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EP-4403190-B1 - SILICA PARTICLES FOR ENCAPSULATING NUCLEIC ACIDS

EP4403190B1EP 4403190 B1EP4403190 B1EP 4403190B1EP-4403190-B1

Inventors

  • LÓPEZ FANARRAGA, Mónica
  • MARÍN CABA, Laura

Dates

Publication Date
20260506
Application Date
20220805

Claims (15)

  1. A method for producing a silica particle characterised in that it comprises at least one nucleic acid encapsulated inside said particle, comprising the following steps: (i) dissolving ethanol (EtOH) in a molar concentration of between 8 and 12 mol/L (M), ammonium hydroxide (NH 4 OH) in a molar concentration of between 0.1 and 3.5 M and a solution of nucleic acids (NAs) in a concentration of between 0.01 and 0.50 µg/µl; (ii) adding to the solution obtained in (i) tetra-ethyl-orthosilicate (TEOS) in a molar concentration of between 0.10 and 0.40M; and (iii) stirring the solution obtained in (ii) between 100 and 1000 rpm for 1 to 3 hours at a temperature between 5 and 40°C, obtaining a colloidal suspension of silica particles comprising the nucleic acids encapsulated therein.
  2. The method according to claim 1, wherein the solvent of the nucleic acid solution of step (i) is deionised water (dH 2 O) between 2 and 18M, Milli-Q water, 16% v/v Tris EDTA (TE) buffer or ethanol, preferably dH 2 O or Milli-Q water.
  3. The method according to claim 1 or 2, wherein the concentration of the nucleic acid solution of step (i) is between 0.15 and 0.35 µg/µl, preferably 0.225 µg/µl.
  4. The method according to any one of claims 1 to 3, wherein in step (i) the molar concentration of EtOH is between 9.7M and 10M, that of NH 4 OH is 0.34 or 1.06 or 2.02M and that of dH 2 O is 9.77M and/or in step (ii) the molar concentration of TEOS is 0.25M.
  5. The method according to any one of claims 1 to 4 further comprising a step (iv), wherein the particles obtained in step (iii) are washed with ethanol for 1 to 5 cycles, wherein each cycle comprises (i) centrifugation between 3500 rpm and 8000 rpm for 1 to 15 minutes and (ii) redispersion with ethanol and stirring for 20 to 80 seconds between 100 and 300 rpm.
  6. The method according to any one of claims 1 to 5, wherein step (iii) is performed for 2 hours.
  7. The method according to any one of claims 1 to 6, wherein step (iii) is performed at between 15°C and 30°C, preferably at 22°C.
  8. The method according to any one of claims 1 to 7 further comprising a step (v), wherein the surface of the silica particle is functionalised with at least one functional group that is selected from the list consisting of: peptides, proteins or protein fragments, nucleotides, enzymes, antibodies, dendrimeric nanoantibodies, molecules targeting receptors and aptamers.
  9. A silica particle characterised in that it comprises at least one nucleic acid encapsulated inside said particle, obtained by the method according to any one of claims1 to 8.
  10. In vitro use of the particle according to of claim 9 in gene transfer or gene editing.
  11. In vitro use of the particle according to any of claims 9 for cell marking.
  12. The particle according to claim 9 for use as a medicinal product.
  13. The particle for use according to claim 12, wherein the medicinal product is a vaccine.
  14. The particle according to claim 9 for use as a medicinal product for protein/enzyme replacement therapy.
  15. Use of the particle according to claim 9 as a contrast agent for the observation of cells.

Description

The present invention belongs to the technical field of nanomedicine and nanotechnology, specifically to particles for gene transfer. The present invention relates to silica particles comprising nucleic acids encapsulated inside the same. Furthermore, the present invention relates to a method for producing said particles and the uses thereof in gene transfer or cell marking and as a medicinal product, specifically as a medicinal product for protein/enzyme replacement therapy and as an immunisation system. BACKGROUND OF THE INVENTION At present, there is a growing interest in the development of systems that allow the vectorisation or distribution of nucleic acids. Among the applications of these systems, various therapeutic approaches stand out, such as the use thereof in the treatment of genetic diseases, in which gene transfer makes it possible to correct or replace genes that cause certain diseases in the tissues and cells of patients. Another therapeutic approach based on the distribution of nucleic acids encompasses the production of vaccines, for example, the SARS-CoV-2 vaccine against COVID19, which is based on a system for the gene transfer of lipid particles that encapsulate mRNA encoding the 'S' protein of the virus backbone, which is critical for its binding to human cells. However, the distribution of biologically active agents, such as nucleic acids, to cells and tissues, presents associated technical difficulties, among others, its stability having a limited duration. At present, methods used for nucleic acid distribution include viral and non-viral vectors. Viral vectors have high efficiency in the distribution of genetic material to cells, being systems widely used in biomedicine. Among them, the use of vectors derived from adeno-associated viruses especially stands out, which is widely used in gene therapy due to its biosafety, low toxicity and selective tropism. Nevertheless, viral vectors have problems for their clinical application, such as limitations in the size of the gene to be transduced, lack of specificity, immunogenicity and possible oncogenic effects. In turn, the use of non-viral vectors overcomes many of these limitations, with there being a growing interest in systems for gene transfer of this type. The most commonly used systems for the distribution of nucleic acids comprise positively charged carrier molecules that neutralise the negative charge of nucleic acids. Among them, cationic liposomes, which do not present limitations in the size of the DNA to be transduced, have low immunogenicity and toxicity, and can be managed in vivo, intravenously, with the particles generally being retained mechanically in natural filters, such as the lungs and liver, or intramuscularly, stand out. Nevertheless, their low stability and rapid degradation in the body pose barriers to their logistical distribution and clinical implementation. New strategies have been developed in fields such as nanomedicine for the design and production of new systems capable of vectorising nucleic acids with greater potential. Nanomedicine generally refers to the medical application of nanotechnology, an interdisciplinary field that exploits the distinctive characteristics of materials, the molecular and cellular size range of which, as well as the versatility thereof, make them systems of interest for the distribution of drugs and nucleic acids. Specifically, the particles show outstanding properties as systems for gene transfer, among others, the ability to target specific tissues or cells, protection against nuclease degradation, improvement of DNA stability and increased transformation efficiency and safety. In fact, they are increasingly more accepted from a clinical point of view as they are considered one of the most promising vectorisation systems due to their biological behaviour and versatility in shapes and sizes (Chen et al., 2016, Molecular Therapy - Methods & Clinical Development, 3, 16023). There are numerous examples of particles capable of vectorising or distributing different therapeutic compounds and nucleic acids for use in gene transfer (Dizaj et al., 2014). Different types of particles have been developed for the distribution of nucleic acids: polymeric types (such as micelles, nanogels, linear polymers, dendrimers, polymersomes), inorganic types (such as gold particles, quantum dots, silica particles, carbon-based nanomaterials), liposomes, exosomes and nanostructure-based DNA carriers, as well as hybrids that integrate the advantages of different materials. All of these systems are primary structures that can in turn be combined with organic or inorganic ligands on the surface thereof to improve targeting or prevent capture by the macrophages of the mononuclear phagocytic system. Nevertheless, most of these systems are very unstable and, to that end, must be prepared minutes before use or stored in extreme cold conditions, which complicates the logistics thereof at an industrial level an