Nucleic Acid Vector Compositions

Abstract

A nucleic acid vector composition comprising: one or more enzymes and / or one or more fragments thereof, a nucleic acid, one or more particles comprising hydrolyzable silicon, and one or more lipids, as well as related products, medical uses, and methods.

Description

[Technical field]

[0001] The present invention relates to nucleic acid vector compositions, as well as related methods and products. The nucleic acid vector compositions are particularly useful, but not limited to, in the field of pharmaceuticals. The compositions of the present invention are particularly useful for protecting nucleic acids from enzymatic degradation. [Background technology]

[0002] There is a need for improved excipients for bioactive agents. The provision of suitable excipients is required for the full translation of advances in biomedical research into effective, safe and cost-effective therapeutics.

[0003] As an illustrative example, nucleic acids such as RNA have been proposed as therapeutic agents. Nucleic acid delivery for therapeutic or other purposes is well known, particularly for the treatment of diseases such as cystic fibrosis and certain cancers, and mRNA has recently been used in an effective vaccine against SARS-CoV-2. The term "gene therapy" may be used to refer to the delivery of a gene or part of a gene to a cell to produce a therapeutic effect, such as by repairing, reconstructing, or compensating for defective genetic material. More broadly, the term "nucleic acid delivery" may be used to refer to any introduction of nucleic acid material into a target cell. As non-limiting examples, nucleic acid delivery includes mRNA vaccination and the production of commercially useful proteins in so-called cell factories.

[0004] Delivery systems for delivering nucleic acids into cells fall into three broad classes: (i) A class involved in direct injection of naked nucleic acid; (ii) A class in which a virus or a genetically modified virus is used; (iii) a class that uses a non-viral delivery agent; It is divided into:

[0005] Each has its own advantages and disadvantages. Virus as a delivery agent has the advantages of high efficiency and high cell selectivity, but has the disadvantages of toxicity and induction of inflammatory response, and is not very suitable for delivery of large DNA fragments.Therefore, mRNA vaccines can advantageously comprise injectable naked mRNA or non-viral delivery systems, such as polyplex vectors or lipid nanoparticle vectors.

[0006] Unfortunately, it has been observed that non-viral nucleic acid delivery systems have low transfection efficiency. Non-viral nucleic acid delivery systems are based on the compression of nucleic acids into nanometer particles by electrostatic interactions between the negatively charged phosphate backbone of the nucleic acid and charged polymers, typically cationic lipids and / or peptides (Erbacher, P. et al, Gene Therapy, 1999, 6, 138-145). The mechanism by which these species are introduced into cells is proposed to involve endocytosis of the intact complex, where the complex formed between the nucleic acid and the lipid binds to the cell surface and then enters the cell by endocytosis. The complex then remains localized in vesicles or endosomes for some time, and the nucleic acid component is released into the cytoplasm.

[0007] The polymer components of the non-viral delivery system associate electrostatically to form a vector complex. The lipid components shield both the nucleic acid and, to some extent, any peptide component(s) from degradation, endosomes, or otherwise. For example, the lipid components may form a lipid bilayer shell that encapsulates the other components of the delivery system, including the nucleic acid molecule. Cationic lipids for such use were developed by Felgner in the late 1980s and reported in Proc. Natl. Acad. Sci. USA 84, 7413-7417, 1987; and U.S. Patent No. 5,264,618. Felgner developed the currently commercially available cationic liposomes known under the trademark "Lipofectin". "Lipofectin" liposomes are spherical vesicles with a lipid bilayer of the cationic lipid DOTMA (2,3-dioleyloxypropyl-1-trimethylammonium) and the neutral phospholipid DOPE (phosphatidylethanolamine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) in a 1:1 ratio. Since then, various other cationic liposome formulations have been devised, most of which combine synthetic cationic lipids with neutral lipids. In addition to DOTMA analogues, mention may also be made of complex alkylamines / alkylamides, cholesterol derivatives such as DC-cholesterol, as well as synthetic derivatives of dipalmitol, phosphatidylethanolamine, glutamate, imidazole, and phosphonate. However, cationic vector systems are highly variable in their transfection efficiency in the presence of serum, which clearly affects their potential use for in vivo gene therapy and / or nucleic acid delivery.

[0008] Peptide components for use in such complexes typically have two functional groups: a "head group" that contains a cell surface receptor sequence, e.g., an integrin sequence, recognition sequence, and a "tail" that can be non-covalently bound to a nucleic acid. Peptide components can be designed to be cell type specific or cell surface receptor specific. For example, a degree of integrin specificity can confer a degree of cell specificity to the complex. Specificity can result from targeting to cell surface receptors (e.g., integrin receptors) and can achieve transfection efficiencies comparable to some adenoviral vectors (Jenkins et al. Gene Therapy 7, 393-400, 2000).

[0009] So far, non-viral delivery of messenger RNA (mRNA) to cells has been particularly problematic and limited due to the lack of efficient vectors. Attempts to deliver mRNA using known non-viral vehicles that have been successfully used with DNA or siRNA have resulted in suboptimal levels of protein expression. Furthermore, known non-viral vehicles have poor storage stability when packaged with mRNA. Overcoming the lipid bilayer to deliver RNA to cells has remained a major obstacle to the widespread development of RNA therapeutics.

[0010] Therefore, there is a need for vectors specifically tailored for the delivery of mRNA that deliver high levels of mRNA to cells and result in good levels of protein expression.There is also a need for compositions tailored for the delivery of mRNA that maintain good stability upon storage, particularly mRNA delivery complexes that maintain their structure and functionality upon storage at moderate temperatures.Similar considerations apply to the delivery of other nucleic acid therapeutics, such as plasmid-derived DNA, in that there are challenges in maintaining good storage stability at moderate temperatures, such as temperatures of about -5°C to about 25°C.

[0011] Some mRNA vaccines against SARS-CoV-2, including the Pfizer and BioNTech vaccine BNT162b2 ("Comirnaty") and the Moderna CX-024414 vaccine, require cold chain storage and transportation. This limits access to the vaccine in low-income countries and adds cost and logistical complexity in all markets. It would be advantageous if the vaccine could be stored and transported at standard refrigerator temperatures (approximately -5°C) or room temperature (approximately 20°C). It would also be beneficial if the vaccine could withstand higher temperatures (e.g., 30°C, 40°C, or 50°C) for storage or at least in the very short term for transport and distribution purposes.

[0012] Maintaining the stability of nucleic acids (e.g., mRNA or DNA) in injectable compositions, such as vaccine compositions, by low temperature, and also poses logistical challenges, has the technical limitation that the nucleic acid must be thawed before injection, and must remain stable in the body for a sufficient time after injection to exhibit sufficient biological activity. This may require maintaining stability during transit to the body environment and / or during escape from endosomal compartments. In vivo stability must also be maintained long enough for sufficient translation into protein to occur.

[0013] Nucleic acids (and especially mRNA) are susceptible to degradation, especially enzymatic degradation. Degradation can be slowed down by low temperature and / or lyophilization of the nucleic acid (e.g., mRNA), but each of these options has drawbacks. Short lengths of nucleic acid can be produced in a fully synthetic production environment that can be kept free of degradative enzymes. Such short nucleic acids can be useful for certain therapeutic applications, such as siRNA. Longer nucleic acids cannot be produced cost-effectively in a fully synthetic production environment, and are therefore typically produced in a production environment that includes biologically sourced material. For example, mRNA can be produced by in vitro transcription using a transcription enzyme derived from biological material (biologically sourced material is either material produced in a cell culture-based system or material produced in vitro using synthetic enzymes (e.g., enzymes produced in a cell culture-based system), specifically referring to nucleic acids). DNA (e.g., plasmid DNA or pDNA) can be extracted from a culture of cells. The use of biological material increases the possibility that biologically sourced degradative enzymes are unintentionally present in the nucleic acid preparation. This may require extensive purification processes (which are costly and result in loss of yield) and it is not possible or cost-effective to completely remove all degradative enzymes from biologically derived nucleic acid preparations, such as preparations of in vitro transcribed (IVT) mRNA or preparations of DNA such as pDNA. Furthermore, during entry into the body and / or escape from endosomal compartments, nucleic acids are typically exposed to physiological and intracellular conditions, including contact with degradative enzymes.

[0014] Encapsulation with lipids or condensation with peptides or cationic polymers (such as protamine) have been used in the prior art to protect nucleic acids (e.g., mRNA for gene therapy or vaccination) from degradation. Condensation with peptides or other polymers relies on the peptide or polymer remaining intact and retaining its charge. There is a need for improved additives that increase the stability of nucleic acids (e.g., mRNA) and improve resistance to enzymatic degradation, especially without the need to freeze the nucleic acid preparation. There is also a need for improved additives to increase the stability of multiple components of compositions that include biologically derived nucleic acids and lipids, and / or peptides or other polymers. Summary of the Invention

[0015] The present invention is based on the recognition that nucleic acids such as mRNA or DNA (e.g., in vitro transcribed mRNA or plasmid DNA) can be stabilized by using hydrolysable silicon in the presence of lipids to protect the nucleic acid from enzymatic degradation.

[0016] This may include, for example, stabilizing the nucleic acid in a pharmaceutical composition (e.g., a vaccine) during storage before the pharmaceutical composition is administered to a subject in need of the pharmaceutical composition, This may include stabilizing the nucleic acid during transit into the body and / or escape from endosomal compartments (when the nucleic acid is exposed to physiological conditions that typically include contact with degradative enzymes).

[0017] Thus, according to a first aspect of the invention there is provided a nucleic acid vector composition comprising: one or more enzymes and / or one or more fragments thereof, nucleic acid, one or more particles comprising hydrolyzable silicon, and One or more lipids A nucleic acid vector composition is provided, comprising:

[0018] The one or more particles comprising hydrolyzable silicon may remove or sequester water molecules, thereby preventing the one or more enzymes from decomposing nucleic acid, based on the fact that enzymes require an aqueous environment to catalyze the degradation of nucleic acid. Optionally, the one or more particles remove water molecules by reaction of the water molecules with silicon or silicon-containing moieties on the surface of the one or more particles. Optionally, the one or more particles sequester water molecules by trapping them in pores present on the surface of the one or more particles. Optionally, the one or more particles sequester water molecules in both of these ways. Thus, the water molecules available to react with nucleic acid in an enzyme-catalyzed reaction may be reduced or eliminated. Thus, overall, the one or more particles comprising hydrolyzable silicon increase the stability of nucleic acid compared to a composition without the one or more particles.

[0019] The one or more enzymes may comprise one or more enzymes for forming an enzyme-substrate complex with the nucleic acid. In the composition of the first aspect of the invention, the enzyme-substrate complex may be formed, but due to the reduced availability of water molecules, the steps required for the enzyme-catalyzed reaction may not occur or may occur at a reduced rate.

[0020] The one or more enzymes may optionally include one or more nucleases. The one or more enzymes may optionally include one or more polymerases. Thus, in certain embodiments, the one or more enzymes may optionally include one or more RNA polymerases, which may be selected from one or more of T7, SP6, and T3 RNA polymerases. The one or more enzymes may optionally include both one or more nucleases and one or more polymerases.

[0021] As used herein, the term nuclease refers to an enzyme that can cleave the phosphodiester bond between nucleotides in a nucleic acid molecule. Nucleases can perform single-stranded cleavage of a target molecule. Nucleases can perform double-stranded cleavage of a target molecule. As used herein, the term exonuclease refers to an enzyme that can digest a nucleic acid molecule by cleaving nucleotides one by one starting from the end (exo) of a polynucleotide chain. As used herein, the term endonuclease refers to an enzyme that can digest a nucleic acid molecule by cleaving nucleotides one by one starting from the middle (endo) of a polynucleotide chain. A nuclease may be a deoxyribonuclease, also known as DNase, that acts on DNA. A nuclease may be a ribonuclease, also known as RNase, that acts on RNA.

[0022] In general terms, a polymerase is an enzyme that catalyzes the formation of a polymer. Thus, the term DNA polymerase as used herein refers to an enzyme that catalyzes the formation of a DNA polymer. As used herein, an RNA polymerase is an enzyme that catalyzes the formation of an RNA polymer. Nucleic acid molecules have a sugar-phosphate backbone, which is typically exposed to the hydrophilic surface of the molecule and is therefore the part of the molecule that is particularly vulnerable to hydrolysis. Nucleophilic cleavage of the phosphodiester bond in the backbone can occur, for example, via intermolecular reaction (with a nucleophile present in the solution, e.g., H2O) and / or intramolecular attack (such as by the 2'-OH group in RNA or other nucleophilic groups). Other modes of degradation are also possible, for example, by oxidation of one or more nucleobases and / or one or more sugar moieties.

[0023] In the nucleic acid vector composition according to the first aspect of the invention, the weight ratio of the enzyme and / or fragment thereof to the nucleic acid is optionally 1:1×10 12 ~1:1, e.g., 1:1×10 11 ~1:1, 1:1×10 10 ~1:1, 1:1×10 9~1:1, 1:1×10 7 ~1:1, 1:1×10 6 ~1:1, 1:1×10 5 ~1:1, 1:1×10 4 The ratio may be within the range of 1:1, 1:1000 to 1:1, or 1:100 to 1:1.

[0024] As used herein, the term enzyme activity refers to the rate of reaction catalyzed by the enzyme in question. The enzyme or enzymes present in the composition of the first aspect of the invention can optionally have an activity of at least 1 nmol / min on a nucleic acid (e.g., mRNA) substrate at a pH of 7.4 and a temperature of 25° C., for example, an activity of at least 10 nmol / min, at least 100 nmol / min, at least 1 μmol / min, at least 2 μmol / min, at least 5 μmol / min, at least 10 μmol / min, or at least 50 μmol / min under these conditions.

[0025] The enzyme fragment(s) may have an activity on a nucleic acid (e.g., mRNA) substrate at pH 7.4 and at 25° C. that is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, and in particular at least 80% of the activity of the corresponding whole enzyme molecule under the same pH and temperature conditions. Thus, the enzyme fragment(s) may retain one or more intact active sites of the corresponding whole enzyme molecule. The enzyme fragment(s) may retain one or more intact secondary structures of the corresponding whole enzyme molecule, such as one or more intact beta sheets and / or alpha helices.

[0026] Optionally, the nucleic acid vector composition is in the form of a lipid nanoparticle, which optionally has a core encapsulated by a shell, where the shell comprises one or more lipids, e.g., in a lipid bilayer, and the core comprises the nucleic acid, and one or more particles comprising hydrolyzable silicon.

[0027] Without wishing to be bound by theory, conventional lipid nanoparticles formulated with one or more lipids and nucleic acids (but without hydrolyzable silicones) are typically believed to have a core encapsulated by a shell. The shell is often a lipid bilayer (e.g., DSPC, PEG, 2000 The core is typically amorphous and is encapsulated by the shell, which, depending on the formulation employed, contains nucleic acid, water, and other components such as cationic lipids and / or cholesterol. This may protect the nucleic acid (e.g., mRNA) from the external medium, but the nucleic acid molecules in the core may still be in contact with water molecules. For example, it has been suggested that water pores surrounded by reverse cationic lipids exist in the core (Viger-Gravel et al., J. Phys. Chem. B 122(7), 2073-2081 (2018)). It has been suggested that the core may have a water content of 10-40% by volume, such as 20-30% by volume, for example 23-25% by volume. The mRNA may be located within water cylinders of a disordered reverse hexagonal phase, as reported by Areta et al., Proc. Natl. Acad. Sci., 115(15), E3351-E3360 (2018).

[0028] Thus, the composition of the present invention may be in the form of lipid nanoparticles, and may have some similarities to conventional lipid nanoparticles, which may have a core encapsulated by a shell, where the shell comprises one or more lipids, for example in a lipid bilayer, and the core (which is optionally amorphous) comprises nucleic acid and one or more particles comprising hydrolyzable silicon.However, in contrast to conventional lipid nanoparticles, in the composition according to the first aspect of the present invention, the particles comprising hydrolyzable silicon may stabilize the nucleic acid in the lipid nanoparticles by isolating water molecules from the nucleic acid, thereby delaying or preventing the reaction of nucleic acid with water in enzyme-catalyzed reactions, for example the reaction of sugar phosphate backbone with water.

[0029] Optionally, components of the delivery system, such as lipid and / or peptide molecules, may be bound to one or more of the silicon particles, thereby resulting in a stabilized complex in which both the hydrolyzable silicon and other components of the delivery system, such as lipids and / or peptides, are stabilized. Positively charged species, such as cationic lipids (and other lipid components that carry a positive charge, including but not limited to phospholipids), may be bound to one or more particles comprising hydrolyzable silicon, thereby stabilizing these positively charged components.

[0030] Thus, preferably, degradation of nucleic acids (e.g., mRNA or pDNA) at room temperature (20°C) is reduced by at least half, more preferably one-fold, or at least 5-fold, 10-fold, 35-fold, 50-fold, 100-fold, 500-fold, or 1000-fold, compared to an equivalent composition without the silicon particles.

[0031] The nucleic acid may be or comprise RNA, such as mRNA. The nucleic acid may be or comprise DNA. If the nucleic acid is or comprises mRNA, the mRNA may be in vitro transcribed mRNA. If the nucleic acid is or comprises DNA, the DNA may be plasmid DNA. If the nucleic acid is or comprises mRNA, the mRNA may comprise an open reading frame encoding a protein and, optionally, 5' Cap and a poly(A) tail; one or more untranslated regions; and one or more of:

[0032] Optionally, the open reading frame of the mRNA encodes an antigen of a pathogen. Optionally, the open reading frame of the mRNA encodes a spike protein antigen of SARS-CoV-2.

[0033] Optionally, in certain preferred embodiments of all aspects of the invention, the open reading frame of the mRNA encodes a tumor-specific antigen. As used herein, the term tumor-specific antigen may refer to an antigen that arises from a non-synonymous somatic mutation (resulting in a neo-antigen) or a viral integration mutation (resulting in a cancer virus antigen) in one or more malignant cancer cells. Thus, a tumor-specific antigen may refer to an antigen that is completely absent (not expressed) in non-cancerous (healthy, normal) cells.

[0034] Optionally, the open reading frame of the mRNA encodes a tumor-associated antigen. As used herein, the term tumor-associated antigen may refer to an antigen that is overexpressed in malignant cancer cells compared to non-cancerous (healthy, normal) cells, for example due to gene amplification or post-translational modification. The term tumor associated antigen can encompass overexpressed antigens (this term can refer to proteins that are moderately expressed in non-cancerous (healthy, normal) cells, but abundantly expressed in malignant cancer cells); differentiation antigens (this term can refer to proteins that are preferentially expressed by the cell lineage from which malignant cells arise, an example being prostate specific antigen); and cancer germline antigens (this term can refer to antigens that are normally restricted to reproductive tissues, but are aberrantly expressed in malignant cancer cells; e.g., Melanoma Antigen Family A3 (MAGE-A3); New York Esophageal Squamous Cell Carcinoma-1 Antigen (NY-ESO-1); and Preferentially Expressed Antigen in Melanoma (PRAME)).

[0035] If the open reading frame of the mRNA encodes a cancer-associated or cancer-specific antigen, the nucleic acid vector composition may be suitable for use in a prophylactic or therapeutic vaccine composition.

[0036] Optionally, the open reading frame of the mRNA encodes an allergen (one or more nut allergens; which in turn include, but are not limited to, one or more seed storage proteins, such as vicilin, legumin, albumin, one or more plant defense-related proteins; and one or more profilins).

[0037] Optionally, the open reading frame of the mRNA encodes a protein that modulates an immune disease, an autoimmune disease, or an inflammatory disease, including, but not limited to, lupus, atherosclerosis, chronic obstructive pulmonary disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatic disease, uveitis, atopic dermatitis, and pulmonary fibrosis.

[0038] Optionally, the nucleic acid vector composition according to the first aspect of the invention further comprises an amino acid, such as glycine. Additionally or alternatively, the composition further comprises one or more disaccharides, such as trehalose.

[0039] According to an alternative aspect of the invention there is provided a nucleic acid vector composition comprising: one or more enzymes; A nucleic acid, one or more particles comprising hydrolyzable silicon; One or more lipids; A nucleic acid vector composition is provided, comprising:

[0040] According to a second aspect of the invention there is provided a method for preparing a nucleic acid vector composition according to the first aspect of the invention comprising the steps of: Obtaining the nucleic acid by in vitro transcription from a DNA template or by purification from a biological source; and combining said nucleic acid with one or more particles comprising hydrolyzable silicon and one or more lipids; A method is provided, comprising:

[0041] According to a third aspect of the present invention there is provided a pharmaceutical composition comprising the nucleic acid vector composition of the present invention, wherein the pharmaceutical composition is a vaccine composition.

[0042] Preferably, the nucleic acid (e.g. mRNA or pDNA) in the pharmaceutical composition according to the third aspect of the invention, including the nucleic acid vector composition of the first aspect of the invention, has a half-life at 4°C of at least 3 months, at least 6 months, or at least 12 months.

[0043] According to a fourth aspect of the invention there is provided a pharmaceutical composition according to the third aspect of the invention for use as a medicament.

[0044] According to a fifth aspect of the present invention there is provided a method of treating or preventing a disease or disorder comprising administering to a subject in need of such treatment or prevention a pharmaceutical composition according to the third aspect of the present invention.

[0045] According to a sixth aspect of the present invention there is provided a method of providing vaccination to a subject comprising subcutaneous or intramuscular administration of a pharmaceutical composition according to the third aspect of the invention (when it is in a form suitable for intramuscular injection).

[0046] According to a seventh aspect of the present invention there is provided the use of a pharmaceutical composition according to the third aspect of the present invention as a lipopolyplex transfection vector.

[0047] According to an eighth aspect of the invention there is provided the use of a pharmaceutical composition according to the third aspect of the invention in the manufacture of a medicament. [Brief description of the drawings]

[0048] [Figure 1] Figure 1 compares the zeta potential of siRNA-loaded and siRNA-unloaded compositions. Each formulation is labeled F1-F5. [Diagram 2]A comparison of the zeta potential of siRNA-loaded (light dotted columns) and siRNA-unloaded (dark diagonal filled columns) compositions is shown, where the silicon nanoparticles are surface-treated with stearylamine (Figure 2), PC (Figure 3), or lecithin (Figure 4), and the amine is arginine. [Diagram 3] Same as above. [Figure 4] Same as above. [Diagram 5] 5 shows the results of an experiment to measure the transfection efficiency of compositions in HCES cells. The cells in the first column are stained with DAPI and fluoresce (originally) blue, indicating the nucleus. The cells in the second column fluoresce (originally) green, indicating successful transfection with formulations F2-F5 of the invention. [Figure 6] FIG. 6 shows the results of a gel electrophoresis experiment, which demonstrates that silicon nanoparticles prepared according to the present invention successfully capture mRNA, especially at ratios of silicon nanoparticles to mRNA of 2:1 or greater. [Figure 7] FIG. 7 shows the results of spectrophotometric experiments, which confirm that silicon nanoparticles prepared according to the present invention successfully capture mRNA, especially at silicon nanoparticle to mRNA ratios of 2:1 or greater. [Figure 8] FIG. 8 shows the results of an experiment to measure the transfection efficiency of a siRNA-loaded silicon nanoparticle delivery system. [Figure 9] FIG. 9 shows the results of an experiment measuring the post-transfection viability of cells treated with a siRNA-loaded silicon nanoparticle delivery system. [Figure 10] FIG. 10 shows the results of experiments to measure the extent of siRNA-induced gene silencing when siRNA was delivered to cells using a silicon nanoparticle delivery system. [Figure 11]FIG. 11 shows the results of experiments evaluating in vivo ocular siRNA delivery by topical silicon nanoparticle formulations. [Figure 12] FIG. 12 shows the results of an experiment measuring luciferase expression in mouse corneas in vivo by imaging in live animals during treatment with a siRNA-loaded silicon nanoparticle delivery system. [Figure 13] FIG. 13 shows the results of an experiment measuring luciferase expression in mouse corneas in vivo by imaging in live animals during treatment with a siRNA-loaded silicon nanoparticle delivery system. [Figure 14] FIG. 14 shows the results of an experiment to measure the nucleic acid binding efficiency for hsDNA for Si nanoparticle-containing formulations and liposomal formulations without Si nanoparticles. [Figure 15] FIG. 15 shows the appearance of a silicon wafer before and after milling into powder. [Figure 16] FIG. 16 shows SEM images of silicon particles that were used to assess the size of manually milled silicon particles. [Figure 17] FIG. 17 shows a TEM image of hand milled silicon particles. [Figure 18] FIG. 18 shows a TEM image of the silicon particles of FIG. 17 after complexing with components of the invention, including RNA. [Figure 19] Figure 19 shows a TEM image of a silicon particle after complexing with a component of the invention, including RNA, similar to the particle shown in Figure 18, except made of boron-doped silicon. [Figure 20] FIG. 20 shows the nucleic acid binding efficiency (mRNA) measured for a "Biocourier" silicone particle-containing formulation of the invention (filled bars) and for the corresponding "liposome" formulation in the absence of silicone particles (open bars). [Figure 21]FIG. 21 shows the nucleic acid binding efficiency (hsDNA) measured for a "Biocourier" silicone particle-containing formulation of the invention (filled bars) and for the corresponding "liposome" formulation in the absence of silicone particles (open bars). [Figure 22] Figure 22 shows the results of gel retardation assays of (A) mRNA-SIS0012 incubated with bovine serum (BS) for various periods of time, and (B) mRNA extracted from mRNA-SIS0012 before and after incubation with bovine serum. Naked mRNA in nuclease-free water was used as a control. A DNA ladder was used as a size guide. [Figure 23] Figure 23 shows the results of gel retardation assays of (A) mRNA-SIS0013 incubated with bovine serum (BS) for various periods of time, and (B) mRNA extracted from mRNA-SIS0013 before and after incubation with bovine serum. Naked mRNA in nuclease-free water was used as a control. A DNA ladder was used as a size guide. [Figure 24] Figure 24 shows the results of gel retardation assays of (A) mRNA-LNPs formulated without silicone and incubated with bovine serum (BS) for various periods of time, and (B) mRNA extracted from LNPs before and after incubation with bovine serum. Naked mRNA in nuclease-free water was used as a control. A DNA ladder was used as a size guide. [Diagram 25] Figure 25 shows the results of a gel retardation assay of naked mRNA incubated with bovine serum (BS) for various periods of time. Naked mRNA in nuclease-free water was used as a control. A DNA ladder was used as a size guide. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] Biologically derived nucleic acids Optionally, the present invention relates to biologically sourced nucleic acids according to certain embodiments. This means either nucleic acids produced in a cell culture-based system or nucleic acids produced in vitro using enzymes produced in a cell culture-based system. Biologically sourced nucleic acids may include RNA (such as mRNA) and DNA (such as pDNA) extracted from cellular material or transcribed in vitro (including with subsequent chemical modification). Nucleic acids produced by a completely chemical synthetic route, such as phosphoramidite chemical synthesis, are not included in biologically sourced nucleic acids.

[0050] In vitro transcribed mRNA In certain preferred embodiments of all aspects of the invention, the nucleic acid vector compositions of the invention comprise a preparation of in vitro transcribed (IVT) mRNA.

[0051] The term "mRNA" is used herein to refer to messenger RNA. This term encompasses unmodified and modified mRNA. In this regard, it will be understood that the mRNA according to certain embodiments of all aspects of the invention may be chemically modified to enhance its therapeutic properties, such as enhanced activity, increased serum stability, reduced off-targeting, and reduced immunological activation.

[0052] Chemical modifications to RNA include any modification generally known in the art, e.g., modification with labels known in the art; methylation; caps, e.g., a 5' cap (e.g., Cap 0, which requires a 7-methylguanosine linked by a triphosphate bridge to the first nucleotide; Cap 1, which requires methylation of the 2'-hydroxyl group of the first cap-proximal nucleotide; or Cap 2, which has an additional 2'-O-methylation of the second nucleotide; or a modified Cap 1 structure m 7 G + m 3’These may include modified cap 0, cap 1, or cap 2 structures, such as 5'-5'-ppp-5'-Am; a poly(A) tail; substitution of one or more of the naturally occurring nucleosides with analogs, for example, replacement of uridine with N1-methyl-pseudouridine; and modifications of the internucleoside linkages.

[0053] In some embodiments, the mRNA can be at least 100 base pairs, at least 200 base pairs, at least 300 base pairs, at least 500 base pairs, or at least 1000 or 2000 base pairs in length.

[0054] Plasmid DNA (pDNA) In other embodiments, the nucleic acid vector composition comprises a preparation of DNA. Preferably, this is plasmid DNA, which includes any DNA in the form of a plasmid obtained from a plasmid (e.g., by excision from a plasmid).

[0055] In vitro mRNA transcription In certain preferred embodiments, the method of the second aspect of the invention for preparing a nucleic acid vector composition entails synthesising mRNA by in vitro transcription (IVT) from a linear DNA template.

[0056] The linear DNA template may be pre-generated. For example, in some embodiments, the DNA template may be pre-generated by linearization of a purified plasmid or by amplification of the region of interest using PCR.

[0057] Transcription can be performed using an RNA polymerase. For example, transcription can be performed in some embodiments using T7, SP6, or T3 RNA polymerase. In certain preferred embodiments, for example, when the open reading frame of the mRNA encodes the spike protein antigen of SARS-CoV-2, T7 RNA polymerase can be used.

[0058] Thus, reactants used for transcription can include a linear DNA template; an RNA polymerase (such as T7, SP6, or T3 RNA polymerase); and a nucleoside triphosphate substrate. The nucleoside triphosphate substrate can include a modified nucleoside triphosphate substrate, such as N1-methyl-pseudouridine. In some embodiments, a polymerase cofactor MgCl2 can be included. In some embodiments, a pH buffer solution (e.g., a pH buffer solution containing a polyamine and an antioxidant) can be included.

[0059] 5'-capping of mRNA can be optionally performed during the IVT reaction, i.e., 5'-capping can be co-transcriptional. This can be achieved by replacing some of the guanosine triphosphate substrates with cap analogs. Optionally, co-transcriptional capping can be performed using CleanCap™ reagent.

[0060] As an alternative to co-transcriptional capping, the mRNA may in some embodiments be capped in a second reaction (post-transcriptionally) catalyzed by vaccinia capping enzyme (VCC) together with a methyl donor. In certain preferred embodiments, for example when the open reading frame of the mRNA encodes the spike protein antigen of SARS-CoV-2, 5'-capping is performed using vaccinia capping enzyme and vaccinia 2'O-methyltransferase.

[0061] In some embodiments, the 5' cap is cap 0, cap 1, or cap 2, or a modified cap 0, modified cap 1 (e.g., m 7 G + m 3’ -5'-ppp-5'-Am cap), or modified cap 2.

[0062] Purification of mRNA and pDNA To prepare the nucleic acid vector compositions of the invention, it is necessary to purify the nucleic acid (e.g., mRNA or pDNA). In embodiments where the nucleic acid is mRNA, this may require purifying the mRNA from the transcription reaction mixture (which in some embodiments contains impurities including one or more of enzymes, residual nucleoside triphosphates, DNA template, and aberrant mRNA formed during IVT) to form a preparation of in vitro transcribed mRNA.

[0063] Optionally, purification may include, but is not limited to, one or more of size exclusion chromatography (SEC); ion pair reverse-phase chromatography (IPC); ion exchange chromatography (IEC); affinity-based separation; tangential flow filtration (TFF); core bead chromatography; hydroxyapatite chromatography; mRNA precipitation in combination with TFF (during TFF, a membrane captures the precipitated mRNA product while other impurities are removed by diafiltration); DNA template removal by performing digestion with immobilized DNase; and the use of tagged DNA templates that can be removed after IVT using affinity chromatography. In certain preferred embodiments, for example, where the open reading frame of the mRNA encodes the spike protein antigen of SARS-CoV-2, purification may include oligo-dT affinity purification, buffer exchange by tangential flow filtration into sodium acetate (pH 5.0), and sterile filtration.

[0064] It will be appreciated that even when purification is complete, the in vitro transcribed mRNA preparation may still contain at least trace amounts of one or more of linear DNA, one or more RNA polymerases, and one or more nucleoside triphosphates. For example, the in vitro transcribed mRNA preparation may contain at least 0.01 v / v% linear DNA. The in vitro transcribed mRNA preparation may contain at least 0.01 v / v% RNA polymerase. The in vitro transcribed mRNA preparation may contain at least 0.01 v / v% nucleoside triphosphates.

[0065] pDNA can be purified from extracts derived from cells (including those derived from microbial cells, such as from bacterial or yeast cells) by similar methods.

[0066] Preparation of Nucleic Acid Vector Compositions In a method for preparing a nucleic acid vector composition according to the second aspect of the invention, a preparation of nucleic acid (e.g., pDNA or in vitro transcribed mRNA) is then combined with water, particles containing hydrolyzable silicon, and one or more lipids. The composition may optionally further comprise one or more amino acids (e.g., glycine, or a mixture of nucleic acids containing glycine) and optionally one or more non-reducing disaccharides such as trehalose. A suitable preparation method may include dispersing the lipid components in a solvent such as methanol; producing a thin film of lipids by evaporating the solvent, for example in a rotary evaporator; hydrating the lipids with an aqueous solution containing activated hydrolyzable silicon particles, for example particles having an average particle size of less than 100 nm, a non-reducing disaccharide such as trehalose, and one or more amino acids such as glycine. The composition may optionally be passed through a filter, for example a 0.4 and 0.1 μm filter, to achieve particle complexation and dispersion. The composition may optionally be stored at 4° C. to allow further complexation to occur. The carrier thus prepared can then be complexed with an aqueous solution of nucleic acid (such as plasmid DNA or mRNA in ratios ranging from 1:6 to 1:16, where 1 represents the nucleic acid). A preferred ratio is 1:8 to 1:12, where 1:8 usually allows for a small excess of biological and 1:12 allows for a small excess of carrier.

[0067] mRNA structure In some embodiments of all of the aspects of the invention where the nucleic acid is an mRNA, the mRNA molecule may optionally include, in addition to an open reading frame encoding a protein, one or more of the following: a 5' cap; a poly(A) tail; and one or more untranslated regions.

[0068] In some embodiments, the open reading frame encoding the protein of the mRNA encodes an antigen, thereby providing a formulation that is a vaccine. The antigen can be a viral antigen, such as an antigen of SARS-CoV-2, such as an antigen derived from the spike protein of SARS-CoV-2 or a portion thereof. Thus, the open reading frame of the mRNA can, in some embodiments, encode a spike protein antigen of SARS-CoV-2. In some embodiments, the open reading frame can encode a mutant form of a naturally occurring protein. For example, in some embodiments, when the open reading frame encodes the spike protein of SARS-CoV-2 or a portion thereof, it can contain two mutations in which the original amino acid is replaced with a proline. Without wishing to be bound by theory, it is believed that this can ensure that the resulting S glycoprotein remains in an antigenically optimal pre-fusion conformation.

[0069] In some embodiments, the open reading frame of the mRNA encoding a protein may encode multiple proteins. For example, the open reading frame of the mRNA encoding a protein may encode a viral antigen and an adjuvanted protein, or multiple viral antigens. In certain preferred embodiments, for example, when the open reading frame of the mRNA encodes the spike protein antigen of SARS-CoV-2, the open reading frame may additionally encode a signal peptide, such as the S glycoprotein signal peptide.

[0070] 5' Cap As described above, the mRNA can be modified by having a 5' cap. A 5' cap requires a 7-methylguanosine linked to the first nucleotide by a triphosphate bridge, Cap0 (m 7 The 5' cap may have the structure Cap1 (mGpppN), which requires methylation of the 2'-hydroxyl group of the first cap-proximal nucleotide.7 The 5' cap may have the structure Cap2(m GpppNm) with an additional 2'-O-methylation of the second nucleotide. 7 GpppNmpN m The cap may have a modified cap 0 structure, a modified cap 1 structure, or a modified cap 2 structure. In certain preferred embodiments, for example, when the open reading frame of the mRNA encodes a spike protein antigen of SARS-CoV-2, the 5' cap is cap 1 or the 5' cap is m 7 G + m 3’ and modified cap 1 having a -5'-ppp-5'-Am cap structure.

[0071] [ka]

[0072] [ka]

[0073] Without wishing to be bound by theory, it is believed that the 5' cap increases mRNA stability by protecting the mRNA from degradation by 5' exonucleases; the 5' cap enables ribosomes to recognize the beginning of the mRNA; and the 5' cap may improve translation efficiency by binding to eukaryotic translation initiation factor 4E (eIF4E).

[0074] 3' poly(A) tail Without wishing to be bound by theory, it is also believed that modification of mRNA by addition of a 3' poly(A) tail can improve translation activity and mRNA stability, including protecting the mRNA from nuclease degradation. A poly(A) tail can be added to the mRNA during in vitro transcription of the mRNA by including a poly(A) sequence in the DNA template in a preferred embodiment (e.g., when the open reading frame of the mRNA encodes the spike protein antigen of SARS-CoV-2). The size of the tail can be selected to optimize mRNA stabilization and expression. Advantageously, in vitro transcription of the mRNA from a DNA template can generate an mRNA with a defined poly(A) tail length. In some embodiments, the poly(A) tail can have a length of 100 nucleotides to 200 nucleotides, for example, a length of 120 nucleotides to 150 nucleotides. In certain preferred embodiments, for example where the mRNA open reading frame encodes the spike protein antigen of SARS-CoV-2, a poly(A) tail of 110 nucleotides is provided, which comprises 30 adenosine residues separated from a further 70 adenosine residues by a linker sequence of 10 nucleotides.

[0075] Untranslated region (UTR) Untranslated regions are non-coding regions of an mRNA sequence located in the upstream (5'UTR) and downstream (3'UTR) domains of the mRNA coding region. Without wishing to be bound by theory, it is believed that UTRs can aid in transcriptional regulation as well as mRNA stability, and that UTRs affect translation efficiency by participating in translation machinery recognition, recruitment, and mRNA transport. It is believed that UTRs can alter mRNA decay and translation efficiency by reacting with RNA-binding proteins.

[0076] In certain preferred embodiments, for example, when the open reading frame of the mRNA encodes the spike protein antigen of SARS-CoV-2, a 5'UTR derived from human alpha globin RNA with an optimized Kozak sequence is provided, and / or a 3'UTR is provided that includes an amino-terminal enhancer of a split (AES) mRNA and two sequence elements derived from the mitochondrially encoded 12S ribosomal RNA. Without wishing to be bound by theory, it is believed that such UTRs confer RNA stability and high total protein expression.

[0077] Open reading frame (ORF) As the term is used herein, an open reading frame (ORF) refers to a protein coding region of a nucleic acid (e.g., an mRNA or pDNA), or one of them. In some embodiments, the ORF sequence may contain synonymous common codons (and / or codons with higher tRNA abundance) as replacements for rarer codons. In this way, it is believed that highly expressed genes can be translated using the same codons of the host and / or ensure the abundance of the associated tRNA during expression of the nucleic acid. However, having a higher translation rate of a nucleic acid is not always preferred, since some proteins require a low translation rate for proper folding. In these situations, using rare codons in the ORF may result in a higher quality protein product.

[0078] Modified Nucleosides and Nucleotides As used herein, the terms "nucleoside" and "nucleotide" are understood to include moieties that contain not only known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides also include modifications to the sugar moiety, for example, where one or more of the hydroxyl groups have been replaced with halogens, aliphatic groups, or functionalized as ethers, amines, and the like. Other modifications to nucleotides or polynucleotides involve rearranging, adding, substituting, or otherwise changing the functional groups on the purine or pyrimidine bases that form hydrogen bonds to the respective complementary pyrimidines or purines, e.g., isoguanine, isocysteine, and the like. In some embodiments, the mRNA comprises at least one, two, three, or four modified nucleotides. For example, in some embodiments, the mRNA can include at least one N1-methyl-pseudouridine nucleoside, such as at least 2, 3, 4, 5, 10, 15, or 20 N1-methyl-pseudouridine nucleosides.

[0079] In some embodiments, a nucleic acid (e.g., an mRNA) comprises one or more universal bases. As used herein, the term "universal base" refers to a nucleotide analog that can hybridize to two or more nucleotides selected from A, U / T, C, and G. In some embodiments, the universal base may be selected from the group consisting of deoxyinosine, 3-nitropyrrole, 4-nitroindole, 6-nitroindole, and 5-nitroindole.

[0080] Without wishing to be bound by theory, it is believed that the introduction of modified nucleosides may help to modulate the innate immune response and / or increase nucleic acid stability. For example, in some embodiments, the uridine content of the mRNA may be partially or entirely replaced by N1-methyl-pseudouridine. In certain preferred embodiments, for example, when the open reading frame of the mRNA encodes the spike protein antigen of SARS-CoV-2, the uridine content of the mRNA may be completely replaced by N1-methyl-pseudouridine.

[0081] Enzymes and their fragments or fragments According to all aspects of the invention relating to nucleic acid vector compositions, the composition may comprise one or more enzymes, and / or one or more fragments thereof. The composition may comprise, at least in trace amounts, linear DNA; one or more RNA polymerases, and / or one or more fragments thereof; and one or more nucleoside triphosphates. Thus, in particular, the composition may comprise one or more RNA polymerases, and / or one or more fragments thereof. Optionally, the one or more RNA polymerases may be selected from one or more of T7, SP6, and T3 RNA polymerase. When the nucleic acid is or comprises DNA, the composition may comprise at least in trace amounts of linear DNA.

[0082] The composition may contain at least trace amounts of an enzyme and / or one or more fragments thereof capable of degrading nucleic acids. However, the one or more particles containing hydrolyzable silicon may remove or sequester water molecules, thereby preventing the one or more enzymes and / or one or more fragments thereof from degrading nucleic acids, based on the fact that enzymes require an aqueous environment to catalyze the degradation of nucleic acids.

[0083] One or more particles containing hydrolyzable silicon According to all aspects of the present invention, the particle or particles containing hydrolyzable silicon can be pure silicon or another hydrolyzable silicon-containing material. If the particle is not pure silicon, the particle contains at least 50% silicon by weight, i.e., at least 50% silicon atoms by weight based on the total mass of atoms in the particle. For example, the silicon particles can contain at least 60%, 70%, 80%, 90%, or 95% silicon. The silicon particles preferably exhibit a rate of hydrolysis of at least 10% of that of a pure silicon particle of the same size, for example in PBS buffer at room temperature. Assays for hydrolysis of silicon-containing materials are widely known in the art (see, for example, WO 2011 / 001456, which is incorporated herein by reference). The particles of the present invention may contain some silica, but the silica is not hydrolyzable silicon, and at least half of the silicon atoms in the particle are in the form of elemental silicon (or doped elemental silicon).

[0084] According to all aspects of the present invention, the particles containing hydrolyzable silicon may be nanoparticles. The nanoparticles have a nominal diameter of 5-400 nm, such as 50-350 nm, such as 80-310 nm, such as 100-250 nm, such as 120-240 nm, such as 150-220 nm, such as about 200 nm. The nanoparticles may be made of either pure silicon or a hydrolyzable silicon-containing material. The nanoparticles are preferably porous. The nominal diameter mentioned above may refer to the average diameter, and at least 90% of the total mass of the particles in a sample of the particles may fall within the specified size range. Particle size may be confirmed or confirmed by transmission electron microscopy (TEM), for example, using the NIST-NCL Joint Assay Protocol, PCC-X, Version 1.1, Revised February 2010, "Measuring the size of nanoparticles using TEM" (https: / / tsapps.nist.gov / publication / get_pdf.cfm?pub_id=854083).

[0085] In some preferred embodiments, the nominal diameter may be less than 100 nm, or less than 80 nm, or less than 70 nm, 50 nm, or less than 30 nm. In some embodiments, the nominal diameter may be about 30 nm (e.g., 20 nm to 40 nm). Particles containing hydrolyzable silicon can be made porous by standard techniques, such as contacting the particles with a hydrofluoric acid (HF) / ethanol mixture and applying an electric current. By varying the HF concentration as well as the current density and exposure time, the density of the pores and their size can be controlled and monitored by scanning electron microscopy and / or nitrogen adsorption-desorption volumetric isotherm measurements.

[0086] When particles are porous, their total surface area increases thanks to their porosity. For example, the surface area may increase by at least 50% or at least 100% over the surface area of ​​the corresponding non-porous particle. In many circumstances, porous particles according to all aspects of the invention actually have a much larger increase in total surface area due to their porosity. According to certain embodiments, the porosity is at least 30%, 40%, 50%, or 60%. This means that 30%, 40%, 50%, or 60% of the particle volume, respectively, is in the pore space. Preferred pore sizes range from 1 nm to 50 nm, for example from 5 nm to 25 nm.

[0087] Optional Silicon Doping All aspects of the invention optionally relate to doped silicon containing materials. Silicon may be n-doped or p-doped as desired. The invention includes embodiments of all aspects in which silicon is doped with one or more elements selected from Mg, P, Cu, Ga, Al, In, Bi, Ge, Li, Xe, N, Au, Pt. Most preferably, the dopant is a p-type dopant. Most preferably, the dopant is boron. P-doped silicon is particularly suitable for stabilizing negatively charged nucleic acids. N-doped silicon may also be useful for indirectly stabilizing negatively charged nucleic acids, since it can protect lipids from degradation, which may indirectly increase the stabilization and protection of nucleic acids.

[0088] The production of doped silicon is well understood in the semiconductor industry and includes ion implantation and diffusion methods. Thus, doped silicon is readily available. If desired, silicon can be doped by using a diffusion method to increase the amount of dopant present in the silicon. As an example of the diffusion method, silicon powder and a doping reagent (e.g., B2O3 for boron doping) are placed in a bowl, mixed, placed under N2 atmosphere, and the dopant (e.g., boron) is diffused into the silicon at temperatures between 1050°C and 1175°C for several minutes. Figures 1 and 2 show boron-doped silicon produced by this method.

[0089] According to a particular embodiment, the doping of the silicon is heavy. Heavy boron doping is particularly preferred. Heavy doping is cm 3 At least 1 x 10 per 15 doping with cm dopant atoms. In some embodiments, the dopant is 3 At least 1 x 10 per 16 dopant atoms, cm 3 At least 1 x 10 per 17 dopant atoms, cm 3 At least 1 x 10 per 18dopant atoms, cm 3 At least 1 x 10 per 19 dopant atoms, or cm 3 At least 1 x 10 per 20 The dopant concentration is present at the level of individual dopant atoms.

[0090] When boron is used as a dopant, preferably, cm 3 1x10 15 dopant atoms, and cm 3 1x10 20 doping levels of 1000 and 1000 dopant atoms correspond to resistivities of 20 mohm-cm and 1 mohm-cm, respectively. The various aspects of the invention in which boron is the preferred dopant do not exclude silicon that is doped, e.g., heavily doped, with boron, as well as doped with other elements. According to preferred embodiments of all aspects of the invention, the majority dopant is boron.

[0091] Silicon to nucleic acid ratio Preferably, the ratio of silicon to nucleic acid (e.g. mRNA or pDNA) is 0.01:1 to 1:8, such as 1:1 to 1:6, 1:1 to 1:5, 1:1 to 1:4, or 1:1 to 1:3. Preferably, the ratio of silicon to nucleic acid is 1:1 to 1:3. Advantageously, this ratio of silicon to nucleic acid further stabilizes the nucleic acid carried by the particle, and therefore also optionally affects the release rate of the nucleic acid.

[0092] Optional Association of Nucleic Acid with Silicon In some embodiments of all aspects of the invention, at least 70% by weight, such as at least 80% by weight, such as at least 90% by weight, of the nucleic acid present (e.g., mRNA or pDNA) is associated with one or more particles comprising hydrolyzable silicon. This means that the nucleic acid is non-covalently associated with one or more particles comprising hydrolyzable silicon. Without wishing to be bound by theory, it is hypothesized that when this occurs, the random movement of the nucleic acid (also referred to as Brownian motion) may be reduced in some embodiments. As a result, the chances of the nucleic acid being degraded are further reduced.

[0093] The optional association of nucleic acid with one or more particles that contain hydrolyzable silicon can be correlated with and / or controlled by the hydrolysis of silicon.Therefore, the rate at which nucleic acid becomes bioavailable can also be correlated with the hydrolysis of silicon, thereby avoiding dose-dumping and / or ensuring the sustained release of nucleic acid over a suitable long period of time.In some embodiments, the association of nucleic acid with one or more particles can further stabilize nucleic acid.For example, the association can result in charge stabilization of nucleic acid.

[0094] Lipids According to all aspects of the invention, the at least one lipid may comprise a cationic lipid; a helper lipid, such as a phospholipid; a structural lipid, such as a cholesterol-based lipid; and / or a polyethylene glycol (PEG) lipid.

[0095] The type of lipid or lipids used can affect the rate of degradation of one or more silicon particles in vivo. For example, one or more lipid molecules may be non-covalently associated with the surface or surfaces of one or more silicon particles, which can be expressed as surface treatment of one or more silicon particles with lipid. For example, it has been found that the presence of at least one lipid allows the hydrolysis rate of silicon to be controlled, so that silicon hydrolyzes into bioavailable orthosilicic acid (OSA) degradation products rather than insoluble polymer hydrolysis products. In particular, it has been found that surface treatment of silicon particles with lipid has a beneficial effect on the surface charge of silicon particles, which provides the silicon particles with the necessary zeta potential to allow improved stabilization of nucleic acids (e.g., mRNA), and optionally controls the release rate of nucleic acids at target sites.

[0096] The lipid may comprise one or more of phosphatidylcholine (PC), hydrogenated PC, stearylamine (SA), dioleoylphosphatidylethanolamine (DOPE), cholesteryl 3β-N-(dimethylaminoethyl)carbamate hydrochloride (DC)-cholesterol, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and any derivatives thereof. In certain embodiments, the lipid comprises or consists of DOTAP. The type of lipid used to treat the surface of the nanoparticles may affect the stabilization and, optionally, the release rate of the nucleic acid (e.g., mRNA or pDNA). In particular, when there is an association between lipid molecules and one or more silicon particles, there is a beneficial effect on the surface charge of the silicon particles, which provides the silicon particles with the zeta potential required to improve the stabilization of the nucleic acid and, optionally, control the release rate of the nucleic acid (e.g., mRNA or pDNA) at the target site. The presence of at least one lipid can allow the hydrolysis rate of silicon to be controlled so that silicon hydrolyzes into bioavailable orthosilicic acid (OSA) degradation products rather than into insoluble polymer hydrolysis products. Controlling the hydrolysis rate of silicon affects the time that nucleic acid protection is sustained.

[0097] According to all aspects of the invention, the lipid or lipids may have an average molecular weight in the range of 500-1000 (e.g., when the lipid contains one or more of a cationic lipid (e.g., DTDTMA (ditetradecyltrimethylammonium), DOTMA (2,3-dioleyloxypropyl-1-trimentylammonium), DHDTMA (dihexadecyltrimethylammonium)), DOTAP, helper lipid, structural lipid, and PEG lipid, or when the lipid contains one or more of PC, hydrogenated PC, SA, DOPE, DOTAP, DTDTMA, DHDT MA, DC-cholesterol, and derivatives thereof), the ratio of lipid to silicone (i.e. total lipid components) before any extrusion or filtration process is carried out is 1:1 to 20:1, e.g., 1:1 to 18:1, 1:1 to 16:1, 1:1 to 11:1, 1:1 to 10:1, 1:1 to 9:1, 1:1 to 8:1, 1:1 to 13:1, 2:1 to 12:1, 2:1 to 11:1, 2:1 to 10:1, 2:1 to 9:1, 2:1 to 8:1, e.g., 1:1 to 7:1, 2:1 to 7:1, 3:1 to 6:1, 4:1 to 5:1. Without wishing to be bound by theory, this ratio of lipid to silicon may provide a vesicular system that can optionally control and stabilize the release of nucleic acids (such as mRNA or pDNA) in contact with the particles of hydrolyzable silicon, as well as promote the controlled release of OSA, a bioavailable degradation product of silicon.

[0098] Advantageously, lipid compounds have a significant effect on the surface charge of silicon particles. Particles containing hydrolyzable silicon treated with phosphatidylcholine (PC), phosphatidylethanolamine (PE) DOTAP, and lecithin exhibited a negative surface charge when subjected to zeta potential analysis (range of -60 to -20 mV, silicon:lipid ratios ranging from 1:1 to 1:3). Particles surface treated with stearylamine exhibited a positive zeta potential (range of 0 to 40 mV, silicon:lipid ratios ranging from 1:1 to 1:3). The use of doped silicon (e.g., p-doped silicon, e.g., boron-doped silicon as described elsewhere herein) can contribute to making the zeta potential even more negative. By controlling the surface charge of silicon particles in this way, their ability to remove or sequester water molecules and thus protect nucleic acids from degradation can be controlled.

[0099] The lipid or lipid component may be or may include a phospholipid in some embodiments. The term "phospholipid" refers to a lipid that includes a fatty acid chain and a phosphate group. Phospholipids are typically neutral molecules in that they may have no overall charge or may have a negative charge, unlike positively charged cationic lipids. Phospholipids are typically zwitterionic compounds that include both positively and negatively charged components, but do not include an overall charge. Thus, phospholipids are typically classified as neutral lipids. Particularly suitable phospholipids are glycerophospholipids. Particularly suitable phospholipids are those in which the polar head group is attached to a quaternary ammonium moiety, such as phosphatidylcholine (PC) or hydrogenated phosphatidylcholine. Another example of a phospholipid is DOPE (phosphatidylethanolamine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). The type of lipid may be selected depending on the nature of the formulation, with neutral or negatively charged phospholipids being preferred for aprotic formulations, while positively charged cationic lipids and small CH3 chain lipids are preferred for protic formulations. The phospholipid may be or be derived from lecithin.

[0100] Preferably, the side chain(s) of the phospholipid is an aliphatic side chain(s) with 15 or more carbon atoms, or an ether side chain with 6 or more repeating ether units, such as a polyethylene glycol or polypropylene glycol chain. A lipid with an ether side chain can be referred to as a "PEG lipid" or a "PEGylated" lipid. The PEG lipid can be a phospholipid such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) with a PEG side chain, such as DSPE-mPEG2000.

[0101] The lipid component may include one or more of phosphatidylcholine (PC), hydrogenated PC, stearylamine (SA), dioleoylphosphatidylethanolamine (DOPE), DOTAP, cholesteryl 3β-N-(dimethylaminoethyl)carbamate hydrochloride (DC)-cholesterol, and derivatives thereof. In certain embodiments, the lipid component may consist essentially of phosphatidylcholine, hydrogenated phosphatidylcholine, stearylamine, or combinations thereof.

[0102] Doping of silicon changes the surface charge. The use of p-dopants according to certain preferred embodiments, such as boron (preferred in many embodiments of the present invention in all its various aspects), makes the zeta potential more positive (i.e., less negative). A typical value of -40 mV for pure silicon becomes about -25 mV when silicon is doped with boron. It can therefore be seen that boron-doped silicon can achieve a positive zeta potential more easily when treated with cationic lipids. For example, treatment with stearylamine or DOTAP can achieve values ​​of about +20 mV to +60 mV. This means that a positive surface zeta potential can be achieved with less cationic lipid or with a wider range of cationic lipids, including those that are less cationic than stearylamine and DOTAP. This also means that the surface zeta potential of the particles will remain in the fully positive range for a longer period of time, even if the cationic lipids degrade ("age") during storage, resulting in a partial loss of the lipid's positive charge.

[0103] Molar ratios of lipid to boron-doped silicon of 0.8:1 to 20:1 have proven to be particularly advantageous, for example 1:1, 6:1, 8:1 or 10:1 or 12:1 or 16:1.

[0104] The lipid or lipid component may be or include a phospholipid in some embodiments. The term "phospholipid" refers to a lipid that includes a fatty acid chain and a phosphate group. Phospholipids are typically neutral molecules in that they have no overall charge or may carry a negative charge, unlike positively charged cationic lipids. Phospholipids are typically zwitterionic compounds that include both positively and negatively charged components, but do not include an overall charge. Thus, phospholipids are typically classified as neutral lipids. Particularly suitable phospholipids are glycerophospholipids. Particularly suitable phospholipids are those in which the polar head group is attached to a quaternary ammonium moiety, such as phosphatidylcholine (PC) or hydrogenated phosphatidylcholine. Another example of a phospholipid is DOPE (phosphatidylethanolamine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). The type of lipid may be selected depending on the nature of the formulation, with neutral or negatively charged phospholipids being preferred for aprotic formulations, while positively charged cationic lipids and small CH3 chain lipids are preferred for protic formulations. The phospholipid may be or be derived from lecithin.

[0105] Preferably, the side chain(s) of the phospholipid is an aliphatic side chain(s) having 15 or more carbon atoms, or an ether side chain having 6 or more repeating ether units, such as a polyethylene glycol or polypropylene glycol chain. Lipids with ether side chains may be referred to as "PEG lipids" or "PEGylated" lipids.

[0106] The lipid or lipid component may be or include a cationic lipid in some embodiments. The term "cationic lipid" refers to a positively charged molecule with a cationic head group attached to a hydrophobic tail via a suitable spacer. Examples include DTDTMA (ditetradecyltrimethylammonium), DOTMA (2,3-dioleyloxypropyl-1-trimentylammonium), DOTAP, DHDTMA (dihexadecyltrimethylammonium), and stearylamine (SA). The positive charge is typically stabilized by a negative counterion. In a preferred embodiment, particularly with respect to vaccine compositions, the cationic lipid is or includes DOTAP.

[0107] In certain embodiments, the lipid is selected from the group consisting of phosphatidylethanolamine (PE), phosphatidylcholine (PC), stearylamine (SA), or any combination thereof.

[0108] In certain embodiments, the lipid may consist essentially of phosphatidylcholine, hydrogenated phosphatidylcholine, stearylamine, or combinations thereof.

[0109] In certain embodiments, the lipid may be composed of at least 5% by weight of hydrogenated phosphatidylcholine, for example at least 20% by weight, typically at least 30% by weight, particularly at least 50% by weight of hydrogenated phosphatidylcholine, based on the total weight of the particle.The molar ratio of hydrogenated phosphatidylcholine to silicon of 0.8:1 to 5:1 has been found to be particularly advantageous, for example 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1 or 4.5:1.

[0110] In certain embodiments, the lipid may be composed of at least 5% by weight of phosphatidylcholine, for example at least 20% by weight, typically at least 30% by weight, in particular at least 50% by weight of phosphatidylcholine, based on the total weight of the particle. Molar ratios of phosphatidylcholine to silicon of 0.8:1 to 5:1 have been found to be particularly advantageous, for example 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1 or 4.5:1.

[0111] In certain embodiments, lipid can be composed of at least 5% by weight of stearylamine, for example at least 20% by weight, typically at least 30% by weight, particularly at least 50% by weight of stearylamine, based on the total weight of particle.The molar ratio of stearylamine to silicon of 0.8:1 to 5:1, for example 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1 or 4.5:1 has been found to be particularly advantageous.

[0112] In certain embodiments, the lipids may consist of PC and SA, preferably in a weight ratio of PC:SA of 1:1 to 20:1, more preferably 7:1 to 10:1, such as a weight ratio of PC:SA of 72:8.

[0113] In certain embodiments, the lipids may consist of DOPE, SA, and DC-cholesterol. The weight ratio of DOPE:SA may be in the range of 1:1 to 10:1, such as 4:1 to 8:1. The weight ratio of DOPE:DC-cholesterol may be in the range of 1:1 to 5:1, such as 1:1 to 3:1. The weight ratio of SA:DC-cholesterol may be in the range of 1:1 to 1:5, such as 1:2 to 1:4. In some embodiments, the weight ratio of DOPE:SA:DC-cholesterol may be 48:8:24.

[0114] In certain preferred embodiments, the lipids may consist of DOTAP, DOPE, and PEG lipids (such as mPEG2000-DSPE). The weight ratio of DOTAP:DOPE may be 1:2 to 2:1, such as about 1:1. The ratio of DOTAP:PEG lipid and DOPE:PEG lipid may be 10:1 to 5:1, such as about 7:1. The total weight ratio of total lipid to silicone may be 20:1 to 10:1, such as about 16:1.

[0115] The lipid component may be or may include an ionizable lipid. The term "ionizable lipid" refers to a lipid having a group that can be positively charged, typically having a Lewis base (hydrogen acceptor) head group attached to a hydrophobic tail via some spacer. Ionizable lipids typically include a head group that contains a tertiary amine moiety. Ionizable lipids may be neutral at physiological pH, but become cationic at lower pH, e.g., below pH 6.5, such as the pH found within the vacuole that is part of endosomal escape. Ionizable lipids are described, for example, in Nano Lett. 2020 Mar 11; 20(3): 1578-1589. Many vaccine platforms use lipid systems that are neutral at physiological pH, then endocytosed into endosomes, and become cationic only when the pH is reduced to about pH 6.2, and the lipids that become cationic then allow endosomal escape. A delivery system containing neutral lipids allows more particles to travel from muscle to lymph nodes and be phagocytosed by dendritic cells. Ionizable lipids can be stabilized by association with solid biocompatible particulate materials, for example by association with hydrolyzable silicones.

[0116] In certain embodiments, the lipid is selected from the group consisting of phosphatidylethanolamine (PE), phosphatidylcholine (PC), stearylamine (SA), or any combination thereof.

[0117] In certain embodiments, the lipid may consist essentially of phosphatidylcholine, hydrogenated phosphatidylcholine, stearylamine, or combinations thereof.

[0118] In certain embodiments, the lipid may be composed of at least 5% by weight of hydrogenated phosphatidylcholine, for example at least 20% by weight, typically at least 30% by weight, particularly at least 50% by weight of hydrogenated phosphatidylcholine, based on the total weight of the particle.The molar ratio of hydrogenated phosphatidylcholine to silicon of 0.8:1 to 5:1 has been found to be particularly advantageous, for example 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1 or 4.5:1.

[0119] In certain embodiments, the lipid may be composed of at least 5% by weight of phosphatidylcholine, for example at least 20% by weight, typically at least 30% by weight, in particular at least 50% by weight of phosphatidylcholine, based on the total weight of the particle. Molar ratios of phosphatidylcholine to silicon of 0.8:1 to 5:1 have been found to be particularly advantageous, for example 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1 or 4.5:1.

[0120] In certain embodiments, lipid can be composed of at least 5% by weight of stearylamine, for example at least 20% by weight, typically at least 30% by weight, particularly at least 50% by weight of stearylamine, based on the total weight of particle.The molar ratio of stearylamine to silicon of 0.8:1 to 5:1, for example 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1 or 4.5:1 has been found to be particularly advantageous.

[0121] In certain embodiments, the lipids may consist of PC and SA, preferably in a weight ratio of PC:SA of 1:1 to 20:1, more preferably 7:1 to 10:1, such as a weight ratio of PC:SA of 72:8.

[0122] In certain embodiments, the lipids may consist of DOPE, SA, and DC-cholesterol. The weight ratio of DOPE:SA may be in the range of 1:1 to 10:1, such as 4:1 to 8:1. The weight ratio of DOPE:DC-cholesterol may be in the range of 1:1 to 5:1, such as 1:1 to 3:1. The weight ratio of SA:DC-cholesterol may be in the range of 1:1 to 1:5, such as 1:2 to 1:4. In some embodiments, the weight ratio of DOPE:SA:DC-cholesterol may be 48:8:24.

[0123] Polycationic nucleic acid binding component The composition of the present invention optionally further comprises a polycationic nucleic acid binding component. The term "polycationic nucleic acid binding component" is well known in the art and refers to a polymer having at least three repeating cationic amino acid residues or other cationic units carrying positively charged groups, which can form a complex with nucleic acid under physiological conditions. An example of a nucleic acid binding polycationic molecule is an oligopeptide containing one or more cationic amino acids. Such an oligopeptide can be, for example, an oligolysine molecule, an oligohistidine molecule, an oligoarginine molecule, an oligoornithine molecule, an oligodiaminopropionic acid molecule or an oligodiaminobutyric acid molecule, or a complex oligomer containing or consisting of any combination of histidine, arginine, lysine, ornithine diaminopropionic acid, and diaminobutyric acid residues. Further examples of polycationic components include dendrimers and polyethyleneimines.

[0124] amino acid All aspects of the present invention may include the additional optional presence of one or more amino acids. In its broadest sense, the term "amino acid" encompasses any artificial or naturally occurring organic compound containing amine (-NH2) and carboxyl (-COOH) functional groups. The term "amino acid" includes alpha amino acids, beta amino acids, gamma amino acids, and delta amino acids. The term "amino acid" includes amino acids of any chiral configuration. According to some embodiments (e.g., when the silicon-containing particles of the invention are combined with one or more of PC, hydrogenated PC, SA, DOPE, DOTAP, DC-cholesterol, and derivatives thereof), the amino acid is preferably a naturally occurring alpha amino acid. It may be a proteinogenic or non-proteinogenic amino acid, such as carnitine, levothyroxine, hydroxyproline, ornithine, or citrulline. In a preferred embodiment, the amino acid comprises arginine, histidine, or glycine, or a mixture of arginine and glycine. In a particularly preferred embodiment, the amino acid comprises glycine. Such amino acids may function to stabilize the silicon particles and control hydrolysis of silicon, both during storage and in vivo.

[0125] Treating lipid-treated silicon particles with amino acid can also provide beneficial stabilizing effect on nucleic acid.Treating lipid-treated silicon particles with amino acid has been shown to stabilize nucleic acid in biological fluids, such as ocular tissue and plasma and tissue fluid.Lipid-treated particles formulated with amino acid in this manner can be particularly suitable for delivery to the body, for example, by transdermal injection, intravitreal injection.

[0126] peptide In addition to the amino acids listed above, all embodiments of the invention may include peptides that contain cell surface receptor, e.g., integrin recognition sequences, that confer some degree of cell specificity to the particle. The peptides may have a "head group" that contains the cell surface receptor recognition sequence and an additional "tail" that may non-covalently bind to nucleic acid (e.g., mRNA or pDNA) and / or may bind to silicon.

[0127] Ratio of amino acid(s) to silicon Preferably, when amino acid(s) are present, the ratio of amino acid to silicon is 0.05:1 to 2:1, for example 0.05:1 to 1.8:1, 0.05:1 to 1.6:1, 0.05:1 to 1.4:1, 0.05:1 to 1.2:1, 0.05:1 to 1:1, 0.05:1 to 0.9:1, 0.05:1 to 0.8:1, 0.05:1 to 0.6:1, 0.05:1 to 0.5:1, 0.05:1 to 0.4:1, 0.05:1 to 0.3:1, 0.05:1 to 0.2:1, preferably 0.2:1 to 0.8:1, in particular 0.3:1 to 0.7:1. The ratio of amino acids to silicon may be 0.05:1 to 0.4:1, for example 0.08:1 to 0.35:1, in particular 0.09:1 to 0.32:1. Advantageously, by adjusting the ratio of amino acids to silicon, the nucleic acid (for example, mRNA or pDNA) is further stabilized.

[0128] In some embodiments, the amino acids are a combination of arginine and glycine, with the ratio of Arg:Gly being 1:0.6-3:1, such as 1:0.8-2.5:1, such as 1:1-2:1.

[0129] According to another embodiment of all aspects of the invention the particles are formulated with arginine, preferably in a ratio of arginine to silicon of 0.05:1 to 0.4:1, such as 0.08:1 to 0.35:1, in particular 0.09:1 to 0.32:1.

[0130] According to another embodiment of all aspects of the invention the particles are formulated with glycine, preferably in a ratio of glycine to silicon of 0.05:1 to 0.5:1, such as 0.08:1 to 0.45:1, in particular 0.09:1 to 0.42:1.

[0131] Preferred amino acids for use in all aspects of the present invention include arginine, glycine, proline, lysine, and histidine, and mixtures of two or more thereof.

[0132] Non-reducing disaccharides All aspects of the invention optionally include compositions and related products and methods of at least one non-reducing disaccharide, optionally selected from sucrose, trehalose, raffinose, stachyose, and verbascose, or any mixtures thereof, and most preferably the non-reducing disaccharide is trehalose or a mixture comprising trehalose.

[0133] The non-reducing disaccharide (e.g., trehalose, or a mixture comprising trehalose) is optionally present in a weight ratio to the silicon of at least 1:1000, at least 1:100, at least 1:50, at least 1:10, at least 1:1, or at least 1:0.5. Preferably, the non-reducing disaccharide is trehalose, optionally present in a weight ratio to the silicon of at least 1:1000, at least 1:100, at least 1:50, at least 1:10, at least 1:1, or at least 1:0.5.

[0134] It is hypothesized that non-reducing disaccharides, especially trehalose, may act as dehydration protectants. Non-reducing disaccharides may act as cocoons to trap nucleic acid molecules within a glassy sugar matrix so that their movement is restricted by the sugar matrix. Trehalose may be particularly effective due to its ability to pass between one crystalline form and another without relaxing its structural integrity, and / or due to its particularly high glass transition temperature compared to other disaccharides such as sucrose. In addition, amorphous trehalose has localized pockets of crystalline dihydrate that trap residual water molecules and immobilize them, which is valuable for preventing nucleic acid degradation, especially when water is relatively scarce. Thus, it may enhance the water sequestration effect of silicon particles. An additional advantage of including a non-reducing disaccharide such as trehalose is that the presence of a non-reducing disaccharide facilitates the resuspension of pharmaceutical compositions that are powder materials.

[0135] Particularly preferred combinations According to all aspects of the invention, a particularly preferred embodiment relates to doping which is boron doping (in particular high concentration boron doping as defined above), wherein the nucleic acid is RNA, in particular mRNA encoding an antigen for an mRNA vaccine, and the lipid is or comprises an ionizable lipid, such as one or more cationic lipids, such as DOTAP, and / or one or more zwitterionic lipids, such as one or more phospholipids, such as DOPE; or both (e.g. the lipid comprises DOTAP, or the lipid comprises both an ionizable lipid and DOTAP).

[0136] Further components and features According to preferred embodiments of all aspects of the present invention, one or more further components may additionally be present, including a transfection reagent.

[0137] In its broadest sense, a "transfection agent" is an agent that facilitates the introduction of naked or purified nucleic acid into a eukaryotic cell. For example, some transfection agents are agents that facilitate the introduction of mRNA into a eukaryotic cell.

[0138] According to other embodiments of all aspects of the invention, the transfection reagent may be a lipofection (liposomal transfection) reagent, a dendrimer, HEPES-buffered saline (HeBS) containing phosphate ions combined with calcium chloride solution, or a cationic polymer such as diethylaminoethyl-dextran (DEAE-dextran) or polyethyleneimine (PEI).

[0139] In a preferred embodiment, the transfection reagent is a lipofection reagent, such as lipofectamine.

[0140] Pharmaceutical Compositions According to a third aspect of the present invention there is provided a pharmaceutical composition comprising the nucleic acid vector composition of the first aspect of the present invention, wherein the pharmaceutical composition is a vaccine composition.

[0141] In some embodiments, the nucleic acid (e.g., in vitro transcribed mRNA or pDNA) has a half-life at 4° C. in the pharmaceutical composition of at least 3 months, e.g., at least 4 months, 5 months, or 6 months.

[0142] In some embodiments, the pharmaceutical and vector compositions of the present invention are in a form suitable for intramuscular injection. The pharmaceutical and vector compositions of the present invention may include additives, including, but not limited to, preservatives, cryoprotectants, and immune adjuvants. As used herein, the term adjuvant may refer to a substance that modulates, e.g., increases, a subject's immune response to a vaccine composition. As non-limiting examples, an adjuvant may be or include one or more lipids, proteins, CpG oligodeoxynucleotides, and / or other molecular adjuvants. The pharmaceutical and vector compositions, in some embodiments, may include one or more buffer components. The pharmaceutical and vector compositions, in some embodiments, may include trometamol. The pharmaceutical and vector compositions, in some embodiments, may include trometamol hydrochloride. The pharmaceutical and vector compositions, in some embodiments, may include acetic acid. The pharmaceutical and vector compositions, in some embodiments, may include sodium acetate trihydrate. The pharmaceutical and vector compositions, in some embodiments, may include potassium chloride. The pharmaceutical and vector compositions, in some embodiments, may include potassium dihydrogen phosphate. The pharmaceutical and vector compositions, in some embodiments, may comprise sodium chloride. The pharmaceutical and vector compositions, in some embodiments, may comprise disodium hydrogen phosphate dehydrate. The pharmaceutical and vector compositions, in some embodiments, may comprise sucrose. It will be appreciated that the pharmaceutical vector compositions of the present invention may be diluted with saline prior to administration by intramuscular injection, if desired. In some embodiments, the pharmaceutical and vector compositions may be a powder. In some embodiments, the pharmaceutical and vector compositions may comprise a liquid.

[0143] In the pharmaceutical and vector compositions of the invention, nucleic acids (e.g., in vitro transcribed mRNA or pDNA) can be protected from enzymatic degradation by one or more particles that contain hydrolyzable silicon, which sequester or remove water as described herein.

[0144] Preparation of particles containing hydrolyzable silicon Particles for use in the present invention can be conveniently prepared by conventional techniques in the art, such as by milling processes or other known techniques for particle size reduction. Silicon-containing particles can be made from sodium silicate particles, colloidal silica, or silicon wafer materials. Macro-, micro-, or nano-scale particles can be ground in a ball mill, a planetary ball mill, or other size reduction mechanisms. The resulting particles can be air classified or sieved to recover particles of the required uniform size. It is also possible to use plasma methods and laser ablation for particle generation. Porous particles can be prepared by conventional methods in the art, including the methods described herein.

[0145] Preparation of particles containing doped hydrolyzable silicon Exemplary specifications for boron doped silicon for use in accordance with the present invention are: single-sided polished wafer, CZ diameter: 150±0.2 mm, orientation: (100)±1°, type: p / boron resistivity: 0.014±25% Ohmcm. cm3 Primary flat: 57.50±2.5mm Primary flat 1 position: D <100> ∼{110} Thickness: 675±15 μm Packing: Ultrapak Shipping Cassette TTV: ≦18 μm TIR: ≦5 μm Such boron doped silicon is commercially available, for example, from Nanografi, Jena, Germany, or Si-Mat, Germany.

[0146] Preparation of pharmaceutical and vector compositions The pharmaceutical and vector compositions of the present invention can be prepared by combining components. In some embodiments, this combining can simply include mixing solutions of the components, for example under conditions that result in their complexation. Combining can include contacting the nucleic acid with particles of silicon-containing material before adding lipid components. Adding lipid components after contacting the nucleic acid with silicon-containing particles can promote the formation of the lipid shell described herein that encapsulates the core, the core comprising both one or more particles and nucleic acid.

[0147] In some embodiments, it has been found to be advantageous, and is therefore preferred, for the silicon-containing particles of the present invention to be activated prior to contact with other components of the present invention. Activation can be carried out by dispersing the particles (e.g., porous doped silicon nanoparticles) in a volatile alcohol or other volatile solvent (such as chloroform, methanol, ethanol, or propanol, e.g., methanol) prior to contact with other components of the present invention.

[0148] Typically, the silicone particles are mixed with the non-reducing disaccharide, nucleic acid, if present, and optionally amino acids, and then contacted with the lipid components to form lipid silicone particles in which the nucleic acid is associated with the particle within the lipid compartment.

[0149] It is hypothesized that delivery systems comprising silicon-containing particles, in particular particles comprising porous hydrolyzable doped silicon, increase the stability of nucleic acids by sequestering water molecules that would otherwise react with the nucleic acid in a hydrolysis reaction catalyzed by enzymes present in the composition.

[0150] Freeze drying In certain embodiments, the pharmaceutical composition or vector composition is optionally lyophilized, optionally with a cryoprotectant and / or lyoprotectant, such as one or more sugars, such as sucrose and / or trehalose, to form, for example, a powder. Lyophilization removes water and allows the production of a dry powder. Such a dry powder can be optionally dispersed, for example, in a hydrogel. Other physical forms of the compositions of the present invention include liquid and frozen forms. In some embodiments, the composition can be provided in a delivery device, for example, an injection device, such as a syringe or multiple microneedles. EXAMPLES

[0151] Various aspects and embodiments of the present invention are illustrated with reference to the following non-limiting examples.

[0152] Example 1: Preparation of Silicon Nanoparticles (SiNPs) Single-side polished silicon wafers were purchased from Si-Mat, Germany. All cleaning and etching reagents were cleanroom grade. Etched silicon wafers were etched in a 1:1 (v / v) mixture of pure ethanol and 10% aqueous HF acid at 80 mA / cm for 2–10 min. 2 The samples were prepared by anodic etching of Si at an anodic current density of 1000 nm. After etching, the samples were rinsed with pure ethanol and dried under a stream of dry high-purity nitrogen before use.

[0153] Etched silicon wafers, either P+ type or N- type, were crushed using milling balls and / or a pestle and mortar. Silicon Wafer Specifications -Single-sided polished wafer, CZ -Diameter: 150±0.2mm -Orientation: (100)±1° -Type: p / boron -Resistivity: 0.014±25%Ohmcm. -5×10^ 18 atoms / cm 3 Close to - Primary flat: 57.50±2.5mm - Primary flat 1 position: D <100> ~{110} -Thickness: 675±15μm -Packing: Ultrapak Shipping Cassette -TTV:≦18μm -TIR:≦5μm The wafer has a porosity of approximately 40% and a thickness of less than 50 μm.

[0154] The above ingredients were milled manually using a ball mill or a pestle and mortar. The appearance before and after milling is shown in Figure 15. Figure 16 shows SEM images of milled silicon particles used for size assessment. Figures 17, 18, and 19 are TEM images of the particles (Figure 17, silicon alone; Figure 18, silicon particles + lipids and other components such as "Biocourier" (see below for definition) and siRNA (SIS0012); Figure 18, similar to Figure 17 but made with boron doped silicon as SIS0013).

[0155] The fine powder was sieved using a Retsch™ sieve shaker AS 200, 38 μm gauge. A uniform and selected particle size selection (20-100 μm) was achieved by the opening size of the sieve. Particle size was measured by quantachrome system and PCS from Malvern instruments. Samples were kept in a closed container until further use.

[0156] Nanosilicon powders were also obtained from Sigma Aldrich and Hefei Kaier, China. Before loading and etching, the particle size was measured by PCS and the size of the particles was recorded (the size ranged from 20 to 100 nm).

[0157] 500 mg of porous silicon nanoparticles with a diameter of 100 nm were mixed with 250 mL of ethanol and stirred with a magnetic bar for 30 minutes. The solution was then centrifuged at 3000 rpm for 30 minutes. The supernatant was discarded and the nanoparticles were washed with 5 mL of distilled water and transferred to a round-bottom flask. The contents of the flask were frozen (-25°C for 2 hours). The frozen nanoparticles were lyophilized overnight using a freeze dryer. The resulting dry powder activated the silicon nanoparticles.

[0158] Example 2: Preparation of lipid-functionalized siRNA-SiNP formulations material Activated silicon nanoparticles (SiNPs) prepared according to Example 1. Nuclease free water. Chloroform. Lipids: stearylamine (SA), catalogue number 305391 (Sigma-Aldrich); L-α-phospatidylcholine (PC) from egg yolk, catalogue number 61755 (Sigma-Aldrich); dioleoyl L-α-phosphatidylethanolamine (DOPE), catalog number P1223 (Sigma-Aldrich); N-(2-dimethylaminoethyl)carbamate cholesterol (DC-cholesterol), catalog number 92243 (Sigma-Aldrich). siRNA: non-specific NSC4 (customized siRNA duplex, Eurogentec); targeted siLUC (customized siRNA oligonucleotide duplex, Eurogentec); FAM-tagged siRNA (green siGlo, Dharmacon). device Rotary evaporation system, vortex, bath sonicator, water bath, round bottom flask, universal test tube, Eppendorf tube, micropipette, freeze dryer system, Zetasizer, high speed centrifuge, NanoDrop spectrophotometer, fluorescence reader.

[0159] procedure NOTE. For fluorescent siRNA (siGlo), light exposure was avoided during the procedure (e.g., cover flasks and tubes with aluminum foil).

[0160] Preparation of component 1: siRNA-SiNP mixture 1. Prepare a filtered solution of SiNPs (0.2 mg / mL) in nuclease-free water. 2. Dispense 700 μL of the above solution into 8 Eppendorf microtubes, each containing 140 μg of SiNPs. 3. Add siRNA and glycine to each aliquot and adjust the volume to 1.4 mL with nuclease-free water.

[0161] [Table 1]

[0162] 4. Mix the tube thoroughly and incubate the sample for 1 hour at room temperature with agitation.

[0163] Preparation of component 2: lipid film 1. Each lipid component (SA, PC, DOPE, DC-cholesterol) is dissolved in chloroform at a concentration of 0.2 mg / mL. 2. Transfer the desired amount of each lipid into a small round bottom flask and mix thoroughly. Prepare each lipid mixture in 8 replicates (in 8 flasks). a. Lipid base DS61 68 μg DOPE (340 μL) 12 μg SA (60 μL) B lipid base PS91 72 μg PC (360 μL) 8 μg SA (40 μL) c. Lipid-based DSC613 48 μg DOPE (240 μL) 8 μg SA (40 μL) 24 μg DC-cholesterol (120 μL) d. lipid base PDS1051 50 μg PC (250 μL) 25 μg DOPE (125 μL) 5 μg SA (25 μL) e. lipid base PDS1052 48 μg PC (240 μL) 23 μg DOPE (115 μL) 9 μg SA (45 μL) f. Lipid base PDSC10514 40 μg of PC (200 μL) 20 μg DOPE (100 μL) 4 μg SA (20 μL) 16 μg DC-cholesterol (80 μL) 3. Carefully evaporate the solvent using a rotary evaporation system to form a thin lipid film. Place the dried lipid under vacuum to remove any residual solvent.

[0164] Functionalization of siRNA-SiNPs (component 1) with lipids (component 2) 1. Dissolve the thin lipid film (component 2) with the siRNA-loaded nanoparticle sample or the empty Si nanoparticle sample (component 1). Add 200 μL of either the first, second, third, or fourth silicon nanoparticle / siRNA mixture to each flask containing the lipid film (a, b, c, d, e, f).

[0165] [Table 2]

[0166] 2. Adjust the volume of the solution to 400 μL by adding 200 μL of nuclease-free water to each flask. 3. Cover each flask with parafilm, mix contents thoroughly and incubate at room temperature for 1 hour. 4. Vortex to completely dissolve the lipid film. Bath sonicate the flask for 15 seconds to aid in dissolving the lipids. 5. Transfer the entire contents of each flask to separate Eppendorf microtubes. 6. Place all tubes in the freezer (-20°C) and keep for at least 3 hours (or overnight). 7. Remove samples from the freezer and place them in a 30° C. water bath for 10 minutes. Cool at room temperature and vortex thoroughly. 8. Repeat the freeze-thaw process (steps 5 and 6) two more times. 9. All samples are kept in the freezer (-20°C) until assayed. 10. Each load sample contains [10 μg siRNA: 20 μg SiNP (silicon nanoparticles): 80 μg lipid base: 10 μg glycine] in a 400 μL volume.

[0167] The components of the resulting formulation are shown in Table 3 below.

[0168] [Table 3]

[0169] Preparation of amino acid, lipid and lipofectamine-loaded siRNA nanoparticles Formulations SIS005-PS91 and SIS005-DS61 (containing glycine) were each prepared by dissolving a thin film of liposome-forming material in an aqueous suspension of silicon nanoparticles containing siRNA. The mixtures were then triple-frozen and thawed and tested for transfection efficiency on HCES cells in vitro. In parallel, the SIS005-PDS1051 formulation was tested after further modification with the aim of introducing more positive charges (shown to have a negative zeta potential) that would be favorable for an siRNA delivery system for the cornea. For this purpose, the proportion of cationic lipids in the formulation was increased.

[0170] Colloidal stability was assessed by dynamic light scattering in parallel with zeta potential measurements for both unloaded and loaded formulations. Additionally, encapsulation efficiency of siRNA was measured by spectrophotometry. Transfection efficiency (internalization into cells) was assessed in human corneal epithelial cells by flow cytometry, followed by a dual luciferase assay to measure in vitro knockdown after treatment with siRNA-loaded formulations.

[0171] Characterization of silicon-nanoparticle / lipid formulations Encapsulation Efficiency siRNA encapsulation efficiency was examined by separating the loaded particles from free (unbound) siRNA by high speed centrifugation and then measuring the encapsulation efficiency.

[0172] Collect 1.50 μL of each sample into a centrifuge microtube. 2. Centrifuge all samples at 21,000g for 30 minutes. 3. Transfer 25 μL (top half) of supernatant from each sample to a separate tube (designated as S1) and store at 4° C. (avoid light exposure for siGlo-loaded samples) until assayed. 4.25 μL of 2% SDS is added to the pellet sample (including remaining supernatant) to disrupt the lipid bilayer and release bound siRNA. 5. The tubes are centrifuged again at 21,000 g for 30 minutes and the supernatant (denoted as S2) is collected. 6. Measure the optical density (OD) at 260 nm of all S1 and S2 supernatant samples using a NanoDrop spectrophotometer. For siGlo-loaded samples, measure the fluorescence intensity (FI) of both S1 and S2 supernatant samples. 7. Calculate the encapsulation efficiency (EE%):

[0173]

number

[0174] In the formula, O.D. S1 - Absorbance of Supernatant S1 (260 nm) (after the first centrifugation) O.D. S2 - Absorbance (260 nm) of the supernatant S2 (after the second centrifugation) For siGlo-loaded samples, OD S1 and O.D. S2 Instead of FI, S1 and F.I. S2 Use. 8. The concentration of siRNA in the supernatant samples and the amount of siRNA entrapped by the silicon nanoparticle formulation can be determined using the siRNA calibration curve (as described above).

[0175] Zetasizer measurement Colloidal stability was assessed by dynamic light scattering in parallel with zeta potential measurements, which were performed on unloaded (empty) formulation samples and on samples loaded with siLUC / NSC4. 1. Collect 200 μL of each sample and dilute with nuclease-free ultrapure water to a total volume of 1 mL. 2. Load the sample into the folded capillary cell. 3. Read the samples using the Zetasizer: particle size, polydispersity index, and zeta potential. Each measurement is performed in triplicate. The data are shown in Figures 1, 2, 3, and 4.

[0176] Transfection efficiency in HCES cells The transfection efficiency (internalization into cells) was evaluated in human corneal epithelial cells by flow cytometry. For the purpose of this study, we use a formulation sample loaded with a fluorescently tagged siRNA probe (siGlo). 1. 2 x 10 cells in 1 mL of DMEM enriched with 10% FBS 5 HCES cells / well (on a 12-well plate) are seeded and grown in standard conditions for 24 hours (until 80% confluence). 2. Replace the medium with 950 μL / well of fresh DMEM enriched with 10% FBS. 3. Allow the siGlo-loaded silicon nanoparticle / lipid sample to adjust to room temperature. Prepare a control sample using Lipofectamine transfection reagent by mixing 4.6 μL of Lipofectamine RNAiMAX Reagent with 3 μL of siGlo stock solution (100 μM) in 151 μL of OptiMEM and incubating at room temperature for 15 minutes. 5. Treat cells in 3 replicates by adding 53.3 μL per well of each siGlo loading formulation or LPF control to obtain a siGlo concentration of 0.1 μM. 6. Grow cells under standard conditions (37° C., 5% CO2) for 24 hours. 7. Discard the medium and wash the wells with 500 μL of PBS. 8. Add 300 μL of Trypsin-EDTA and incubate the plate at 37° C. for 10 minutes. 9. Immediately add 300 μL of DMEM enriched with 10% FBS to stop trypsinization. 10. Transfer cells to separate microfuge tubes and centrifuge at 1000-2000 rpm for 5 minutes. 11. Discard the supernatant and carefully suspend the cells in 500 μL of PBS. 12. Centrifuge the samples at 1000-2000 rpm for 5 minutes. 13. Discard the supernatant and carefully resuspend the cells in 600 μL of FACS buffer containing PI dye. 14. Analyze the samples using a flow cytometer.

[0177] Knockdown efficiency To measure the efficiency of knockdown induction, dual luciferase assays were performed with test formulation samples loaded with specific formulation (siLUC), non-specific formulation (NSC4), and unloaded formulation.

[0178] 1. 6.5 x 10 cells per well in 100 µL of DMEM enriched with 10% FBS 3 HCES cells (on 96-well plates) are seeded and grown for 24 hours in standard conditions. Duplicate plates are prepared in 5 replicates each for studying all formulation samples. 2. Transfect the cells with Renilla and Luc2p plasmids using Lipofectamine 2000 according to the manufacturer's protocol. Add 50 μL of reagent mixture to each well, containing 0.3 μL Lipofectamine 2000, 1 ng Renilla plasmid, and 5 ng Luc2p plasmid diluted in OptiMEM medium to a total volume of 50 μL. 3. Cells are grown for 24 hours at 37° C., 5% CO2. 4. Replace the medium with 90 μL of fresh DMEM per well. 5. Allow formulation samples to reach room temperature. Mix 32 μL of each sample with 28 μL of OptiMEM. 6. Prepare control samples using Lipofectamine transfection reagent. Mix 1.2 μL of Lipofectamine RNAiMAX reagent with 55.8 μL of OptiMEM and incubate at room temperature for 5 min, then add 3 μL of siLUC stock solution and continue incubation for 10 min. Mix 1.2 μL of Lipofectamine RNAiMAX reagent with 55.8 μL of OptiMEM and incubate at room temperature for 5 min, then add 3 μL of NSC4 stock solution and continue incubation for 10 min. Mix 1.2 μL of Lipofectamine RNAiMAX Reagent with 58.8 μL of OptiMEM and incubate at room temperature for 15 minutes.

[0179] 7. Cells are treated by adding 10 μL per well of load formulation or blank formulation or LPF control in 5 replicates. 8. Grow cells under standard conditions (37°C, 5% CO2). 9. After 48 hours, discard the medium and wash the wells with PBS. 10. Add 20 μL of Passive Lysis Buffer per well. 11. Incubate the plate on an orbital shaker (900 rpm) at room temperature for 15 minutes. 12. Read luminescence levels using the Dual-Luciferase Reporter Assay kit and a LUMIstar OPTIMA plate reader according to the manufacturer's protocol. The results are shown in Figure 5.

[0180] Example 3: Combination of siRNA-loaded silicon nanoparticles with Lipofectamine (SIS005-LPF) 1. Aliquot the silicon nanoparticle suspension into 6 Eppendorf microtubes, each containing 20 μg SiNPs. 2. To the above SiNP aliquot, add 10 μg of siRNA: Add 37 μL of 20 μM siLuc2p = target siRNA to two microtubes b. Add 37 μL of 20 μM NSC4 to two microtubes = non-specific siRNA c. Unloaded control: Leave the remaining two microtubes containing SiNPs without siRNA to prepare an empty control. 3. Incubate the samples with agitation at room temperature for 1 hour, then vortex and place the tubes in the freezer (-20°C) and store for 2-3 hours. 4. Connect the samples to the freeze drying system for overnight free drying. 5. Prior to further analysis, siRNA-loaded (or unloaded control) SiNP samples are dissolved in Lipofectamine solution (Lipofectamine RNAiMAX Reagent, Cat. No. 13778075, ThermoFisher Scientific), diluted with nuclease-free water to a total volume of 150 μL, vortexed, and incubated at room temperature for 1 h.

[0181] [Table 4]

[0182] SIS005-LPF1 and SIS005-LPF2 used lipofectamine (as lipid) in combination with silicon nanoparticles in two different ratios. LFP1 used 15 μL of 20 μM lipofectamine solution combined with 20 μg silicon nanoparticles. LPF2 used twice the amount of lipofectamine. The purpose of this experiment was to demonstrate that the combination of lipofectamine and silicon particles can boost transfection facilitated by lipofectamine. The results showed that silicon nanoparticles improved transfection by lipofectamine.

[0183] siRNA loading had the greatest effect on the surface charge of stearylamine-treated silicon nanoparticles, inducing a high negative charge. As previous analysis showed, SA and SA + arginine loaded blank Si-NPs had positive or nearly neutral surface charges, and therefore they effectively attracted anionic siRNA molecules.

[0184] Example 4: Effect of loading ratio on mRNA capture efficiency Samples were prepared according to the protocol described above for SIS005-DSC613G (Example 2). Each sample consisted of silicon nanoparticles:dioleoylphosphatidylethanolamine (DOPE):stearylamine:cholesterol 3β-N-(dimethylaminoethyl)carbamate hydrochloride (DC-cholesterol):glycine in a weight ratio of 10:24:4:12:5. Samples were prepared with different ratios of nucleic acid to silicon nanoparticles, as shown in Table 5 below.

[0185] [Table 5]

[0186] Example 5: Evaluation of mRNA capture by silicon nanoparticle formulations using gel electrophoresis The effect of the loading ratio of silicon nanoparticles to mRNA on the mRNA capture efficiency was examined by gel electrophoresis. 1% E-Gel EX precast agarose gels were used. Gels were visualized using a Gel Logic 100 Imaging System (Kodak). The results are shown in Figure 6.

[0187] FIG. 6 shows unloaded mRNA (U0) in line 1. Silicon nanoparticles loaded with mRNA (L0.5, L1, L2, L3, L4, L5, L6, L8) are in lines 2-9 in increasing silicon nanoparticle to mRNA ratio from left to right. Equal amounts of mRNA (100 ng) were loaded in each of lines 1-9. Invitrogen E-Gel 1 Kb Plus Express DNA Ladder (80 ng) was loaded in line M as a marker. Line 10 was left empty (as a water blank).

[0188] Electrophoresis demonstrated that the SIS005-DSC613G formulation of the present invention successfully captured mRNA, especially at higher silicon nanoparticle to mRNA ratios, such as ratios above 2:1. The control lane in FIG. 6 (unloaded mRNA, U0) shows a single fast-migrating band as expected. A band was also seen in line 2 (loaded L0.5) and line 3 (loaded L1). This visible band corresponds to unbound mRNA. The intensity of the bands in lines 2 and 3 (especially line 3) was lower compared to the control (U0). This indicates that even in samples with very low silicon nanoparticle to mRNA ratios, some mRNA is still captured by the silicon nanoparticles and is therefore not visible in the band.

[0189] Efficiently captured mRNA cannot migrate through the gel pores and remains in the well (as no band appears, unlike U0, L0.5, and L1). Figure 6 shows that samples L2-L8, which have an increased content of silicon nanoparticles compared to lines 1-3, successfully captured mRNA. This is evidenced by the absence of bands seen in U0, L0.5, and L1. This demonstrates that the silicon nanoparticle formulations of the present invention can successfully capture mRNA. The results suggest that the optimal loading ratio (the ratio at which the capture of mRNA is maximal while minimizing the amount of nanoparticles used) is L2, corresponding to ratios of 2:1 (silicon nanoparticles:mRNA) and 11:1 (all other components of the delivery system:mRNA). The same loading ratios were found to be effective for siRNA loading. This ratio corresponds to an N / P charge ratio of approximately 2.5.

[0190] Example 6: Evaluation of capture efficiency using spectrophotometric measurements To estimate the capture efficiency of mRNA capture (EE expressed as a percentage), SIS005-DSC613G samples (U0-L8) were also centrifuged to separate unbound mRNA. The nucleic acid content in the supernatant was measured by spectrophotometry, and the capture efficiency was calculated using the following formula:

[0191]

number

[0192] O.D. unloaded is the absorbance of the unloaded mRNA control (U0). loaded is the absorbance of each of the samples L0.5 to L8.

[0193] The results are shown in Figure 7 and confirm the results of the gel electrophoresis experiments. As the ratio of silicon nanoparticles to mRNA increases, the capture efficiency increases until it reaches a plateau and then levels off at silicon to mRNA ratios above 2:1.

[0194] Example 7: Evaluation of the activity of silicon nanoparticle formulations in vivo Samples were prepared according to the protocols of SIS005-PS91G and SIS005-DSC613G in Example 2 above, containing the following weight ratios of siRNA, silicon nanoparticles, lipids, and glycine:

[0195] [Table 6]

[0196] SA is stearylamine, DOPE is dioleoylphosphatidylethanolamine, PC is phosphatidylcholine, and DC-cholesterol is cholesteryl 3β-N-(dimethylaminoethyl)carbamate hydrochloride.

[0197] Samples containing specific siRNA (siLUC) and non-specific siRNA (NSC4) were prepared. Both siRNAs (siLUC and NSC4) were designed as 21-mers with a central 19 bp duplex region and symmetric dTdT dinucleotide overhangs at each 3' end. siRNAs were provided by Eurogentec (Belgium).

[0198] To prepare the samples, siRNA dissolved in nuclease-free water was added to the aqueous solution of nanoparticles and incubated for 60 min at room temperature. A 2:1 ratio of silicon nanoparticles to mRNA was used (as this was found to be the optimal ratio in gel electrophoresis and spectrophotometric experiments, see above).

[0199] Live animal imaging Animals were used for the following experiments with ethical approval by the Home Office (Scotland) and the Department of Health, Social Services and Public Safety (Northern Ireland) in accordance with the UK Animal Welfare Act. Experiments to evaluate delivery of fluorescent siRNA (DY-547-labeled siGLO, Dharmacon, UK) to the cornea were performed in wild-type C57BL / 6 mice. To evaluate the siRNA bioavailability and silencing activity of the formulations, a reporter knock-in mouse line (Krt12+ / luc2) was used, expressing firefly luciferase specifically in the corneal epithelium (under the control of the endogenous Krt12 promoter). This animal model was developed on a C57BL / 6 background as previously reported and provides a reliable model for the in vivo evaluation of siRNA delivery methods using reporter gene expression monitoring. For in vivo imaging, mice were anesthetized with 1.5–2% isoflurane (Abbott Laboratories Ltd., UK) under oxygen flow of approximately 1.5 L / min. At the measured time points after topical application, siGlo fluorescence was detected by a Xenogen IVIS Spectrum with LivingImage 3.2 software (both Perkin Elmer, UK) using a DsRed filter combination (excitation 535 nm, emission 570 nm). To measure luciferase reporter gene expression, luciferin (30 mg / mL D-luciferin potassium salt; Gold Biotechnology, USA) mixed 1:1 w / w with Viscotears gel (Novartis, UK) was instilled into the eyes of anesthetized mice immediately prior to imaging. Bioluminescence readings were acquired by the IVIS Spectrum for approximately 10 minutes and quantified using LivingImage software after ensuring that the signal remained stable within the acquisition time. For quantification of signal intensity, regions of interest (ROIs) were selected separately for each eye keeping constant ROI parameters (size and shape) throughout the experiment.Values ​​are expressed as right eye / left eye ratio (RE / LE) using a split-body controlled measurement regime.

[0200] In vivo siRNA treatment Split body control experiments were performed by treating one eye of the same animal under test and comparing it with the other eye, which was a negative control. During treatment, mice were anesthetized as described above. Silicon nanoparticle formulations containing 25 μM siRNA complexed at a weight ratio of 2:1 SiNP to mRNA were prepared and applied topically as drops to the intact cornea in a total volume of 4 μL per eye. After application, mice were kept anesthetized for an additional 15 minutes to allow absorption and maximize uptake. After treatment, fluorescence and luminescence experiments were performed as described below.

[0201] Assessment of siRNA penetration into the cornea To investigate the delivery of siRNA to the cornea, in vivo fluorescence studies were performed in wild-type mice using eye drops containing siGlo. Fluorescent siRNA silicon nanoparticle formulations were applied to the right eye, while the same amount of naked siGlo was topically applied to the left eye of each mouse as a control. Fluorescent live imaging was acquired using an IVIS Spectrum 15 minutes after siGlo application (i.e., immediately after the treatment procedure) and 3, 6, and 24 hours later, and signal intensity was normalized to background fluorescence measured before treatment (i.e., untreated eye) and quantified as previously described. After measurements at either 3 or 24 hours, mice were sacrificed and eyes were enucleated and fixed in 4% paraformaldehyde in PBS for 30 minutes at room temperature, immersed in PolyFreeze (Sigma-Aldrich, UK), and immediately frozen at -80°C. Five-micrometer sections were cut using a cryostat (CM 1850, Leica) and mounted on APES-coated slides (3-aminopropyltriethoxysilane, Sigma Aldrich, UK) with DAPI-containing mounting medium (DAPI I, Vysis, USA), and fluorescence was visualized using an AxioScope A1 microscope equipped with a 20x / 40x N Archoplan lens on an AxioCam MRc camera (Carl Zeiss, Germany).

[0202] Evaluation of siRNA-mediated gene silencing Luciferase reporter mice (n=7) were used to measure the bioavailability of siRNA in the cornea after topical delivery with silicon nanoparticles of the invention. In split-body control experiments, luciferase-targeted siLuc complexed with silicon nanoparticle formulations was applied topically as drops to the intact cornea of ​​the right eye (RE) of anesthetized mice, while the left eye (LE) was similarly treated with NSC4 complexed with silicon nanoparticle formulations as a negative control. Treatments were repeated daily for 8 consecutive days, and in vivo ocular luminescence measurements were performed approximately 4-5 hours later. The effect of treatment on luciferase reporter gene expression was confirmed by measuring luciferase bioluminescence activity (as above) daily throughout the treatment regimen and for an additional 8 days after treatment cessation to monitor the washout period. A baseline luminescence was defined for each experimental animal by monitoring luciferase activity in the eye at 24-h intervals for 4 days prior to treatment. Relative RE / LE luciferase bioluminescence activity was quantified using IVIS LivingImage software and plotted as mean values ​​± standard deviation.

[0203] statistical analysis Data are presented as mean ± 1 standard deviation and represent representative values ​​of at least three independent measurements unless otherwise stated. Statistical significance was assessed by one-way or two-way ANOVA followed by Tukey's HSD post-hoc test at the 95% confidence level. For in vitro dual luciferase assays, two-tailed Student's t-tests were performed separately for each formulation to analyze knockdown levels (siLuc vs. NSC4 control). For in vivo luciferase experiments, statistical analysis was performed by comparing the mean right / left ratio for all seven mice in the first 4 days before the start of treatment (set as baseline) with the R / L ratio measured on the subsequent days. Statistical analysis was performed using GraphPad Prism software (GraphPad Software, USA).

[0204] result Characteristics of silicone-based siRNA delivery systems Two replicates of the silicon nanoparticle delivery system of the present invention (SIS005-PS91G and SIS005-DSC613G) were formulated by surface functionalization of silicon with cationic lipids, stearylamine and DC-cholesterol, commonly used for nucleic acid delivery, which resulted in hybrid particles with similar hydrodynamic sizes of about 350 nm and relatively positive zeta potential values. Complexation of cationic silicon nanoparticle formulations with siRNA, studied by gel electrophoresis, showed complete entrapment of nucleic acids at a minimum SiNP-siRNA w / w ratio of 2:1. The percentage of complexed siRNA for various w / w ratios was measured by spectrophotometry and calculated from the difference in the amount of siRNA added to the carrier and the concentration of siRNA in solution after particle separation. Higher siRNA entrapment efficiency was observed for complexation with nanoparticles containing cationic cholesterol derivatives compared to particles functionalized with stearylamine. However, both variants showed siRNA loading capacities in the range of 13-48 nmol per mg of silicon nanoparticles. Following the siRNA loading studies described above, a fixed SiNP / siRNA ratio of 2:1 was selected for all further experiments.

[0205] Since the physicochemical properties of nanoparticles play an important role in drug delivery, the particle size and surface charge of the siRNA-loaded complexes were measured. SIS005-DSC613G showed no significant difference in size or zeta potential when comparing the empty and loaded carriers, whereas SIS005-PS91G showed an increase in average particle size and negative surface charge when complexed with siRNA, suggesting the absorption of nucleic acid molecules on the surface of the hybrid particles in addition to internal siRNA entrapment (see table below). The formulations examined in this study were compared to the gold standard for gene silencing, Lipofectamine™ RNAiMAX, a commercially available lipid-based carrier specifically designed for siRNA delivery. Zetasizer analysis of empty siRNA-loaded RNAiMAX also showed an increase in particle size and a reversal of surface charge from positive to negative values ​​after complexation with nucleic acids.

[0206] [Table 7]

[0207] Example 8: Evaluation of in vitro siRNA delivery to corneal cells For initial in vitro screening, a human corneal epithelial cell line (HCE-S) was used to evaluate the efficiency of the silicon nanoparticle delivery system of the present invention in cell transfection, along with its potential cytotoxicity. Transfection efficiency was quantified by flow cytometry analysis performed 24 hours after treatment with fluorescent oligonucleotide duplexes loaded onto a silicon carrier system (see FIG. 8). SIS005-DSC613G showed 55±2% and SIS005-PS91G showed 65±6% FAM-positive cells, compared to 84±1% observed for the RNAiMAX reagent.

[0208] Cell viability after transfection was assessed based on live / dead staining with propidium iodide (PI), a common indicator of membrane disruption. This showed that the silicon nanoparticle formulation was well tolerated by corneal epithelial cells, in contrast to lipofectamine (see FIG. 9). Although lipofectamine is highly effective in delivering exogenous nucleic acids to cells in vitro, it is not suitable for clinical use. More than 50% of the cells transfected with lipofectamine RNAiMAX were shown to have damaged membranes, as evidenced by internal staining with the membrane-impermeable dye PI, whereas more than 86% and more than 98% of the cells after treatment with silicon nanoparticle formulations were observed to be intact cells.

[0209] The bioavailability of siRNA was evaluated in gene expression studies using a dual luciferase reporter assay. After treating HCE-S cells with 0.1 μM siLuc complexed with a silicon-based delivery system, the knockdown achieved by SIS005-PS91G and SIS005-DCS613G was 46±5% (p<0.01, siLuc vs. NSC4 control) and 38±8% (p<0.01), respectively, while siLuc transfected with RNAiMAX reduced luciferase reporter gene expression by 66±9% (p<0.001). See FIG. 10. Thus, the silicon nanoparticle delivery system was demonstrated to be up to 70% as effective as commercial siRNA transfection reagents, while being safer and better tolerated by cells.

[0210] Example 9: Evaluation of in vivo ocular siRNA delivery via topical silicon nanoparticle formulations Following the demonstration of successful siRNA delivery and gene knockdown in vitro, two silicon nanoparticle formulations (SIS005-PS91G and SIS005-DCS613G) were evaluated in vivo by topical administration to the anterior segment of the eye. First, SIS005-PS91G and SIS005-DCS613G were complexed with fluorescent siGlo and applied as eye drops to wild-type mice after unilateral treatment with naked siGlo control instilled in the contralateral eye. Ocular fluorescence was monitored for up to 24 hours using an in vivo imaging system. The first measurement was taken 15 minutes after administration while the mice were still under anesthesia after treatment, and the following measurements were repeated at 3, 6, and 24 hours after administration. Equal amounts of siGlo were topically applied to each eye, and measured after 15 minutes, the maximum fluorescence intensity was observed in the SIS005-DSC613G treatment group, slightly lower in SIS005-PS91G, and 2-fold lower in naked siGlo (p<0.05) (see FIG. 11). This indicated increased ocular surface adhesion of the formulated drug compared to the naked oligonucleotide, and improved residence time for the two silicon nanoparticle formulations. After 3 hours, the in vivo fluorescence signal was reduced 3-fold in siGlo-SIS005-DSC613G treated eyes due to active ocular clearance mechanisms, while returning to baseline levels for siGlo-SIS005-PS91G and unformulated siGlo eye drops. Although a further gradual decrease in in vivo signal intensity was observed, the fluorescence in eyes treated with siGlo-SIS005-DSC613G persisted up to 24 hours and was significantly higher than eyes treated with unformulated naked siGlo at all time points (p<0.01 at 3 and 6 hours, p<0.05 at 24 hours). This suggests that the topically administered siRNA drug formulated with the silicon nanoparticles of the present invention was effectively taken up. To verify nanoparticle penetration into tissues, the distribution of siRNA throughout the corneal layers was examined by fluorescence microscopy of corneal sections after treatment. Red siGlo fluorescence was detected throughout all corneal layers in all treated sections of eyes collected 3 hours after eye drop application, while no fluorescence above background was observed in the naked siGlo control.24 hours after administration of the siRNA formulation, fluorescence was observed only in corneal sections treated with SIS005-DSC613G.

[0211] Following in vivo uptake studies, SIS005-DSC613G siRNA delivery was further investigated in functional assays using a mouse reporter model with luciferase expression exclusively restricted to the corneal epithelium. Prior to in vivo treatment, basal corneal luciferase activity in reporter mice was quantified every 24 h for 4 days to confirm consistent left-right ratios in split-body control experiments. SIS005-DSC613G complexed with siLuc or control siRNA was applied topically as eye drops to the contralateral eye of the same animals 8 times at daily intervals, and corneal luciferase expression was assessed daily by imaging in live animals throughout the treatment regimen and for 8 days thereafter. A decrease in luciferase expression was observed within 24 h of treatment initiation, with maximum inhibition (41% ± 13, p < 0.001) achieved on day 11. Significant gene silencing effects persisted throughout the treatment regimen and continued for 4 days after treatment termination. As expected, the reduced bioluminescence levels in the eyes gradually returned to baseline after treatment was discontinued, indicating successful recovery from gene silencing. See Figures 12 and 13 for these results. Importantly, gross examination of the treated eyes and daily visual inspection of the animals after topical treatment revealed no adverse effects from the eye drops, suggesting that the silicon nanoparticle formulation was well tolerated in vivo.

[0212] Increased nucleic acid binding efficiency of Si-containing formulations compared to known liposomes Sample formulations containing silicon nanoparticles were prepared according to the protocol above. Formulations without silicon nanoparticles were also prepared. The formulations had the compositions shown in the table below.

[0213] [Table 8]

[0214] Each of these compositions was examined for nucleic acid binding efficiency to hsDNA, and the results are shown in Figure 14. As Figure 14 shows, the binding efficiency observed for the Si nanoparticle-containing formulations showed improvement over the liposomal formulations without silicon nanoparticles.

[0215] Example 10: Further investigations with Biocourier formulations "Biocourier" as used in these examples refers to a vector composition according to the invention, comprising silicone, lipids, and other components as optionally used according to the invention, onto which nucleic acids may be loaded.

[0216] Preparation of starting materials (a) Non-activated boron-doped silicon wafer: obtained from BS Silicon and manually milled (b) Non-activated AE silicon: provided by American Elements, average diameter 30 nm, porous (c) Activated AE silicon: American Elements, average diameter 30 nm, porous. 1 g of material was suspended in 50 mL of MeOH and then subjected to a slow evaporation process under a fume hood. (d) Activated BS silicon: porous. 1 g of material was suspended in 50 mL of MeOH and then subjected to a process of slow evaporation under a fume hood. (e) DOTAP-Cl solution: DOTAP was dissolved in methanol at a concentration of 5 mg / mL. In particular, 50 mg of DOTAP was dissolved in 10 mL of methanol and sonicated until completely dissolved. (f) DOPE solution: DOPE was dissolved in methanol at a concentration of 5 mg / mL. In particular, 60 mg of DOPE was dissolved in 12 mL of methanol and sonicated until completely dissolved. (g) mPEG2000-DSPE solution: mPEG2000-DSPE was dissolved in methanol at a concentration of 5 mg / mL. In particular, 40 mg of mPEG2000-DSPE was dissolved in 8 mL of methanol and sonicated until completely dissolved. (h) Trehalose (THR): Powder provided by Sigma Aldrich. (i) Glycine (GLY): Powder provided by Sigma Aldrich. (j) SiNP AE+GLY+THR solution: Activated SiNPs (AE), glycine, and trehalose were suspended in nuclease-free water. In particular, 50 mg of activated SiNPs, 50 mg of trehalose, and 25 mg of glycine were suspended in 50 mL of nuclease-free water and sonicated for 60 min. (k) SiNP BS+GLY+THR solution: Activated SiNP(BS), glycine, and trehalose were suspended in nuclease-free water. In particular, 50 mg of activated SiNP, 50 mg of trehalose, and 25 mg of glycine were suspended in 50 mL of nuclease-free water and sonicated for 60 min.

[0217] Preparation of pDNA-Biocourier aliquots for immunization studies Select Biocourier type and calculate ratio for optimal pDNA complexation Biocourier Formulation Type 1 prepared with American Elements (AE) silicone (porous, activated, average particle size <100 nm). The ingredients and proportions are as follows:

[0218] [Table 9]

[0219] Lipid:Si (before extrusion) ratio = 16:1 16mg total lipid / 10mL Biocourier, 1.6mg / mL lipid, and 0.1mg / mL silicon, equivalent to: Biocourier stock solution=1.6μg / μL pDNA stock solution=2μg / μL Each vial must contain: 80 μg pDNA Total volume of 400 μL 80 µg pDNA equivalent to 40 µL - remaining volume = 400 µL (total volume) - 40 µL (pDNA volume) = 360 µL 360μL can contain up to 1.6μg / μL x 360μL = 576μg of Biocourier Biocourier:pDNA ratio = 576:80 = 7.2:1 Using this format, 50 μL (single dose / mouse) of SiSaf Biocourier / pDNA complex contains: 10 μg pDNA and 72 μg Biocourier

[0220] All samples are provided as extruded samples suspended in nuclease-free water. Extrusion is considered the gold standard technique for preparing injectable samples and refers to passing suspended particles through filter membranes multiple times to ensure that the particles are not unacceptably aggregated.

[0221] Biocourier injectable id / im was extruded through 400 nm and 100 nm polycarbonate membranes and ready to be complexed with a substantial amount of pDNA.

[0222] Preparation of Biocourier Formulation Type 1: Liquid Form (A) Lipid film preparation (1) Transfer the lipids in the amounts shown in Table 9 above from the stock solutions into a clean glass round-bottom flask and mix. (2) Carefully evaporate the solvent using a rotary evaporator with a 40° C. water bath and vacuum. (B) Rehydration of the film (3) Add the Si-NPs+GLY+THR solution to the film and adjust the final volume to 10 mL with nuclease-free water, if necessary. (4) Cover the flask with Parafilm, rehydrate the film, and agitate the flask in a water bath (60°C) for 5 minutes. (5) Divide 1 mL of sample into a total of 10 Eppendorf tubes without RNA. Store the suspension in the refrigerator. (C) Extrusion process (6) The suspension is passed through membrane filters with pore sizes of 0.4 μm and 0.1 μm for 10 cycles, then through 0.1 μm for 10 cycles, at 60° C. After extrusion is complete, the suspension is stored in a refrigerator. (D) Loading of Biocourier with pDNA (7) Thaw the pDNA samples stored at -40°C and leave the tubes under a fume hood until they reach room temperature (estimated time: 10 minutes). (8) Equilibrate the extruded Biocourier sample by transferring it from the refrigerator to a fume hood and placing it at room temperature (estimated time 6 minutes). (9) Load an amount of Biocourier with pDNA into a sterile Eppendorf tube. In particular, mix 360 μL of Biocourier with 40 μL of pDNA solution. Seal the Eppendorf. (10) Gently vortex the Eppendorf for 15 seconds and leave the sealed sample to equilibrate at room temperature for 30 minutes. Transfer the sample to the refrigerator until use. Follow any pre-injection instructions provided. Prepare four aliquots as above.

[0223] Preparation of Biocourier Formulation Type 1: Lyophilized Form (A) Lipid film preparation (1) Transfer the lipids in the amounts shown in Table 9 above from the stock solutions into a clean glass round-bottom flask and mix. (2) Carefully evaporate the solvent using a rotary evaporator with a 40° C. water bath and vacuum. (B) Rehydration of the film (3) Add the Si-NPs+GLY+THR solution to the film and adjust the final volume to 10 mL with nuclease-free water, if necessary. (4) Cover the flask with Parafilm, rehydrate the film, and agitate the flask in a water bath (60°C) for 5 minutes. (5) Divide 1 mL of sample into a total of 10 Eppendorf tubes containing no RNA. Store the suspension in the refrigerator. (C) Extrusion process (6) The suspension is passed through membrane filters with pore sizes of 0.4 μm and 0.1 μm for 10 cycles, then through 0.1 μm for 10 cycles, at 60° C. After extrusion is complete, the suspension is stored in a refrigerator. (D) Loading of Biocourier with pDNA (7) Thaw the pDNA samples stored at -40°C and leave the tubes under a fume hood until they reach room temperature (estimated time: 10 minutes). (8) Equilibrate the extruded Biocourier sample by transferring it from the refrigerator to a fume hood and placing it at room temperature (estimated time 6 minutes). (9) Load an amount of Biocourier with pDNA into a sterile Eppendorf. In particular, mix 360 μL of Biocourier with 40 μL of pDNA solution. Seal the Eppendorf. (10) Gently vortex the Eppendorf for 15 seconds and leave the sealed samples to equilibrate at room temperature for 30 minutes. Transfer samples to the refrigerator for 3 hours. (11) Transfer the samples to -40°C for freezing (5 hours). (12) Start the freeze-dryer and set the thermostatic chamber to -40° C. Transfer the sample to the freeze-dryer and evacuate it. (13) Freeze the dried samples overnight. (14) Seal the resulting sample and place it in a refrigerator. Prepare four aliquots as above.

[0224] Biocourier Formulation Type 2 (Porous, Active Ingredients, and Ratios) Prepared with Boron-Doped Silicon

[0225] [Table 10]

[0226] Lipid:Si (before extrusion) ratio = 16:1 16mg total lipid / 10mL Biocourier, 1.6mg / mL lipid, and 0.1mg / mL silicon, equivalent to: Biocourier stock solution=1.6μg / μL pDNA stock solution=2μg / μL Each vial contains: 80 μg pDNA Total volume of 400 μL

[0227] 80 µg pDNA equivalent to 40 µL - remaining volume = 400 µL (total volume) - 40 µL (pDNA volume) = 360 µL 360μL can contain up to 1.6μg / μL x 360μL = 576μg of Biocourier Biocourier:pDNA ratio = 576:80 = 7.2:1 Using this format, 50 μL (single dose / mouse) of Biocourier / pDNA complex contains: 10 μg pDNA and 72 μg Biocourier

[0228] All samples are provided as extruded samples suspended in nuclease-free water. Extrusion is considered the gold standard practice for preparing injectable samples.

[0229] Biocourier id / im for injection was extruded through 400 nm and 100 nm polycarbonate membranes. The Biocourier then extruded samples complexed with a corresponding amount of pDNA.

[0230] Preparation of Biocourier Formulation Type 2: Liquid Form (A) Lipid film preparation (1) Transfer the lipids in the amounts shown in Table 10 above from the stock solutions into a clean glass round-bottom flask and mix. (2) Carefully evaporate the solvent using a rotary evaporator with a 40° C. water bath and vacuum. (B) Rehydration of the film (3) Add the Si-NPs+GLY+THR solution to the film and adjust the final volume to 10 mL with nuclease-free water, if necessary. (4) Cover the flask with Parafilm, rehydrate the film, and agitate the flask in a water bath (60°C) for 5 minutes. (5) Divide 1 mL of sample into a total of 10 Eppendorf tubes without RNA. Store the suspension in the refrigerator. (C) Extrusion process (6) The suspension is passed through membrane filters with pore sizes of 0.4 μm and 0.1 μm for 10 cycles, then through 0.1 μm for 10 cycles, at 60° C. After extrusion is complete, the suspension is stored in a refrigerator. (D) Loading of Biocourier with pDNA (7) Thaw the pDNA samples stored at -40°C and leave the tubes under a fume hood until they reach room temperature (estimated time: 10 minutes). (8) Equilibrate the extruded Biocourier sample by transferring it from the refrigerator to a fume hood and placing it at room temperature (estimated time 6 minutes). (9) Load an amount of Biocourier with pDNA into a sterile Eppendorf. In particular, mix 360 μL of Biocourier with 40 μL of pDNA solution. Seal the Eppendorf. (10) Gently vortex the Eppendorf for 15 seconds and leave the sealed sample to equilibrate at room temperature for 30 minutes. Transfer the sample to the refrigerator until use. Follow the pre-injection instructions provided.

[0231] Prepare four aliquots as above.

[0232] Preparation of Bio-courier Formulation Type 2: Lyophilized Form (A) Film preparation (1) Transfer the lipids in the amounts listed in Table 3 above from stock solutions into a clean glass round-bottom flask and mix. (2) The solvent is carefully evaporated under vacuum using a rotary evaporator and a water bath at 40°C. (B) Rehydration of the film (3) Add the Si-NPs+GLY+THR solution onto the film and adjust the final volume to 10 mL with nuclease-free water, if necessary. (4) Cover the flask with Parafilm, rehydrate the film, and agitate the flask in a water bath (60°C) for 5 minutes. (5) Divide 1 mL of the sample in the RNA-free Eppendorf into a total of 10 Eppendorfs. Store the suspension in the refrigerator. (C) Extrusion process (6) The suspension is passed through membrane filters with pore sizes of 0.4 μm and 0.1 μm for 10 cycles, then through 0.1 μm for 10 cycles, at 60° C. After extrusion is complete, the suspension is stored in a refrigerator. (D) Loading of Biocourier with pDNA (7) Thaw the pDNA samples stored at -40°C and leave the tubes under the fume hood until they reach room temperature (estimated time 10 minutes). (8) Equilibrate the extruded Biocourier sample by transferring it from the refrigerator to a fume hood and placing it at room temperature (estimated time 6 minutes). (9) Load an amount of Biocourier with pDNA into a sterile Eppendorf. In particular, mix 360 μL of Biocourier with 40 μL of pDNA solution. Seal the Eppendorf. (10) Gently vortex the Eppendorf for 15 seconds and leave the sealed samples to equilibrate at room temperature for 30 minutes. Transfer samples to the refrigerator for 3 hours. (11) Transfer the samples to -40°C for freezing (5 hours). (12) Start the freeze-dryer and set the thermostatic chamber to -40° C. Transfer the sample to the freeze-dryer and evacuate it. (13) Freeze the dried samples overnight. (14) Seal the resulting sample and place it in a refrigerator. Prepare four aliquots as above.

[0233] General recommendations for sample reconstitution and handling Instructions for reconstitution of lyophilized powder samples Transfer the Eppendorf tube to a fume hood and resuspend the contents of the Eppendorf using 400 µL of nuclease-free water. Resuspension is carried out at room temperature under a fume hood with appropriate sterile protection equipment, thus intending to avoid any cross-contamination of the contents. After resuspension, the Eppendorf tube is closed, gently vortexed for 15 seconds, and the sealed sample is allowed to equilibrate at room temperature for 30 minutes. Transfer the samples to a refrigerated container and allow to come to room temperature a few minutes before administration. Gently invert the Eppendorf twice before sampling. Rehydrate only those vials intended for use / administration on the same day.

[0234] Liquid Sample Instructions Transfer the Eppendorf to a fume hood. Equilibrate at room temperature for 3 minutes, gently vortex for 15 seconds, and leave the sealed sample to equilibrate at room temperature for 30 minutes. Transfer the samples to a refrigerated container and allow to come to room temperature a few minutes before administration. Gently invert the Eppendorf twice before sampling.

[0235] Example 11: Considerations regarding extrusion and how this process step may change the optimal starting ratio of ingredients. Extrusion can result in a loss of silicon particles relative to the initial amount used. For the present purposes, a final silicon content after extrusion of approximately 2 mg / L was determined, which corresponds to 2 μg / mL.

[0236] To produce 1 mL of finished vector composition meeting these requirements, it is generally recommended to start with 1.6 mg of lipid components, 0.1 mg of trehalose (if present), 0.05 mg of glycine (if present), and 2 μg of silicone.

[0237] For convenience, when Biocourier:nucleic acid ratios are referred to herein, this is the molar ratio between the lipid component of the Biocourier and the nucleic acid.

[0238] Exemplary compositions are shown in Table 11 below.

[0239] [Table 11]

[0240] Example 12: Evaluation of Zeta Potential Formulations were produced having the ingredients shown in Table 12. The measured zeta potential of these formulations is shown in Table 13. The effect of ingredients on zeta potential can be assessed and the change in zeta potential versus RNA loading can be used as a simple quality control marker as evidence of successful complexation.

[0241] [Table 12]

[0242] [Table 13]

[0243] Example 13: Demonstration of the benefits of silicon on nucleic acid binding efficiency and complex stability A series of Biocourier formulations (prepared as described in the previous examples) were screened to assess whether the presence of silicon had a beneficial effect on nucleic acid binding efficiency and stability in comparison to lipid nanoparticle compositions formulated in the same manner with the same components as each Biocourier composition, but without the silicon-containing particles.

[0244] In particular, studies were carried out with Biocourier formulations DSC613G, DS61G, DS6G, and D6G to assess the effect of silicon particles on nucleic acid binding efficiency and stability, using crude extract of herring sperm (hsDNA) as an example of DNA and yeast mRNA as an example of mRNA.

[0245] Table 14:

[0246] [Table 14]

[0247] The results are shown in Figures 20 and 21. The binding efficiency (and thus infers stability) of nucleic acids (hsDNA or mRNA) is clearly improved when silicon particles are present. Thus, as demonstrated in these figures, nucleic acid binding and stability were found to be superior for formulations containing silicon particles than the corresponding formulations made without silicon particles.

[0248] Example 14 - Stability of Dasher GFP mRNA complexed with Si material mRNA A solution of 200 μg (200 μL) of Dasher GFP mRNA in nuclease-free water (1 mg / mL) was obtained from Aldevron, 4055 41st Avenue South Fargo, North Dakota 58104, USA. The mRNA was further diluted with 200 μL of nuclease-free water to a final concentration of 0.5 mg / mL and then dispensed into 50 μL aliquots in 0.2 mL PCR tubes.

[0249] Bovine serum Bovine serum was obtained in frozen liquid form from Merck (The Old Brickyard, New Rd, Gillingham, Dorset, SP8 4XT). Upon receipt, the serum was thawed and aliquots were prepared therefrom in 15mL and 50mL sterile Falcon tubes, which were then stored at -20°C. 1mL of serum was mixed with 4mL of nuclease-free water to obtain 5mL of 20% serum. From this sample, 4% serum was prepared by mixing 1mL of 20% serum with 4mL of nuclease-free water. The 20% and 4% serum samples were then dispensed into 1mL aliquots in nuclease-free 2mL PCR tubes (nuclease- and protease-free) and stored at -20°C until use in the assay.

[0250] SIS0012, SIS0013, and lipid nanoparticles Following the above protocol, SIS0012 (undoped Si) and SIS0013 (boron-doped Si; 5 × 10 18 Boron atoms / cm 3 ) samples were provided. Lipid nanoparticle samples containing all the components of SIS0012 and SIS0013 except silicon were also provided.

[0251] Following the protocol above, mRNA was loaded into SIS0012, SIS0013, and lipid nanoparticle samples. The weight ratio of mRNA to each of SIS0012, SIS0013, and LNP was 1:12.

[0252] Experimental procedure Each of the following samples was added to eight 0.2 mL PCR tubes (30 μL / tube). ·mRNA-SIS0012 complex ·mRNA-SIS0013 complex mRNA-lipid nanoparticle (except silicon) complexes ("mRNA-X") Control sample of mRNA in nuclease-free water

[0253] All samples were mixed with bovine serum, incubated at 37° C., and then analyzed by gel electrophoresis according to the following procedure:

[0254] 30 μL of bovine calf serum (4%) was added to each tube, which was then mixed thoroughly by pipetting up and down 4-5 times. The caps were tightly closed. The tubes were oversealed with parafilm and placed in a microtube rack in a 37°C water bath. As a serum-free control, one tube was mixed with 20 μL of nuclease-free water only.

[0255] Aliquots were taken at 0, 0.5, 1, 2, 4, 6, and 24 hours for gel electrophoresis analysis (see Figures 22-25 below).

[0256] For the purpose of gel electrophoresis analysis, samples of mRNA extracted from SIS0012, SIS0013, and lipid nanoparticle formulations were also collected at each time point. To extract mRNA, samples were mixed with sodium dodecyl sulfate (1% solution in nuclease-free water), mixed thoroughly by vortexing, and incubated at room temperature for 20 minutes. At the end of the incubation, KCl (0.1 M solution in nuclease-free water) was added to the mixture, mixed thoroughly by vortexing, and then incubated on ice for 10 minutes. The mixture was then centrifuged at 19000×g (14224 rpm) for 15 minutes at 4° C. The supernatant was carefully removed without disturbing the pellet and transferred to a new PCR tube.

[0257] Gel electrophoresis analysis was performed using E-Gel™ Power Snap electrophoresis devices and E-Gel™ agarose gels (1%), both available from ThermoFisher Scientific, 168 Third Avenue, Waltham, MA USA 02451.

[0258] Discussion of results Gel electrophoresis results of mRNA-SIS0012, mRNA-SIS0013, mRNA-X (silicon-free LNP), and naked mRNA after incubation with 2% bovine serum for 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, and 24 h are presented in Figures 22 to 25.

[0259] Figures 22A and 23A show that no mRNA migrated from the mRNA-SIS0012 and mRNA-SIS0013 wells, either before or after incubation with serum, at any of the time points examined.

[0260] In the mRNA-SIS0012 and mRNA-SIS0013 wells, there was a strong mRNA signal before any treatment with serum. After exposure to serum, the signal in each of the mRNA-SIS0012 and mRNA-SIS0013 wells gradually decreased over time. Finally (i.e., for samples incubated for 6 and 24 hours), a relatively weak signal was observed in the mRNA-SIS0012 and mRNA-SIS0013 wells. The lack of mRNA migration away from the SIS0012 and SIS0013 wells indicates that SIS0012 and SIS0013 are sufficiently associated with the mRNA. It is believed that over time, in the serum environment at 37°C (mimicking the in vivo environment to which the mRNA may be exposed upon administration to a subject, where nuclease enzymes are present), the mRNA may be gradually degraded, resulting in a fainter signal in the wells. Thus, the association of the mRNA with SIS0012 and SIS0013 is sufficient. The mRNA is protected from degradation over time, even in an in vivo-like environment where nuclease enzymes are present and the temperature is kept at 37°C.

[0261] To confirm the above results, the mRNA extracted from mRNA-SIS0012 and mRNA-SIS0013 was subjected to gel electrophoresis analysis at all time points examined. As shown in Figures 22B and 23B, the mRNA was observed to migrate through the gel. There was a strong and sharp band present in the mRNA dissociated from SIS0012 and SIS0013 before incubation with serum (no serum control), which was as strong and migrated the same distance as the band observed in the naked mRNA control sample, indicating the integrity of the SIS0012- and SIS0013-bound mRNA. In the case of the mRNA recovered from the mRNA-SIS0012 and mRNA-SIS0013 samples mixed with serum, there was a strong band present in the mRNA extracted from the 0-6 hour time point. However, the intensity of the band then began to decrease over time, and smearing was observed from 4 hours onwards.

[0262] The gel electrophoresis results of the LNP counterpart formulated without silicone, called mRNA-X, are shown in Figure 24. Figure 24B (extracted mRNA) shows more pronounced smearing than SIS0012 and SIS0013, indicating that LNP-associated mRNA is more susceptible to degradation upon exposure to serum than SIS0012- or SIS0013-associated mRNA. The LNP sample shows a weak signal as early as the 0.5 hour band, indicating the presence of small fragments of mRNA that migrated in the gel.

[0263] Thus, the gel retardation assay confirmed the successful complexation of mRNA with SIS0012 and SIS0013.

[0264] In summary, these gel electrophoresis results demonstrate the protective effect of SIS0012 and SIS0013 on mRNA against degradation by nuclease enzymes present in bovine serum. In contrast, in the case of mRNA associated with silicon-free LNPs (Figure 24), the gel electrophoresis results show strong smearing of the mRNA. For naked mRNA, Figure 25) shows that fragmentation started immediately after incubation with serum (0.5 hours) and degradation was observed almost immediately.

Claims

1. One or more enzymes and / or one or more fragments thereof, nucleic acid, one or more particles comprising hydrolysable silicon, and one or more lipids A nucleic acid vector composition comprising

2. The nucleic acid vector composition according to claim 1, wherein the one or more enzymes comprise one or more enzymes for forming a nucleic acid-enzyme substrate complex.

3. The nucleic acid vector composition according to claim 1, wherein the one or more enzymes comprise one or more nucleases and / or one or more polymerases.

4. The weight ratio of the enzyme and / or its fragment to the nucleic acid is in the range of 1:1×10 １２ to 1:1, and the nucleic acid vector composition according to claim 1.

5. The nucleic acid vector composition according to claim 1, wherein the one or more enzymes comprise one or more enzymes having an activity of at least 1 μmol / min with respect to a nucleic acid substrate at pH 7.4 and a temperature of 25°C.

6. In the form of lipid nanoparticles, having a core encapsulated by a shell, the shell comprising one or more lipids, and the core comprising the nucleic acid and the one or more particles comprising hydrolysable silicon, the nucleic acid vector composition according to claim 1.

7. The nucleic acid vector composition according to claim 1, wherein the nucleic acid is mRNA.

8. The nucleic acid vector composition according to claim 1, wherein the nucleic acid is DNA.

9. The mRNA comprises an open reading frame encoding a protein, a 5' cap, a poly(A) tail, and one or more untranslated regions, The nucleic acid vector composition according to claim 7, comprising one or more of

10. The open reading frame of the mRNA is an antigen of a pathogen; a tumor-associated antigen or a tumor-specific antigen; an allergen; or a regulator of an immune disease, an autoimmune disease, or an inflammatory disease The nucleic acid vector composition according to claim 9, encoding

11. The nucleic acid vector composition according to claim 1, further comprising an amino acid and / or further comprising one or more disaccharides.

12. Obtaining the nucleic acid by in vitro transcription from a DNA template or by purification from a biological source, and Combining the nucleic acid with the one or more particles and the one or more lipids A method for preparing the nucleic acid vector composition according to claim 1, comprising

13. A method for preparing the nucleic acid vector composition according to claim 12, wherein the nucleic acid is mRNA and the mRNA is obtained by in vitro transcription from a DNA template.

14. A prophylactic or therapeutic vaccine composition comprising the nucleic acid vector composition according to claim 1.

15. The vaccine composition according to claim 14, wherein the nucleic acid has a half-life of at least 3 months at 4°C in the vaccine composition.

16. The vaccine composition according to claim 14, wherein the nucleic acid has a half-life of at least 6 months at 4°C in the vaccine composition.

17. The vaccine composition according to claim 14, further comprising an adjuvant.

18. The vaccine composition according to claim 14, which is in a form suitable for intramuscular injection.

19. A pharmaceutical comprising the vaccine composition according to claim 14.

20. The vaccine composition according to claim 14, which is administered orally, subcutaneously, or intramuscularly.

21. The vaccine composition according to claim 14, wherein the nucleic acid vector composition functions as a lipopolyplex transfection vector.

22. The vaccine composition according to claim 21, wherein the nucleic acid is targeted to cells.

23. A method for manufacturing a pharmaceutical, comprising using the vaccine composition according to claim 14.

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