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    • 专利摘要:
    Biodegradable scaffold comprising messenger rna. This invention relates to a biodegradable scaffold comprising messenger rna. More particularly, it refers to the scaffolding, the method of preparation and the use thereof. A biodegradable scaffold comprises a biodegradable polymer, an isolated mrna that encodes a transcription factor and transfection agents. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2584107A1
    申请号:ES201630565
    申请日:2016-05-02
    公开日:2016-09-23
    发明作者:Marcos Garcia Fuentes；Adriana MARTINEZ LEDO；Anxo Vidal Figueroa
    申请人:Universidade de Santiago de Compostela；
    IPC主号:
    专利说明:

    DESCRIPTION
    Biodegradable scaffolding comprising messenger RNA Field of the Invention
    This invention relates to a biodegradable scaffold comprising messenger RNA. More particularly, it refers to scaffolding, the method of preparation and use thereof. 5
    Background of the invention
    Transcription factors (TFs) are proteins capable of inducing large changes in gene expression (Graf, 2009, Nature 462, 587-594). Since intracellular protein transport is a major challenge, one of the main concerns is how to transfer these 10 TFs to their place of action inside the nucleus and how to integrate these transfer technologies into tissue or device engineering systems. implantable For these applications, the assignment is usually made through gene therapy, using scaffolds activated with viral vectors. However, using a viral vector can cause safety problems, including dangerous immune reactions and immune suppression of viral vectors. The activation of scaffolds with plasmid DNA (pDNA) is also possible (Fang, 1996, PNAS 93, 5753-5758), but the transfection efficiency is low. Scaffolds have also been activated with other oligonucleotides such as microRNA and siRNA, but these molecules can only produce gene expression inhibition, not forced protein expression (Andersen et al., 2010, Mol Ther 18, 2018- 2027). 20 Furthermore, these strategies still have difficulties, especially in the case of in vivo transfection performed on tissue scaffolds.
    Recent publications have shown that messenger RNA (mRNA) can induce forced protein expression, and have been applied for the coding of growth factors. Thus, Lui et al. they used mRNA for 25 VEGF growth factor coding for tissue vascularization, and implanted this therapy together with the cells in a commercial tumor protein scaffold (Matrigel®) (Lui, 2013, Cell Research 23, 1172-1186). However, this technology cannot be applied in therapy since Matrigel is based on tumor proteins.
    On the other hand, Balmayor et al. they have yielded an mRNA that codes for growth factors to a femur defect in a rat model with good results (Balmayor, 2016, Biomaterials 87, 131-146).
    However, there is still a need to provide technologies for the transfer of mRNAs, and more specifically mRNAs encoding transcription factors that are strictly regulated intracellular proteins, with a very short half-life. The mRNAs must continue to be effective after cession, and achieve transfection in the three-dimensional environment, which generally shows lower efficiency than in 2D. These devices must be able to produce a clear biological effect induced by overexpression of the transcription factor. This effect could be measured by an overexpression of the transcription factor itself, but even more importantly, of other target genes regulated by said growth factor. In addition, the 3D structure of the scaffold must be able to house adhered cells and, if necessary, allow proliferation.
     fifteen Brief Description of the Invention
    The authors of the present invention have developed a biodegradable scaffold that is activated with mRNA sequences encoding transcription factors. This biodegradable scaffolding can lead to the pronounced forced expression of the transcription factor, greater than that achieved with plasmid DNA. We have also confirmed that this forced expression of a transcription factor induces changes in the expression levels of other genes, indicating a clear biological effect. In addition, this scaffolding has the advantage of avoiding security problems, in particular it avoids viral vectors.
    Thus, one aspect of the invention relates to a biodegradable scaffold comprising a biodegradable polymer, an isolated mRNA encoding a transcription factor and a transfection agent.
    Another aspect of the invention relates to a biodegradable scaffold of the invention for use as a medicament. In a particular embodiment, the biodegradable scaffold of the invention is for use in tissues or organs of regenerative therapy, preferably the tissue is cartilage, muscle or nerve tissue. In another particular embodiment, the biodegradable scaffold of the invention is for use in the treatment of a cartilage defect, muscle damage or nerve tissue damage.
    In another particular embodiment, the invention relates to the use of the biodegradable scaffold of the invention to prepare a medicament. In another particular embodiment, the invention relates to the use of the biodegradable scaffold of the invention to prepare a medicament for use in tissues or organs in regenerative therapy, preferably the tissue is cartilage, muscle or nerve tissue. In another particular embodiment, the invention relates to the use of the biodegradable scaffold of the invention to prepare a medicament for use in the treatment of a cartilage defect, muscle damage or nerve tissue damage.
    Another aspect of the invention relates to a pharmaceutical composition comprising the biodegradable scaffold of the invention described previously.
    A further aspect of the invention relates to a cosmetic composition comprising the biodegradable scaffold of the invention described previously.
    A further aspect of the invention relates to a method for preparing the biodegradable scaffold described above, which comprises
    (i) mixing a biodegradable polymer, an isolated mRNA encoding a transcription factor and a transfection agent, optionally adding cells selected from the group consisting of primary cells and immortalized cell lines,
    (ii) incubate the prepared mixture in (i),
    (iii) induce coagulation of the mixture prepared in (ii).
     twenty
    Detailed description of the figures

    Figure 1: (A) Structure of the plasmid vector used for in vitro transcription of the mRNA encoding the YFP fluorescent protein. (B) Fluorescence image of U87MG cells, 24 h after transfection with YFP-encoding mRNA using 25 lipofectamine 2000 (right image). The experiment was performed in a 24-well plate. A transmitted light image of the same cells is shown as a reference (left image).

    Figure 2: (A) A diagram illustrating scaffolds, cells and mRNA with a transfection agent 30 is shown in Figure 2A. (B) Optical microscopy images of human mesenchymal stem cells cultured in scaffolds activated with 3DFectIN / mRNA complexes. Scaffolds were prepared at two fibrin concentrations (2 and 4
    mg / mL) (C) Fluorescence image of plasmid activated scaffolds, in 3DFectIN / pDNA complexes. Three 3DFectIN / pDNA ratios were tested: 2: 1, 3: 1 and 4: 1. The pDNA was labeled with SYBERGold for observation by fluorescence microscopy.
     5
    Figure 3: Scanning electron microscopy images of fibrin scaffolds (2 mg / mL), both without cells (Control) or with cells (Sown) and at different magnifications. The scale bars are integrated into each image and referenced on the side.

    Figure 4: Scanning electron microscopy images of fibrin scaffolds (4 10 mg / mL), both without cells (Control) or with cells (Sown) and at different magnifications. The scale bars are integrated into each image and referenced on the side.

    Figure 5: Biodegradable scaffolds prepared from alginate and polyarginine. On the left, the macroscopic image of the gels formed in the conical lower part of 15 Eppendorf tubes, keeping in position after the inversion of the tube. On the right, the optical microscopy images of two representative examples of these scaffolds seeded with U87MG cells.

    Figure 6: Transfection of U87MG cells in fibrin scaffolds activated with mRNA 20 (2 μg mRNA, 3DFectIN / mRNA 4: 1 ratio). A YFP coding mRNA was used. The transfection of the cells was evaluated at 24 h, 48 h, 72 h and at 5 days. Each panel shows as reference images of transmitted light (left) and fluorescence (right) of the same area.
     25
    Figure 7: Transfection of U87MG cells in fibrin scaffolds activated with mRNA (1 μg of mRNA, 3DFectIN / mRNA 3: 1 ratio). A YFP coding mRNA was used. The transfection of the cells was evaluated at 24 h, 48 h, 72 h and at 5 days. Each panel shows as reference images of transmitted light (left) and fluorescence (right) of the same area. 30

    Figure 8: Transfection of U87MG cells in fibrin scaffolds activated with mRNA (1 µg of mRNA, 3DFectIN / mRNA 2: 1 ratio). A YFP coding mRNA was used.
    The transfection of the cells was evaluated at 24 h, 48 h and at 5 days. Each panel shows as reference images of transmitted light (left) and fluorescence (right) of the same area.

    Figure 9: Transfection of U87MG cells in fibrin scaffolds activated with pDNA 5 (1 μg pDNA, 3DFectIN / pDNA 3: 1 ratio). A YFP coding pDNA was used. The transfection of the cells was evaluated at 48 h and at 5 days. Each panel shows as reference images of transmitted light (left) and fluorescence (right) of the same area.
     10
    Figure 10: Cytotoxicity study with U87MG cells cultured in fibrin scaffolds (4 mg / mL fibrin) activated with 3DFectIN / mRNA complexes in 2: 1 and 3: 1 ratios. Non-activated scaffolds were used as control (labeled "C" in the figure). The cytotoxicity of the scaffolds was measured by an MTT test at 24 h (A) and 48 h (B). (C) The ability of fibrin scaffolds activated with 3DFectin / mRNA to support cell proliferation was confirmed by quantifying the amount of DNA in the culture at 0, 3 and 7, measured by a PicoGreen assay.

    Figure 11: (A) Transfection of human mesenchymal stem cells (hMSCs) in the 20 fibrin scaffolds activated with mRNA complexes at 24 h (1 μg of mRNA, 3DFectIN / mRNA 3: 1 ratio). YFP coding mRNA was used. Cell transfection was evaluated in scaffolds prepared from fibrin solutions of 2 and 4 mg / mL. Each panel shows the fluorescence image on the left, and as a reference, the transmitted light image of the same area on the right. (B) Study of the cytotoxicity 25 of hMSC cultured in fibrin scaffolds (2 or 4 mg / ml fibrin) activated with mRNA (3DFectIN / mRNA 3: 1 ratio). Scaffolds not activated were used as control (labeled "C" in the figure). The cytotoxicity of the scaffolds was measured by an MTT test at 24 h and 48 h. (C) The ability of mRNA activated scaffolds (2 and 4 mg / ml fibrin) to support cell proliferation was confirmed by quantifying the mass of DNA in the culture at 0, 3, 7 and 10 weeks, measured by a PicoGreen test. In each graph, proliferation in activated scaffolding
    With mRNA ("Treatment") it is compared with the same scaffolds without 3DFectIN / mRNA ("Control") complexes.

    Figure 12: (A) Structure of the plasmid vector used for in vitro transcription of mRNA encoding the transcription factor SOX9. (B) After transfection of 5 HEK293 cells with this mRNA and with lipofectamine, the expression of SOX9 was evaluated by extracting the proteins at 12 and 24 h and performing a western blot. Untransfected cells were used as a negative control (C-). Cells transfected with pDNA encoding SOX9 were used as a positive control (C +). The α-tubulin protein was used as a reference in the western blot. 10

    Figure 13: (A) Relative expression of Sox9 24 h after transfection of the U87MG cell line in a fibrin scaffold (4 mg / mL) activated with mRNA or pDNA (1 μg, 3DFectIN / mRNA and 3DFectIN / pDNA ratio 2: 1 and 3: 1). An mRNA encoding Sox9 was used. The relative expression of Sox9 was quantified by quantitative RT-PCR (qRT-PCR) analysis of transfected cells with respect to the GAPDH and β-actin reference genes. (B) A replica of the previous experiment of Sox9 expression at 24 h post-transfection in U87MG, but focused on fibrin scaffolds activated with mRNA or pDNA (1 μg) with a 3: 1 ratio. (C) The same comparative study of panel (B), but performed on human mesenchymal stem cells. twenty

    Figure 14: Expression kinetics of Sox9 mRNA after transfection of hMSCs in fibrin scaffolds of (A) 2 mg / mL and (B) 4 mg / mL. The scaffolds were activated with 3DFectIN / mRNA or 3DFectIN / pDNA (1 μg, 3: 1 ratio). An mRNA encoding Sox9 was used. Gene expression was measured after 12, 24 and 48 h. 25

    Figure 15: Relative gene expression of chondrogenic differentiation markers in hMSC transfected into fibrine scaffolds (4 mg / mL) activated with mRNA or pDNA, or not activated ("C"). The scaffolds were grown for 21 days in incomplete chondrogenic medium (ICM) or complete chondrogenic medium (CCM). The relative gene expression of the markers (A) Sox9 (A) and (B) aggrecan (ACAN) was measured under these conditions after 21 days of culture.

    Figure 16: Relative gene expression of chondrogenic differentiation markers in hMSC transfected into fibrin scaffolds (2 mg / mL and 4 mg / mL) activated with mRNA, pDNA or not activated ("C"). The scaffolds were grown for 28 days in incomplete chondrogenic medium (ICM) or complete chondrogenic medium (CCM). The relative gene expression of the markers (A) Sox9 (A) and (B) aggrecan (ACAN) was measured under these conditions after 28 days of culture.
    Detailed description of the invention
    In one aspect, the invention relates to a biodegradable scaffold comprising a biodegradable polymer, an isolated mRNA encoding a transcription factor and a transfection agent.
    The scaffolds of the invention have the advantage that they are biocompatible and do not exert significant toxicity to resident cells (example 4 and figure 10). In addition, the scaffolds of the invention can support cell proliferation (example 4) and can lead to high levels of forced expression of transcription factors by cells 15 (example 7, figure 13); even transfection was effective in human mesenchymal cells (example 5 and figure 11).
    The scaffolds of the invention activated with mRNA encoding SOX9 are capable of achieving transfection in a three-dimensional environment, and achieving significant expression of SOX9 in U87MG and also in human mesenchymal cells (hMSCs). This expression is much higher than that obtained when a plasmid DNA (pDNA) was used (example 6, Figures 13A and 13C).
    In addition, we have confirmed that mRNA-activated scaffolds of the invention can modify the gene expression profile of resident cells, leading per se to the differentiation of hMSCs into a chondrogenic lineage (examples 8 and 9). 25
    "Scaffolding" means a temporary structure used to support cells in three dimensions, while reconstructing a tissue or organ or performing other biological functions. Tissue scaffolds are widely described in the literature, and can have two possible structures, or intermediate structures between ends: (i) a solid matrix-shaped structure that has interconnected pores large enough (> 50 30 μm) to allow cell penetration and lodging or (ii) a hydrogel structure where cells can be encapsulated. The scaffolds of the invention are biodegradable and thus suitable to be replaced by natural tissue.
    The term "biocompatibility" is understood to refer to the ability of a material to perform with an appropriate response in its host in a specific situation. To be considered biocompatible, a device should comply with ISO 10993 or a similar standard, and be tested in animals and in clinical trials.
    Materials that are susceptible to the preparation of the scaffolds of the invention, which are biocompatible and biodegradable and are well described in the literature, can be classified into inorganic materials, enzymatically degradable polymers and hydrolytically degradable polymers. Examples of biodegradable inorganic materials are, but are not limited to, ceramic materials such as apatites, for example hydroxyapatite, and porous silicon. Preferably, the material used should be free of residues of pathogens of animal or human origin.
    "Biodegradable" in relation to the present invention means that the material is completely reabsorbed when it is in the environment of an organism after 24 hours.
    "Biodegradable polymer" means a polymer that is completely reabsorbed after implantation after 24 hours, and which is suitable for accommodating or for cell growth. Preferably, the biodegradable polymer is free of pathogenic materials and / or not derived from pathogenic samples. The biodegradable polymers in this invention can be enzymatically degradable polymers and hydrolytically degradable polymers. Enzymatically degradable polymers as understood in the present invention are, for example, collagen, elastin, elastin-like peptides, albumin, fibrin, silk fibroin, chitosan, alginate, hyaluronic acid and chondroitin sulfate. Hydrolytically degradable polymers as understood in the present invention are, for example, polyesters, polyurethanes, poly (ester amides), poly (ortho esters), polyanhydrides, poly (anhydro-co-imide), crosslinked polyanhydrides, poly (propylene fumarate), poly (pseudoamino acids), poly (alkyl cyanoacrylates), polyphosphazenes, polyphosphoesters. Examples of useful polyesters include, but are not limited to, polyglycolic, polylactic, poly (lactic-co-glycolic), polydioxanone, polycaprolactone and poly (trimethylene carbonate).
    In a preferred embodiment of the invention the biodegradable polymer is selected from fibrin, alginate and mixtures thereof. 30
    "Isolated mRNA" is understood as a polymeric molecule made of nucleic acids capable of being translated in ribosomes to a specific amino acid sequence, and therefore, to express one or more proteins, which has been isolated by procedures.
    Technicians of a biological medium or have been previously synthesized to be used in the scaffolding of the present invention. This term also includes mRNA that can be chemically modified. Some examples of chemical modifications of mRNA nucleic acids, but not limited, are: 5-methyl-cytidine, 2-thio-uridine, 5-methoxyuridine, N-1-methylpseudo-uridine and pseudo-uridine. 5
    The term "messenger RNA" ("mRNA) is understood as a polymeric molecule made of nucleic acids capable of being translated in ribosomes into a specific amino acid sequence, and therefore, to express one or more proteins. In the context of this invention, the mRNA is encoding at least one transcription factor (TF). Preferably, the mRNA employed in this invention is optimized for translation in eukaryotic cells. Preferably, the mRNA employed in this invention is synthesized for a specific purpose and with specific sequences. Therefore, sets of mRNA extracted from natural, non-manipulated living organisms or parts thereof are not preferred for the present invention.
    Although not limited to these procedures, the mRNA of the invention is preferably synthesized by in vitro transcription reactions, from a plasmid template, or alternatively, by solid phase chemical synthesis.
    Although not limited to the structures described below, the effectiveness of the invention benefits from the use of mRNA with high translatability. The structural characteristics of mRNA such as a 5 ’Cap, a 3’ polyadenin tail are some of the 20 most important to ensure high translation capacity. In addition, a short 3 'section of oligouridine is also potentially useful. Other structures that could improve mRNA translation are untranslated regions, which could be located at 5 'and / or at the 3' end in relation to the coding region.
    Therefore, a particular embodiment of the invention is directed to an mRNA having a 5'-Cap. Another particular embodiment relates to an mRNA having a polyadenine tail. Another particular embodiment of the invention is directed to an mRNA having a 5 'untranslated region. And another particular embodiment refers to an mRNA having a 3 'untranslated region.
    The mRNA sequences can generate cellular immunity, thus, for the present invention it is preferred that some of the nucleic acids be chemically modified to reduce their immune recognition. A particular embodiment of the invention is directed to an mRNA having chemically modified nucleic acids.
    selected from the list consisting of 5-methyl-citidine, 2-thio-uridine, 5-methoxyuridine, N1-methylpseudo-uridine and pseudo-uridine.
    The term "transcription factor" ("TF") means a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to mRNA. Sometimes TFs are also called "trans-activators" in 5 bibliography, both terms being synonyms. The TFs for the present invention preferably have one or more DNA binding domains. TFs have been classified by their superclass into: (1) basic domains, (2) zinc-coordinated DNA binding domains, (3) helix-spin-helix, (4) beta structure factors and minor cleft contacts , (5) other transcription factors. Several reviews of the function and structure of TF are available in the literature (Latchman, 1997, Int J Biochem Cell Biol 29, 1305-1312).
    The Medical Subject Headings (MeSH) descriptor database identifies TFs through the three numbers D12.776.930. There are several TF databases available to search for TF sequences and functions, for example, JASPAR ( http://jaspar.genereg.net). fifteen
    In a preferred embodiment of the invention, the mRNA encodes a chondrogenic transcription factor.
    In a preferred embodiment of the invention, the encoded TFs activate genetic programs responsible for cell differentiation or dedifferentiation. In a preferred embodiment of the invention, the mRNA encodes a transcription factor selected from the group consisting of SOX9, MyoD, NeuroD1, c-Myc, Klf4, Nanog, Oct4, SOX2, C / EBP-β, PPAR-γ , Brn2, Lmx1a, Nurr1, Mash1, Myt1l and NeuroG2. In a more preferred embodiment of the invention, the mRNA encodes for the transcription factors selected from SOX9, MyoD, NeuroD1, SOX2, Oct4, Klf4 and c-Myc. In a more preferred embodiment of the invention, the TF is SOX9. 25
    The term "transfection agent" is understood as a compound capable of improving the cession of the messenger RNA sequence (mRNA) to the cytoplasm. Thus, the presence of a transfection agent is evidenced by a marked increase in protein expression. The transfection agent, also called the gene transfer system, gene transfer vehicle, or activated gene matrices, has been described in many 30 publications (Borrajo, 2015, In: Polymers in Regenerative Medicine, 285-336).
    Transfection agents can be made of inorganic materials, lipid materials and polymeric materials. Although not limited to these, a possible list of
    Inorganic transfection agents are calcium phosphate salts and cationic silicon nanoparticles.
    Lipid transfection agents can be classified into condensing and non-condensing lipids, the condensers being often referred to as lipoplexes. Non-condensing lipids are emulsions, nanoemulsions and liposomes that can encapsulate the genetic material. Lipoplejos are formed by lipids with an aliphatic chain and one or more cationic groups. Although not necessarily limited to these, these cationic groups are often primary, secondary or tertiary amines, or structures with a mixture of these. The net lipid charge in lipoplexes should be positive at physiological pH, as a measure of du zeta potential, and should be able to bind to genetic material by electrostatic forces.
    Polymeric transfection agents can also be classified as condensing and non-condensing. Non-condensing generally binds to genetic material through some encapsulation technique or through weak forces.
    The condensing polymeric vehicles are formed by polymers that show a positive net charge at physiological pH, as a measure of zeta potential, and that can be attached to the genetic material, by electrostatic forces.
    In a preferred embodiment of the invention, the transfection agent is selected from cationic lipids, cationic polymers, and a calcium phosphate salt.
    In a preferred embodiment of the invention, the transfection agent. In a preferred embodiment of the invention, the transfection agent is a lipid condensing agent. In a more preferred embodiment of the invention, the lipid condensing agent is Lipofectamine or 3DFectin.
    In a preferred embodiment of the invention, the transfection agent is a polymeric condensing agent. In a more preferred embodiment of this invention, the condensing polymeric agent is polyarginine. In another more preferred embodiment of the invention, the condensing polymeric agent is poloxamine. In another more preferred embodiment of the invention, condensing polymeric agent is a cationic polyphosphazene.
    In a particular embodiment of the invention, the biodegradable anadamium further comprises cells. Although a variety of cells could benefit from the ability of this invention to exert control over their functions, primary cells are of first interest. Among them, progenitor cells with high plasticity such as adult stem cells and induced pluripotent stem cells could be the best candidates to be
    included to this invention. These cells have the ability to proliferate and can recapitulate different differentiation pathways. The cells incorporated into the scaffolding of the invention can proliferate and form biological structures in 3D form including tissues in these scaffolds.
    In a particular embodiment, the cells are selected from the group consisting of primary cells and immortalized cell lines. In a particular embodiment, the cells are not embryonic stem cells. In a preferred embodiment, the scaffold of the invention is clinically useful since it is biodegradable and biocompatible.
    In a more preferred embodiment of the invention, the primary cells are progenitor cells. In a more preferred embodiment of the invention, the primary cells are adult stem cells or induced pluripotent stem cells. In a preferred embodiment of the invention, adult stem cells are mesenchymal stem cells.
    In another preferred embodiment of the invention, the primary cells are fibroblasts or chondrocytes.
    In another embodiment, the invention is directed to a pharmaceutical composition comprising a scaffold as described above.
    In a particular embodiment, the pharmaceutical composition further comprises pharmaceutically acceptable carriers.
    In another particular embodiment, the pharmaceutical composition further comprises at least one additional active pharmaceutical ingredient. Thus, other drugs or compounds can be incorporated into the compositions to improve their performance or improve their presentation for final use. In a preferred embodiment, the additional active ingredient is selected from drugs, such as antibiotics, immunosuppressants, anti-inflammatories, biologics such as growth factors, cytokines, morphogens, proteins, extracellular matrix polysaccharides; of compounds for modifying the mechanical and gelling properties of scaffolds such as additional crosslinking agents.
    In another particular embodiment, the pharmaceutical composition is an injectable solution, suspension, hydrogel or a solid porous matrix.
    In another particular embodiment, the pharmaceutical composition is for use as a vaccine. 30
    In another particular embodiment, the invention relates to a method for preparing the scaffolding of the invention as described above, comprising:
    (i) Mixing a biodegradable polymer, an isolated mRNA encoding a transcription factor and a transfection agent, and optionally selected cells from the group consisting of primary cells and immortalized cell lines,
    (ii) Incubate the prepared mixture in (i), 5
    (iii) Induce coagulation of the mixture prepared in (ii).
    In another particular embodiment, the invention relates to an alternative method for preparing the scaffold of the invention as described above, comprising:
    (i) Prepare a scaffold,
    (ii) Mix an isolated mRNA encoding a transcription agent and a transfection agent,
    (iii) Incubate the mixture prepared in (ii) on the scaffold prepared in (i), and optionally add cells.
    In a particular embodiment, the biodegradable polymer is selected from fibrin, alginate and mixtures thereof. In a preferred embodiment, the fibrin concentration is between 1 mg / mL and 5 mg / mL. In a more preferred embodiment, the fibrin concentration is between 2 mg / mL and 4 mg / mL.
    In a particular embodiment, the coagulation of step (iii) is carried out by the addition of a coagulation agent. In a preferred embodiment, the coagulation agent is selected from thrombin, calcium salt and polyphosphate salt. twenty
    In a particular embodiment of the invention, when thrombin is used as a coagulation agent, the thrombin range used is between 0.2 U and 1.2 U per mg of the fibrinogen used.
    In the alternative method, the interaction of the scaffold and the mRNA / transfection agent in step (iii) can be reinforced by drying or lyophilization of the system. 25
    In another embodiment, the invention relates to a biodegradable scaffold obtained by the method described above.
    In another embodiment, the invention relates to the use of a scaffold of the invention, as an in vitro differentiation reagent or as a cosmetic implant.
    Another aspect of the invention relates to the use of a biodegradable scaffold as described above as a device for tissue and organ regeneration. In a preferred embodiment of the invention, the biodegradable scaffold is used as a device for cartilage regeneration.
    Another aspect of the invention relates to the use of a biodegradable scaffold defined above for cosmetic purposes.
    A final aspect of the invention relates to the use of a biodegradable scaffold as defined above as a drug for preventing, alleviating or curing diseases.
     5
    Some illustrative examples of the invention are described below; however, they should not be considered as limitations imposed on it.


    EXAMPLES 10

    Example 1
    Synthesis of YR fluorescent protein encoding mRNA: A plasmid for in vitro transcription of mRNA was designed based on plasmid pBluescript KS (pBSK KS, Stratagene, USA), with a T7 transcription promoter. In this plasmid, the YFP sequence and a polyadenylation signal was cloned from a YFP pIRES plasmid (Clontech, Germany), using the SmaI and XhoI restriction sites. The correct design was verified through its cleavage at restriction sites, and analysis by gel migration and sequencing assays. The structure of the plasmid used is depicted in Figure 1A. The mRNA was synthesized with an anti-reverse analogue Cap 20 (ARCA) through the ultra mMACHINE T7 kit (Ambio), following the manufacturer's instructions. The mRNA can be isolated by a standard phenol-chloroform extraction method. However, better reproducibility between batches of mRNA is achieved if the extraction is performed with a Pharme Lock Gel Light tube (5Prime, Germany), following the manufacturer's instructions. 25
    To verify mRNA activity, U87MG cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. U87MG cells were seeded in 96-well plates at a density of 26300 cells / cm2 the day before transfection. The lipoplexes were then prepared in 100 µl of OptiMEM (Gibco), with 0.5 µg of mRNA and with a mRNA: lipid ratio of 2: 1; The prepared complexes were added to the cells. After 6 h of incubation, the medium with lipoplexes was removed and replaced with fresh culture medium. The presence of fluorescent cells was verified by a fluorescence microscope (Olympus)
    24 h after transfection. The results confirmed that a high fraction of the cells that can be observed with transmitted light were successfully transfected (Fig. 1B).
    U87MG cells were routinely cultured in complete medium, consisting of Dulbecco's Modified Eagle's Medium with high glucose (D5671 Sigma) supplemented with 5 10% fetal bovine serum, 2 mM glutamine and 100 mg / L penicillin-streptomycin (Sigma- Aldrich). The culture was maintained at 37 ° C and under an atmosphere of 5% CO2. A scheme of scaffolding, cells, mRNAs and the transfection agent is depicted in Figure 2A; The illustration depicts a biodegradable scaffold activated with mRNA that codes for transfection factors and complexed this to a transfection agent. The inclusion of the cells in the scaffolding could be an interesting option for some applications, but it is considered as optional in the present invention.
    Preparation of fibrin scaffolds activated with 1 or 2 μg of mRNA and 3DFectIN as a transfection agent: First, 1 μg or 2 μg of mRNA were diluted up to 25 μl in OptiMEM (for scaffolds activated with 1 or 2 μg of mRNA , respectively). 15 Next, this mRNA solution was mixed with another 25 μl phase of 3DFectin (OZ Biosciences, France) in OptiMEM. For scaffolds with 1 μg of mRNA, this second phase had 2, 3 or 4 μl of 3DFectIN (corresponding to the 2: 1, 3: 1 or 4: 1 ratios, respectively) diluted to 25 μL in OptiMEM. For scaffolds with 2 μg of mRNA, this second phase had 4, 6 or 8 μl of 3DFectIN (corresponding to 20 2: 1, 3: 1 or 4: 1 ratios, respectively) and diluted up to 25 μL in OptiMEM. The mRNA and 3DFectIN phases were mixed and allowed to interact for 20 minutes. This reaction results in 50 μL of 3DFectIN / mRNA phase.
    In addition to this 3DFectIN / mRNA phase, two other solutions were prepared. A fibrinogen solution of 20 μL at 10 or 20 mg / mL was prepared to generate scaffolds of 2 or 4 mg / mL of final concentration. A thrombin solution of 20 μL at 12.5 U / mL was also prepared as a fibrinogen crosslinker. The fibrinogen solution is then pipetted into a culture well or in the place where it is intended to generate the scaffolds. The 3DFectIN / mRNA complexes are then mixed with 10 μL of OptiMEM and the resulting suspension is mixed with the fibrinogen by pipetting. Then, this phase is mixed with a thrombin solution for gelation. After 1 h of incubation at 37 ° C with thrombin, all these possible combinations of systems have formed a hydrogel.
    Cells can be integrated into this composition, changing the 10 μL of OptiMEM added to the 3DFectIN / mRNA complexes for the same volume of cell suspension in OptiMEM. A number of 1.5x105 cells can easily be incorporated into this volume. When the cells are to be cultured in the scaffolds, complete cell media can be added to the scaffolds after their formation, that is, after 5 hours of incubation of fibrinogen and thrombin at 37 ° C.
    Human mesenchymal stem cells (hMSC) were incorporated into fibrin scaffolds activated with YFP-encoding mRNA. Observation by optical microscopy shows that hMSCs are perfectly integrated into these 3D matrices, and can be grown both in 4 mg / mL fibrin scaffolds and in 2 10 mg / mL scaffolds (Figure 2B).
    Plasmid DNA activated scaffolds (pDNA): as a reference, fibrin scaffolds activated with YFP-encoding pDNA were also prepared. The plasmid used for these experiments was the same as we used for in vitro transcription of mRNA (Figure 1), since plasmid pBSK KS also has a promoter of eukaryotic transcription. These scaffolds can be prepared in exactly the same way as those activated with mRNA, but by changing mRNA for the same amount of pDNA. The scaffolds activated with pDNA showed exactly the same morphological and mechanical properties. We expect that 3Dfectin / pDNA complexes will have a similar distribution in scaffolds to 3Dfectin / mRNA complexes. 20 Because of this, we mark the scaffolds activated with pDNA by SYBRGold (Thermo Fisher Scientific, Inc.), and observe the distribution of fluorescence in scaffolds without cells (Figure 2C). Fluorescence was present in all areas of the scaffolding. However, it was observed that the 2: 1 3DFectin / pDNA ratios resulted in fluorescence agglomerations. This agglomeration was less prominent in the 3: 1 ratio and not detectable in the 4: 1 ratio.
    Fibrin scaffolds (2 mg / mL and 4 mg / mL) were prepared as described above, and loaded with 1.5x105 U87MG cells.
    Fibrin scaffolds activated with mRNA were seeded with hMSC cells, and cultured for a week at 37 ° C with complete medium (90% humidity, 5% 30 CO2). After one week, the scaffolds were lyophilized, metallized with gold-palladium in vacuo, and studied by scanning electron microscopy (SEM, LEO 435VP-SEM, SEMTECH Solutions, United Kingdom). SEM images confirmed
    that fibrin hydrogels form a highly porous structure (Figure 3 and 4). Contrary to our expectations, the pore size was larger in fibrin hydrogels of 4 mg / mL than in those of 2 mg / mL. Micrographs suggest a different structure for scaffolds seeded with cells compared to control scaffolds. This could be related to the mechanical contraction of the scaffolding induced by cell adhesion and by the deposition of extracellular matrix by said cells.

    Example 2
    This example describes the synthesis of alginate scaffolds activated with mRNA and two cationic polymers, polyarginine and protamine. For the current examples, mRNA encoding YFP was used, prepared as described in example 1. In a first step, 1 or 2 μg of mRNA was mixed with a solution of 10 μl of polyarginine or protamine (1 or 2 mg ). The solution was allowed to interact for 5 minutes at room temperature. Then, 50 μl of alginate (8 or 16 mg) was added to this suspension and mixed. Then, a 10 µl suspension with 1.5x105 U87MG cells was added to the solution. The system formed a hydrogel-like structure after the addition of 70 µl of the first mixture over 30 µl of a solution of CaCl2 (243 or 486 mM). The hydrogels are stabilized by incubation at 37 ° C, 5% CO2 and with 95% humidity for 5 minutes. After this point, complete cell culture medium 20 can be added on the scaffolds.
    All hydrogels formed stable and mechanically competent structures, as evidenced by tube inversion tests (Figure 5, left). The cells were successfully integrated into the scaffolds, and could be cultured for at least four days in these structures (Figure 5, right). 25
    It was found that the prototype with 0.2% protamine and 1.6% alginate resulted in the forced expression of YFP.

    Example 3
    Fibrin scaffolds (4 mg / mL) activated with 3DFectIN / mRNA (1 or 2 μg 30 mRNA, ratios 2: 1, 3: 1 and 4: 1) were prepared using a YFP coding mRNA according to the method described in Example 1, but before the addition of thrombin, instead of 10 μl of OptiMEM, the same volume of this medium containing 1.5x105 cells was added
    U87MG As a reference, a fibrin scaffold (4 mg / mL) activated with 3DFectin / pDNA (1 μg of pDNA, 3: 1 ratio) and with the same concentration of U87MG cells was prepared. After hydrogel formation, complete culture medium was added and the cells were cultured for 5 days (37 ° C, 5% CO2), with changes in the medium every two days. 5
    Cellular transfection was evaluated through a fluorescence microscope at 24 h, 48 h, 72 h and 5 days (5 d) after scaffolding preparation. Optical images of the same area were taken as a reference. The results showed high cellular transfection in all mRNA activated scaffolds regardless of the 3DFectin / mRNA ratio and the observation time point (Figures 6-8). The transfection of 10 mRNA activated scaffolds was clearly greater than that observed for pDNA activated scaffolds (Figure 9). This result was a first indication that mRNA activated scaffolds could achieve forced gene expression levels higher than those obtained with pDNA activated scaffolds.
     fifteen
    Example 4
    Fibrin scaffolds (4 mg / mL) activated with 3DFectIN / mRNA (1 µg of mRNA, ratios 2: 1 and 3: 1) were prepared encoding YFP as described in example 1, but before adding thrombin, in Instead of 10 μl of OptiMEM, the same volume of this medium containing 1.5x105 U87MG cells was added. As a negative control (C), a non-activated scaffold was prepared using the same procedure, but adding only OptiMEM instead of the 50 μl 3DFectIN / mRNA suspension in OptiMEM. After hydrogel formation, complete culture medium was added and the cells were cultured for 24, 48 h, 3 weeks and 7 weeks (37 ° C, 5% CO2).
    Cell viability at 24 and 48 h was measured by an MTT assay following the manufacturer's instructions. The ability of scaffolds to sustain cell proliferation was evaluated by measuring the DNA content in the cultures at the beginning (week 0), after 3 weeks and after 7 weeks. Scaffolds with a 3: 1 ratio were the prototypes selected for this proliferation test. The DNA content in the scaffolds was measured, after DNA extraction, by a PicoGreen assay (Thermo 30 Fisher Scientific, Inc.), following the manufacturer's instructions. DNA extraction for quantification was performed by incubating the scaffolds with a solution of 100 μL trypsin (5%) for 30 minutes, and then, using the
    incubation of the resulting suspension for 20 minutes in 0.1% SDS under intense agitation.
    The MTT test results showed that scaffolds activated with the 2: 1 complex were not toxic under any of the conditions (Figure 10). The scaffolds activated with the 3: 1 ratio showed no toxicity at 24 h and showed signs of less toxicity at 48 h. The DNA content test confirmed that 3: 1 scaffolding can support proliferation and cell culture over a period of 7 weeks, although no additional proliferation was observed after week 3.

    Example 5 10
    Fibrin scaffolds (2 and 4 mg / mL) activated with mRNA (1 µg of mRNA, 3: 1 ratio) encoding YFP were prepared as described in example 1, but before adding thrombin, instead of 10 μl of OptiMEM, the same volume of this medium containing 1.5x105 hMSCs was added. As a negative control (C), a non-activated scaffolding was prepared for the same procedure, but using only OptiMEM instead of the 50 μl of the 3DFectIN / mRNA suspension. After hydrogel formation, complete culture medium was added and the cells were cultured for 24 and 48 h (37 ° C, 5% CO2). Transfection of hMSCs was evaluated at 24 h as described in example 3. Scaffolding toxicity was evaluated at 24 and 48 h by an MTT assay as described in example 4. Scaffolding capacity for supporting the proliferation of 20 cells at 0, 3, 7 and 10 days was evaluated by a DNA quantification assay as described in example 4.
    The experiment showed that mRNA activated scaffolds can generate an effective transfection in hMSCs, both for prototypes with 2 mg / mL of fibirin and those of 4 mg / mL of fibrin. MTT tests indicated that all fibrin 25 scaffolds showed less toxicity at 24 h, and no toxicity at 48 h. DNA quantification supports the ability of these scaffolds to support cell proliferation. This was particularly visible for the 2 mg / mL scaffolds of fibrin (Figure 11).
     30
    Example 6
    Synthesis of mRNA encoding SOX9: a plasmid was designed for in vitro transcription of mRNA based on a plasmid pCMVTnT®, into which the SOX9 gene was introduced
    together with a Kozak consensus sequence to initiate translation, a 5 'UTR region of β-globin and a 3' UTR region of α-globin. A polyimine tail or a late polyadenylation signal of SV40 in 3 'was well added for mRNA synthesis with a polyadenine tail. The designed plasmid also had a eukaryotic transcription site, since it was used as a control in the activated scaffolds pDNA encoding 5 SOX9. The structure of the plasmid used is shown in Figure 12. The synthesis and isolation of mRNA from the plasmid was performed by the method described in example 1. To validate the bioactivity of the SOX9 sequence, U87MG cells were cultured in a plate 24-well culture and transfected with 1 μg of this mRNA. The expression of SOX9 in cultured cells 12 and 24 h after transfection was validated by western blotting after protein extraction (anti-SOX9 antibodies, Santa Cruz Biotech, USA). Untransfected cells were used as a negative control (C-) and cells transfected with the plasmid were used as a positive control (C +). The western blot results confirmed the bioactivity of the synthesized mRNA. fifteen
    Fibrin scaffolds (4 mg / mL) activated with 3DFectIN / mRNA or 3DFectIN / pDNA (1 μg of mRNA / pDNA, ratios 2: 1 and 3: 1) encoding SOX9 were prepared as described in example 1, but before if thrombin was added, instead of 10 μl of OptiMEM, the same volume of this medium containing 1.5x105 U87MG cells was added. As a negative control (C-), a scaffold without mRNA / pDNA or 3DFectIN was prepared by the same procedure, but using only OptiMEM instead of the 50 μl of 3DFectIN / mRNA suspension. After hydrogel formation, complete culture medium was added, and the cells were cultured for 24 h (37 ° C, 5% CO2).
    The ability of mRNA or pDNA activated scaffolds to induce forced expression of SOX9 was measured by a quantitative real-time polymerase chain reaction (qRT-PCR, C1000 thermal cycler, Bio-Rad Laboratories, Inc., USA). .), using probes for the SOX9, GAPDH and β-actin genes (Taqman, Thermo Fisher Scientific, Inc.). Relative expression was evaluated using the 2ΔΔCt method was used to evaluate relative expression, using GAPDH and β-actin (ACTB) as the reference genes. The relative expression of the control (C) is 1 in all the graphs, but it is not visible in the graphs due to the scale required to represent the rest of the data.
    The experiment demonstrated the ability to generate extreme positive regulation of SOX9 expression with scaffolds activated with mRNA. The expression with the
    mRNA activated scaffolding with a 2: 1 ratio was about 5000 times that of the control, while the 3: 1 ratio reached 20,000 times the control. The positive regulation generated with scaffolds activated with pDNA were orders of magnitude smaller than that obtained with mRNA, although significantly higher than the negative control (Figure 13A). 5
    The same experiment was repeated, with a greater number of replicates, but only for scaffolds activated with mRNA or pDNA in the 3: 1 ratio (Figure 13B). Finally, the same experiment was repeated for scaffolds activated with mRNA and pDNA in the 3: 1 ratio, but using hMSCs instead of U87MG cells (same seeding density). The results showed an overexpression of SOX9 in scaffolds 10 activated with mRNA 40,000 times higher than the control, notably better than that achieved with pDNA (Figure 13C).

    Example 7
    In this experiment, we tried to establish the expression kinetics of SOX9 at short times after transfection of hMSC in scaffolds activated with mRNA or pDNA. For this, scaffolds with 2 and 4 mg / mL of fibrin and activated with 1 µg of mRNA or pDNA (3: 1 ratio of 3DFectIN / mRNA or 3DFectIN / pDNA) were prepared with hMSCs following the procedure described in example 1, but using mRNA encoding Sox9 (see synthesis in example 7). The scaffolds were grown for 48 hours in complete medium, at 37 ° C, with 90% relative humidity and 5% CO2. Gene expression of SOX9 in the cells was assessed by qRT-PCR as described in example 6. The results showed a different behavior for scaffolds of 2 and 4 mg / mL fibrin. PDNA activation was as efficient or even more efficient in some cases than mRNA activation in scaffolds with 2 mg / mL fibrin. On the other hand, mRNA activation was clearly more efficient in scaffolds with 4 mg / mL fibrin (Figure 15).
    The kinetics of gene expression seem to be affected both by the polynucleotide used for activation, mRNA or pDNA, and by the concentration of fibrin in scaffolds. For mRNA, maximum values were reached at 12 and 24 h after 30 cell seeding, regardless of conditions. For pDNA, the expression was more constant, but a peak could be observed at 24 h for the 2 mg / mL scaffolds and at 48 h for the 4 mg / mL scaffolds (Figure 14).

    Example 8
    In this experiment, the ability of mRNA activated scaffolds to induce direct cell differentiation towards a chondrogenic lineage was tested. Fibrin scaffolds (4 mg / mL) were activated with mRNA encoding SOX9 (3: 1 ratio) and seeded with hMSCs, following the procedure described in example 6. As a reference, scaffolding seeded with hMSCs was used and activated with pDNA (also 3: 1 ratio).
    After the formation of the cross-linking scaffolding (1 h at 37 ° C after the addition of the thrombin), cell culture medium was added to the culture plates where the scaffolds were placed. The experiment was carried out by culturing the cells in the scaffolds in two different media: (i) incomplete chondrogenic medium (ICM) and (ii) complete chondrogenic medium (MCC). The ICM contained DMEM with high glucose (Sigma), 100 nM dexamethasone, 50 μg / mL ascorbic acid 2-phosphate, 40 μg / mL L-proline, 1% Premix ITS supplement (Becton Dickinson), 1 mM pyruvate sodium (Sigma) and 1% 15 penicillin / streptomycin (Sigma). The CCM contained ICM and 10 ng / mL of TGF-β3 (Peprotech, UK). The scaffolds were grown for 21 days, with 3 changes of medium per week, at 37 ° C, with 90% humidity and 5% CO2. After 21 days, the expression of chondrogenic differentiation marker genes Sox9, aggrecan (ACAN) and type II collagen (Col2a1) was evaluated. twenty
    The results showed that scaffolds, both activated with mRNA and pDNA, produce much larger quantities than the controls of the master chondrogenic regulator SOX9 after 21 days, regardless of the culture medium (ICM or CCM). The mRNA activated scaffolds were also able to induce ACAN expression compared to the controls in ICM, although their effect appeared to be negative for this gene in the scaffolds grown in CCM (Figure 15).
    The expression of Col2a1 was also measured. However, because the gene was not detected in the control scaffolds, we were not able to use the 2ΔΔCt calculation procedure. Instead, Table 2 presents the Ct of Col2a1 and the reference genes (GAPDH, ActB) for each sample. It is noteworthy that the scaffolds activated with mRNA were the only 30 type of sample where Col2a1 was consistently expressed, and that this result was independent of the culture medium used (ICM or MCP).
    In general, this data confirms that mRNA activated scaffolds can induce hMSC differentiation towards a chondrogenic lineage.

    Table 2: Ct values of Col2a1 and reference genes (GAPDH, ActB) expressed by hMSCs cultured in ICM or MCP for 21 days in fibrin scaffolds (4 mg / mL) 5 not activated (C-), activated with 3DFectIN / mRNA (3: 1 ratio) or activated with 3DFectIN / pDNA (3: 1 ratio). N / A = Not detected.

     C- mRNA 3: 1 pDNA 3: 1
     ICM  Col2a1 ActB GAPDH Col2a1 ActB GAPDH Col2a1 ActB GAPDH
     N / A  15,9893 17,57854 37,82298 16,05839 17,91439 N / A 16,05839 17,91439
     N / A  15,95448 17,54497 39,31226 16.09343 17.98782 N / A 16.09343 17.98782
     N / A  16,06559 17.65261 32.98505 16.0598 17.81497 35.54242 16.0598 17.81497
     CCM  N / A 15,00767 17.28146 31,41614 15,41693 17,09681 N / A 14.95397 16,98424
     N / A  14.99462 17.26188 29.41751 15.44408 17.28308 N / A 15,04821 17,0261
     N / A  15,06974 17,27417 30.63256 15.42 17.09684 28.43756 15,07596 17.07207

     10
    Example 9
    In this test, the same experiment as in example 8 was repeated, but using scaffolds with two fibrin concentrations (2 mg / mL and 4 mg / mL), prepared as in example 6. In addition, in this experiment the markers of Differentiation were analyzed after 28 days of culture. fifteen
    At 28 days, Sox9 was clearly overexpressed in mRNA activated scaffolds compared to controls, and this result was independent of the fibrin concentration in the scaffolding (2 mg / mL or 4 mg / mL) and the culture medium (ICM or CCM). At 28 days, ACAN was also overexpressed compared to the control in scaffolds activated with 2 mg / mL mRNA, and this result was also independent of the culture medium used. For the 4 mg / mL scaffolds, mRNA activated prototypes showed similar levels to the controls with both media (Figure 16).
    The analysis of the Col2a1 gene expression was only performed with scaffolds grown in CCM. The results confirmed that the scaffolds activated with mRNA were 25
    unique capable of producing consistent expression of this gene. In this experiment, all scaffolds with 4 mg / mL of fibrin were able to express Col2a1. However, only mRNA activated scaffolds showed mRNA expression in prototypes with 2 mg / mL fibrin (Table 3). This data confirms that mRNA activated scaffolds can induce the differentiation of hMSC towards a chondrogenic lineage, either by itself, or in combination with classical means of differentiation with growth factors.

    Table 3: The Ct values of Col2a1 and the reference gene (GAPDH) expressed by hMSCs cultured in CCM for 28 days in scaffolds of 2 or 4 mg / mL of fibrin, not activated (C-), activated with 3DFectIN / mRNA (3: 1 ratio), or activated with 3DFectIN / pDNA (3: 1 ratio). N / A = Not detected.

     C- RNA 3: 1 DNA 3: 1
     2 mg / mL  Col2a1 GAPDH Col2a1 GAPDH Col2a1 GAPDH
     N / A  19,80034 N / A 19,96768 N / A 20,62754
     N / A  19.78157 38.71413 20.16157 N / A 20.70585
     N / A  19.78686 38.71413 19.96851 N / A 20.68075
     4 mg / mL  26,98096 18,42884 38,62078 17.79144 39.60755 17.98131
     26,98096  18,33812 35,16769 17,75767 39,60755 18,12178
     38,62078  18,22016 35,16769 17,81862 N / A 18,13876

      fifteen
    权利要求:
    Claims (26)
    [1]
    1. Biodegradable scaffold comprising a biodegradable polymer, an isolated mRNA encoding a transcription factor and a transfection agent.
    [2]
    2. Scaffolding according to claim 1, further comprising cells selected from the group consisting of primary cells and immortalized cell lines, with the proviso that said cells are not embryonic stem cells.
    [3]
    3. Scaffolding according to any of the preceding claims, wherein the mRNA encodes for a chondrogenic transcription factor.
    [4]
    4. Scaffolding according to claims 1-2, wherein the mRNA encodes a transcription factor selected from the group consisting of SOX9, MyoD, NeuroD1, c-10 Myc, Klf4, Nanog, Oct4, SOX2, C / EBP-β , PPAR-γ, Brn2, Lmx1a, Nurr1, Mash1, Myt1l and NeuroG2.
    [5]
    5. Scaffolding according to claims 1-2, wherein the mRNA encodes for dedifferentiation transcription factor.
    [6]
    6. Scaffolding according to any of the preceding claims, wherein the mRNA 15 codes for a transcription factor selected from SOX9, MyoD, NeuroD1, SOX2, Oct4, Klf4 and c-Myc.
    [7]
    7. Scaffolding according to claims 2-6, wherein the primary cells are induced pluripotent stem cells or adult stem cells.
    [8]
    8. Scaffolding according to claims 2-6, wherein the primary cells are fibroblasts or chondrocytes.
    [9]
    9. Scaffolding according to any of the preceding claims, wherein the biodegradable polymer is selected from fibrin, alginate and mixtures thereof.
    [10]
    10. Scaffolding according to any of the preceding claims, wherein the transfection agent is selected from a cationic lipid, a cationic polymer, and a calcium phosphate salt.
    [11]
    11. Scaffolding according to claim 10, wherein the cationic polymer is polyarginine or a cationic polyphosphazene.
    [12]
    12. Scaffolding, according to any of claims 1-11, for use as a medicament. 30
    [13]
    13. Scaffolding according to any of claims 1-11, for use in regenerative therapy of tissues or organs.
    [14]
    14. Scaffolding according to any of claims 1-11, for use according to claim 13 wherein the tissue is cartilage, muscle or nerve tissue.
    [15]
    15. Scaffolding according to any one of claims 1-11, for use in the treatment of a cartilage defect, muscle damage or nerve tissue damage.
    [16]
    16. Pharmaceutical composition comprising the scaffold described in any of the claims 1 to 11.
    [17]
    17. Pharmaceutical composition according to claim 16, wherein it further comprises acceptable pharmaceutical vehicles.
    [18]
    18. Pharmaceutical composition according to any of claims 16-17, further comprising at least one additional active pharmaceutical ingredient. 10
    [19]
    19. Pharmaceutical composition according to claims 16-18, which is an injectable solution, suspension, hydrogel or a solid porous matrix.
    [20]
    20. Pharmaceutical composition according to any of claims 16-19, for use as a vaccine.
    [21]
    21. Cosmetic composition comprising the scaffold as described in any one of claims 1-11.
    [22]
    22. A method of preparing the scaffold as described in any of claims 1-11, comprising:
    (i) Mixing a biodegradable polymer, an isolated mRNA encoding a transcription factor and a transfection agent, and optionally 20 cells selected from the group consisting of primary cells and immortalized cell lines,
    (ii) Incubate the prepared mixture in (i),
    (iii) Induce coagulation of the mixture prepared in (ii).
    [23]
    23. Method according to claim 22, wherein the coagulation step (iii) is carried out by the addition of a coagulation agent.
    [24]
    24. Method for preparing the scaffold as described in any of claims 1-11, comprising:
    (i) Prepare a scaffold,
    (ii) Mix an isolated mRNA encoding a transcription agent and a transfection agent,
    (iii) Incubate the mixture prepared in (ii) on the scaffold prepared in (i), and optionally add cells.
    [25]
    25. Biodegradable scaffolding obtained by the method according to claims 22-23 or by the method according to claim 24.
    [26]
    26. Use of a scaffold as described in any of claims 1-11, as an in vitro differentiation reagent or as a cosmetic implant.
      5
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    US20070031465A1|2003-10-15|2007-02-08|Japan Science And Technology Agency|Implant for regenerating bone or cartilage with the use of transcriptional factor|
    US20100189794A1|2009-01-05|2010-07-29|Cornell University|Nucleic acid hydrogel via rolling circle amplification|
    US20140341870A1|2011-12-29|2014-11-20|"Nextgen" Company Limited|Biocomposite for regeneration of injured tissue and organs, a kit for making the biocomposite, a method of making the biocomposite and a method of treating injuries|
    US20150010501A1|2012-03-27|2015-01-08|Parcell Laboratories, Llc|Intervertebral disc repair compositions and methods|
    ES2809348B2|2020-10-27|2021-09-28|Univ Santiago Compostela|POLYMERS FOR GENE THERAPY|
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