DETAILED ACTION
Claim Status
As of the Non-Final Office Action mailed 12/29/2025, claims 1, 6-10, 12-14, and 16 were pending.
In Applicant's Response filed on 2/20/2026, claim 1 was amended.
As such, claims 1, 6-10, 12-14, and 16 are pending and have been examined herein.
Withdrawn Objections/Rejections
The objections and rejections presented herein represent the full set of objections and rejections currently pending in this application. Any objections or rejections not specifically reiterated are hereby withdrawn.
Claim Rejections - 35 USC § 112(a) Scope of Enablement – Necessitated by Amendments
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1, 6-10, 12-14, and 16 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a method for generating an invitro cardiac disease tissue model via electrostimulation protocol, wherein the cardiomyocytes comprise human induced pluripotent stem cell-derived cardiomyocytes derived from a human subject with hypertension with clear echocardiographic evidence of left ventricular hypertrophy, does not reasonably provide enablement for generating an in vitro cardiac disease model with hiPSCs-derived cardiomyocytes from healthy human subjects or subjects with other diseases. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims. Please note that this rejection is necessitated by amendment, as the claims now require that the electrostimulation protocol as claimed produces a disease state in the cardiac tissue construct.
While determining whether a specification is enabling, one considered whether the claimed invention provides sufficient guidance to make and use the claimed invention, if not, whether an artisan would have required undue experimentation to make and use the claimed invention and whether working examples have been provided. When determining whether a specification meets the enablement requirement, some of the factors that need to be analyzed are: the breadth of the claims, the nature of the invention, the state of the prior art, the level of one of ordinary skill, the level of predictability in the art, the amount of direction provided by the inventor, the existence of working examples, and whether the quantity of any necessary experimentation to make or use the invention based on the content of the disclosure is “undue” (In re Wands, 858 F.2d at 737, 8 USPQ2d 1400, 1404 (Fed. Cir.1988)).
Furthermore, the USPTO does not have laboratory facilities to test if an invention with function as claimed when working examples are not disclosed in the specification, therefore, enablement issues are raised and discussed based on the state of knowledge pertinent to an art at the time of the invention, therefore, skepticism raised in the enablement rejection are those raised in the art by artisans of expertise.
Nature of the invention:
A method for generating an in vitro cardiac disease tissue model, comprising: forming an elongated tissue by disposing a plurality of cardiomyocytes within a culture plate; culturing the tissue such that each end of the elongated tissue contacts one of a pair of attachment wired adhered to the culture plate; and inducing a disease state in the elongated tissue by electrically stimulating the elongated tissue in culture, comprising: applying an electric field along a long axis of the elongated tissue, applying the electric field at an initial frequency of 2 Hz for 1 week, incrementally increasing the electric field to a peak frequency of 6 Hz over a period of 4 weeks, and decreasing the electric field to a maintenance frequency of 3 Hz, and applying the electric field at the maintenance frequency of 3 Hz for six months, wherein the cardiomyocytes comprise human induced pluripotent stem cell-derived cardiomyocytes.
The state of the prior art:
The state of the prior art for inducing a disease state in cardiomyocyte tissue culture via chronic electrostimulation, wherein the cardiomyocytes are derived from healthy subjects was unpredictable before the effective filing date of the claimed invention.
The state of the prior art for inducing a disease state in cardiomyocyte tissue culture via chronic electrostimulation, wherein the cardiomyocytes are derived from subjects with various diseases (other than hypertension with clear echocardiographic evidence of left ventricular hypertrophy) was unpredictable before the effective filing date of the claimed invention.
The breadth of the claims:
The claims encompass inducing a disease state in cardiomyocyte tissue culture via electrostimulation, wherein the cardiomyocytes are either from healthy individuals or individuals with various diseases.
The level of skill in the art:
The level of skill is high that requires a researcher with a PhD degree.
The working examples and guidance provided:
The specification discloses working examples in which iPSC-derived cardiomyocytes were constructed into engineered tissue strips. The cells were derived from two patient populations: hypertensive patients with clear echocardiographic evidence of left ventricular hypertrophy (“affected” herein) and healthy individuals (“non-affected” herein). Both populations were subjected to long-term electrical stimulation protocol used to mimic chronic increased workload in ventricular tissue: 1 Hz step-up electrical conditioning to 6Hz, maintained 6Hz for 1 week, then decreased down to 3Hz and maintained for up to 6 months. Fig. 3C shows enrichment in cardiac genes associated with cardio-functional categories and cardiac-related canonical pathways post-stimulation protocol. Active force was significantly reduced in tissues derived from the patients that exhibited higher level of left ventricular hypertrophy in response to prolonged hypertension at the 6 week mark. Active force was absent in all tissues from affected patients after the 8 month period, but still present in non-affected patients.
The specification fails to provide any working examples in which electrostimulation protocol was able to recapitulate any phenotype (i.e., disease state) in tissue culture derived from hiPSC-derived cardiomyocytes from healthy individuals or individuals with any other diseases other than those with hypertension with clear echocardiographic evidence of left ventricular hypertrophy.
The unpredictable nature of the art:
Post-dated Sesena-Rubfiaro et al (ACS Biomater Sci Eng. 2023 Mar 13;9(3):1644-1655. doi: 10.1021/acsbiomaterials.2c01370. Epub 2023 Feb 10) discusses membrane remodeling of human engineered cardiac tissue by chronic electric stimulation. It states that electrical pacing on ECTs has been shown to greatly improve the maturation process by upregulating cardiac markers, promoting sarcomere alignment, increasing the contractile force, and improvement of calcium handling. hECTs are formed from early-stage hiPSC-CMs shortly after the initiation of spontaneous contractions, following previously reported procedures15. The hiPSC-CMs are mixed with fibrin hydrogel components and Matrigel in a millimeter size well containing two flexible PDMS posts (referred to hereafter as milli-tug device). After matrix polymerization and CM-mediated contraction, the mixture forms a hECT suspended between two posts, which providing the static mechanical load (see Figure 1B). The typical size of the tissue is approximately 1.5 mm in length, 0.28 mm in thickness, and 0.6 mm for the narrowest width in the middle (see Figure 1B (iii)). The narrower width in the middle is mainly due to the relative tension differences in the matrix16. ES with monophasic square wave pulses at 5 Hz is applied between two carbon plates in the capacitive coupling configuration (see Figure 1B(ii)). With the ES, the regular crest/valley patterns with a spatial period close to 2 μm appeared on the cellular surface of hiPSC-CMs at the edges of hECTs, demonstrating better development of the t-tubule network. The better-developed membrane structures of the t-tubule system are consistent with the sarcomere structure development, enabling effective electrical signal propagation as revealed by the calcium handling measurements. Accordingly, the hECT under ES generates a more prominent twitch force with a reduced beating frequency.
Post-dated Sterckel et al (Front Bioeng Biotechnol. 2025 Oct 9;13:1686342. doi: 10.3389/fbioe.2025.1686342) discusses promoting maturation of hiPSC-derived cardiomyocytes in 3D cardiac tissues. Exogenous electrical stimulation has emerged as complementary strategy to further drive maturation. 3D tissue culture systems enhance CM maturation by displaying an increase in cell size, elongation, and notably, sarcomere organization and alignment. The addition of electrical stimulation further promotes maturation of cell shape and size toward the adult CM phenotype. Desmosomes, crucial for cardiomyocyte adhesion, are absent in immature hPSC-CMs, however, the presence of desmosomes is seen in electrically stimulated tissues. T-tubules, which are also rarely observed in hPSC-CMs (were detected following electrical stimulation. The presence of these structures indicates improved tissue organization and suggests enhanced excitation-contraction coupling. Electrical stimulation elevates the expression of genes encoding contractile proteins like TNNT2 and MYH7, reflecting enhanced assembly of the contractile apparatus. Furthermore, cell-cell junction proteins, including Cx43 and N-cadherin, are upregulated, underscoring their roles in electrical and mechanical coupling of CMs.
Post-dated Cho et al (Theranostics. 2022 Mar 14;12(6):2758-2772. doi: 10.7150/thno.67661) states that 3D cultures of hPSC-CMs can better induce CM maturation, which is a critical challenge in regenerative medicine and drug testing. Various approaches have been developed to improve hPSC-CM maturity. Co-culture with non-cardiac cells was the most frequently used method for CM maturation. Soluble factors secreted from human mesenchymal stem cells (hMSCs) co-cultured with hiPSC-CMs were employed to impact hiPSC-CM maturation. Extended culture periods also increased iPSC-CM maturity. Electromechanical stress was also reported for better CM maturation. Mechanical stress improved cardiac maturation via stretching CM. Electrical stimulation with gradually increasing frequency over weeks also matured hiPSC-CMs. For example, physical conditioning with increasing intensity allowed hiPSC-CMs to have a transcriptionally and structurally advanced mature identity. Cardiac disease models were investigated with 2D-cultured hiPSC-CMs carrying genetic mutation(s) or induced pathological cardiac conditions. Diseased hiPSC-CMs and electrophysiological experiment systems are appropriate to investigate disorders caused by abnormal ion channel activities but are not suitable for cardiac maladaptations caused by mechano-structural problems. Hypertrophic cardiomyopathy (HCM) is a polygenic disease that is strongly influenced by environmental factors and usually associated with mutations in contractile components of the sarcomere. Increased expression of hypertrophic markers, aberrant calcium handling, and thickening of myocardium are characteristics of HCM patients. hEHT models of HCM were generated with hiPSC-CMs containing BRAF, or PRKAG2 mutations, or electrical stimulation on hiPSC-CMs derived from a hypertension patient. BRAF encodes a serine/threonine kinase regulating the RAS/MARK pathway, which has diverse roles in cell cycle, cell growth, differentiation, and senescence. hEHT generated with hiPSC-CMs with a BRAF mutation exhibited hypertrophic characteristics including a trend of shorter twitch duration and higher passive Young's modulus, indicating tissue stiffness. However, the pathological phenotypes were diminished only after 11 days from the hEHT formation, suggesting the need for extra stimulations or further sophisticated development of hEHT to recapitulate the cardiac hypertrophy shown in patients. PRKAG2 mutations can cause inherited autosomal dominant left ventricular hypertrophy. HCM-hEHT with a PRKAG2 mutation exhibited HCM phenotypes with increased AMPK activity and reduced adverse remodeling and arrhythmia with AMPK agonist. In addition, hypertrophic hEHT was generated with hiPSC-CMs derived from hypertension patients and application of electrical stimulation for up to 8 months. The hypertrophic hEHT showed enriched gene expression related to pathological remodeling, cardiac enlargement and dysfunction, heart failure, and cardiac hypertrophy. Chronic electrical stimulation and a long period of hEHT culture might be essential for generation of human hypertrophic heart in a dish.
Hirt et al (Journal of Molecular and Cellular Cardiology. Volume 74, September 2014, Pages 151-161.) states that continuous pacing improved structural and functional properties of rEHTs and hEHTs to an unprecedented level. Electrical stimulation appears to be an important step toward the generation of fully mature EHT. The effect of constant pacing with a low frequency was tested. In the natural time course of rEHTs there is no beating at day 0, start of uncoordinated beating around day 4 and coherent beating of the entire tissue construct around day 8. At day 4 we initiated constant pacing at 0.5 Hz which could be attained throughout the lifetime of a rEHT without evidence of toxicity even during a maximum pacing duration of 32 days. Continuously paced (cp) rEHTs developed increasingly higher forces than control EHTs. The curves started to deviate from each other at 7 days of pacing (11 days of culture). After 18 days of pacing cp-rEHTs generated 85–90% higher forces than control rEHTs. The higher contractile forces were observed independent of whether rEHTs in the pacing group were actively paced (cp-p) or beat spontaneously during the measurements (pacer off, cp-s; Figs. 3A–B). Throughout pacing diameters of paced rEHTs decreased to greater extents than that of non-paced rEHTs. hEHTs differed in their spontaneous beating pattern, showing very regular beating ~ 1.5 Hz in the beginning and a drop to around 1 Hz during the first 14 days of culture (Fig. 7A). For this reason we tested two pacing protocols, pacing at 0.5 Hz for the entire period and pacing at 2 Hz during the first week and 1.5 Hz thereafter, thus ensuring permanent overstimulation of the spontaneous beating activity (Fig. 7B). Culture medium was changed daily assuming more hypochlorous acid to be produced by the higher pacing rate. Stimulated hEHTs developed higher forces than controls after 4–6 days of stimulation (Fig. 7C). Paced hEHTs compared to unpaced controls exhibited a much better muscular network of longitudinally oriented cardiomyocytes with higher cytoplasm-to-nucleus ratio (Fig. 8B). Staining with the MLC2v antibody (Figs. 8C–D) confirmed that cardiomyocytes were longer both in absolute (Fig. 8E) and relative terms (expressed as ratio to cardiomyocyte width, Fig. 8F), were slightly slimmer i.e. less round, (Supplemental Fig. S5), were more stringently aligned in paced hEHTs (smaller scatter in Fig. 8G) and exhibited clearer cross-striation. Furthermore, paced hEHTs had more contractile mass relative to cell-free matrix than control hEHTs (Fig. 8H). Continuous pacing of rat (at 0.5 Hz) and human EHT (2 Hz/1.5 Hz) was continued for more than 4 weeks without obvious signs of toxicity. Human stimulated EHTs (at 2 Hz/1.5 Hz) surpassed their controls after 4–5 days and reached ~ 50% higher forces after 10 days of continuous pacing, showing improved function after chronic pacing. The reference concludes that hydrogel-based EHTs benefit from long-term electrical stimulation and reach an unprecedented cardiac tissue structure and function, including considerably higher force, denser cardiomyocyte network with improved gap junction coupling, a more physiological response to external Ca2 +, increased inotropic response to isoprenaline and a well-developed sarcomeric ultrastructure. Electrical stimulation induces electrochemical stress and that the improved phenotype of chronically paced EHTs is likely the net outcome of beneficial and detrimental effects of pacing, opening the way for further improvement.
Post-dated Stenzig et al (J Mol Cell Cardiol. 2022 Feb;163:97-105. doi: 10.1016/j.yjmcc.2021.10.001. Epub 2021 Oct 8) discusses recapitulation of dyssynchrony-associated contractile impairment. Cardiomyocytes were differentiated from human induced pluripotent stem cells from a healthy donor and cast into a fibrin matrix to produce engineered heart tissue (EHT). EHTs were either field stimulated in their entirety (symmetrically) or excited locally from one end (asymmetrically) or they were allowed to beat spontaneously. Human EHTs were cultured on two different types of racks which only differed in the silicone stiffness. A very soft version with a stiffness of k = 0.28 mN/mm led to rather low final contractile forces, while a stiffer version with k = 0.80 mN/mm led to high force EHTs. For some experiments high force EHTs were heavily mechanically challenged during the last week of culture in a procedure called afterload enhancement (AE). The two silicone posts representing the anchoring points and providing the elastic resistance for each attached EHT were stiffened by metal braces. This procedure increased the afterload of EHTs by a factor of 14 (from k = 0.80 mN/mm to 11.5 mN/mm) and led to contractile dysfunction (i.e., contractile dysfunction was recapitulated by afterload enhancement and not the electrical stimulation alone). The first exploratory long-term pacing experimental series were performed in rEHTs. Rat EHTs beat in an uncommon burst (4 Hz) and resting phase-pattern, the two states typically alternating every 10–30 s. They were continuously stimulated at 0.5 Hz, which therefore only had an effect during the resting phases. The contractility results of the rEHTs (Supplemental Fig. S6 A–E) were partially inhomogeneous. There was a clear trend towards higher force in the symmetrically paced EHTs compared to their asymmetrical counterparts or the controls. After these pilot experiments we used hEHTs for all further experiments, which in contrast to rEHTs beat very regularly at frequencies around 0.5–0.8 Hz when cultured in the presence of 300 nM ivabradine. The frequency chosen for continuous pacing was 1 Hz. The results were more uniform and demonstrated moderate but significant superiority in high force EHTs at the end of the long-term pacing (Fig. 3 C) and marked superiority of symmetrical pacing regarding contractile force, velocity of contraction and velocity of relaxation in low force EHTs (Fig. 3 D). Here, after 11 days of pacing (at d15 of culture) forces of symmetrically paced EHTs were 2.86× higher (adj. p < 0.0001) than controls, while this effect was not observed for the asymmetrically paced EHTs (1.48× force of controls, adj. p-value = 0.15). EHTs of all three groups (control, symmetrically paced and asymmetrically paced) were then subjected to an AE procedure during the last week of culture (Fig. 4 A). We have previously shown that this leads to a variety of changes characteristic of pathological cardiac hypertrophy, e.g., contractile dysfunction. As mentioned above, asymmetrically paced EHTs had lower absolute forces than symmetrically paced EHTs, and the relative AE-effect on force (Fig. 4 B), contraction (Fig. 4 C) and relaxation velocity (Fig. 4 D) was most accentuated in the asymmetrically paced group. This shows that other culture constraints (such as mechanical afterloading) is needed to recapitulate pathological cardiac hypertrophy in engineered cardiac tissues from healthy donors.
The art shows that long term or chronic electrical stimulation improves maturation of hiPSC-derived cardiomyocytes in cardiac tissue constructs and other culture constraints such as mechanical afterloading of posts is necessary to produce pathological hypertrophy in healthy cells. This makes it unpredictable for one of ordinary skill in the art to know whether the electrical stimulation protocol as instantly claimed, on its own, is sufficient to recapitulate cardiac disease state in cardiomyocytes derived from healthy donors or cardiomyocytes from those with other diseases other than hypertension with left ventricular hypertrophy. Absent specific guidance, one skilled in the art before the effective filing date of the claimed invention would not know how to practice the claimed invention and would require undue experimentation to practice over the full scope of the invention claimed.
The amount of experimentation necessary:
The specification only shows that disease state can be recapitulated in cardiomyocytes ultimately derived from hypertensive subjects with echocardiographic evidence of left ventricular hypertrophy when subjected to the instantly claimed electrical stimulation protocol. One of ordinary skill in the art could not reasonably take this working example and apply the protocol to healthy cells or cells derived from individuals with other diseases and actually obtain an engineered tissue construct with phenotypic recapitulation of cardiac hypertrophy, especially since healthy cells utilized in the instant specification did not). These teachings do not reasonably support the broad scope of cells as embraced by the claims.
For the reasons set forth above, one skilled in the art before the effective filing date of the claimed invention would not be able to make and/or use the invention as claimed. This is particularly true given the nature of the invention, the state of the prior art, the breadth of the claims, the amount of experimentation necessary, the level of skill which is high, the working examples provided and scarcity of guidance in the specification, and the unpredictable nature of the art.
Response to Arguments
Applicant’s arguments with respect to the previously posited art rejections of claim(s) 1, 6-10, 12-14, and 16 have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument. All previous art rejection of record have been withdrawn due to Applicant’s amendments.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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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/G.R./Examiner, Art Unit 1632 /KARA D JOHNSON/Primary Examiner, Art Unit 1632
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