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WO2017191345A1 - Échafaudage biodégradable comprenant un arn messager - Google Patents

Échafaudage biodégradable comprenant un arn messager Download PDF

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Publication number
WO2017191345A1
WO2017191345A1 PCT/ES2017/070262 ES2017070262W WO2017191345A1 WO 2017191345 A1 WO2017191345 A1 WO 2017191345A1 ES 2017070262 W ES2017070262 W ES 2017070262W WO 2017191345 A1 WO2017191345 A1 WO 2017191345A1
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mrna
scaffolds
scaffolding
cells
activated
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PCT/ES2017/070262
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Spanish (es)
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Marcos Garcia Fuentes
Adriana Martinez Ledo
Anxo VIDAL FIGUEROA
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Universidade De Santiago De Compostela
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Publication of WO2017191345A1 publication Critical patent/WO2017191345A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor

Definitions

  • This invention relates to a biodegradable scaffold comprising messenger RNA. More particularly, it refers to scaffolding, the method of preparation and use thereof.
  • TFs Transcription factors
  • TFs Transcription factors
  • pDNA plasmid DNA
  • RNA messenger RNA
  • Lu ⁇ 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 *) (Lu ⁇ , 2013, Cell Research 23, 1172-1186).
  • this technology cannot be applied in therapy since Matrigel is based on tumor proteins.
  • 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).
  • 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.
  • the 3D structure of the scaffold must be able to house adhered cells and, if necessary, allow proliferation.
  • 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.
  • this forced expression of a transcription factor induces changes in the expression levels of other genes, indicating a clear biological effect.
  • this scaffolding has the advantage of avoiding security problems, in particular it avoids viral vectors.
  • 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.
  • biodegradable scaffold of the invention for use as a medicament.
  • 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.
  • the scaffolding Biodegradable of the invention is for use in the treatment of a cartilage defect, muscle damage or nerve tissue damage.
  • the invention relates to the use of the biodegradable scaffold of the invention to prepare a medicament.
  • 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.
  • 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
  • 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
  • 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 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.
  • FIG. 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 ⁇ g 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 ⁇ g 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 ⁇ g of mRNA / pDNA and the 3 DF ectIN: mRN A / pDN A 3: 1 ratio ( ⁇ .: ⁇ £). 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 ⁇ -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.
  • FIG. 10 (A) Transfection of human mesenchymal stem cells (hMSCs) in fibrin scaffolds activated with mRNA complexes at 24 h (1 ⁇ g 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).
  • FIG. 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 ⁇ -actin expression.
  • 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).
  • ICM incomplete chondrogenic medium
  • CCM complete chondrogenic medium
  • 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.
  • FIG. 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 ⁇ -actin expression and compared to levels in hMSCs before encapsulation. Data are shown as mean and standard deviation of three replicates in one experiment.
  • 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).
  • 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).
  • pDNA plasmid DNA
  • 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).
  • Staffolding 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 ⁇ ) 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.
  • 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.
  • inorganic materials 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.
  • biodegradable inorganic materials are, but are not limited to, ceramic materials such as apatites, for example hydroxyapatite, and porous silicon.
  • 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.
  • useful polyesters include, but are not limited to, polyglycolic, polylactic, poly (lactic-co-glycolic), polydioxanone, polycaprolactone and poly (trimethylene carbonate).
  • 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, ⁇ -1-methylpseudo-uridine and pseudo-uridine.
  • mRNA messenger RNA
  • mRNA messenger RNA
  • TF transcription factor
  • the mR A used in this invention is optimized for translation in eu karyotic cells
  • 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.
  • the mRNA of the invention is preferably synthesized by in vitro transcription reactions, from a plasmid template, or alternatively, by solid phase chemical synthesis.
  • 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.
  • poly (A) after in vitro transcription or during the elongation step of the transcription itself in vitro.
  • 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.
  • ARCA anti-reverse analogue Cap
  • 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 ⁇ and ⁇ -globin proteins.
  • 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, ⁇ and ⁇ -globin.
  • another particular embodiment relates to an mRNA having a 3 'untranslated region, preferably an untranslated region of a highly translated gene such as ⁇ and ⁇ -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.
  • TF transcription factor
  • 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.
  • TF databases available for searching sequences and functions of TFs, for example, JASPAR (http: // j aspar. Enereg.net).
  • JASPAR http: // j aspar. Enereg.net.
  • the mRNA encodes a chondrogenic transcription factor.
  • the encoded TFs activate genetic programs responsible for cell differentiation or dedifferentiation.
  • mRN A codes for a transcription factor selected from the group consisting of SOX9, MyoD, NeuroDl, c-Myc, Klf4, Nanog, Oct4, SOX2, C / ⁇ - ⁇ , PPAR- ⁇ , Brn2, Lmxla, Nurrl, Mashl, Mytll and NeuroG2.
  • the mRNA encodes for the transcription factors selected from SOX9, MyoD, NeuroDl, SOX2, Oct4, Klf4 and c-Myc.
  • the TF is SOX9.
  • 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.
  • 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.
  • the transfection agent is selected from cationic lipids, cationic polymers, and a calcium phosphate salt.
  • the transfection agent 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.
  • the transfection agent is a polymeric condensing agent.
  • the condensing polymeric agent is polyarginine.
  • the condensing polymeric agent is poloxamine.
  • condensing polymeric agent is a cationic polyphosphazene.
  • the biodegradable anadamium further comprises cells.
  • 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.
  • 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.
  • the scaffold of the invention is clinically useful since it is biodegradable and biocompatible.
  • 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.
  • the primary cells are fibroblasts or chondrocytes.
  • the invention is directed to a pharmaceutical composition comprising a scaffold as described above.
  • the pharmaceutical composition further comprises pharmaceutically acceptable carriers.
  • the pharmaceutical composition further comprises at least one additional active pharmaceutical ingredient.
  • 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.
  • the pharmaceutical composition is an injectable solution, suspension, hydrogel or a solid porous matrix.
  • the pharmaceutical composition is for use as a vaccine.
  • 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,
  • the invention relates to an alternative method for preparing the scaffold of the invention as described above, comprising:
  • the biodegradable polymer is selected from fibrin, alginate and mixtures thereof.
  • 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.
  • the coagulation of step (iii) is carried out by the addition of a coagulation agent.
  • the coagulation agent is selected from thrombin, calcium salt and polyphosphate salt.
  • the thrombin range used is between 0.2 U and 1.2 U per mg of the fibrinogen used.
  • the interaction of the scaffold and the mRNA / transfection agent in step (iii) can be reinforced by drying or lyophilization of the system.
  • the invention relates to a biodegradable scaffold obtained by the method described above.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ of OptiMEM (Gibco). The lipoplexes were prepared next in 50 ⁇ of OptiMEM (Gibco), with 0.5 ⁇ g 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 ⁇ g of mRNA and 3DFectIN as a transfection agent First, 1 ⁇ g or 2 ⁇ g of mRNA were diluted up to 25 ⁇ in OptiMEM (for scaffolds activated with 1 or 2 ⁇ g of mRNA , respectively). Next, this mRNA solution was mixed with another 25 ⁇ phase of 3DFectin (OZ Biosci enees, France) in OptiMEM. For scaffolds with 1 ⁇ g of mRNA, this second phase had 2, 3 or 4 ⁇ of 3DFectIN (corresponding to the 2: 1, 3: 1 or 4: 1 ratios, respectively) diluted up to 25 ⁇ in OptiMEM.
  • 3DFectin OZ Biosci enees, France
  • this second phase had 4, 6 or 8 ⁇ of 3DFectIN (corresponding to the 2: 1, 3: 1 or 4: 1 ratios, respectively) and diluted up to 25 ⁇ in OptiMEM.
  • the mRNA and 3DFectIN phases were mixed and allowed to interact for 20 minutes. This reaction gives rise to 50 ⁇ , phase 3DFectIN / mRNA.
  • a fibrinogen solution of 20 ⁇ 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 ⁇ 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 ⁇ ⁇ 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 ⁇ ⁇ 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.
  • 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.
  • hMSC Human mesenchymal stem cells
  • 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.
  • 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.
  • mRNA encoding YFP was used, prepared as described in example 1.
  • 1 or 2 ⁇ g of mRNA was mixed with a solution of 10 ⁇ of polyarginine or protamine (1 or 2 mg ). The solution was allowed to interact for 5 minutes at room temperature. Then, 50 ⁇ of alginate (8 or 16 mg) was added to this suspension and mixed. Then, a 10 ⁇ 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 ⁇ of the first mixture over 30 ⁇ 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.
  • Fibrin scaffolds (4 mg / mL) activated with 3DFectIN / mRNA (1 or 2 ⁇ g 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 ⁇ of OptiMEM, the same volume of this medium containing 1.5 x 10 5 U87MG cells was added.
  • a fibrin scaffold (4 mg / mL) activated with 3DFectin / pDNA (1 ⁇ g 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.
  • Fibrin scaffolds (4 mg / mL) activated with 3DFectIN / mRNA (1 ⁇ g 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 ⁇ of OptiMEM, the same volume of this medium containing 1.5 x 10 5 U87MG cells was added.
  • a negative control a non-activated scaffold was prepared using the same procedure, but adding only OptiMEM instead of the 50 ⁇ 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 ⁇ , 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.
  • Fibrin scaffolds (2 and 4 mg / mL) activated with mRNA (1 ⁇ g of mRNA, 3: 1 ratio) encoding YFP were prepared as described in example 1, but before adding thrombin, instead of 10 ⁇ of OptiMEM, the same volume of this medium containing 1.5 x 10 5 hMSCs was added.
  • a negative control (C) a non-activated scaffolding was prepared for the same procedure, but using only OptiMEM instead of 50 ⁇ 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.
  • 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 ⁇ -globin. 5 'UTR of ⁇ -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.
  • HEK293 cells were cultured in a 6-well culture plate and were transfected with 4 ⁇ g 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 ⁇ g 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 ⁇ of OptiMEM, the same volume of this medium containing 1.5 x 10 5 U87MG cells was added.
  • 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 ⁇ 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 ⁇ -actin genes (Taqman, Thermo Fisher Scientific, Inc.). Relative expression was evaluated using method 2 AACt was used to evaluate relative expression, using GAPDH and ⁇ -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).
  • scaffolds with 2 and 4 mg / mL of fibrin and activated with 1 ⁇ g of mRNA or 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 h in complete medium, at 37 ° C, with 90% relative humidity and 5% C0 2 .
  • 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.
  • hMSCs 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.
  • 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 ⁇ g / mL ascorbic acid 2-phosphate, 40 ⁇ g / 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.
  • 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).
  • Col2al 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).
  • the expression of SOX9 did not change when compared to the expression found at 7 days, except for scaffolds activated with 0.25 ⁇ g of DNA, which reached the same levels of expression as scaffolds of 1 ⁇ g 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 ⁇ g of RNA and 0.25 ⁇ g of DNA were seeded to promote greater expression of Col21Al ( Figure 15, right).
  • 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 ⁇ g 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.
  • 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.
  • 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 ⁇ ) were activated with 0.5 ⁇ g 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 ⁇ ).
  • 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.
  • DMEM serum DMEM high glucose + 1% Horse Serum and 1% Penicillin-streptomycin
  • 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.
  • 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.

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Abstract

Échafaudage biodégradable comprenant un ARN messager. L'invention concerne un échafaudage biodégradable comprenant un ARN messager. Plus particulièrement, elle concerne cet échafaudage, son procédé de préparation et ses utilisations. Un échafaudage biodégradable comprend un polymère biodégradable, un ARNm isolé codant pour un facteur de transcription et des agents de transfection.
PCT/ES2017/070262 2016-05-02 2017-04-28 Échafaudage biodégradable comprenant un arn messager WO2017191345A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2809348A1 (es) * 2020-10-27 2021-03-03 Univ Santiago Compostela Polimeros para terapia genica

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070031465A1 (en) * 2003-10-15 2007-02-08 Japan Science And Technology Agency Implant for regenerating bone or cartilage with the use of transcriptional factor
US20100189794A1 (en) * 2009-01-05 2010-07-29 Cornell University Nucleic acid hydrogel via rolling circle amplification
US20140341870A1 (en) * 2011-12-29 2014-11-20 "Nextgen" Company Limited Biocomposite for regeneration of injured tissue and organs, a kit for making the biocomposite, a method of making the biocomposite and a method of treating injuries
US20150010501A1 (en) * 2012-03-27 2015-01-08 Parcell Laboratories, Llc Intervertebral disc repair compositions and methods

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070031465A1 (en) * 2003-10-15 2007-02-08 Japan Science And Technology Agency Implant for regenerating bone or cartilage with the use of transcriptional factor
US20100189794A1 (en) * 2009-01-05 2010-07-29 Cornell University Nucleic acid hydrogel via rolling circle amplification
US20140341870A1 (en) * 2011-12-29 2014-11-20 "Nextgen" Company Limited Biocomposite for regeneration of injured tissue and organs, a kit for making the biocomposite, a method of making the biocomposite and a method of treating injuries
US20150010501A1 (en) * 2012-03-27 2015-01-08 Parcell Laboratories, Llc Intervertebral disc repair compositions and methods

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HASTINGS CONN L ET AL.: "Drug and cell delivery for cardiac regeneration", ADVANCED DRUG DELIVERY REVIEWS NETHERLANDS, vol. 84, April 2015 (2015-04-01), pages 85 - 106, XP029224307, ISSN: 1872-8294 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2809348A1 (es) * 2020-10-27 2021-03-03 Univ Santiago Compostela Polimeros para terapia genica
WO2022090598A1 (fr) * 2020-10-27 2022-05-05 Universidade De Santiago De Compostela Polymères pour thérapie génique

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