Drug efficacy testing

Abstract

The present invention discloses a method for evaluating the efficacy of a pharmaceutical agent suspected to be effective in treating a disease, comprising the steps of: (i) incubating an artificial tissue with the pharmaceutical agent, wherein the artificial tissue is or has been modified to express the disease; (ii) determining at least one physical parameter of the artificial tissue obtained in step (i), wherein the physical parameter is an indicator of the functionality of a physiological bodily function; and (iii) determining whether the value of the at least one physical parameter determined in step (ii) satisfies a predetermined threshold value for the physical parameter, thereby determining whether the pharmaceutical agent is effective; and if the value of the at least one physical parameter exceeds the predetermined threshold value, it is indicated that the pharmaceutical agent is effective in treating the disease by restoring at least a portion of the functionality of the physiological bodily function.

Description

[Technical Field]

[0001] The present invention discloses a method for evaluating the efficacy of a pharmaceutical agent suspected to be effective in treating a disease, the disease being preferably a genetic or non-genetic disease that causes abnormalities in physiological bodily functions in patients with the disease, the method comprising: (i) incubating engineered tissue with the pharmaceutical agent, the engineered tissue being modified or having been modified to express the disease; and (ii) determining at least one physical parameter of the engineered tissue obtained in step (i), the physical parameter being an indicator of the functionality of the physiological bodily function. [Background technology]

[0002] To evaluate novel therapeutics, particularly gene and cell therapies for muscle diseases, as well as the clinical application of small molecule compounds, it is particularly important from a regulatory perspective to standardize and verify the specific effects of products through so-called "potency tests." Regulatory authorities (e.g., EMA, PEI, FDA) require the establishment and validation of potency tests to verify the quality of medicinal products, especially dose-dependent effects. Potency variations between manufacturing batches are not uncommon, especially for advanced therapeutic medicinal products (ATMPs), and can have significant impacts on clinical applications / studies or the interpretation of clinical data. Therefore, potency tests should be performed under conditions as close as possible to the ATMP's expected mechanism of action.

[0003] Preclinical validation of ATMPs has been limited by traditional models (animal models, cell lines) and is often limited to surrogate markers that lack sufficient evidence of their mechanism of action (mode of action). Such surrogates typically include protein and gene expression. However, some ATMPs, such as eteplirsen (Exondys51), have been approved by the FDA despite the agency no longer accepting surrogate markers; for example, golodirsen (Vyondys53) was approved only on appeal due to safety concerns and little-to-no evidence of efficacy. The only surrogate marker used was truncated dystrophin expression, the correlation of which with muscle function is unclear.

[0004] Thus, for example, there remains a need for improved potency tests, e.g., potency tests suitable for regulatory procedures that require demonstrating efficacy of a compound under investigation, such as ATMP. The technical challenge is to meet this need. Summary of the Invention

[0005] The technical problem is solved by the subject matter of the invention defined in the claims. The present invention relates to a method for assessing the efficacy of a pharmaceutical agent for treating a disease, preferably a genetic or non-genetic disease. The disease is preferably characterized by a loss of tissue bodily function, the function being determined or characterized by physical parameters such as contractile force, stiffness, and / or one or more multi-electrode array (MEA) measurements. For this purpose, an artificial tissue representing the disease is incubated with the pharmaceutical agent. After incubation, at least one physical parameter is determined. Since the physical parameter correlates with the tissue bodily function, the physical parameter is a direct measure of the efficacy of the pharmaceutical agent to restore the bodily function, i.e., to treat the disease.

[0006] Therefore, the present invention relates to a method for evaluating the efficacy of a pharmaceutical agent suspected to be effective in treating a disease, said disease being preferably a genetic or non-genetic disease, said disease causing abnormalities in physiological bodily functions in a patient having said disease, said method comprising: (i) incubating an artificial tissue with a pharmaceutical agent, the artificial tissue being modified or having been modified to express the disease; (ii) determining at least one physical parameter of the artificial tissue obtained in step (i), wherein said physical parameter is an indicator of the functionality of a physiological body function; (iii) determining whether the value of the at least one physical parameter determined in step (ii) satisfies a predetermined threshold value for the physical parameter, thereby determining whether the pharmaceutical product is efficacious; If the value of the at least one physical parameter exceeds the predetermined threshold, it indicates that the pharmaceutical agent is effective in treating the disease by restoring at least part of the functionality of a physiological body function.

[0007] The predetermined threshold value is preferably based on or obtained by a standard (experiment) using statistical methods, more preferably a value that deviates from the (average) standard value obtained for at least one physical parameter in the standard (experiment) by (at least) 0.5 to 2.5 standard deviations (SD), and / or by (at least) 0.5 x SD, 1 x SD, 1.5 x SD or 2 x SD, optionally expressed as a % change from the (average) standard value.

[0008] The artificial tissue preferably comprises one or more of cardiac tissue, cardiac muscle, muscle tissue, skeletal muscle tissue, nerve tissue or connective tissue.

[0009] The engineered tissue is preferably gene-edited to contain a mutation that is implicated in a (genetic) disease, or alternatively, or in addition, the engineered tissue is preferably subject to non-genetic intervention to express the disease.

[0010] The pharmaceutical agent is preferably an advanced medical product such as a gene therapy pharmaceutical (GTMP), a cell therapy pharmaceutical (CTMP), or a tissue engineered product (TEP); a small molecule, a peptide, a protein, a nucleic acid, a synthetic RNA, a non-coding RNA, a ribonucleoprotein particle (RNP) complex, or a read-through enhancer.

[0011] The GTMP is preferably a viral vector, preferably a nanoparticle containing a nucleic acid and / or a protein, preferably encapsulated in an RNP, or a nucleic acid such as an antisense oligonucleotide. The GTMP is preferably a synthetic vector or a viral vector such as a retrovirus or lentivirus, such as an adenovirus or adeno-associated virus (AAV), more preferably the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrhlO, AAVrhl74, or any combination thereof, preferably AAV2 or AAV9.

[0012] The pharmaceutical preferably contains a nucleic acid encoding a protein for gene editing, such as an endonuclease, such as a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a clustered regularly interspaced short palindromic repeats (CRISPR) nuclease, or an expression thereof.

[0013] The GTMP preferably contains an endonuclease such as Cas9 or Cas13, preferably Streptococcus pyogenes Cas9 (SpCas9), S. aureus Cas9, S. auricularis Cas9, S. lugdunensis Cas9, or N. meningitides Cas9, and a guide RNA (gRNA).The GTMP preferably contains a nucleic acid encoding an endonuclease such as Cas9 or Cas13, preferably Streptococcus pyogenes Cas9 (SpCas9), S. aureus Cas9, S. auricularis Cas9, S. lugdunensis Cas9, or N. meningitides Cas9, and one or more guide RNAs (gRNA).

[0014] The at least one physical parameter is one or more selected from the group consisting of length, mass, time, current, temperature, light intensity, and physical parameters derived therefrom.

[0015] The at least one physical parameter is preferably one or more selected from the group consisting of force, movement, current, mass, tissue structure (expansion / condensation, transparency), and one or more biomechanical parameters (tissue compression, contraction, stiffness, tensile strength, elasticity, extensibility, excitability, resilience, toughness, etc.).

[0016] The at least one physical parameter is preferably one or more selected from tissue contraction, stiffness, force (stretch), spontaneous beating frequency, contraction time and / or velocity, relaxation time and / or velocity, (contraction) force, contraction force, and one or more multi-electrode array (MEA) measurements; preferably measured as one or more selected from the group consisting of pole deflection, Young's modulus (EHM) shortening, spontaneous beating frequency, contraction time and / or velocity, relaxation time and / or velocity, force of contraction (FOC), twitch tension (TT), resting tension (RT), and (weighted) mean firing rate, burst frequency (Hz), inter-spike interval within burst (ms), network burst (NB) frequency, inter-spike interval within NB (ms), and NB duration (s).

[0017] At least one of said physical parameters is preferably not the expression level of a gene or protein.

[0018] The artificial tissue preferably contains cells obtained from a patient who is to be treated with the pharmaceutical agent. Alternatively, or in addition, the artificial tissue preferably contains cells that are not obtained from a patient who is to be treated with the pharmaceutical agent.

[0019] In one embodiment, the artificial tissue used herein is cardiac muscle. Preferably, the at least one physical parameter is the force and / or movement of the artificial tissue. Preferably, the one or more conditions include a state in which the artificial tissue exerts a force of 0.01 mN or more, 0.05 mN or more, 0.1 mN or more, or 1 mN or more. Preferably, the disease is a genetic cardiac condition, preferably a genetic cardiac condition selected from the group consisting of genetic forms of dilated, hypertrophic, and arrhythmogenic cardiomyopathy (ACM), or fibroblastosis, and cardiomyopathy associated with a congenital metabolic disorder, more preferably a condition selected from the group consisting of Duchenne muscular dystrophy (DMD), Noonan syndrome, dilated cardiomyopathy, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, long / short QT syndrome, takotsubo cardiomyopathy, lysosomal storage disorders, titinopathy, and Barth syndrome. Alternatively or additionally, the disease is preferably a non-genetic cardiac condition, more preferably induced by mimicking neurohormonal and / or pharmacological stimuli (e.g., by catecholamines, angiotensin, and / or transforming growth factor (TGF) β), by drugs such as doxorubicin, tyrosine kinase inhibitors and / or cardiotoxic drugs, by mechanical injury such as crush injury, by temperature injury such as frostbite or thermal injury, by biophysical injury such as radiation injury, by infection such as viral infection with cardioactive virions such as Coxsackievirus, SARS coronavirus, cytomegalovirus and dengue virus, or by parasitic infection such as Chagas disease caused by Trypanosoma cruzi, optionally by bloodstream such as serum from a patient with cardiac disease, and / or by cells such as mononuclear cells (e.g., T cells, B cells, NK cells, macrophages) derived from the blood of a patient with cardiac disease.

[0020] In one embodiment, the artificial tissue is a neural tissue, preferably a neural organoid. Preferably, the at least one physical parameter is current and / or activity. Preferably, one or more of the conditions include localized and coordinated electrical bursts and / or clusters. Preferably, the disease is a neuronal disorder, preferably selected from the group consisting of neurodegenerative diseases (e.g., dementia, Parkinson's disease, Huntington's disease), neuroinflammatory diseases (e.g., multiple sclerosis), encephalitis (e.g., meningitis), channelopathies (e.g., epilepsy), and psychiatric disorders (including autism spectrum disorders and schizophrenia).

[0021] In one embodiment, the artificial tissue is muscle tissue, preferably skeletal muscle tissue. Preferably, the at least one physical parameter is force or movement. Preferably, the one or more conditions include a state in which the artificial tissue exerts a force of 0.01 mN or more, 0.05 mN or more, 0.1 mN or more, or 1 mN or more. Preferably, the disease is a genetic muscle disease, preferably a genetic muscle disease selected from the group consisting of Duchenne muscular dystrophy, facioscapulohumeral dystrophy, Becker dystrophy, Emery-Dreifuss dystrophy, myotonic dystrophy, limb dystrophy, oculopharyngeal muscular dystrophy, congenital dystrophy, congenital myopathy, myotonic dystrophy, inflammatory myopathy, familial periodic paralysis, and hereditary connective tissue diseases such as Marfan syndrome, Ehlers-Danlos syndrome, or Loeys-Dietz syndrome.

[0022] In one embodiment, the artificial tissue is a hybrid of different types of tissue, preferably a hybrid of (i) nerve cells and skeletal muscle, or (ii) nerve cells and cardiac muscle; more preferably, the hybrid of different types of tissue forms one or more neuromuscular junctions.

[0023] In one particular embodiment, The artificial tissue is cardiac muscle or skeletal muscle, the pharmaceutical product is an AAV2 and / or AAV9 viral vector comprising a nucleic acid encoding Cas9, preferably S. aureus Cas9, S. auricularis Cas9, S. lugdunensis Cas9, or N. meningitides Cas9, and one or two guide RNAs (gRNAs), wherein the Cas9 gene is under the control of a muscle-specific promoter such as CK8e or TNNT2; the disease is Duchenne muscular dystrophy, the physical parameter is a force; Preferably, the artificial tissue contains cells obtained from a patient who is to be treated with the pharmaceutical agent.

[0024] In one embodiment, the artificial tissue is a connective tissue. Preferably, the at least one physical parameter is a biomechanical parameter, such as tissue compression, contraction, stiffness, tensile strength, elasticity, extensibility, excitability, resilience, or toughness. Preferably, the disease is (i) a cardiac disease associated with myocardial fibrosis, such as heart failure with reduced or preserved left ventricular ejection fraction (due to unknown and / or genetic causes), myocardial infarction, congenital heart disease, cardiomyopathies with known genetic mutations, myocardial fibrosis induced by aging and / or senescence, drug-induced heart disease, metabolic heart disease, diseases (monogenic or polygenic) involving the heart and myocardial fibrosis, or diseases associated with reactive or replacement fibrosis in organs such as the kidney, liver, lung, and / or skin; (ii) a disease associated with scar formation and impaired wound healing, such as diabetes; and / or (iii) a hereditary connective tissue disease, such as Marfan syndrome, Ehlers-Danlos syndrome, or Loeys-Dietz syndrome.

[0025] The invention will be better understood by reference to the detailed description in conjunction with the non-limiting examples and the accompanying drawings, in which: [Brief explanation of the drawings]

[0026] [Figure 1]Figure 1 illustrates an efficacy test. Pluripotent stem cells or their differentiated products can be used as cellular starting material and reconstituted with a biologically derived or synthetic extracellular matrix (preferably collagen type I) to promote the formation of desired artificial tissue and / or organoid types (e.g., engineered human myocardium (EHM), engineered skeletal muscle (ESM), bioengineered neural organoids (BENO), artificial connective tissue (ECT), etc.). The artificial tissues are subject to specific functional assays using physical readouts such as contractile force (measured in Newtons, typically μN to mN), network activity (measured in volts, typically μV), viscoelasticity, and elastic modulus (measured in Pascals, typically kPa), which can be accurately measured, for example, as the effect of experimental drug exposure after delivery of nucleic acid drugs via viral (e.g., AAV) or non-viral (synthetic lipid nanoparticles or extracellular vesicles) particles or small molecules. [Figure 2] Figure 2 shows pole deflection analysis to evaluate the antifibrotic efficacy of drugs. A) iPSC-StC / fibroblast cells were used to generate ECTs. 5 ng / ml TGF-β1 was added on day 4. Pole deflection was analyzed daily. A biphasic contractile response was observed. B) An exemplary experimental design using a drug with a known IC50. C) Theoretical and / or illustrative results of an "ideal" antifibrotic drug in the absence and / or presence of TGF-β1. D) E) Schematic representation of the thresholds for antifibrotic activity obtained in the absence (dotted line) and presence (dashed line) of TGF-β1 during the drug efficacy test period. In Figure 2, the reference curves represent experimental data, with "Reference" representing the untreated ECT reference and "TGFβ-1" representing the TGFβ-1 ECT reference. [Figure 3]Figure 3 shows tensile tests to evaluate the antifibrotic efficacy of drugs. ECTs were generated using iPSC-StC / fibroblast cells. Once the plateau phase was reached, 5 ng / ml TGF-β1 and drugs were administered. Tensile tests were performed on a portion of the untreated ECTs before drug administration, and the remainder were analyzed 10 days later. A) Representative stress-strain curves are shown along with key biomechanical parameters. Young's modulus (the slope of the curve within the elastic region) indicates the stiffness of the ECT. B) Theoretical and / or example results for the stiffness of an "ideal" antifibrotic drug in the absence (left bar) and presence (right bar) of TGF-β1, preferably obtained at approximately day 10 of the drug efficacy test period. The significance level for the examples is indicated by dashed lines. In Figure 3, plateau and baseline stiffness are example data; "Baseline" indicates the untreated ECT baseline; and "TGFβ-1" indicates the TGFβ-1 ECT baseline. [Figure 4] Figure 4 shows gene correction in a Duchenne muscular dystrophy (DMD) EHM model. Force of contraction (FOC; mN) normalized by muscle content in EHM adjusted to contain 0%, 10%, 30%, 50%, or 100% gene-corrected cardiomyocytes; n=4-8, *p<0.05 (by 2-way ANOVA and Tukey's multiple comparison test). Adapted from Long et al. (2018), Sci Adv, 4:eaap9004. [Figure 5] Figure 5 shows a representative example of physical efficacy measurements in EHM, focusing on measuring contractile volume as force of contraction (FOC; mN) versus external calcium concentration (mM). As can be seen, FOC is significantly reduced in the DMD disease model (DMD - black circles) compared to the reference EHM (black triangles). The EDIT 1-6 curves show EHM in the DMD model after "therapeutic" genome editing, with complete functional recovery in EDIT 1-4 (filled gray symbols) and only minimal functional recovery in EDIT 5-6 (empty gray symbols), providing strong evidence for the "efficacy" of CRISPR editing in the EDIT 1-4 model and only limited "efficacy" of CRISPR editing in the EDIT 5-6 model. [Figure 6A-C]Figure 6 shows an example of a raster plot of neural network activity in BENO (day 60). A) An example raster plot, showing electrode / channel activity in a separate row (left and center). Spikes are indicated by black lines at the top of each plot, bursts are indicated by black lines within the main body of each plot, and network bursts, indicating synchronization between bursts, are highlighted by dashed rectangles. Parameters such as firing, burst, network burst duration, inter-firing interval, and inter-burst interval are graphically depicted in Figure 6A. Additionally, on the right, a diagram of BENO placed in a multi-electrode array well and covered with a coil that stabilizes the tissue above the electrodes is shown. As a representative example, three electrodes (EL1-3) are shown, which record local field potentials. The component activity recorded from each electrode over time is displayed in the raster plot (center), where each electrode is represented by a separate row. B) Representative activity raster plots of the same well before and after picrotoxin treatment. Increases in firing rate and burst frequency were observed, characteristic of a proconvulsant. Figure C shows a representative example of the effect of PIC on two network activity parameters listed in Table 14 (day 60): (weighted) mean firing rate (left bar) and burst frequency (right bar). The horizontal lines indicate the mean values ​​for PIC-treated BENO (center), CNQX-treated BENO (right), and blank vehicle-treated BENO (left). Statistics: n = 4 tissues / group, one-way ANOVA or nonparametric Student's t-test. *p < 0.05. The GABA receptor antagonist picrotoxin (PIC) was administered as a proconvulsant compound, resulting in increases in (weighted) mean firing rate and mean burst frequency. On the other hand, compared with baseline and PIC BENO, incubation in the presence of the AMPA / kainate receptor antagonist cyanquizaline (CNQX) reduced both the weighted mean firing rate and the mean burst frequency, indicating an anticonvulsant effect. DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention is described in detail below and is further illustrated by the accompanying examples and drawings.

[0028] The present invention provides for the use of artificial tissues in pharmaceutical efficacy testing. These efficacy tests may be used, for example, to demonstrate the claimed efficacy of drug candidate molecules to meet requirements in the drug approval process. These efficacy tests may also be used, for example, as efficacy tests in the GMP manufacturing process of approved drugs after the drug is approved. In this case, for example, if a batch of a pharmaceutical product meets a predetermined threshold (value) described herein, the batch may be released for use as a pharmaceutical product. In this case, the threshold described herein may serve as the release standard in the GMP pharmaceutical manufacturing process. Therefore, the above disclosure may be relevant to a method for evaluating the efficacy of a pharmaceutical product for treating a disease, such as a genetic or non-genetic disease. The disease may be characterized by the loss of a tissue's bodily function, and the tissue's function can be determined or characterized by a physical parameter, such as force. To this end, an artificial tissue expressing the disease is contacted (incubated) with a pharmaceutical product. After incubation, at least one physical parameter is determined. Because the physical parameter correlates with the tissue's bodily function, the physical parameter is a direct measure of the pharmaceutical product's efficacy in restoring the bodily function, i.e., treating the disease.

[0029] Therefore, the present invention relates to a method for evaluating the efficacy of a pharmaceutical agent suspected to be effective in treating a disease, said disease being preferably a genetic or non-genetic disease that causes abnormalities in physiological bodily functions in patients having said disease, said method comprising: (i) incubating an artificial tissue with a pharmaceutical agent, the artificial tissue being modified or having been modified to express the disease; (ii) determining at least one physical parameter of the artificial tissue obtained in step (i), said physical parameter being indicative of the functionality of a physiological body function.

[0030] The disease is preferably, but not limited to, a genetic disease and is characterized by an abnormality in physiological bodily function. As used herein, "physiological bodily function" typically refers to a function associated with a tissue, organ, or cell. Specific examples include muscle (which generates force and / or electrical signals as a physiological bodily function. More specific examples include cardiac muscle, which pumps blood through the circulatory system through the contraction of muscle cells; skeletal muscle, which is essential for all voluntary movement and is determined by the contractile force generated by skeletal muscle myofibrils); connective tissue, which is produced primarily by interstitial cells (fibroblasts outside the brain, glial cells within the brain) and provides mechanical stability to tissues through the production of extracellular matrix (such as collagen) and intrinsic contractility (e.g., known as wound healing); or nerve cells, whose activity as individual nerve cells and neural networks provides distinct electrical currents, which are important components of their physiological bodily function. In the context of the present invention, a disease causes partial or complete loss of physiological bodily function. "Abnormal function," as used herein, refers to a state in which an organ, tissue, or cell is unable to perform its normal physiological bodily function. In the context of the present invention, "abnormal" may refer to a complete loss of function (100% loss), or a partial loss of function, for example, a loss of about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 95% or more of the physiological bodily function (of the artificial tissue) of a healthy subject. The functionality of the physiological bodily function can be expressed by a physical parameter. For example, the physical parameter is muscle force. Thus, the abnormal functionality of the physiological bodily function may refer to a decrease in the value of at least one physical parameter, for example, a decrease of about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 4% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, or about 100% compared to a physical parameter determined using (the artificial tissue of) a healthy subject. A "healthy subject," as used herein, refers to a subject that is not suffering from a disease, preferably a subject that is not suffering from a disease that a pharmaceutical agent is suspected to be effective in treating.

[0031] "Efficacy," as used herein, refers to the ability of a pharmaceutical agent to exert an effect of a given intensity. Depending on the intensity (expressed in the context of the invention by at least one physical parameter), partial or complete recovery of bodily function may be observed as an effect. Such partial or complete recovery of bodily function may also be considered successful treatment. In other words, "therapeutically effective" may refer to partial or complete recovery of a physiological bodily function. "Treating" or "treatment" or "alleviating" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent, delay (alleviate), or at least partially alleviate or eliminate an abnormal condition (including a pathological condition) in an organism. Subjects in need of treatment include those already suffering from the disease, those susceptible to the disease, or those in whom the disease should be prevented (preventive measures). Thus, efficacy can be described, for example, as increasing or decreasing the value of at least one physical parameter by a value corresponding to at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% of the value of the at least one physical parameter determined, for example, using a (prosthetic tissue of) a healthy subject. Herein, efficacy of a pharmaceutical agent is assessed in step (iii) by determining whether the value of the at least one physical parameter determined in step (ii) satisfies a predetermined threshold value for the physical parameter, thereby determining whether the pharmaceutical agent is efficacious, wherein a value of the at least one physical parameter exceeding the predetermined threshold value indicates that the pharmaceutical agent is effective in treating the disease by restoring at least a portion of the functionality of a physiological body function. Detailed information regarding determining the efficacy of pharmaceuticals is disclosed further herein below.

[0032] <Pharmaceuticals> As described herein, the methods of the present invention are useful for determining whether a pharmaceutical product has effective efficacy in treating a particular disease. The methods of the present invention are applicable to a variety of different pharmaceutical products. However, the methods of the present invention are particularly useful for pharmaceutical products involving gene therapy or pharmaceutical products that alter (i.e., increase or decrease) gene expression or the expression of the protein encoded by the gene, such as ATMPs. Thus, the pharmaceutical product is preferably an advanced therapeutic medicinal product (ATMP), such as a gene therapy medicinal product (GTMP), a cell therapy medicinal product (CTMP), or a tissue engineered product (TEP); a small molecule, a peptide, a protein, a nucleic acid, a synthetic RNA, a non-coding RNA, a ribonucleoprotein granule (RNP) complex, or a read-through enhancer. Alternatively, or in addition, the term "pharmaceutical product" preferably also includes substances (chemical and / or pharmaceutical) under development, i.e., drug candidates in preclinical or clinical development.

[0033] In a preferred embodiment, the medicinal product is an advanced therapeutic medicinal product (ATMP).ATMP is, for example, as described and defined in Article 2 of Regulation (EC) No. 1394 / 2007 of the European Parliament and of the Council of November 13, 2007 on advanced therapeutic medicinal products, and amending Directive 2001 / 83 / EC and Regulation (EC) No. 726 / 2004.In particular, ATMP includes gene therapy medicinal products (preferably defined in Annex I, Part IV of Directive 2001 / 83 / EC), somatic cell therapy medicinal products (preferably defined in Annex I, Part IV of Directive 2001 / 83 / EC), tissue engineering products (TEP).Also, ATMP is described in Goula et al. 2020, J Clin Med Res, 12(12):780-786.

[0034] In one embodiment, the pharmaceutical is a GTMP. As used herein, a "gene therapy pharmaceutical" (GTMP) refers to a biological pharmaceutical having the following characteristics: (a) an active ingredient containing or consisting of a recombinant nucleic acid used or administered to a subject or patient for the purpose of regulating, repairing, replacing, adding, or deleting a genetic sequence; and (b) its therapeutic, prophylactic, or diagnostic effect is directly related to the sequence of the recombinant nucleic acid it contains or the gene expression product of this sequence. Gene therapy pharmaceuticals preferably do not include vaccines against infectious diseases. GTMPs are particularly useful in treating diseases such as inherited / genetic disorders, cancer, and tissue regeneration (e.g., vision loss). Representative GTMPs include, but are not limited to, idecbutagen viclucel (trade name: Abecma®), talimogene laherparepvec (trade name: Imlizic®), tisagenlecleucel (trade name: Kymriah®), atidarsagene autotemcel (trade name: Remmerdi®), boretigene neparvovec (trade name: Luxturna®), Strimvelis (an autologous CD34+ cell-enriched fraction containing CD34+ cells transduced with a retroviral vector encoding a human ADA cDNA sequence), brexcabtagene outtemcel (trade name: Tecartus®), axicabtagene ciloreucel (trade name: Yescarta®), onasemnogene abeparvovec (trade name: Zolgensma®), or vetibegrogen outtemcel (trade name: Zyntegro®).

[0035] In one embodiment, the pharmaceutical product is a CTMP. As used herein, a "cell therapy pharmaceutical product" (CTMP) includes recombinant CTMPs and / or SCTMPs. An example of a recombinant CTMP is CART-T cell therapy. CART-T cell therapy may be particularly useful in treating diseases such as lymphoma. As used herein, a "somatic cell therapy pharmaceutical product" (SCTMP) refers to a biological pharmaceutical product that (a) contains or consists of cells or tissues that have been significantly manipulated to alter their biological, physiological, or structural characteristics suitable for the intended therapeutic use, or cells or tissues that are not intended to be used for the same essential function in the recipient and donor, and (b) is presented as having properties, or is used, or is administered to humans for the treatment, prevention, or diagnosis of disease through the pharmacological, immunological, or metabolic effects of the cells or tissues. SCTMPs are particularly effective in treating diseases such as immune disorders, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, and cartilage defects, and can also be used for cardiac repair, skin replacement, or cancer immunotherapy. Representative SCTMPs include, but are not limited to, Allophycel or AMESANAR.

[0036] In one embodiment, the pharmaceutical product is a TEP. As used herein, "tissue engineered product" (TEP) and "artificial tissue" refer to a product containing or consisting of engineered cells or tissues, presented with properties for, or used or administered to, a human for the purpose of, regenerating, repairing, or replacing human tissue. A tissue engineered product may contain cells or tissues, including human or animal cells, or both. The cells or tissues may be viable or nonviable. They may also contain additional substances, such as cellular products, biomolecules, biomaterials, chemicals, scaffolds, or matrices. Cells or tissues are considered "artificial" if they meet at least one of the following conditions: they have been significantly manipulated to achieve biological properties, physiological functions, or structural characteristics appropriate for the intended regeneration, repair, or replacement; and / or they are not intended to be used in the recipient for the same essential function or set of functions as in the donor. This definition excludes products composed solely of nonviable human or animal cells and / or tissues, that do not contain viable cells or tissues, and that do not act primarily through pharmacological, immunological, or metabolic mechanisms. TEPs can be used in small-diameter vascular grafts, tracheal replacements, tissue-engineered esophageal, liver, or kidney transplants, or nerve conduits. Representative TEPs include, but are not limited to, Holoclar, MACI, MukCell, NOVOCART 3D, NOVOCART Inject, Obnitix, Spherox, and t2c001. Alternatively or optionally, representative TEPs include, but are not limited to, TEPs designed to release bioactive substances, such as growth factors, cytokines, and non-coding RNA, directly or via exosomes (e.g., Zimmermann WH, Soong PL: Pouch-like structure with paracrine activity and method for its preparation, European Patent Publication 2842581 and / or US Patent 9801817).

[0037] Nonessential manipulations include, for example, cutting, polishing, shaping, centrifugation, soaking in antibiotic or antimicrobial solutions, sterilization, irradiation, cell separation, concentration or purification, filtration, lyophilization, freezing, cryopreservation, and vitrification.

[0038] In one embodiment, the pharmaceutical agent is a small molecule. Small molecules or micromolecules are organic compounds with low molecular weights (≦1000 Daltons, preferably ≦500 Daltons) that may modulate biological processes, preferably with a size of about 1 nm. Larger structures such as nucleic acids and proteins, as well as many polysaccharides, are not small molecules, but their building blocks, the monomers (ribonucleotides or deoxyribonucleotides, amino acids, and monosaccharides, respectively), are generally classified as small molecules. Small molecules can be used as research tools to elucidate biological functions and as leads in the development of novel therapeutic agents. Some substances can inhibit specific protein functions or interfere with protein-protein interactions. As a specific example, the pharmaceutical agent is AMX0035 (Elybrio®), a composition containing sodium phenylbutyrate and taurursodiol, which is useful for the treatment of amyotrophic lateral sclerosis (ALS), Alzheimer's disease, and Wolfram syndrome. When efficacy is assessed using the efficacy tests described herein, the artificial tissue can be neural tissue or a neuromuscular junction. In such tests, the physical parameter may be electrical activity, or, if a motor plate is used, the force exerted by muscle tissue in contact with the neuromuscular junction. Further, representative small molecules include tafamidis in transthyretin amyloidosis, ataluren in nonsense-mutation-induced Duchenne muscular dystrophy (DMD), and migalastat in Fabry disease.

[0039] In one embodiment, the pharmaceutical agent is a peptide. In another embodiment, the pharmaceutical agent is a protein. The distinction between peptides and proteins is controversial, but for purposes of the present invention, peptides contain 20 or fewer amino acids in the amino acid chain, while proteins contain more than 20 amino acids in the amino acid chain. Exemplary peptides include insulin. Exemplary proteins include proteins in enzyme replacement therapy, for example, in Fabry disease (agalsidase alpha / beta) or Pompe disease (also known as type II glycogen storage disease (alglucosidase alpha)).

[0040] In one embodiment, the pharmaceutical agent is a nucleic acid. In the present invention, the term "nucleic acid" generally encompasses both DNA and RNA. However, nucleic acids in which nucleotides are replaced with artificial derivatives or modified nucleic acids (artificial or synthetic DNA or RNA) derived from natural DNA or RNA are also included in the context of the present invention. The pharmaceutical agent may be RNA. The pharmaceutical agent may be DNA. The term nucleic acid may also include phosphorodiamidate morpholino oligonucleotides (PMOs). In one embodiment, the pharmaceutical agent is eteplirsen, which is sold, for example, by Sarepta Therapeutics as Exondys51®. In this particular embodiment, the artificial tissue may be (cardiac) muscle tissue, and the physical parameter may be force. The term nucleic acid may also include peptide-conjugated PMOs (PPMOs). The term nucleic acid may also include synthetic RNAs. The term nucleic acid may also include non-coding RNAs. The term nucleic acid may also include locked nucleic acids (LNAs) and aptamers. The term nucleic acid may also include single-stranded nucleic acids (Anti-miR™) designed to bind and inhibit endogenous microRNA (miRNA) molecules. The term nucleic acid may also include circular RNA. The term nucleic acid may also include decoy RNA. The pharmaceutical may be a ribonucleoprotein particle (RNP). The RNP may be associated with a complex formed between RNA and an RNA-binding protein. Alternatively, or in addition, the RNP may be associated with a gapmer, such as a short (antisense) DNA strand flanked by strands of one or more RNA-like segments and / or RNA mimics (e.g., locked nucleic acid (LNA)).

[0041] As a specific example, the pharmaceutical product is WVE-004, available from Wave Life Sciences USA. WVE-004 is an antisense oligonucleotide (ASO) targeting C9ORF72 mutations that cause amyotrophic lateral sclerosis and frontotemporal dementia. The ASO mediates the degradation of C9ORF72 mRNA containing hexanucleotide expansions. Both mRNA and dipeptide proteins generated from hexanucleotide repeats are known to be toxic to neurons, and therefore reducing them may be beneficial. WVE-004 can be tested in the efficacy studies described herein, in which the artificial tissue is neural tissue or a neuromuscular junction, preferably one containing a human C9ORF72 gene with a repeat expansion. In such studies, the physical parameter may be electrical activity, or, if a neuromuscular junction is used, the force exerted by muscle tissue in contact with the motor plate.

[0042] Pharmaceuticals may also function as read-through enhancers. It has been shown that the binding of aminoglycoside antibiotics (e.g., gentamicin and G418) to eukaryotic ribosomes alters the structure of the ribosomal decoding center, allowing near-cognate aminoacyl-tRNA complexes to bind to premature termination codons (PTCs) and incorporate amino acids in place of premature translation termination. This process, called PTC read-through (PTCR), allows the formation and restoration of full-length proteins from PTC-bearing mRNAs. Examples of read-through enhancers include 2-aminothiazole-4-carboxamides such as amlexanox, PTC124, RTC13, RTC14, RTC204, RTC219, GJ071, GJ072, or ataluren, which are described, for example, in Rabea et al. (2019), ACS Med. Chem. Lett., 10(5):726-731, the chemical structures corresponding to which compounds are incorporated herein by reference.

[0043] In a preferred embodiment, GTMP may be a viral vector. The viral vector may include a lentivirus, adenovirus, or retrovirus such as adeno-associated virus (AAV). Preferably, the viral vector is an AAV, more preferably an AAV selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or any combination thereof, and most preferably AAV2 or AAV9. Alternatively, AAV (e.g., rhAAV) or synthetic vectors having a natural host other than humans may be used.

[0044] The GTMP may be a nanoparticle, preferably containing a nucleic acid and / or a protein, preferably encapsulated in an RNP. The nanoparticle may be a synthetic nanoparticle or a nanoparticle of biological origin. Biological nanoparticles may include, for example, microvesicles, exosomes, etc. Nanoparticles are described, for example, in Li et al. (2007), Pharm Res, 24(3):438-49, which is incorporated herein by reference. The pharmaceutical agent may be a nucleic acid, such as an antisense oligonucleotide. The pharmaceutical agent may also be an extracellular vesicle (e.g., isolated from the supernatant of cells, such as iPSCs), in which the cells have been modified to produce a specific cargo encapsulated in the extracellular vesicle.

[0045] To achieve a therapeutic, prophylactic, or diagnostic effect, the pharmaceutical agent may contain a protein for gene editing, such as an endonuclease, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPRs), nuclease-dead endonucleases, or endonucleases modified to contain base and prime editing protein components. Alternatively, or in addition, the pharmaceutical agent may contain a nucleic acid, where expression of the nucleic acid results in the production of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPRs), nuclease-dead endonucleases, or endonucleases modified to contain base and prime editing protein components. Preferably, the pharmaceutical comprises GTMP, which comprises an endonuclease such as Cas9 or Cas13, preferably Streptococcus pyogenes Cas9 (SpCas9), S. aureus Cas9, Staphylococcus auricularis Cas9, Staphylococcus lugdunensis Cas9, or Neisseria meningitides Cas9, and a guide RNA (gRNA); or comprises a nucleic acid encoding an endonuclease such as Cas9 or Cas13, preferably Streptococcus pyogenes Cas9 (SpCas9), S. aureus Cas9, Staphylococcus auricularis Cas9, Staphylococcus lugdunensis Cas9, or Neisseria meningitides Cas9, and a guide RNA (gRNA).

[0046] <Physical parameters> As described herein, a key advantage of the present invention is that, rather than solely using surrogate markers such as protein or gene expression, the physiological functionality of an artificial tissue or organ is directly measured by incubating the artificial tissue with a pharmaceutical agent and then determining physical parameters, such as the force exerted by artificial (cardiac and / or skeletal) muscle or connective tissue, or network activity in artificial neural tissue, such as BENO. This allows for direct assessment of the efficacy of a pharmaceutical agent in restoring the functionality of a physiological bodily function, compared to pathologies such as those seen in muscular dystrophy, fibroblastosis, or neurodegenerative diseases. The methods of the present invention provide multimodal information regarding disease phenotypes and their pharmacological remediation, using a "precision medicine" approach, without precluding further analysis of biomarkers, such as gene or protein expression of biomarkers. Thus, the methods of the present invention preferably further comprise analysis of biomarkers, such as gene or protein expression of biomarkers, which are preferably clinically relevant. However, it is preferred that at least one physical parameter is not gene or protein expression.

[0047] "Physical parameter" or "physical quantity," both used interchangeably throughout this specification, refer to a physical property of a substance or system that can be quantified by measurement. A physical parameter can be expressed as a value that is the algebraic multiplication of a number and a unit. For example, the physical parameter mass can be quantified as n kg, where n is a number and kg is a unit.

[0048] Physical quantities are divided into fundamental parameters and derived parameters. The fundamental parameters of a given system of physical quantities are a subset of those physical quantities; no fundamental quantity can be expressed in terms of other physical quantities, but all physical quantities in the system can be expressed in terms of fundamental quantities. Preferred fundamental quantities can be selected from the group consisting of length, mass, time, current, temperature, and luminous intensity. Thus, the physical parameters are preferably one or more selected from the group consisting of length, mass, time, current, temperature, luminous intensity, quantity of substance, and physical parameters derived therefrom. The physical parameters are more preferably one or more selected from the group consisting of length, mass, time, current, temperature, luminous intensity, and physical parameters derived therefrom. Preferably, at least one physical parameter is not the amount of a gene product or protein.

[0049] A derived quantity or physical parameter is a quantity defined in a quantity system in terms of only the base quantities of that quantity system. Thus, the physical parameter is preferably one or more selected from the group consisting of force, motion, current, mass, tissue structure (expansion / condensation, transparency), or a biomechanical parameter such as tissue compression, contraction, stiffness, tensile strength, elasticity, extensibility, excitability, resilience, toughness, etc. As used herein, rheological parameters encompass the biomechanical parameters disclosed herein. Thus, alternatively and / or optionally, a physical parameter may also be a rheological parameter.

[0050] The at least one physical parameter is preferably tissue contraction, stiffness, force (stretch), spontaneous beating frequency, (contraction) force, contractile force, and one or more MEA measurements; preferably measured as one or more selected from the group consisting of pole deflection, Young's modulus, (EHM) shortening, spontaneous beating frequency, force of contraction (FOC), twitch tension, and (weighted) mean firing rate, burst frequency (Hz), inter-burst spike interval (ms), network burst (NB) frequency, inter-NB spike interval (ms), and NB duration (s). Alternatively, or in addition, the at least one physical parameter is preferably one or more selected from the group consisting of pole deflection, Young's modulus, (EHM) shortening, spontaneous beating frequency, force of contraction (FOC), twitch tension, and one or more selected from the group consisting of (weighted) mean firing rate, burst frequency (Hz), inter-burst spike interval (ms), NB frequency, inter-NB spike interval (ms), and / or NB duration (s).

[0051] Alternatively, or in addition, the physical parameters may be one or more selected from the following list:

[0052] [Table 1]

[0053] The physical parameters may also be one or more selected from the following list:

[0054] [Table 2] JPEG2026501000000003.jpg255161JPEG2026501000000004.jpg255168JPEG2026501000000005.jpg25516 1JPEG2026501000000006.jpg255161JPEG2026501000000007.jpg255167JPEG2026501000000008.jpg78168

[0055] Further stiffness moduli are expressed in the following formulas: G' = G × cos(δ), which is the "storage" or "elastic" modulus; G'' = G × sin(δ), which is the "loss" or "plastic" modulus; and tan δ = G'' / G', hereafter a measure of elasticity (tan δ < 1) or plasticity (tan δ > 1). These may also be employed and measured as physical parameters in the context of the present invention. Examples of further physical parameters include strain rate, which is expressed as dF / dt max = increase in maximum force over time of contraction amplitude (a measure of contraction), or dF / dt min = decrease in maximum force over time of contraction amplitude (a measure of relaxation).

[0056] The method of the present invention may comprise a step of comparing whether the (value of the) physical parameter obtained in step (ii) of the method corresponds to a certain predetermined threshold value. Thus, the present invention relates to a method for evaluating the efficacy of a pharmaceutical agent suspected to be effective in treating a disease, said disease being preferably a genetic or non-genetic disease, which causes abnormalities in physiological body functions in patients having the disease, said method comprising: (i) incubating an artificial tissue with a pharmaceutical agent, the artificial tissue being modified or having been modified to express the disease; (ii) determining at least one physical parameter of the artificial tissue obtained in step (i), wherein said physical parameter is an indicator of the functionality of a physiological body function; (iii) determining whether the value of the at least one physical parameter determined in step (ii) satisfies a predetermined threshold value for the physical parameter, thereby determining whether the pharmaceutical product is efficacious; If the value of the at least one physical parameter exceeds the predetermined threshold, it indicates that the pharmaceutical agent is effective in treating the disease by restoring at least part of the functionality of a physiological body function.

[0057] Depending on the nature of the disease and the physical parameters, a pharmaceutical product may aim to increase a physical parameter (e.g., strength in muscular dystrophy) or decrease a physical parameter (e.g., heart rate in tachycardia). Such distinctions are within the capabilities of those skilled in the art. Therefore, it can be said that the value of at least one physical parameter fulfilling, satisfying, and / or matching a predetermined threshold indicates that the pharmaceutical product has efficacy for treating the disease. More specifically, a value of at least one determined physical parameter exceeding a predetermined threshold herein preferably refers to a value of at least one physical parameter that is at least equal to, (preferably significantly) higher, or lower than the predetermined threshold, depending on the disease, pharmaceutical product, and / or physical parameter under study, indicating that the pharmaceutical product has efficacy for treating the disease by at least partially restoring the functionality of a physiological body function. For example, if the artificial tissue is a bioengineered neuronal organoid (BENO), incubation with an AMPA / kainate receptor antagonist, such as cyanoquinoxaline (CNQX), that is competitive with glutamate receptors (AMPA) is expected to result in a decrease in the physical parameter "weighted average firing rate" due to silencing of excitatory networks, and / or a decrease in synaptic activity, which can be exemplified by an increase in the physical parameter "inter-burst spike interval" (see, for example, Figure 6C and Experiment 9). Thus, satisfying each predetermined threshold (e.g., a threshold for weighted average firing rate) involves, for example, comparing the determined weighted average firing rate value with the predetermined threshold value for the physical parameter, where the determined weighted average firing rate value exceeds the respective predetermined threshold, and the determined weighted average firing rate value falls below the respective predetermined threshold, indicating the efficacy of the receptor antagonist. For at least one physical parameter (e.g., including or consisting of inter-burst spike intervals), efficacy of the burst antagonist is indicated when the determined inter-burst spike interval values ​​exceed a respective predetermined threshold, and thus efficacy of the receptor antagonist is indicated when the determined inter-burst spike interval values ​​are higher than a respective predetermined threshold.

[0058] Here, the term "predetermined" refers to an a priori determination, preferably in the context of a threshold. As an example, such a predetermined threshold may relate to the value of at least one physical parameter determined a priori by a person skilled in the art, preferably based on a standard (experiment). Thus, the predetermined threshold may be determined a priori in a standard (experiment). Alternatively, or in addition, the predetermined threshold may relate to the value of at least one physical parameter determined by a person skilled in the art based on a standard (experiment) conducted in parallel and / or at least partially overlapping with the efficacy measurement described herein. Thus, the standard (experiment) and efficacy test can be conducted at least partially overlapping and / or parallel. Regarding the standard (experiment), the standard (experiment) preferably includes steps (i) and (ii), and preferably mirrors the efficacy test described herein in the sense that in step (i), no pharmaceutical agent is used and / or a buffer and / or an empty vector is used instead of the pharmaceutical agent. The value(s) of at least one physical parameter obtained in the reference (experiment) is preferably investigated to determine a respective predetermined threshold value that can be used in the method of the present invention to test the efficacy of a pharmaceutical agent under investigation for treating a disease. The described reference (experiment) has the advantage that the experimental settings and / or conditions between the reference (experiment) and the efficacy test are equivalent, preferably identical, except for incubation with the pharmaceutical agent. Thus, the effect of the pharmaceutical agent on at least one physical parameter can be evaluated without or with only limited bias that may arise from differences in the experimental settings and / or conditions. Therefore, such a predetermined threshold value can be considered to be optimized taking into account the characteristics of the artificial tissue, disease, pharmaceutical agent, and / or physical parameter(s) used in the method of the present invention for evaluating the efficacy of a pharmaceutical agent. Exemplary and / or preferred examples of suitable combinations of artificial tissue, disease, and physical parameter are described in the respective artificial tissue sections below.

[0059] Said predetermined threshold is determined based on standard (experiment), and at least one physical parameter of artificial tissue and / or artificial organoid is determined by comparing the value of at least one physical parameter obtained in the absence of pharmaceutical agent or in the absence and presence of pharmaceutical agent respectively.Said standard (experiment) can include one or more conditions, and therefore refers to the standard (experiment) based on: i) ia) derived from a healthy individual, and / or ib) well-characterized iPSCs that represent a healthy wild-type; ii) unprocessed artificial tissues and / or unprocessed artificial organoids, iia) which have been modified or altered to express a disease, or iib) which are derived from a patient with a disease, preferably a genetic or non-genetic disease, for the treatment of which the efficacy of a pharmaceutical agent is to be assessed using the method according to the invention; and / or iii) Artificial tissues and / or organoids modified by permanent genetic repair of disease-associated, preferably disease-causing, mutations.

[0060] Option i) is also referred to herein as the "normal" standard. This option may be advantageous for investigating the effect of a pharmaceutical agent on healthy wild-type individuals, and therefore does not involve disease-specific confounding effects. Such an effect may be an increase or decrease in a physical parameter, such as contractile force, due to treatment compared to the "normal" standard. Alternatively or optionally, option i) may be advantageous in studies that focus on, for example, assessing the extent to which treatment with a given drug restores the functionality of a physiological physical function of a research subject compared to healthy individuals. Option ii) is also referred to herein as the "diseased" standard. This option may be advantageous in studies that focus on, for example, the extent to which treatment with a drug restores the functionality of a physiological physical function of a research subject compared to untreated patients suffering from a disease. Alternatively or additionally, the above option may be advantageous for assessing time-course information regarding the efficacy of a pharmaceutical agent, such as the time to assess efficacy of a treatment and / or the duration of effect. Therefore, such "disease" standards may be the same or different artificial tissues and / or organoids as those used in efficacy tests as disclosed herein. Taking these factors into consideration, option ii) is considered preferable herein as a standard (experiment) for determining a predetermined threshold for efficacy tests as described herein, particularly when the primary objective is to ensure symptomatic relief and / or improvement of physiological bodily functions upon treatment with a pharmaceutical. Thus, pharmaceuticals with the efficacy of shifting the (average) value of a physical parameter from the (average) value of the physical parameter observed in the "disease" standard can be identified. Option iii) may be advantageous for determining the maximum dynamic range of pharmaceuticals and / or substances that can be investigated as a standard (experiment). Regarding options ia) and / or iia), investigating treated and untreated artificial tissues modeling a disease may be advantageous for evaluating the efficacy of a drug on a patient population that shares a specific mutation underlying the disease. In such a scenario, each mutation(s) is preferably introduced de novo into well-characterized iPSC cells.This has the advantage that the effect of a drug on a disease caused by the specific mutation can be evaluated in a well-defined setting. Option ib) and / or iib) may be particularly advantageous when evaluating the efficacy of a drug in personalized patient treatment. In such a case, taking artificial connective tissue (ECT) as an example, iPSC-derived stromal cells for ECT production are preferably generated from somatic cells obtained from the patient. Thus, the effect of a specific drug treatment, more specifically, the efficacy of the drug, can be evaluated in the patient's unique genetic background, thereby reflecting the potential in vivo effects of the drug. Therefore, the predetermined threshold is preferably determined based on and / or obtained by a reference experiment, and preferably, at least one physical parameter of the artificial tissue is determined at least in the absence of the drug. The artificial tissue used in the reference experiment is preferably derived from healthy individuals and / or well-characterized iPSCs representing healthy wild-type individuals. Alternatively or additionally, the artificial tissue used in the reference (experiment) is preferably unprocessed artificial tissue modified to express a disease or derived from a subject with the disease, and the disease is preferably a genetic or non-genetic disease, for which the efficacy of a pharmaceutical product for treatment is evaluated using the efficacy test disclosed herein. In all cases described herein, it is assumed that a pharmaceutical product associated with a change (preferably a statistically significant change) in the (average) value of at least one physical parameter compared to the respective (average) parameter value in at least one reference (experiment and / or condition), preferably at least the "diseased" reference (experiment and / or condition), is preferably evaluated as having efficacy for treating the respective disease. Preferably, in the case of two or more references (experiments), the efficacy of the pharmaceutical product is indicated when the value of at least one physical parameter exceeds a predetermined threshold value for the parameter in at least one reference (experiment and / or condition), preferably at least the "diseased" reference (experiment and / or condition).

[0061] Regarding the predetermined threshold value, those skilled in the art can determine the predetermined threshold value. An exemplary method includes determining at least one physical parameter of an artificial tissue obtained from a healthy subject, i.e., a subject not affected by the respective disease. Advantageously, in such a comparison, the artificial tissue does not contain mutations that are thought to be the cause of the disease (see isogenic). The value of at least one physical parameter obtained from such a reference artificial tissue can be considered as the predetermined threshold. In some cases, complete treatment, i.e., restoring physiological bodily functions to those of a healthy subject, is (yet) impossible. In such cases, the predetermined threshold can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, or 90%, preferably at least 20%, 25%, 30%, 35%, and / or 40% of the value of at least one physical parameter of a healthy subject.

[0062] Preferably, the predetermined threshold is determined based on and / or obtained by a reference (experiment), and the reference (experiment) is repeated at least once, i.e., at least once, more preferably at least three, four, five, six, seven, eight, nine, or ten times. This has the advantage that statistical methods can be performed and the efficacy of the pharmaceutical under study can be statistically evaluated while minimizing the influence of potential bias. Alternatively or additionally, the efficacy test described herein is repeated at least once, i.e., at least once, more preferably at least three, four, five, six, seven, eight, nine, or ten times. Ideally, the reference experiment(s) and the efficacy test(s) are repeated at least once, i.e., at least once, more preferably at least three, four, five, six, seven, eight, nine, or ten times.

[0063] Preferably, the predetermined threshold is determined using a statistical method. For example, the statistical method may be a significance test such as a t-test, an f-test, a chi-square test, a (non-parametric) Student's t-test, a one-way analysis of variance (preferably combined with Dunnett's multiple comparison test), or a two-way analysis of variance (preferably combined with Tukey's multiple comparison test). This is preferably the case when the predetermined threshold is determined based on and / or obtained by a standard (experiment), in which at least one physical parameter of the artificial tissue is determined both in the presence (treated) and absence (untreated) of a pharmaceutical agent, and the respective physical parameter values ​​are compared to determine the predetermined threshold. In such a scenario, the predetermined threshold can represent the minimum value of at least one physical parameter determined to be significantly different between values ​​obtained from treated artificial tissue and untreated artificial tissue under equivalent, or preferably identical, experimental conditions. As another example, the statistical method may include determining the standard deviation (SD). This is preferably the case when the predetermined threshold is determined based on a reference (experiment) and / or based on values ​​obtained by the reference (experiment), in which at least one physical parameter of the artificial tissue is determined in the absence of a pharmaceutical agent, and the reference (experiment) may be repeated at least 3, 4, 5, 6, 7, 8, 9, or 10 times. In such a scenario, the predetermined threshold preferably represents a value of at least one physical parameter that differs from the respective (average) physical parameter value observed in the repeated reference (experiment) by, for example, 1 × SD, 1.5 × SD, 2 × SD, 2.5 × SD, or 3 × SD. Alternatively, or optionally, the predetermined threshold determined using statistical methods can be expressed as a percentage change (% change) relative to the respective (average) reference value of the physical parameter. In any of these scenarios, the value of at least one physical parameter that exceeds the predetermined threshold preferably significantly deviates from the respective (average) value obtained in the reference (experiment), and the direction of deviation from the respective (average) reference (experimental) value is evaluated by a person skilled in the art in terms of the disease, artificial tissue, and / or physical parameter being studied.

[0064] The predetermined threshold value is based on or obtained by a standard (experiment) using statistical methods, preferably repeated at least once, i.e. at least once, more preferably at least 3, 4, 5, 6, 7, 8, 9 or 10 times. It is particularly preferred that the predetermined threshold value deviates from the (average) reference value obtained for at least one physical parameter in the standard (experiment) by (at least) 0.5 to 2.5 standard deviations (SD), and / or by (at least) 0.5 x SD, 1 x SD, 1.5 x SD or 2 x SD. Alternatively or additionally, the predetermined threshold value may be (at least) 0.5 to 2.5 standard deviations (SD), and / or (at least) 0.5 x SD, 1 x SD (standard deviation), 1.5 x SD or 2 x SD from the (average) reference value obtained for at least one physical parameter in a reference (experiment), expressed as a % change from the (average) reference value.

[0065] Those skilled in the art will recognize that a pharmaceutical agent whose efficacy is to be evaluated using the method of the present invention may affect one or more physical parameters. In this case, those skilled in the art will be able to identify the most relevant physical parameters to be determined for efficacy testing. Those skilled in the art will also be able to interpret results obtained when only a portion of the determined physical parameter values, e.g., one, two, three, half, etc., meet or correspond to a respective predetermined threshold. For example, the magnitude of the effect of a particular drug treatment on a physical parameter may vary depending on the target of each physical parameter. For example, if a pharmaceutical agent is a general excitatory neuron inhibitor, investigating the therapeutic effect may reveal suppression of excitatory networks as a primary effect. This may be reflected, for example, by at least one physical parameter being and / or including (weighted) mean firing rate, burst frequency, and / or network burst (NB) frequency. The treatment may also have secondary effects arising from the primary effect, which may be reflected by at least one physical parameter being and / or including, for example, inter-burst spike intervals. Those skilled in the art will recognize that such secondary effects may have smaller effect sizes than the primary effects, for example, which may result in the physical parameters associated with detecting the primary effects exceeding their respective thresholds, as opposed to the physical parameters associated with the secondary effects. A similar scenario is observed in the case of secondary effects that exhibit larger effect sizes than the primary effects. Thus, depending on the disease, pharmaceutical product, artificial tissue, and at least one physical parameter determined for potency measurement, those skilled in the art may consider that the efficacy of a pharmaceutical product is indicated when one or more values ​​of at least one physical parameter exceed their respective thresholds. Thus, in the case of one or more physical parameters, the value of at least one of the one or more physical parameters is preferably required to exceed its respective threshold to indicate efficacy, at least in terms of the "disease" criteria (condition and / or experiment).

[0066] Those skilled in the art will recognize that the efficacy of a drug presumed to be effective in treating a disease may be evaluated under constraints such as the concentration of the drug used during incubation and / or the duration of incubation. Generally, the appropriate concentration to be investigated using an efficacy test as disclosed herein may be a clinically meaningful concentration, and the efficacy of a drug is tested when at least a clinically meaningful concentration of the drug is associated with at least (mean) values ​​of physical parameters that exceed respective predetermined thresholds obtained by at least one criterion (experiment) of the drug, preferably at least the "disease" criterion. For example, for small molecules, EC50 / IC50 may be used as an indicator of the appropriate concentration, since drugs preferably not only exhibit efficacy within their biophysical solubility but also within a range from, for example, 100-fold higher to 100-fold lower than the EC50 / IC50. For non-classical pharmaceutical products, e.g., gene therapy drugs such as AAV-mediated therapeutic products, a concentration of, for example, 10 per kg may be used. 12 ~10 14 To determine a clinically meaningful dosage (concentration), such as vector genome (vg / kg), a potency-based approach may be advantageous. Accordingly, those skilled in the art will recognize that the method according to the present invention may be used to determine, for example, the minimum amount, concentration, incubation time, and / or treatment time of a pharmaceutical product, these factors by which a pharmaceutical product may be assessed as efficacious for treating a disease. Consequently, the method according to the present invention may advantageously be used to determine a suitable (e.g., minimum) potent drug concentration and / or a suitable (e.g., minimum) treatment duration for the intended treatment of a disease, where the drug is assessed as efficacious for treating a disease by at least partial restoration of physiological bodily functions.

[0067] <Artificial tissue> To be suitable for efficacy testing, the artificial tissue used in the methods of the present invention is representative of the disease to be treated with the pharmaceutical agent. As used herein, "representing a disease" refers to an artificial tissue that has a deviation from a healthy state. This deviation leads to an abnormality in physiological bodily function that can be measured by determining at least one physical parameter. As a specific example, a mutation ("deviation") causes the expression of a non-functional dystrophin protein in Duchenne muscular dystrophy (DMD), resulting in a loss of muscle strength ("abnormal physiological bodily function"). Such a mutation can be considered to be "causative of the (genetic) disease." This loss of muscle strength can be determined by measuring the force exerted by the patient's muscles ("at least one physical parameter") and / or by mimicking it using an artificial tissue. In this specific example, a pharmaceutical agent can be designed to restore the mutation, e.g., by gene therapy, so that the non-functional dystrophin is functional again. Turning now to an exemplary efficacy test: the artificial tissue is a tissue that represents a disease, such as DMD. Although there are several methods for producing artificial (DMD) muscle tissue that lacks or exhibits reduced force-producing capacity or other measurable physical functions, such treatments do not reflect the actual or true cause of the disease. Such tissue may not represent the disease because the abnormality in physiological bodily function (here, reduced or lost force-producing capacity) is not causally related to the deviation from a healthy state, but is merely a non-disease-related therapeutic effect. In a corresponding exemplary efficacy test, to represent the disease, the artificial tissue contains a disease-causing mutation, e.g., DMD. In this way, the efficacy of pharmaceuticals aimed at eliminating (treating) the deviation (e.g., by repairing a dysfunctional dystrophin gene) or at least bringing the subject to a state in which the original deviation no longer has an effect (e.g., by introducing a functional replacement of the dystrophin gene or a correction of the gene reading frame) can be measured and assayed for efficacy.Thus, there is an interplay between the drug, its mechanism of action, the disease, and how the engineered tissue mimics the disease. A typical efficacy test method is shown schematically in Figure 1.

[0068] Such artificial tissues are known to those skilled in the art. Artificial tissues suitable for the present invention include, for example, cardiac muscle (see, e.g., International Publication No. 2015 / 040142), skeletal muscle (see, e.g., International Publication No. 2021 / 0741236), connective tissue (see, e.g., European Patent No. 3945133), neurons or neuronal organoids (see, e.g., International Publication No. 2018 / 228948), or human cardiac muscle (see, e.g., International Publication No. 2015 / 025030), all of which are incorporated herein by reference. Furthermore, methods for genetically engineering tissues or pluripotent stem cells prior to differentiation into engineered tissues are well known to those skilled in the art. Exemplary procedures are described in Long et al. (2018), Sci Adv, 4:eaap9004.

[0069] Thus, the artificial tissue may comprise one or more of cardiac tissue, cardiac muscle tissue, muscle tissue, skeletal muscle tissue, nerve tissue or connective tissue.

[0070] As outlined herein, the engineered tissue is or has been engineered to express the disease. This can be achieved by a variety of means. In one embodiment, the engineered tissue is gene-edited to contain a mutation believed to be causative of the (genetic) disease.

[0071] In addition to genetic diseases, the efficacy testing method of the present invention can also be used to analyze the efficacy of treatment for non-genetic diseases. Gene therapy is also useful for such non-genetic diseases. Therefore, the artificial tissue may be subjected to non-genetic intervention to express the disease. Such non-genetic intervention may be drug exposure, factor exposure (e.g., growth factors, cytokines), body fluid exposure (e.g., patient serum), mechanical intervention (e.g., cutting injury), biophysical intervention (e.g., heat or radiation injury), and / or cell exposure (e.g., PBMCs isolated from a patient).

[0072] The biological species of the cells of the artificial tissue is not particularly limited. However, it is advantageously the same as the species of the subject to be treated with the pharmaceutical. The artificial tissue may be derived from humans, non-human primates such as macaques or marmosets, pigs, or rodents such as mice, rats, or guinea pigs. In a preferred embodiment, the artificial tissue is human. However, non-human animal models remain important as translational research models. Therefore, the artificial tissue may also be derived from a humanized animal model.

[0073] A further advantage of the present invention is that the engineered tissue can be universal, i.e., derived initially from a specific donor for use in various efficacy studies for different subjects, or personalized, i.e., derived directly from the patient undergoing pharmaceutical treatment, thus providing great flexibility.

[0074] In one embodiment, the artificial tissue contains cells obtained from a patient who will be treated with the pharmaceutical agent. This does not necessarily exclude the presence of extracellular matrix, such as a collagen matrix, obtained from a subject other than the patient. In other words, all cells contained in the artificial tissue may be originally obtained from the patient who will be treated with the pharmaceutical agent. This approach is a further step toward personalized medicine. It allows for the direct evaluation of the efficacy of pharmaceutical agents in specific patients. This allows for the examination of other influencing factors, such as novel mutations that may affect (genetic) diseases. Furthermore, the efficacy of pharmaceutical agents can be more thoroughly evaluated in multifactorial genetic diseases, such as asthma, autoimmune diseases such as multiple sclerosis, cancer, ciliosis, diabetes, heart disease, hypertension, inflammatory bowel disease, intellectual disability, mood disorders, obesity, refractive errors, and infertility.

[0075] As outlined above, the methods of the present invention also allow for the universal application of artificial tissue derived from cells of a subject for efficacy measurements, and therefore, artificial tissue may contain cells that are not obtained from the patient who is to be treated with the pharmaceutical agent.

[0076] <Disease> The methods of the present invention are suitable for a variety of diseases, including genetic diseases, but also non-genetic diseases. In one embodiment, the disease is a genetic disease. In another embodiment, the disease is a non-genetic disease.

[0077] As used herein, the terms "disorder" and "disease" are used interchangeably. Furthermore, as used herein, a "genetic disorder" refers to a health problem caused by one or more abnormalities in the genome. This can be caused by mutations in a single gene (monogenic) or multiple genes (polygenic), or by chromosomal abnormalities. The causative mutation can occur spontaneously before embryonic development (de novo mutation), be inherited from two parents who are carriers of the defective gene (autosomal recessive inheritance), or be inherited from both parents who have the disorder (autosomal dominant inheritance). Genetic disorders inherited from one or both parents are also classified as genetic disorders. Some diseases are caused by mutations on the X chromosome and are X-linked. Few diseases are inherited on the Y chromosome or mitochondrial DNA (due to their size). Genetic disorders exist before birth. The opposite of T-gene disorders is acquired disorders, or "non-genetic disorders." Most cancers involve genetic mutations in a small percentage of cells in the body and are acquired diseases. However, some cancer syndromes, such as BRCA mutations, are inherited genetic disorders.

[0078] In one embodiment, the disease is an inherited cardiac condition, preferably an inherited cardiac condition selected from the group consisting of genetic forms of dilated, hypertrophic, and arrhythmogenic cardiomyopathy (ACM), or fibroblastosis, and cardiomyopathy associated with a congenital metabolic disorder, more preferably Duchenne muscular dystrophy (DMD), Noonan syndrome, dilated cardiomyopathy (preferably involving myosin heavy chain (e.g., MHY7), troponin (e.g., TNNT2, TNNI3, TNNC1), dystrophin (DMD), desmin (DES), phospholamban (PLB), Presenilin (e.g., PSEN1 and PSEN2), actinin (e.g., ACTN2), tropomyosin (e.g., TPM1), titin (e.g., TTN), actin (e.g., ACTC1), laminin (e.g., LMNA)), hypertrophic cardiomyopathy (preferably associated with mutations in myosin heavy chain (e.g., MHY7), troponin (e.g., TNNT2, TNNI3), myosin-binding protein (e.g., MYBPC3), tropomyosin (e.g., TPM1), myosin light chain (e.g., MYL2, MYL3), titin (e.g., TTN), actin (e.g., associated with mutations in ACTC1, and / or AMP-activated protein kinase (e.g., PRKAG2)), arrhythmogenic right ventricular cardiomyopathy (preferably associated with mutations in desmoplakin (DSP), plakophilin (e.g., PKP2), plakoglobin (JUP), desmoglein (e.g., DSG2), desmocollin (e.g., DSC2), ryanodine receptor (e.g., RYR2), and / or laminin (e.g., LMNA)), long / short QT syndrome (preferably associated with mutations in sodium channels (e.g., SCN5A, S CN4B, SNTA1), potassium channels (e.g., KCNH2, KCNQ1, KCNJ2, KCNJ5, KCNE1, KCNE2) and / or calcium channels (e.g., CACNA1A, CACNA1C, CACNA2D1), bicarbonate / chloride exchangers (e.g., SLC4A3), caveolins (e.g., CAV3), and / or calmodulins (e.g., CALM1, CALM2, CALM3), Takotsubo cardiomyopathy, lysosomal storage disorders, titinopathy and Barth syndrome (preferablyAlternatively, or in addition, the disease is a condition selected from the group consisting of myosin heavy chain (e.g., MHY7), troponin (e.g., TNNT2, TNNI3, TNNC1), dystrophin (DMD), desmin (DES), phospholamban (PLB), presenilin (e.g., PSEN1 and PSEN2), actinin (e.g., ACTN2), tropomyosin (e.g., TPM1), titin (e.g., TTN), actin (e.g., ACTC1), laminin (e.g., LMNA), myosin-binding protein (e.g., MYBPC3), myosin light chain (e.g., MYL2, MYL3), AMP-activated protein kinase (e.g., PRKAG2), desmoplakin (DSP), plakophilin (e.g., PKP2), plakoglobin (JUP), desmog associated with and / or caused by mutations in genes and / or proteins selected from the group consisting of rhein (e.g., DSG2), desmocollin (e.g., DSC2), ryanodine receptor (e.g., RYR2), sodium channels (e.g., SCN5A, SCN4B, SNTA1), potassium channels (e.g., KCNH2, KCNQ1, KCNJ2, KCNJ5, KCNE1, KCNE2), calcium channels (e.g., CACNA1A, CACNA1C, CACNA2D1), bicarbonate / chloride exchangers (e.g., SLC4A3), caveolin (e.g., CAV3), calmodulin (e.g., CALM1, CALM2, CALM3), and tafazzin (TAZ).

[0079] In a further embodiment, the disease is a non-genetic cardiac condition, preferably induced by mimicking neurohormonal and / or pharmacological stimuli (e.g., by catecholamines, angiotensin, and / or transforming growth factor (TGF) β), by drugs such as doxorubicin, tyrosine kinase inhibitors and / or cardiotoxic drugs, by mechanical injury such as crush injury, by temperature injury such as frostbite or thermal injury, by biophysical injury such as radiation injury, by infection such as viral infection with cardioactive virions such as Coxsackievirus, SARS coronavirus, cytomegalovirus and Dengue virus, or by parasitic infection such as Chagas disease caused by Trypanosoma cruzi, optionally by bloodstream such as serum from a patient with cardiac disease, and / or by cells such as mononuclear cells (e.g., T cells, B cells, NK cells, macrophages) derived from the blood of a patient with cardiac disease. Examples of cardiotoxic drugs are well known to those skilled in the art, see, for example, Mladenka et al. 2018 (Med Res Rev 2018;38:1332-1403, PMID: 29315692, DOI: 10.1002 / med.21476).

[0080] In one embodiment, the disease is a neuronal disorder, preferably selected from the group consisting of neurodegenerative diseases (e.g., dementia, Parkinson's disease, Huntington's disease), neuroinflammatory diseases (e.g., multiple sclerosis), cerebroinflammatory diseases (e.g., meningitis), channelopathies (e.g., epilepsy), and psychiatric disorders (including autism spectrum disorders and schizophrenia).

[0081] In one embodiment, the disease is a genetic muscle disease, preferably selected from the group consisting of Duchenne muscular dystrophy, facioscapulohumeral dystrophy, Becker dystrophy, Emery-Dreifuss dystrophy, myotonic dystrophy, limb dystrophy, oculopharyngeal muscular dystrophy, congenital dystrophies, congenital myopathies, myotonic dystrophy, familial periodic paralysis, and inherited connective tissue diseases such as Marfan syndrome, Ehlers-Danlos syndrome, or Loeys-Dietz syndrome.

[0082] In one embodiment, the disease is (i) a cardiac disease associated with myocardial fibrosis, such as heart failure (due to unknown and / or genetic causes) with reduced or preserved left ventricular ejection fraction, myocardial infarction, congenital heart disease, cardiomyopathies with known genetic mutations, aging and / or age-induced myocardial fibrosis, drug-induced heart disease, metabolic heart disease, diseases (monogenic or polygenic) involving the heart and myocardial fibrosis, diseases with reactive or replacement fibrosis in organs such as the kidney, liver, lung, and / or skin; (ii) a disease associated with scar formation and impaired wound healing, such as diabetes; and / or (iii) a hereditary connective tissue disease, such as Marfan syndrome, Ehlers-Danlos syndrome, or Loeys-Dietz syndrome.

[0083] <heart tissue> The method of the present invention can be used to test the efficacy of pharmaceuticals suspected to be effective in treating diseases affecting myocardium or myocardial tissue. Thus, the artificial tissue can be myocardium or myocardial tissue.

[0084] The physiological bodily function of myocardium (tissue) is to exert force to contract the ventricles, thereby pumping blood through the vessels of the circulatory system. To this end, cardiomyocytes present in the myocardium contract in a coordinated, synchronized manner. Therefore, the method of the present invention is particularly useful when the at least one physical parameter determined in the method is a force, e.g., expressed in Newtons (N), or a change in volume. Thus, in one embodiment, the at least one physical parameter is a force. In another embodiment, the at least one physical parameter is a change in volume, e.g., a change in the volume of a heart chamber formed by the artificial tissue. In another embodiment, the at least one physical parameter is the rate of muscle contraction-relaxation cycles.

[0085] Those skilled in the art can prepare an artificial heart (myocardium). Methods for producing artificial human myocardium are described, for example, in International Publication No. 2015 / 025030, International Publication No. 2015 / 040142, or Long et al. (2018), Sci Adv, 4:eaap9004. An exemplary method for obtaining cardiomyocytes from pluripotent stem cells is as follows: iPSCs are applied to TESR-E8 (STEMCELL Technologies) on 1:120 Matrigel in a PBS-coated plate and maintained in this medium, subcultured twice a week using EDTA solution (Versene, Thermo Fisher Scientific). For cardiac differentiation, iPSCs are cultured at 5x10 4 ~1×10 5 cells / cm 2and incubated for 3 days in RPMI, 2% B27, 200 mM L-ascorbic acid-2-phosphate sesquimagnesium salt hydrate (Asc; Sigma-Aldrich), activin A (9 ng / ml; R&D Systems), BMP4 (5 ng / ml; R&D Systems), 1 mM CHIR99021 (Stemgent), and FGF-2 (5 ng / ml; Miltenyi Biotec); then, after washing with fresh RPMI medium, cells can be cultured with 5 mM IWP4 (Stemgent) in RPMI supplemented with 2% B27 and 200 mM ascorbic acid from day 4 to day 13. Cardiomyocytes can then be metabolically purified by glucose deprivation in glucose-free RPMI (Thermo Fisher Scientific) supplemented with 2.2 mM sodium lactate (Sigma-Aldrich), 100 mM β-mercaptoethanol (Sigma-Aldrich), penicillin (100 U / ml), and streptomycin (100 mg / ml) from day 13 to day 17. To generate defined, serum-free engineered human myocardium (EHM), purified cardiomyocytes are mixed with HFF (American Type Culture Collection) at a 70%:30% ratio. The cell mixture can be reconstituted with pH-neutralized medical-grade bovine collagen (0.4 mg / EHM; LLC Collagen Solutions) and concentrated serum-free medium [2x RPMI, 8% B27 (without insulin), penicillin (200 U / ml), streptomycin (200 mg / ml)] supplemented with 4% B27 (without insulin), 1% non-essential amino acids, 2 mM glutamine, 300 mM ascorbic acid, IGF1 (100 ng / ml; AF-100-11), FGF-2 (10 ng / ml; AF-100-18B), VEGF165 (5 ng / ml; AF-100-20), TGF-b1 (5 ng / ml; AF-100-21C; all growth factors available from Pepro Tech), penicillin (100 U / ml), and streptomycin (100 mg / ml). The cells were cultured in medium for 3 days.After a 3-day aggregation period, the EHM may be transferred to a flexible holder to support stress-induced contractions. The manufacturers listed are intended to indicate that all components are commercially available and should not be construed as limiting.

[0086] For example, in tests described in International Publication No. WO 2015 / 025030, International Publication No. WO 2015 / 040142, Tiburcy et al. (2017), Circulation, 135(19): 1832-1847, or Long et al. (2018), Sci Adv, 4:eaap9004, artificial (cardiac) muscle tissues, when healthy, are typically capable of exerting a force of 0.01 mN or greater. Accordingly, the one or more conditions preferably include conditions in which the artificial tissue exerts a force of 0.01 mN or greater, 0.05 mN or greater, 0.1 mN or greater, or 1 mN or greater. However, what is more important is the restoration of muscle function relative to a healthy baseline. The force exerted by artificial (cardiac) muscle tissue can be measured using the following exemplary experiment: Contraction experiments can be performed under isometric conditions, e.g., in a 37 °C organ bath in gassed (5% CO2 / 95% O2) Tyrode's solution (containing 120 mM NaCl, 1 mM MgCl2, 0.2 mM CaCl2, 5.4 mM KCl, 22.6 mM NaHCO3, 4.2 mM NaH2PO4, 5.6 mM glucose, and 0.56 mM ascorbic acid). The artificial tissue can be subjected to electrical stimulation at 1.5 Hz with 5 ms rectangular pulses of 200 mA. The artificial tissue can be mechanically stretched at 125 mm intervals until maximal contractile force amplitude (FOC) is observed, according to the Frank-Starling mechanism. The response to increases in extracellular calcium (e.g., 0.2 to 6 mM) can be examined to determine maximum inotropic capacity. Muscle strength can be normalized to muscle mass (sarcomer α-actinin positive cell mass determined by flow cytometry).

[0087] The physiological bodily functions of (cardiac) muscle tissue can be abnormal due to various diseases, which can be hereditary (genetic) or non-hereditary. In one embodiment, the disease is a hereditary cardiac condition, preferably a hereditary cardiac condition selected from the group consisting of genetic forms of dilated, hypertrophic, and arrhythmogenic cardiomyopathy (ACM), or fibroblastosis, and cardiomyopathy associated with a congenital metabolic disorder, more preferably a condition selected from the group consisting of Duchenne muscular dystrophy (DMD), Noonan syndrome, dilated cardiomyopathy, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, long / short QT syndrome, takotsubo cardiomyopathy, lysosomal storage disorders, titinopathy, and Barth syndrome. Preferably, the condition is Duchenne muscular dystrophy. In other embodiments, the disease is a non-genetic cardiac condition, preferably induced by mimicking neurohormonal and / or pharmacological stimuli (e.g., by catecholamines, angiotensin, and / or transforming growth factor β), by drugs such as doxorubicin, tyrosine kinase inhibitors and / or cardiotoxic drugs, by mechanical injury such as crush injury, by temperature injury such as frostbite or thermal injury, by biophysical injury such as radiation injury, by infection such as viral infection with cardioactive virions such as Coxsackievirus, SARS coronavirus, cytomegalovirus, and Dengue virus, or by parasitic infection such as Chagas disease caused by Trypanosoma cruzi, optionally by bloodstream such as serum from a patient with cardiac disease, and / or by cells such as mononuclear cells (e.g., T cells, B cells, NK cells, macrophages) derived from the blood of a patient with cardiac disease.

[0088] Preferably, the at least one physical parameter is selected from the group consisting of (EHM) shortening, spontaneous beating frequency, contraction time / rate, relaxation time / rate, RT, FOC and TT.

[0089] Preferably, the at least one physical parameter is selected from the group consisting of (EHM) shortening, spontaneous beating frequency, and FOC. Preferably, the at least one physical parameter is determined about 14 days after incubation of the artificial tissue, and the artificial tissue is preferably EHM. More preferably, the predetermined threshold is determined based on and / or obtained by a standard (experiment), and preferably, at least one physical parameter of the artificial tissue is determined in the absence of at least a pharmaceutical agent. The predetermined threshold is based on or obtained by a standard (experiment) using statistical methods, and is preferably 1 x SD (standard deviation), preferably 2 x SD, away from the (average) reference value obtained for at least one physical parameter in the standard (experiment). Alternatively, or in addition, it is expressed as a percentage change from the (average) reference value.

[0090] Preferably, at least one physical parameter comprises or is (EHM) shortening, and the predetermined threshold is 0.5x to 2.5x standard deviations (SD), and / or (at least) 0.5x, 1x, 1.5x, or 2x SD, preferably 1x or 2x SD, away from the (average) reference value obtained for the at least one physical parameter in a reference (experiment). Alternatively, or in addition, the predetermined threshold is a change of at least 15%, preferably at least 20%, more preferably at least 25%, at least 35%, or at least 40% from the (average) reference value obtained for the at least one physical parameter in a reference (experiment), and (EHM) shortening is preferably determined in the absence of pharmaceutical agents using artificial tissues derived from healthy individuals or patients with disease and / or well-characterized iPSCs that are modified to represent healthy wild-type or disease. Thus, as disclosed herein, a pharmaceutical product is evaluated and / or determined to be efficacious for treating a disease, preferably by at least partially restoring functionality of a physiological bodily function, if the value of at least one physical parameter determined in an efficacy test exceeds a predetermined threshold, preferably 2×SD and / or (approximately) 35% or 40% change from the respective (mean) baseline value.

[0091] Preferably, the at least one physical parameter is spontaneous beating frequency (in Hz), and the predetermined threshold is 0.5x to 2.5x standard deviations (SD), and / or (at least) 0.5x, 1x, 1.5x, or 2x SD, preferably 1x or 2x SD, away from the (average) reference value obtained for the at least one physical parameter in a reference (experiment). Alternatively, or in addition, the predetermined threshold is a change of at least 10%, preferably at least 15%, more preferably at least 20%, at least 25%, or at least 30% from the (average) reference value obtained for the at least one physical parameter in a reference (experiment), and the spontaneous beating frequency is preferably determined in the absence of pharmaceutical agents using artificial tissue derived from healthy individuals or patients with a disease and / or well-characterized iPSCs that are modified to represent healthy wild-type or disease. Thus, as disclosed herein, a pharmaceutical product is evaluated and / or determined to be effective in treating a disease, preferably by at least partially restoring the functionality of a physiological bodily function, if the value of at least one physical parameter determined in an efficacy test exceeds a predetermined threshold, preferably 2×SD and / or (approximately) 20%, 25% or 30% change of the respective (mean) reference value.

[0092] Preferably, at least one physical parameter is a FOC, and the predetermined threshold value is 0.5x to 2.5x standard deviations (SD), and / or (at least) 0.5x, 1x, 1.5x, or 2x SD, preferably 1x or 2x SD, from the (average) reference value obtained for the at least one physical parameter in a reference (experiment). Alternatively or additionally, the deviation is expressed as a % change from the (average) reference value obtained for the at least one physical parameter in a reference (experiment), and the FOC is preferably determined in the absence of pharmaceutical agents using healthy individuals or patients with a disease and / or artificial tissues derived from well-characterized iPSCs that are either healthy wild-type or modified to represent a disease.

[0093] <Nervous tissue> The methods of the present invention can be used to evaluate the efficacy of pharmaceuticals suspected to be effective in treating diseases involving the nervous system. Therefore, the artificial tissue can be neural tissue, such as a neural organoid, or a neural cell. The neural organoid can also be a bioengineered neural organoid (BENO). "Nervous tissue" and "neuronal tissue" are sometimes used interchangeably. Nervous tissue, also known as neuronal tissue or tissue of neurons, is the primary tissue component of the nervous system. The nervous system regulates and controls bodily functions and activities. The nervous system consists of two parts: the central nervous system (CNS), which includes the brain and spinal cord, and the peripheral nervous system (PNS), which includes the branching peripheral nerves. The nervous system typically consists of neurons, also known as nerve cells (which receive and transmit impulses), and neuroglia, also known as glial cells or glia. Neuroglia, more specifically, include macroglia (e.g., astrocytes, oligodendrocytes, ependymal cells, radial cells, Schwann cells, satellite glial cells, and enteric glial cells) and microglia (i.e., specialized macrophages of the central nervous system; they assist in the propagation of nerve impulses, provide nutrients to neurons, and / or perform phagocytic and immunoregulatory functions). Nervous tissue is composed of various types of neurons, all of which have axons—the long, stalk-like part of the cell that transmits action potentials to the next cell. Bundles of axons make up the nerves of the PNS and the tracts of the CNS. The physiological bodily functions of the nervous system include sensory input, integration, muscle and gland control, homeostasis, and mental activity.

[0094] Numerous protocols exist in the prior art for inducing neural differentiation of human stem cells. One such method involves the induction of neuroectoderm through dual SMAD (Sma and Mad Related Family) signaling inhibition (i.e., BMP and TGF-beta inhibition) in culture for approximately 8–12 days (Chambers, Fasano et al., Nat. Biotechnol., 2009). At this point, the majority of stem cells transform into neural progenitor cells (NPCs). After day 12, some protocols (Lancaster and Knoblich, Science, 2014) allow spontaneous differentiation into various neuronal and glial cell types, while others (Qian, Nguyen et al., Cell, 2016; Birey, Andersen et al., Nature, 2017) apply various patterning factors to pattern the tissue or neurotrophic factors (BDNF, GDNF) to enhance neuronal survival. Furthermore, the addition of dbcAMP or inhibition of Notch expression by DAPT has been shown to promote neural differentiation of these pluripotent cells (Crawford and Roelink, Dev. Dyn., 2007; Kriks, Shim et al., Nature, 2011). The differentiation potential of these treatments is determined by the factors used for neural induction and differentiation, as well as the timing of the treatments. In another report, human cerebral organoids were generated as a model of microcephaly, a condition that is difficult to reproduce in mouse models (Lancaster et al., Nature, 2013; Qian, Nguyen et al., Cell, 2016).

[0095] Protocols for producing neural organoids are also well known to those skilled in the art. (See, for example, International Publication No. 2018 / 228948 or Zafeiriou et al. (2020), Nature Communications, 11:3791, both of which are incorporated herein by reference.) An exemplary method for preparing BENO is described below. GMP hiPS line (TC1133, Lonza) or cell line hiPS-G1 can be used for BENO production. One day before neural differentiation, a 1:1 mixture of acid-solubilized bovine collagen I (Collagen Solutions) and serum-free 2xDMEM (Thermoscientific) can be prepared and neutralized by adding 0.1M NaOH. An iPSC suspension (4500 cells / μl) can be prepared in StemMACS™ iPS-Brew XF medium (Miltenyi) supplemented with 20 ng / ml FGF-2 (Miltenyi) and 10 μmol / L Y-27632 (Stemgent). iPSCs were added to the collagen / DMEM mixture to a final concentration of 3,000 cells / μl and 1 mg / ml type I collagen. Thirty microliters of the cell-collagen mixture was dispensed into a 96-well plate (U-bottom, low-attachment) and incubated in an incubator for 30 minutes. After collagen polymerization, 250 μl of StemMACS™ iPS-Brew XF supplemented with 10 ng / ml FGF and 10 μmol / L Y-27632 was added to each well. From day 0 to day 10, BENOs are cultured in neuronal commitment medium (NCM, basal medium [Neurobasal-A containing 2 mmol / L glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 2% B27, 1% N2 supplement, 200 μmol / L ascorbic acid] supplemented with 10 μmol / L SB431542 [Tocris], 50 ng / ml Noggin [R&D systems], and 1 μmol / L RA [Sigma]). On day 3, BENOs can be transferred to 6-well plates (10 BENOs / well).From days 10 to 15, BENOs can be cultured in neural progenitor cell expansion medium (basal medium supplemented with NPEM, 10 ng / ml FGF-2, and 5 ng / ml TGFB1 [Peprotech]). Finally, from days 15 to 28, BENOs can be cultured in neural differentiation medium (basal medium supplemented with 2.5 μmol / L DAPT [Tocris] and 5 ng / mL TGFB1). From day 29 onward, BENOs can be cultured in basal medium. Medium changes can be performed every two days. The manufacturers listed are intended to indicate that all components are commercially available and should not be interpreted as limiting.

[0096] As used herein, the term "organoid" refers to a tissue culture that forms a three-dimensional aggregate that at least partially mimics the structure and / or function of an organ, such as a human organ. Organoids can be generated, for example, from pluripotent stem cells in a three-dimensional (3D) environment. One such 3D environment for organoids is a spheroidal 3D environment. Organoids can also be considered a miniaturized or simplified version of an organ.

[0097] As used herein, the term "bioengineered neuronal organoid" (BENO) refers to an organoid derived from nervous tissue produced according to the method of the present invention. BENO can be considered as a miniaturized and simplified model of nervous organs, including the brain, and / or nervous tissues present within / controlling organs, such as the nervous tissues present within the heart (e.g., the sympathetic nervous system) and the nervous tissues present within skeletal muscle (e.g., the nicotinic nerve endings at the skeletal neuromuscular junction). Here, BENO can preferably be considered as a miniaturized and simplified model of nervous organs, including the brain. BENO is particularly suitable as a forebrain model. BENO self-patterns into the midbrain and hindbrain, and is therefore also suitable as a midbrain and / or hindbrain model.

[0098] It is known that nerve tissue or nervous system tissue transmits information via electric current or chemical compounds. Therefore, the at least one physical parameter is preferably electric current and / or electrical activity. In one embodiment, the at least one physical parameter is electric current, which can be expressed in amperes (A). In another embodiment, the at least one physical parameter is electrical activity. It is also possible to monitor changes in electrical potentials (action potentials) caused by neuronal activity. Therefore, the at least one physical parameter can be an electrical potential, which can be expressed in volts (V).

[0099] As used herein, a "neural network" refers to a group of one or more interconnected neurons. The connections between neurons in a neural network allow information to be transmitted from one neuron to another. In a neural network, the connections between neurons are made via synapses. The existence of a neural network can be easily confirmed, for example, by calcium imaging of neurons or multi-electrode array analysis. In the presence of a disease, the neural network may be impaired or absent.

[0100] As used herein, a "functional neural network" refers to a neural network that exhibits the transmission of electrochemical information from one neuron to another. A functional neural network is characterized by a pattern of network activity, including synchronized electrical activity of one or more neurons in the network, or patterns of neuronal activation or inhibition, which indicate functional interdependence among the neurons in the network. The existence of a neural network can be confirmed, for example, by calcium imaging of neurons. In particular, the presence of a functional neural network can be confirmed by reaction with a set of neuronal signaling molecules or inhibition of receptors for signaling molecules (e.g., GABA receptors). One example of a functional neural network is when neurons in the network exhibit synchronized calcium signals (e.g., calcium spikes), which become desynchronized upon the addition of a receptor inhibitor that blocks the neurotransmitter and resynchronize upon removal of the inhibitor. Therefore, the functionality of physiological bodily functions is characterized by the presence of a functional neural network.

[0101] Functional neural networks are also characterized by the presence of localized and coordinated electrical bursts and / or clusters. Therefore, preferably, one or more of the above conditions include localized and coordinated electrical bursts and / or clusters. This can be assessed, for example, by multielectrode arrays, as described in Zafeiriou et al. (2020), Nature Communications, 11:3791. An exemplary method is described below: Prior to seeding, a 6-well plate with 64 platinum microelectrode arrays per well (MEA; 0.04 MΩ per microelectrode, 30 μm microelectrode diameter, 200 μm spacing) can be coated with Matrigel™ diluted 1:120 in PBS for 1 hour at room temperature. One or two fragments (300 μm thick) can be placed into the MEA wells and secured with concentrically wrapped tungsten rings. Recordings of 10–15 min / 2 h are performed every other day for up to 60 days using the Maestro Pro MEA System (Axion Biosystems). Data recording can be performed automatically with Axion software (AxIS Navigator) using the manufacturer's Spontaneous Neural Configuration. Data analysis can be performed using the manufacturer's standalone tools, the Neural Metric Tool, and the AxIS Metric Plotting Tool (Axion Biosystems). A spike detection threshold was set at 5.5 standard deviations, and electrodes detecting at least five spikes per minute were classified as active. Spike bursts were identified using an ISI threshold requiring a minimum of five spikes with a maximum ISI of 100 ms. NBs were identified by an envelope algorithm using a threshold of 1.25, a minimum IBI interval of 100 ms, and a 75% burst inclusion rate with a minimum of 10% active electrodes. Firing synchrony was estimated by the area under normalized synchronous cross-correlation over a 20 ms time window.For potentiation measurements, multichannel fEPSP recordings can be performed using the MEA2100 device (MC_Rack 3.2.1.0 software, Multi Channel Systems, Reutlingen, FRG). To reduce noise, the bath can be earthed via a custom-made Ag / AgCl electrode attached to the ground socket of the MEA amplifier. LTP is induced by three consecutive HFS (20 pulses of 100 Hz, 50 μs, 100 μA) with a 20 s interval. Before HFS, neurons can be stimulated with a single pulse (100 μA, 50 μs pulse width, 30 s interpulse interval). After stimulation, the same protocol can be performed for 1 h to quantify differences in synaptic strength. The listed manufacturers are intended to indicate that all components are commercially available and should not be interpreted as limiting.

[0102] As used herein, "neuronal network function" refers to the transmission of electrochemical information from one neuron to another. Furthermore, as used herein, "network bursts" refers to the synchronized firing of multiple neurons in different regions of the brain and represents a measure of network bursts. Network bursts or oscillations are defined as alternating periods of high and low synchronized activity and are characteristic of functional networks.

[0103] Preferably, the at least one physical parameter is an MEA measure, preferably one or more selected from the group consisting of (weighted) mean firing rate, burst frequency (Hz), inter-burst spike interval (ms), network burst (NB) frequency, inter-NB spike interval (ms), and NB duration (s). Preferably, the at least one physical parameter is determined about 10 minutes after incubation of the artificial tissue, and the artificial tissue is preferably BENO. More preferably, the predetermined threshold is determined based on and / or obtained by a reference (experiment), and preferably, the at least one physical parameter of the artificial tissue is determined at least in the absence of a pharmaceutical agent. The predetermined threshold is based on or obtained by a standard (experiment) using statistical methods and preferably deviates from the (average) reference value obtained in the standard (experiment) for at least one physical parameter by 0.5x to 2.5x standard deviation (SD), and / or by (at least) 0.5x, 1x, 1.5x or 2x SD, preferably 1x or 2x SD. Alternatively or additionally, the deviation is expressed as a % change from the (average) reference value. Alternatively or additionally, the at least one physical parameter is plasticity.

[0104] For assessing the proconvulsant efficacy of a pharmaceutical product, the at least one physical parameter is one or more selected from the group consisting of (weighted) mean firing rate, burst frequency (Hz), network burst (NB), and NB duration (s), and the predetermined threshold is preferably 0.5× to 2.5× standard deviation (SD), and / or (at least) 0.5×SD, 1×SD, 1.5×SD, or 2×SD, preferably 1×SD or 2×SD, from the (average) reference value obtained for the at least one physical parameter in a reference (experiment). Alternatively or additionally, the predetermined threshold is a change of at least 5% or 10%, preferably at least 25% or 30%, more preferably at least 50% or 55%, at least 60% or 100% of the (average) baseline value obtained for at least one physical parameter in a reference (experiment), and the at least one physical parameter is preferably determined in the absence of a pharmaceutical agent using healthy individuals or patients with a disease and / or artificial tissue derived from well-characterized iPSCs that are expressed as healthy wild-type or modified to express a disease. Thus, as disclosed herein, when the value of at least one physical parameter determined in an efficacy test exceeds a predetermined threshold, preferably 2×SD and / or (approximately) 10%, 50%, 55%, 60% or 100% of the (average) baseline value, the pharmaceutical agent is preferably evaluated and / or determined to be effective in treating a disease, preferably by at least partially restoring the functionality of physiological body functions, and preferably has proconvulsant properties.More specifically, if at least one physical parameter is a (weighted) mean firing rate, the predetermined threshold is preferably 2×SD and / or a change of (approximately) 55% or 60% of the (average) reference value; if at least one physical parameter is burst frequency (Hz), the predetermined threshold is preferably 2×SD and / or a change of (approximately) 50% of the (average) reference value; if at least one physical parameter is network burst (NB), the predetermined threshold is preferably 2×SD and / or a change of (approximately) 100% of the (average) reference value; if at least one physical parameter is NB duration (s), the predetermined threshold is preferably 2×SD and / or a change of (approximately) 10% of the (average) reference value.

[0105] Convulsant-inducing drugs, for example, offer an opportunity to evaluate the potential scope of efficacy and / or determine potential effects in contraindication studies. Such contraindication studies can be performed, for example, by administering the drug to patients without neurological disease or when the drug is to be administered to patients without neurological disease. On the other hand, the efficacy measurement method disclosed herein is particularly advantageous for evaluating the anticonvulsant efficacy of drugs. Examples of (new) anticonvulsant drugs include carbamazepine, eslicarbazepine acetate, sodium valproate, levetiracetam, lamotrigine, lacosamide, perampanel, topiramate, pregabalin, and / or cenobamate. When evaluating the anticonvulsant efficacy of drugs, standards (experiments) including healthy BENO standards and / or permanently gene-corrected BENO standards (e.g., using CRISPR-modified cells) may be particularly suitable.

[0106] For assessing the anticonvulsant efficacy of a pharmaceutical agent, the at least one physical parameter is one or more selected from the group consisting of (weighted) mean firing rate and inter-burst spike interval (s), and the predetermined threshold is preferably 0.5× to 2.5× standard deviation (SD), and / or (at least) 0.5×, 1×, 1.5×, or 2×SD, preferably 1× or 2×SD, from the (average) baseline value for the at least one physical parameter obtained in a reference (experiment). Alternatively or additionally, the predetermined threshold is a change of at least 15%, preferably at least 25%, 30%, and more preferably at least 55% or 60%, from the (average) baseline value for the at least one physical parameter obtained in a reference (experiment), and the at least one physical parameter is preferably determined in the absence of the pharmaceutical agent using artificial tissue derived from healthy individuals or patients with a disease and / or well-characterized iPSCs that are modified to represent healthy wild-type or disease-representing cells. Therefore, as disclosed herein, a pharmaceutical product is evaluated and / or determined to be efficacious for treating a disease, preferably by at least partially restoring the functionality of a physiological bodily function, and preferably to have anticonvulsant properties, when the value of at least one physical parameter determined in an efficacy test exceeds a predetermined threshold, preferably 2×SD and / or (about) 15%, 25%, 30%, 55%, or 60% change from the (average) reference value. More specifically, when at least one physical parameter is a (weighted) mean firing rate, the predetermined threshold is preferably 2×SD and / or (about) 55% or 60% change from the (average) reference value; when at least one physical parameter is an intra-burst interspike interval, the predetermined threshold is preferably 2×SD and / or (about) 30% change from the (average) reference value.

[0107] It is particularly preferred that the at least one physical parameter is or comprises a (weighted) mean firing rate and that the predetermined threshold deviates from the (mean) reference value obtained for the at least one physical parameter in the reference (experiment) by 2 × SD and / or (approximately) 60% variation of the respective (mean) reference value.

[0108] <Muscle tissue> The method of the present invention can also be used to evaluate the efficacy of pharmaceuticals that are suspected to be effective in treating diseases involving muscle tissue. Thus, the artificial tissue may be muscle tissue, preferably skeletal muscle tissue, or muscle.

[0109] Those skilled in the art can prepare artificial muscle tissue. Methods for producing muscle tissue are disclosed, for example, in International Publication No. 2021 / 074126, the entire contents of which are incorporated herein by reference. In particular, in exemplary embodiments, artificial skeletal muscle tissue can be produced from pluripotent stem cells as follows: (i) inducing mesodermal differentiation of pluripotent stem cells by culturing the pluripotent stem cells in a basal medium containing effective amounts of (a) FGF2, (b) a GSK3 inhibitor, (c) a SMAD inhibitor, and (d) a serum-free additive containing transferrin, insulin, progesterone, putrescine, and selenium or a bioavailable salt thereof; (ii) culturing the cells obtained in step (i) in a basal medium containing effective amounts of (a) a γ-secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive similar to that in (i), and then continuing the culture in the above medium supplemented with an effective amount of (d) HGF, and then culturing the cells in the above medium supplemented with effective amounts of (a) a γ-secretase / NOTCH inhibitor, (b) HGF, (c) a serum-free additive similar to that in (i), and (d) a knockout serum substitute ( (iii) culturing the cells obtained in step (ii) in a basal medium containing effective amounts of (a) HGF, (b) a serum-free additive similar to that in step (i), and (d) knockout serum replacement (KSR) to expand and mature the cells into skeletal myoblasts and satellite cells; and (iv) culturing the cells obtained in step (iii) dispersed in an extracellular matrix under mechanical stimulation in a basal medium containing effective amounts of (a) a serum-free additive similar to that in step (i), and (b) a serum-free additive containing albumin, transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, a fatty acid additive, and triiodo-L-thyronine (T3) to mature the cells into skeletal myotubes and satellite cells, thereby producing artificial skeletal muscle tissue. The pluripotent stem cells may be obtained from a patient who is to receive a pharmaceutical treatment. The pluripotent stem cells do not have to be obtained from a patient who is to receive a pharmaceutical treatment.

[0110] The physiological bodily function of (skeletal) muscle tissue is to exert force in order to move a body part. Therefore, the method of the present invention is particularly useful when the at least one physical parameter determined in the method is force, e.g., expressed in Newtons (N), or movement, e.g., expressed as the velocity of a moving object in meters per second (m / s). Thus, in one embodiment, the at least one physical parameter is force. In another embodiment, the at least one physical parameter is movement. In another embodiment, the at least one physical parameter is velocity.

[0111] Methods for determining the force exerted by (skeletal) muscle tissue are known to those skilled in the art and are described in WO 2021 / 074126, Tiburcy et al. (2019, FASEB Bioadv, 1(12):731-746), or Shahriyari et al. (2022 Cachexia Sarcopenia Muscle, 13(6):3106-3121, doi: 10.1002 / jcsm.13094), which are incorporated herein by reference. Preferably, the force is determined isometrically. In an exemplary embodiment, measurements can be performed as follows: Contractile function of engineered skeletal muscle tissue can be measured isometrically in an organ bath filled with gassed (5% CO2 / 95% O2) Tyrode's solution (containing, in mmol / L: 120 NaCl, 1 MgCl2, 0.2 CaCl2, 5.4 KCl, 22.6 NaHCO3, 4.2 NaH2PO4, 5.6 glucose, and 0.56 ascorbic acid) at 37°C. To verify the force-length relationship, ESM can be electrically stimulated with 200 mA, 5 ms rectangular pulses at 1 Hz, and mechanical stretches can be applied at 125 μm intervals to increase muscle length until maximum contractile force is observed. Quadriceps contractile force can be assessed at the length where maximum force is generated under defined stimulation frequencies (10, 20, 40, 60, 80, and 100 Hz for 4 seconds).

[0112] Artificial (skeletal) muscle tissue typically exerts a force of 0.01 mN or greater under normal conditions, and therefore the one or more conditions preferably include the artificial tissue exerting a force of 0.01 mN or greater, 0.05 mN or greater, 0.1 mN or greater, or 1 mN or greater.

[0113] The physiological bodily functions of (skeletal) muscle tissue can be abnormal due to various diseases. For example, the disease is a genetic muscle disease, preferably selected from the group consisting of Duchenne muscular dystrophy, facioscapulohumeral dystrophy, Becker dystrophy, Emery-Dreifuss dystrophy, myotonic dystrophy, limb dystrophy, oculopharyngeal muscular dystrophy, congenital dystrophy, congenital myopathy, myotonic dystrophy, familial periodic paralysis, and hereditary connective tissue diseases such as Marfan syndrome, Ehlers-Danlos syndrome, or Loeys-Dietz syndrome. Preferably, the disease is Duchenne muscular dystrophy. Alternatively, or in addition, the disease may be a non-genetic muscle disease.

[0114] In one particular embodiment, the artificial tissue is cardiac muscle; the pharmaceutical product is an AAV2 and / or AAV9 viral vector comprising a nucleic acid encoding Cas9, preferably S. aureus Cas9, S. auricularis Cas9, S. lugdunensis Cas9, or N. meningitides Cas9, and one or two guide RNAs (gRNAs), wherein the Cas9 gene is under the control of a muscle-specific promoter such as CK8e or TNNT2; the disease is Duchenne muscular dystrophy, the physical parameter is a force; Preferably, the artificial tissue contains cells obtained from a patient who is to be treated with the pharmaceutical agent.

[0115] In one particular embodiment, The artificial tissue is skeletal muscle, the pharmaceutical product is an AAV2 and / or AAV9 viral vector comprising a nucleic acid encoding Cas9, preferably S. aureus Cas9, S. auricularis Cas9, S. lugdunensis Cas9, or N. meningitides Cas9, and one or two guide RNAs (gRNAs), wherein the Cas9 gene is under the control of a muscle-specific promoter such as CK8e or TNNT2; the disease is Duchenne muscular dystrophy, the physical parameter is a force, Preferably, the artificial tissue contains cells obtained from a patient who is to be treated with the pharmaceutical agent.

[0116] In some embodiments, the artificial tissue is muscle tissue and the disease is a muscle tissue disease, preferably DMD.

[0117] Preferably, the at least one physical parameter is the contractile force of the tissue, preferably determined as twitch tension and / or using a stimulation frequency of 40 Hz or 100 Hz. The at least one physical parameter is preferably determined on about day 10 or 14 of the drug effect test period, thus about day 10 or 14 of incubation of the artificial tissue, and the artificial tissue is preferably ESM. More preferably, the predetermined threshold is determined based on and / or obtained by a standard (experiment), preferably, at least one physical parameter of the artificial tissue is determined in the absence of a pharmaceutical agent. Preferably, the predetermined threshold is based on or obtained by a standard (experiment) using a statistical method, and deviates from the (average) standard value for the at least one physical parameter in the standard (experiment) by 0.5× to 2.5× standard deviation (SD), and / or by (at least) 0.5×SD, 1×SD, 1.5×SD, or 2×SD, preferably 1.5×SD or 2×SD. Alternatively, or in addition, the deviation is expressed as a % change from the (average) baseline value.

[0118] <Nerve Junction> The versatility of the method of the present invention also allows the efficacy of pharmaceuticals for treating diseases to be measured based on a mixture of different types of artificial tissue. In one example, such a mixture contains nerve cells and skeletal muscle tissue or cardiac muscle tissue. This is similar to a neuronal junction, and the method of the present invention can be used in this embodiment to measure the efficacy of treating diseases present at the neuromuscular junction or more specific diseases that impair the function of the neuromuscular junction. Here, the artificial tissue can include nerve cells, such as BENO described herein, and skeletal muscle tissue or cardiac muscle tissue co-cultured during the preparation of the artificial tissue (see also International Publication No. 2018 / 228948, incorporated herein by reference in its entirety).

[0119] Thus, the artificial tissue may be a hybrid of different types of tissue. Preferably, the artificial tissue may be a hybrid of nerve cells and skeletal muscle tissue. Preferably, the artificial tissue may be a hybrid of nerve cells and cardiac muscle tissue. Preferably, the hybrid of different types of tissue forms one or more neuromuscular junctions. A "neuromuscular junction" (or myoneural junction) is a chemical synapse between a motor neuron and a muscle fiber.

[0120] <Connective tissue> Connective tissue is one of many fundamental types of animal tissue, along with epithelial tissue, muscle tissue, and nervous tissue. It develops from the mesoderm during embryology. Connective tissue is typically present between other tissues throughout the body, including the nervous system. The three outer membranes (meninges) surrounding the brain and spinal cord are composed of dense, inert connective tissue. Connective tissue typically contains three main components: fibers (elastic and collagen fibers), matrix, and cells, all of which are immersed in body water. Connective tissue cells may include fibroblasts, adipocytes, macrophages, mast cells, and leukocytes. Thus, in one embodiment, the artificial tissue is connective tissue.

[0121] Connective tissue may be subject to diseases treatable by the pharmaceutical products defined herein, such as (i) heart diseases associated with myocardial fibrosis, such as heart failure (due to unknown and / or genetic causes) with reduced or preserved left ventricular ejection fraction, myocardial infarction, congenital heart disease, cardiomyopathies with known genetic mutations, aging and / or age-induced myocardial fibrosis, drug-induced heart disease, metabolic heart disease, diseases (monogenic or polygenic) involving the heart and myocardial fibrosis, diseases with reactive or replacement fibrosis in organs such as the kidney, liver, lung, and / or skin, (ii) diseases associated with scar formation and impaired wound healing, such as diabetes, and / or (iii) hereditary connective tissue diseases, such as Marfan syndrome, Ehlers-Danlos syndrome, or Loeys-Dietz syndrome.

[0122] Connective tissue has a wide variety of physiological bodily functions, depending on the cell types and fiber types involved. Loose, dense, irregular connective tissue, primarily formed by fibroblasts and collagen fibers, plays an important role in providing a medium for oxygen and nutrients to diffuse from capillaries to cells and for carbon dioxide and waste products to return from cells to the circulation. It also allows organs to resist stretching and tearing forces. Dense connective tissue, which forms tissue structures, is the primary functional component of tendons, ligaments, and periosteum and is also found in highly specialized organs such as the cornea. Elastic fibers, made from elastin and fibrillin, also provide resistance to stretching forces. Elastic fibers are typically found in the walls of large blood vessels and in certain ligaments, particularly the femoral ligament. In hematopoietic and lymphatic tissues, reticular fibers produced by reticular cells provide the interstitium (structural support) for the parenchyma (or functional parts) of organs. Mesenchyme is a type of connective tissue found in developing organs during embryonic development and can differentiate into any type of mature connective tissue. Another type of relatively undifferentiated connective tissue is the mucous connective tissue known as Wharton's jelly, found in the umbilical cord. Many different types of specialized tissues and cells fall under the connective tissue category, including brown and white adipose tissue, blood, cartilage, and bone.

[0123] Wound healing is characterized by connective tissue formation, i.e., scar formation as a replacement for damaged organ tissue, also known as replacement fibrosis.Therefore, the disease can be fibrosis, such as fibrosis within or of an organ.Replacement fibrosis of connective tissue is observed, for example, after myocardial infarction, cardiomyopathy, muscle damage such as replacement fibro-fatty tissue in DMD, stroke patients, toxic liver damage, kidney disease, skin injury, etc.

[0124] Connective tissue may be characterized by its biomechanical parameters. Examples of biomechanical parameters include tissue compression, contraction, stiffness, tensile strength, elasticity, extensibility, excitability, resilience, or toughness. Thus, the at least one physical parameter may be tissue compression. The at least one physical parameter may be tissue contraction. The at least one physical parameter may be stiffness. Stiffness is the degree to which an object resists deformation in response to an applied force. The at least one physical parameter may be tensile strength. The at least one physical parameter may be elasticity. Elasticity refers to the ability of an object to resist a distorting influence and return to its original size or shape when the influence or force is removed. The at least one physical parameter may be extensibility. Extensibility refers to the ability of an object to resist a distorting influence before failure (and return to its original size or shape when the influence or force is removed). The at least one parameter may be excitability. The at least one physical parameter may be resilience. The at least one physical parameter may be toughness. Toughness is the ability of a material to absorb energy and deform elastically and plastically without fracture.

[0125] Methods for producing artificial connective tissue are known to those skilled in the art. Cardiac interstitial cells are a type of connective tissue and can be produced as described in WO 2022 / 023451 or Santos et al. (2021), J. Vis. Exp., 174:e62700, which are incorporated herein by reference in their entirety. An exemplary method for producing artificial connective tissue is as follows: First, interstitial cells or fibroblasts are prepared, for example, as described in WO 2022 / 023451. Then, the separated interstitial cells or fibroblasts are cast into a suitable mold together with an extracellular matrix, such as collagen.

[0126] In some embodiments, the artificial tissue is a connective tissue, and the disease is (i) a cardiac disease associated with myocardial fibrosis, such as heart failure with reduced or preserved left ventricular ejection fraction (due to unknown and / or genetic causes), myocardial infarction, congenital heart disease, cardiomyopathies with known genetic mutations, aging and / or age-induced myocardial fibrosis, drug-induced heart disease, metabolic heart disease, diseases (monogenic or polygenic) involving cardiac and myocardial fibrosis, diseases associated with reactive or replacement fibrosis in organs such as the kidney, liver, lung, and / or skin, (ii) a disease associated with scar formation and impaired wound healing, such as diabetes, and / or (iii) a hereditary connective tissue disease, such as Marfan syndrome, Ehlers-Danlos syndrome, or Loeys-Dietz syndrome.

[0127] Regarding the selection of suitable standard (experiment), those skilled in the art will recognize two scenarios.In the case of non-genetic diseases such as non-genetically caused (cardiac) fibrosis, the efficacy of a pharmaceutical product with anti-fibrotic effect is shown when the difference between at least one physical parameter value, preferably statistically significant, is observed between treated artificial tissue or organoid and at least the standard (experiment) that is or comprises healthy wild-type artificial tissue or organoid.However, when genetic mutation is associated with the disease and the pharmaceutical product may act in this respect, and / or when it is a personalized therapeutic pharmaceutical product, the difference between at least one physical parameter value, preferably statistically significant, can be shown to demonstrate anti-fibrotic efficacy.

[0128] Preferably, the at least one physical parameter is tissue contraction and / or stiffness, preferably measured as pole deflection and / or Young's modulus. The at least one physical parameter is preferably determined approximately during the drug effect test period, i.e., approximately on day 10 of incubation of the artificial tissue, and the artificial tissue is preferably ECT. More preferably, the predetermined threshold is determined based on and / or obtained by a standard (experiment), in which case, preferably, at least one physical parameter of the artificial tissue is determined in the absence of a pharmaceutical agent. Preferably, the predetermined threshold is based on or obtained by a standard (experiment) using a statistical method, and deviates from the (average) reference value for at least one physical parameter in the standard (experiment) by 0.5× to 2.5× standard deviation (SD), and / or by (at least) 0.5×SD, 1×SD, 1.5×SD, or 2×SD, preferably 1.5×SD or 2×SD. Alternatively or additionally, the deviation is expressed as a percentage change from the (average) reference value.

[0129] To assess the pro-fibrotic efficacy of a pharmaceutical agent, preferably, at least one physical parameter is measured as pole deflection and / or Young's modulus, and the predetermined threshold is 1×SD or 1.5×SD deviation from the (average) baseline value obtained for at least one physical parameter in a reference (experiment). Alternatively, or in addition, the predetermined threshold is a change of at least 30%, 35%, 55%, or 60% from the (average) baseline value obtained for at least one physical parameter in a reference (experiment) in the absence of TGF-β1, and the at least one physical parameter is preferably determined in the absence of the pharmaceutical agent using artificial tissue derived from healthy individuals or patients with a disease and / or well-characterized iPSCs that have been modified to represent healthy wild-type or disease. Therefore, when the at least one physical parameter is or includes pole deflection, if the value of the at least one physical parameter determined in an efficacy test as disclosed herein exceeds a predetermined threshold, preferably 1×SD and / or (about) 35% change from the (average) baseline value in the absence of TGF-β1, the pharmaceutical product is preferably evaluated and / or determined to be effective in treating a disease by at least partially restoring the functionality of a physiological bodily function, and preferably to be pro-fibrotic. When the at least one physical parameter is or includes Young's modulus, if the value of the at least one physical parameter determined in an efficacy test as disclosed herein exceeds a predetermined threshold, preferably 1.5×SD and / or (about) 60% change from the (average) baseline value in the absence of TGF-β1, the pharmaceutical product is preferably evaluated and / or determined to be effective in treating a disease by at least partially restoring the functionality of a physiological bodily function, and preferably to be pro-fibrotic.

[0130] To evaluate the antifibrotic efficacy of a pharmaceutical agent, preferably, at least one physical parameter is measured as pole deflection and / or Young's modulus, and the predetermined threshold value is 1×SD or 1.5×SD from the (average) reference value obtained for at least one physical parameter in a reference (experiment). Alternatively, or in addition, the predetermined threshold value is a change of at least 30%, 35%, 55%, or 60% from the (average) reference value obtained for at least one physical parameter in a reference (experiment) in the presence and / or absence of TGF-β1, and the at least one physical parameter is preferably determined in the absence of the pharmaceutical agent using artificial tissue derived from healthy individuals or patients with a disease and / or well-characterized iPSCs that are modified to represent a healthy wild type or a disease. Therefore, when the at least one physical parameter is or includes pole deflection, as disclosed herein, when the value of the at least one physical parameter determined in an efficacy test exceeds a predetermined threshold, which is preferably 1×SD and / or (approximately) a 35% change from the (average) baseline value in the absence of TGF-β1 and / or (approximately) a 30% change from the (average) baseline value in the presence of TGF-β1, the pharmaceutical is evaluated and / or determined to be effective in treating the disease by at least partially restoring the functionality of physiological bodily functions, and preferably to have anti-fibrotic properties. When the at least one physical parameter is or includes Young's modulus, as disclosed herein, if the value of the at least one physical parameter determined in an efficacy test exceeds a predetermined threshold, which is preferably a change of 1.5 x SD and / or (approximately) 60% of the above (average) baseline value in the absence of TGF-β1, and / or a change of (approximately) 55% or 60% of the respective (average) baseline value in the presence of TGF-β1, the pharmaceutical is evaluated and / or determined to be effective in treating the disease by at least partially restoring the functionality of physiological body functions, and preferably to have anti-fibrotic properties.

[0131] It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art that may be modified or substituted for the methods described herein.

[0132] Unless otherwise specified, the term "at least" preceding a series of elements is understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by this invention.

[0133] As used herein, the term "and / or" includes the meaning of "and," "or," "all or any other combination of the elements connected by that term."

[0134] The terms "less than" and "greater than" do not include specific numbers.

[0135] For example, less than 20 means less than the indicated number. Similarly, greater than or equal to means greater than or equal to the indicated number, for example, greater than 80% means greater than or equal to 80% of the indicated number.

[0136] Throughout this specification and the claims that follow, unless the context clearly indicates otherwise, the word "comprise," and variations thereof, such as "comprises" and "comprising," are understood to mean the inclusion of the stated objects or steps or group of objects or steps, but not the exclusion of other objects or steps or group of objects or steps. As used herein, the term "comprising" can be interchangeable with the terms "containing" or "including," or, as used herein, with the term "having." As used herein, "consisting of" excludes unspecified elements, steps, or ingredients.

[0137] The term "including" means "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0138] As used herein, the terms "about" or "approximately" mean within 20%, preferably within 15%, more preferably within 10%, and even more preferably within 5% of a given value or range. Also, a specific numerical value, i.e., "about 20," includes the numerical value 20.

[0139] It is to be understood that this invention is not limited to the particular methodology, protocols, materials, reagents, substances, etc. described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0140] All publications cited throughout the text of this specification (including all patents, patent applications, scientific publications, manuals, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent that material incorporated by reference is inconsistent or contradictory with the present specification, the present specification will control for such material.

[0141] The contents of all publications and patent documents cited herein are incorporated by reference in their entirety. [Example]

[0142] A better understanding of the present invention and its advantages will be apparent from the following examples, which are given for illustrative purposes only and are not intended to limit the scope of the invention.

[0143] Example 1: Drug Effect Test for ECT Using Pole Deflection (background) Artificial connective tissue (ECT) can be generated using various types of fibroblasts, including induced human pluripotent stem cell-derived stromal cells (iPSC-StCs) and primary human fibroblasts from various sources, such as skin, gum, and heart. Depending on the disease context under study, pro- and anti-fibrotic drugs can be tested to identify their efficacy. For example, in myocardial fibrosis, anti-fibrotic drugs are required primarily to prevent excessive extracellular matrix deposition in the myocardium by cardiac fibroblasts. Conversely, diabetes is typically associated with abnormalities in skin wound healing. Therefore, drugs that can temporarily convert dermal fibroblasts into more contractile myofibroblasts may be of interest as they may aid and / or accelerate wound healing.

[0144] (ECT production) The general experimental procedure was the same for all cells used, including primary fibroblasts and iPSC-StCs. Supporting information regarding the ECT model, particularly its generation, can be found in Santos et al. (2021, Fibroblast Derived Human Engineered Connective Tissue for Screening Applications. J. Vis. Exp. e62700, doi:10.3791 / 62700, PMID: 34487119), Santos et al. (2022, Using different geometries to modulate the cardiac fibroblast phenotype and the biomechanical properties of engineered connective tissues. Biomater Adv. 139:213041, PMID: 35909053), and European Patent Application 20188364. Specifically, acid-solubilized type 1 collagen was mixed 1:1 with 2x DMEM containing 20% ​​fetal calf serum and antibiotics. The pH of the collagen mixture was neutralized with NaOH before combining with iPSC-StCs or fibroblasts and carefully mixing to obtain 0.3 mg of collagen and 0.75 × 10 cells per ECT and per 180 µL. 6 ECTs consisting of 1000 cells were prepared. The cell-collagen mixture was dispensed into a 48-well myrPlate mold (Tiburcy et al., 2020 STAR Protocols and International Publication No. 2017 / 207431) containing two poles made of flexible polymer (e.g., TM5MED) material. After seeding, the ECTs were allowed to solidify for 1 hour in a cell incubator, after which the appropriate cell culture medium was added and replaced every 2 days.

[0145] (Pole deflection analysis) Pole deflection measurements are well established in the art, see for example WO 2017 / 207431 (also registered as German Patent 102016110328). In the examples presented herein, the poles were imaged under UV light immediately after the first medium addition, and subsequently once daily. Pole deflection as a function of ECT compression was calculated as follows: 100 × (distance between poles) 0日目 -Pole distance X日目 ) / Pole distance 0日目 .

[0146] (Research design) Fibrotic processes are triggered by mechanical and / or biochemical triggers, which induce specific, partially connected signaling cascades. Therefore, an ideal antifibrotic drug would interfere with both types of stressors. Therefore, it is desirable to test both conditions, where mechanical restraint alone induces cell activation, and where mechanical activation is combined with the application of a profibrotic cytokine, preferably TGF-β1. Drug testing is preferably performed once stable ECTs have formed and early processes unrelated to fibrosis have been completed. Therefore, drug testing is preferably performed when previously 2D cultured cells have adapted to a 3D environment, as in the ECT model. In the ECT model, cellular adaptation can occur within the first few days, paralleling ECT compression and systole I. Therefore, drug administration is preferably performed before the ECT reaches a contractile plateau and systole II, which indicates cellular reactivation, is initiated. According to current understanding of fibrotic mechanisms, administering a profibrotic cytokine, such as TGF-β1, once the ECT reaches a contractile plateau can further enhance mechanically driven systole II.

[0147] (Methodological aspects) The absolute detection limits for pole deflection were 100% (upper limit) and 0% (lower limit). However, without being bound by theory, the detection limits actually observed were approximately 60-70% (upper limit) and 2% (lower limit), respectively. The relative detection limits were approximately 5-10 times the plateau value (upper limit) and approximately 0.25 times the plateau value (lower limit), respectively.

[0148] (ECT treatment) Baseline ECTs treated with drug vehicle in the absence or presence of TGF-β1 were analyzed along with TGF-β1 application, an additional measure of profibrotic drug properties. Both baseline conditions were replicated six times. Pole deflection was observed to be maximal between days 3 and 5. Accordingly, mechanically driven phase II contraction was further enhanced in the TGF-β1 baseline ECTs with 5 ng / ml TGF-β1 applied on day 4 (Figure 2A).

[0149] In the treatment condition, the drug of interest is administered at the time of maximum deflection between days 3 and 5. Here, the day on which the drug is added during the plateau phase is also referred to as day 1 of the drug effect test period. Thus, in the exemplary embodiment disclosed herein, day 1 of the drug effect test period represents day 4 of ECT culture. The drug effect test period may be as long as the ECT culture time. Drug concentrations are determined by the IC of each drug, if available. 50 / EC 50 The concentration used is chosen based on the IC 50 / EC50 The range is x ±30 times, and it is applied in half log steps. For example, IC 50 For a 1 μM drug, concentrations of 0.03, 0.1, 0.3, 1, 3, 10, and 30 μM are tested. A reference ECT treated with drug solvent (absence of TGF-β1) is also analyzed as above. All conditions are run in six replicates. A second plate uses the same experimental setup, but samples are spiked with 5 ng / ml TGF-β1 (Figure 2B, right). The IC of a given drug is then calculated. 50 / EC 50If this value is unknown, the highest concentration should be 100 times the drug's solubility. For example, if the maximum soluble concentration is 100 mM, the highest final concentration used should be 1 mM. Additionally, six log-step dilutions and solvents should be tested. The drug-containing medium should be changed every two days for the next incubation period, e.g., 10 days.

[0150] (Classification of drugs based on their effects on systolic phase II) Statistical analysis of the baseline ECT revealed that a decrease in contraction of at least 1 SD was significant in a two-way analysis of variance with Tukey's multiple comparison test. Table 3 summarizes the mean contraction (%) determined by pole deflection analysis for the baseline ECT without TGFβ-1 and the baseline ECT with TGFβ-1. A deviation of 1 SD equates to a change in inhibition of contractile increase between plateau and day 13 (absolute values; day 10 of the drug effect test period): 35% for the baseline without TGFβ-1 and 30% for the baseline with TGFβ-1.

[0151] [Table 3]

[0152] The theoretical and / or exemplary predicted interference of an "ideal" antifibrotic drug on systole II in the absence or presence of TGF-β1 is shown in Figures 2C and 2D, respectively. 50 Administration of a concentration of TGF-β1 results in a 50% inhibition of systole II without inhibiting the degree of contraction resulting from systole I. Thus, the inhibition of systole II is assessed based on the pole deflection observed at the end of the culture and at the plateau, i.e., herein, on days 10 and 1, respectively, of the drug effect test period, and on the difference in the results compared between the ECT model with drug treatment (treatment condition(s)) and the ECT model without drug treatment (reference condition(s)). In this exemplary example, pole deflection at day 13 is significantly inhibited by about 30% and about 35% with and without TGF-β1 administration, respectively, already exceeding IC 50It is expected to be 0.3 times the concentration. The 13th day corresponds to the last culture day and / or the 10th day of the drug effect test period in this exemplary embodiment.

[0153] Therefore, a drug having anti-fibrotic activity is expected to show at least 1xSD inhibition of systolic phase II in at least one of both settings (+ / - TGF-β1) compared to their respective baselines, for example, on about the 10th day of the (approximate) drug effect test period in the ECT model (Figure 2E). In the ECT model systolic phase II in the absence of TGF-β1, for example, on about the 10th day of the drug efficacy test phase, a drug that enhances contraction by at least 1×SD compared to their respective baselines is considered to have a fibrotic promoting effect.

[0154] In this exemplary embodiment and the following embodiments, the ECT culture time during the study ends on the 13th day, which corresponds to the last culture day and / or the 10th day of the drug effect test period. However, those skilled in the art will recognize that the culture time depends on the cell type and ECT under study, and therefore the last culture day and / or the 10th day of the drug effect test period used herein may be earlier or later than that mentioned herein. For example, if the response of the cells and / or ECT under study is slower than that observed herein, a longer culture period may be advantageous.

[0155] <Example 2: Evaluation of Drug Effects in ECT Using Tensile Tests> (Background) Interference with the contraction characteristics of fibroblasts has shown the potential effects of anti-fibrotic or fibrotic promoting drugs. However, it does not necessarily show that the drug also interferes with the production or degradation of the extracellular matrix. Therefore, drugs are tested for their effects on ECT stiffness by evaluating the plateau and reference stiffness and performing a follow-up analysis of the tensile test in Example 1.

[0156] <Preparation of ECT and Study Design> Reference ECTs were prepared and processed as described in Example 1 herein. In contrast to the pole deflection analysis disclosed in Example 1 herein, eight ECTs were harvested for tensile testing immediately after reaching a plateau, before administration of drug vehicle with or without TGF-β1. The remaining ECTs were cultured for an additional 10 days. Macroscopic images were then taken of the ECTs on the poles to estimate their cross-sectional area based on the observed ECT thickness. Tensile testing was performed using a dynamic mechanical analyzer (DMA). To do this, the ECTs were loaded onto two hooks clamped to a DMA in an organ bath filled with PBS and conditioned at 37°C. Uniaxial extension was then applied at a constant linear rate of approximately 1% (0.03 mm / s) until the ECTs broke. The change in force was recorded and converted into a stress-strain curve. Young's modulus, which represents the stiffness of the ECTs, was analyzed by determining the slope of the linear portion within the elastic region (Figure 3A). All measurements were repeated eight times.

[0157] In the treatment conditions, the study design is as described in the paragraph above for the reference ECT, except that once a plateau is reached, TGF-β1 is added or not added at an effective drug concentration. 50 Concentrations ranging from 0.3 to 30 times the concentration were considered. All measurements were repeated eight times.

[0158] (Methodological aspects) The absolute detection limits for Young's modulus were 1 MPa (upper limit) and 10 kPa (lower limit), respectively. The relative detection limits were approximately 50 times the plateau value (upper limit) and approximately 0.5 times the plateau value (lower limit), respectively.

[0159] Classification of drugs based on their effect on stiffness (Young's modulus) Statistical analysis of the baseline ECT revealed a 1.5 × SD reduction in contraction, which was significant by one-way analysis of variance with Dunnett's multiple comparison test. Table 4 summarizes the (mean) Young's modulus (kPa) values ​​determined by tensile testing for the baseline ECT without TGFβ-1 and the TGFβ-1 baseline ECT. The 1.5 × SD deviation is equivalent to the change in inhibition of Young's modulus increase between the plateau and day 13 (absolute values; day 10 of the drug effect test period): 59% for the baseline without TGFβ-1 and 57% for the baseline with TGFβ-1.

[0160] [Table 4]

[0161] Tensile testing of treated ECTs predicts an increase in stiffness at the end of the drug-effect test period compared to measurements taken during the plateau phase. This increase is further enhanced by administering 5 ng / ml TGF-β1 at the beginning of the drug-effect test period. The theoretical and / or exemplary predicted interference of an "ideal" antifibrotic drug on ECT stiffness in the absence and presence of TGF-β1 is shown in Figure 3B. IC 50 Administration of 0.3 times the concentration inhibited stiffness increase by approximately 30%, and the IC 50 The administration of IC concentrations is expected to suppress stiffness increase by 50%, and such suppression occurs between the plateau and the end of the culture. For example, IC 50 In the case of concentration, the inhibition of stiffness increase is determined as follows: I C 50 Stiffness = Stiffness プラトー +(rigidity 終了 -rigidity プラトー ) / 2 In this exemplary embodiment, stiffness プラトー was determined on day 1 of the drug effect test period, and stiffness 終了 is determined in this illustrative example on day 10 of the drug efficacy study period.

[0162] Thus, a drug with antifibrotic activity would be one that suppresses stiffness increase in at least one of both settings (+ / - TGF-β1) by at least 1.5xSD compared to the respective baseline in the ECT model, e.g., at (approximately) day 10 of the drug efficacy test period. Drugs that increase stiffness increase in the absence of TGF-β1 in the ECT model by at least 1.5xSD compared to the respective baseline, e.g., at day 10 of the drug efficacy test period, are considered to have profibrotic activity.

[0163] Example 3: Effect of pirfenidone on heart failure with preserved left ventricular ejection fraction using ECT, pole deflection, and tensile tests (background) Based on the results obtained in Examples 1 and 2, an efficacy study of pirfenidone will be conducted. Pirfenidone is an orally administered synthetic small molecule pyridine derivative approved for the treatment of idiopathic pulmonary fibrosis. It is believed to have broad effects on inflammation and other fibrotic pathways, as well as inhibitory effects on TGFβ signaling. Pirfenidone is currently undergoing clinical trials as an antifibrotic drug for patients with preserved left ventricular ejection fraction (HEF), characterized by cardiac diastolic dysfunction that correlates with the severity of myocardial fibrosis. Therefore, myocardial fibrosis is an important therapeutic target for HEF. Therefore, pirfenidone will be studied using an ECT-based strength measurement method. Pole deflection and tensile tests were selected as representative physical parameters because both the contractile force and stiffness of the myocardial extracellular matrix are considered essential for reliably studying the biochemical properties of ECT following pirfenidone administration in vitro. Therefore, pole deflection and / or tensile tests represent physiological bodily function in the efficacy studies herein.

[0164] (Research design) ECTs are prepared as in Examples 1 and 2, and pole deflection and tensile tests are performed, except that ECTs are prepared from well-characterized iPSCs representing healthy wild-type or healthy individuals as a reference ("healthy" + / - TGF-β1) and iPSCs obtained from patients with heart failure with preserved left ventricular ejection fraction. More specifically, patient-derived ECTs are divided into an additional reference ("diseased" + / - TGF-β1), a "low" treatment group with 0.3 mg / ml pirfenidone treatment (+ / - TGF-β1), and a "high" treatment group with 1 mg / ml pirfenidone treatment (+ / - TGF-β1). For treatment, pirfenidone is dissolved in the respective culture medium at the concentration given for each group. The IC of the drug is 50 was determined in vitro to be 0.43 mg / ml, so the "high" treatment group contained at least the IC 50 Pirfenidone treatment will be performed at concentrations above the typical therapeutic range. Because pirfenidone is generally less potent in vitro than in vivo, in vitro pirfenidone analysis will investigate concentrations beyond the typical therapeutic range. All physical parameter values ​​will be obtained with 8-10 replicates per group on Day 10 (absolute Day 13) of the drug efficacy study period. The predetermined threshold for at least one physical parameter will be 1xSD for pole deflection and 1.5xSD for tensile testing relative to the respective (mean) value of that parameter observed in each baseline ECT. Pirfenidone is inferred to be effective in treating heart failure with preserved left ventricular ejection fraction if the value of at least one physical parameter (pole deflection and / or tensile strength) exceeds a predetermined threshold value for said parameter obtained in at least one of two settings (+ / - TGF-β1) and in at least two respective reference conditions ("healthy" + / - TGF-β1 and "diseased" + / - TGF-β1, respectively), preferably at least in the reference condition of "diseased" + / - TGF-β1.

[0165] (Expected) Results Pirfenidone is expected to be equally effective in both settings (+ / - TGF-β1). Furthermore, at the end of the culture, i.e., herein, on day 10 of the drug effect test period, pirfenidone treatment is expected to significantly inhibit phase II systole as determined by pole deflection and / or significantly inhibit stiffness increase as determined by tensile testing, at least in the "high" treatment group. The (mean) contraction value (%) determined by pole deflection analysis in at least one of the two settings is expected to meet the predetermined threshold obtained in at least one of the reference conditions in at least one of the two settings, indicating that pirfenidone may be effective in treating heart failure with preserved ejection fraction by, at least in part, restoring physiological function in terms of (ECT) tissue contraction. However, if the (mean) contraction (%) values ​​determined by pole deflection analysis do not meet the predetermined thresholds in either of the two settings and / or for either of the respective reference conditions, pirfenidone may not be assessed as having efficacy for treating heart failure with preserved left ventricular ejection fraction (HEF) by at least partially restoring physiological bodily function in terms of (ECT) tissue contraction. Furthermore, the (mean) Young's modulus values ​​determined by tensile testing are expected not to meet the respective predetermined thresholds obtained in at least one reference condition in either of the two settings, indicating that pirfenidone may not be effective for treating HEF with preserved left ventricular ejection fraction (HEF) in terms of ECT stiffness. However, if the (mean) Young's modulus values ​​meet the predetermined thresholds obtained in at least one of the reference conditions in at least one of the two settings, pirfenidone may be assessed as having efficacy for treating HEF with preserved left ventricular ejection fraction (HEF) by at least partially restoring physiological bodily function in terms of (ECT) stiffness.

[0166] Example 4: Genetic rescue of muscle function in tissue engineered human muscle - titration experiments Several muscle diseases are associated with the dysfunction and / or deficiency of specific proteins. To address the efficacy of potential therapeutic approaches, a human muscle model was created in which dystrophin was depleted as an exemplary target protein. Therefore, this human muscle model can be considered a DMD model. Subsequently, genetic rescue experiments were applied to determine the degree of gene repair required to confirm a discernible effect on muscle function, as described in detail in Long et al. (2018), Sci Adv, 4:eaap9004.

[0167] Artificial myocardium (more specifically, engineered human myocardium; EHM) was generated from dystrophin-deficient induced pluripotent stem cell (iPSC)-derived cardiomyocytes and genetically corrected isogenic cardiomyocytes. EHM was titrated to contain 0%, 10%, 30%, 50%, and 100% corrected cardiomyocytes. EHM functionality was assessed by contraction experiments after 4 weeks of maturation on a flexible holder. Therefore, contraction experiments were performed in organ bath experiments. Organ bath experiments and multi-wall plate experiments are routine methods well known to those skilled in the art (see, e.g., Tiburcy et al., 2017, Defined Engineered Human Myocardium with Advanced Maturation for Applications in Heart Failure Modeling and Repair. Circulation 135:1832-1847; Tiburcy et al., 2020, Generation of Engineered Human Myocardium in a Multi-well Format. STAR Protoc. 1:100032; Zimmermann et al., 2005, Engineering Heart Tissue for In Vitro and In Vivo Studies, In: Dhein, S., Mohr, FW, Delmar, M. (eds) Practical Methods in Cardiovascular Research, Springer, Berlin, Heidelberg, https: / / doi.org / 10.1007 / 3-540-26574-0_34).

[0168] Specifically, contraction experiments were performed under isometric conditions in organ baths at 37°C in gassed (5% CO2 / 95% O2) Tyrode's solution (120 mM NaCl, 1 mM MgCl2, 0.2 mM CaCl2, 5.4 mM KCl, 22.6 mM NaHCO3, 4.2 mM NaH2PO4, 5.6 mM glucose, and 0.56 mM ascorbic acid). EHMs were electrically stimulated with 200 mA, 5 ms rectangular pulses at 1.5 Hz. EHMs were mechanically stretched at 125 mm intervals according to the Frank-Starling mechanism until maximal contractile force amplitude (FOC) was observed. Responses to increases in extracellular calcium (0.2–4 mM) were examined to determine maximal inotropic capacity. Where indicated, force was normalized to muscle mass (sarcomer α-actinin-positive cell mass determined by flow cytometry).

[0169] Our results indicate that approximately 30% of the cardiomyocytes must be repaired to partially rescue contractile force, and approximately 50% of the cardiomyocytes must be repaired to fully rescue the phenotype. Restoring approximately 10% of the cardiomyocytes did not affect force production (Figure 4; see also Long et al. (2018), Sci Adv, 4:eaap9004).

[0170] Example 5: Genetic rescue of muscle function in EHM - genome editing After determining the degree of gene repair required to see a clear effect on muscle function, genome editing conditions were compared considering their impact in the context of DMD. DMD is often caused and / or affected by frameshift mutations resulting from the deletion of one or more exons in the dystrophin gene. This prevents translation of functional (wild-type) dystrophin protein, producing structurally impaired dystrophin, which often leads to muscle cell degeneration, inflammation, and fibrosis. However, there may be exons within the dystrophin gene that, when skipped, can convert DMD to a milder phenotype. Therefore, various genome editing conditions were tested to evaluate the potential of exon skipping to correct frameshift mutations in human DMD.

[0171] Engineered human myocardium (EHM) was generated from induced pluripotent stem cells obtained from patients with Duchenne muscular dystrophy using the method described by Tiburcy et al. (2017, Defined Engineered Human Myocardium with Advanced Maturation for Applications in Heart Failure Modeling and Repair. Circulation 135:1832-1847). Organ bath and multi-wall plate experiments are routine methods familiar to those skilled in the art (e.g., Tiburcy et al., 2017, Defined Engineered Human Myocardium with Advanced Maturation for Applications in Heart Failure Modeling and Repair. Circulation 135:1832-1847; Tiburcy et al., 2020, Generation of Engineered Human Myocardium in a Multi-well Format. STAR Protoc. 1:100032). We performed these experiments to obtain data on the force of contraction (FOC) of EHMs derived from DMD patients and compared them with EHMs derived from induced pluripotent stem cells obtained from healthy donors. EHMs derived from DMD patients exhibited significantly reduced contractile force. In an attempt to rescue the DMD phenotype and validate potential therapeutic strategies, we applied genome editing by targeted CRISPR / Cas9 insertion of insertion / deletion mutations (indels).

[0172] A total of six different editing conditions were compared, identifying four edits associated with normalization of contractile function and two edits that did not significantly rescue contractile function (Figure 5). Thus, this example demonstrates the application of artificial muscle tissue to identifying therapeutically efficient genome editing strategies. At least one physical parameter may differ by (at least) 1 × SD or 1.5 × SD from a predetermined (mean) threshold value obtained in a reference (experiment; i.e., for example, patient-derived DMD-EHM). Alternatively, or in addition, the predetermined threshold may be a change of at least 30%, 35%, 55%, or 60% from the (mean) value obtained for at least one physical parameter obtained in the reference (experiment). The reference experiment may be performed, for example, using a DMD model, which may be patient-derived or genetically engineered to resemble a clinically relevant DMD model.

[0173] Thus, this example illustrates two extremes: healthy and diseased. Any treatment using a therapeutic agent, such as a biological agent, drug, ATMP, LNP, or all-in-one AAV-CAS9-gRNA vector, is expected to result in a physical parameter value between the above extremes in the respective efficacy tests disclosed herein. A preferably statistically significant change in the value of a physical parameter compared with the respective parameter in an untreated diseased model is preferably evaluated as an indicator (threshold) of the efficacy of the respective drug in treating the disease under study. Therefore, an ideal pharmaceutical product is expected to result in at least partial, preferably complete, recovery (i.e., return of the determined (mean) value of at least one physical parameter to the respective (mean) value obtained under the "healthy" reference (experiment) and / or optimally edited reference conditions (such as EDITs 1-4 in this example)). When applying the efficacy tests according to the present invention, a change of about 10%, preferably at least about 12.5% ​​or 15%, is expected to be found to be statistically significant. Deviations from this assumption may be caused by low sample size and / or technical detection limits, but can be recognized and appreciated by those skilled in the art.

[0174] Example 6: Screening for drugs with unknown cardioactive properties using EHM Mutations in the titin (TTN) gene can contribute to dilated cardiomyopathy (DCM). Based on this finding, in this example, we generated mutant EHMs and used them to evaluate the efficacy of several substances with unknown cardiac activity profiles. More specifically, the TTNtv iPSC-derived cardiomyocytes used in this example had a mutation in exon 326 of the TTN gene (c.43628insAT, p.Ser14450fsX). The mutations in the sarcomeric protein titin can cause the accumulation of truncated titin proteins, which can contribute to (mainly dilated) cardiomyopathy (e.g., Fomin et al., Science Translational Medicine, 2021, Vol 13, Issue 618, doi: 10.1126 / scitranslmed.abd3079; and Gramlich et al., EMBO Mol Med (2015) 7:562-576 https: / / doi.org / 10.15252 / emmm.201505047). Therefore, the mutations may be interesting candidates for targeted therapy to treat DCM.

[0175] Engineered human myocardium (EHM) was generated in 48-well plates from TC1133 TTN-wt (wild-type, unedited), TTNtv iPSC-derived cardiomyocytes (edited with a mutation in exon 326 of the TTn gene (c.43628insAT, p.Ser14450fsX) that causes dilated cardiomyopathy (DCM)), and human fibroblasts as described in Tiburcy et al. STAR Protocols (2020). EHMs were matured for 28 days and then treated with 10 μM of each substance (2 EHMs per substance; treatment condition) or vehicle (0.2% DMSO; reference condition) for 14 days (until day 42) with daily medium changes. EHM function was monitored weekly by optically recording pole bending. Therefore, day 29 can be considered day 1 of the drug effect testing period, and day 42 can be considered day 14 of the drug effect testing period applied in this illustrative example. The substances tested in this example were developed with the aim of reducing the amount of abnormal protein aggregates, and therefore their efficacy in treating DCM caused by the TTN c.43628insAT mutation was investigated using the EHM model.

[0176] The effects of 19 substances (Z1-19; provided by Prof. C. Griesinger, Max-Planck for Multidisciplinary Sciences; Department of NMR-based Structural Biology) on EHM function were determined by assessing the EHM contraction rate as a parameter of force generation, as well as the spontaneous beating frequency. EHM contraction rate, as used herein, refers to the pole deflection per contraction, calculated as 100 × (the pole distance). 完全弛緩 -Pole distance 最大収 ) / Pole distance 完全弛緩 Thus, the EHM contraction rate is determined as a physical parameter, and thus as a measure of physiological bodily function, which determines whether a given substance in this example has efficacy in treating DCM by at least partially restoring the function of the measured physiological bodily function.

[0177] The mean EHM contraction rates (units: %) for vehicle-treated baseline EHMs are summarized in Tables 5 and 6. To determine effects related to EHM contraction rate, thresholds of 1 standard deviation (SD) or 2 standard deviations were defined, corresponding to a change in EHM contraction rate of 18% (1 × SD) or 36% (2 × SD) for TTN-wt EHMs and 20% (1 × SD) or 40% (2 × SD) for TTNtv EHMs, respectively.

[0178] [Table 5]

[0179] [Table 6]

[0180] By applying a threshold of 2 standard deviations (SD) of each reference EHM sample, a number of substances were identified that increased or decreased EHM contraction rate, as summarized in Table 7. A substance was inferred to be effective in treating DCM if the value of at least one physical parameter (EHM contraction rate) exceeded the predetermined threshold for that parameter obtained in at least one of the two reference conditions (unedited / "healthy" and / or edited / "diseased"), preferably at least in the "diseased" reference.

[0181] [Table 7]

[0182] The spontaneous beating frequencies of vehicle-treated baseline EHMs are summarized in Tables 8 and 9. To determine the relevant effect on EHM beating frequency, thresholds of 1 SD or 2 SD were defined, corresponding to a change in EHM beating frequency of 15% (1 × SD) or 30% (2 × SD) for TTN-wt EHMs and 11% (1 × SD) or 22% (2 × SD) for TTNtv EHMs, respectively.

[0183] [Table 8]

[0184] [Table 9]

[0185] Substances that increase or decrease spontaneous beating frequency are identified by applying a threshold of 2 × SD for each reference EHM sample. In this case, a substance is inferred to be effective in treating DCM if the value of at least one physical parameter (spontaneous beating frequency) exceeds a predetermined threshold value for said parameter obtained in at least one of the two reference conditions (unedited TTN-wt and / or edited TTNtv), preferably at least in the "diseased" reference condition. Considering spontaneous beating frequency and / or EHM contraction rate as physical parameters to be determined to assess the potency of a substance, a substance is identified as increasing or decreasing these parameters. In this case, a substance is inferred to be effective in treating DCM if the value of at least one physical parameter (EHM contraction rate and / or spontaneous beating frequency) exceeds a predetermined threshold value for said parameter obtained in at least one of the two reference conditions (unedited and / or edited), preferably at least in the "diseased" reference condition.

[0186] Example 7: Efficacy test of DMD rescue using EHM (background) Considering the results obtained in Examples 4 to 6, the exon skipping approach may at least partially alleviate some DMD phenotypes. Because skipping exon 51 of the dystrophin gene may correct more than 10% of DMD phenotypes, one candidate will be further investigated. Eteplirsen is a marketed drug based on the exon 51 skipping approach and has received accelerated FDA approval based on a "surrogate endpoint" rather than the direct indication of clinical benefit required for traditional FDA approval. Therefore, efficacy testing for DMD rescue will be performed on eteplirsen to illustrate the commercial, regulatory, and scientific validity of the method according to the present invention in the context of DMD using EHM. For the assay of eteplirsen in the DMD EHM model, the physical parameters exemplarily evaluated herein are EHM contraction rate (see Example 6), spontaneous beating frequency (see Example 6), and maximum force of contraction amplitude (FOC) (see Examples 4 and 5). The physical parameter of focus will be FOC, as contractile force represents the expected primary target of eteplirsencen.

[0187] Eteplircensen is chosen as an exemplary drug, specifically as an example of a nucleotide therapeutic. Eteplircensen is indicated for patients with DMD who have a confirmed mutation in the dystrophin gene that allows exon 51 skipping. This indication has received accelerated FDA approval based on the observed increase in skeletal muscle dystrophin in some patients treated with eteplirsen. Continued approval may be contingent on verification of clinical benefit through time-consuming and costly confirmatory trials. Currently published clinical data indicate that eteplirsen administration can only increase dystrophin protein expression by approximately 1% (e.g., EMA / 691796 / 2018; and / or Aartsma-Rus and Krieg, Nucleic Acid Therapeutics, 2017, Vol. 27, Number 1, doi: 10.1089 / nat.2016.0657). Therefore, in this example, the DMD EHM model does not appear to show significant improvement for any of the selected physical parameters, including the putative primary target physical parameter, FOC.

[0188] (Research design) Engineered human myocardium (EHM) was generated from induced pluripotent stem cells obtained from healthy individuals as well as from DMD patients capable of exon 51 skipping, as described in Tiburcy et al. (2017, Defined Engineered Human Myocardium with Advanced Maturation for Applications in Heart Failure Modeling and Repair. Circulation 135:1832-1847). To assess three physical parameters, five groups of seven replicates each were investigated, representing baseline and treatment conditions. Specifically, EHM from healthy individuals ("healthy" baseline) and EHM from DMD patients not receiving eteplirsenten ("diseased" baseline) represented the baseline (baseline condition), while treatment conditions included EHM from DMD patients treated with "low-dose," "standard-dose," and "high-dose" eteplirsenten. To determine the respective concentrations, serum concentrations following clinically administered eteplirsen concentrations, e.g., as reported in the CHMP assessment report (EMEA / H / C / 004355 / 0000), can be used as an estimate of the "standard" treatment. Additionally, a broader range, e.g., from about 1 log higher to about 1 log lower than such (reported) serum concentrations and / or clinically effective concentrations, can be tested. Thus, for example, a "high" treatment can be 1 log higher than the concentration used for the "standard" treatment, and a "low" treatment can be 1 log lower. For efficacy testing, EHMs are incubated with eteplirsen at the indicated concentrations for the designated groups for 14 days. Physical parameter measurements are performed as described in Examples 4-6. All physical parameter values ​​are obtained after 14 days of incubation with or without eteplirsen. The predetermined threshold for at least one physical parameter (EHM contractility, spontaneous beating frequency and / or FOC) is 2 × SD of the (mean) value of said parameter observed in the respective reference EHM not incubated with the pharmaceutical agent eteplirsen.

[0189] All three physical parameters are expected to show elevated values ​​within the treatment concentrations, with eteplirsen at the "standard" treatment concentration showing higher (mean) values ​​than the "low" treatment concentration, but lower than the "high" treatment concentration. Overall, incubation with eteplirsen, regardless of the concentration selected, is not expected to result in (mean) EHM contraction rate values ​​that meet the respective predetermined thresholds obtained in at least one of the two reference conditions, preferably at least in the "diseased" reference condition, which would indicate that eteplirsen has no efficacy in treating DMD (by at least partially restoring functionality of physiological body functions measured as EHM contraction rate). However, if at least the "high" treatment group exhibits a (mean) EHM contraction rate that meets the respective predetermined threshold obtained in at least one of the two reference conditions, preferably at least in the "diseased" reference condition, eteplirsen is assessed as having efficacy for treating DMD by at least partially restoring the functionality of the physiological body function measured as EHM contraction rate. Also, in the case of spontaneous beat frequency (Hz), incubation with eteplirsen is not expected to result in any determined (mean) spontaneous beat frequency value that meets the respective predetermined threshold obtained in at least one of the two reference conditions, preferably at least in the "diseased" reference condition, indicating that eteplirsen is not effective for treating DMD by at least partially restoring the functionality of the physiological body function measured as spontaneous beat frequency (Hz). However, if at least the "high" treatment group exhibits (mean) spontaneous beat frequency values ​​that meet the respective predetermined thresholds obtained in at least one of the two reference conditions, preferably at least in the "diseased" reference condition, then eteplirsen will be assessed as having efficacy in treating DMD by at least partially restoring functionality of physiological bodily functions measured as spontaneous beat frequency.Furthermore, in the case of FOC, incubation with any of the investigated concentrations of eteplirsen is not expected to result in a determined (mean) FOC value that meets the predetermined threshold obtained in at least one of the two reference conditions, preferably at least in the "diseased" reference condition, which would indicate that eteplirsen is not effective in treating DMD by at least partially restoring the functionality of physiological body functions measured as FOC. However, if at least the "high" treatment group shows a (mean) FOC value that meets the respective predetermined threshold obtained in at least one of the two reference conditions, preferably at least in the "diseased" reference condition, eteplirsen will be evaluated as having efficacy in treating DMD by at least partially restoring physiological body functions measured as FOC. Even if the expectation is met, eteplirsen is not considered to be effective in treating DMD. In particular, it is not expected that the value of any one of at least one physical parameter (EHM contractility, spontaneous beat frequency and / or FOC) would exceed a predetermined threshold value for said parameter obtained in at least one of the two reference conditions, preferably at least in the "diseased" reference condition, and therefore eteplirsen would not be presumed to have efficacy in treating DMD.

[0190] In general, a pharmaceutical product (such as eteplirsen) is preferably considered to have efficacy for treating a disease of interest (such as DMD) using the efficacy tests described herein if a concentration-dependent effect is observed and / or if the (mean) value of at least one determined physical parameter exceeds a respective predetermined threshold value at one or more (tested) concentrations of the pharmaceutical product, preferably at least in the "diseased" reference condition.

[0191] Furthermore, without being bound by theory, it is believed that eteplirsen's lack of efficacy may be due to its failure to reach the intended target cells (at least not in effective amounts) to exert its effects. Alternative formulations (e.g., via chemical and / or nanopharmacological modifications, such as packaging into nanoparticles, with or without target organ / cell specificity) or alternative nucleotide therapeutics may be able to enhance eteplirsen's therapeutic activity in situ. However, identifying such formulations requires suitable platforms and approaches, which have been lacking until now. This problem is addressed by the efficacy tests described herein. The efficacy tests disclosed herein provide an opportunity to test the potency of pharmaceuticals and / or their alternative formulations, for example, in artificial skeletal muscle or cardiac muscle. This allows for the determination of individualized efficacy and dosage for patients. With regard to eteplirsen as an illustrative example, this may be enabled and / or supported by the fact that the efficacy tests disclosed herein may use a physical parameter (e.g., contractile force) as a readout rather than currently investigated surrogates such as dystrophin protein levels. The importance of a direct readout is also demonstrated in Example 5, where DMD was "re-expressed" in EDIT5-6, but without clear and / or detectable functional improvement.

[0192] Example 8: Efficacy test using artificial skeletal muscle (ESM) Another major effect of DMD is observed in skeletal muscle, therefore, artificial skeletal muscle (ESM) will be used in a separate eteplirsen efficacy study in relation to DMD.

[0193] iPSCs from healthy subjects and DMD patients capable of exon 51 skipping were used to generate ESMs as described in Shahriyari et al. (Engineered skeletal muscle recapitulates human muscle development, regeneration, and dystrophy, 2022, Journal of Cachexia, Sarcopenia and Muscle, DOI: 10.1002 / jcsm.13094). The ESMs were incubated with different concentrations of eteplirsen as described in Experiment 7. The physical parameter evaluated in the efficacy test of eteplirsen in the ESM model of DMD is contractile force, as described in Shahriyari et al. 2022, preferably determined using a stimulation frequency of 40 Hz or 100 Hz.

[0194] Shahriyari et al. (2022) disclosed that dystrophin deletion in DMD ESM caused a significant decrease in contractile force (-35±7%, P<0.05), determined as twitch tension, compared to the rescued phenotype after genome editing using the genome editing approach described in Example 5. Therefore, a change of about 40% is expected in the gene-corrected model. However, the change in pharmacologically relevant physical parameters is expected to be much lower, such as about 10%, compared to the reference (experimental) model. With this in mind, the predetermined threshold for twitch tension values ​​observed and determined in eteplirsen-incubated DMD ESM models is expected to be associated with an increase in twitch tension of approximately 0.5x SD or 1x SD, and / or x-fold SD in the range of approximately 0.5x SD to 1x SD, relative to the respective twitch tension values ​​obtained for genetically rescued, and therefore permanently gene-repaired, ESMs, untreated DMD ESM baseline ("diseased" baseline) and / or healthy subjects ("healthy" baseline).

[0195] As in Example 7, eteplirsen is not expected to significantly alter physical parameters, and the determined twitch tension values ​​in the eteplirsen-treated "standard" and / or "high" concentration ESM models are not expected to meet and / or exceed the predetermined thresholds obtained in at least one of the reference conditions, preferably at least the "diseased" reference condition, which would not indicate that eteplirsen has efficacy in treating DMD. This is consistent with what was expected given that the mean re-expression of dystrophin in muscle tissue under eteplirsen administration was shown to be 1% or less (see, e.g., Figure 6 in the CHMP Assessment Report (EMEA / H / C / 004355 / 0000)). As shown in Figure 4, restoration of dystrophin expression by 10% or more was determined to be essential for recovery of muscle function. However, at least the "high" treatment group (unexpectedly) exhibits (mean) twitch tension values ​​that meet the respective predetermined thresholds obtained in at least one of the reference conditions, preferably at least in the "diseased" reference condition, and eteplirsen is assessed as having efficacy in treating DMD by at least partially restoring functionality of physiological body functions measured as twitch tension in the ESM model.

[0196] <Example 9: Evaluation of efficacy against BENO using MEA> Cerebral organoids are advantageous for modeling developmental malformations such as microcephaly and dicephaly. BENO exhibits excitatory-inhibitory interactions, which are advantageous for testing proconvulsant and anticonvulsant drugs.

[0197] (method) Bioengineered neural organoids (BENO) were generated according to the BENO generation protocol described by Zafeiriou et al. (Nature Communications, 2020, 11:3791, https: / / doi.org / 10.1038 / s41467-020-17521-w).

[0198] The method used in this illustrative example was multielectrode array (MEA) recording. Specifically, prior to seeding, 6-well plates containing 64 platinum microelectrode arrays per well (0.04 MΩ / microelectrode, 30 μm microelectrode diameter, 200 μm spacing) were coated with Matrigel™ diluted 1:120 in PBS for 1 hour at room temperature. One or two fragments (300 μm thick) were placed into MEA wells and secured with concentrically wrapped tungsten rings. Recordings of 10–15 min / 2 h were performed every other day for up to 60 days using the Maestro pro MEA system (Axion Biosystems). Data recording was performed automatically with Axion software (AxIS Navigator) using the manufacturer's Spontaneous Neural Configuration. Data analysis was performed using the manufacturer's standalone tools, the Neural Metric Tool, and the AxIS Metric Plotting Tool (Axion Biosystems). The spike detection threshold was set at 5.5 standard deviations, and electrodes detecting at least five spikes per minute were classified as active. Spike bursts were identified using an ISI threshold requiring a minimum of five spikes with a maximum ISI of 100 ms. Network bursts (NBs) were identified by an envelope algorithm using a threshold of 1.25, a minimum IBI interval of 100 ms, and a 75% burst inclusion rate with a minimum of 10% active electrodes.

[0199] (process) Picrotoxin (PIC) was used as an exemplary proconvulsant. Cyanoquizaline (CNQX) was used as an example of a general excitatory neuron inhibitor. As disclosed herein, PIC and CNQX can also be used as positive controls in efficacy assays when evaluating the efficacy of pharmaceuticals with potentiating and reducing activity, respectively. This is particularly true in diseases such as epilepsy. Using PIC and / or CNQX as positive controls may be advantageous for examining the construction of interconnected BENO cells and / or functional integrated networks. However, those skilled in the art will understand that the selection of an appropriate positive control will depend on the disease. For example, in the case of Parkinson's disease, the positive control may advantageously be dopamine production / release and / or dopamine neuron levels.

[0200] Table 10 shows the processing details of Example 9.

[0201] [Table 10]

[0202] The investigated parameters and their respective observation ranges are shown in Table 11.

[0203] [Table 11]

[0204] (result) Table 12 shows the physical parameters investigated and their respective thresholds, expressed as both standard deviations (SD) and percentage changes, determined for untreated baseline BENO, as well as their respective thresholds for subsequent use as predetermined thresholds for treated BENO to assess the efficacy of PIC and CNQX.

[0205] [Table 12]

[0206] By applying a threshold of 2 × SD of the reference BENO (thus requiring physical parameter values ​​that deviate from the reference BENO by more than 2 × SD in this example), picrotoxin (PIC) and cyanquizarin (CNQX) were found to increase or decrease at least some of the determined BENO activity parameters (summarized in Table 13).

[0207] [Table 13]

[0208] The theoretically predicted range of parameter values ​​and the observed parameter values ​​are summarized in Table 14. Figure 6C plots the observed parameter values ​​listed in Table 14. Note that for two physical parameters, inter-burst interspike interval and inter-NB interspike interval, the ranges are shown from large to small because smaller values ​​are more suitable for the functionality of BENO.

[0209] [Table 14]

[0210] Picrotoxin treatment increased spontaneous activity. Weighted-average firing rate, burst frequency, and network burst frequency increased when the inhibitory GABAergic network was inhibited. Network burst complexity, as measured by network burst duration, decreased. This suggests that the GABAergic network may contribute to network complexity.

[0211] CNQX binding to glutamate receptors (AMPA) inhibited glutamate binding, resulting in decreased synaptic activity. This was exemplified by a decrease in the weighted average firing rate (NB frequency). Burst frequency decreased to, but did not exceed, the predetermined threshold of 2x SD required in this study. The interspike intervals within bursts increased after treatment due to silencing of excitatory networks. The few spikes still detected were from spontaneously firing neurons. NBs were not observed in BENO treated with CNQX, and therefore NB frequency was zero, and NB-related parameters could not be assessed.

[0212] Figure 6 shows an example raster plot of neural network activity in BENO at day 60. (A) An example raster plot showing all electrode (El) / channel activity. Each spike is represented by a black line at the top of each plot, bursts are represented by a black line within the main body of each plot, and network bursts, indicating inter-burst synchronization, are highlighted by a dashed rectangle. Parameters such as spike, burst, network burst duration, interspike interval, and interburst interval are all shown in the figure. (B) Representative activity raster plot of the same well before and after picrotoxin treatment, showing an increase in firing rate and burst frequency. This indicates that picrotoxin blocks inhibitory networks and induces hyperactivity. (C) Bar graphs show weighted average firing rate (Hz) (left) and burst frequency (Hz) (right). PIC (picrotoxin) increases both locomotor activity and CNQX decreases both locomotor activity. Horizontal lines indicate the "healthy" baseline reference value (vehicle - solid line), and the change from baseline reference value with PIC and CNQX (striped line). Efficacy of therapeutic drugs can be identified when drug-induced increases or decreases in activity are normalized to the "healthy" baseline reference value (vehicle - solid line).

[0213] (Discussion) The data obtained demonstrate that neural network function measured by BENO with MEA can be altered by proconvulsant drugs such as picrotoxin, and therefore, BENO-based efficacy testing may be highly advantageous for assessing the effectiveness of treatments and / or drugs.

[0214] This illustrative example relates to the potential application of efficacy testing as disclosed herein. In principle, genetic and non-genetic factors can lead to changes in function (preferably compared to healthy reference values), and significant changes, preferably (at least partial) normalization to healthy reference (average) values, can be considered a sign of efficacy. The efficacy of a pharmaceutical product is indicated, for example, when (at least partial or complete) normalization of function occurs.

[0215] The present invention also has the features described in the following paragraphs.

[0216] 1. A method for evaluating the efficacy of a pharmaceutical agent suspected to be effective in treating a disease, wherein the disease is preferably a genetic or non-genetic disease, and the disease causes abnormalities in physiological bodily functions in patients having the disease, and the method comprises: (i) incubating an artificial tissue with a pharmaceutical agent, the artificial tissue being modified or having been modified to express the disease; (ii) determining at least one physical parameter of the artificial tissue obtained in step (i), wherein said physical parameter is an indicator of the functionality of a physiological body function; (iii) determining whether the value of the at least one physical parameter determined in step (ii) satisfies a predetermined threshold value for the physical parameter, thereby determining whether the pharmaceutical product is efficacious; If the value of the at least one physical parameter exceeds the predetermined threshold, it indicates that the pharmaceutical agent is effective in treating the disease by restoring at least part of the functionality of a physiological body function.

[0217] 2. The predetermined threshold is based on or obtained by a standard (experiment) using statistical methods, and the predetermined threshold is preferably 1 x SD (standard deviation), 1.5 x SD or 2 x SD away from the (average) reference value obtained for at least one physical parameter in the standard (experiment), optionally expressed as a % change from the (average) reference value; The method according to paragraph 1.

[0218] 3. The artificial tissue comprises one or more of cardiac tissue, cardiac muscle, muscle tissue, skeletal muscle tissue, nerve tissue, or connective tissue; 10. The method of any one of the preceding clauses.

[0219] 4. The engineered tissue is genetically edited to contain a mutation that is known to cause a disease. 10. The method of any one of the preceding clauses.

[0220] 5. The artificial tissue has been subjected to non-genetic intervention to express the disease. 10. The method of any one of the preceding clauses.

[0221] 6. The pharmaceutical is an advanced therapeutic medicinal product (ATMP), such as a gene therapy medicinal product (GTMP), a somatic cell therapy medicinal product (SCTMP), or a tissue engineered product (TEP); a small molecule, a peptide, a protein, a nucleic acid, a synthetic RNA, a non-coding RNA, a ribonucleoprotein particle (RNP) complex, or a read-through enhancer; 10. The method of any one of the preceding clauses.

[0222] 7. The GTMP is a viral vector, preferably a nanoparticle containing a nucleic acid and / or a protein, preferably encapsulated in an RNP, or a nucleic acid such as an antisense oligonucleotide; The method according to paragraph 6.

[0223] 8. The GTMP is a synthetic vector or a viral vector, such as a retrovirus or lentivirus, such as an adenovirus or adeno-associated virus (AAV), more preferably wherein the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrhlO, AAVrhl74, or any combination thereof, preferably AAV2 or AAV9; 8. The method according to paragraph 6 or 7.

[0224] 9. The pharmaceutical contains a nucleic acid encoding a protein for gene editing, such as an endonuclease, such as a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a clustered regularly interspaced short palindromic repeats (CRISPR) nuclease, or an expression thereof; 10. The method of any one of the preceding clauses.

[0225] 10.GMTP is an endonuclease such as Cas9 or Cas13, preferably Streptococcus pyogenes Cas9 (SpCas9), S. aureus Cas9, S. auricularis Cas9, S. lugdunensis Cas9, or N. meningitides Cas9, and a guide RNA (gRNA); or a nucleic acid encoding an endonuclease such as Cas9 or Cas13, preferably Streptococcus pyogenes Cas9 (SpCas9), S. aureus Cas9, S. auricularis Cas9, S. lugdunensis Cas9, or N. meningitides Cas9, and one or more guide RNAs (gRNAs); Item 7. The method according to any one of items 7 to 9.

[0226] 11. The physical parameter is one or more selected from the group consisting of length, mass, time, current, temperature, light intensity, and physical parameters derived therefrom; 10. The method of any one of the preceding clauses.

[0227] 12. The physical parameter is one or more selected from the group consisting of force, movement, current, mass, tissue structure (swelling / condensation, transparency) or one or more biomechanical parameters (tissue compression, contraction, stiffness, tensile strength, elasticity, extensibility, excitability, resilience, toughness, etc.); iii) at least one selected from the group consisting of tissue contraction, stiffness, force (stretch), spontaneous beating frequency, contraction time and / or velocity, relaxation time and / or velocity, (contraction) force, contractile force, and one or more MEA measurements; preferably measured as one or more selected from the group consisting of pole deflection, Young's modulus, (EHM) shortening, spontaneous beating frequency, contraction time and / or velocity, relaxation time and / or velocity, contractile force, twitch tension, resting tension, and (weighted) mean firing rate, burst frequency (Hz), interspike interval within burst (ms), network burst (NB) frequency, interspike interval within NB (ms), and NB duration (s), 10. The method of any one of the preceding clauses.

[0228] 13. At least one of the physical parameters is not a gene or protein expression level; 10. The method of any one of the preceding clauses.

[0229] 14. The artificial tissue is derived from a non-human primate such as a human, macaque, or marmoset, a pig, or a rodent such as a mouse, rat, or guinea pig. 10. The method of any one of the preceding clauses.

[0230] 15. The artificial tissue contains cells obtained from a patient who is to be treated with the pharmaceutical agent. 10. The method of any one of the preceding clauses.

[0231] 16. The artificial tissue contains cells not obtained from a patient who is to be treated with the pharmaceutical agent. 15. The method according to any one of items 1 to 14.

[0232] 17. Further comprising analyzing the biomarker, such as gene or protein expression of the biomarker; 10. The method of any one of the preceding clauses.

[0233] 18. The artificial tissue is cardiac muscle. 10. The method of any one of the preceding clauses.

[0234] 19. The at least one physical parameter is the force of the artificial tissue and / or the movement of the artificial tissue; 10. The method of any one of the preceding clauses.

[0235] 20. One or more of the conditions include a condition in which the artificial tissue exerts a force of 0.01 mN or more, 0.05 mN or more, 0.1 mN or more, or 1 mN or more. 19. The method of claim 18 or 19.

[0236] 21. The disease is an inherited cardiac condition, preferably an inherited cardiac condition selected from the group consisting of genetic forms of dilated, hypertrophic, and arrhythmogenic cardiomyopathy (ACM), or fibroblastosis, and cardiomyopathy associated with a congenital metabolic disorder, more preferably a condition selected from the group consisting of Duchenne muscular dystrophy (DMD), Noonan syndrome, dilated cardiomyopathy, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, long / short QT syndrome, Takotsubo cardiomyopathy, lysosomal storage disorders, titinopathy, and Barth syndrome, 10. The method of any one of the preceding clauses.

[0237] 22. The disease is a non-genetic cardiac condition, preferably induced by mimicking neurohormonal and / or pharmacological stimuli (e.g., by catecholamines, angiotensin, and / or transforming growth factor β), by drugs such as doxorubicin, tyrosine kinase inhibitors and / or cardiotoxic drugs, by mechanical injury such as crush injury, by temperature injury such as frostbite or thermal injury, by biophysical injury such as radiation injury, by infection such as viral infection with cardioactive virions such as Coxsackievirus, SARS coronavirus, cytomegalovirus and Dengue virus, or by parasitic infection such as Chagas disease caused by Trypanosoma cruzi, optionally by bloodstream, e.g., serum from a patient with cardiac disease, and / or by cells, e.g., mononuclear cells (e.g., T cells, B cells, NK cells, macrophages), optionally derived from the blood of a patient with cardiac disease, 21. The method according to any one of items 1 to 20.

[0238] 23. The artificial tissue is a neural tissue, preferably a neural organoid; 18. The method according to any one of items 1 to 17.

[0239] 24. The at least one physical parameter is current and / or activity; 23. The method according to any one of paragraphs 1 to 17 and 22.

[0240] 25. One or more of the above conditions include localized and coordinated electrical bursts and / or clusters; 22. The method according to claim 23.

[0241] The disease is a neuronal disorder, and is preferably selected from the group consisting of neurodegenerative diseases (e.g., dementia, Parkinson's disease, Huntington's disease), neuroinflammatory diseases (e.g., multiple sclerosis), cerebroinflammatory diseases (e.g., meningitis), channelopathies (e.g., epilepsy), and psychiatric disorders (including autism spectrum disorders and schizophrenia). 26. The method according to any one of paragraphs 23 to 25.

[0242] 27. The artificial tissue is muscle tissue, preferably skeletal muscle tissue. 18. The method according to any one of items 1 to 17.

[0243] 28. The at least one physical parameter is force or movement; 28. The method according to any one of paragraphs 1 to 17 and 27.

[0244] 29. One or more of the conditions include a condition in which the artificial tissue exerts a force of 0.01 mN or more, 0.05 mN or more, 0.1 mN or more, or 1 mN or more. 29. The method of claim 27 or 28.

[0245] 30. The disease is an inherited or hereditary muscle disease, preferably an inherited and / or genetic muscle disease selected from the group consisting of Duchenne muscular dystrophy, facioscapulohumeral dystrophy, Becker dystrophy, Emery-Dreifuss dystrophy, myotonic dystrophy, limb dystrophy, oculopharyngeal muscular dystrophy, congenital dystrophies, congenital myopathies, myotonic dystrophy, familial periodic paralysis, and hereditary connective tissue diseases such as Marfan syndrome, Ehlers-Danlos syndrome, or Loeys-Dietz syndrome. 30. The method according to any one of paragraphs 27 to 29.

[0246] 31. The artificial tissue is a hybrid of different types of tissue, preferably: (i) neurons and skeletal muscle, or (ii) Neurons and cardiac muscle It is a mixture of; More preferably, the hybrid of different types of tissue forms one or more neuromuscular junctions. 31. The method according to any one of paragraphs 1 to 17 and paragraphs 27 to 30.

[0247] 32. The artificial tissue is cardiac or skeletal muscle; the pharmaceutical product is an AAV2 and / or AAV9 viral vector comprising a nucleic acid encoding Cas9, preferably S. aureus Cas9, S. auricularis Cas9, S. lugdunensis Cas9, or N. meningitides Cas9, and one or two guide RNAs (gRNAs), wherein the Cas9 gene is under the control of a muscle-specific promoter such as CK8e or TNNT2; the disease is Duchenne muscular dystrophy, the physical parameter is a force; Preferably, the artificial tissue contains cells obtained from a patient who is to be treated with the medicament. 18. The method according to any one of items 1 to 17.

[0248] 33. The artificial tissue is a connective tissue. 18. The method according to any one of items 1 to 17.

[0249] 34. The at least one physical parameter is a biomechanical parameter such as tissue compression, contraction, stiffness, tensile strength, elasticity, extensibility, excitability, resilience, or toughness. 34. The method according to any one of paragraphs 1 to 17 and 33.

[0250] 35. The method of claim 33 or 34, wherein the disease is (i) a heart disease associated with myocardial fibrosis, such as heart failure (due to unknown and / or genetic causes) with reduced or preserved left ventricular ejection fraction, myocardial infarction, congenital heart disease, cardiomyopathies with known genetic mutations, myocardial fibrosis induced by aging and / or ageing, drug-induced heart disease, metabolic heart disease, diseases (monogenic or polygenic) involving the heart and myocardial fibrosis, diseases associated with reactive or replacement fibrosis in organs such as the kidney, liver, lung, and / or skin; (ii) a disease associated with scar formation and impaired wound healing, such as diabetes; and / or (iii) a hereditary connective tissue disease, such as Marfan syndrome, Ehlers-Danlos syndrome, or Loeys-Dietz syndrome.

[0251] It will be readily apparent to those skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0252] The invention illustratively described herein can suitably be practiced in the absence of any element(s), limitation(ies), or both, not specifically disclosed herein. Moreover, the terms and expressions employed herein are used as terms of description and not to imply limitation, and the use of such terms and expressions is not intended to exclude equivalents of the features shown and described, or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, while the present invention has been specifically disclosed in terms of preferred embodiments and optional features, it should be understood that modifications and variations of the invention disclosed and embodied therein may be employed by those skilled in the art, and that such modifications and variations are considered to be within the scope of the present invention. The invention has been described broadly and generically herein. Each narrower species and subgeneric grouping falling within the generic disclosure also forms part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, whether or not specifically described herein. Furthermore, when features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is thereby also described in terms of any individual element or subgroup of elements of the Markush group. Further embodiments of the present invention will become apparent from the following claims.

[0253] <Cross-reference to related applications> This application claims the benefit of priority from European Patent Application No. 22213839.8, filed December 15, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes.

Claims

1. A method for evaluating the efficacy of a pharmaceutical agent that is suspected to be effective in treating a disease, wherein the disease is preferably a genetic disease, and the disease causes abnormalities in physiological bodily functions in patients having the disease, and the method comprises: (i) incubating an artificial tissue with the pharmaceutical agent, the artificial tissue being modified or having been modified to express the disease; (ii) determining at least one physical parameter of the artificial tissue obtained in step (i), wherein the physical parameter is an indicator of the functionality of a physiological body function; (iii) determining whether the value of the at least one physical parameter determined in step (ii) satisfies a predetermined threshold value for the physical parameter, thereby determining whether the pharmaceutical product is efficacious; a value of the at least one physical parameter exceeding the predetermined threshold indicates that the pharmaceutical product is effective in treating the disease by restoring at least part of the functionality of a physiological body function; The predetermined threshold is determined based on a standard (experiment), wherein the at least one physical parameter of the artificial tissue is determined in the absence of the pharmaceutical agent or by comparing values ​​of the at least one physical parameter obtained in the absence and presence of the pharmaceutical agent, and the standard (experiment) includes a standard (condition and / or experiment) based on an untreated artificial tissue that has been modified or altered to represent the disease. method.

2. said predetermined threshold value is based on or obtained by a standard (experiment) using statistical methods, said predetermined threshold value preferably being (at least) 0.5 to 2.5 standard deviations (SD) and / or (at least) 0.5 x SD, 1 x SD, 1.5 x SD or 2 x SD from the (average) standard value obtained for said at least one physical parameter in said standard (experiment), optionally expressed as a % change of the (average) standard value; The method of claim 1.

3. The artificial tissue comprises one or more of cardiac tissue, cardiac muscle tissue, muscle tissue, skeletal muscle tissue, nerve tissue or connective tissue; 3. The method according to claim 1 or 2.

4. The artificial tissue of i) is gene-edited to contain a mutation that is believed to cause the (genetic) disease. The method according to any one of claims 1 to 3.

5. The engineered tissue has been subjected to non-genetic intervention to express the disease. The method according to any one of claims 1 to 4.

6. The pharmaceutical is an advanced therapeutic medicinal product (ATMP), such as a gene therapy medicinal product (GTMP), a cell therapy medicinal product (CTMP) or a tissue engineered product (TEP); a small molecule, a peptide, a protein, a nucleic acid, a synthetic RNA, a non-coding RNA, a ribonucleoprotein particle (RNP) complex, or a read-through enhancer; The method according to any one of claims 1 to 5.

7. The GTMP is a viral vector, preferably a nanoparticle containing a nucleic acid and / or a protein, preferably encapsulated in an RNP, or a nucleic acid such as an antisense oligonucleotide; The method of claim 6.

8. The GTMP is a synthetic vector or a viral vector, such as a retrovirus or lentivirus, such as an adenovirus or an adeno-associated virus (AAV), more preferably the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrhlO, AAVrh74 or any combination thereof, preferably AAV2 or AAV9; 8. The method according to claim 6 or 7.

9. The pharmaceutical product contains a nucleic acid encoding a protein for gene editing, such as a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or an endonuclease such as a clustered regularly interspaced short palindromic repeats (CRISPR) nuclease, or an expression thereof. The method according to any one of claims 1 to 8.

10. The GTMP is containing an endonuclease such as Cas9 or Cas13, preferably Streptococcus pyogenes Cas9 (SpCas9), S. aureus Cas9, S. auricularis Cas9, S. lugdunensis Cas9, or N. meningitides Cas9, and a guide RNA (gRNA); or a nucleic acid encoding an endonuclease such as Cas9 or Cas13, preferably Streptococcus pyogenes Cas9 (SpCas9), S. aureus Cas9, S. auricularis Cas9, S. lugdunensis Cas9, or N. meningitides Cas9, and one or more guide RNAs (gRNAs); The method according to any one of claims 7 to 9.

11. The physical parameter is one or more selected from the group consisting of length, mass, time, current, temperature, light intensity, and physical parameters derived therefrom; The method according to any one of claims 1 to 10.

12. the physical parameters are measured as one or more selected from the group consisting of force, movement, current, mass, tissue structure (expansion / condensation, transparency) or one or more biomechanical parameters (tissue compression, contraction, stiffness, tensile strength, elasticity, extensibility, excitability, resilience, toughness, etc.); iii) at least one selected from the group consisting of tissue contraction, stiffness, force (stretch), spontaneous beating frequency, contraction time and / or velocity, relaxation time and / or velocity, (contraction) force, contraction force, and one or more multi-electrode array (MEA) measurements; preferably one or more selected from the group consisting of pole deflection, Young's modulus, (EHM) shortening, spontaneous beating frequency, contraction time and / or velocity, relaxation time and / or velocity, contraction force, twitch tension, resting tension, and (weighted) mean firing rate, burst frequency (Hz), time interval between firings in a burst (ms), network burst (NB) frequency, time interval between firings in a NB (ms), and NB duration (s), The method according to any one of claims 1 to 11.

13. at least one of said physical parameters is not a gene or protein expression level; The method according to any one of claims 1 to 12.

14. The artificial tissue is derived from a human, a non-human primate such as a macaque or a marmoset, a pig, or a rodent such as a mouse, a rat, or a guinea pig, and preferably the artificial tissue is human. The method according to any one of claims 1 to 13.

15. The artificial tissue contains cells obtained from a patient who is to be treated with the pharmaceutical agent. The method according to any one of claims 1 to 14.

16. The artificial tissue contains cells that are not obtained from a patient who is to be treated with the pharmaceutical agent. The method according to any one of claims 1 to 14.

17. further comprising analyzing said biomarkers, such as gene or protein expression of said biomarkers. The method according to any one of claims 1 to 16.

18. The artificial tissue is cardiac muscle. The method according to any one of claims 1 to 17.

19. The at least one physical parameter is the force of the artificial tissue and / or the movement of the artificial tissue; The method according to any one of claims 1 to 18.

20. The one or more conditions include a state in which the artificial tissue exerts a force of 0.01 mN or more, 0.05 mN or more, 0.1 mN or more, or 1 mN or more.

20. The method of claim 18 or 19.

21. The disease is an inherited cardiac condition, preferably an inherited cardiac condition selected from the group consisting of genetic forms of dilated, hypertrophic, and arrhythmogenic cardiomyopathy (ACM), or fibroblastosis, and cardiomyopathy associated with a congenital metabolic disorder, more preferably a condition selected from the group consisting of Duchenne muscular dystrophy (DMD), Noonan syndrome, dilated cardiomyopathy, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, long / short QT syndrome, Takotsubo cardiomyopathy, lysosomal storage disorders, titinopathy, and Barth syndrome, The method according to any one of claims 1 to 20.

22. the disease is a non-genetic cardiac condition, preferably induced by mimicking neurohormonal and / or pharmacological stimuli (e.g. by catecholamines, angiotensin, and / or transforming growth factor beta), by drugs such as doxorubicin, tyrosine kinase inhibitors and / or cardiotoxic drugs, by mechanical injury such as crush injury, by temperature injury such as frostbite or thermal injury, by biophysical injury such as radiation injury, by infection such as viral infection with cardioactive virions such as Coxsackievirus, SARS coronavirus, cytomegalovirus and dengue virus, or by parasitic infection such as Chagas disease caused by Trypanosoma cruzi, optionally by bloodstream such as serum from a patient with a cardiac disease, and / or by cells such as mononuclear cells (e.g. T cells, B cells, NK cells, macrophages) derived from the blood of a patient with a cardiac disease, The method according to any one of claims 1 to 20.

23. The artificial tissue is a neural tissue, preferably a neural organoid. The method according to any one of claims 1 to 17.

24. the at least one physical parameter is current and / or activity; The method according to any one of claims 1 to 17 and 22.

25. one or more of said conditions include localized and coordinated electrical bursts and / or clusters; 24. The method of claim 22 or 23.

26. The disease is a neuronal disorder, and is preferably selected from the group consisting of neurodegenerative diseases (e.g., dementia, Parkinson's disease, Huntington's disease), neuroinflammatory diseases (e.g., multiple sclerosis), cerebroinflammatory diseases (e.g., meningitis), channelopathies (e.g., epilepsy), and psychiatric disorders (including autism spectrum disorders and schizophrenia). The method according to any one of claims 23 to 25.

27. The artificial tissue is muscle tissue, preferably skeletal muscle tissue. The method according to any one of claims 1 to 17.

28. the at least one physical parameter is force or movement; The method of any one of claims 1 to 17 and 27.

29. The one or more conditions include a state in which the artificial tissue exerts a force of 0.01 mN or more, 0.05 mN or more, 0.1 mN or more, or 1 mN or more.

29. The method of claim 27 or 28.

30. The disease is a genetic muscle disease, preferably selected from the group consisting of Duchenne muscular dystrophy, facioscapulohumeral dystrophy, Becker dystrophy, Emery-Dreifuss dystrophy, myotonic dystrophy, limb dystrophy, oculopharyngeal muscular dystrophy, congenital dystrophies, congenital myopathies, myotonic dystrophy, familial periodic paralysis, and hereditary connective tissue diseases such as Marfan syndrome, Ehlers-Danlos syndrome, or Loeys-Dietz syndrome, 30. The method according to any one of claims 27 to 29.

31. The artificial tissue is a composite of different types of tissue, preferably (i) nerve cells and skeletal muscle, or (ii) Neurons and cardiac muscle It is a mixture of; More preferably, the method according to any one of claims 1 to 17 and 27 to 30, wherein the hybrid of different types of tissue forms one or more neuromuscular junctions.

32. The artificial tissue is cardiac muscle or skeletal muscle, the pharmaceutical product is an AAV2 and / or AAV9 viral vector comprising a nucleic acid encoding Cas9, preferably S. aureus Cas9, S. auricularis Cas9, S. lugdunensis Cas9, or N. meningitides Cas9, and one or two guide RNAs (gRNAs), wherein the Cas9 gene is under the control of a muscle-specific promoter such as CK8e or TNNT2; the disease is Duchenne muscular dystrophy, the physical parameter is a force; Preferably, the artificial tissue contains cells obtained from a patient who is to be treated with the medicament. The method according to any one of claims 1 to 17.

33. The artificial tissue is a connective tissue. The method according to any one of claims 1 to 17.

34. The at least one physical parameter is a biomechanical parameter such as tissue compression, contraction, stiffness, tensile strength, elasticity, extensibility, excitability, resilience, or toughness.

34. The method of any one of claims 1 to 17 and 33.

35. The disease is (i) a cardiac disease associated with myocardial fibrosis, such as heart failure with reduced or preserved left ventricular ejection fraction (due to unknown and / or genetic causes), myocardial infarction, congenital heart disease, cardiomyopathies with known genetic mutations, myocardial fibrosis induced by aging and / or ageing, drug-induced heart disease, metabolic heart disease, diseases (monogenic or polygenic) involving the heart and myocardial fibrosis, diseases associated with reactive or replacement fibrosis in organs such as the kidney, liver, lung, and / or skin, (ii) a disease associated with scar formation and impaired wound healing, such as diabetes, and / or (iii) a hereditary connective tissue disease, such as Marfan syndrome, Ehlers-Danlos syndrome, or Loeys-Dietz syndrome, 35. The method of claim 33 or 34.

36. The criteria (experiments) further include: i) artificial tissues and / or artificial organoids derived from ia) healthy individuals, and / or ib) well-characterized iPSCs representing a healthy wild type, and / or ic) well-characterized iPSCs representing a disease genotype, and / or id) well-characterized iPSCs representing a disease phenotype, and / or ii) artificial tissues and / or organoids modified by permanent genetic repair of disease-associated, preferably disease-causing, mutations; Include criteria (conditions and / or experiments) based on 36. The method according to any one of claims 1 to 35.

37. The predetermined threshold value is i) determined on the basis of or obtained by a standard (experiment), said standard (experiment) being repeated at least once; and / or ii) based on or obtained by a standard (experiment) using statistical methods, said standard (experiment) being repeated at least once; 37. The method according to any one of claims 1 to 36.

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