 2018 (more than 1 year prior to the effective filing date of the presently application), the methods of Hirayama et al. for producing the gelatin nanoparticles containing nucleic acid auxiliary components, described as an improvement over the “conventional” gelatin particles referenced by Hirayama et al., would have been known to one of ordinary skill in the art.
The response argues that although “the office indicates that Hirayama’s gelatin particles “may contain nucleic acids (Hirayama et al., paragraph 0044),” but Hirayama does not explicitly teach or suggest applying its specific distribution-design requirements to molecular beacons (nucleic acid probes) for the purpose of long-term intracellular visualization.
This argument has been thoroughly reviewed and is not persuasive.
First, “long-term intracellular visualization” is not recited by the rejected or present claims. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Second, the argument that Hirayama does not explicitly teach or suggest all of the claimed limitations is not persuasive at least because this argument addresses Hirayama individually. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Finally, the response asserts “a replacement of Wiraja’s PLGA nanoparticles with gelatin particles would still not have been straightforward or predictable” because “Wiraja’s long-term monitoring depends on multiple parameters… for PLGA particles”.
This argument has been thoroughly reviewed and is not persuasive.
First, as described above, Hirayama et al. teach methods of producing gelatin nanoparticles carrying an auxiliary component in the interior of the particle, wherein the auxiliary component may expressly be a nucleic acid, including siRNA, shRNA, DNA, etc. (Hirayama et al., paragraph 0044) and using said particles to release the auxiliary component into the cytoplasm of a cell over time. Wiraja et al. teach analogous methods comprising introducing molecular beacons (i.e. a nucleic acid probe) into living cells over time utilizing a different particle (PLGA) containing the nucleic acid auxiliary component. As described above, the ordinary artisan would have used the methods of producing and using “non-toxic”, “biodegradable”, “easy-uptake gelatin nanoparticles” taught by Hirayama et al. to introduce nucleic acids (such as the molecular beacons taught by Wiraja et al.) into cells with reasonable expectation that the nucleic acid probes would function (i.e. anneal to complementary nucleic acids) when released into the cytoplasm of target cells because Wiraja et al. show molecular beacons function after introduction into cells and/or because Hirayama et al. teach the easy-uptake gelatin particles may contain siRNAs and shRNAs for pharmaceutical applications (i.e. the siRNAs or shRNAs anneal to complementary target nucleic acids). As such, the ordinary artisan would not need to be concerned with the PLGA particle optimization parameters, but rather would have followed the guidance of Hirayama et al. for making molecular-beacon containing “easy-uptake gelatin nanoparticles”.
Second, “long-term monitoring” is not recited by the rejected or present claims. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
All of the arguments in the response filed March 2, 2026 have been fully considered but are not persuasive. Therefore, the 103 rejections of record are maintained.
Conclusion
No claim is allowed.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/Z.M.T./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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