Non-viral vectors comprising polypropyleneimine

Abstract

The present invention relates to the field of non-viral vectors and pharmaceutical compositions comprising a polypropyleneimine and a nucleic acid and their use in human or veterinary medicine. More specifically, the present invention relates to pharmaceutical compositions comprising polymers or copolymers of polypropyleneimine for 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

[0001] This application is a divisional, the application number of the parent application is 202080088144.0, the application date is December 17, 2020, and the name of the invention is "Non-viral vector containing polypropylene imine". Field of the Invention

[0002] The present invention relates to the field of non-viral vectors and pharmaceutical compositions comprising polypropylene imine and nucleic acids and their uses in humans or veterinary medicine. More specifically, the present invention relates to pharmaceutical compositions of polymers or copolymers comprising polypropylene imine for delivery or transfection of nucleic acids (e.g., RNA). The pharmaceutical compositions described herein are particularly suitable 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 of the Invention

[0004] For many years, especially in view of the progress of gene therapy, the introduction of exogenous nucleic acids encoding one or more polypeptides for prevention and treatment purposes has been the target of biomedical research. However, it has been proved that the introduction of exogenous nucleic acids is useful, more specifically in the context of nucleic acid-based vaccination, protein therapy, protein replacement therapy, gene editing, base editing, cell therapy, immunotherapy, stem cell therapy, regenerative medicine, gene silencing, RNA inhibition or protein inhibition. Nucleic acid delivery is a promising new tool with several applications, which can treat some currently incurable diseases, such as genetic disorders, cancer diseases and some retinal diseases, and can also be used for vaccination purposes. Nucleic acid delivery includes introducing nucleic acid (such as RNA and DNA) into cells. Because naked nucleic acid itself can not be effectively internalized by cells usually, nucleic acid delivery requires a carrier system (carrier). The introduction of exogenous nucleic acids in cells changes according to the type and / or delivery system of target cells or organisms, nucleic acid molecules.

[0005] Affected by the safety and effectiveness issues associated with the use of deoxyribonucleic acid (DNA) molecules, ribonucleic acid (RNA) molecules have received increasing attention in recent years. Various methods for delivering RNA have been proposed, such as non-viral or viral delivery vectors. In viruses and viral delivery vectors, nucleic acids are usually encapsulated by proteins and / or lipids (virions). For example, engineered RNA virus particles derived from RNA viruses have been proposed as delivery vectors for treating plants or for mammalian vaccination. A variety of compounds for nucleic acid vectorization, so-called transfection reagents, have been previously described. These compounds are typically polycations or compositions comprising cationic lipids or lipid-like compounds (such as lipidoids). The complex of nucleic acid and polycations is referred to as a polymer complex (polyplexes), and those complexes with cationic lipids are referred to as cationic liposomes (lipoplexes).

[0006] Although viruses are the most effective delivery vectors currently available, their possible use raises safety concerns. The medical and veterinary communities are reluctant to administer RNA virus particles to humans or animals. Due to all of the above reasons, other types of vectors that do not contain virus particles are currently being studied. Non-viral vectors currently under investigation include polymers, which have been found to have advantages due to their chemical flexibility, ease of synthesis, potential for biocompatibility, simplicity, and inexpensive synthesis.

[0007] Prior art discloses the use of polymers (such as PEI and PGA) for delivering biomacromolecules (WO2018 / 156617A2). It also describes the use of polymer micelles for delivering various therapeutic drugs (WO2018 / 002382A1). However, compositions in the prior art generally exhibit disadvantages such as low transfection efficiency or being limited by their cytotoxicity. Therefore, even though non-viral vectors have been extensively studied in the context of nucleic acid delivery, due to various reasons, i.e., toxicity, unsatisfactory transfection efficiency, technology and regulatory issues, it is not very successful to convert non-viral vector methods into clinical practice. Therefore, there is a need for alternative pharmaceutical compositions for delivery and transfection of nucleic acids. In the present invention, we have identified novel non-viral vectors that are effective in nucleic acid delivery and transfection and overcome the above problems. These vectors are characterized in that they contain PPI, preferably with a low degree of polymerization, more preferably linear PPI.

[0008] A specific finding of the present invention is that L-PPI monomers and L-PPI / L-PEI copolymers having high PPI content were found to more efficiently complex RNA than L-PEI monomers and L-PPI / L-PEI polymers having low PPI content. SUMMARY OF THE INVENTION

[0010] 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 degree of polymerization of the PPI is about 20-1000, preferably about 100-500, and most preferably about 200-300.

[0011] In a preferred embodiment, the PPI is linear.

[0012] In a further embodiment, the composition further comprises a polyethyleneimine polymer (PEI). In another embodiment, the PEI is linear.

[0013] In a further embodiment, the degree of polymerization of the PEI is about 20-1000, preferably about 100-500, and most preferably about 200-300.

[0014] In one embodiment, the PPI and the PEI form a copolymer, which may be a random copolymer. Therefore, the present invention also provides a pharmaceutical composition comprising a PPI / PEI copolymer.

[0015] In one embodiment according to the present invention, the degree of polymerization of the copolymer is about 20-1000, preferably about 100-500, and most preferably about 200-300.

[0016] In a specific embodiment, in the composition or copolymer of the present invention, the degree of polymerization of the PEI and the degree of polymerization of the PPI are in the range of about 1:1-1:500, preferably about 1:1-1:100, and most preferably about 1:2-1:10.

[0017] In one embodiment, the pharmaceutical composition further comprises a lipid.

[0018] In another embodiment, the nucleic acid is an RNA or DNA molecule; preferably selected from mRNA, self-replicating mRNA (replicon), circular mRNA, circular RNA, mRNA or replicon whose translation can be controlled by external or internal molecules, non-coding RNA, siRNA, sense RNA, antisense RNA, ribozymes, RNA aptamers, RNA aptazymes, saRNA, pDNA, minicircles, closed linear DNA, genomic DNA, cDNA, single-stranded and / or double-stranded DNA, and any combination or chemically modified form thereof.

[0019] In yet another embodiment, the N / P ratio is less than 40; preferably less than 20; more preferably less than 10.

[0020] In another embodiment, the pharmaceutical composition according to the present invention is used in human medicine or veterinary medicine, more specifically, the pharmaceutical composition is used 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.

[0021] One advantage of the present invention is that the compositions have high transfection efficiency, low cytotoxicity compared to prior art non-viral vectors, and another advantage is that their small size makes them suitable for in vivo use.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] With specific reference now to the accompanying drawings, it is emphasized that the details shown are only by way of example and only for the purpose of illustrative discussion of different embodiments of the present invention. They are presented in order to provide what are considered to be the most useful and easy descriptions of the principles and conceptual aspects of the present invention. In this regard, no attempt is made to illustrate the structural details of the present invention in more detail than is necessary for a basic understanding of the present invention. The description in conjunction with the accompanying drawings makes it clear to those skilled in the art how to embody several forms of the present invention in practice.

[0024] Figure 1 (Figure 1), also abbreviated as Figure 1 (Fig. 1) shows the transfection efficiency of a composition comprising a linear PPI (L-PPI) and a DP of 250 according to the present invention,

[0025] Figure 2 , also abbreviated as Figure 2 , showing the transfection efficiency of a composition comprising linear PEI (L-PEI) and a DP of 250.

[0026] Figure 3 , also abbreviated as Figure 3 , shows the transfection efficiency of a composition comprising a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) with a DP of 50 / 200.

[0027] Figure 4 , also abbreviated as Figure 4 , shows the transfection efficiency of a composition comprising a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) with a DP of 200 / 50.

[0028] Figure 5 , also abbreviated as Figure 5 , shows the transfection efficiency of a composition comprising a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) with a DP of 100 / 150.

[0029] Figure 6 , also abbreviated as Figure 6 , showing the in vitro transfection efficiency of a composition comprising a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) with a DP of 150 / 100.

[0030] Figure 7 , also abbreviated as Figure 7 , shows the transfection efficiency of a composition comprising modified mRNA and linear PEI (L-PEI) with a DP of 250.

[0031] Figure 8 , also abbreviated as Figure 8 , shows the gene silencing efficiency of a composition comprising siRNA and a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) with a DP of 50 / 200 in HeLa cells.

[0032] Fig. 9 , also abbreviated as Fig. 9 , shows the gene silencing efficiency of a composition comprising siRNA and a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) with a DP of 50 / 200 in SKOV3-Luc cells.

[0033] Fig.10 , also abbreviated as Fig.10 , shows the determination of the zeta potential of a composition comprising a linear PPI (L-PPI) having a DP of 250.

[0034] Fig.11 , also abbreviated as Fig.11 , shows the determination of the zeta potential of a composition comprising a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) having a DP of 50 / 200.

[0035] Fig.12 , also abbreviated as Fig.12 , shows the size determination of a composition comprising a linear PPI (L-PPI) with a DP of 250 and replicon RNA.

[0036] Fig.13 , also abbreviated as Fig.13 , shows the size determination of a composition comprising a linear PPi (L-PPi) with a DP of 250 and a modified non-replicating mRNA.

[0037] Fig.14 , also abbreviated as Fig.14 , shows the size determination of a composition comprising a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) with a DP of 50 / 200 and replicon RNA.

[0038] Fig.15 , also abbreviated as Fig.15 , showing the cell viability of a composition comprising a linear PPI with a DP of 250.

[0039] Fig.16 , also abbreviated as Fig.16 , showing the cell viability of a composition comprising a linear PEI with a DP of 250.

[0040] Fig.17 , also abbreviated as Fig.17 , showing the cell viability of a composition comprising a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) having a DP of 50 / 200 (ratio 1:4).

[0041] Fig.18 , also abbreviated as Fig.18 , shows the cell viability if the composition comprises a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) with a DP of 200 / 50 (ratio 4:1).

[0042] Fig.19 , also abbreviated as Fig.19 , showing the cell viability of a composition comprising a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) having a DP of 100 / 150 (ratio 2:3).

[0043] Fig. 20 , also abbreviated as Fig. 20 , showing the cell viability of a composition comprising a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) having a DP of 150 / 100 (ratio 3:2).

[0044] Fig.21 , also abbreviated as Fig.21 , shows the transfection results of an in vitro test of transfection efficiency using the prior art transfection reagent lipofectamine Messenger Max (MM).

[0045] Fig. 22 , also abbreviated as Fig. 22 , shows the cell viability of a composition comprising lipofectamine MessengerMax (MM) at a ratio of 2:1 (μl MM: μg mRNA). DETAILED DESCRIPTION OF THE INVENTION

[0047] The present invention will now be further described. In the following paragraphs, different aspects of the present invention are defined in more detail. Unless clearly indicated to the contrary, each aspect so defined can be combined with any other one or more aspects. In particular, any feature indicated as being preferred or advantageous can be combined with any other one or more features indicated as being preferred or advantageous.

[0048] When describing the compounds of the present invention, the terms used are to be interpreted in accordance with the following definitions, unless the context dictates otherwise.

[0049] The present invention describes a pharmaceutical composition comprising: (a) a polyethyleneimine polymer PPI; and (b) a nucleic acid, wherein the PPI has a degree of polymerization of about 20-1000, preferably about 100-500, and most preferably about 200-300. We have found that the composition according to the present invention has a higher transfection efficiency than other currently available non-viral vectors. In addition, the composition has a small particle size making it suitable for in vivo use. Therefore, one advantage of the present invention is that the composition has a high transfection efficiency, and another advantage is that its small size makes it conducive to in vivo use. In addition, the composition causes less cytotoxic effects than compositions based on non-viral vectors of the prior art.

[0050] The pharmaceutical composition according to the present invention may be or comprise a polymer blend, a copolymer, a homopolymer, a block copolymer, a gradient copolymer and a random copolymer. The pharmaceutical composition may further comprise an active pharmaceutical ingredient and other excipients.

[0051] The term "nucleic acid" refers to a biological molecule composed of a 5-carbon sugar, a phosphate group and a nitrogenous base. The term nucleic acid includes single-stranded and / or double-stranded DNA and RNA, and any combination or chemically modified form thereof.

[0052] Unless otherwise indicated, the term "degree of polymerization" or "DP" as used herein refers to the number-average degree of polymerization. It can be calculated using the following equation: 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, PPI consists of repeated propylamine units, so propylamine is the monomer unit of PPI. The molecular weight of the monomer unit of the PPI is about 57.1 g / mol. Based on the above formula, the number average molecular weight of the PPI polymer with a DP of 200 can be calculated to be equal to about 11.400 g / mol in its free base form. The polymer can also be composed of a protonated form, where the mass of the repeating unit increases with the mass of the salt, such as for the HCl salt, the mass of the PPI-HCl repeating unit is about 93.6 g / mol.

[0053] As used herein, the term "about" or "approximately" when referring to a measurable value such as a parameter, amount, duration, etc., is meant to encompass variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and still more preferably + / -0.1% or less of the specified value, provided that such variations are suitable for performing the disclosed invention. It should be understood that the value to which the modifier "about" or "approximately" refers itself is also specifically and preferably disclosed.

[0054] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. By way of example, "a compound" means one compound or more than one compound.

[0055] According to a specific embodiment of the present invention, the composition may further include polyethyleneimine polymer (PEI).

[0056] Polyethyleneimine (PEI) and polypropyleneimine (PPI) are organic macromolecules with a high cationic charge density. Polyethyleneimine, also known as PEI or poly(ethylene imine), is a polymer composed of repeated ethylamine units. Polypropyleneimine, also known as PPI or poly(propylene imine), is a polymer composed of repeated n-propylamine units. Due to the presence of charged amino groups, they have the potential to be protonated, especially in their linear form, so that polymeric compositions containing PEI and / or PPI can bind and compress nucleic acids. PEI and PPI can compress nucleic acids into positively charged particles, which can interact with anionic proteoglycans on the cell surface and promote the entry of particles.

[0057] According to a specific embodiment of the present invention, PPI or PEI or both are linear. In the present application, linear PPI is also referred to as L-PPI, and linear PEI is also referred to as L-PEI. The advantage of using linear PPI and / or linear PEI is that the resulting pharmaceutical composition can be positively charged, and the complex formed with nucleic acid has a small size. Another advantage of polymers is that they are short and therefore will be more easily cleared by the kidneys.

[0058] Furthermore, it was found that L-PPI monomers and L-PPI / L-PEI copolymers with high PPI content complexed RNA more efficiently than L-PEI monomers and L-PPI / L-PEI polymers with low PPI content.

[0059] According to a specific embodiment of the present invention, the degree of polymerization of the PEI is about 20-1000, preferably about 100-500, and most preferably about 200-300.

[0060] The term "mean diameter" refers to the average hydrodynamic diameter of the particles determined by dynamic light scattering, using the so-called cumulant algorithm for data analysis, which provides as a result the so-called "Z-average value" with a length dimension. Herein, the "mean diameter", "diameter" or "size" of the particles is used synonymously with the Z-average value.

[0061] According to the present invention, "N / P ratio" refers to the molar ratio of nitrogen atoms (N) in a polymer to phosphorus atoms (P) in a nucleic acid. The N / P ratio reflects the input molar ratio of nitrogen in a given amount of polymer to phosphate in a given amount of nucleic acid. In a specific embodiment, the N / P ratio is less than 40; preferably less than 20; more preferably less than 10, such as 5, 4, 3, 2, 1 or less, such as 0.5 or 0.2. In a specific embodiment, the N / P ratio can be, for example, approximately between 0.2 and 10; for example, between 1 and 10, or between 1 and 5. Alternatively, the ratio can also be between 1 and 20. Specifically, a lower N / P ratio can be beneficial in reducing the toxicity of the composition used. This can be the case, for example, in a composition with a relatively high PPI content.

[0062] According to another specific embodiment of the present invention, the PPI and the PEI or the L-PPI and L-PEI form a copolymer, preferably a random copolymer.

[0063] The copolymer of L-PPI and L-PEI can be represented, for example, as follows:

[0064]

[0065] The inventors surprisingly found that RNA was effectively complexed and transfected into cells when the copolymer preferably had a degree of polymerization of about 20-1000, more preferably about 100-500, and most preferably about 200-300. More specifically, higher RNA complexation can be achieved in copolymers with a relatively high ratio of L-PPI to L-PEI.

[0066] Particularly preferred compositions according to the invention are characterized in that they contain one or more of the following substances:

[0067] -PPE / PEI copolymers with high PPI content

[0068] -DP is 250 PPI

[0069] -PPI / PEI copolymer with DP of 250

[0070] - PPI / PEI copolymers having a PPI / PEI ratio of at least 1.5:1, preferably at least 4:1

[0071] - N / P ratio greater than 1; preferably greater than 5; more preferably between 5 and 20

[0072] Particularly preferred compositions of the invention comprise:

[0073] -DP of 250 L-PPI, and

[0074] -RNA, the N / P ratio is between about 0.2 and 10; preferably between 1 and 10; most preferably between 1 and 5.

[0075] These compositions are characterized in particular by high transfection efficiency, small particle size, good stability and low toxicity.

[0076] Another particularly preferred composition of the present invention comprises:

[0077] -Copolymer of L-PEI and L-PPI, with a DP of 250

[0078] -RNA, N / P ratio between about 1 and 20

[0079] -PPI / PEI ratio of at least 1.5:1; preferably at least 4:1

[0080] These compositions are characterized in particular by high transfection efficiency, small particle size, good stability and low toxicity.

[0081] The term "random copolymer" as used herein refers to a statistical copolymer in which the probability of finding a monomer residue of a given type at a particular point in the chain is similar to the mole fraction of that monomer residue in the chain. This is usually described by the reactivity ratio of the statistical copolymerization of the parent polymer used as the L-PEI / PPI precursor. Here, we define a random copolymer as a copolymer with a reactivity ratio (r1 = k p1,1 / k p1,2 ; r2=k p2,2 / k p2,1 , where monomer 1 is more reactive) r1<1.35 and r2>0.7. The copolymers of the present invention may contain other polymers besides PPI and / or PEI.

[0082] In the context of the present invention, copolymers of L-PEI and L-PPI with different molar ratios of L-PEI to L-PPI were synthesized and tested. According to an embodiment of the present invention, the degree of polymerization (DP) of the PEI and the degree of polymerization (DP) of the PPI are in the range of 1:1-1:500, preferably about 1:1-1:100, and most preferably about 1:2-1:10. According to a specific embodiment of the present invention, it has been found that a composition comprising PPI-enriched PEI and PPI shows a higher transfection efficiency than other nucleic acid vectors. A composition rich in PPI is a composition in which the amount of PPI exceeds the amount of any other polymer component (e.g., PEI) in the composition. According to the present invention, the nucleic acid is an RNA or DNA molecule; preferably selected from mRNA, self-replicating mRNA (replicon), circular mRNA, circular RNA, mRNA or replicon whose translation can be controlled by external or internal molecules, non-coding RNA, siRNA, sense RNA, antisense RNA, ribozymes, RNA aptamers, RNA aptazymes, saRNA, pDNA, minicircles, closed linear DNA, genomic DNA, cDNA, single-stranded and / or double-stranded DNA, and any combination or chemically modified form thereof.

[0083] The term "RNA" refers to a molecule comprising ribonucleotide residues and preferably consisting entirely or substantially of ribonucleotide residues and comprises all RNA types as described herein. The term "RNA" includes double-stranded RNA, single-stranded RNA, separated RNA such as partially or completely purified RNA, substantially pure RNA, synthetic RNA and recombinantly produced RNA such as RNA different from the modified RNA of naturally occurring RNA by the addition, deletion, substitution and / or change of one or more nucleotides. Such a change can include the addition of non-nucleotide materials, such as being added to the end or inside of RNA, such as at one or more nucleotides of RNA. The nucleotides in the RNA molecule can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. The RNA of these changes can be referred to as analogs, particularly analogs of naturally occurring RNA. The RNA used according to the present invention can have a known composition, or the composition of the RNA can be partially or completely unknown.

[0084] The term "DNA" refers to a molecule comprising deoxyribonucleotide residues and preferably consisting entirely or substantially 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 chemically modified form thereof.

[0085] In one embodiment, the pharmaceutical composition further comprises a lipid.In order to further improve the properties of the pharmaceutical composition according to the invention, co-formulation with a lipid and / or a negatively charged polymer coating may be achieved.

[0086] The term "lipid" refers to fatty substances that are insoluble in water, including fats, oils, waxes, and related compounds. Lipids can be produced in the blood (endogenous) or ingested in the diet (exogenous). Lipids are necessary for normal body function, and whether produced from exogenous or endogenous sources, they must be transported and then released for use by cells. The production, transportation, and release of lipids for use by cells is called lipid metabolism. Although there are several classes of lipids, the two main categories are cholesterol and triglycerides. Cholesterol can be ingested in the diet and is produced by cells in most organs and tissues in the body, primarily in the liver. Cholesterol can exist in its free form or, more commonly, in combination with fatty acids, so-called cholesterol esters.

[0087] The pharmaceutical composition according to the present invention can be used in humans or veterinary medicine. According to another embodiment, the pharmaceutical composition according to the present invention can be used in methods useful for nucleic acid delivery, such as 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. Example

[0088] Materials and methods

[0089] Synthetic mRNA production

[0090] use Kit (Thermo Fisher Scientific, Massachusetts, US) synthesizes self-amplification RNA or replicon of coding luciferase derived from Venezuelan equine encephalitis virus (VEEV) by in vitro transcription (IVT).I-SceI linearized plasmid is used as template.After purification using silica-based column (RNeasy Mini Kit, Qiagen, Hilden, Germany), RNA is capped using ScriptCapTM Cap 1 capping system kit (Cellscript, Wisconsin, US) according to the manufacturer's instructions.Finally, RNA is purified again using silica-based column and concentration is determined by spectrophotometry (NanoDrop, Thermo Fisher Scientific, Massachusetts, US).

[0091] N1-methylpseudouridine (1mΨ)-modified non-replicating mRNA (mod-mRNA) encoding luciferase was generated by IVT from an I-SceI linearized plasmid by replacing all uridine-5'-triphosphates in the IVT mixture with N1-methylpseudouridine-5'-triphosphate (Trilink Biotechnologies, San Diego, USA). Next, the mRNA was purified and capped using vaccinia virus capping enzyme and 2'-O-methyltransferase (Cellscript, Wisconsin, USA) to produce cap1, and then purified again using the RNeasy mini kit (Qiagen, Germany). The poly(A) tails of these mod-mRNAs (which were 40 adenosines in length) were extended to approximately 200 adenosines using the A-Plus Poly(A) polymerase tailing kit (Cellscript) and then purified. Finally, the mod-mRNA concentration was determined by spectrophotometry (NanoDrop, Thermo Fisher Scientific, Massachusetts, US).

[0092] Small interfering RNA

[0093] Small interfering RNA (siRNA) targeting firefly luciferase (pGL3) or control siRNA was purchased from Dharmacon (Lafayette, USA) and dissolved in RNase-free water at a concentration of 16.5 μM and stored in 20 μl aliquots at -20°C.

[0094] Polymer production

[0095] The polymer L-PEI with a DP of 250, the polymer L-PPI with a DP of 250 and its copolymer (L-PEI / L-PPI with a DP of 200 / 50-150 / 100-100 / 150-50 / 200) were synthesized as follows. First, methyl toluenesulfonate was used as an initiator, the ratio of total monomer to initiator was 250, and a copolymer of 2-ethyl-2-oxazoline (EtOx) and 2-isopropyl-2-oxazine (iPrOzi) was prepared at a total monomer concentration of 4M (different ratios of monomers (EtOx:iPrOzi=250:0, 0:250, 200:50, 150:100, 100:150 and 50:200) to prepare different copolymers) to obtain a polymer with a DP of 250. The polymerization was carried out in a Biotage microwave reactor at 140°C, and complete monomer conversion was confirmed by gas chromatography. Size exclusion chromatography confirmed the formation of fairly defined copolymers with a dispersity below 1.4, and 1H NMR spectrum reveals the target composition. Subsequently, by dissolving 1 gram of polymer in 7.5mL demineralized water (demi water) and 7.5mL hydrochloric acid, these copolymerization (2-oxazoline) are hydrolyzed to obtain L-PPI and L-PEI / PPI polymers. The closed vial is then heated to 140°C in a Biotage microwave for 9 hours to be hydrolyzed. Thereafter, the polymer is diluted with demineralized water, and HCl and demineralized water are evaporated under reduced pressure. The sample is neutralized with a 2M sodium hydroxide (NaOH) aqueous solution and freeze-dried. 1 H NMR analysis confirmed near quantitative hydrolysis.

[0096] Preparation and characterization of self-amplifying and modified mRNA nanocomplexes

[0097] To prepare nanocomplexes, an equal volume of RNA solution was 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). Different polymer to mRNA ratios were used to produce nanocomplexes. For self-amplifying mRNA nanocomplexes, the N / P ratios were N / P 40-20-10-5-1 and 0.2, and for mod-mRNA nanocomplexes, the N / P ratios were 30-15-8-4-0.8-0.2. Dynamic light scattering (Zetasizer Nano, Malvern Instruments, Malvern, UK) was then used to determine the size and zeta potential of the self-amplifying and modified mRNA nanocomplexes. The zeta potential is a typical measure of surface charge and therefore a typical measure of the stability of charged particles in suspension. Typically, a zeta potential of at least about 20 indicates that the particles have good stability.

[0098] Cell culture and transfection procedures

[0099] HeLa cells were cultured in culture medium and maintained in a humidified incubator at 37°C and 5% CO2. The culture medium consisted of Dulbecco's modified Eagle's medium (DMEM) (Gibco, Thermo Fisher Scientific, Massachusetts, US) supplemented with 10% fetal bovine serum (Biowest, California, US), 5.000 units / mL penicillin and 5.000 μg / mL streptomycin (Thermo Fisher Scientific, Massachusetts, US). One day before transfection with self-amplified or modified mRNA, HeLa cells were seeded in 24-well plates at a density of 50,000 cells / well. The next day (i.e., 24 hours later), the culture medium was replaced with Opti-MEM, and 20 μL of a polymer: RNA complex solution containing 500 ng RNA was added to each well. 24 hours after transfection, the expression of luciferase was analyzed by bioluminescence imaging. To this end, the cells were trypsinized and a portion of the neutralized cell suspension (60%) was transferred to a black 96-well plate. D-luciferin solution (50 mg / mL; 10% of the final pore volume) was added to each well and incubated for 10 minutes. Subsequently, the emitted bioluminescence was determined using IVIS Lumina II (Xenogen Corporation, Alameda, California, US). 500 ng RNA was used per 24 wells, and transfection with reference vector Lipofectamine Messenger Max (Thermo Fischer Scientific) at different ratios was performed in a similar manner.

[0100] Preparation of siRNA nanocomplexes and in vitro silencing efficiency

[0101] In order to evaluate the ability of polymer to deliver siRNA, siRNA-nano complexes were prepared by adding an equal volume of siRNA solution to the polymer solution and gently mixing and incubating at room temperature for 30 minutes. Both polymer and siRNA were dissolved in 20mM sodium acetate buffer (pH=5.2). The final concentration of siRNA was 10nM. Different polymers (DP is PEI / PPI of 50 / 200) and siRNA ratios were used to produce siRNA-nano complexes. Subsequently, these siRNA-nano complexes were tested in two schemes.

[0102] In the first set of experiments, we performed co-transfection of luciferase replicon with PEI / PPI siRNA nanocomplexes in HeLa cells. HeLa was cultured as described above. One day before co-transfection with PEI / PPI (50 / 200)-based replicon mRNA and siRNA nanocomplexes, HeLa cells were seeded in 24-well plates at a density of 50,000 cells / well. The next day (i.e., 24 hours later), the culture medium was replaced with Opti-MEM and cells were transfected with 500ng of the replicon encoding luciferase using PEI / PPI (DP50 / 200) at an N / P of 5. After 30 minutes, PEI / PPI siRNA-nanocomplexes containing 6pmol siRNA were added to the cells. After 24, we measured luciferase using the IVIS Lumina II imaging system for mRNA as described above. As controls, we used cells treated with luciferase replicon alone or with luciferase replicon plus PEI / PPI nanocomplexes containing control siRNA prepared at the highest N / P ratio studied (scrambled siRNA). The latter ratio is expected to have the highest cytotoxicity.

[0103] In the second set of experiments, SKOV3-luc cells stably expressing firefly luciferase were used. These cells were cultured in a humidified incubator at 37°C and 5% CO2 using McCoy's 5A (modified) medium (Gibco, Thermo Fisher Scientific, Massachusetts, US) supplemented with 10% fetal bovine serum (Biowest, California, US), 5.000 units / mL penicillin and 5.000 μg / mL streptomycin (Thermo Fisher Scientific, Massachusetts, US). SKOV-3-Luc cells were seeded at a density of 50,000 cells / well in 24-well plates one day before transfection with PEI / PPI (50 / 200)-based siRNA-nanocomplexes. The next day (i.e., 24 hours later), the medium was replaced with Opti-MEM, and 20 μL of PEI / PPI (50 / 200) polymer siRNA-nanocomplex solution containing 6 pmol siRNA was added to each well. 36 hours after transfection, luciferase expression was analyzed using the IVIS Lumina II imaging system for mRNA as described above.

[0104] Cell viability

[0105] Cell viability was determined 24 h after transfection using the cell proliferation reagent WST-1 (Roche). After trypsin digestion, a portion of the neutralization volume (6.66%) was transferred to a clear ELISA plate and the WST-1 solution was added according to the manufacturer's instructions. After 30 min of incubation, the plate was shaken on a plate shaker for 1 min and the absorbance at 450 nm (620 nm reference) was measured using an EZ Read 400 microplate reader (Biochrom).

[0106] In vivo transfection

[0107] After local injection in the neck or wings of chicken, the in vivo transfection efficiency of the nanocomplex comprising self-amplification mRNA and L-PEI / PPI polymer (DP is 50 / 200) was studied in chicken. For this reason, as described in the in vitro experiment, the self-amplification mRNA-PEI-PPI nanocomplex was prepared with an N / P ratio of 5. Subsequently, the nanocomplex containing 5 μg of self-amplification mRNA (encoding luciferase) was injected into the neck or wings. Two days later, the chicken was injected with D-luciferin, then euthanized and imaged with IVIS Lumina II.

[0108] result

[0109] Determination of transfection efficiency

[0110] Figures 1 to 7 Results of in vitro tests of transfection efficiency with self-amplifying mRNA nanocomplexes or modified mRNA nanocomplexes are shown. Compositions comprising polymers and nucleic acids with different N / P ratios were prepared. More specifically, 0.2, 1, 5, 10, 20 and 40. Control solutions (control, ctrl) with only buffer and thus no nanocomplexes were also prepared.

[0111] Figure 1 The transfection results of compositions comprising self-amplifying mRNA and a linear PPI (L-PPI) with a DP of 250 are shown, showing that the transfection efficiency is increased in each case compared to the control. For compositions with N / P ratios between 1 and 10; in particular between 5 and 10, a particularly increased transfection efficiency is achieved.

[0112] Figure 2 The transfection results of compositions comprising self-amplifying mRNA and linear PEI (L-PEI) with a DP of 250 are shown. It is important to note that the bioluminescence intensity and therefore the transfection efficiency of the first composition with an N / P of 10 and an N / P of 5 significantly exceeds the fluorescence intensity of the second composition. In contrast to the results obtained with PPI, for PEI, no increase in transfection efficiency was observed for the different compositions compared to the control, except for an N / P of 40.

[0113] Figure 3 The transfection results of the composition comprising self-amplified mRNA and a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) with a DP of 50 / 200 are shown. Thus, the ratio of the degree of polymerization of the PEI to the degree of polymerization of the PPI is 1:4. Again, compared to the control, an increase in the transfection efficiency of all compositions was observed, with a particular increase in the transfection efficiency of compositions with an N / P ratio higher than 5.

[0114] Figure 4 The transfection results of a composition comprising a self-amplifying mRNA and a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) with a DP of 200 / 50 (ratio 4:1) are shown. It is important to note that for each N / P ratio tested, the bioluminescence intensity of the first composition enriched with PPI significantly exceeded the bioluminescence intensity of the second composition. Thus, the first composition enriched with L-PPI showed a higher bioluminescence intensity than the composition comprising L-PPI but not L-PEI (the results of which are shown in FIG. Figure 1 Left side) higher transfection efficiency.

[0115] Figure 5 The transfection results of a composition comprising self-amplifying mRNA and a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) with a DP of 100 / 150 (ratio 2:3) are shown. The figure again shows that excess PPI has a beneficial effect on the transfection efficiency of the tested composition.

[0116] Figure 6 The transfection results of a composition comprising self-amplifying mRNA and a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) with a DP of 150 / 100 (ratio 3:2) are shown. The figure demonstrates that excess PEI in the composition does not appreciably affect the transfection efficiency of the tested composition.

[0117] Figure 7 The transfection results of a composition comprising a modified mRNA and a linear PPI (L-PPI) with a DP of 250 are shown, indicating that the transfection efficiency is increased when the N / P is between 0.8 and 30 compared to the control (Ctrl). For compositions with N / P ratios between 1 and 10, a particularly increased transfection efficiency is achieved. The figure also shows the results of in vitro transfections performed with the prior art transfection reagent lipofectamine Messenger Max (MM). Compositions comprising this lipid carrier and modified mRNA (at a ratio of 2 μl MM: 1 μg mod-mRNA) generally result in a transfection efficiency of 1x10 6 and 1x10 7When using modified non-replicating mRNA, we can conclude that the composition of the invention is at least as effective as MM.

[0118] Figure 8 The transfection results of the composition of the copolymer (L-PEI / L-PPI) of linear PEI and linear PPI comprising siRNA and DP of 50 / 200 in HeLa cells are shown, showing that when compared with control siRNA or the HeLa cells only receiving the replicon (neg.ctrl.) encoding luciferase, the silencing mediated by siRNA is most effective between N / P of 1 and 0.2. By adding siRNA-nano complexes into the HeLa cells cotransfected with the replicon encoding luciferase to obtain data. Lower expression (total flux) shows the good intracellular delivery of siRNA and the signal transduction of target luciferase mRNA subsequently.

[0119] Fig. 9 The results of transfection of a composition comprising siRNA and a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) with a DP of 50 / 200 in SKOV-3-Luc cells are shown, indicating that siRNA-mediated silencing is most effective at an N / P ratio of 5 or less. SKOV-3-Luc cells stably express luciferase. The lower expression (total flux) indicates good intracellular delivery of siRNA and subsequent signaling of the target luciferase mRNA.

[0120] In summary, the results showed that the L-PPI-enriched composition had a higher transfection efficiency than the L-PEI-enriched composition. In addition, the results showed that the composition was also effective in vivo and on modified mRNA and siRNA.

[0121] Physicochemical properties of compounds

[0122] Zeta potential

[0123] Fig.10 Shown is the determination of the zeta potential of compositions comprising self-amplifying mRNA and a linear PPI (L-PPI) having a DP of 250. As shown, compositions having an N / P ratio of at least 5 have excellent zeta potential and are considered to be stable formulations.

[0124] Fig.11 Determination of the zeta potential of a composition comprising self-amplifying mRNA and a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) having a DP of 50 / 200 (ratio 1:4) is shown. As shown, a composition having an N / P ratio of at least 5 has an excellent zeta potential and is considered to be a stable formulation.

[0125] Dimensional determination

[0126] Fig.12 Sizing of a composition comprising a linear PPI (L-PPI) with a DP of 250 and a replicon RNA (self-amplifying mRNA) is shown. Compositions with an N / P ratio of 1 or less are shown to have a higher Z-average value than compositions with higher N / P ratios. For some applications, a low average diameter of the particles may be beneficial.

[0127] Fig.13 Sizing of compositions comprising modified mRNA and a linear PPI (L-PPI) having a DP 250 is shown. Compositions having an N / P ratio of 0.2 or less are shown to have a higher Z-average value than compositions having a higher N / P ratio. For some applications, a low average diameter of the particles may be beneficial.

[0128] Fig.14 Sizing of compositions comprising self-amplifying mRNA and copolymers of linear PEI and linear PPI (L-PEI / L-PPI) with a DP of 50 / 200 (ratio 1:4) and replicon RNA is shown. For the PPI-rich copolymers, compositions with N / P of 5-20 yielded particles of low average diameter.

[0129] Cell viability

[0130] Fig.15 Shown is the cell viability 24 hours after transfection with a composition comprising self-amplifying mRNA and a linear PPI (L-PPI) with a DP of 250. As is apparent from the figure, the lower the N / P ratio, the less toxic the composition.

[0131] Fig.16 Shown are cell viability 24 hours after transfection with a composition comprising self-amplifying mRNA and linear PEI (L-PEI) with a DP of 250. In contrast to the results obtained with PPI, the N / P ratio does not significantly affect the toxicity of compositions comprising high amounts of PEI.

[0132] Fig.17 Cell viability after 24 h of transfection with a composition comprising self-amplifying mRNA and a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) with a DP of 50 / 200 (ratio 1:4) is shown. For the copolymer, the lower the N / P ratio, the less toxic the composition.

[0133] Fig.18 Cell viability after 24 h of transfection with a composition comprising self-amplifying mRNA and a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) with a DP of 200 / 50 (ratio 4:1) is shown. In contrast to the results obtained with PPI, the N / P ratio does not significantly affect the toxicity of compositions comprising a large amount of PEI.

[0134] Fig.19 Cell viability after 24 hours of transfection with a composition comprising self-amplifying mRNA and a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) with a DP of 100 / 150 (ratio 2:3) is shown. For the copolymers, also the lower the N / P ratio, the less toxic the composition.

[0135] Fig. 20 Cell viability after 24 h of transfection with a composition comprising self-amplifying mRNA and a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) with a DP of 150 / 100 (ratio 3:2) is shown. In contrast to the results obtained with PPI, the N / P ratio does not significantly affect the toxicity of compositions comprising a large amount of PEI.

[0136] Fig.21 Transfection results of an in vitro test of transfection efficiency with the prior art transfection reagent lipofectamine MessengerMax (MM) are shown. Compositions comprising the lipid carrier and nucleic acid (in different ratios) were prepared. The ratios indicated hereby are μl MM: μg mRNA. A control solution (control) having only buffer and thus no nanocomplexes was also prepared. The typical transfection efficiency observed with MM is in the range of 1×10 6 and 1×10 7 Between. From Figure 1 and Figure 3 It is evident from the results that the compositions of the present invention are at least as effective, or even better, reaching 1×10 7 and 1×10 8 The transfection efficiency between .

[0137] Fig. 22 Cell viability after 24 hours of transfection with a composition comprising lipofectamine MessengerMax (MM) at a ratio of 2:1 (μl MM: μg mRNA) is shown. MM showed only about 30% cell viability, in contrast, and as Figures 11 to 16 As shown, cell viability higher than 50%, even close to 100%, can be achieved by the composition of the present invention. It is important to note that cell viability was determined after a 24-hour transfection period.

[0138] In vivo experiments

[0139] In addition, we performed in vivo transfection experiments in chickens using a composition comprising a self-amplifying mRNA encoding luciferase and a copolymer of linear PEI and linear PPI (L-PEI / L-PPI) with a DP of 50 / 200. Bioluminescent images were taken shortly after euthanasia because visible bioluminescent signals are usually an underestimate of the true signal, as the luminescent enzymatic conversion of D-luciferin requires ATP, which is known to show a rapid decline after euthanasia. The results showed a clear bioluminescent signal in transfected chickens compared to uninjected control chickens (data not shown).

Claims

1. A pharmaceutical composition comprising: (a) polypropyleneimine polymer (PPI); and (b) nucleic acid, and Features The PPI has a degree of polymerization of about 20 to 1000, preferably about 100 to 500, and most preferably about 200 to 300.

2. The pharmaceutical composition according to claim 1, wherein the PPI is linear.

3. The pharmaceutical composition according to any one of claims 1 or 2, further comprising a polyethyleneimine polymer (PEI).

4. The pharmaceutical composition according to claim 3; wherein the degree of polymerization of the PEI is about 20 to 1000, preferably about 100 to 500, most preferably about 200 to 300.

5. The pharmaceutical composition according to any one of claims 3 or 4, wherein the PEI is linear.

6. The pharmaceutical composition according to any one of claims 1 to 2, wherein the PPI is in the form of a PPI / PEI copolymer.

7. The pharmaceutical composition according to claim 6, wherein the copolymer is a random copolymer.

8. The pharmaceutical composition according to any one of claims 6 or 7, wherein the degree of polymerization of the copolymer is about 20 to 1000, preferably about 100 to 500, most preferably about 200 to 300.

9. The pharmaceutical composition according to any one of claims 3 to 8, wherein the degree of polymerization of the PEI and the degree of polymerization of the PPI are in the range of about 1:1 to 1:500, preferably about 1:1 to 1:100, most preferably about 1:2 to 1:

10.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the pharmaceutical composition further comprises a lipid.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the nucleic acid is an RNA or DNA molecule; preferably selected from mRNA, self-replicating mRNA (replicon), circular mRNA, circular RNA, mRNA or replicon whose translation can be controlled by external or internal molecules, non-coding RNA, siRNA, sense RNA, antisense RNA, ribozymes, RNA aptamers, RNA aptazymes, saRNA, pDNA, minicircles, closed linear DNA, genomic DNA, cDNA, single-stranded and / or double-stranded DNA, and any combination or chemically modified form thereof.

12. A pharmaceutical composition according to any one of claims 1 to 11; wherein the N / P ratio is less than 40; preferably less than 20; more preferably less than 10.

13. A pharmaceutical composition according to any one of claims 1 to 12 for use in human or veterinary medicine.

14. A pharmaceutical composition according to any one of claims 1 to 13, for use 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.

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