WO2017191345A1

WO2017191345A1 - Biodegradable scaffold comprising messenger rna

Info

Publication number: WO2017191345A1
Application number: PCT/ES2017/070262
Authority: WO
Inventors: Marcos Garcia Fuentes; Adriana Martinez Ledo; Anxo VIDAL FIGUEROA
Original assignee: Universidade De Santiago De Compostela
Priority date: 2016-05-02
Filing date: 2017-04-28
Publication date: 2017-11-09
Also published as: ES2584107B2; ES2584107A1

Abstract

The invention relates to a biodegradable scaffold comprising messenger RNA. More specifically, the invention relates to the scaffold, the production method thereof and the use of same. A biodegradable scaffold comprising a biodegradable polymer, an isolated mRNA encoding a transcription factor and transfection agents.

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.

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 TFs to their place of action inside the nucleus and how to integrate these transfer technologies into tissue engineering systems or implantable devices. . For these applications, the assignment is usually carried out 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, 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 (niRNA) can induce forced protein expression, and have been applied to the coding of growth factors. Thus, Luí 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 (Matrigei *) (Luí, 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.

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 scaffolding Biodegradable 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).

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 coding mRNA using Lipofectamine 2000 (right image). The experiment was performed on 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 is shown in Figure 2A. (B) Optical microscopy images of human mesenchymal stem cells cultured in complex-activated scaffolds of 3DFectIN / mRNA. Scaffolds were prepared at two concentrations of fibrin (2 and 4 mg / mL). (C) Fluorescence image of plasmid activated scaffolds, in 3DFectIN / pDNA complexes. Three proportions 3 DF ectIN / pDN A were tested: 2: 1, 3: 1 and 4: 1. The pDNA was labeled 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.

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.

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, 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: Evaluation of 3DFectIN: mRNA / pDNA ratios with the YFP reporter sequence and the U87MG cell line in 4 mg / mL fibrin gels. Representative fluorescence and visible micrographs corresponding to transfection experiments carried out with 1 μg mRNA (left) and pDNA (right) complexed with 3DFectIN using the 3DFectIN: mRNA / pDNA ratios of 2: 1 (above), 3: 1 ( medium), 4: 1 (below).

Figure 7: Evaluation of the mRNA dose by scaffolding carried out on 4 mg / mL fibrin gels seeded with U87MG cells. Representative fluorescence and visible micrographs corresponding to transfection experiments carried out with 1 and 2 μg of mRNA complexed with 3DFectIN in proportions 3DFectIN: mRNA 3: 1 (left) and 4: 1 (right).

Figure 8: Protein expression kinetics of the YFP reporter sequence in scaffolds activated with mRNA and pDNA. Representative fluorescence and visible micrographs corresponding to transfection experiments carried out with 1 μg of mRNA / pDNA and the 3 DF ectIN: mRN A / pDN A 3: 1 ratio (ύ .: μ £). Cell transfection was evaluated at 24 h, 48 h, 72 h and at 5 days. Figure 9: Cytotoxicity study with U87MG cells cultured in fibrin scaffolds (4 mg / mL fibrin) activated with 3DFectIN / mRNA or pDNA complexes in 2: 1 and 3: 1 ratios were tested to mark the expression of the SOX9 gene (A ). Non-activated scaffolds were used as control (labeled "C" in the figure). The best 3: 1 condition was selected to compare the transfection of mRNA and pDNA (B). The expression of SOX9 was quantified by qRT-PCR analysis of the transfected cells relative to the expression of GAPDH and β-actin. The cytotoxicity of scaffolds was measured by a MTT assay at 24 h and 48 h (C) and 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 test.

Figure 10: (A) Transfection of human mesenchymal stem cells (hMSCs) in 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 of hMSC cultured in fibrin scaffolds (2 or 4 mg / mi 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 through a PicoGreen trial. In each graph, proliferation in mRNA activated scaffolding ("Treatment") is compared with the same scaffolds without 3DFectIN / mRNA ("Control") complexes.

Figure 11: (A) Structure of the plasmid vector used for in vitro transcription of mRNA encoding the transcription factor SOX9. (B) After transfection of FIEK293 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-). Transfected cells with pDNA encoding SOX9 were used as a positive control (C +). The tubulin protein was used as a reference in the western blot.

Figure 12: Effect of fibrinogen concentration and type of genetic material on the kinetics of SOX9 expression in human mesenchymal cells (hMSCs) encapsulated in fibrin scaffolds. (A) SOX9 expression 24 h after encapsulation of hMSCs in 2 and 4 mg / mL fibrin gels activated with SOX9. (B) 48 h proliferation curves for 2 and 4 mg / mL fibrin gels. (C) SOX9 expression kinetics in hMSCs encapsulated in fibrin gels with 2 mg / mL and (D) 4 mg / mL activated with mRNA and pDNA. Gene expression levels were measured by qRT-PCR and normalized to β-actin expression. Maximum expression levels were assigned arbitrarily as 100% in graphs C and D. Data are shown as mean and standard deviation of three independent experiments with two replicates per experiment. Figure 13: 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 14: 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.

Figure 15: Chondrogenic marker expression of hMSCs encapsulated in fibrin 2 and 4 mg / mL hydrogels activated with mRNA and pDNA cultured for 7 and 21 days in complete chondrogenic medium. Expression of (A) SOX9, (B) aggrecan, (C) type II collagen, at 7 and 21 days. Gene expression levels were measured by qRT-PCR, normalized to GAPDH expression and compared to levels in hMSCs before encapsulation. Data are shown as mean and standard deviation of two replicates in one experiment. A standard pellet culture is shown as comparative. Figure 16. Expression of myogenic marker after transfection of hMSCs in fibrin scaffolds 2 mg / mL (left) and 4 mg / mL (right) activated with MyoD mRNA (1 μg, 3: 1 3DFectIN / mRNA) maintained for 48 h in the middle of growth. Gene expression levels were measured at 12, 24 and 48 h by means of qRT-PCR normalized to GAPDH expression and compared to the levels in hMSCs before encapsulation.

Figure 17. Expression of myogenic markers of hMSCs encapsulated in fibrin hydrogels 2 and 4 mg / mL activated with MyoD mRNA. The cells were cultured for 14 days in reduced serum medium and the expressions of MYOD (A) and MYOG (B) were measured by qRT-PCR. Gene expression levels were normalized to β-actin expression and compared to levels in hMSCs before encapsulation. Data are shown as mean and standard deviation of three replicates in one experiment.

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 9C, 10C and 10D). 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 6, Figure 9A and 9B); even transfection was effective in human mesenchymal cells (examples 6 and 7 and figure 12A).

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 9 A, 9B and 12A).

In addition, we have confirmed that mRNA-activated scaffolds of the invention can modify the gene expression profile of resident cells, leading per the differentiation of hMSCs into a chondrogenic lineage (examples 8, 9 and 10; figures 13, 14 and 15) and myogenic lineages (examples 11 and 12; figures 16 and 17).

"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 μιη) 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. Polymers Hydrophilically degradable as understood in the present invention are, for example, polyesters, polyurethanes, poly (ester amides), poly (ortho esters), polyanhydrides, poly (anhydrous-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.

"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 technical means procedures. biological or has been synthesized above 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, Ν-1-methylpseudo-uridine and pseudo-uridine.

The term "messenger RNA" ("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. In the context of In this invention, the mRNA is encoding at least one transcription factor (TF). Preferably, the mR A used in this invention is optimized for translation in eu karyotic cells Preferably, the mRNA used in this invention is synthesized for a purpose specific 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 sequences with high stability and translatability. The structural characteristics of mRNA such as a 5 'Cap, a 3' polyadenine tail are some of the most important to ensure proper stability and thus allow High translation capacity This polyadenine tail can be synthesized by polymerization of poly (A) after in vitro transcription or during the elongation step of the transcription itself in vitro. For the second option, the inclusion of a 3 'stretch of oligothimidine in the template plasmid is preferred. This option is potentially useful to avoid the synthesis of polyadenine tails of different lengths that can cause reproducibility problems. A preferred option is the use of 5'-Cap, in particular the use of an anti-reverse analogue Cap (ARCA) is preferred, which allows synthesizing exclusively weathered RNAs in the correct orientation (Stepinski, 2001, RNA 7 (10)): 1486-95 and Peng, 2002, Org Lett 4 (2): 161-4) and improve the performance of in vitro transcription reactions.

As for the translation, it is preferred to include a strong signal of initiation of the Kozak translation before the open reading frame (ORF) of the gene of interest, and weakening this sequence with non-translatable regions (UTRs) of high translation genes in the 3'and 5 'ends. .

Some high-translation gene UTRs include, but are not limited to those encoding the α and β-globin proteins.

Therefore, a particular embodiment of the invention is directed to an mRNA having a 5'-Cap, more preferably a 5'-ARK. 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, preferably an untranslated region of a highly translated gene, such as, for example, α and β-globin. And another particular embodiment relates to an mRNA having a 3 'untranslated region, preferably an untranslated region of a highly translated gene such as α and β-globin.

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-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 information transcription rate. DNA genetics to mRNA. Sometimes TFs are also called "trans-activators" in 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 for searching sequences and functions of TFs, for example, JASPAR (http: // j aspar. Enereg.net). 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, mRN A codes for a transcription factor selected from the group consisting of SOX9, MyoD, NeuroDl, c-Myc, Klf4, Nanog, Oct4, SOX2, C / ΕΒΡ-β, PPAR-γ , Brn2, Lmxla, Nurrl, Mashl, Mytll and NeuroG2. In a more preferred embodiment of the invention, the mRNA encodes for the transcription factors selected from SOX9, MyoD, NeuroDl, 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 cession of the messenger RNA sequence (mRNA) to the cytoplasm. Thus, the presence of a transfection agent is evidenced by a marked increase in the expression of the target gene. 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 may 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 as 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 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 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.

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),

(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 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.

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

EXAMPLES 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 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 depicted 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 Phase 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 78125 cells / cm ² the day before transfection. 4 h before transfection, the culture medium was removed and replaced with 50 μΕ of OptiMEM (Gibco). The lipoplexes were prepared next in 50 μΐ of OptiMEM (Gibco), with 0.5 μg of mRNA and with a ratio 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) 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. IB).

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% C0 ₂ . 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 transcription 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 μΐ in OptiMEM (for scaffolds activated with 1 or 2 μg of mRNA , respectively). Next, this mRNA solution was mixed with another 25 μΐ phase of 3DFectin (OZ Biosci enees, France) in OptiMEM. For scaffolds with 1 μg of mRNA, this second phase had 2, 3 or 4 μΐ of 3DFectIN (corresponding to the 2: 1, 3: 1 or 4: 1 ratios, respectively) diluted up to 25 μΕ in OptiMEM. For scaffolds with 2 μg of mRNA, this second phase had 4, 6 or 8 μΐ of 3DFectIN (corresponding to the 2: 1, 3: 1 or 4: 1 ratios, respectively) and diluted up to 25 μΕ in OptiMEM. The mRNA and 3DFectIN phases were mixed and allowed to interact for 20 minutes. This reaction gives rise to 50 μΐ, phase 3DFectIN / mRNA.

In addition to this 3DFectIN / mRNA phase, two other solutions were prepared. A fibrinogen solution of 20 μΕ 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 μΕ 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 scaffolding The 3DFectIN / mRNA complexes are then mixed with 10 μΐ _^ OptiMEM and the resulting suspension is mixed with the fibrinogen by pipetting. This phase is then 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.

The cells can be integrated into this composition, changing the 10 μΐ _^ of OptiMEM added to the 3DFectIN / mRNA complexes by the same volume of cell suspension in OptiMEM. A number of 1, ⁵ x 10 ⁵ cells can be easily 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 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 in vitro transcription of mRNA (Figure 1), since 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 for both scaffolds (2 and 4 mg / mL fibrinogen). However, greater aggregation was observed for higher proportions of 3DFectIN / pDNA that were consistent with the larger complex size measured by correlation spectroscopy. of photon at these proportions (table 1). The smaller size of 3DFectin / mRNA complexes makes it difficult to observe their distribution in scaffolds.

Table 1: Physicochemical characterization of transfectant complexes based on pDNA and mRNA. Polydispersion size and index (PDI) obtained by photonic correlation microscopy.

Fibrin scaffolds (2 mg / mL and 4 mg / mL) were prepared as described above, and loaded with 1.5 x 10 ⁵ U87MG cells.

Fibrin scaffolds activated with mRNA were seeded with hMSC cells, and cultured for one week at 37 ° C with complete medium (90% humidity, 5% C0 ₂ ). 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 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 scaffold 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 μΐ of polyarginine or protamine (1 or 2 mg ). The solution was allowed to interact for 5 minutes at room temperature. Then, 50 μΐ of alginate (8 or 16 mg) was added to this suspension and mixed. Then, a 10 μΐ suspension was added to the solution with 1.5 x 10 ⁵ U87MG cells. The system formed a hydrogel-like structure after the addition of 70 μΐ of the first mixture over 30 μΐ of a solution of CaCl ₂ (243 or 486 mM). The hydrogels are stabilized by incubation at 37 ° C, 5% C0 ₂ 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 scaffolds, and could be cultured 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 µg 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 μΐ of OptiMEM, the same volume of this medium containing 1.5 x 10 ⁵ U87MG cells was added. 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% C0 ₂ ), 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 scaffolding preparation. Optical images of the same area were taken as a reference. The results showed a high cellular transfection in all scaffolds activated with mRNA 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 AB, Figure 12A). This result was a first indication that mRNA activated scaffolds could achieve levels of forced gene expression higher than those obtained with pDNA activated scaffolds.

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 μΐ of OptiMEM, the same volume of this medium containing 1.5 x 10 ⁵ 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 μΐ 3DFectIN / mRNA suspension in OptiMEM. After hydrogel formation, complete culture medium was added and the cells were cultured for 24, 48 h, 3 days and 7 days (37 ° C, 5% C0 ₂ ).

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 (day 0), after 3 days and after 7 days. 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 Fisher Scientific, Inc.), following the manufacturer's instructions. DNA extraction for quantification was performed by incubating the scaffolds with a solution of 100 μΐ, trypsin (2.5%) for 30 minutes, and then, by incubating the resulting suspension for 20 minutes in SDS 0, 1% and 10 minutes in Triton X-100 1% (both solutions in PBS) 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 9C). 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 the scaffolding 3: 1 can withstand proliferation and cell culture over a period of 7 days, although no additional proliferation was observed after day 3.

Example 5

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 μΐ of OptiMEM, the same volume of this medium containing 1.5 x 10 ⁵ hMSCs was added. As a negative control (C), a non-activated scaffolding was prepared for the same procedure, but using only OptiMEM instead of 50 μΐ 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% C0 ₂ ). 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 cell proliferation at short times (0, 12, 24 and 48 h) and long term (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 at short and long-term times (Figures 10E, F and 12B).

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 along with a Kozak consensus sequence to initiate translation, and an untranslated region 3 'of α-globin. 5 'UTR of β-globin was already present in the vector. A polyimidine tail or a late polyadenylation signal of SV40 in 3 'was added either for the synthesis of mRNA 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 SOX9. The structure of the plasmid used is shown in Figure 11. 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, HEK293 cells were cultured in a 6-well culture plate and were transfected with 4 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.

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 μΐ of OptiMEM, the same volume of this medium containing 1.5 x 10 ⁵ 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 μΐ 3DFectIN / mRNA suspension. After hydrogel formation, complete culture medium was added, and the cells were cultured for 24 h (37 ° C, 5% C0 ₂ ).

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 method 2 ^AACt 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 9A). 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 9B). 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 150000 times higher than the control, notably better than that achieved with pDNA (Figure 12A).

Example 7

In this experiment we tried to establish the kinetics of SOX9 expression 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 µg of mRNA or pDNA (3: 1 ratio of 3 DF ectIN / mRNA or 3 DF ectIN / pDN A) 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 h in complete medium, at 37 ° C, with 90% relative humidity and 5% C0 ₂ . Gene expression of SOX9 in the cells was assessed by qRT-PCR as described in example 6 at 12, 24 and 48 h. The maximum expression values of the gene were adjusted to 100% arbitrarily to compare the kinetics of mRNA and pDNA at the same scale. SOX9 expression values were expressed as relative to cell levels before encapsulation. The results showed a similar behavior for scaffolds of 2 and 4 mg / mL of fibrin. Maximum levels of expression were obtained at short times in the case of scaffolds activated with mRNA and at longer times for scaffolds activated with pDNA (Figure 12 C and D).

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). Scaffolds seeded with hMSCS and not activated were used as control.

After the formation of the cross-linking scaffolding (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 μg / mL ascorbic acid 2-phosphate, 40 μg / 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% C0 ₂ . After 21 days, the expression of chondrogenic differentiation marker genes Sox9, aggrecan (ACAN) and type II collagen (Col2al) 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 13).

The expression of Col2al was also measured. However, we because the gene was not detected on Scaffolds control were not able to use the calculation method 2 ^AACT. Instead, Table 2 presents the Ct of Col2al and the reference genes (GAPDH, ActB) for each sample. It is noteworthy that mRNA activated scaffolds were the only type of sample where Col2al 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: C2 values of Col2al and reference genes (GAPDH, ActB) expressed by hMSCs cultured 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 3 DF ectIN / pDN A (3: 1 ratio). N / A = Not detected.

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.

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, the mRNA activated prototypes showed similar levels to the controls with both media (Figure 14).

The analysis of the Col2al gene expression was only performed with scaffolds grown in CCM. The results confirmed that mRNA activated scaffolds were the only ones capable of producing consistent expression of this gene. In this experiment, all scaffolds with 4 mg / mL of fibrin were able to express Col2al. However, only mRNA activated scaffolds showed mRNA expression in prototypes with 2 mg / mL fibrin (Table 3). This data confirms that the scaffolding activated with mRNA 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 Col2al and the reference gene (GAPDH) expressed by 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 3 DF ectIN / pDN A (3: 1 ratio). N / A = Not detected.

Example 10

In this differentiation test, we repeat the same experiment as in example 9, but the expression of the chondrogenic marker was analyzed after 7 and 21 days in culture. Two doses of mRNA / pDNA were also tested per scaffolding (100 μΌ} 1 and 0.25 μg. All scaffolds were grown in CCM with some differences in the preparation of the medium as compared in previous experiments. No scaffolding was included. Control and gene expression levels at the end of the experiment were compared with hMSCs levels before encapsulation.

At 7 days, SOX9 expression was regulated in all activated scaffolds: scaffolds activated with ^ g of DNA produced the highest positive regulation of genes followed by scaffolds activated with ^ g RNA. The lowest expression of SOX9 was produced by 0.25 μg DNA and 0.25 μg RNA although its expression levels were still higher than those observed in standard cultures of micromassage (Pellet). The highest ACAN expression at this time was obtained for both RNA-activated scaffolds, with no statistical differences between them. ACAN levels for DNA scaffolds and cell pellets were many lower than RNA scaffolds. Finally, in relation to the expression of Col2Al, RNA scaffolds obtained the highest gene expression again, followed by DNA scaffolds, and finally micromassage cultures, with a very low expression in comparison. There were no statistical differences between 2 and 4 mg / mL scaffolds at this point (Figure 15, left).

At 21 days, the expression of SOX9 did not change when compared to the expression found at 7 days, except for scaffolds activated with 0.25 μg of DNA, which reached the same levels of expression as scaffolds of 1 μg DNA . Differences between 2 and 4 mg / mL scaffolds of fibrin appeared at this point: 2 mg / mL scaffolds induced ACAN and Col2Al expression higher than 4 mg / mL for almost all conditions evaluated. In fact, the 4 mg / mL scaffolds did not induce detectable levels of Col2Al for most conditions. The RNA / DNA doses did not have a significant impact on the induction of ACAN, achieving the same levels of ACAN with both doses. In contrast, scaffolds activated with 1 μg of RNA and 0.25 μg of DNA were seeded to promote greater expression of Col21Al (Figure 15, right).

In addition, these results confirm the ability of SOX9 activated scaffolds to induce the expression of key chondrogenic markers ACAN and Col21Al.

Example 11

This example constitutes an experiment analogous to that presented in Example 7. This time, we tried to determine the kinetics of MyoD expression on the transfection of hMSCs within a scaffold activated with MyoD-mRNA. Scaffolds with 2 and 4 mg / mL of fibrin were activated with 0.5 μg of MyoD mRNA (3: 1 3DFectIN / mRNA) following the procedure described in example 1, but using mRNA encoding MyoD (Miltenvi Biotec, Germany). This is a chemically modified commercial mRNA, resistant to degradation by RNAsas, that induces myogenic differentiation to fibroblasts and hMSCs after transfection. The scaffolds were grown for 48 hours in a complete medium, at 37 ° C, with 90% humidity and 5% C02. The forced expression of MyoD in hMSCs were followed by quantitative real-time PCR at 12, 24 and 48 h. As compared in the experiment described in Example 7, in this experiment we also evaluated the expression kinetics of myogenic markers MyoG and CDH15, genes that positively regulate once myogenic cascade differentiation has begun. Figure 16 shows a very high positive regulation of the transfected transcription factor, MyoD, and the MyoG myogenic transcription factor, in the hMSCs encapsulated in the scaffolds of the invention. This positive regulation is observed at 12 h post-transfection. More importantly, the expression of an early myogenic gene, CDH15, was also detected at 12 h post-transfection. No significant differences were found in the expression of myogenic markers between the 2 and 4 mg / mL fibrin scaffolds. These results suggest the induction of myogenic differentiation in hMSCs encapsulated with fibrin scaffolds activated with MyoD mRNA. Example 12

This experiment is related to the myogenic differentiation of hMSCs encapsulated in fibrin gels activated with MyoD mRNA. 2 and 4 mg / mL fibrin gels (200 μΕ) were activated with 0.5 μg of MyoD mRNA and hMSCs were encapsulated within them. The gels were prepared as in example 1 but the amount of all components was increased 2 times and the gel was prepared at twice the volume (200 instead of 100 µΕ). Complete growth medium (a-MEM + 10% Fetal Bovine Serum, 1% Penicillin-streptomycin and 10 ng / mL FGF-2) was added on the gels during the first 24 h to promote transfection. The day after transfection, the medium was changed to DMEM serum (DMEM high glucose + 1% Horse Serum and 1% Penicillin-streptomycin) to promote differentiation. The cells were cultured for 14 days, changing the medium every 3 days, and removed at the end of the experiment to allow quantification of myogenic marker expression by quantitative real-time PCR. As shown in Figure 17, the expression of MyoD and MyoG was positively regulated in hMSCs encapsulated within fibrin scaffolds activated with MyoD. MyoG expression was higher in scaffolds of 4 mg / mL compared to 2 mg / mL.

Claims

1. Biodegradable scaffold comprising a biodegradable polymer, an isolated mRNA encoding a transcription factor and a transfection agent.

2. Scaffolding according to claim 1, further comprising cells selected from the group consisting of primary cells and immortalized cell lines.

3. Scaffolding according to any 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, NeuroDl, c-Myc, Klf4, Nanog, Oct4, SOX2, C / ΕΒΡ-β, PPAR-γ, Brn2, Lmxla, Nurrl, Mashl,

Mytll 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, NeuroDl,

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. Scaffolding, according to any of claims 1-11, for use according to claim 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 further 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 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).

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.

Biodegradable scaffolding obtained by the method according to claims 22-23 or by the method according to claim 24.

Use of a scaffold as described in any of claims 1-11, as an in vitro differentiation reagent or as a cosmetic implant.