Transfection and transduction systems

Abstract

The present invention relates to the preparation and use of silica organic nanoparticles as delivery vehicles for viruses and virus-like species into the body. The nanoparticles typically have a hollow core and a surface morphology that allows for effective attachment of the species to the surface for delivery into the body. In particular, the present invention is useful for transfection and transduction.

Description

[Technical Field]

[0001] The present invention relates to compositions comprising nanoparticles and one or more delivery components, and to methods for introducing said delivery components into cells. The present invention also relates to the use of said compositions in therapy. [Background technology]

[0002] Gene therapy has entered clinical use after significant pharmaceutical investment. Clinical trial applications are also on the rise, with over 1,800 applications approved between 2015 and 2017 using virus-based products, such as Stremvellis (GSK), Glybera® (Uniqure), and Kymriah® (Novartis), to deliver therapeutic genes for the treatment of genetic diseases in humans. Gene therapy relies on these vectors to deliver functional copies of therapeutic genes. However, the need to mass-produce these vectors within a clinically relevant budget poses a major limitation to the success of gene therapy. An example of this limitation is the discontinuation of Glybera®, a therapy intended to treat lipoprotein lipase deficiency. The overall cost of this therapy was determined to be too high due to a number of factors, including its orphan indication, research and development costs, and manufacturing costs.

[0003] Over the past 40 years, several protocols for producing viral vectors for gene therapy have been developed, reducing costs. For safety reasons, vectors are typically generated as replication-incompetent viruses. To create these vectors, genes normally present in wild-type viral genomes that are necessary for production are removed. These genes, including those for viral structure and envelope (required for cell attachment), are placed on a plasmid vector. These plasmid vectors are transfected into specialized producer cells, enabling the cells to generate recombinant, infectious but replication-incompetent versions of the virus. Thus, the producer cells can only package the product encoded by the plasmid for the therapeutic recombinant viral genome, which contains a specialized packaging signal for selection. One example is a retroviral vector that has the structural gag, pol, and env genes removed from its own genome and placed on two plasmid vectors. A third plasmid carries the therapeutic genome. Transfection of all three plasmids into human cells generates a therapeutic, infectious, replication-defective retroviral vector.

[0004] The main drawback is that some envelopes are toxic to the producing cells, resulting in transient virus production. This transient transfection method is now the "gold standard" method used for generating not only retroviruses and adenoviruses, but also adeno-associated viruses. Over the years, several research groups have searched for the optimal transfection agent to transfect cells. Some commonly used transfection agents include polyethyleneimine (PEI), FuGENE®, Lipofectamine®, and Transfectam®.

[0005] Ideally, a transfection agent that could bind all three plasmids at once would be most likely to deliver all three plasmids to the same cell, resulting in good virus production. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2019 / 097226 Summary of the Invention

[0007] In a first aspect of the present invention, there is provided a composition comprising inorganic nanoparticles; and one or more delivery components; wherein the nanoparticles have protrusions on their surface; the nanoparticles have a diameter in the range of 50 nm to 3000 nm; the inorganic nanoparticles are at least partially coated with a transfection agent; and the one or more delivery components are selected from a vector, a viral vector, a plasmid, a virus, a viroid, a prion, a virus-like particle, a virus-derived component, and mixtures thereof.

[0008] The composition is suitable for carrying out transfection and transduction.

[0009] The term "nanoparticle" is intended to be interpreted in the usual sense in the art, and thus includes particles that are nanoscale, i.e., have an average diameter in the range of 1 nm to 1000 nm, when the particles are approximated as spheres.

[0010] The term "diameter" as used herein is intended to describe the diameter of a nanoparticle when the nanoparticle is approximated as a sphere, including any protrusions provided on the nanoparticle surface.

[0011] For the avoidance of doubt, the term "inorganic" is intended to refer to materials that do not contain carbon.

[0012] Typically, the nanoparticles comprise an inorganic material selected from silica, titania, alumina, or a combination thereof. However, in many instances, the nanoparticles will comprise silica. Silica has been found to function well as nanoparticles in the present invention because it can be fully functionalized as needed in many different species. The nanoparticles may consist of, or consist essentially of, the inorganic material. A composition consisting essentially of a specific component includes the specific component, with any other component present being provided in an essentially negligible amount (e.g., less than 0.5 wt%). That is, any added component or components do not substantially affect the function of the specific component.

[0013] The nanoparticles are typically hollow. While many delivery systems use vesicle or micelle structures to internalize a delivery component and then release the component upon reaching the target site, hollow nanoparticles typically do not contain a delivery component within their hollow structure. However, in some embodiments, the hollow structure of the nanoparticle may contain a delivery component or other materials useful for inducing transfection. Other components not useful for inducing transfection, such as pharmaceutically active agents, may also be contained within the hollow structure of the nanoparticle, either alone or in combination with other components or delivery components. The components may be released to act separately from or in conjunction with the effect caused by transfection.

[0014] In some embodiments, the nanoparticles may comprise a shell comprising an inorganic compound, a hollow core having a volume defined by the interior surface of the shell, and a plurality of protrusions comprising the inorganic compound disposed on the exterior of the shell. Typically, the protrusions are integral with the shell.

[0015] The term "mesoporous" is intended to be interpreted in its ordinary sense in the art. In particular, it refers to mesopores, i.e., pores having a width (i.e., pore size) of 2 nm to 50 nm, more typically 5 nm to 40 nm, and in some instances 10 nm to 30 nm; The pores are typically located in the shell of the particle and typically extend through the entire thickness of the shell. In some embodiments, materials can be loaded into the hollow core of the nanoparticle through the pores. However, in many instances, the core remains empty; i.e., no material is specifically introduced into the core.

[0016] Typically, the nanoparticles are at least partially coated with the one or more delivery components. The delivery components typically coat the outer surface of the nanoparticles. However, the nanoparticles may be functionalized with one or more functional groups to facilitate attachment of a delivery agent.

[0017] The inorganic nanoparticles are at least partially coated on their outer surface with a transfection agent. The term "transfection agent" is intended to be interpreted in its usual sense in the art. In particular, it refers to a compound or substance that enhances the ability of an active agent to be transfected. This may be by enhancing the ability of the delivery system itself (in this case, inorganic nanoparticles) or the active material to penetrate the cell membrane and / or by improving the ability of the active ingredient to be incorporated into the genetic material of the target cell. In this case, the transfection agent is typically provided to mask the charge on the inorganic material and facilitate the binding of the delivery agent to the nanoparticles.

[0018] The combination of a transfection agent with the inorganic nanoparticles described herein has been shown to be particularly effective in promoting transfection.

[0019] Often, the transfection agent substantially covers at least half of the nanoparticle surface, more typically at least 75% of the nanoparticle surface, and most typically substantially all of the nanoparticle surface, with "substantially all" as used herein typically meaning 95% or more of the nanoparticle surface.

[0020] The transfection agent is often a polymer. Polymeric materials provide good coating for the particles. Furthermore, the mechanism of the transfection agent often utilizes polymer structure to maximize efficacy. The polymer may be a copolymer. For example, the copolymer may be a block copolymer, an alternating copolymer, a statistical copolymer, or a combination thereof.

[0021] The choice of transfection agent is not particularly limited. Typically, the transfection agent is cationic. Cationic compounds, and in particular cationic polymers, are advantageous because they promote the binding of negatively charged species such as nucleic acids. The cationic polymer typically comprises a polyamine. The cationic polymer may be a polypeptide, such as polyarginine, polylysine, or polyhistidine.

[0022] In some embodiments, the cationic compound is chitosan or a derivative thereof. In some cases, a proportion of the amino groups of the chitosan are alkylated, often tri-alkylated (i.e., alkylated with three alkyl groups, e.g., three methyl groups). Alternatively, the cationic polymer may be polyamidoamine (PAMAM), PAMAM dendrimer, polylysine, DEAR-dextran, or polybrene. Commercially available transfection agents, such as FuGENE®, Lipofectamine, and Transfectam, may also be used.

[0023] The transfection agent may include a polyalklylimine. As one skilled in the art will appreciate, in some circumstances, the transfection agent may Advantageously, transfection agents contain a series of amine linkages (-NH-). Such groups have been shown to react with nucleic acids in a manner that enhances delivery into cells. Transfection agents for use in the present invention may also be combinations of two or more of the above.

[0024] The type of polyalkylimine used is not particularly limited, and may be linear, branched, or dendritic polyalkylimine, or a combination thereof. Branched or dendritic polyalkylimines are often used. The polyalkylimine may have a weight average molecular weight in the range of 2000 da to 40,000 da, more typically in the range of 10,000 da to 25,000 da. In some embodiments, the polyalkylimine has a weight average molecular weight in the range of 3000 da to 7000 da, more typically about 5000 da.

[0025] Typically, the nanoparticles contain at least 1.0% by weight of transfection agent. More typically, the nanoparticles contain at least 2.0% by weight, and in some instances at least 5.0% by weight of transfection agent. Typically, the nanoparticles contain in the range of 6.0-15% by weight of transfection agent.

[0026] Typically, the polyalkylimine is polyethyleneimine (PEI). PEI has been shown to function well as a transfection agent. PEI may be linear or branched, and the PEI is generally branched.

[0027] Typically, the nanoparticles also contain one or more moieties for binding the transfection agent thereto. As those skilled in the art will appreciate, different transfection agents exhibit different binding properties to nanoparticles constructed from different inorganic materials. Therefore, various moieties can be used to ensure good binding between the nanoparticles and the transfection agent. However, typically, the outer surface of the nanoparticles is at least partially covered with one or more acidic groups. Typical examples of acidic groups include, but are not limited to, phosphonates, phosphates, sulfates, carboxylates, alpha-ketocarboxylates, and combinations thereof. Of these groups, phosphates, phosphonates, and sulfates are most frequently used. Typically, the acidic groups are phosphonates, such as methylphosphonates (e.g., 3-(trihydroxysilyl)propylmethylphosphonate). Providing acidic groups on the surface of the nanoparticles is advantageous because these groups are typically negatively charged. This increases the negative charge on the charged surface of the nanoparticles, thereby improving binding of the transfection agent to the nanoparticle surface.

[0028] The core, i.e., the space within the shell, typically has a diameter in the range of 50 nm to 500 nm, for example, 75 to 300 nm, and the shell typically has a thickness in the range of 10 nm to 200 nm.

[0029] The particle size of the nanoparticles is typically in the range of 50 nm to 3000 nm, more typically 75 nm to 2000 nm, even more typically 100 nm to 1000 nm, and often 100 nm to 500 nm. In some cases, the nanoparticles are in the range of 100 nm to 300 nm. For the avoidance of doubt, the term "particle size" herein is intended to describe the diameter of a nanoparticle when it is approximated as a sphere. Furthermore, the average particle size of a plurality of nanoparticles is typically in the range of 50 nm to 3000 nm, more typically 75 nm to 2000 nm, even more typically 100 nm to 1000 nm, and often 100 nm to 500 nm. In some cases, the nanoparticles are in the range of 100 nm to 300 nm. The term "average" is intended to refer to an average value. Particle sizes referred to herein are typically measured using dynamic light scattering or by reference to SEM images.

[0030] The nanoparticles typically have a rough or "spiky" surface morphology. For example, the nanoparticles may be rambutan-like or morning star-like. In particular, the protrusions on the surface of the particles often form multiple spike- or finger-like structures on the surface that can trap materials between them.

[0031] The protrusions generally extend radially outward from the shell and are typically made of the same inorganic material as the shell. The protrusions typically increase the surface area of ​​the hollow nanoparticles. Typically, the protrusions have a length equal to or less than the diameter of the core. In many cases, the protrusion length is in the range of 5 nm to 1000 nm, more typically in the range of 10 nm to 200 nm, and even more often in the range of 50 nm to 150 nm. The protrusion length is typically substantially uniform, although variations in protrusion length are acceptable. As used herein, the term "substantially uniform" typically refers to a range of ±15% from the average protrusion length.

[0032] The protrusions typically have a diameter in the range of 2 nm to 50 nm, more typically 5 nm to 25 nm, and even more typically about 10 nm to 20 nm. The diameter referred to in this specification refers to the diameter at the base of the protrusion, i.e., the part where the protrusion abuts the shell.

[0033] The nanoparticles may be highly monodisperse. Typically, the polydispersity index (PDI; also known as dispersion index) of the nanoparticles is 0.3 or less, more typically 0.15 or less, even more typically 0.1 or less, and in some cases 0.05 or less. The dispersion index can be calculated as the ratio of the quadratic average (i.e., the average value d of the squares of the measured diameters) to the square of the arithmetic mean of the measured diameters. The dispersion index can be calculated as defined in ISO standard documents 13321:1996E and ISO22412:2008.

[0034] The nanoparticles typically have a large surface area, for example, at least 120 cm 2 / g, more typically e.g., at least 150 cm 2 / g. In some embodiments, the nanoparticles may have a BET surface area of ​​at least 140 cm 2 The hollow nanoparticles have an average particle size in the range of 160 to 250 nm and a BET surface area of ​​at least 120 cm 2 / g. The BET surface area may be measured, for example, using the ISO 9277 standard. The BET surface area may be measured based on nitrogen adsorption and desorption.

[0035] The nanoparticles typically comprise at least 70 wt% of the inorganic material, based on the total weight of the nanoparticles, and in some cases the inorganic material may comprise at least 90 wt% of the nanoparticles, more typically at least 95 wt% of the nanoparticles.

[0036] Without wishing to be bound by theory, the present inventors have found that nanoparticles with protrusions, or "spiky" nanoparticles, improve transfection and transduction efficiency.

[0037] The term "delivery moiety" is intended to refer to a species that is introduced into a cell to cause or assist in transfection or transduction.

[0038] The one or more delivery moieties are typically attached to the exterior surface of the nanoparticles. Without being bound by theory, it is believed that the "spiky" structure can effectively entangle the delivery moiety and facilitate its transport into target cells, causing transfection or transduction.

[0039] In many cases, the composition contains at least two, and sometimes at least three, delivery components. It has been shown that the nanoparticles of the present invention can carry multiple different delivery components on a single nanoparticle. This is particularly useful because it allows simultaneous delivery in combination therapy. For example, the present inventors have shown that multiple delivery components can be bound to the spiky nanoparticles and delivered to cells.

[0040] In some embodiments, the nanoparticles may be at least partially coated with the one or more delivery components. The nanoparticles may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% coated with the one or more delivery components. The nanoparticles may be completely coated (i.e., 95% or more coated) with the one or more delivery components.

[0041] The amount of delivery component relative to the nanoparticles can vary depending on the choice of delivery component and the dosage required for a given application, but typically the weight ratio of delivery component to nanoparticles ranges from 1:100 to 100:1, more typically from 1:50 to 5:1, even more typically from 1:30 to 1:1, and most typically from 1:20 to 1:2.

[0042] Typically, the one or more delivery components are independently selected from vectors, viruses, viroids, prions, virus-like particles, virus-derived components, and mixtures thereof. Exemplary vectors include viral vectors and plasmids.

[0043] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting other nucleic acids linked thereto. Vectors are well known in the art, and any type of vector may be used. A "plasmid" is a type of vector and refers to a circular double-stranded DNA into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector, in which additional nucleic acid segments can be ligated into the viral genome. Other types of vectors include cosmids. Other types of vectors include artificial chromosomes, including yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), and human artificial chromosomes (HACs).

[0044] Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., mammalian non-episomal vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of controlling the expression of genes to which they are operatively linked. In general, expression vectors utilized in recombinant DNA techniques are often in the form of plasmids. Some vectors used in accordance with the practice of the inventions described herein may be vectors well known in the art, such as plasmids derived from pBR322, pUC, pCMV, pMDG, or pHR, or mixtures thereof, or viral vectors derived from retroviruses, adenoviruses, or adeno-associated viruses, or mixtures thereof.

[0045] Non-limiting examples of the types of modifications to vectors that may be suitable for practicing the present invention include, but are not limited to, modifications such as the addition of one or more enhancers, one or more promoters, one or more ribosome binding sites, or one or more origins of replication. In some non-limiting embodiments, the expression vectors used to practice the present invention may contain one or more enhancer elements selected to improve expression of the protein of interest in the transient expression system. The selected enhancer element may be located 5' or 3' to the expressible nucleic acid sequence used to express the protein of interest. .

[0046] Preferably, the one or more delivery components comprise a vector. More preferably, the one or more delivery components comprise a viral vector, preferably a lentiviral vector; a plasmid, preferably a plasmid derived from pCMV, pMDG, or pHR; or a mixture thereof.

[0047] The vector may be empty or contain an insert or transgene. The insert or transgene may contain foreign DNA, RNA, small interfering RNA, microRNA, or small hairpin RNA. Preferably, the insert or transgene contains DNA.

[0048] The delivery component can be a virus. Typical examples of viruses include but are not limited to retroviruses such as lentiviruses; adenoviruses; adeno-associated viruses (AAV); herpes simplex viruses; and combinations thereof.Preferably, the virus is a lentivirus.

[0049] Alternatively, the delivery moiety may be a viroid. Exemplary viroids include those belonging to the Pospiviroidae and Avsunviroidae families.

[0050] In some embodiments, the delivery moiety is a prion. Exemplary prions include those derived from PrP.

[0051] In some embodiments, the one or more delivery components may comprise a viral component, which may be selected from a virion, a capsid, a viral nucleic acid, a viral DNA, a viral RNA, or a viral protein.

[0052] In various embodiments, the virus-derived components can be made from DNA viruses or RNA viruses.The virus-derived components can be derived from adenovirus, adeno-associated virus, herpes simplex virus, retrovirus such as lentivirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus, or picornavirus.The virus-derived components are preferably derived from lentivirus.

[0053] In some embodiments, the one or more delivery components may comprise virus-like particles (VLPs), which closely resemble viruses but do not contain viral genetic material. VLPs may be produced from components of a wide variety of virus families, including Parvoviridae (such as Adeno-associated Virus), Retroviridae (such as HIV), Flaviviridae (such as Hepatitis C), Paramyxoviridae (such as Nipah), and bacteriophages (such as Qβ and AP205).

[0054] In some embodiments, the composition further comprises a cell. The cell may be a eukaryotic cell or a prokaryotic cell. Preferably, the cell is a eukaryotic cell. The cell may be a primary cell or a cell derived from a cell line, such as an immortalized cell line or a transformed cell line. The cell may be a plant cell or an animal cell. Preferably, the cell is a mammalian cell. More preferably, the cell is a human cell.

[0055] The cell can be any type of cell. Preferably, the cell is a white blood cell. More preferably, the cell is an antigen-presenting cell or a lymphocyte, such as a B cell or a T cell. More preferably, the cell is a T cell. The cell may be a CD4+ T cell or a CD8+ T cell. The cell may be a helper T cell, a cytotoxic T cell, or a regulatory T cell. Alternatively, the cell may be a stem cell, such as an induced pluripotent stem cell (iPSC).

[0056] The cells may be genetically modified or transfected. For example, the cells may be chimeric antigen receptor T cells (CAR-T cells) or T cell receptor (TCR)-modified T cells (TCR-T cells). The cells may be genetically modified by any known method, including, but not limited to, viral vectors, liposomes, and electroporation. The cells may be genetically modified by the following methods:

[0057] The composition may further comprise one or more pharmaceutically acceptable excipients, such as carriers, diluents, and / or other medicinal agents, pharmaceutical agents, or adjuvants.

[0058] As used herein, the term "transfection" refers to the non-viral delivery of a foreign nucleic acid, protein, or other polymer into a target cell so that the foreign nucleic acid, protein, or other polymer is expressed or biologically functional in the cell. Non-limiting examples of foreign nucleic acids include DNA, RNA, siRNA, miRNA, shRNA, mRNA, and mixtures thereof. Typically, the foreign nucleic acid is DNA, more typically supercoiled plasmid DNA. The transfection of the cell may be transient, i.e., the foreign nucleic acid is present in the cell for a limited time and is not integrated into the target cell genome. Alternatively, the transfection of the cell may be stable, i.e., the foreign nucleic acid is integrated into the target cell genome or maintained as an episomal plasmid, resulting in long-term maintenance of the foreign nucleic acid in the target cell and its progeny.

[0059] As used herein, the term "transduction" refers to the delivery of exogenous nucleic acid to a target cell by a virus or viral vector so that the exogenous nucleic acid is expressed or biologically functional in the cell. Non-limiting examples of exogenous nucleic acid include DNA, RNA, siRNA, miRNA, shRNA, mRNA, and mixtures thereof. Typically, the exogenous nucleic acid is DNA, more typically supercoiled plasmid DNA. The transfection of the cell may be transient, i.e., the exogenous nucleic acid is present in the cell for a limited time and is not integrated into the target cell genome. Alternatively, the transfection of the cell may be stable, i.e., the exogenous nucleic acid is integrated into the target cell genome or maintained as an episomal plasmid, resulting in long-term maintenance of the exogenous nucleic acid in the target cell and its progeny.

[0060] The transfection or transduction may be performed in vitro or ex vivo. For example, the transfection or transduction may be performed on cultured cell lines or isolated primary cells. Preferably, the transfection or transduction is performed on cells isolated from a patient.

[0061] In a second aspect of the present invention, there is also provided a composition according to the first aspect of the present invention for use in therapy. The inventors have found that the composition of the first aspect of the present invention is highly effective in transfecting or transducing cells. Therefore, there is no particular limitation on the cells to be transfected or transduced. The genetic makeup of the cells can be varied. It is envisioned that a variety of different cells could be treated with a variety of different delivery components to induce structural changes. Thus, the treatment is typically gene therapy. Administration of cells treated according to the present invention, or in vivo transfection or transduction with the composition of the first aspect of the present invention, can both be used to treat or prevent a wide range of diseases. However, non-limiting examples of typical diseases that may be treated or prevented include genetic diseases, cancer, infectious diseases, and autoimmune diseases. Examples of typical genetic diseases include cystic fibrosis, heart disease, diabetes, hemophilia, and retinitis pigmentosa.

[0062] The cancer may be any cancer, including blood cancer, leukemia, lymphoma, and multiple myeloma, as well as solid tumors and other non-blood and non-hematological cancers. The infectious disease may be any infectious disease caused by a pathogen, including infections caused by viruses, bacteria, fungi, parasites, and prions. The autoimmune disease may be any autoimmune disease, including type 1 diabetes, rheumatoid arthritis, psoriasis, psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, Addison's disease, Graves' disease, Sjögren's syndrome, Hashimoto's disease, myasthenia gravis, autoimmune vasculitis, pernicious anemia, pemphigus vulgaris, and celiac disease.

[0063] Typically, the disease is cancer, an infectious disease, or an autoimmune disease.

[0064] When the disease is "treated" in the use or method, it means that one or more symptoms of the disease are alleviated. It does not mean that the symptoms are completely cured so that the patient no longer has the symptoms, although this may be the case in some methods. As a result of treating the disease, the severity of one or more symptoms of the disease is reduced compared to before treatment. As a result of treatment, the severity of multiple symptoms of the disease may be reduced compared to before treatment. The present invention can also be used for prophylactic purposes to prevent the onset of a disease.

[0065] In a third aspect of the invention, there is also provided the use of a composition according to the first aspect of the invention in the manufacture of a medicament for treatment, said treatment typically as described for the second aspect of the invention. Again, as in the second aspect of the invention, the method comprises in vivo transfection or transduction and delivery of transfected or transduced cells using a composition according to the first aspect of the invention.

[0066] Also provided in a fourth aspect of the present invention is a method of treating a disease in a patient, comprising administering to the patient a composition according to the first aspect of the present invention. Preferably, the patient is human. Preferably, a therapeutically effective amount of the composition is administered to the patient. A "therapeutically effective amount" refers to an amount effective, at a dosage and for a period of time necessary, to achieve the desired therapeutic result. It is also intended that the treatment include delivering to the patient cells that have been transfected or transduced with a composition according to the first aspect of the present invention.

[0067] There is also provided, as a fifth aspect of the present invention, a method for producing a composition according to the first aspect of the present invention, the method comprising mixing nanoparticles according to the first aspect of the present invention with one or more delivery components according to the first aspect of the present invention.

[0068] The nanoparticles of the present invention can be prepared using a Stober-like process, such as the method described on pages 24-28 of Patent Document 1. In the case of application of a transfection agent, a further step is performed following the preparation of the inorganic nanoparticles to coat the nanoparticles. In this case, the inorganic nanoparticles are typically first functionalized with a linker moiety to facilitate coating of the nanoparticles with the transfection agent. As explained above, the linker may be, but is not limited to, a phosphonate. Typically, the inorganic nanoparticles are mixed with the linker and stirred for at least 5 minutes (usually 30 minutes to 10 hours, most often 1 hour to 4 hours), often at a temperature above room temperature, typically in the range of 20°C to 70°C.

[0069] In some embodiments, the mixing step may last less than 60 minutes. Typically, the mixing step lasts for 10 to 45 minutes, more typically 10 to 30 minutes. This process is typically carried out at room temperature, i.e., 15°C to 35°C, and most typically in a polar solvent, usually water. Often, a buffer solution may be used, such as phosphate buffered saline.

[0070] In a sixth aspect of the present invention, there is also provided a method of transduction or transfection using a composition according to the first aspect of the present invention, comprising the steps of (i) providing a composition according to any one of claims 1 to 14; and (ii) incubating said composition with cells.

[0071] In one embodiment, a method for transducing cells is provided, wherein one or more delivery components comprise a viral vector. In particular, the method includes: (i) providing nanoparticles having protrusions on their surface, the nanoparticles having a diameter in the range of 100 nm to 3000 nm; (ii) providing one or more delivery components, the delivery components being viral vectors; and (iii) incubating the nanoparticles and the one or more delivery components with cells.

[0072] In a further embodiment, a method for transfecting a cell is provided, wherein one or more delivery components comprise a plasmid. In particular, the method includes: (i) providing nanoparticles having protrusions on their surface, the nanoparticles having a diameter in the range of 100 nm to 3000 nm; (ii) providing one or more delivery components, the delivery components being plasmids; and (iii) incubating the nanoparticles and the one or more delivery components with cells.

[0073] In the above transfection and transduction embodiments, the incubation step typically lasts for less than 48 hours, more typically less than 24 hours, and even more typically less than 5 hours. Often, the incubation step lasts for less than 3 hours, and even more typically less than 2 hours. Often, the incubation step lasts for between 5 minutes and 1 hour, usually between 10 minutes and 30 minutes.

[0074] As explained above, the nanoparticles may contain a transfection agent to aid in the transfection of the transduction process.

[0075] Both methods can be performed in vivo or in vitro. Typically, the methods are performed in vitro.

[0076] Those skilled in the art will understand that any aspect of the present invention is equally applicable to all other aspects of the present invention, whether it relates to the composition, its use, or a method of treatment, etc. In particular, for example, the composition aspect may be described in more detail than other aspects of the present invention, such as the use. However, even if more detailed information is given for a particular aspect of the present invention, the skilled artisan will understand that this information is equally applicable to other aspects of the present invention in general.

[0077] All patent and literature references cited herein are incorporated by reference in their entirety.

[0078] The invention will now be described in detail by way of example only with reference to the following drawings, in which: [Brief explanation of the drawings]

[0079] [Figure 1] Cell viability after 4 hours of Nuvec treatment. Viability of cells treated with various concentrations of Nuvec (negative control (NC), 1 μg, 10 μg, 40 μg, 60 μg, 80 μg) for 4 hours. After 4 hours, the medium was changed. Cell viability and recovery were recorded at the indicated time points for 72 hours after the medium change. [Figure 2] Cell viability after 4 hours of Nuvec treatment. (A, B) Cell viability over time (every 24 hours) of HEK293T cells treated with a range of Nuvec concentrations for 4 hours (n=1). See Table 3 below. [Figure 3]Cell viability after 24 hours of Nuvec treatment. Viability of cells treated with various concentrations of Nuvec (negative control (NC), 1 μg, 10 μg, 40 μg, 60 μg, 80 μg) for 24 hours. After 24 hours, the medium was changed. Cell viability and recovery were recorded at the indicated time points for 72 hours after the medium change. [Figure 4] Cell viability after 24 hours of Nuvec treatment. (A, B) Cell viability over time for HEK293T cells treated with a range of Nuvec concentrations for 24 hours. See Table 4. [Figure 5] Cell viability after 48 hours of Nuvec treatment. Viability of cells treated with various concentrations of Nuvec (negative control (NC), 1 μg, 10 μg, 40 μg, 60 μg, 80 μg) for 48 hours. After 48 hours, the medium was changed. Cell viability and recovery were recorded at the indicated time points for 72 hours after the medium change. [Figure 6] Cell viability after 48 hours of Nuvec treatment. (A, B) Cell viability over time for HEK293T cells treated with a range of concentrations of Nuvec for 48 hours. [Figure 7] Cell viability and GFP expression after transfection with 1 μg of Nuvec and 5 μg of GFP vector alone for 4, 24, and 48 hours. Analysis was performed 72 hours after transfection. [Figure 8] Cell viability and GFP expression after transfection with 10 μg of Nuvec and 5 μg of GFP vector alone for 4, 24, and 48 hours. Analysis was performed 72 hours after transfection. [Figure 9] Cell viability and GFP expression after transfection with 40 μg of Nuvec and 5 μg of GFP vector alone for 4, 24, and 48 hours. Analysis was performed 72 hours after transfection. [Figure 10] Cell viability and GFP expression after transfection with 60 μg of Nuvec and 5 μg of GFP vector alone for 4, 24, and 48 hours. Analysis 72 hours after transfection. [Figure 11] Cell viability and GFP expression after transfection with 80 μg of Nuvec and 5 μg of GFP vector alone for 4, 24, and 48 hours. Analysis was performed 72 hours after transfection. [Figure 12] Virus was produced and titered against indicator cells for later use to test binding to Nuvec. High-titer LVs were produced using traditional production methods and titered using various dilutions of LVs. GFP expression rates at various dilutions were measured by flow cytometry analysis, which was used to calculate a titer of 1.18 x 109 TU / ml. The produced LVs were later used in transduction assays with Nuvec. [Figure 13] HEK293T indicator cells were transfected with NV00100028, NV00100026-28, NV0010032, and NV0010033 for 4 or 24 hours, along with 5 μg of plasmid DNA and a 4:3:1 ratio of GFP, GAG-POL, and VSV-G. Viral supernatants were collected every 24 hours for 72 hours. Photographs show transfected cells 72 hours post-transfection. [Figure 14] HEK293T cells were transduced with viral supernatant collected from cells transfected with Nuvec at various concentrations and transfection times. Seventy-two hours after transduction, the percentage of GFP-positive cells was analyzed using flow cytometry. The results are shown below each image. Initially, we were unsure how much lentiviral Nuvec would actually be produced, so we transduced HEK293T cells with 500 μl and 200 μl of viral supernatant. To obtain a more accurate representation of the viral titer, we used a GFP-positive percentage between 1 and 30% for the lentiviral titer calculation. As noted above, even at the lowest dilution used (200 μg), more than 30% of the cells were GFP-positive, so we performed replicates using smaller amounts. [Figure 15]Replicate titrations were performed using smaller amounts of viral supernatant harvested from cells transfected with various concentrations of Nuvec. HEK293T cells were then transduced with 100 μl and 10 μl of viral supernatant, and the percentage of positive green cells was measured by flow cytometry 72 hours after transduction. [Figure 16] Analysis of lentiviral transduction efficiency of indicator cells. Lentivirus (MOI 20) was combined with various concentrations of NV00100028. After various incubation times, lentivirus was added to HEK293T indicator cells. N=4 [Figure 17] Analysis of lentiviral transduction efficiency of indicator cells. Binding of lentivirus (MOI 20) to various concentrations of NV00100026-28. After binding for various incubation times, lentivirus was added to HEK293T indicator cells. N=4 [Figure 18] Analysis of lentiviral transduction efficiency of indicator cells. Binding of lentivirus (MOI 20) to various concentrations of NV0010032. After binding for various incubation times, lentivirus was added to HEK293T indicator cells. N=4 [Figure 19] Analysis of lentiviral transduction efficiency of indicator cells. Binding of lentivirus (MOI 20) to various concentrations of NV0010033. After binding for various incubation times, lentivirus was added to HEK293T indicator cells. N=4 [Figure 20] Transduction efficiency of lentivirus (MOI 20) in indicator cells. Lentivirus was combined with various batches of Nuvec at various concentrations for 10 minutes and then added to HEK293T indicator cells. GFP expression was measured by flow cytometry. N=4 [Figure 21] Transduction efficiency of lentivirus (MOI 20) in indicator cells. Lentivirus was combined with various batches of Nuvec at various concentrations for 20 minutes and then added to HEK293T indicator cells. GFP expression was measured by flow cytometry. N=4 [Figure 22]Transduction efficiency of lentivirus (MOI 20) in indicator cells. Lentivirus was combined with various batches of Nuvec at various concentrations for 30 minutes and then added to HEK293T indicator cells. GFP expression was measured by flow cytometry. N=4 [Figure 23] Mean cell viability after lentiviral transduction (MOI 20) using various concentrations of Nuvec and various incubation times. N=2. [Figure 24] Viral transduction of indicator cells; + / - polybrene. Transduced cells with lentivirus at various limiting dilutions, with or without 5 μg / ml polybrene. See Table 7. [Figure 25] FIG. 25 shows an SEM image of the nanoparticles. DETAILED DESCRIPTION OF THE INVENTION [Example]

[0080] Example 1 - Synthesis of Ram-SNPs.

[0081] Resorcinol (0.4 g) and formaldehyde (37 wt %, 0.56 ml) were added to a solution consisting of aqueous ammonia (28 wt %, 12 ml), deionized water (40 ml), and ethanol (280 ml). The mixture was stirred at room temperature (approximately 25°C) for 8 hours, and then 2.4 ml of tetraethyl orthosilicate (TEOS) was added to the solution and stirred for 8 minutes. After stirring for 1 h, resorcinol (1.6 g) and formaldehyde (37 wt %, 2.24 ml) were added again. The mixture was stirred for 2 h at room temperature, then collected by centrifugation at 4700 rpm (3877 rcf) for 5 min, washed with ethanol, and dried at 50 °C overnight. Finally, the Ram-SNPs were collected by calcination in air at 550 °C for 5 h.

[0082] Example 2 – Synthesis of PEI-modified RNPs

[0083] 0.03 grams of the Ram-SNPs prepared above were dispersed in 10 ml of water under sonication (ensuring no obvious particle clumps were precipitated in the solution). 0.213 ml of 3-(trihydroxysilyl)propylmethylphosphonate (HTPMP, 50 wt % in water) was added to another 10 ml of water, and the 10 ml of nanoparticle solution was mixed with the HTPMP solution and stirred at 40°C for 2 hours. The phosphonate-modified silica nanoparticles were recovered by centrifugation at 12,000 rpm (17,420 rcf) for 5 minutes and washed once with water. These nanoparticles were then directly redispersed in 5 ml of carbonate-bicarbonate buffer (pH = 9.6) by sonication.

[0084] Polyethyleneimine (PEI; branched; average MW 10k; Alfar Aesar) was dissolved in 10 ml of carbonate-bicarbonate buffer by sonication. The particle solution was then mixed with the PEI solution and stirred at room temperature for 4 hours. The PEI-loaded nanoparticles were collected by centrifugation at 12,000 rpm for 5 minutes and washed once with water. The nanoparticles were then resuspended in 3 ml of water, frozen under liquid nitrogen for 30 minutes, and then dried in a freeze dryer for 2 days. The fully dried particles were stored in a desiccator in a refrigerator. These particles are hereafter referred to as "Nuvec."

[0085] Example 3 – Cell viability

[0086] 6×10 5 HEK293T cells were seeded in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (hereinafter referred to as "complete medium") and incubated overnight at 37°C under 5% CO2.

[0087] Cells were treated with Nuvec at various concentrations ranging from 1 μg to 80 μg / 0.5 ml. Treated cells were incubated at 37°C in 5% CO2 for 4, 24, or 48 hours, after which the medium was replaced with fresh complete medium. After medium replacement, cell viability was measured every 24 hours over 72 hours by trypan blue exclusion. A Countess automated cell counter was used according to the manufacturer's instructions.

[0088] Cell viability analysis after 4 hours of incubation with Nuvec is provided in Figures 1 and 2. Cell viability analysis after 24 hours of incubation with Nuvec is provided in Figures 3 and 4. Cell viability analysis after 48 hours of treatment with Nuvec is provided in Figures 5 and 6.

[0089] Example 4 – Single Plasmid Transfection with Nuvec

[0090] The transfection efficiency of Nuvec was evaluated in HEK293T cells by assessing the delivery efficiency of plasmid DNA encoding green fluorescent protein (pDNA-EGFP).

[0091] Fresh HEK293T cells were plated at 1.2 x 10 per well in complete medium in a 12-well plate. 5 Cells were seeded at a density of 700 μg / well and incubated overnight at 37°C under 5% CO2. The mixture was grown to ~90% confluence.

[0092] For various amounts of Nuvec, ranging from 1 to 80 μg per well, bioburden was minimized by resuspending the particles in at least five volumes of 70% ethanol, mixing, briefly centrifuging to collect the particles, and removing the clear supernatant. A suspension of Nuvec in PBS was prepared by adding 50 μl of sterile PBS to each well and sonicating in an ultrasonic bath at a power output of at least 120 W until a uniform suspension was obtained (up to 30 minutes). Any clumps that quickly settled during sonication were dispersed by pipetting.

[0093] Nuvec® at various concentrations ranging from 1 μg to 80 μg / 0.5 ml in PBS was allowed to bind with a total of 1 μg of plasmid DNA for 30 minutes at room temperature. After binding, the pDNA-loaded Nuvec® was suspended in 1 ml of complete medium, which was then used to replace the cell culture medium. Cells were incubated at 37°C and 5% CO2 for 4, 24, or 48 hours, after which the medium was replaced with fresh complete medium. 72 hours after the medium change, cells were analyzed by flow cytometry and confocal microscopy to measure intracellular green fluorescent protein expression. Experiments were performed in triplicate for each group. The results are presented in Figures 7–11.

[0094] Example 5 – Lentivirus Production

[0095] 1.5×10 7 Fresh HEK293T cells were seeded in complete medium in a T175 flask and incubated overnight at 37°C, 5% CO2 to reach confluence.

[0096] A total of 5 μg of plasmid DNA (eGFP, pCMVR8.74, and pMD2.G) (ratio 4:3:1) was bound to polybrene in serum-free medium (Opti-MEM) for 20 minutes. After binding, the pDNA-loaded transfection reagent was suspended in 1 ml of complete medium and used to replace the cell culture medium. Cells were incubated at 37°C and 5% CO2 for 24 hours, after which the medium was replaced with fresh complete medium. After the replacement, supernatants were collected every 24 hours for 72 hours. The conditioned medium was filtered through a 0.45 μM filter to remove cell debris and stored at 4°C for later use.

[0097] The conditioned medium was concentrated by ultracentrifugation at 23,000 rpm at 4°C. The supernatant was discarded and the pellet was air-dried for 10 minutes. The pellet was resuspended in 200 μl of serum-free medium and incubated on ice for 1 hour. The resuspended virus pellet was aliquoted and stored at -80°C for later use.

[0098] Example 6 – Lentiviral titration

[0099] Fresh HEK293T cells were cultured at 2 × 10 in complete medium in a 12-well plate. 5 The cells were seeded at a density of 1000 cells / well and incubated overnight at 37°C under 5% CO2.

[0100] Serial dilutions of virus were prepared and incubated with 5 μg / ml polybrene in complete medium for 20 minutes at room temperature. This virus-polybrene mixture was suspended in 1 ml of complete medium and used to replace the cell culture medium. One well of cells was left untreated, and one well of cells was counted to determine the number of cells present for infection.

[0101] Cells were incubated at 37°C and 5% CO2 for 24 hours, after which the medium was replaced with fresh complete medium. 48 hours after the medium change, cells were analyzed by flow cytometry analysis for GFP expression. Only samples showing 1-30% GFP expression were analyzed as being accurately representative of the virus titer. The virus titer was determined at each dilution point as shown below. The total titer was calculated using the average of the ion points.

[0102] Titer (TU / ml) = ((cell number x (percentage of GFP expression / 100)) / volume) x DF

[0103] TU / ml is transducing units per ml. DF is the dilution factor. Figure 12 shows images of GFP expression at limiting dilution. The titer was 1.18 x 10 9 Calculated as TU / ml.

[0104] Example 7 – Triple Plasmid Transfection of Cells

[0105] Fresh HEK293T cells were plated at 6 x 10 per well in complete medium in a 6-well plate. 5Cells were seeded at a density of 1000 and incubated overnight at 37°C under 5% CO2 to reach confluence.

[0106] Nuvec® at various concentrations, ranging from 1 μg to 80 μg / 0.5 ml, was conjugated with a total of 5 μg of plasmid DNA (eGFP, pCMVR8.74, and pMD2.G) at a 4:3:1 ratio in serum-free medium for 30 minutes. Different batches of Nuvec® were also tested. After conjugation, the pDNA-loaded Nuvec® was suspended in 1 ml of complete medium and used to replace the cell culture medium. Cells were incubated at 37°C and 5% CO2 for 4 or 24 hours, after which the medium was replaced with fresh complete medium. After medium replacement, supernatants were collected every 24 hours for 72 hours. 72 hours after transfection, cells were analyzed for GFP expression by fluorescence microscopy. The conditioned medium was centrifuged at 1500 rpm for 5 minutes, and the collected supernatant was stored at 4°C for later use. Figure 13 shows transfected cells 72 hours after transfection.

[0107] The transfection experiment was repeated a total of three times for NV00100028, NV00100026-28, NV0010032, and NV0010033, including 4-hour and 24-hour transfections. The resulting titers (TU / ml) were calculated and are shown in the following table:

[0108] [Table 1]

[0109] Example 8 – Transduction of cells with conditioned medium

[0110] Fresh HEK293T cells were cultured at 2 × 10 in a 12-well plate. 5 Cells were seeded at a density of 1000 cells / well and incubated overnight at 37°C under 5% CO2 to reach confluence.

[0111] Cells were transduced using 500 μl or 200 μl of conditioned medium collected from the cells transfected in Example 7. Cells were incubated at 37° C. and 5% CO for 24 hours, after which the medium was replaced with fresh complete medium. 72 hours after transduction, cells were analyzed for GFP expression by fluorescence microscopy and flow cytometry. The results are presented in FIG. 14.

[0112] Fresh HEK293T cells were transduced with 100 μl or 10 μl of conditioned medium collected from the cells transfected in Example 7. The cells were incubated at 37° C. and 5% CO for 24 hours, after which the medium was replaced with fresh complete medium. 72 hours after transduction, the cells were analyzed for GFP expression by fluorescence microscopy and flow cytometry. The results are presented in FIG. 15.

[0113] Example 9 – Lentiviral transduction

[0114] Fresh HEK293T cells were plated at 2 x 10 in complete medium in a 12-well plate. 5 Cells were seeded at a density of 1000 cells / well and incubated overnight at 37°C under 5% CO2 to reach confluence.

[0115] Nuvec at various concentrations ranging from 1 μg to 80 μg / 0.5 ml was bound to lentivirus carrying a GFP transgene (MOI 20) for 10, 20, or 30 minutes at room temperature. The lentivirus was produced according to Example 5. Different batches of Nuvec were tested.

[0116] After binding, the lentivirus-Nuvec mixture was suspended in 1 ml of complete medium and used to replace the cell culture medium. Cells were incubated at 37°C and 5% CO2 for 24 hours, after which the medium was replaced with fresh complete medium. 72 hours after transduction, cells were analyzed for GFP expression by fluorescence microscopy and flow cytometry. Figures 16-22 show the GFP expression of cells transduced with various batches of Nuvec.

[0117] The viability of the transduced cells was analyzed using trypan blue exclusion, and the results are presented in Figure 23.

[0118] Example 10 - Comparison with standard transduction protocols

[0119] Fresh HEK293T cells were plated at 2 x 10 in complete medium in a 12-well plate. 5 Cells were seeded at a density of 1000 cells / well and incubated overnight at 37°C under 5% CO2 to reach confluence.

[0120] Various limiting dilutions of lentivirus carrying a GFP transgene (MOI 20) were combined with 5 μg / ml polybrene for 20 minutes at room temperature. Lentivirus incubated without polybrene was used as a control.

[0121] After binding, the lentivirus-polybrene mixture was suspended in 1 ml of complete medium and used to replace the cell culture medium. Cells were incubated at 37°C and 5% CO for 24 hours, after which the medium was replaced with fresh complete medium. 72 hours after transduction, cells were analyzed for GFP expression by fluorescence microscopy and flow cytometry. Figure 24 shows the GFP expression in transduced cells.

[0122] A summary of the transduction efficiencies is provided in the table below.

[0123] [Table 2]

[0124] [Table 3]

[0125] [Table 4]

[0126] Table 5

[0127] Table 6

[0128] Table 7

Claims

1. Inorganic mesoporous nanoparticles comprising silica; and one or more delivery components; A composition comprising: the nanoparticles have protrusions on their surfaces; the nanoparticles have a diameter in the range of 50 nm to 3000 nm; the inorganic nanoparticles are at least partially coated with a transfection agent; the one or more delivery components comprise a viral vector; composition.

2. The composition of claim 1 , wherein the viral vector is an adenoviral vector, an adeno-associated viral vector, or a retroviral vector such as a lentiviral vector.

3. The composition of claim 2 , wherein the viral vector is an adenoviral vector or a lentiviral vector.

4. The nanoparticles are a silica-containing shell; a hollow core having a volume defined by the interior surface of said shell; and The composition of any one of claims 1 to 3, comprising: a plurality of protrusions comprising silica disposed on the exterior of the shell.

5. The composition of claim 4 , wherein the projections are integral with the shell.

6. 6. The composition of claim 4 or 5, wherein the projections extend radially outward from the shell.

7. The composition of any one of claims 1 to 6, wherein the protrusions have a length of from 5 nm to 1000 nm, optionally from 10 nm to 200 nm, optionally from 50 nm to 150 nm.

8. The composition of any one of claims 1 to 7, wherein the nanoparticles are hollow.

9. The composition of any one of claims 1 to 8, wherein the nanoparticles are rambutan-like or morning star-like.

10. The composition of any one of claims 1 to 9, wherein the protrusions comprise fingers or spikes.

11. The composition of any one of claims 1 to 10, wherein the transfection agent is a cationic polymer.

12. 12. The composition of claim 11, wherein the transfection agent is a polyalkylimine, chitosan, polylysine, DEAE-dextran, polybrene, or polyamidoamine (PAMAM) dendrimer.

13. The composition of claim 11 , wherein the transfection agent is a polyalkylimine.

14. The composition of claim 11 , wherein the transfection agent is polyethyleneimine (PEI).

15. The composition of any one of claims 1 to 14, wherein the nanoparticles are at least partially coated with the one or more delivery components.

16. The composition of any one of claims 1 to 15, wherein the composition comprises at least two delivery components.

17. The composition of any one of claims 1 to 16, wherein the composition comprises at least three delivery components.

18. The composition of any one of claims 1 to 17, further comprising cells.

19. 19. The composition of claim 18, wherein the cell is a T cell.

20. A composition according to any one of claims 1 to 19 for use in therapy.

21. 21. The composition for use according to claim 20, wherein the therapy is gene therapy.

22. 21. A composition for use according to claim 20 for use in the treatment or prevention of cancer, an infectious disease, or an autoimmune disease.

23. Use of a composition according to any one of claims 1 to 17 in the manufacture of a medicament for the treatment of a genetic disease, cancer, an infectious disease or an autoimmune disease.

24. A method for producing a composition according to any one of claims 1 to 17, comprising the steps of: i) mixing inorganic mesoporous nanoparticles comprising silica and having protrusions on their surface with one or more delivery components, the nanoparticles have a diameter in the range of 50 nm to 3000 nm; the one or more delivery components comprise a viral vector; the steps of: method.

25. 1. A method for transfecting or transducing a cell, comprising: i) providing a composition according to any one of claims 1 to 17; and ii) incubating the composition with cells; method.

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