Nonviral vectors containing polypropyleneimines

Abstract

The present invention relates to the field of non-viral vectors and pharmaceutical compositions comprising polypropyleneimine and nucleic acids, and their use in human or veterinary medicine. More particularly, the present invention relates to pharmaceutical compositions comprising polypropyleneimine polymers or copolymers for the delivery or transfection of nucleic acids, such as RNA. The pharmaceutical compositions described herein are particularly useful for (nucleic acid) vaccination, nucleic acid-based protein therapy, nucleic acid-based protein replacement therapy, gene editing, base editing, cell therapy, immunotherapy, stem cell therapy, regenerative medicine, gene silencing, nucleic acid inhibition or protein inhibition.

Description

[Technical Field]

[0001] The present invention relates to the field of nonviral vectors and pharmaceutical compositions comprising polypropyleneimines and nucleic acids, and their use in human or veterinary medicine. More specifically, the present invention relates to pharmaceutical compositions comprising polymers or copolymers of polypropyleneimines for the delivery or transfection of nucleic acids, such as RNA. The pharmaceutical compositions described herein are particularly useful for (nucleic acid) vaccination, nucleic acid-based protein therapy, nucleic acid-based protein replacement therapy, gene editing, base editing, cell therapy, immunotherapy, stem cell therapy, regenerative medicine, gene silencing, nucleic acid inhibition, or protein inhibition. [Background technology]

[0002] Introducing exogenous nucleic acids encoding one or more polypeptides for preventive and therapeutic purposes has long been a goal of biomedical research, particularly in light of advances in gene therapy. In this context, the introduction of exogenous nucleic acids has proven useful, more specifically, in relation to nucleic acid-based vaccination, protein therapy, protein substitution therapy, gene editing, base editing, cell therapy, immunotherapy, stem cell therapy, regenerative medicine, gene silencing, RNA inhibition, or protein inhibition.

[0003] Nucleic acid delivery is a promising new tool with several applications, capable of treating several currently incurable diseases, such as genetic disorders, cancers, and some retinal diseases, and can also be used for vaccination purposes. Nucleic acid delivery involves introducing nucleic acids, such as RNA and DNA, into cells. Since naked nucleic acids themselves are typically not efficiently internalized by cells, a carrier system (vector) is required for nucleic acid delivery. The introduction of exogenous nucleic acids into cells varies depending on the target cell or organism, the type of nucleic acid molecule, and / or the delivery system.

[0004] Influenced by concerns regarding the safety and efficacy associated with the use of deoxyribonucleic acid (DNA) molecules, ribonucleic acid (RNA) molecules have attracted increasing attention in recent years. Various approaches for RNA delivery, such as non-viral or viral delivery vehicles, have been proposed. In viruses and viral delivery vehicles, nucleic acids are typically encapsulated by proteins and / or lipids (viral particles). For example, engineered RNA virus particles derived from RNA viruses have been proposed as delivery vehicles for plant therapy or mammalian vaccination. Various compounds for vectorizing nucleic acids, so-called transfection reagents, have been described previously. These compounds are usually either polycations or compositions containing lipid-like compounds such as cationic lipids or lipidoids. Complexes of nucleic acids with polycations are called polyplexes, and complexes with cationic lipids are called lipoplexes.

[0005] While viruses are currently the most efficient delivery vehicle available, their usability has raised safety concerns. The medical and veterinary communities are reluctant to administer RNA viral particles to humans or animals. For all the reasons mentioned above, other types of vectors that do not contain viral particles are currently being investigated. The non-viral vectors currently being investigated include polymers, which have been found to be advantageous due to their chemical flexibility, ease of synthesis, potential biocompatibility, simplicity, and low cost of synthesis.

[0006] Prior art has disclosed the use of polymers such as PEI and PGA for the delivery of biomacromolecules (Patent Document 1). The use of polymer micelles for the delivery of various therapeutic drugs has also been described (Patent Document 2). However, prior art compositions often exhibit drawbacks such as low transfection efficiency or limitations due to their cytotoxicity. Therefore, non-viral vectors have been diligently investigated in relation to nucleic acid delivery, but the bridging of non-viral vector approaches to clinical practice has not been very successful for various reasons, namely toxicity, insufficient transfection efficiency, and technical and regulatory problems. Thus, alternative pharmaceutical compositions for nucleic acid delivery and transfection are needed. In the present invention, the inventors have identified novel non-viral vectors that are efficient in nucleic acid delivery and transfection and overcome the problems described above. These vectors are characterized by containing PPIs, preferably with a low degree of polymerization, and more preferably with linear PPIs.

[0007] In particular, the present invention has found that L-PPI monomers and L-PPI / L-PEI copolymers having a high PPI content are more efficient at conjugating RNA compared to L-PEI monomers and L-PPI / L-PEI polymers having a low PPI content. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] International Publication No. 2018 / 156617 [Patent Document 2] International Publication No. 2018 / 002382 [Overview of the project]

[0009] In a first aspect, the present invention provides a novel composition comprising a polypropyleneimine polymer (PPI) and a nucleic acid. More specifically, the present invention provides a pharmaceutical composition comprising a PPI and a nucleic acid, wherein the PPI has a degree of polymerization of about 20 to 1000, preferably about 100 to 500, and most preferably about 200 to 300.

[0010] In a preferred embodiment, the PPI is linear.

[0011] In further embodiments, the composition further comprises polyethyleneimine polymer (PEI). In another embodiment, the PEI is linear.

[0012] In further embodiments, the PEI has a degree of polymerization of about 20 to 1000, preferably about 100 to 500, and most preferably about 200 to 300.

[0013] In certain embodiments, the PPI and PEI form a copolymer, which may be a random copolymer. Therefore, the present invention also provides pharmaceutical compositions comprising a PPI / PEI copolymer.

[0014] In a particular embodiment of the present invention, the copolymer has a degree of polymerization of about 20 to 1000, preferably about 100 to 500, and most preferably about 200 to 300.

[0015] In certain embodiments, the ratio of the degree of polymerization of PEI to the degree of polymerization of PPI in the composition or copolymer of the present invention is in the range of about 1:1 to 1:500, preferably about 1:1 to 1:100, and most preferably about 1:2 to 1:10.

[0016] In one embodiment, the pharmaceutical composition further comprises lipids.

[0017] In yet another embodiment, the nucleic acid is selected from a list including RNA or DNA molecules, preferably mRNA, self-replicating mRNA (replicon), circular mRNA, circular RNA, mRNA or replicon whose translation can be controlled by an external or internal molecule, non-coding RNA, siRNA, sense RNA, antisense RNA, ribozyme, RNA aptamer, RNA aptazyme, saRNA, pDNA, minicircle, closed linear DNA, genomic DNA, cDNA, single-stranded and / or double-stranded DNA, and any combination or chemical modification thereof.

[0018] In yet another embodiment, the N / P ratio is less than 40, preferably less than 20, and more preferably less than 10.

[0019] In another embodiment, the pharmaceutical composition according to the present invention is used in human or veterinary medicine, and more specifically, the pharmaceutical composition is used in (nucleic acid) vaccination, nucleic acid-based protein therapy, nucleic acid-based protein replacement therapy, gene editing, base editing, cell therapy, immunotherapy, stem cell therapy, regenerative medicine, gene silencing, nucleic acid inhibition, or protein inhibition.

[0020] The present invention has the advantage that the composition has high transfection efficiency and low cytotoxicity compared to non-viral carriers of the current state of the art, and further has the advantage that its small size makes it suitable for in vivo use.

[0021] The following will refer specifically to the drawings, but it is emphasized that the descriptions provided are illustrative and intended solely as a descriptive discussion of various embodiments of the present invention. These drawings are presented to provide what is considered to be the most useful and simplest explanation of the principles and conceptual aspects of the present invention. In this regard, no further details of the structural aspects of the present invention beyond those necessary for a basic understanding of the present invention are to be shown. This description, together with the drawings, will make to those skilled in the art how several embodiments of the present invention can be actually implemented. [Brief explanation of the drawing]

[0022] [Figure 1] A diagram (also abbreviated as FIG. 1) showing the transfection efficiency of the composition according to the present invention containing linear PPI (L-PPI) having a DP of 250. [Figure 2] A diagram (also abbreviated as FIG. 2) showing the transfection efficiency of the composition containing linear PEI (L-PEI) having a DP of 250. [Figure 3] A diagram (also abbreviated as FIG. 3) showing the transfection efficiency of the composition containing a copolymer (L-PEI / L-PPI) of linear PEI and linear PPI having DPs of 50 / 200. [Figure 4] A diagram (also abbreviated as FIG. 4) showing the transfection efficiency of the composition containing a copolymer (L-PEI / L-PPI) of linear PEI and linear PPI having DPs of 200 / 50. [Figure 5] A diagram (also abbreviated as FIG. 5) showing the transfection efficiency of the composition containing a copolymer (L-PEI / L-PPI) of linear PEI and linear PPI having DPs of 100 / 150. [Figure 6] A diagram (also abbreviated as FIG. 6) showing the in vitro transfection efficiency of the composition containing a copolymer (L-PEI / L-PPI) of linear PEI and linear PPI having DPs of 150 / 100. [Figure 7] A diagram (also abbreviated as FIG. 7) showing the transfection efficiency of the composition containing modified mRNA and linear PEI (L-PEI) having a DP of 250. [Figure 8] A diagram (also abbreviated as FIG. 8) showing the gene silencing effect in HeLa cells of the composition containing siRNA and a copolymer (L-PEI / L-PPI) of linear PEI and linear PPI having DPs of 50 / 200. [Figure 9] A diagram (also abbreviated as FIG. 9) showing the gene silencing effect in SKOV3-Luc cells of the composition containing siRNA and a copolymer (L-PEI / L-PPI) of linear PEI and linear PPI having DPs of 50 / 200. [Figure 10] This figure (also abbreviated as Figure 10) shows the measured Z potential of a composition containing a linear PPI (L-PPI) having 250 DPs. [Figure 11] This figure (also abbreviated as Figure 11) shows the measured Z potential of a composition containing a copolymer (L-PEI / L-PPI) of linear PEI and linear PPI having a DP of 50 / 200. [Figure 12] This figure (also abbreviated as Figure 12) shows the size measurement results of a composition containing a linear PPI (L-PPI) having 250 DPs and replicon RNA. [Figure 13] This figure (also abbreviated as Figure 13) shows the size measurement results of a composition containing a linear PPI (L-PPI) with 250 DPs and modified non-replicating mRNA. [Figure 14] This figure (also abbreviated as Figure 14) shows the size measurement results of a composition containing a copolymer of linear PEI and linear PPI having 50 / 200 DP (L-PEI / L-PPI) and replicon RNA. [Figure 15] This figure (also abbreviated as Figure 15) shows the cell viability of a composition containing a linear PPI having 250 DPs. [Figure 16] This figure (also abbreviated as Figure 16) shows the cell viability of a composition containing linear PEI having 250 DPs. [Figure 17] This figure (also abbreviated as Figure 17) shows the cell viability of a composition containing a copolymer (L-PEI / L-PPI) of linear PEI and linear PPI having a DP ratio of 50 / 200 (1:4). [Figure 18] This figure (also abbreviated as Figure 18) shows the cell viability of a composition containing a copolymer (L-PEI / L-PPI) of linear PEI and linear PPI having a DP ratio of 200 / 50 (4:1 ratio). [Figure 19] This figure (also abbreviated as Figure 19) shows the cell viability of a composition containing a copolymer (L-PEI / L-PPI) of linear PEI and linear PPI having a DP ratio of 100 / 150 (2:3). [Figure 20]This figure (also abbreviated as Figure 20) shows the cell viability of a composition containing a copolymer (L-PEI / L-PPI) of linear PEI and linear PPI having a DP ratio of 150 / 100 (3:2). [Figure 21] This figure (also abbreviated as Figure 21) shows the transfection results of an in vitro transfection efficiency test conducted using lipofectamine MessengerMax (MM), a transfection agent representing the current level of technology. [Figure 22] This figure (also abbreviated as Figure 22) shows the cell viability of a composition containing lipofectamine MessengerMax (MM) in a ratio of 2:1 (μl MM:μg mRNA). [Modes for carrying out the invention]

[0023] The present invention will now be described further. The following sections will further specify various aspects of the present invention. Each of the aspects specified herein may be combined with any other aspect(s) unless it is explicitly stated otherwise. In particular, any feature shown to be preferred or advantageous may be combined with any other feature(s) shown to be preferred or advantageous.

[0024] When describing the compounds of the present invention, terms used shall be interpreted according to the following definitions unless otherwise indicated by the context.

[0025] This invention describes a pharmaceutical composition comprising (a) a polypropylene imine polymer PPI and (b) a nucleic acid, wherein the PPI has a degree of polymerization of about 20 to 1000, preferably about 100 to 500, and most preferably about 200 to 300. The inventors have found that the composition according to the present invention exhibits higher transfection efficiency than other non-viral carriers currently available. Furthermore, this composition has a small particle size, making it suitable for in vivo use. Thus, this invention has the advantage of the composition having high transfection efficiency and the further advantage of being suitable for in vivo use due to its small size. Moreover, the cytotoxic effect induced by this composition is lower than that of non-viral carrier-based compositions of the current state of the art.

[0026] The pharmaceutical composition according to the present invention may be or may comprise polymer blends, copolymers, homopolymers, block copolymers, gradient copolymers, and random copolymers. The pharmaceutical composition may further comprise an active pharmaceutical ingredient and other excipients.

[0027] The term "nucleic acid" refers to a biomolecule composed of pentose sugars, phosphate groups, and nitrogen bases. The term nucleic acid includes single-stranded and / or double-stranded DNA and RNA, as well as any combination thereof or chemically modified thereof.

[0028] In this specification, the terms “degree of polymerization” or “DP” refer to the number-average degree of polymerization unless otherwise specified. This can be calculated using the formula Mn / M0, where Mn is the number-average molecular weight of the polymer and M0 is the molecular weight of the monomer unit. For example, since PPI is composed of repeating propylamine units, propylamine is the monomer unit of PPI. This monomer unit of PPI has a molecular weight of approximately 57.1 g / mol. Based on the above formula, the number-average molecular weight of a polymer of PPI with a DP of 200 can be calculated to be approximately 11,400 g / mol in its free base form. The polymer may also consist of a protonated form, in which case the mass of the repeating units increases with the mass of the salt. For example, in the case of the HCl salt, the mass of the PPI-HCl repeating units is approximately 93.6 g / mol.

[0029] In this specification, the terms “about” and “approximately” when referring to measurable values ​​such as parameters, quantities, and periods mean that variations of + / -10%, preferably + / -5%, more preferably + / -1%, and even more preferably + / -0.1% or less from a specified value are appropriate for carrying out the disclosed invention. It is understood that the values ​​themselves referred to by the modifying phrases “about” or “approximately” are also to be specifically and preferably disclosed.

[0030] As used herein and in the appended claims, the singular nouns ("a," "an," and "the") refer to multiple subjects unless otherwise specified in the context. For example, "a compound" means one compound or two or more compounds.

[0031] According to certain embodiments of the present invention, the composition may further comprise polyethyleneimine polymer (PEI).

[0032] Polyethyleneimine (PEI) and polypropyleneimine (PPI) are organic polymers with high cationic charge density. Polyethyleneimine, also called PEI or poly(ethyleneimine), is a polymer composed of ethylamine repeating units. Polypropyleneimine, also called PPI or poly(propyleneimine), is a polymer composed of n-propylamine repeating units. Since PEI and PPI can be protonated, particularly in their linear forms, given the presence of charged amino groups, polymer compositions containing PEI and / or PPI can bind to and compress nucleic acids. PEI and PPI can interact with anionic proteoglycans on the cell surface to compress nucleic acids into positively charged particles that can facilitate particle penetration.

[0033] According to certain embodiments of the present invention, either PPI or PEI, or both thereof, are linear. In this application, linear PPI is also referred to as L-PPI, and linear PEI is also referred to as L-PEI. The use of linear PPI and / or linear PEI has the advantage that the resulting pharmaceutical composition can be positively charged and that the complex formed with the nucleic acid has a small size. It also has the other advantage that, due to the short polymer length, it is easier to remove by the kidneys.

[0034] Furthermore, it was found that L-PPI monomers and L-PPI / L-PEI copolymers with a high PPI content complex RNA more efficiently than L-PEI monomers and L-PPI / L-PEI polymers with a low PPI content.

[0035] According to a particular embodiment of the present invention, the above PEI has a degree of polymerization of about 20 to 1000, preferably about 100 to 500, and most preferably about 200 to 300.

[0036] The term "average particle diameter" refers to the average hydrodynamic diameter of a particle measured by dynamic light scattering with data analysis using the so-called cumulant algorithm, which consequently provides a so-called "Z-mean" with a length dimension. Here, the "average particle diameter," "particle diameter," or "size" of a particle are used synonymously with this Z-mean value.

[0037] According to the present invention, the "N / P ratio" refers to the molar ratio of nitrogen atoms (N) in the polymer to phosphorus atoms (P) in the nucleic acid. The N / P ratio reflects the input molar ratio of a given amount of nitrogen in the polymer to a given amount of phosphate in the nucleic acid. In certain embodiments, this N / P ratio is less than 40, preferably less than 20, more preferably less than 10, for example, 5, 4, 3, 2, 1 or less, for example, 0.5 or 0.2. In certain embodiments, the N / P ratio can be, for example, about 0.2 to 10, for example, 1 to 10 or 1 to 5. Alternatively, this N / P ratio can also be 1 to 20.

[0038] Specifically, a lower N / P ratio can be beneficial in reducing the toxicity of the composition used. This may be the case, for example, with compositions that have a relatively high PPI content.

[0039] According to a further specific embodiment of the present invention, the above PPI and the above PEI or the above L-PPI and L-PEI form a copolymer, preferably a random copolymer.

[0040] The copolymer of L-PPI and L-PEI can be represented, for example, as follows: [ka]

[0041] To our surprise, we have found that RNA is efficiently complexed and transfected into cells when the copolymer has a degree of polymerization of preferably about 20 to 1000, more preferably about 100 to 500, and most preferably about 200 to 300. More specifically, higher RNA complexation can be achieved in copolymers having a relatively high L-PPI / L-PEI ratio.

[0042] A particularly preferred composition of the present invention is characterized by comprising one or more of the following: PPI / PEI copolymer with high PPI content PPI with 250 DP PPI / PEI copolymer with 250 DP PPI / PEI copolymer having a PPI / PEI ratio of at least 1.5:1, preferably at least 4:1. N / P ratio greater than 1, preferably greater than 5, more preferably 5 to 20

[0043] Particularly preferred compositions of the present invention are L-PPI with 250 DP, It contains RNA with an N / P ratio of approximately 0.2 to 10, preferably 1 to 10, and most preferably 1 to 5. These compositions are characterized in particular by having high transfection efficiency, small particle size, good stability, and low toxicity.

[0044] Another particularly preferred composition of the present invention is, A copolymer of L-PEI and L-PPI having 250 DP, It contains RNA with an N / P ratio of approximately 1-20. It has a PPI / PEI ratio of at least 1.5:1, preferably at least 4:1. These compositions are characterized in particular by having high transfection efficiency, small particle size, good stability, and low toxicity.

[0045] As used herein, the term “random copolymer” refers to a statistical copolymer in which the probability of finding a given type of monomer residue at a specific location in the chain is similar to the mole fraction of that monomer residue in the chain. This is typically described by the reactivity ratio to the statistical copolymerization of the parent polymer used as a precursor of L-PEI / PPI, where the reactivity ratio r1 is less than 1.35 and the reactivity ratio r2 is greater than 0.7 (r1=k p1,1 / k p1,2 , r²=k p2,2 / k p2,1 A copolymer in which monomer 1 is more reactive is defined as a random copolymer. The copolymers of the present invention may include other polymers other than PPI and / or PEI.

[0046] In the context of the present invention, L-PEI and L-PPI copolymers with various molar ratios of L-PEI to L-PPI were synthesized and tested. According to embodiments of the present invention, the ratio of the degree of polymerization (DP) of PEI to the degree of polymerization (DP) of PPI is in the range of 1:1 to 1:500, preferably about 1:1 to 1:100, and most preferably about 1:2 to 1:10. According to certain embodiments of the present invention, compositions containing PEI and PPI, and being PPI-rich, were found to exhibit higher transfection efficiency than other nucleic acid vectors. A PPI-rich composition is one in which the amount of PPI is greater than the amount of any other polymer component (such as PEI) in the composition. According to the present invention, the nucleic acid is selected from a list including RNA or DNA molecules, preferably mRNA, self-replicating mRNA (replicon), circular mRNA, circular RNA, mRNA or replicon whose translation can be controlled by an external or internal molecule, non-coding RNA, siRNA, sense RNA, antisense RNA, ribozyme, RNA aptamer, RNA aptazyme, saRNA, pDNA, minicircle, closed linear DNA, genomic DNA, cDNA, single-stranded and / or double-stranded DNA, and any combination or chemical modification thereof.

[0047] The term "RNA" refers to a molecule containing, and preferably entirely or substantially composed of, ribonucleotide residues, and includes all RNA types described herein. The term "RNA" includes isolated RNA such as double-stranded RNA, single-stranded RNA, partially or completely purified RNA, essentially pure RNA, synthetic RNA, and RNA produced by recombination, such as modified RNA which differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may include the addition of non-nucleotide material, for example, at one or more nucleotides of RNA, to the terminal(s) or internally of the RNA. Nucleotides in the RNA molecule may also include non-standard nucleotides, such as non-naturally occurring nucleotides, chemically synthesized nucleotides, or deoxyribonucleotides. These modified RNAs may be referred to as analogs, in particular analogs of naturally occurring RNA. The RNA used in accordance with the present invention may have a known composition, or the composition of the RNA may be partially or completely unknown.

[0048] The term "DNA" refers to a molecule comprising, and preferably entirely or substantially composed of, deoxyribonucleotide residues, and includes all DNA types described herein. The term "DNA" includes pDNA, minicircles, closed linear DNA, genomic DNA, cDNA, single-stranded and / or double-stranded DNA, and any combination or chemical modifications thereof.

[0049] In one embodiment, the pharmaceutical composition further comprises lipids. To further improve the properties of the pharmaceutical composition according to the present invention, co-formulations with lipids and / or negatively charged polymer coatings can be realized.

[0050] The term "lipids" refers to non-water-soluble fatty substances, including fats, oils, waxes, and related compounds. Lipids can be produced in the blood (endogenous) or obtained through diet (exogenous). Lipids are essential for normal bodily functions and, whether produced from exogenous or endogenous sources, need to be transported and then released for use by cells. The production, transport, and release of lipids for use by cells is called lipid metabolism. There are several classes of lipids, but the two main classes are cholesterol and triglycerides. Cholesterol is obtained through diet and can be produced by cells in most organs and tissues in the body, mainly in the liver. Cholesterol can be found in free form or, more often, in combination with fatty acids called cholesterol esters.

[0051] The pharmaceutical compositions according to the present invention may be used in human or veterinary medicine. According to further embodiments, the pharmaceutical compositions according to the present invention may be used in methods in which nucleic acid delivery is useful, for example, but not limited to, (nucleic acid) vaccination or nucleic acid-based protein therapy, nucleic acid-based protein replacement therapy, gene editing, base editing, cell therapy, immunotherapy, stem cell therapy, regenerative medicine, gene silencing, nucleic acid inhibition or protein inhibition, etc. [Examples]

[0052] Materials and methods Synthetic mRNA production Self-amplified RNA or replicons encoding luciferase derived from Venezuelan equine encephalitis virus (VEEV) were synthesized by in vitro transcription (IVT) using the MEGAscript® kit (Thermo Fisher Scientific, Massachusetts, USA). I-SceI linear plasmid was used as a template. After purification using a silica column (RNeasy Mini Kit, Qiagen, Hilden, Germany), the RNA was capped using the ScriptCap® Cap 1 Capping System Kit (Cellscript, Wisconsin, USA) according to the manufacturer's instructions. Finally, the RNA was purified again using a silica column, and its concentration was measured by spectrophotometric analysis (Nanodrop, Thermo Fisher Scientific, Massachusetts, USA).

[0053] N1-methylpseudridine (1mΨ) modified non-replicating mRNA (mod-mRNA) encoding luciferase was prepared by IVT from I-SceI linear plasmid by replacing all uridine-5'-triphosphates in the IVT mix with N1-methylpseudridine-5'-triphosphate (Trilink Biotechnologies, San Diego, USA). The mRNA was then purified and capped using vaccinia virus capping enzyme and 2'-O-methyltransferase (Cellscript, Wisconsin, USA) to produce cap 1, which was then purified again using the RNeasy mini kit (Qiagen, Germany). The poly(A) tails of these mod-mRNAs, which were 40 adenosines long, were extended to approximately 200 adenosines using the A-plus Poly(A) polymerase tailing kit (Cellscript) and then purified. Finally, mod-mRNA concentration was measured by spectrophotometric method (Nanodrop, Thermo Fisher Scientific, Massachusetts, USA).

[0054] Small interfering RNA Small interfering RNAs (siRNAs) targeting firefly luciferase (pGL3) or control siRNA were purchased from Dharmacon (Lafayette, USA), dissolved in RNase-free water at a concentration of 16.5 μM, and stored in 20 μl aliquots at -20°C.

[0055] Polymer fabrication Polymers L-PEI DP 250, polymer L-PPI DP 250, and their copolymers (L-PEI / L-PPI DP 200 / 50, 150 / 100, 100 / 150, 50 / 200) were synthesized as follows. First, copolymers of 2-ethyl-2-oxazoline (EtOx) and 2-isopropyl-2-oxazine (iPrOzi) (with various monomer ratios (EtOx:iPrOzi = 250:0, 0:250, 200:50, 150:100, 100:150, and 50:200)) were prepared at a total monomer concentration of 4 M, using methyl tosylate as an initiator, with a total monomer-to-initiator ratio of 250 to obtain polymers with a DP of 250. Polymerization was carried out in a Biotage microwave reactor at 140°C until the monomers were completely converted, as confirmed by gas chromatography. Size exclusion chromatography confirmed the formation of fairly distinct copolymers with a dispersion degree of less than 1.4, and 1H NMR spectroscopy confirmed the acquisition of the desired composition. Subsequently, these copolymers (2-oxazoline) were hydrolyzed by dissolving 1 gram of polymer in 7.5 mL of demineralized water and 7.5 mL of hydrochloric acid (HCl) to obtain L-PPI and L-PEI / PPI polymers. The closed vials were then heated in a Biotage microwave reactor to 140°C for 9 hours to hydrolyze the polymers. The polymers were then diluted with demineralized water, and the HCl and demineralized water were evaporated under reduced pressure. The samples were neutralized with 2 M sodium hydroxide (NaOH) aqueous solution and lyophilized. 1H NMR analysis confirmed nearly quantitative hydrolysis.

[0056] Preparation and Characterization of Self-Amplified and Modified mRNA Nanocomplexes To prepare the nanocomplexes, equal volumes of RNA solution were added to the polymer solution, gently mixed, and incubated at room temperature for 30 minutes. Both the polymer and RNA were dissolved in 20 mM sodium acetate buffer (pH=5.2). Nanocomplexes were prepared using various polymer-to-mRNA ratios. The N / P ratios were 40, 20, 10, 5, 1, and 0.2 for the self-amplified mRNA nanocomplexes, and 30, 15, 8, 4, 0.8, and 0.2 for the mod-mRNA nanocomplexes. Subsequently, the size and zeta potential of the self-amplified and modified mRNA nanocomplexes were measured using dynamic light scattering (Zetasizer Nano, Malvern Instruments, Malvern, UK). Zeta potential is a typical measure of surface charge and, consequently, the stability of charged particles in suspension. Typically, a zeta potential of at least about 20 indicates that the particles have good stability.

[0057] Cell culture and transfection procedures HeLa cells were cultured in medium and maintained in a humidified incubator at 37°C and 5% CO2. The medium consisted of Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Thermo Fisher Scientific, Massachusetts, USA) supplemented with 10% fetal bovine serum (Biowest, California, USA), 5,000 units / mL of penicillin, and 5,000 μg / mL of streptomycin (Thermo Fisher Scientific, Massachusetts, USA). One day prior to transfection with self-amplified or modified mRNA, HeLa cells were seeded at a density of 50,000 cells / well in 24-well plates. The following day (i.e., 24 hours later), the medium was changed to opti-MEM, and 20 μL of polymer:RNA complex solution containing 500 ng of RNA was added to each well. Luciferase expression was analyzed by bioluminescence imaging 24 hours after transfection. For this purpose, cells were trypsinized, and a portion (60%) of the neutralized cell suspension was transferred to a black 96-well plate. D-luciferin solution (50 mg / ml; 10% of the final well volume) was added to each well and incubated for 10 minutes. Subsequently, the emitted bioluminescence was measured using IVIS Lumina II (Xenogen Corporation, Alameda, California, USA). Transfection with the reference carrier Lipofectamine MessengerMax (Thermo Fischer Scientific) at various ratios was similarly performed using 500 ng of RNA per 24 wells.

[0058] Preparation of siRNA nanocomplexes and their silencing effect in vitro. To evaluate the siRNA delivery capability of the polymer, siRNA nanocomplexes were prepared by adding an equal volume of siRNA solution to the polymer solution, gently mixing, and incubating at room temperature for 30 minutes. Both the polymer and siRNA were dissolved in 20 mM sodium acetate buffer (pH=5.2). The final concentration of siRNA was 10 nM. siRNA nanocomplexes were prepared using various polymer (PEI / PPI DP 50 / 200) and siRNA ratios. Subsequently, these siRNA nanocomplexes were tested using two protocols.

[0059] In the first of a series of experiments, cotransfection of HeLa cells with a luciferase replicon using a PEI / PPI siRNA nanocomplex was performed. HeLa cells were cultured as described above. One day prior to cotransfection with the replicon mRNA and siRNA nanocomplex based on PEI / PPI(50 / 200), HeLa cells were seeded in 24-well plates at a density of 50,000 cells / well. The following day (i.e., 24 hours later), the medium was changed to Opti-MEM, and cells were transfected with a replicon encoding luciferase using PEI / PPI(DP 50 / 200) at an N / P ratio of 5. After 30 minutes, a PEI / PPI siRNA nanocomplex containing 6 pmol of siRNA was added to the cells. After 24 hours, luciferase was measured for mRNA using the IVIS Lumina II imaging system as described above. For control, cells treated with either luciferase replicons alone or with luciferase replicons and PEI / PPI nanocomplexes containing scrambled siRNA prepared with the highest N / P ratio investigated were used. The latter ratio is expected to exhibit the highest cytotoxicity.

[0060] In the second experiment in the series, SKOV3-Luc cells, which stably express firefly luciferase, were used. These cells were cultured in McCoy's 5A (modified) medium (Gibco, Thermo Fisher Scientific, Massachusetts, USA) supplemented with 10% fetal bovine serum (Biowest, California, USA), 5,000 units / mL of penicillin, and 5,000 μg / mL of streptomycin (Thermo Fisher Scientific, Massachusetts, USA) in a humidified incubator at 37°C and 5% CO2. One day prior to transfection with siRNA nanoconjugates based on PEI / PPI(50 / 200), SKOV-3-Luc cells were seeded in 24-well plates at a density of 50,000 cells / well. The following day (i.e., 24 hours later), the medium was changed to opti-MEM, and 20 μL of PEI / PPI(50 / 200) polymer siRNA nanoconjugate solution containing 6 pmol of siRNA was added to each well. 36 hours after transfection, luciferase expression in mRNA was analyzed using the IVIS Lumina II imaging system, as described above.

[0061] Cell viability Cell viability 24 hours after transfection was determined using Cell Proliferation Reagent WST-1 (Roche). After trypsin treatment, a portion of the neutralized volume (6.66%) was transferred to a clear ELISA plate, and WST-1 solution was added according to the manufacturer's instructions. After 30 minutes of incubation, the plate was shaken in a plate shaker for 1 minute, and the absorbance at 450 nm (reference 620 nm) was measured using an EZ Read 400 microplate reader (Biochrom).

[0062] in vivo transfection The in vivo transfection efficiency of a nanocomplex containing self-amplified mRNA and an L-PEI / PPI polymer (DP 50 / 200) was investigated in chickens after local injection into the neck or wing. For this purpose, the self-amplified mRNA-PEI-PPI nanocomplex was prepared with an N / P ratio of 5, as described in the in vitro experiment. Subsequently, the nanocomplex containing 5 μg of self-amplified mRNA (encoding luciferase) was injected into the neck or wing. Two days later, the chickens were injected with D-luciferin, followed by euthanasia and imaging with IVIS Lumina II.

[0063] result Transfection efficiency measurement Figures 1-7 show the results of in vitro transfection efficiency tests performed using self-amplifying mRNA nanocomplexes or modified mRNA nanocomplexes. Compositions containing polymers and nucleic acids were prepared with various N / P ratios. More specifically, the N / P ratios were 0.2, 1, 5, 10, 20, and 40. A control solution containing only the buffer and therefore no nanocomplexes was also prepared (controle, ctrl).

[0064] Figure 1 shows the transfection results of compositions containing self-amplified mRNA and linear PPI (L-PPI) with 250 DPs. From this figure, it can be seen that in each case, the transfection efficiency is higher compared to the control. Particularly high transfection efficiency is achieved for compositions with an N / P ratio of 1-10, especially 5-10.

[0065] Figure 2 shows the transfection results of compositions containing self-amplified mRNA and linear PEI (L-PEI) with 250 DPs. It is important to note that the bioluminescence intensity, and therefore the transfection efficiency, of the first composition at N / P ratios of 10 and 5 significantly exceeds that of the second composition. In contrast to the results obtained for PPI, no increase in transfection efficiency compared to the control was observed for PEI, except for the case with an N / P ratio of 40, for various compositions.

[0066] Figure 3 shows the transfection results for compositions containing self-amplified mRNA and a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) having a DP ratio of 50 / 200. Therefore, the degree of polymerization of the PEI and the degree of polymerization of the PPI are in a ratio of 1:4. In this case as well, an increase in transfection efficiency was observed for all compositions compared to the control, and particularly high transfection efficiency was obtained for compositions with an N / P ratio greater than 5.

[0067] Figure 4 shows the transfection results of compositions containing self-amplified mRNA and a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) with a DP ratio of 200 / 50 (4:1 ratio). It is important to note that the bioluminescence intensity of the first composition, which is rich in PPI, is significantly higher than that of the second composition for each N / P ratio tested. Therefore, the first composition, which is rich in L-PPI, exhibits even higher transfection efficiency than the composition containing L-PPI but not L-PEI (the results are shown on the left side of Figure 1).

[0068] Figure 5 shows the transfection results of a composition containing self-amplified mRNA and a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) with a DP ratio of 100 / 150 (2:3 ratio). This figure again shows that excess PPI has a beneficial effect on the transfection efficiency of the tested composition.

[0069] Figure 6 shows the transfection results of a composition containing self-amplified mRNA and a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) having a DP ratio of 150 / 100 (3:2 ratio). This figure confirms that the excess PEI in the composition does not substantially affect the transfection efficiency of the tested composition.

[0070] Figure 7 shows the transfection results of compositions containing modified mRNA and a linear PPI (L-PPI) with 250 DPs. From this figure, it can be seen that transfection efficiency increases compared to the control (ctrl) at N / P ratios of 0.8 to 30. Particularly high transfection efficiency is achieved for compositions with N / P ratios of 1 to 10. This graph also shows the results of in vitro transfection using lipofectamine MessengerMax (MM), a transfection agent representing the current state of technology. A composition containing this lipid carrier and modified mRNA (in a ratio of 2 μl MM:1 μg mod-mRNA) typically exhibits a transfection efficiency of 1 × 10⁻⁶. 6 ~1 × 10 7 This resulted in transfection efficiency. When using modified non-replicating mRNA, it can be concluded that the composition of the present invention is at least as efficient as MM.

[0071] Figure 8 shows the transfection results in HeLa cells with a composition containing siRNA and a copolymer of linear PEI and linear PPI with 50 / 200 DP (L-PEI / L-PPI). This figure shows that siRNA-mediated silencing is most efficient with an N / P ratio of 1–0.2 compared to HeLa cells receiving only scrambled siRNA or a luciferase-encoding replicon (negative Ctrl). The data were obtained by adding siRNA nanocomplexes to HeLa cells co-transfected with a luciferase-encoding replicon. Lower expression (total flux) indicates good intracellular delivery of siRNA and subsequent signaling of the target luciferase mRNA.

[0072] Figure 9 shows the transfection results in SKOV-3-Luc cells with a composition containing siRNA and a copolymer of linear PEI and linear PPI with 50 / 200 DP (L-PEI / L-PPI). This figure shows that siRNA-mediated silencing is most efficient with an N / P ratio of 5 or less. SKOV-3-Luc cells stably express luciferase. Lower expression (total flux) indicates good intracellular delivery of siRNA and subsequent signaling of target luciferase mRNA.

[0073] Overall, the results indicate that the L-PPI-rich composition exhibits higher transfection efficiency compared to the L-PEI-rich composition. Furthermore, the results show that the composition is effective in vivo in conjunction with modified mRNA and siRNA.

[0074] Physicochemical properties of compounds Zeta potential Figure 10 shows the measured zeta potentials of compositions containing self-amplified mRNA and linear PPI (L-PPI) with 250 DPs. As shown, compositions with an N / P ratio of at least 5 exhibit excellent zeta potentials and are considered to be stable formulations.

[0075] Figure 11 shows the measured Z potentials of compositions containing self-amplified mRNA and a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) having a DP ratio of 50 / 200 (1:4 ratio). As shown in the figure, compositions having an N / P ratio of at least 5 exhibit excellent zeta potentials and are considered to be stable formulations.

[0076] Size measurement Figure 12 shows the size measurement results for compositions containing linear PPI (L-PPI) with 250 DPs and replicon RNA (self-amplified mRNA). Compositions with an N / P ratio of 1 or less were found to have a higher Z-mean compared to compositions with a higher N / P ratio. In some applications, a small average particle size can be beneficial.

[0077] Figure 13 shows the size measurement results for compositions containing modified mRNA and linear PPI (L-PPI) with 250 DPs. Compositions with an N / P ratio of 0.2 or less were found to have a higher Z-mean compared to compositions with a higher N / P ratio. In some applications, a smaller average particle size can be beneficial.

[0078] Figure 14 shows the size measurement results for compositions containing self-amplified mRNA, a copolymer of linear PEI and linear PPI having a DP ratio of 50 / 200 (1:4 ratio) (L-PEI / L-PPI), and replicon RNA. For PPI-rich copolymers, a small average particle size is obtained for compositions with an N / P ratio of 5 to 20.

[0079] Cell viability Figure 15 shows the cell viability after 24 hours of transfection with a composition containing self-amplified mRNA and a linear PPI (L-PPI) with 250 DPs. As is clear from the figure, the lower the N / P ratio, the lower the toxicity of the composition.

[0080] Figure 16 shows the cell viability after 24 hours of transfection with a composition containing self-amplified mRNA and linear PEI (L-PEI) with 250 DPs. In contrast to the results obtained for PPI, the N / P ratio does not significantly affect the toxicity of compositions containing large amounts of PEI.

[0081] Figure 17 shows the cell viability after 24 hours of transfection with a composition containing self-amplified mRNA and a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) having a 50 / 200 DP ratio (1:4 ratio). In the case of copolymers as well, the lower the N / P ratio, the lower the toxicity of the composition.

[0082] Figure 18 shows the cell viability after 24 - hour transfection with a composition containing self - amplifying mRNA and a copolymer (L - PEI / L - PPI) of linear PEI and linear PPI having a DP of 200 / 50 (ratio of 4:1). In contrast to the results obtained for PPI, the N / P ratio does not significantly affect the toxicity of the composition containing a large amount of PEI.

[0083] Figure 19 shows the cell viability after 24 - hour transfection with a composition containing self - amplifying mRNA and a copolymer (L - PEI / L - PPI) of linear PEI and linear PPI having a DP of 100 / 150 (ratio of 2:3). Also in the case of the copolymer, the lower the N / P ratio, the lower the toxicity of the composition.

[0084] Figure 20 shows the cell viability after 24 - hour transfection with a composition containing self - amplifying mRNA and a copolymer (L - PEI / L - PPI) of linear PEI and linear PPI having a DP of 150 / 100 (ratio of 3:2). In contrast to the results obtained for PPI, the N / P ratio does not significantly affect the toxicity of the composition containing a large amount of PEI.

[0085] Figure 21 shows the transfection results of an in vitro transfection efficiency test using lipofectamine MessengerMax (MM), a transfection agent of the current state of the art. Compositions containing this lipid carrier and nucleic acid in various ratios were prepared. The ratio shown is μl MM:μg mRNA. A control solution containing only the buffer and thus no nanocomplex was also prepared (controle). The typical transfection efficiency observed with MM is 1×10 6 ~1×10 7 . As is clear from Figures 1 and 3, the composition of the present invention is at least equally efficient or even better, that is, it reaches a transfection efficiency of 1×10 7 ~1×10 8 .

[0086] Figure 22 shows the cell viability after 24 hours of transfection with a composition containing lipofectamine MessengerMax (MM) in a 2:1 ratio (μl MM:μg mRNA). It was found that MM achieved only about 30% cell viability. In contrast, as is clear from Figures 15-20, the composition of the present invention can achieve cell viability of over 50%, and even close to 100%. It is important to note that cell viability was measured after a 24-hour transfection period.

[0087] in vivo experiments Furthermore, in vivo transfection experiments were performed in chickens using a composition containing self-amplified mRNA encoding luciferase and a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) with 50 / 200 DPs. Since ATP is required for the photosynthetic conversion of D-luciferin and is known to show a rapid decline after euthanasia, and visible bioluminescence signals often underestimate the actual signal, bioluminescence images were captured immediately after euthanasia. The results showed a clear bioluminescence signal in transfected chickens compared to uninjected control chickens (data not shown).

[0088] Drawing translation Figure 1 Total flux (photons / s) controle contrast Figure 2 Total flux (photons / s) controle contrast Figure 3 Total flux (photons / s) controle contrast Figure 4 Total flux (photons / s) controle contrast Figure 5 Total flux (photons / s) controle contrast Figure 6 Total flux (photons / s) controle contrast Figure 7 Total Flux (p / s) ctrl contrast Figure 8 Total Flux [p / s]: Total flux (number of photons per second) Neg.Ctrl Negative Control Scrambled siRNA Polymer N / P 2,5 Figure 9 Total Flux [p / s]: Total flux (number of photons per second) Figure 10 Zeta potential Figure 11 Zeta potential Figure 12 Z-average Figure 13 Z-average Figure 14 Z-average Figure 15 Viability Survival rate Figure 16 Viability Survival rate Figure 17 Viability Survival rate Figure 18 Viability Survival rate Figure 19 Viability Survival rate Figure 20 Viability Survival rate Figure 21 Total flux (photons / second) Ratio controle contrast Figure 22 cell viability cell viability

Claims

1. (a) Linear polypropyleneimine (L-PPI) / linear polyethyleneimine (L-PEI) copolymer, (b) Nucleic acids and, A pharmaceutical composition containing, The copolymer has a degree of polymerization of 200 to 1000. The copolymer has a PPI / PEI ratio of 2:1 to 100:

1. A pharmaceutical composition having an N / P ratio of 5 to 40.

2. The pharmaceutical composition according to claim 1, wherein the copolymer is a random copolymer.

3. The pharmaceutical composition according to claim 1 or 2, wherein the copolymer has a degree of polymerization of 200 to 500.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the copolymer has a degree of polymerization of 200 to 300.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the copolymer has a PPI / PEI ratio of 2:1 to 10:

1.

6. A pharmaceutical composition according to any one of claims 1 to 5, further comprising lipids.

7. The nucleic acid is an RNA or DNA molecule, or The nucleic acid is selected from a list including mRNA, self-replicating mRNA (replicon), circular mRNA, circular RNA, mRNA or replicon whose translation can be controlled by an external or internal molecule, non-coding RNA, siRNA, sense RNA, antisense RNA, ribozyme, RNA aptamer, RNA aptazyme, saRNA, pDNA, minicircle, closed linear DNA, genomic DNA, cDNA, single-stranded and / or double-stranded DNA, and any combination or chemical modifications thereof. A pharmaceutical composition according to any one of claims 1 to 6.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the N / P ratio is 5 to 20.

9. A pharmaceutical composition according to any one of claims 1 to 8, for use in humans or in veterinary medicine.

10. A pharmaceutical composition according to any one of claims 1 to 9, for use in nucleic acid vaccination or immunization, nucleic acid-based protein therapy, nucleic acid-based protein replacement therapy, gene editing, base editing, cell therapy, immunotherapy, stem cell therapy, regenerative medicine, gene silencing, nucleic acid inhibition, or protein inhibition.

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