Apoptosis-based decellularization method

Abstract

The present invention is directed to a method for manufacturing a human cell derived tissue engineered matrix (hTEM) for an implant application, said hTEM based on genetically modified cells expressing an inducible apoptotic initiator gene. The invention further is directed towards an hTEM manufactured by the respective method and to the respective apoptosis-assisted decellularization process.

Description

[0001] F07370

[0002] 1

[0003] TITLE

[0004] APOPTOSIS-BASED DECELLULARIZATION METHOD

[0005] TECHNICAL FIELD

[0006] The present invention relates to a method for manufacturing a human cell derived tissue engineered matrix (hTEM), for production of an implant, especially for production of a cardiovascular implant, e.g. of a vascular or valvular graft, especially of a human cell derived tissue engineered heart valve (hTEHV), comprising an apoptosis-assisted decellularization, as well as an hTEM manufactured by such a method.

[0007] PRIOR ART

[0008] Valvular heart disease is a growing health problem worldwide (Coffey et al. 2016). Patients suffering from severe valvular insufficiency or stenosis require valve replacement (Head et al., 2017). Mechanical heart valves carry a high risk of thromboembolic events due to their non-physiological hemodynamics, requiring a life-long anti-coagulation treatment, which in turn can lead to bleeding complications (Head et al., 2017, Nishimura and Warnes, 2015; Soliman Hamad et al., 2009). On the other and, bioprosthetic heart valves, e.g. based on glutaraldehyde-fixed xenogeneic tissues, are prone to degeneration and calcification over time, and thus have a limited durability (Arsalan and Walther, 2016; Bourguignon et al., 2015; Costa et al. , 2019; David, 2010). To overcome these shortcomings, tissue engineered heart valves (TEHVs) offer a promising alternative to current heart valve prostheses. In classical in vitro tissue engineering, which is not limited to cardiovascular applications, selected autologous cells are seeded on a biocompatible scaffold. The scaffold is then usually cultured in a bioreactor system to produce an extracellular matrix, followed by implantation of the construct in the patient. However, the clinical translation of this approach has shown to be logistically challenging and the reproducibility of ECM production by autologous and allogenic cells has been limited, due to donor-to-donor variability (Biermann et al., 2019; Lisy et al., 2017; Tudorache et al., 2016).

[0009] In situ tissue engineering, which uses the regenerative potential of the host to drive tissue engraftment, repopulation and remodeling, has gained attention in the past decade (Fioretta et al., 2021 ; Poulis et al., 2020; Wissing et al., 2017). Therein, a decellularized tissue or a biodegradable polymer is implanted and subsequently repopulated by endogenous cells which remodel the implant into a functional tissue. With respect to TEHVs, the implanted in F07370

[0010] 2 situ constructs cause an initial inflammatory response, marked by the infiltration of host cells and the deposition of newly synthesized ECM. Then the TEHV undergoes an adaptive remodeling phase, during which the inflammatory cascade gradually resolves, resulting in the integration of the construct (Fioretta et al., 2021 ; Wissing et al., 2017). In case of maladaptive remodeling, the initial inflammatory response persists, resulting in chronic inflammation, deposition of fibrotic tissue, shortening of leaflets and ultimately, failure of the implant (Fioretta et al., 2021).

[0011] Decellularization is crucial for removing cellular components of the construct with minimal impact on the ECM, i.e. keeping the network of proteins and other molecules to surround, support and provide structure to cells and tissues in the body after implantation. Decellularization of a tissue involves a tradeoff between removing cells and immunogenic molecules while preserving the ECM structure and key components such as collagens, GAGs, and growth factors (Crapo et al., 2011). The conservation of these components is crucial for tissue remodeling. For example, GAGs influence the activity of growth factors and modulate inflammation response (Taylor and Gallo, 2006). Furthermore, biophysical features such as material stiffness and topography influence the cell behavior (Ahmed et al., 2012; Ma et al., 2017). Additionally, decellularization enables the creation of an off-the shelf product.

[0012] Decellularized native tissues, such as decellularized heart valve homografts and xenografts have been extensively tested in preclinical and clinical trials (Fioretta et al., 2021). While decellularized pulmonary and aortic valve homografts have shown promising functionality and performance with spontaneous recellularization in early follow up studies (Fioretta et al., 2021 ; Sarikouch et al., 2016), a 5-year follow-up of decellularized homografts for aortic valve replacement showed an increasing rate of peak gradient and regurgitation. Additionally, these decellularized homografts appear to elicit a low-grade immune response in pediatric patients (Horke et al., 2024), and the limited donor availability hinders their widespread use (Cebotari et al., 2011 ; Sarikouch et al., 2016; Tudorache et al., 2016). On the other hand, decellularized xenografts have yielded mixed results in preclinical and clinical trials (Fioretta et al., 2021), with instances of strong inflammatory responses, severe calcification, regurgitation, stenosis, and failure having been reported in patients (Backhoff et al., 2014; Hofmann et al., 2017; Mosala Nezhad et al., 2017; Perri et al., 2012; Zaidi et al., 2014). The poor performance of these valves can be attributed to factors such as high mechanical stress and host-specific immune inflammation response to the implanted material, the latter of which has been repeatedly shown to be caused by incomplete decellularization (Filippo et al., 2013; van Rijswijk et al., 2020; Woo et al., 2016).

[0013] As an alternative to decellularized native tissue, cell-derived tissue engineered matrices F07370

[0014] 3

[0015] (TEMs), either derived from animal cells or from human cells, have been undergoing pre- clinical investigations and have shown promising functionality and remodeling capacity one year post implantation in a large animal model (Weber et al., 2013; Driessen-Mol et al., 2014; Motta et al., 2018; Lintas et al., 2018; Emmert et al., 2018; Motta et al., 2019). Human cell derived tissue engineered matrices (hTEMs) are a hybrid material of bioresorbable polymer and human cell-derived ECM, which is decellularized (Dijkman et al., 2012). State of the art decellularization methods for a TEM, or an hTEM, respectively, rely on physical treatment, such as freeze and thawing cycles, sonication, pressure, or mechanical agitation, and / or on enzymatic digestion, and / or on chemical detergents, such as solutions comprising 0.25% Triton-X 100, 0.255% sodium deoxycholate, and 0.02% EDTA. Usually, such initial decellularization steps are followed by a nuclease treatment step, e.g. with benzonase. While detergent-based methods effectively eliminate cells and reduce DNA content in the hTEM, they inadvertently trigger necrotic cell death. While detergent-based decellularization protocols successfully meet the standardized DNA decellularization criteria (Crapo et al., 2011), i.e. reaching DNA residues of less than 50 ng / mg of ECM dry weight with DNA fragment lengths smaller than 200 base pairs, and the absence of cell nuclei remnants as confirmed by DAPI or H&E staining, this processes overlook the presence of other intracellular damage-associated molecular patterns (DAMPs), including heat shock and cytoskeletal proteins (Matzinger, 2002; Roh and Sohn, 2018).

[0016] The use of detergents for decellularization induces cell lysis, thereby potentially increasing its immunogenicity (Boer et al., 2011). The cell lysis results in the release of intracellular contents, including damage-associated molecular patterns (DAMPs), and their adsorption onto the hTEM. The innate immune system is highly sensitive to DAMPs, which play a significant role in polarizing migrating macrophages towards a pro-inflammatory M1 phenotype (Brown et al., 2012b), thus potentially exacerbating the inflammatory response (Brown et al., 2012a). The activation of innate immune cells can potentially lead to chronic inflammatory responses in the host after transplantation and to an altering of the subsequent remodeling capacities of the respective hTEM. It is for this reason, that the American Society for Testing and Materials (ASTM, F3354-19) recommends the reduction of intracellular proteins including DAMPs for decellularized ECM materials. From a clinical translation perspective, reducing DAMPs in ECM-based in situ biomaterials, such as hTEMs, may be crucial to mitigate host immune reactions towards the implant, enhancing the prospects for successful clinical outcomes.

[0017] It therefore is an object of the present invention to provide, especially for cardiovascular implant applications, a more clinically relevant method for manufacturing an hTEM F07370

[0018] 4 comprising a decellularization approach which, while successfully removing cellular material and nucleic acids, minimizes intracellular DAMPs, and still conserves the composition, the mechanical properties, and the biologic activity of the ECM, which are important for influencing the remodeling capacities of the tissue surrounding the site of implantation of the respective hTEM or an implant based thereon in the host.

[0019] Apoptosis, a regulated process of cell death, plays a crucial role in achieving tissue homeostasis (Mehrotra and Ravichandran, 2022). During apoptosis, various intracellular processes occur, including the reorganization of cytoskeletal proteins, fragmentation of DNA, and degradation of nuclear proteins (Elmore, 2007). The initiation of apoptosis allows for the controlled degradation of cellular components such as cytoskeletal proteins, DNA, and cell organelles. The activation of apoptosis, initiated by the cleavage of effector caspases such as Casp-3, -6, or -7, triggers a cascade of these intracellular events.

[0020] Thus, apoptosis could serve as a decellularization tool for targeting the reduction of DAMPs and intracellular proteins from hTEM-based constructs, thereby decreasing potential immunogenicity risks, thus enhancing their clinical relevance. Several apoptosis-based decellularization methods have been developed for decellularizing tissue engineered cartilage, peripheral nerve, and lungs (Bourgine et al., 2014; Cornelison et al., 2018; Song et al., 2021).

[0021] Additionally, apoptotic cells are known to actively modulate their environment by secreting metabolites, exosomes, and proteins (Beer et al., 2015; Medina et al., 2020). These factors play pivotal roles in influencing processes such as cell proliferation of neighboring cells (Li et al., 2010; Lucas et al., 2022), wound healing, and cytoprotection (Lichtenauer et al., 2011).

[0022] WO2013 / 186264A1 discloses the use of human mesenchymal stem cells (hMSCs) in an inducible apoptosis system for use in tissue engineering. Therein, in various embodiments, a telomerase transgene expression construct and an inducible genetic construct are introduced into hMSCs, leading to apoptosis upon induction.

[0023] In US 10,898,609, various tissue decellularization methods for tissue engineering purposes are disclosed. Among others, apoptosis is induced by an apoptotic agent, followed by an optional DNAse treatment. Implantation of apoptosis-decellularized grafts was compared to implantation of detergent-decellularized grafts, and it was shown that the apoptosis- decellularization method has the potential to perform similarly to detergent-only methods.

[0024] It is a further object of the present invention, to provide a method for producing an hTEM which comprises a decellularization process specifically tailored for cardiovascular F07370

[0025] 5 applications, such as for a vascular or valvular graft.

[0026] SUMMARY OF THE INVENTION

[0027] The abovementioned objects are achieved by a method for manufacturing a human cell derived tissue engineered matrix (hTEM) for production of an implant as described below, preferably for production of a cardiovascular implant, in particular a valvular or vascular implant, such as a human cell derived tissue engineered heart valve (hTEHV).

[0028] The inventive method comprises the following steps:

[0029] - a.) providing a population of genetically modified human cells expressing an inducible apoptotic initiator gene;

[0030] - b.) seeding the genetically modified human cells expressing an inducible apoptotic initiator gene onto a scaffold, resulting in a seeded scaffold;

[0031] - c.) culturing the seeded scaffold in a tissue culture medium until formation of a human extracellular matrix (ECM) on the scaffold, resulting in a human cell derived tissue- engineered matrix (hTEM);

[0032] - d.) decellularizing the hTEM, including the following steps: d1.) inducing apoptosis in the genetically modified human cells of the hTEM by treating the hTEM with an apoptosis-inducing drug; d2.) treating the hTEM resulting from step d1.) with a solution comprising a detergent; d3.) treating the hTEM resulting from step d2.) with a solution comprising a nuclease, in order to reduce a nucleic acid content in the hTEM.

[0033] In step a.), the term "providing" can either mean to obtain or purchase a population of human cells, which have already previously been genetically modified to express the inducible apoptotic initiator gene, as desired according to their intended use. It is, however, also possible that the step of "providing" includes the process of carrying out the genetic modification of the cells, e.g. the transduction or transfection itself. Examples for types of transduction are described below. The "providing" of the cell population can thus also include taking a biopsy or tissue sample from a human subject, which in case of autologous human cells would also be the recipient of the implant, and growing of cell cultures from said biopsy and will further include the genetic modification, i.e. preferably transduction of said human cell, until a population of genetically modified cells is reached which has the F07370

[0034] 6 target gene stably integrated into the genome of the respective cells.

[0035] Preferably, the genetically modified human cells of step a.) are genetically modified human primary cells, preferably selected from a group consisting of fibroblasts, endothelial cells, smooth muscle cells and interstitial cells. More preferably, the genetically modified human primary cells are selected from a group consisting of dermal fibroblasts, cardiac fibroblasts, umbilical cord derived fibroblasts, vascular endothelial cells, valvular endothelial cells, vascular smooth muscle cells and valvular interstitial cells. Most preferably, the genetically modified human primary cells are human dermal fibroblasts (hDFBs).

[0036] The genetically modified human cells are typically allogenic cells, i.e. preferably commercially available human primary cells, however, they can also be autologous human primary cells, i.e. cells originating from the same individual into which the respective implant comprising the hTEM is to be implanted.

[0037] The inducible apoptotic initiator gene expressed in the genetically modified human cells preferably encodes a fusion protein which is able to dimerize and as a result of dimerization is able to trigger apoptosis upon exposure to a dimerization drug. The apoptosis-inducing drug mentioned in step d1.) in that case preferably is such a dimerization drug, preferably a dimerizing small molecule pharmaceutical drug, more preferably AP20187, which is a synthetic cell-permeable dimerizing drug, a chemical inducer of dimerization (CID). Cl Ds are advantageous as they enable selection without causing toxic effects, i.e. adverse effects in cells other than the genetically modified target cells.

[0038] According to a preferred embodiment, the inducible apoptotic initiator gene, also known as an inducible suicide gene, is a modified human Caspase gene, preferably an inducible or drug-inducible Caspase 9 gene (iCasp9). More preferably, the gene encodes a modified human caspase 9 protein which is fused to a human small-molecule binding protein, most preferably to a human FK506 binding protein (FKBP), to allow dimerization by the dimerization drug.

[0039] AP20187 (B / B homodimerizer), as mentioned above, is a cell-permeable ligand used to dimerize FK506-binding protein (FKBP) fusion proteins and initiate biological signaling cascades and gene expression or disrupt protein-protein interactions; i.e. it can induce homodimerization of fusion proteins containing the F36V mutant of an FKBP domain (FKBPF36V), available commercially as the DmrB domain. F07370

[0040] 7

[0041] The human primary cells preferably have been genetically modified via transduction, preferably via retroviral transduction, more preferably via lentiviral transduction. According to an especially preferred embodiment of the present invention, such a lentiviral transduction is conducted by incorporating a plasmid containing the apoptotic initiator gene into a lentivirus backbone. The lentivirus backbone preferably includes a human Cytomegalovirus (hCMV) promoter to drive an expression of the inducible apoptotic initiator gene. The inducible apoptotic initiator gene, as mentioned above, preferably is iCasp9. Alternative genes encoding inducible apoptotic initiator proteins include, for example, Casp8, Casp3 or a genetically modified gene encoding a mutant herpes simplex virus thymidine kinase (e.g. TKA168H), i.e. an enzyme prodrug suicide gene activated by ganciclovir (GCV) (HSV-TK / GCV system).

[0042] Alternatively, the modified cells of step a.) can also be of animal origin, such as ovine or murine cells, preferably of the above-mentioned cell-types. Such cells can form the basis for production of a non-human, i.e. animal cell derived tissue engineered matrix (TEM). The same or similar, i.e. analogous transduction systems as mentioned above, i.e. inducible apoptotic initiator genes of human or animal origin are conceivable for this purpose.

[0043] The choice of method for decellularization of a tissue engineered matrix depends on tissuespecific factors, e.g. on tissue size, thickness and shape, as well as cell type and matrix density. The disruptive effect of the detergent upon the ECM during the decellularization process shall be minimized. Typical detergents for decellularization are non-ionic, ionic, or zwitterionic, or combinations thereof. The detergent used in step d2.) of the present invention preferably is composed of phosphate-buffered-saline (PBS) supplemented with 4- (1 , 1 ,3,3-T etramethylbutyl)-phenyl-polyethylenglykol, (Alkylphenylpolyethylenglykol / Octylphenoxypolyethoxyethanol) (e.g. Triton-X-100 (Merck)), preferably at 0.25%, sodium-deoxycholate (SDS) (e.g. by Sigma-Aldrich), preferably at 0.25%, and ethylenediaminetetraacetic acid (e.g. by Sigma-Aldrich), preferably at 0.02%. The detergent treatment is advantageously carried out for 24 hours and preferably at 37°C.

[0044] The nuclease used in step d3.) preferably is an endonuclease, for example Benzonase Nuclease (e.g. by Novagen, Merck), preferably in an acidic buffer, e.g. TRIS-HCL buffer (pH=8), preferably for 3 days at 37°C.

[0045] The underlying scaffold, on which the genetically modified human cells are seeded and thus F07370

[0046] 8 the inventive hTEM is to be developed is preferably formed of a biodegradable material, especially preferably comprising or consisting of polyglycolic acid (PGA). Especially preferably the scaffold is coated with a solution containing Poly-4-Hydroxybutyrate (P4HB). Alternative scaffold materials include other preferably biodegradable polymeric biomaterials.

[0047] The present invention is also directed to a human cell derived tissue engineered matrix (hTEM), manufactured according to any of the above-mentioned embodiments of the inventive method. If such an hTEM comprises a concentration of double stranded DNA (dsDNA) in the ECM which is less than the industry "gold standard" set by Crapo et al. in 2011 , i.e. max. 50 ng per mg ECM dry weight, the hTEM can be viewed as successfully decellularized. Crapo et al. also suggested that the DNA fragment length should be below 200 bp.

[0048] According to a further preferred embodiment of the present invention, the hTEM manufactured according to the process described above comprises a reduced concentration of at least one intracellular protein, preferably a reduced concentration of at least one intracellular protein selected from a group consisting of H2AC11 (H2A clustered histone 11), ACTB (beta-actin), HBB (hemoglobin beta), PPIB (peptidylprolyl isomerase B), VIM (vimentin), H3-7 (H3.7 histone), HBA1 (hemoglobin alpha 1), H4C1 (H4 clustered histone 1), TLIBA1C (tubulin alpha 1c chain) , ACTA2 (actin alpha 2), H2BC12 (H2B clustered histone 12), TAGLN (transgelin), ACTBL2 (actin beta-like 2), RPL27A (ribosomal protein L27a), EEF1A1 (eukaryotic translation elongation factor 1 alpha 1), UBB (polyubiquitin), CSRP2 (cysteine and glycine-rich protein 2), C9 (complement C9), PFN1 (profilin 1), and PRDX1 (peroxi redoxin 1), as compared to a concentration of said at least one intracellular protein in an hTEM produced from genetically unmodified human cells or in an hTEM produced using a decellularization process not relying on induced apoptosis.

[0049] Said reduction of concentration in the inventive hTEM is preferably at least 10%, more preferably at least 14% lower as compared to a concentration of said at least one intracellular protein in an hTEM which has been produced from genetically unmodified human cells or as compared to a concentration of said at least one intracellular protein in an hTEM produced using a decellularization process not relying on induced apoptosis, such as a decellularization process based on detergent-only treatment. The term "detergent-only" does not exclude a nuclease treatment following the detergent treatment, as the removal of nucleases typically follows a detergent treatment. Advantageously, the concentration of F07370

[0050] 9 more than one, preferably of several of these intracellular proteins is reduced, compared to hTEMs manufactured using standard decellularization methods not relying on a combination of induced apoptosis and detergent treatment.

[0051] The inventive hTEM preferably also comprises a reduced concentration of at least one damage-associated molecular pattern molecule (DAMP), preferably of at least one DAMP selected from a group consisting of ACTA2 (actin alpha 2), ALDH1 L2 (aldehyde dehydrogenase 1 family member L2), CALR (calreticulin), GPC1 (Glypican 1), GPC6 (Glypican 6), H2AC11 (H2A clustered histone 11), H2BC12 (H2B clustered histone 12), HMGB1 (high mobility group box-1), HSPA12A (heat shock protein 12A), HSPA13 (heat shock protein 13), HSP90AA1 (heat shock protein 90kDa alpha (Cytosolic), class A, member 1), HSP90AB1 (heat shock protein 90kDa alpha (cytosolic), class B, member 1 , HSPA1A (inducible heat shock protein A1A), HSPA8 (heat shock 70kDa protein 8), HSPB1 (heat shock protein beta-1), HSPB6 (heat shock protein beta-6), HSPD1 (heat shock protein family D, member 1), LGALS1 (lectin, galactoside-binding, soluble, 1), LGALS3 (lectin, galactoside-binding, soluble, 3), LGALS3BP (lectin, galactoside-binding, soluble, 3 binding protein), MACROH2A1 (macroH2A.1 histone, formerly H2AFY: H2A histone family member Y), MACROH2A2 (macroH2A.2 histone), PRDX1 (peroxiredoxin 1), PRDX2 (peroxi redoxin 2), PRDX6 (peroxi redoxin 6), S100A10 (S100 protein A10), S100A11 (S100 protein A11), S100A4 (S100 protein A4), TXNDC5 (thioredoxin domain containing protein-5), and TXNL1 (thioredoxin-like protein 1 , also called TRP32 (thioredoxin related protein of 32 kDa)), as compared to a concentration of said at least one DAMP in an hTEM produced from genetically unmodified human cells or in an hTEM produced using a decellularization process not relying on induced apoptosis. Preferably, said reduction of concentration is at least 5%, more preferably at least 7% lower as compared to a concentration of said at least one DAMP in an hTEM produced from genetically unmodified human cells or in an hTEM produced using a decellularization process not relying on induced apoptosis, i.e. not including any step of inducing apoptosis.

[0052] Advantageously, the concentration of more than one, preferably of several of these DAMPs is reduced, compared to hTEMs manufactured using standard decellularization methods not relying on a combination of induced apoptosis and detergent treatment.

[0053] The apoptosis-assisted decellularization ideally does not lead to a change or disruption of mechanical function of the resulting hTEM, as compared to an hTEM produced using a decellularization process not including a step of inducing apoptosis. F07370

[0054] 10

[0055] The present invention is further directed towards a human implant, preferably a cardiovascular implant, more preferably a vascular or a valvular implant, most preferably a human cell derived tissue engineered heart valve (hTEHV), which comprises an hTEM produced by a method according to the above-described method.

[0056] The present invention is furthermore directed to a method or process for decellularizing a human cell derived tissue engineered matrix (hTEM) for an implant application, preferably for a production of a cardiovascular implant, more preferably a vascular or valvular implant. The inventive decellularization process comprises the following steps: dO.) providing an hTEM comprising a human extracellular matrix (ECM) produced from a population of genetically modified human cells expressing an inducible apoptotic initiator gene; d1.) inducing apoptosis in the genetically modified human cells of the hTEM by treating the hTEM with an apoptosis-inducing drug; d2.) treating the hTEM resulting from step d1.) with a solution comprising a detergent; d3.) treating the hTEM resulting from step d2.) with a solution comprising a nuclease, in order to reduce a nucleic acid content in the hTEM.

[0057] In the step dO.) the term "providing an hTEM" can either mean to obtain / purchase an hTEM on which an ECM has already developed from genetically modified human cells expressing an inducible apoptotic initiator gene. However, it can also include the process of seeding genetically modified human cells expressing an inducible apoptotic initiator gene on a scaffold and culturing the scaffold until the formation of an ECM based on those genetically modified human cells, thereby producing an hTEM, which then shall be decellularized according to the above-mentioned inventive process according to step d1.)-d3.) prior to implantation into the host.

[0058] The present invention combines the cell- and DNA removal efficiency of detergent and nuclease based decellularization with the advantages of apoptosis-based decellularization, achieving the degradation and thus reduction of intracellular proteins and of DAMPs. An hTEM manufactured according to the inventive method contains fewer potentially immunogenic proteins than an hTEM decellularized according to a detergent-only protocol. The new manufacturing method thus reduces the likelihood of adverse immune reactions or remodeling in the host following implantation of the respective hTEM or implant, respectively. F07370

[0059] 11

[0060] The inventive hTEM, produced by a method as described above, and comprising an ECM produced from genetically modified human cells expressing an apoptotic initiator gene, more preferably expressing iCasp9 comprises an ECM produced from genetically modified human cells expressing an apoptotic initiator gene, more preferably expressing iCasp9, is thus suitable for use as an implant, preferably as a cardiovascular implant, more preferably a valvular implant, most preferably a hTEHV. The present invention therefore is also directed towards an hTEM which has been produced by any above-described embodiment of the inventive method, for use in the treatment of a cardiovascular disease, preferably a vascular or valvular disease, more preferably a valvular heart disease, wherein the hTEM comprises an ECM based on genetically modified human cells expressing an inducible apoptotic initiator gene, preferably iCasp9.

[0061] Further embodiments of the invention are laid down in the dependent claims.

[0062] BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,

[0064] Fig. 1 shows a schematic overview of the apoptosis-assisted decellularization method according to a first preferred embodiment.

[0065] Fig. 2 shows a schematic overview of the process for manufacturing a tissue engineered heart valve, comprising the apoptosis-assisted decellularization according to Fig. 1.

[0066] Fig. 3 shows experimental results reflecting the efficiency of the inducible caspase 9 gene (iCasp9) in hDFBs, wherein in A) microscopic images of cell cultures of hDFBs transduced with the iCasp9 gene (hDFBs-iCasp9+) and treated with AP20187 for 24h at different concentrations are shown, as compared to untreated hDFBs-iCasp9+(control); B) represents results of annexin V / propidium iodide staining of hDFBs-iCasp9+cells treated with 100nM of AP20187 for 24h, and C) represents a Western blot analysis illustrating cleavage of caspase 3 (effector caspase) hDFBs-iCasp9+cells treated with F07370

[0067] 12

[0068] Fig. 4 shows experimental results reflecting the characterization of iCasp9+hTEMs, wherein A) shows macroscopic images and representative microscopic images of hTEMs with H&E, Coll and Col3 staining, respectively, after standard detergent-based decellularization (n=4 per group); B) illustrates a biochemical analysis of hydroxyproline content of untransduced hTEMs vs. iCasp9+hTEMs (n=4 per group); C) illustrates a biochemical analysis of sulphated glycosaminoglycans relative to dry tissue weight (n=4 per group); and C) represents the results of biaxial mechanical testing of hTEMs produced based on hDFBs-iCasp9+cells, as compared to hTEMS produced based on nontransduced hDFBs (n=4 per group).

[0069] Fig. 5 shows experimental results reflecting the efficiency of apoptosis-assisted decellularization in hTEMs, wherein A) illustrates results of H&E staining of decellularized hTEMs decellularized with a detergent-only based method, compared to an apoptosis-assisted detergent method; B) shows results of DNA quantification; C) shows the fold change of the 20 most abundant intracellular proteins in hTEMs decellularized with the detergent-only based vs. the apoptosis-assisted detergent based method; and D) illustrates the fold change of DAMPs present in the respective hTEM.

[0070] Fig. 6 shows experimental results reflecting the characterization of apoptosis-assisted decellularized hTEMs, wherein hTEMs derived from hDFB-iCasp9+were decellularized using either the typical detergent-only based method, or by using the apoptosis-assisted detergent method, respectively, wherein A) shows representative IHC images of Coll and Col3 staining; B) shows a quantification of hydroxyproline and glycosaminoglycans, respectively (n=4 per decellularization group); wherein D)-F) show mass spectrometry data indicating fold change of the most abundant collagens, proteoglycans; and glycosaminoglycans, respectively, and wherein in G) results of biaxial mechanical testing of hTEMs decellularized with the detergent-only method vs. the apoptosis-assisted detergent based method are illustrated (n=4 per group).

[0071] Fig. 7 shows experimental results illustrating the in vitro characterization of apoptosis- assisted decellularized hTEHV for pulmonary application, wherein A)-C) show macroscopic images of pulmonary hTEHV from the side (A), as well as in a pulmonary (B), and in a ventricular view (C); D) shows snapshots l-VI of an apoptosis-assisted decellularized hTEHV tested on an in vitro pulse duplicator tester at pulmonary pressure conditions, and E) shows H&E staining of hTEHVs (n=7) with various decellularization methods: no decellularization (Ei; n=1), F07370

[0072] 13 detergent-only based decellularization (Eii; n=1), apoptosis-only decellularization (Eiii; n=1), and apoptosis-assisted detergent based decellularization (Eiv; n=4).

[0073] DESCRIPTION OF PREFERRED EMBODIMENTS

[0074] For the present invention, hTEHVs derived from, i.e. based on hTEMs were decellularized using an apoptosis-assisted method. For this purpose, human dermal fibroblasts (hDFBs) were transduced with an inducible caspase 9 gene (iCasp9) (Straathof et al., 2005), to develop an apoptosis-assisted decellularization approach with the aim to reduce intracellular DAMP adsorption onto hTEM-based constructs. The decellularization efficiency was evaluated, the influence of apoptosis-assisted decellularization on hTEM morphology was assessed, the ECM composition was analyzed and the mechanical stability was measured.

[0075] The decellularized hTEMs were characterized by (immuno-)histology, biochemical assays, biaxial mechanical testing, and mass spectrometry. hTEHV functionality was evaluated in an in vitro pulse duplicator under pulmonary conditions for up to 1 hour.

[0076] A schematic overview of the inventive method of producing an hTEM for use in tissue engineered constructs is depicted in Fig. 2. As an exemplary cardiovascular application, an hTEHV is schematically shown.

[0077] In Figure 1 , a schematic overview of the inventive apoptosis-assisted decellularization is shown. Therein, as a first step of the inventive decellularization process, apoptosis is induced by activating the inducible caspase 9 gene. This is followed by a second step comprising a treatment of the hTEM with a detergent, e.g. a chemical detergent. Subsequently, as a third step of decellularization, the hTEM is treated with a nuclease, in order to remove any remnants of nucleic acids.

[0078] Characterization of hDFBs-iCasp9+cells:

[0079] To induce rapid and efficient apoptosis for the first step of the decellularization process, an inducible apoptotic initiator gene, caspase 9 (iCasp9), was utilized. A stable overexpression of caspase 9 was achieved in human dermal fibroblasts (hDFBs) via lentivirus infection. To induce apoptosis, the inactive monomeric iCasp9 protein must dimerize into its active form, in order to activate enzymatic activity, which is responsible for the downstream cleavage F07370

[0080] 14 and activation of caspase 3 and 7 to enact cell death. The "iCasp9 suicide switch" is activated through a synthetic dimerization drug, AP20187, which only initiates apoptosis in cells with expression of iCasp9 (Straathof et al., 2005), and thus does not affect other cells. AP20187, a B / B homodimerizer with the formula C82H107N5O20, and the IIIPAC name "2,2 - [[2-[(dimethylamino)methyl]-1 ,3-propanediyl]bis[imino(2-oxo-2,1-ethanediyl)oxy-3,1- phenylene[(1 R)-3-(3,4-dimethoxyphenyl)propylidene]]] ester" is a cell-permeable ligand used to dimerize FK506-binding protein (FKBP) fusion proteins (containing the F36V mutant of an FKBP domain (FKBPF36B)). It is used to initiate biological signaling cascades and gene expression or disrupt protein-protein interactions. Exposure of hDFBs-iCasp9+cells to varying concentrations of the dimerization drug AP20187 revealed that a concentration of 100 nM was sufficient to induce apoptosis in high GFP hDFBs-iCasp9+cells (see Fig. 3A). Treatment with 100 nM AP20187 resulted in the detection of over 97% annexin V-positive cells within 24 hours (see Fig. 3B). Moreover, cleavage of caspase 3 was observed within 30 minutes of treatment with AP20187 (see Fig. 3C). Overall, these results indicate that the hDFBs-iCasp9+cell line is able to rapidly undergo targeted apoptosis.

[0081] Production and characterization of iCasp9+-hTEMs: hDFBs-iCasp9+cells were utilized to generate human cell derived tissue engineered matrices (hTEMs). Gross and morphological analysis of the iCasp9+-hTEMs, demonstrated by H&E staining (Figure 4A), revealed a tissue thickness and an ECM deposition comparable to hTEMs produced with untransduced hDFBs. Additionally, immunostaining for collagen 1 (Coll) and 3 (Col3) showed no discernible differences between hDFBs- iCasp9+cells and control hDFBs (Figure 4A). Quantitative analysis of glycosaminoglycan (GAG) content and hydroxyproline, as a measurement of fibrillar collagen, also revealed no significant differences (Figure 4B and 4C). Furthermore, biaxial mechanical testing revealed that both hDFB- and iCasp9+-hTEMs had no difference in tissue stiffness or strain, suggesting comparable ECM mechanical functionality (Fig. 4D). Taken together, hDFBs- iCasp9+cells do not affect the tissue growth, ECM deposition, nor tissue integrity when generating hTEMs, suggesting that hDFBs-iCasp9+cells are a suitable cell line for tissue engineering.

[0082] Decellularization efficiency of apoptosis-assisted decellularization method:

[0083] To assess the efficiency of the apoptosis-assisted decellularization method, hTEMs were compared to the standard detergent-only based method, i.e. detergent treatment, followed by nuclease treatment, but without prior induction of apoptosis. Both groups were subjected to staining with H&E to visualize cell nuclei, and DNA quantification was performed. No F07370

[0084] 15 visible cell nuclei were observed in the H&E staining of hTEMs decellularized using the apoptosis-assisted method (Figure 5A). Moreover, the results from the DNA quantification indicated that the concentration of DNA in the decellularized hTEMs was lower than 50 ng / mg dry weight (Figure 5B). This threshold of DNA concentration (indicated by a dashed line) aligns with recommended maximum levels for decellularized tissues according to a stanrdard set by Crapo et al., 2011. To assess whether the apoptosis-assisted decellularization method effectively reduces global intracellular proteins, the respective hTEMs were subjected to mass spectrometry. The fold change of the 20 most abundant intracellular proteins and DAMPs in detergent-only versus apoptosis-assisted decellularized hTEMs is reported in Figures 5C and 5D, respectively. A fold change greater than 0 indicates higher protein- or DAMP abundance in the detergent-based decellularized tissue, whereas a fold change less than 0 indicates higher protein- or DAMP abundance in the apoptosis-assisted decellularized hTEMs. Fold change was analyzed by a moderated t-test. * signifies a p-value <0.05, ** a p-value <0.01 , *** a p-value <0.001 , and **** a p-value <0.0001 . Of the 20 most abundant intracellular proteins, it was revealed that the apoptosis- assisted decellularization method effectively reduced the abundance of all proteins compared to detergent-based decellularization (Figure 5C). Notably, proteins such as vimentin (VIM), tubulin alpha 1C (TLIBA1C), H2A Clustered Histone 11 (H2AC11), and Ribosomal Protein L27a (RPL27A) exhibited substantial reductions in the apoptosis- assisted decellularized hTEMs. In summary, of the 20 most abundant intracellular proteins, the concentration of individual proteins in the study was reduced in the range of 1.14 times (PFN1) to 7.516 times (VIM).

[0085] The apoptosis-assisted decellularization method also reduces the abundance of most known DAMPs, as compared to detergent-only decellularization (Figure 5D). Specifically, DAMPs associated with cytoskeletal proteins, such as alpha-smooth muscle actin (ACTA2), proteoglycans like glypican 6 (GPC6), heat shock proteins like HSPA1A, and galectin 3 (LGALS3), were reduced, specifically between 1.07 times (LGALS1) to 3.58 times (GPC6). Conversely, a limited number of DAMPs, such as calcium-binding proteins S100A11 (calgizzarin) and S100A4, exhibited a significant increase in abundance in apoptosis- assisted decellularized hTEMs. However, this is due to the fact that calgizzarin is involved in the translocation of the apoptosis-induced factor into the nucleus, which is necessary for chromatin condensation and DNA fragmentation seen in apoptotic cells (Zhang et al., 2021). In sum, the apoptosis-assisted decellularization method results in a similar decellularization status in hTEMs, but carries the added benefit of reducing the content of intracellular protein and DAMPs present in the matrix. This suggests that the apoptosis-assisted F07370

[0086] 16 decellularization method may be less immunogenic.

[0087] Apoptosis-assisted decellularized hTEM characterization:

[0088] To evaluate the impact of the apoptosis-assisted decellularization process on ECM composition and collagen structure, Coll and Col3 immunohistochemistry was performed. Compared to the detergent-based method, the apoptosis-assisted approach revealed no discernible difference in collagen structure or visible abundance of hTEM patches (Figure 6A). This observation was further supported by biochemical assays measuring glycosaminoglycans and hydroxyproline, which showed no significant difference between the decellularization approaches (Figure 6B and 6C). Hydroxyproline was used as a proxy for collagen abundance. To provide a more detailed analysis of ECM composition, hTEMs decellularized using both approaches underwent mass spectrometry analysis. The fold change of the most abundant collagens, proteoglycans, and glycosaminoglycans were reported (Figure 6D-F). A fold change greater than 0 indicates higher protein abundance in the detergent-only decellularized tissue. Results revealed that apoptosis-assisted decellularization influenced several ECM proteins, with a reduction observed in collagen 1 (COL1A1 and COL1A2) and collagen 3 (COL3A1). To elucidate whether these changes in ECM composition, particularly the reduction in Coll and Col3, resulted in changes in ECM structural integrity, detergent-only and apoptosis-assisted decellularized hTEMs underwent biaxial mechanical testing. However, the results indicated no significant difference in mechanical properties between the detergent-only and apoptosis-assisted decellularized hTEMs (Figure 6G), i.e. the induced apoptosis during the decellularization process did not seem to cause any disruption of the mechanical function of the hTEM.

[0089] Apoptosis-assisted decellularized hTEHVs:

[0090] To translate the findings of the apoptosis-assisted decellularization method from patches to hTEHVs, a proof-of-concept experiment was conducted. In this experiment, hTEHVs (n=7) were produced using a diastolic pulse duplicator bioreactor and subsequently decellularized using various methods: apoptosis-assisted decellularization (n=4), detergent-only decellularization (n=1), apoptosis alone (n=1), and no decellularization (n=1). The apoptosis-assisted decellularized hTEHVs exhibited a shiny morphology at the macroscopic level (Figure 7A-C). These hTEHVs were then subjected to a 1-hour in vitro test under pulmonary-like pressure conditions (mean pressure 16.78 ± 1.62 mmHg). After 1 hour, the hTEHVs demonstrated a good to complete leaflet coaptation, unobstructed opening area, and symmetrical leaflet motion (Figure 7D i-iv). To evaluate ECM morphology and the decellularization, the hTEHVs were stained with H&E. All hTEHVs showed ECM F07370

[0091] 17 deposition throughout the valve (Figure 7E i-iv). The apoptosis-assisted decellularized hTEHVs (Figure 7E iv) displayed similar decellularization to the detergent-only method, with no visible cell nuclei. hTEHVs decellularized using apoptosis alone showed a significant reduction in visible cell nuclei compared to non-decellularized hTEHVs (Figure 7E i and iii). However, a few cells were still visible in the apoptosis-only group, as compared to the detergent-only or apoptosis-assisted decellularized hTEHVs (Figure 7E ii-iv). These results demonstrate that the apoptosis-assisted decellularization method can be applied to hTEHVs, showing complete decellularization and good valvular performance under pulmonary conditions for up to 1 hour.

[0092] Conclusion

[0093] The decellularization process is an essential step in the production of hTEM-based implants. It has been successfully demonstrated that hDFBs with stable upregulation of the suicide switch, iCasp9, were able to achieve drug-induced apoptosis with up to 97% efficiency. Additionally, the hDFB-iCasp9+cells were able to generate hTEMs with tissue deposition and functionality comparable to non-edited hDFBs. Overall, this data demonstrates that hDFB-iCasp9+cells are a good alternative to human primary cells in generating hTEM-based implants. Using the hDFB-iCasp9+cells, an apoptosis-assisted decellularization protocol was thus developed, which can achieve decellularization of hTEMs beyond current norms.

[0094] In addition to being an alternative to standard protocols, the present inventive protocol advantageously significantly reduces the levels of various intracellular proteins (in particular cytoskeletal-, histone-, and ribosomal-related proteins), as well as most known DAMPs, while essentially conserving the ECM structure and mechanics. hTEMs produced using the apoptosis-assisted decellularization protocol still meet the standards for DNA content recommended for decellularized tissue engineered biomaterials (Crapo et al., 2011). The reduction in the concentration of certain specific collagens, proteoglycans, and GAGs may be due to the secretion of MMP-9 by apoptotic cells. However, no significant changes were observed in the quantification of hydroxyproline and GAG levels, suggesting that while the concentration of specific individual proteins may be altered, the overall quantities of ECM remained similar.

[0095] In summary, the efficiency of the apoptosis-assisted decellularization method, coupled with its marginal impact on ECM structure, suggests its potential as a viable decellularization method for clinically available prostheses. Biaxial mechanical testing revealed no significant difference in the mechanical properties between the hTEMs decellularized using detergent- only and apoptosis-assisted methods. Additionally, it was demonstrated that the inventive F07370

[0096] 18 apoptosis-assisted decellularization protocol is applicable to hTEM-based hTEHVs. Such hTEM-based hTEHVs, which had been decellularized using the apoptosis-assisted method exhibited excellent mechanical stability and sustained valve performance in the in vitro heart valve tester under pulmonary conditions / pressure for up to 1 hour.

[0097] The study underlying the present invention demonstrated that the apoptosis-assisted decellularization method can significantly reduce various intracellular proteins, including cytoskeletal, histone, and ribosomal proteins, as well as many known immunogenic DAMPs in hTEMs, and could potentially contribute to an improved in situ tissue remodeling of a respective hTEM-based implant.

[0098] Materials and Methods

[0099] Generation of the hDFBs-iCasp9+cell line

[0100] Human dermal fibroblasts (hDFBs; CellSystems Biotechnology Vertrieb GmbH), were transduced with the inducible caspase 9 gene by using the iCasp9 plasmid (Addgene #15567, see also Straathof et al., 2005) which was incorporated into a lentivirus backbone using the Gibson Assembly Cloning Kit (New England Biolabs). This lentivirus backbone included a human Cytomegalovirus (CMV) promoter to drive the expression of the iCasp9 gene and GFP. hDFBs were transduced at a multiplicity of infection (MOI) rate of 0.6. GFP- positive cells were isolated on the third day post-transfection through FACS cell sorting (BD FACS Aria III). Subsequently, hDFBs-iCasp9+were expanded in advanced DMEM medium (Gibco), supplemented with 10% fetal bovine serum (FBS, Gibco), 1 % GlutaMax (Gibco), and 1 % penicillin-streptomycin (Lonza).

[0101] Apoptosis induction

[0102] Varying concentrations of the iCasp9 dimerization drug (AP20187, Sigma-Aldrich) were administered to hDFBs-iCasp9+. The efficacy of apoptosis induction was confirmed by assessing the presence of cleaved, i.e. active, caspase 3 (Abeam, ab32042) through Western blot analysis and annexin V / propidium iodide staining (Invitrogen) using flow cytometry.

[0103] Western blot analysis

[0104] Cells were lysed with RIPA buffer and a protease inhibitor mixture (complete, Roche). Extracted proteins were loaded onto a Mini-Protean TGX gel (Biorad), transferred to a F07370

[0105] 19

[0106] PVDF membrane (Biorad), and immunoblotted with cleaved-caspase 3 and p-actin antibodies (both from Abeam). Protein bands were detected using the ECL chemiluminescent system (Thermo Fisher).

[0107] Flow cytometry

[0108] Apoptotic cells were detached and pelleted at 300g for 5 minutes. After removing the supernatant, the cells were washed once with cold PBS (Gibco) and resuspended in annexin-binding buffer (Invitrogen) containing 10 mM HEPES, 140 mM NaCI, and 2.5 mM CaCI2. Annexin V conjugate (Invitrogen) and propidium iodide (Invitrogen) were added to a 100 pl aliquot of the resuspended cells and incubated for 15 minutes. The samples were then analyzed using an SP6800 Spectral Cell Analyzer (Sony). Flow cytometry data were processed using FlowJo software. hTEM production

[0109] Human cell derived tissue engineered matrices (hTEMs) were produced by coating nonwoven polyglycolic-acid meshes (PGA; thickness: 1.0 mm; specific; Cellon) with 1 % poly- 4-hydroxybutyrate (P4HB; TEPHA Inc.) in liquid tetrahydrofuran (Sigma-Aldrich) according to previous methods (Lintas et al., 2018; Motta et al., 2019). Briefly, the coated patches were sutured onto stainless steel rings (28 mm diameter) prior to cell seeding. hDFBs or hDFBs-iCasp9+, respectively, were seeded onto the scaffolds at a density of 106cells / cm2using fibrin as a cell carrier (Mol et al., 2005). The hTEMs were cultured on an orbital shaker starting from the second day in tissue culture medium composed of advanced DM EM medium (Gibco), supplemented with 10% fetal bovine serum (FBS, Gibco), 1% GlutaMax (Gibco), and 1 % penicillin-streptomycin (Lonza) supplemented with L-ascorbic acid 2- phosphate (0.25 mg / mL; Sigma-Aldrich). After one week, an additional 5 ng / mL of TGFpi (Peprotech) was introduced. hTEMs (n=41 total) were cultured for a duration of 4 weeks.

[0110] Decellularization

[0111] After the tissue culture, which resulted in a formation of an ECM on the scaffold, hTEMs underwent decellularization using either a detergent-only (i.e. detergent treatment followed by nuclease treatment) or the apoptosis-assisted method (i.e. induction of apoptosis, followed by detergent treatment and then nuclease treatment). The detergent-based method (Dijkman et al., 2012), was adapted to hTEMs being washed in PBS and then incubated in a detergent solution composed of PBS supplemented with 0.25% Triton-X-100 (Merck), 0.25% sodium-deoxycholate (Sigma-Aldrich), and 0.02% ethylenediaminetetraacetic acid (Sigma-Aldrich) for 24 hours at 37°C. hTEMs subsequently F07370

[0112] 20 underwent treatment with Benzonase Nuclease (Novagen, Merck) in TRIS-HCL buffer (pH=8) for 3 days at 37°C. In the apoptosis-assisted decellularization procedure, apoptosis was induced in hDFBs-iCasp9+using 100 nM of AP20187 for 24 hrs at 37°C. Following this, the previously described detergent-based decellularization protocol with 48h of chemical detergent and 72h of nuclease treatment was carried out (see Fig.1).

[0113] Histology and immunohistochemistry hTEMs were fixed with 4% formalin, paraffin-embedded, and cut into 5 pm thick slices. The presence of cell nuclei and tissue morphology was examined through hematoxylin and eosin (H&E) staining. The collagenous matrix was assessed using collagen-1 (Coll , Abeam, ab34710) and collagen-3 (Col3, Abeam, ab7778) immunohistochemistry. Stained hTEMs were imaged through brightfield microscopy (Mirax Midi Microscope, Carl Zeiss GmbH) and analyzed using Panoramic Viewer software.

[0114] Biochemical assays

[0115] Glycosaminoglycans (GAGs) and hydroxyproline (HYP) were quantified using previously developed biochemical assays (Dijkman et al., 2012). DNA content in the digested samples was quantified using Qubit (Model 3, Thermo Fisher). The measured values for all samples were normalized to their respective digested dry weights.

[0116] Mass spectrometry analysis (LC-MS / MS)

[0117] Proteomics of detergent-only and apoptosis-assisted decellularized hTEMs were determined through mass spectrometry analysis. An initial quality control using three samples from detergent-only decellularized hTEMs was used to ensure sample size. Subsequently, a sample size of n=4 per decellularization group was chosen for the comprehensive analysis. Samples underwent processing, analysis, and statistical analysis at the Functional Genomics Center Zurich. The LC-MS / MS method (Evosep TIMS-TOF) was utilized for this experiment. Acquired data underwent processing with DIA-NN (Version 16, PMID:31768060), and normalization as well as fold change calculations were performed using the R-Package prolfqua (Wolski et al., 2023) for proteins identified with a minimum of 2 peptides. The fold change was statistically analyzed using a moderated t-test (Smyth, 2004). The most abundant ECM proteins were defined with a normalized MS1 intensity above 15. The Matrisome Annotator web tool (Shao et al., 2023) was employed for annotating proteins into categories such as collagens, glycoproteins, proteoglycans, ECM- affiliated proteins, ECM regulators, and ECM-secreted factors. DAMPs were annotated using the PRRDB2.0 database (Kaur et al., 2019). F07370

[0118] 21

[0119] Biaxial mechanical testing

[0120] To analyze the mechanical properties of hTEMs produced by hDFBs and hDFBs-iCasp9+(detergent-only and apoptosis-assisted decellularization, respectively, n=4 for each group), biaxial mechanical testing was performed. Square samples measuring 9x9 mm were cut from the hTEMs, and their thickness was measured using a digital caliper between two thin metal plates. The samples were then mounted on a planar biaxial mechanical tester (BioTester 5000, Cellscale) with the BioRake sample mounting system (Cellscale; tine diameter 254 pm, tine spacing 0.7 mm, puncture depth 1.4 mm). The samples were subjected to stretching at 25% strain in both equibiaxial and non-equibiaxial directions (ratios of 25:12.5 and 12.5:25 for (X:Y). Each loading cycle comprised 5 seconds of stretching followed by 5 seconds of unloading, repeated 5 times. The final measurement was the average of two subsequent cycles.

[0121] Tissue engineered heart valve (TEHV) manufacturing hTEHVs were fabricated by sewing a non-woven polyglycolic acid (PGA) scaffold to a nitinol stent (28 mm diameter) as previously described (Driessen-Mol et al., 2014). The scaffold was then coated with 1 % P4HB (TEPHA Inc.) dissolved in tetrahydrofuran (Sigma-Aldrich), dried overnight, and sterilized according to previous methods (Driessen-Mol et al., 2014). Next, hTEHVs were seeded with hDFBs-iCasp9+cells following the established procedure. One day post-seeding, the hTEHV was placed in a modified diastolic pulse duplicator bioreactor system, providing adequate nutrient delivery and hydrostatic pulsatile pressure over a period of 4 weeks (Mol et al., 2005). Tissue culture medium was further enriched with 5 ng / mL of TGFpi (Peprotech) after one week. To shape the heart valve leaflet, a custom-made mold was inserted 10 days post-seeding (Emmert et al., 2018). The hTEHVs were maintained in the diastolic pulse duplicator bioreactor system for a total of 4 weeks. The hTEHVs were decellularized after 4 weeks using the apoptosis-assisted, detergent- only or apoptosis-only decellularization method as described above. hTEHV in vitro functionality assessment hTEHV functionality was tested and visualized using a pulse duplicator system (HDT-500, BDC Laboratories) as previously described (Sanders et al., 2016). hTEHVs (n=4) were subjected to physiological pulmonary pressure conditions with a peak systolic pressure of 30 mmHg and an end-diastolic pressure of 5 mmHg for up to 1 hour. Following the in vitro functionality test, the hTEHV were fixed in 4% formalin and stained with H&E as previously described. F07370

[0122] 22

[0123] Statistical analysis

[0124] Data are presented as mean ± standard deviation. Quantitative measurements of glycosaminoglycans, hydroxyproline, and DNA were averaged per sample (technical replicates) and subsequently per group (n=4, unless stated otherwise). Data visualization was conducted using GraphPad Prism (version 9.0.1), and graphics were created with BioRender.

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Claims

F0737028CLAIMS1 . Method for manufacturing a human cell derived tissue engineered matrix (hTEM) for production of an implant, preferably for production of a cardiovascular implant, comprising the following steps: a.) providing a population of genetically modified human cells expressing an inducible apoptotic initiator gene; b.) seeding the genetically modified human cells expressing an inducible apoptotic initiator gene onto a scaffold, resulting in a seeded scaffold; c.) culturing the seeded scaffold in a tissue culture medium until formation of a human extracellular matrix (ECM) on the scaffold, resulting in a human cell derived tissue-engineered matrix (hTEM); d.) decellularizing the hTEM, including the following steps: d1 .) inducing apoptosis in the genetically modified human cells of the hTEM by treating the hTEM with an apoptosis-inducing drug; d2.) treating the hTEM resulting from step d1.) with a solution comprising a detergent; d3.) treating the hTEM resulting from step d2.) with a solution comprising a nuclease, in order to reduce a nucleic acid content in the hTEM.

2. Method according to claim 1 , wherein the genetically modified human cells of step a.) are genetically modified human primary cells, preferably selected from a group consisting of fibroblasts, endothelial cells, smooth muscle cells and interstitial cells, wherein the genetically modified human primary cells more preferably are selected from a group consisting of dermal fibroblasts, cardiac fibroblasts, umbilical cord derived fibroblasts, vascular endothelial cells, valvular endothelial cells, vascular smooth muscle cells and valvular interstitial cells, wherein the genetically modified human primary cells most preferably are human dermal fibroblasts (hDFBs).

3. Method according to one of the preceding claims, wherein the inducible apoptotic initiator gene encodes a fusion protein which is able to dimerize and as a result of dimerization is able to trigger apoptosis upon exposure to a dimerization drug, and wherein in step d1 .), the apoptosis-inducing drug is such a dimerization drug, preferably a dimerizing small molecule pharmaceutical drug, more preferablyF0737029AP20187.

4. Method according to claim 3, wherein the inducible apoptotic initiator gene is a modified human Caspase gene, preferably an inducible Caspase 9 gene (iCasp9), more preferably encoding a modified human caspase 9 protein which is fused to a human small-molecule binding protein, most preferably to a human FK506 binding protein (FKBP), to allow dimerization by the dimerization drug.

5. Method according to one of the preceding claims, wherein the genetically modified human cells expressing an apoptotic initiator gene have been genetically modified via transduction, preferably via retroviral transduction, more preferably via lentiviral transduction, most preferably by incorporating a plasmid containing the apoptotic initiator gene into a lentivirus backbone, wherein the lentivirus backbone preferably includes a human Cytomegalovirus (hCMV) promoter to drive an expression of the inducible apoptotic initiator gene.

6. Method according to one of the preceding claims, wherein the scaffold is formed of a biodegradable material, especially preferably comprising or consisting of polyglycolic acid (PGA), wherein especially preferably the scaffold is coated with a solution containing Poly-4-Hydroxybutyrate (P4HB).

7. Human cell derived tissue engineered matrix (hTEM), manufactured according to one of the preceding claims.

8. Human cell derived tissue engineered matrix (hTEM) according to claim 7, wherein a concentration of double stranded DNA (dsDNA) in the ECM is less than an industry standard of 50 ng per mg ECM dry weight.

9. Human cell derived tissue engineered matrix (hTEM), manufactured according to one of claims 1-6, wherein the hTEM comprises a concentration of at least one intracellular protein, preferably of at least one intracellular protein selected from a group consisting of H2AC11 , ACTB, HBB, PPIB, VIM, H3-7, HBA1 , H4C1 , TUBA1C, ACTA2, H2BC12, TAGLN, ACTBL2, RPL27A, EEF1A1 , UBB, CSRP2, C9, PFN1 , and PRDX1 , which is at least 10%, preferably at least 14% lower as compared to a concentration of said at least one intracellular protein in an hTEM produced from genetically unmodified human cells or in an hTEM produced usingF0737030 a decellularization process not relying on induced apoptosis.

10. Human cell derived tissue engineered matrix (hTEM), manufactured according to one of claims 1-6, wherein the hTEM comprises a concentration of at least one damage-associated molecular pattern molecule (DAMP), preferably of at least one DAMP selected from a group consisting of ACTA2, ALDH1 L2, CALR, GPC1 , GPC6, H2AC11 , H2BC12, HMGB1 , HSPA12A, HSPA13, HSP90AA1 , HSP90AB1 , HSPA1A, HSPA8, HSPB1 , HSPB6, HSPD1 , LGALS1 , LGALS3, LGALS3BP, MACROH2A1 , MACROH2A2, PRDX1 , PRDX2, PRDX6, S100A10, S100A11 , S100A4, TXNDC5, and TXNL1 , which is at least 5%, preferably at least 7% lower as compared to a concentration of said at least one DAMP in an hTEM produced from genetically unmodified human cells or in an hTEM produced using a decellularization process not relying on induced apoptosis.11 . Human implant, preferably a cardiovascular implant, more preferably a vascular or a valvular implant, most preferably a human cell derived tissue engineered heart valve (hTEHV), comprising an hTEM produced by a method according to one of claims 1-6.

12. Method for decellularizing a human cell derived tissue engineered matrix (hTEM) for production of an implant, preferably for production of a cardiovascular implant, comprising the following steps:- dO.) providing an hTEM comprising a human extracellular matrix (ECM) produced from a population of genetically modified human cells expressing an inducible apoptotic initiator gene;- d1.) inducing apoptosis in the genetically modified human cells of the hTEM by treating the hTEM with an apoptosis-inducing drug;- d2.) treating the hTEM resulting from step d1.) with a solution comprising a detergent;- d3.) treating the hTEM resulting from step d2.) with a solution comprising a nuclease, in order to reduce a nucleic acid content in the hTEM.

13. Human cell derived tissue engineered matrix (hTEM), produced by a method according to one of claims 1-6, for use in the treatment of a cardiovascular disease, preferably a vascular or valvular disease, more preferably a valvular heart disease, wherein the hTEM comprises an ECM based on geneticallymodified human cells expressing an inducible apoptotic initiator gene.

14. Human cell derived tissue engineered matrix (hTEM), produced by a method according to one of claims 1-6, for use as an implant, preferably as a cardiovascular implant, more preferably a valvular implant, most preferably an hTEHV.

15. Human cell derived tissue engineered matrix (hTEM), produced by a method according to one of claims 1-6, wherein the hTEM comprises an ECM produced from genetically modified human cells expressing an apoptotic initiator gene, more preferably expressing iCasp9.

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