ES2584107B2 - Biodegradable scaffolding comprising messenger RNA - Google Patents

Abstract

Biodegradable scaffolding comprising messenger RNA. This invention relates to a biodegradable scaffold comprising messenger RNA. More particularly, it refers to scaffolding, the method of preparation and the use thereof. A biodegradable scaffold comprises a biodegradable polymer, an isolated mRNA encoding a transcription factor and transfection agents.

Description

DESCRIPTION

Biodegradable scaffolding comprising messenger RNA Field of the invention

This invention relates to a biodegradable scaffold comprising messenger RNA. 5 More particularly, it refers to scaffolding, the method of preparation and use thereof.

Background of the invention

Transcription factors (TFs) are proteins capable of inducing large changes in gene expression (Graf, 2009, Nature 462, 587-594). Since the intracellular transport of 10 proteins is a great challenge, one of the main concerns is how to transfer these 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 performed through genetic therapy, using scaffolds activated with viral vectors. However, using a viral vector can cause 15 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 efficiency of transfection is low. Scaffolds have also been activated with other oligonucleotides such as microRNA and siRNA, but these molecules can only produce inhibition of gene expression, and not forced protein expression (Andersen et al., 2010, Mol Ther 18, 2018 -2027). In addition, 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 codification of growth factors. Thus, Lui et al. they used mRNA for the coding of the VEGF growth factor for tissue vascularization, and implanted this therapy together with the cells in a commercial tumor protein scaffold (Matrigel®) (Lui, 2013, Cell Research 23, 11721186). However, this technology cannot be applied in therapy since Matrigel is based on tumor proteins.

5

10

fifteen

twenty

25

30

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 assignment of mRNAs, and more specifically mRNAs that code for transcription factors that are strictly regulated intracellular proteins, with a very short half-life. The mRNAs must remain effective after their assignment, and achieve transfection in the three-dimensional environment, which generally shows a lower efficiency than in 2D. These devices must be capable of producing 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 its proliferation.

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 safety 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 defect in cartilage, muscle damage or nerve tissue damage.

5

10

fifteen

twenty

25

30

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).

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 lipofectamine 2000 (right image). The experiment was performed in a 24-well plate. For reference, an image of transmitted light from the same cells is shown (left image).

Figure 2: (A) A diagram illustrating scaffolds, cells and mRNA with a transfection agent 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

5

10

fifteen

twenty

25

30

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 framed with SYBERGold for observation by fluorescence microscopy.

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 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 Eppendorf tubes, staying 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 (2 pg 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.

Figure 7: Transfection of U87MG cells in fibrin scaffolds activated with mRNA (1 pg 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.

Figure 8: Transfection of U87MG cells in fibrin scaffolds activated with mRNA (1 pg of mRNA, 3DFectIN / mRNA 2: 1 ratio). A YFP coding mRNA was used.

5

10

fifteen

twenty

25

30

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 (1 pg 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.

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 3DFectin / mRNA activated fibrin scaffolds 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 fibrin scaffolds activated with mRNA complexes at 24 h (1 pg of mRNA, 3DFectIN / mRNA 3: 1 ratio). YFP encoding 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 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 a 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 through a PicoGreen trial. In each graph, the proliferation in the activated scaffolding

5

10

fifteen

twenty

25

30

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 SOX9 transcription factor. (B) After transfection of HEK293 cells with this mRNA and with lipofectamine, the expression of SOX9 was evaluated by extraction of the proteins at 12 and 24 h and the realization of a western blot. Untransfected cells were used as a negative control (C-). Cells transfected with SOX9 encoding pDNA were used as a positive control (C +). The tubulin protein was used as a reference in the western blot.

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 pg, 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 P-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 pg) with 3: 1 ratio. (C) The same comparative study of panel (B), but conducted in human mesenchymal stem cells.

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

Figure 15: Relative genetic 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 genetic expression of the markers (A) Sox9 (A) and (B) aggrecan (ACAN) was measured under these conditions after 21 days of culture.

5

10

fifteen

twenty

25

30

Figure 16: Relative genetic 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 genetic 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 (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 the scaffolds of the invention activated with mRNA 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).

"Scaffolding" means a temporary structure used to hold cells in three dimensions, while reconstructing a tissue or organ or performing other biological functions. Tissue scaffolds are widely described in the literature, and may have two possible structures, or intermediate structures between ends: (i) a solid matrix-shaped structure that has interconnected pores large enough (> 50 pm) 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.

5

10

fifteen

twenty

25

30

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 pathogen residues 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 is 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 fibroma, chitosan, alginate, hyaluronic acid and chondroitin sulfate. Hydrolytically degradable polymers as understood in the present invention are, for example, polyesters, polyurethanes, poly (ester amide), poly (ortho esters), polyanhydrides, poly (anhydrocoimide), crosslinked polyanhydrides, poly (propylene fumarate), poly (pseudoamino acids), poly (alkyl cyanoacrylates), polyphosphazenes, polyphosphoresters. 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.

"Isolated mRNA" is understood as a polymeric molecule made of nucleic acids capable of being translated in the ribosomes to a specific amino acid sequence, and therefore, to express one or more proteins, which has been isolated by procedures

5

10

fifteen

twenty

25

30

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 to, are: 5-methyl-cytidine, 2-thio-uridine, 5- methoxyuridine, N-1-methylpseudo-uridine and pseudo-uridine.

The term "messenger RNA" ("mRNA) is understood as a polymer 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. In the context of this invention, the mRNA is encoding at least one transcription factor (TF). Preferably, the mRNA used in this invention is optimized for translation into 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 most important to ensure high translation capacity. In addition, a short 3 'section of oligouridine is also potentially useful. Other structures that could improve the translation of the mRNA are untranslated regions, which could be located at 5 'and / or at the 3' end in relation to the codification region.

Therefore, a particular embodiment of the invention is directed to an mRNA having a 5'-Cap. Another particular embodiment relates to an mRNA that has a polyadenin tail. Another particular embodiment of the invention is directed to an mRNA that has a 5 'untranslated region. And another particular embodiment refers to an mRNA that has a 3 'untranslated region.

The mRNA sequences can generate cellular immunity, so, for the present invention it is preferred that some of the nucleic acids are chemically modified to reduce their immune recognition. A particular embodiment of the invention is directed to an mRNA having chemically modified nucleic acids.

5

10

fifteen

twenty

25

30

selected from the list consisting of 5-methyl-cytidine, 2-thio-uridine, 5-methoxyuridine, N-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 bibliography, both terms being synonymous. 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) helice-gyro-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 using 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).

In a preferred embodiment of the invention, the mRNA encodes a chondrogenic transcription factor.

In a preferred embodiment of the invention, the coded 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-P, PPAR-y, 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.

The term "transfection agent" is understood as a compound capable of improving the assignment 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 gene activated matrices, has been described in many 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

5

10

fifteen

twenty

25

30

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, often these cationic groups are primary, secondary or tertiary amines, or structures with a mixture of these. The net charge of lipids in lipoplexes should be positive at physiological pH, as a measure of du zeta potential, and should be able to bind to genetic material through electrostatic forces.

Polymeric transfection agents can also be classified as condensing and non-condensing. Non-condensers generally bind 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 the 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 exercise 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

5

10

fifteen

twenty

25

30

included in this invention. These cells have the ability to proliferate and can recapitulate different ways of differentiation. 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 vehicles.

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, from biologicals such as growth factors, cytokines, morphogens, proteins, polysaccharides from the extracellular matrix; 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.

In another particular embodiment, the invention relates to a method for preparing the scaffolding of the invention as described above, comprising:

5

10

fifteen

twenty

25

30

(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),

(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.

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.

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 the regeneration of tissue and organs. In a preferred embodiment of the invention, the biodegradable scaffold is used as a device for cartilage regeneration.

5

10

fifteen

twenty

25

30

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.

Below are some illustrative examples of the invention; however, they should not be considered as limitations imposed on it.

EXAMPLES Example 1

Synthesis of mRNA encoding the YFP fluorescent protein: A plasmid for in vitro transcription of mRNA was designed based on the 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 Smal and Xhol 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 represented in Figure 1A. The mRNA was synthesized with an anti-reverse analogue Cap (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.

To verify the activity of mRNA, 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 / cm the day before transfection. The lipoplexes were then prepared in 100 pl of OptiMEM (Gibco), with 0.5 pg of mRNA and with a proportion of mRNA: lipid 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)

5

10

fifteen

twenty

25

30

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.

1 B).

U87MG cells were routinely cultured in complete medium, consisting of Dulbecco's Modified Eagle's Medium with high glucose (D5671 Sigma) supplemented with 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 represents 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 activated fibrin scaffolds with 1 or 2 pg of mRNA and 3DFectIN as a transfection agent: First, 1 pg or 2 pg of mRNA were diluted until

25 pl in OptiMEM (for scaffolds activated with 1 or 2 pg of mRNA, respectively). Next, this mRNA solution was mixed with another 25 pl phase of 3DFectin (OZ Biosciences, France) in OptiMEM. For scaffolds with 1 pg of mRNA, this second phase had 2, 3 or 4 pl of 3DFectIN (corresponding to the 2: 1, 3: 1 or 4: 1 ratios, respectively) diluted to 25 pL in OptiMEM. For scaffolds with 2 pg of mRNA, this second phase had 4, 6 or 8 pl of 3DFectIN (corresponding to the 2: 1, 3: 1 or 4: 1 ratios, respectively) and diluted up to 25 pL in OptiMEM. The mRNA and 3DFectIN phases were mixed and allowed to interact for 20 minutes. This reaction results in 50 pL of 3DFectIN / mRNA phase.

In addition to this 3DFectIN / mRNA phase, two other solutions were prepared. A fibrinogen solution of 20 pL 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 pL at 12.5 U / mL was also prepared as a fibrinogen crosslinker. The fibrinogen solution is then pipetted into a culture well or where it is intended to generate the scaffolds. The 3DFectIN / mRNA complexes are then mixed with 10 pL of OptiMEM and the resulting suspension is mixed with the fibrinogen by pipetting. Next, 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.

5

10

fifteen

twenty

25

30

The cells can be integrated into this composition, changing the 10 pL of OptiMEM added to the 3DFectIN / mRNA complexes for the same volume of cell suspension in OptiMEM. A number of 1.5x105 cells can be easily incorporated into this volume. When the cells are going to be cultured in the scaffolds, complete cell media can be added to the scaffolds after their formation, that is, after 1 h of incubation of the 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 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 the one we used for the in vitro transcription of mRNA (Figure 1), since the plasmid pBSK KS also has a eukaryotic transcription promoter. 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. 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.

The mRNA activated fibrin scaffolds were seeded with hMSC cells, and grown for a week at 37 ° C with complete medium (90% humidity, 5% CO2). After one week, the scaffolds were lyophilized, metallized with vacuum palladium, and studied by scanning electron microscopy (SEM, LEO 435VP-SEM, SEMTECH Solutions, United Kingdom). SEM images confirmed

5

10

fifteen

twenty

25

30

that fibrin hydrogels form a highly porous structure (Figure 3 and 4). Contrary to our expectations, the pore size was higher in fibrin hydrogels of 4 mg / mL than in those of 2 mg / mL. The 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 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 pg of mRNA was mixed with a solution of 10 pl of polyarginine or protamine (1 or 2 mg ). The solution was allowed to interact for 5 minutes at room temperature. Then, 50 pl of alginate (8 or 16 mg) was added to this suspension and mixed. Then, a 10 pl suspension with 1.5x105 U87MG cells was added to the solution. The system formed a hydrogel-like structure after the addition of 70 pl of the first mixture over 30 pl 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 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 scaffolding, and could be grown for at least four days in these structures (Figure 5, right).

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 pg of mRNA, ratios 2: 1, 3: 1 and 4: 1) were prepared using a YFP coding mRNA according to the method described in the Example 1, but before the addition of thrombin, instead of 10 pl of OptiMEM, the same volume of this medium containing 1.5x105 cells was added

5

10

fifteen

twenty

25

30

U87MG As a reference, a fibrin scaffold (4 mg / mL) activated with 3DFectin / pDNA (1 pg of pDNA, ratio 3: 1) and with the same concentration of U87MG cells was prepared. After hydrogel formation, complete culture medium was added and the cells were grown for 5 days (37 ° C, 5% CO2), with changes in the medium every two days.

Cellular transfection was evaluated through a fluorescence microscope at 24 h, 48 h, 72 h and 5 days (5 d) after the scaffolding preparation. Optical images of the same area were taken as a reference. The results showed a 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 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 levels of forced genetic expression higher than those obtained with pDNA activated scaffolds.

Example 4

Fibrin scaffolds (4 mg / mL) activated with 3DFectIN / mRNA (1 pg 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 pl of OptiMEM, the same volume of this medium containing 1.5x105 U87MG cells was added. As a negative control (C), a non-activated scaffolding was prepared using the same procedure, but adding only OptiMEM instead of the 50 pl 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. Scaffolding 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 Fisher Scientific, Inc.), following the manufacturer's instructions. DNA extraction for quantification was carried out by incubation of the scaffolds with a solution of 100 pL trypsin (5%) for 30 minutes, and then, by

5

10

fifteen

twenty

25

30

incubation of the resulting suspension for 20 minutes in 0.1% SDS under intense agitation.

The MTT test results demonstrated that scaffolds activated with the 2: 1 complex were not toxic under any of the conditions (Figure 10). Scaffolds activated with the 3: 1 ratio showed no toxicity at 24 h and showed signs of toxicity less than 48 h. The DNA content test confirmed that 3: 1 scaffolding can withstand proliferation and cell culture over a period of 7 weeks, although no additional proliferation was observed after week 3.

Example 5

Fibrin scaffolds (2 and 4 mg / mL) activated with mRNA (1 pg of mRNA, ratio 3: 1) YFP coding were prepared as described in example 1, but before adding thrombin, instead of 10 OptiMEM pl, 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 pl of the 3DFectIN / mRNA suspension. After hydrogel formation, complete culture medium was added and the cells were grown 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 to supporting cell proliferation 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 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).

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

5

10

fifteen

twenty

25

30

together with a Kozak consensus sequence to initiate translation, a 5 'UTR region of P-globin and a 3' UTR region of a-globin. A polyimine tail or a late polyadenylation signal of SV40 in 3 'was well added for the synthesis of mRNA with a polyadenine tail. The plasmid designed also had a eukaryotic transcription site, since it was used as a control in the activated scaffolds pDNA encoding SOX9. The structure of the plasmid used is shown in Figure 12. The synthesis and isolation of the 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 pg 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.

Fibrin scaffolds (4 mg / mL) activated with 3DFectIN / mRNA or 3DFectIN / pDNA (1 pg 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 pl 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 pl of 3DFectIN / mRNA suspension. After the formation of the hydrogel, 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 P-actin genes (Taqman, Thermo Fisher Scientific, Inc.). Relative expression was evaluated using the 2AACt method was used to evaluate relative expression, using GAPDH and P-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 mRNA activated scaffolds. The expression with the

5

10

fifteen

twenty

25

30

mRNA activated scaffolding with a 2: 1 ratio was around 5000 times that of the control, while the 3: 1 ratio reached 20000 times the control. The positive regulation generated with scaffolds activated with pDNA was orders of magnitude smaller than that obtained with mRNA, although significantly higher than the negative control (Figure 13A).

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 activated with mRNA 40000 times higher than the control, significantly better than that achieved with pDNA (Figure 13C).

Example 7

In this experiment we tried to establish the kinetics of expression of SOX9 at short times after the 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 pg 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. The genetic 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 of 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 appear 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 cell seeding, regardless of conditions. For the 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).

5

10

fifteen

twenty

25

30

Example 8

In this experiment, the ability of mRNA activated scaffolds to induce direct cell differentiation to 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 scaffolding by reticulation (1 h at 37 ° C after the addition of 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 pg / mL ascorbic acid 2-phosphate, 40 pg / mL L-proline, 1% Premix ITS supplement (Becton Dickinson), 1 mM pyruvate sodium (Sigma) and 1% penicillin / streptomycin (Sigma). The CCM contained ICM and 10 ng / mL of TGF-P3 (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 Sox9 chondrogenic differentiation marker genes, aggrecan (ACAN) and type II collagen (Col2a1) was evaluated.

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 2AACt calculation procedure. Instead, Table 2 presents the Ct of Col2a1 and the reference genes (GAPDH, ActB) for each sample. It is reasonable that the mRNA activated scaffolds were the only type of sample where Col2a1 was consistently expressed, and that this result was independent of the culture medium used (ICM or MCP).

5

10

fifteen

twenty

25

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 grown in ICM or MCP for 21 days in fibrin scaffolds (4 mg / mL) 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

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. Also, in this experiment the markers of Differentiation were analyzed after 28 days of culture.

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 the

only 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 5 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 10 hMSCs cultured in CCM for 28 days in scaffolds of 2 or 4 mg / mL 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

Claims (23)

10 fifteen twenty 25 30 1. Biodegradable scaffolding comprising a biodegradable polymer, an isolated mRNA encoding a transcription factor and a transfection agent. 2. Scaffolding, according to claim 1, which further comprises 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. Scaffolding according to any one of the preceding claims, wherein the mRNA encodes for a chondrogenic transcription factor. 4. Scaffolding according to claims 1-2, wherein the mRNA encodes a transcription factor selected from the group consisting of SOX9, MyoD, NeuroD1, c-Myc, Klf4, Nanog, Oct4, SOX2, C / EBP-P, PPAR-y, Brn2, Lmx1a, Nurr1, Mash1, Myt1l and NeuroG2. 5. Scaffolding according to claims 1-2, wherein the mRNA encodes for dedifferentiation transcription factor. 6. Scaffolding according to any of the preceding claims, wherein the mRNA encodes for a transcription factor selected from SOX9, MyoD, NeuroD1, SOX2, Oct4, Klf4 and c-Myc. 7. Scaffolding according to claims 2-6, wherein the primary cells are induced pluripotent stem cells or adult stem cells. 8. Scaffolding according to claims 2-6, wherein the primary cells are fibroblasts or chondrocytes. 9. Scaffolding according to any of the preceding claims, wherein the biodegradable polymer is selected from fibrin, alginate and mixtures thereof. 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. Scaffolding, according to claim 10, wherein the cationic polymer is polyarginine or a cationic polyphosphazene. 12. Scaffolding, according to any of claims 1-11, for use as a medicament. 13. Scaffolding, according to any of claims 1-11, for use in regenerative therapy of tissues or organs. 14. 15. 5 16. 17. 18. 10 19. twenty. 15 21. 22 twenty 25 23. 24. 30 Scaffolding, according to any of claims 1-11, for use according to revindication 13 wherein the tissue is cartilage, muscle or nerve tissue. Scaffolding, according to any of claims 1-11, for use in the treatment of a cartilage defect, muscle damage or nerve tissue damage. Pharmaceutical composition comprising the scaffold described in any of claims 1-11. Pharmaceutical composition according to claim 16, wherein it also comprises acceptable pharmaceutical vehicles. Pharmaceutical composition according to any of claims 16-17, which further comprises at least one additional active pharmaceutical ingredient. Pharmaceutical composition according to claims 16-18, which is an injectable solution, suspension, hydrogel or a solid porous matrix. Pharmaceutical composition according to any of claims 16-19, for use as a vaccine. Cosmetic composition comprising the scaffold as described in any of claims 1-11. A method of preparing the scaffold as described in any of claims 1-11, comprising: (i) Mixing a biodegradable pod, 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), (iii) Induce coagulation of the mixture prepared in (ii). Method, according to claim 22, wherein the coagulation step (iii) is carried out by the addition of a coagulation agent. 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. Biodegradable scaffolding obtained by the method according to claims 22-23 or by the method according to claim 24. 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