Transfection method and compositions for intracellular uptake of nucleic acids into target cells

Abstract

The invention relates to transfection methods and agents to facilitate the uptake of nucleic acids in target cells with improved efficiency.

Description

[0001] Transfection method and compositions for intracellular uptake of nucleic acids into target cells

[0002] The disclosure relates to transfection methods, uses and transfecting compositions for the uptake of nucleic acids in target cells, such as eukaryotic target cells, with improved efficiency.

[0003] Prior Art

[0004] Several methods for transfection, the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells, have been developed. Known methods include physical, chemical and viral methods.

[0005] There are various types of transfection agents, each with its own set of advantages and disadvantages. Some examples include:

[0006] • Lipofection (or liposome transfection), which is a technique used to inject genetic material into a cell by means of liposomes, which are vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer. Lipofection generally uses a positively charged (cationic) lipid (cationic liposomes or mixtures) to form an aggregate with the negatively charged (anionic) genetic material. The efficiency of lipofection can be improved by treating transfected cells with a mild heat shock. However, liposomes can cause toxicity in the target cells and immune responses in the body.

[0007] • Cationic polymers, which are positively charged molecules that can interact with negatively charged nucleic acids. They are used as transfection agents because they can protect nucleic acids from degradation and facilitate their entry into cells. However, cationic polymers can also cause toxicity in the target cells and inflammation in the body.

[0008] • Viral vectors, which are viruses that have been modified to carry nucleic acids into cells. They are used as transfection agents because they can infect a wide range of cell types and deliver nucleic acids with high efficiency. However, viral vectors can cause immune responses and have the potential to integrate into the host genome, which can lead to mutagenesis and other problems. Further, experiments with viral vectors often need to be carried out in specific laboratories with at least biosafety level 2 and are often cumbersome to employ in every-day tasks.

[0009] • Electroporation, which involves the use of an electric field to create temporary pores in cell membranes, allowing nucleic acids to enter. It is a simple and efficient transfection method, but it can cause cell damage and is not suitable for all cell types. • Calcium phosphate, which is a commonly used transfection agent that works by forming a precipitate with nucleic acids. It is inexpensive and easy to use, but it can be toxic to cells and has low transfection efficiency.

[0010] However, most transfection methods, especially RNA transfection, work only under in-vitro conditions and render a systemic delivery of the nucleic acid difficult or even impossible under in- vivo-conditions.

[0011] Thus, overall, nucleic acid transfection agents have advantages and disadvantages that must be carefully considered when selecting a method for a particular application. Furthermore, not each cell, especially eukaryotic cell, can be transfected with same efficiency. Lymphocytes, which constitute an integral part of the immune system, are considered notoriously hard to transfect in vitro or / in vivo with non-viral methods. Overcoming the barriers of RNA delivery to leukocytes requires intense research and further development of novel delivery strategies.

[0012] Thus, despite a number of transfection methods, there is still a need for new methods, which allow a high transfection efficiency, even in “difficult” cells. Such methods are presented herein- under.

[0013] Description of the invention

[0014] In a first aspect a method for transfecting nucleic acid into a target cell comprising the steps of: a) Providing a transfecting composition of a nucleic acid and a transfection enhancing agent, the transfection enhancing agent comprising a conjugate of a cell penetrating peptide (CPP) and a lipid; b) Bringing the transfecting composition of step a) into contact with a target cell;

[0015] In one embodiment said method may comprise before step a) the steps of: aa) Providing a transfection enhancing agent comprising a conjugate of a cell penetrating peptide (CPP) and a lipid; ab) Bringing the transfection enhancing agent and a nucleic acid into contact; ac) Obtaining a transfecting composition comprising the nucleic acid and the transfection enhancing agent.

[0016] In one embodiment said methods may comprise additional step c), which comprises incubating said target cell with said transfecting composition. In one embodiment said method may comprise additional step c) or d), which comprises obtaining a target cell transfected with the nucleic acid.

[0017] The term “transfection” is defined herein as the process of introducing foreign nucleic acids, such as DNA or RNA, into cells, especially eukaryotic cells, in order to alter the genetic information or manipulate gene expression. The introduced nucleic acids can be from a variety of sources, including plasmids, viral vectors, or synthetic oligonucleotides.

[0018] In a second aspect a transfection enhancing agent for transfecting nucleic acids into a target cell, such as a prokaryotic and / or eukaryotic cell, is disclosed, comprising:

[0019] - conjugate of a cell penetrating peptide (CPP) and a lipid,

[0020] - an oily component,

[0021] - optionally, a surfactant, such as a cationic surfactant,

[0022] - optionally, at least one solubilizer.

[0023] In one embodiment, the transfection enhancing agent may be a micellar composition, in another embodiment a self-emulsifying composition (SEC).

[0024] In a third aspect the use of the method as disclosed hereinunder, or the transfection enhancing agent as disclosed hereinunder, or the CPP-lipid-conjugate as disclosed hereinunder, for the transfection of a nucleic acid into a target cell is disclosed, optionally for the transfection in a method according as disclosed hereinunder.

[0025] The present disclosure pertains to new methods and uses of transfecting compositions comprising the disclosed transfection enhancing agent and a nucleic acid for transfection of target cells, such as prokaryotic and / or eukaryotic cells, with improved efficiency. It shows an improved efficiency even in difficult to transfect cell-types, such as lymphoma cells, such as for example P388. P388 cells are mouse lymphoma cells which can be identified by RRID:CVCL 7222, and were first described in Dawe C.J., Potter M., “Morphologic and biologic progression of a lymphoid neoplasm of the mouse in vivo and in vitro.”, Am. J. Pathol. 33:603-603(1957).

[0026] The transfection methods and uses may be performed inside or outside an animal or human body. In one embodiment a method and a use are disclosed, where the cell is not brought back into the animal and / or the human body.

[0027] In one embodiment, the incubation time of the transfection enhancing agent with the nucleic acid in step ab) of the method is optionally at least 1 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, or at least 20 minutes. In another embodiment the incubation time in step ab) of the method is optionally up to 2 hours, up to 1 hour, up to 50 minutes, or up to 40 minutes. In another embodiment the incubation time in step b) of the method is optionally more than 5 minutes and less than 2 hours, or more than 10 minutes and less than 40 minutes.

[0028] In one embodiment, the target cell, such as a prokaryotic and / or eukaryotic cell, which is brought into contact with transfecting composition of a nucleic acid and a transfection enhancing agent, the transfection enhancing agent comprising a conjugate of a cell penetrating peptide (CPP) and a lipid, of present disclosure may be present in cell culture in a petri-dish or multiwell plate. Optionally, the cells are free floating, or, in another embodiment the cells are optionally adherent. In one embodiment the cells are optionally sub-confluent, i.e. cover the surface at 50-70%. In another embodiment the cells may be a tissue, such as a eukaryotic tissue.

[0029] “Confluent cells” refer to a layer of cells that have grown together to form a continuous, singlelayered sheet. This term is commonly used in cell culture to describe a monolayer of cells that have reached confluence, which means that the surface of the culture vessel is completely covered by a layer of cells and there is no space left for further growth. When cells become confluent, they typically stop dividing and become quiescent or enter into a state of cell cycle arrest, which can affect their behaviour and function. Confluent cells can also exhibit changes in cell shape, cell-cell contacts, and gene expression compared to cells in sub-confluent cultures.

[0030] Therefore, the term "sub confluent" refers to a state where the cells have not yet grown to cover the entire surface of the culture vessel, and there are still some spaces between the cells. This can occur when cells are first seeded onto a culture dish or flask, or if they have been passaged at a low density. The term “sub confluent” refers to the state before the cells reach the “conflu- ency”, given in percentage of the area which is already overgrown.

[0031] In one embodiment, the incubation time of the transfecting composition with the target cell in step c) of the method or use is optionally at least 2 hours, at least 3 hours, at least 4 hours. In another embodiment the incubation time in step d) of the method is optionally up to 12 hours, up to 10 hours, or up to 8 hours. In another embodiment the incubation time in step c) of the method or use is optionally more than 2 hours and less than 8 hours, more than 3 hours and less than 7 hours, or more than 3.5 hours and less than 5 hours. In one embodiment the incubation time may be over night.

[0032] The transfection method and use are surprisingly efficient as compared to other prior art methods. The transfection efficiency can be measured as fluorescence of a GFP -reporter assay. In such an assay the transfection efficiency is determined by using Green Fluorescent Protein (GFP) as a reporter. Commercially available GFP-mRNA with an appropriate promotor can be used in order to transfect the target cells and measure the expression of GFP of those cells. The transfection efficiency is then determined as the percentage of cells expressing GFP in the entire viable cell population. Overall number of cells can be determined for example by using Hoechst 33342 (blue), the number of viable cells may be determined by different means, for example by using an MTT-Test, wherein cells are incubated with yellow, water-soluble 3-(4,5-di- methylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) which is reduced by living cells into a blue-violet, water-insoluble formazan, therefore the percentage of living cells can be derived by counting blue-violet cells; the percentage of GFP expressing cells (green) can easily be determined by the fluorescent signal.

[0033] The disclosed methods and uses may achieve in one embodiment a transfection efficiency of at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of all viable cells. In another embodiment they may achieve a transfection efficiency of up to 100%, up to 98%, up to 94%, up to 86%, up to 84%, or up to 83% of all viable cells. This is significantly higher than prior art transfection methods and agents, for example such which rely on a modified peptide with a cellpenetrating activity whose amino acid composition enables it to interact with a protein cargo and transport this protein across the cell membrane barrier, such as X-fect® (Takara Bio) Thus, in one embodiment of the methods, uses and compositions of the present disclosure, the transfection efficiency of the transfecting composition comprising the enhancing agent is increased by at least 1.2-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 5-fold, or at least 6-fold. Optionally, the transfection efficiency is increased up to 10-fold, up to 15-fold, or even up to 20-fold, as compared to the transfection efficiency of X-fect® under the same conditions of incubation, temperature and pH. Optionally, the transfection efficiency of the transfecting composition comprising the enhancing agent is increased from 1.2-fold to 20-fold, from 1.5-fold to 15-fold, from 2-fold to 10-fold, or from 5-fold to 10-fold.

[0034] In one embodiment the disclosed methods and uses may achieve a transfection efficiency in the difficult to transfect P388 cells of at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% of all viable cells. Optionally, they may achieve a transfection efficiency of up to 40%, up to 45%, or up to 50% of all viable cells. Optionally, the transfection efficiency in the difficult to transfect P388 cells is from 10% to 50%, from 15% to 45%, or from 20% to 40%. Prior art transfection methods achieve not more than 5-6% efficiency under the same conditions.

[0035] In one embodiment results of the maxGFP fluorescence measurements can be blank-corrected with signal from non-transfected but Hoechst-stained cells and normalized to 10,000 cells with the corresponding cell density value from the same well. Efficiency-values may be presented as maxGFP relative fluorescence units (rfu) per 10 cells. The methods and agents of the present disclosure may show a maxGFP relative fluorescence ((rfu) * 10'1cells) of at least 2 rfu * 10'1cells, at least 3 rfu * 10'1cells, at least 4 rfu * 10'1cells, or at least 5 rfu * 10'1cells. In one embodiment the methods and agents of the present disclosure may show a maxGFP relative fluorescence ((rfu) * 10'1cells) of up to 10 rfu * 10'1cells, up to 15 rfu * 10'1cells, or up to 20 rfu * 10'1cells. In one aspect of the invention from 2 rfu * 10'1cells to 30 rfu * 10'1cells, from 3 rfu * 10-1cells to 15 rfu * 10'1cells, or from 4 rfu * 10'1cells to 10 rfu * 10'1cells show a maxGFP relative fluorescence ((rfu) * 10'1cells).

[0036] Thus, in another embodiment of the disclosed methods and uses the maxGFP relative fluorescence ((rfu) * 10-1cells) of the transfection efficiency of the transfecting composition comprising the enhancing agent is increased by at least 2-fold, at least 5-fold, at least 10-fold, at least 25- fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 50-fold, or at least 60-fold as compared to the transfection efficiency of the same transfecting composition but without the transfection enhancing agent under the same conditions of incubation, temperature and pH. Optionally, the transfection efficiency is increased up to 100-fold, up to 150-fold, or even up to 200- fold. Optionally, the transfection efficiency of the transfecting composition comprising the enhancing agent is increased from 2-fold to 200-fold, from 5-fold to 150-fold, from 10-fold to 100- fold, from 20-fold to 90-fold, from 20-fold to 80-fold, from 25-fold to 70-fold, from 30-fold to 60- fold, or from 35-fold to 50-fold.

[0037] In yet another embodiment the maxGFP relative fluorescence ((rfu) * 10'1cells) of the transfection efficiency of the transfecting composition comprising the enhancing agent is increased as compared to the transfection efficiency of the transfecting composition comprising calcium phosphate precipitates instead of the transfection enhancing agent under the same conditions of incubation, temperature and pH by at least 1.2-fold, at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 3.5-fold, at least 4.0-fold, at least 5.0-fold, or at least 6.0-fold. Optionally, the transfection efficiency of the transfecting composition comprising the enhancing agent is increased up to 10-fold, up to 15-fold, or even up to 20-fold. Optionally, the transfection efficiency of the transfecting composition comprising the enhancing agent is increased as compared to the transfection efficiency of the transfecting composition comprising calcium phosphate precipitates instead of the transfection enhancing agent under the same conditions of incubation, temperature and pH from 1.2-fold to 20-fold, from 1.5-fold to 18-fold, from 1.7-fold to 16-fold, from 2.0-fold to 15-fold, from 5.0-fold to 13-fold.

[0038] In one embodiment at least 0.25 pg, at least 0.5 pg, at least 1 pg, at least 2.5 pg, at least 5 pg, at least 10 pg, at least 12 pg, or at least 15 pg of the CPP-lipid-conjugate per 1 pg nucleic acid may be used for transfection. In another embodiment up to 100 pg, up to 75 pg, up to 60 pg, up to 30 pg, or up to 20 pg of the CPP-lipid-conjugate per 1 pg nucleic acid may be used for transfection. Optionally, the amount of CPP-lipid-conjugate per 1 pg nucleic acid is from 0.25 pg to 100 pg, from 1 pg to 30 pg, or from 5 pg to 20 pg.

[0039] In one embodiment, the disclosed methods, uses and / or transfection enhancing agents are surprisingly versatile with respect to the nucleic acid to be transfected, thus the nucleic acid may be selected from the group consisting of cyclic or linear oligonucleotides, such as ribonucleic acid (RNA) (such as small-interfering RNA (siRNA), messenger RNA (mRNA), micro RNA (miRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), long non-coding RNA (IncRNA), etc) desoxyribonucleic acid (DNA), xenonucleic acid (XNA), peptide-nucleic acids (PNA) and locked nucleic acid (LNA), as well as any combination thereof.

[0040] In one embodiment, the disclosed methods, uses and / or transfection enhancing agents are surprisingly versatile for transfecting eukaryotic cells. Thus, the eukaryotic cell can be selected from animal cells, i.e. , cells that make up the tissues and organs of animals, including humans, mice, dogs, cats, etc.; plant cells; fungal cells; protist cells, e.g., including unicellular organisms such as amoebae and paramecia, as well as multicellular organisms such as algae; archaea cells, such as those with eukaryotic- 1 ike features, including a membrane-bound nucleus.

[0041] In one embodiment, the target cell which is transfected, is a leukocyte cell. In some embodiments, the leukocyte cell is selected from neutrophils, lymphocytes, monocytes, eosinophils and basophils, as well as any combination thereof.

[0042] The uptake of nucleic acids into target cells is significantly improved as compared to prior art methods. Thus, in one embodiment only little amounts of conjugate with respect to 1 ug nucleic acid are needed for a successful transfection, such as 100 ug or more of conjugate with respect to 1 ug nucleic acid, 150 ug or more ug of conjugate, 200 ug or more of conjugate, 250 ug or more of conjugate, 300 ug or more of conjugate, 350 ug or more of conjugate, or 400 ug or more of conjugate. Optionally, the amount of conjugate with respect to 1 ug nucleic acid are from 100 ug to 400 ug, from 150 ug to 350 ug, or from 200 ug to 300 ug.

[0043] Thus, in one embodiment the agent can be used as a very effective transformation agent in vitro, although in vivo applications, such as a vaccine- or gene-vector, are also encompassed.

[0044] In one embodiment the agent of the present invention does not form a phospholipid bilayer and therefore differs from conventional transfection enhancing agents.

[0045] CPP-Conjugated lipids

[0046] The expressions “CPP-conjugated lipid” and “lipid-CPP conjugate” are used interchangeably in this description.

[0047] The lipid conjugates of this invention may have the following general structure:

[0048] L - [LINKER]n- CPP (Formula I)

[0049] In this formula, L represents the lipid, LINKER represents a linker of n-units, wherein n can be any integer from 1 to 5000, from 1 to 1000, from 1 to 100, from 1 to 40, or from 1 to 15, but also may be 0 in cases in which the CPP is directly covalently bound to the lipid, and CPP represents the cell penetrating peptide. The hyphens represent covalent bonds. In other words, the lipid conjugate may comprise the lipid covalently attached to the CPP directly and / or via a linker.

[0050] Preferred lipid conjugates have molecular weights in the range of 1.000 to 10.000 g / mol, from 1.200 to 5.000 g / mol, or from 1.500 to 3.500 g / mol.

[0051] Optionally, the lipid conjugate may carry a positive charge and / or the net charge of the lipid conjugate may be positive. A lipid conjugate has a positive net charge, if the number of positive charges is larger than the number of negative charges in the conjugate.

[0052] In some embodiments a part of the CPP-lipid conjugate within the transfection enhancing agent may be exchanged with any lipid-PEG, such as for example DSPE-PEG; e.g., - DSPE-PEG- 2000, or DSPE-PEG-5000, or DSPE-PEG-10000. Thus, in some embodiments part of the lipid conjugate is replaced by DSPE-PEG in order to achieve a ratio between the lipid conjugate and DSPE-PEG from 10:1 to 1 :10, from 5:1 to 1:5, or from 2:1 to 1:2. For example 1 :3, 1:4, 1:5, or 1 :10. Or on other words, compositions wherein up to 1%, up to 5%, up to 10%, up to 20%, up to 25%, up to 30%, up to 40%, up to 50%, up to 60%, up to 70%, up to 80%, up to 90%, or up to 99% of the lipid-conjugate in the agent are replaced by DSPE-PEG. However, it seems that although higher contents of DSPE-PEG- do increase the survivability of the cells, they do decrease the transformation efficiency of the nucleic acid. Without being bound to theory, this may be in association with the zeta potential, which decreases with elevated contents of DSPE-PEG. In one embodiment a zeta potential of more than 4 mV may be present, in such a case a content of more than 50% lipid conjugate, more than 75% lipid conjugate, more than 90% lipid conjugate, more than 95% lipid conjugate, or more than 98% lipid conjugate and up to 100 % may be present.

[0053] Cell penetrating peptides

[0054] The cell penetrating peptides (CPPs) in the conjugate may be selected from penetratin, TAT (transactivator of transcription), MAP (model amphiphatic peptide), polyarginines (including R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12), pVEC, transportan, MPG, and combinations thereof. The CPPs may be cyclized or linear, dimerized or un-dimerized. The CPPs may consist of the following sequences, or comprise the following sequences: Penetratin may include SEQ ID NO: 1; TAT may include SEQ ID NO: 2; MAP may include SEQ ID NO: 3; R9 may include SEQ ID NO: 4; pVEC may include SEQ ID NO: 5; transportan may include SEQ ID NO: 6; and / or MPG may include SEQ ID NO: 7. The listed CPPs include functional derivatives and peptide-mimetics of the mentioned sequences. Functional derivatives include CPPs that consist of or comprise the above-mentioned sequences, or sequences having at least 90%, or at least 95% sequence identity therewith. Optional functional derivatives have the sequences disclosed above with up to one, up to two or up to three amino acids replaced by other amino acids. Optionally, the functional derivative may include additional amino acids.

[0055] In an embodiment the CPPs used in this invention are positively charged and / or cyclized. Cyclized CPPs have the advantage of being less reactive and more stable than linear CPPs, which is advantageous within the concept of this invention. Cyclic peptides are more stable towards enzymatic cleavage than linear CPPs. As used herein, the term "cyclized" is not to be construed as relating to a peptide having one ring system only, i.e. , the present invention is not limited to monocyclic peptides. Accordingly, the present disclosure includes cyclopeptides wherein two or more ring systems are covalently linked to each other. Furthermore, the cyclopeptides may also comprise amino acids that are not part of the ring system, i.e., the invention includes branched cyclopeptides. Optionally, the cyclopeptides are monocyclic peptides, such as unbranched monocyclic peptides. Further, the CPPs can be composed of L-amino acids, D-amino acids, or mixtures thereof, wherein for linear CPPs, D-amino acids may be chosen.

[0056] In an embodiment, the CPPs comprise a majority of lysine and / or arginine moieties, which have isoelectric points of around 9.5 and 11 , respectively. Due to their additional amino or guanidine group, these two amino acids are positively charged under neutral and even under weakly basic conditions. Accordingly, a CPP mostly comprising moieties of said two specific amino acids is positively charged under neutral and weakly basic conditions as well. Herein, the term "majority" means that at least 30%, at least 50%, more at least 60%, and particularly at least 70% of the amino acids forming the CPP molecule are lysine and / or arginine moieties.

[0057] Thereby, it is ensured that the CPPs have a positive charge under neutral and weakly basic conditions, i.e., have an isoelectric point of more than 7.0. Therefore, in a specific embodiment of the present invention, the CPPs have an isoelectric point of more than 7.0, of more than 7.5, more of more than 8.0, or of more than 8.5. In this context, the isoelectric point of the CPP is the arithmetic mean of the isoelectric points of the amino acids forming the CPP. Optionally, the isoelectric point of the CPPs is from 7.0 to 8.5, or from 7.5 to 8.0.

[0058] In a specific embodiment of the present invention, the CPPs comprise between 2 to 19, between 3 to 16, between 4 to 14, or between 6 to 12 arginine moieties as well as one or more moieties selected from the group consisting of tyrosine, threonine, serine, lysine, aspartic acid, glutamic acid, glutamine, asparagine and cysteine.

[0059] For example, the CPP may comprise nine arginine moieties and one cysteine moiety in a ring system and are referred to as a cyclic cysteine R9C derivative (such as SEQ ID NO: 8; RRRRRRRRRC). Another example is a cyclopeptide comprising nine arginine moieties and one lysine moiety in the ring system, which is referred to as cyclic R9K derivative (SEQ ID NO: 9; RRRRRRRRRK). The CPPs of this invention include dimerized CPPs, wherein homo- and heterodimers are within the scope of this disclosure. Dimerization of CPPs can be affected by any means known in the art. In a particular embodiment, CPPs are dimerized via the tripeptide KAK.

[0060] The amino acids forming the cyclopeptides are not limited to proteinogenic amino acids. Herein, the amino acids may be selected from any amino acids known in the art, and may include the respective D-enantiomer, L-enantiomer, or any mixture thereof.

[0061] CPPs may include peptide-mimetics of the CPPs mentioned above. Peptido-mimetics include depsipeptides and peptoids of the CPPs disclosed herein. A depsipeptide CPP is a CPP wherein at least one peptide bond was replaced by an ester bond.

[0062] Lipids

[0063] The lipid may be selected from the group consisting of steroids, fatty acids, fatty alcohols, fatty amines, hydrocarbons with carbon chain lengths of at least eight carbon atoms (e.g. liquid paraffin), phospholipids, sphingolipids, ceramides, glycolipids, etherlipids, polyethers, carotenoids, and glycerides (mono-, di- and / or triglycerides) and combinations thereof. Steroids include compounds having a sterane structure. Steroids include cholesterol and its derivatives. Triglycerides include medium chain triglycerides (e.g. Ce to C12 fatty acids). Mono-, di- or triglycerides and / or fatty acids may be modified, such as PEGylated, ethoxylated, esterified (e.g. with propylene glycol, sorbitol or sorbitan) and / or in salt form. Mono-, di- or triglycerides include vegetable oils.

[0064] The lipids in the conjugated lipid may be selected from amphiphilic compounds / surfactants. Optional lipids with surfactant properties may be selected from the group consisting of mono and / or diglycerides, ethoxylated mono-, di- or triglycerides (e.g. Kolliphor® EL), medium chain mono- and / or diglycerides (e.g. Ce to C12 fatty acids), ethoxylated plant oil (e.g. ethoxylated castor oil), monoglycerides of C12 to C20 saturated or unsaturated fatty acids (e.g. C14 to Cis), esters of saturated or unsaturated fatty acids with diols (such as Cs to C20 fatty acids; e.g. 2-hydroxypropyl octanoate; propylene glycol monolaurate), mono- and di-esters esters of polyethylene glycol with medium chain fatty acids (e.g. Ce to C10 fatty acids), sorbitol or sorbitan esters, esters of sorbitol or sorbitan with polyethylene glycol and / or fatty acids (e.g. polysorbates), transesterified ethoxylated vegetable oils (e.g. Labrafil® M1944CS), polyethers (e.g. copolymers of polyalkylene glycols, such as Poloxamer®), salts of fatty acids, cetrimonium bromide, bis(2- ethylhexyl) sulfosuccinate and / or mixtures thereof.

[0065] The hydrocarbons comprise at least one activating group for chemical coupling, such as malei- mide active ester, amine alcohol, halogenide, thiol, ketone or aldehyde, triple and / or double carbon bonds.

[0066] In an embodiment, the fatty acids, fatty amines, fatty alcohols and / or hydrocarbons may have carbon chain lengths of from 8 to 24 carbon atoms, from 12 to 20 carbon atoms, or from 16 to 20 carbon atoms. The fatty acids, fatty amines, fatty alcohols and / or hydrocarbons may be saturated or unsaturated. Saturated compounds have the advantage of being chemically more stable than the unsaturated ones.

[0067] Preferred lipids include phospholipids and fatty acids.

[0068] In an embodiment, the phospholipids may be synthetic, semi-synthetic or natural phospholipids, or combinations thereof. Preferred phospholipids include phosphatidylcholines, phosphatidylethanolamines, phosphatidylinosites, phosphatidylserines, cephalines, phosphatidylglycerols, lyso- phospholipids, and combinations thereof. Preferred lipids may be selected from 1 ,2-dioleoyl- snglycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (sodium salt), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (Sodium Salt), 1,2-dioleoyl-sn-glyc- ero-3-phosphoethanolamine-N-(succinyl) (sodium salt), 1,2-distearoyl-sn-glycero-3-phosphoeth- anolamine-N-[maleimide(polyethylene glycol)-2000] (ammonium salt), and combinations thereof.

[0069] In one embodiment the lipid is selected from:

[0070] - the group consisting of a phospholipid, optionally selected from phosphatidylcholines, phosphatidylethanolamines, phosphatidylinosites, phosphatidylserines, cephalines, phosphatidylglycerols, lysophospholipids, and combinations thereof, and / or

[0071] - the group consisting of steroids (including cholesterol and its derivatives), fatty acids, fatty alcohols, fatty amines, hydrocarbons with carbon chain lengths of at least eight carbon atoms, sphingolipids, ceramides, glycolipids, etherlipids, carotenoids, glycerides and combinations thereof.

[0072] Linker

[0073] The cell penetrating peptide may be conjugated to the lipid via a linker of n-number of units length.

[0074] In one embodiment, the lipid is covalently bound to the CPP via a linker moiety, e.g., polyethylene glycol (PEG). In one embodiment the linker may comprise less than 15 units, less than 12 units, less than 10 units, but at least 5 units, at least 8 units. In one embodiment, the polyeth- ylene-glycol (PEG) linker comprises between 5 to 15 units, between 9 to 14 units. A short linker of less than 15 units (n= 15) does not trigger autoimmune responses in a subject. Accordingly, a PEG polymeric part of an activated polymeric group may in one embodiment have a length of 1 to 15 individual PEG units, of 5 to 13 individual PEG units. In one embodiment n may be zero, that is, the CPP is directly covalently linked to the lipid. Exemplary conjugates

[0075] Exemplary lipid conjugates are shown in figures 4 to 10.

[0076] An optional lipid conjugate is the following compound A (peptide part - SEQ ID NO: 1):

[0077] RQIKIWFQNRRMKWKK

[0078] An optional lipid conjugate is the following compound B (peptide part - SEQ ID NO: 10):

[0079] An optional lipid conjugate is the following compound C (peptide part - SEQ ID NO: 11):

[0080] An optional lipid conjugate is the following compound D (peptide part - SEQ ID NO: 12):

[0081] An optional lipid conjugate is the following compound E (peptide part - SEQ ID NO: 5):

[0082] An optional lipid conjugate is the following compound F (peptide part - SEQ ID NO: 4):

[0083] An optional lipid conjugate is the following compound G:

[0084] An optional lipid conjugate is the following compound H (peptide part - SEQ ID NO: 13):

[0085] An optional lipid conjugate is the following compound I, wherein R represents arginine and n may be an integer of > 3:

[0086] An optional lipid conjugate is the following compound J (peptide part - SEQ ID NO: 11):

[0087] In an embodiment, the transfection enhancing agent of this disclosure is comprised of particles. The particles may form spontaneously without any need for specialized equipment or expertise. The particles may be present in liquid form.

[0088] The term “particle” means a micelle, vesicle, droplet, self-emulsifying composition (SEC) or particle of colloidal dimensions that exists in equilibrium with the molecules or ions in solution from which it is formed. In the context of this description, “colloidal dimension” means a particle size of less than 450 nm, less than 400 nm, less than 350 nm, less than 300 nm, less than 250 nm, less than 200 nm less than 100 nm, less than 75 nm, or less than 50 nm. Optionally, the particle size may be from 50 nm to 450 nm, from 75 nm to 400 nm, or from 100 to 350. A particle may form spontaneously.

[0089] The term “particle” means in one embodiment a self-emulsifying composition (SEC), i.e. , the transfection enhancing agent may comprise SECs. SECs may enhance the uptake of the nucleic acid.

[0090] In one embodiment the disclosed SECs comprise the CPP-lipid-conjugate; an oily-component, such as for example corn oil; a surfactant such as hexa-decyltrimethylammoniumchloride (CTAC); and a solubilizer, such as propylene glycol.

[0091] Optional compositions of the SECs comprise at least 50 mol%, at least 75 mol%, at least 85 mol%, or at least 90 mol% of CPP-lipid conjugate relative to the total amount of oily components in the composition. In one embodiment the ratio of CPP-lipid conjugate to the oily component is between 10:1 and 1:10. In one embodiment the ratio of CPP-lipid conjugate to the surfactant is between 10:1 and 1:10. In one embodiment the amount of conjugated lipid is from 5.0 to 50 mol% relative to the total amount of oily components in the composition.

[0092] In another embodiment the term “particle” means a micelle. A micelle is a small, spherical structure composed of amphiphilic molecules, such as phospholipids or surfactants. These molecules have both a hydrophilic (water-friendly) and a hydrophobic (water-fearing) part. In an aqueous solution, the hydrophilic ends of the molecules face outward, while the hydrophobic ends face inward. When the concentration of amphiphilic molecules in a solution exceeds the critical micelle building concentration, the molecules can aggregate to form micelles. In a micelle, the hydrophobic ends of the molecules are shielded from the surrounding water molecules and form a core, while the hydrophilic ends face outward and interact with the water.

[0093] The particles may form spontaneously, i.e., simply by combining the components with water.

[0094] The particles may be very homogeneous. The “polydispersity index” (PDI) as used herein, is a measure of homogeneity and may be measured (as well as particle size and zeta potential) at room temperature using a Zetasizer Nano ZS from Malvern™ (Malvern Instruments Ltd., Worcestershire, United Kingdom).

[0095] The particles according to the invention have a polydispersity index (PDI) of less than 0.35, less than 0.25, or of less than 0.20. The term „particle“ does not include „liposome“. As opposed to liposomes, the agents of disclosed herein typically do not contain cholesterol in amounts of more than 1.0 mol% relative to the total amount of the oily component. The amount of cholesterol may even be limited to less than 0.5 mol%, or less than 0.1 mol% relative to the total amount of oily component.

[0096] The term "liposome" refers to artificially prepared vesicles composed of lipid bilayers. Liposomes can be used for delivery of APIs due to their unique property of encapsulating a portion of an aqueous solution inside a lipophilic bilayer membrane. Lipophilic compounds can be dissolved in the lipid bilayer, and in this way, liposomes can carry both lipophilic and hydrophilic compounds. To deliver the molecules to sites of action, the lipid bilayer can fuse with other bilayers such as cell membranes, thus delivering the liposome contents. In an embodiment, the agents of this disclosure are essentially free of liposomes, particularly of liposomes and other particles having an average particle size of at least 450 nm, or at least 250 nm. Optionally, from 100nm to 300 nm.

[0097] “Essentially free of liposomes” optionally means that the number of liposomes within the composition is less than 10% relative to the total number of particles in the composition. Preferably, the number of liposomes is less than 5%, less than 3%, or less than 1% of the total number of particles in the composition. In an embodiment, the composition contains less than 0.1% by weight of liposomes, or less than 0.05% by weight of liposomes relative to the total weight of the composition.

[0098] Transfection Enhancing Agent

[0099] In this disclosure, the term “composition” is used for any type of mixture of different components.

[0100] In this disclosure, the terms “transfection enhancing agent” and “agent” are used interchangeably. They relate to any composition comprising a conjugate of a cell penetrating peptide (CPP) and a lipid, but without any nucleic acid.

[0101] These terms are not to be confused with the “transfecting composition”, which comprises at least the transfection enhancing agent and a nucleic acid. The “transfecting composition” is brought into contact with the target cell(s).

[0102] The “transfection enhancing agent” or the “transfecting composition” may be in one embodiment a liquid.

[0103] In a further embodiment the “transfection enhancing agent” or the “transfecting composition” may be a freeze-dried lyophilizate. In such a case lyophilizing the method of making the transfection enhancing agent or the transfecting composition may include the step of: preparing a mixture of the transfection enhancing agent or the transfecting composition and at least one cryoprotectant.

[0104] It has been shown that there is an optimum amount range for cryoprotectant in relation to the amount of lipid in the mixture. Preferably, the amount of cryoprotectant ranges from 0.01 to 2 g cryoprotectant per gram of lipid, preferably from 0.02 to 1 g per gram lipid, or from 0.03 to 0.5 g per gram of lipid. Optionally, a minimum value is at least 0.03 g per 1 g of lipid. A maximum value may be 0.3, 0.2, or 0.1 g cryoprotectant per g of lipid. In this context, “lipid” refers to the total amount of conjugated, or un-conjugated first and second lipid in the composition. It was found that a lower amount of cryoprotectant will destabilize liposomes, reduce the Z-average value and thereby affect product uniformity. Higher amounts of the amount of cryoprotectant are not desirable because higher amounts would not further increase the positive effects, and many cryoprotectants are not suitable for certain applications so that the amount should be limited. Thus, the amount of cryoprotectant should preferably not exceed 0.5 g per 1 g of lipid.

[0105] The cryoprotectant may be selected from the group of saccharides, preferably monosaccharides or disaccharides, including sugars and sugar alcohols. The cryoprotectant may be selected from sucrose, mannitol, glucose, trehalose, lactose, palatinose and combinations thereof. In some embodiments a cryoprotectant may be selected from sucrose, trehalose, mannitol, glycerol and / or DMSO (Dimethyl sulfoxide).

[0106] The transfection enhancing agent may include solubilizers, such as water, in amounts of at least at least 25.0 wt.-%, at least 30.0 wt.-%, at least 40.0 wt.-%, at least 50.0 wt.-%, at least 75.0 wt.- %, or at least 90.0 wt.-%. Optionally, the enhancing agent may include solubilizers, such as water, in amounts from 25.0 wt.-% to 75.0 wt.-%, from 30.0 wt.-% to 50.0 wt.-%, or from 35.0 wt.-% to 40.0 wt.-%.

[0107] The relative weight amount of lipid conjugates in the transfection enhancing agent may be in the area of 1 to 25 wt.-% relative to the total weight of the composition. Preferred lower limits include 2 wt.-%, 3 wt.-%, or 5 wt.-%. Preferred upper limits include 20 wt.-%, 15 wt.-%, or 10 wt.- %. Optionally, the relative weight amount of lipid conjugates in the transfection enhancing agent may be from 2 wt.-% to 20 wt.-%, from 3 wt.-% to 15 wt.-%, or from 5 wt.-% to 10 wt.-%.

[0108] The weight amount of lipid conjugate in the transfection enhancing agent may be at least as high as the amount of nucleic acid. In an embodiment, the weight amount of lipid conjugate exceeds the amount of nucleic acid by a factor of at least 1.5, at least 2.0, at least 2.5, or at least 3.0. Optionally, the weight amount of CPP-lipid conjugate may be limited to about 100 times the amount of nucleic acid, up to about 50 times, up to about 25 times, or up to about 15 times. Optionally, the weight amount of lipid conjugate in the transfection enhancing agent exceeds the amount of nucleic acid from 1.5 to 100 times, from 2.0 to 50 times, from 2.5 to 25 times, or from 3.0 to 15 times.

[0109] In an embodiment, the amount of CPP-lipid conjugate in the composition is at least 0.1 mg / g relative to the agent. Optionally, the amount may be at least 0.3 mg / g, at least 0.5 mg / g, or at least 0.7 mg / g. The amount may be limited to up to 700 mg / g, up to 500 mg / g, up to 300 mg / g, up to 200 mg / g, or up to 100 mg / g. In embodiments, the amount of lipid conjugate is up to 70 mg / g, up to 50 mg / g, or up to 35 mg / g of the agent. In an exemplary embodiment, the amount is from 0.1 to 700 mg / g, from 0.3 to 300 mg / g, from 0.5 to 100 mg / g or from 0.7 to 35 mg / g. The amount may range up to 1000 mg / g. In an embodiment of a transfection enhancing agent, the amount of CPP-lipid-conjugate in the composition may be selected in a range of from 0.1 to 2.0 mg / ml, from 0.3 to 1.5 mg / ml, or from 0.5 to 1.0 mg / ml. Optionally, the amount is at least 0.7 mg / ml. It was found that these concentrations are best suited for particle formation.

[0110] Optionally, the transfection enhancing agent comprises an aqueous solution with particles therein, wherein the particles comprise the CPP-lipid conjugate. It was found that the CPP-lipid conjugates described herein have the capability of forming particles upon contact with aqueous media, such as buffers. The aqueous solution may be a buffer, such as a citrate buffer or a phosphate buffer or a mixture thereof. Because the particles form spontaneously upon mixing the CPP-lipid conjugates with aqueous media (self-emulsifying), the difficult preparation of liposomes is not necessary. Optionally, the particles have an average particle size of less than 450 nm, less than 350 nm less, less than 300 nm, than 250 nm, less than 200 nm, less than 150 nm, less than 100 nm, or less than 50 nm. The particles may be much smaller than liposomes.

[0111] Typically, liposomes of the CPP-lipid-conjugates used herein may be larger than 50 nm, larger than 75 nm, larger than 100 nm, larger than 125 nm, larger than 150 nm, larger than 175 nm, larger than 200 nm, larger than 225, or larger than 250 nm. The zeta potential of the particles may be positive, on one embodiment between 0.5 mV and 8.0 mV, such as at least 0.8 mV, at least 1.0 mV, at least 2.0 mV, at least 3.0 mV, at least 4.0 mV, or at least 5.0 mV. In another embodiment up to 6.0 mV, up to 7.0 mV, or up to 8.0 mV. Optionally, the zeta potential of the particles may be from 0.5 mV to 7.0 mV, from 0.8 mV to 6.0 mV, or from 1.0 to 5.0 mV. Liposomes typically have negative zeta potentials because of the negative charges of the lipids in their bilayers, however, the positive zeta potential masks the negative charge of nucleic acids and seems to facilitate the uptake of the nucleic acid into cells.

[0112] In an embodiment, the invention includes transfection enhancing agents comprising one or more oily components in an amount of not more than 10.0 wt.-% relative to the agent, one or more lipid conjugates in a total amount of at least 25.0 mol% relative to the total amount of oily components, and one or more nucleic acid(s).

[0113] In another embodiment, the invention includes transfection enhancing agents comprising one or more oily components in a total amount of at least 50.0 wt.-% relative to the agent, one or more lipid conjugates in a total amount of at least 1.0 mol% relative to the total amount of oily components, and one or more nucleic acids.

[0114] The amount of cholesterol in the transfection enhancing agent may be limited to not more than 1 .0 mol% relative to the total amount of oily component. The agent may be a liquid composition, such as an emulsion, a micelle and / or a SEC. Optionally, the agent may be essentially free of liposomes and / or nanoparticles and / or essentially free of any particles of more than 450 nm, more than 400 nm, more than 250 nm particle size, more than 200 nm particle size, more than 150 nm particle size, or more than 100 nm particle size. In this context, “essentially free” means that the content of the mentioned constituents is less than 0.5 wt.%, less than 0.1 wt.%, or even less than 0.01 wt.% of the composition.

[0115] Optionally, the lipid conjugate is not part of a liposome’s lipid double layer. Particularly, the cell penetrating peptide may be attached to a compound that is not part of a liposome’s lipid double layer. Optionally, the agents are free of tetraether lipids.

[0116] Nucleic acid

[0117] Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main classes of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). If the sugar is ribose, the polymer is RNA; if the sugar is the ribose derivative deoxyribose, the polymer is DNA.

[0118] However, also artificial nucleic acids may be encompassed.

[0119] In general, at least the following nucleic acids are encompassed: deoxyribonucleic acid (DNA), ribonucleic acid (RNA) (including messenger RNA (mRNA), small-interfering RNA (siRNA), mi- croRNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), antisense RNA (RNAi), etc.), xeno nucleic acid (XNA), peptide-nucleic acids (PNA) and locked nucleic acid (LNA).

[0120] The transfected nucleic acid may be between a few bases, such as 20, 50 or 100 bases and 2 Mb (megabases) large, in one embodiment at least 10 bases, at least 50 bases, at least 100 bases, at least 500 bases, at least 1 kb (kilobases), at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb. In another embodiment up to 100 kb, up to 200 kb, up to 300 kb, up to 500 kb, up to 1 Mb, up to 2 Mb. In one embodiment from 100 bases to 2 Mb, from 1 kb to 1 Mb, or from 5 kb to 500 kb.

[0121] In one embodiment are also encompassed for example RNA aptamers, ASO (antisense oligonucleotides), and / or RNAi (siRNA and miRNA). Both linear as well as circular forms of the nucleic acid are encompassed. In other embodiments nucleic acid stabilizing proteins or peptides, for example heat-shock proteins may be employed in order to enhance the stability of the nucleic acid.

[0122] Further excipients

[0123] The transfection enhancing agent may contain excipients, including emulsifier, surfactant, and solubilizers.

[0124] “Emulsifier” and “surfactant” (or “co- surfactant”) are used herein interchangeably. Surfactant is an amphiphilic compound useful for achieving the self-emulsifying property of the agent. Exemplary surfactants are selected from the group consisting of polyethylene glycol (PEG), p-tert-oc- tylphenole-derivative (with 7 - 10 ethylene-oxide-units), dioleoyl-3-trimethylammonium propane (DOTAP), PEG-lipids, and / or a cationic surfactant (Lipoplex-building agent), such as 10 mol-%, 20 mol-%, 30 mol-%, 40 mol-%, or 50 mol-% hexadecyltrimethylammoniumchloride.

[0125] In one embodiment the surfactants in the transfection enhancing agent do not exceed 100 pg / ml, do not exceed 75 pg / ml, do not exceed 50 pg / ml, or do not exceed 30 pg / ml. Optionally, the surfactants in the transfection enhancing agent are from 5 pg / ml to 100 pg / ml, from 10 pg / ml to 50 pg / ml, or from 15 pg / ml to 25 pg / ml. In one embodiment the transfection enhancing agent is free of surfactants.

[0126] A solubilizer is defined herein as a solvent other than water, used to increase capability of composition to dissolve lipophilic compounds. The molecular weight of the solubilizer will typically be less than 400 g / mol, less than 300 g / mol, or less than 200 g / mol. Optionally, the molecular weight of the solubilizer may be from 20 g / mol to 400 g / mol, from 30 g / mol to 300 g / mol, or from 50 g / mol to 200 g / mol. Solubilizers may include alcohols, such as polyalcohols. Exemplary solubilizers include 2-(2-ethoxyethoxy)ethanol, propylene glycol, glycerol, tetraglycol and combinations thereof. Other solubilizers besides water, may include dimethyl sulfoxide, ethanol, isopropanol and combinations thereof. Generally, solubilizers may be present in the agent in amounts of up to 60 wt.-%, up to 40 wt.-%, or up to 30 wt.-%. Optionally, the amount may be at least 15 wt.-%, or at least 20 wt.-%. Optionally, the amount may be from 10 wt.-% to 60 wt.-%, from 15 wt.-% to 40 wt.-%, or from 20 wt.-% to 30 wt.-%. In one embodiment the transfection enhancing agent is free of solubilizers.

[0127] Oily components are lipid and amphiphilic substances comprised in the composition. The oily components include only compounds that are not conjugated to a cell penetrating peptide, i.e. , the lipid conjugate does not form part of the oily components. Oily components may be used in pharmaceutical compositions, e.g., in emulsions, micelles, SECs or other compositions. In an embodiment, the oily components include all ingredients of the agent, except the conjugated lipids, having an n-octanol / water partition coefficient (Log P) of at least 1.0, at least 2.0, or at least 3.0 at 25°C. Oily components may be the components of the composition, except the conjugated lipids, that are immiscible with water at 25°C. Oily components may have saturated or unsaturated carbon chain lengths of more than 6, more than 8, or more than 10 carbon atoms. Optionally, the oily components may have saturated or unsaturated carbon chain lengths from 2 to 10 carbon atoms, from 4 to 8 carbon atoms, or from 5 to 7 carbon atoms. Optional oily components are modified or unmodified fatty acids.

[0128] Oily components may be selected from the group consisting of steroids, fatty acids, fatty alcohols, fatty amines, hydrocarbons with carbon chain lengths of at least eight carbon atoms (e.g. liquid paraffin), phospholipids, sphingolipids, ceramides, glycolipids, etherlipids, polyethers, carotenoids, and glycerides (mono-, di- and / or triglycerides) and combinations thereof. Steroids may include compounds having a sterane structure. Steroids may include cholesterol and its derivatives. Triglycerides include medium chain triglycerides (e.g., C6 to C12 fatty acids). Mono- , di- or triglycerides and / or fatty acids may be modified, such as PEGylated, ethoxylated, esteri- fied (e.g., with propylene glycol, sorbitol or sorbitan) and / or in salt form. Mono-, di- or triglycerides include vegetable oils.

[0129] Oily components may include amphiphilic compounds / surfactants. Optional oily components with surfactant properties may include mono- and / or diglycerides, ethoxylated mono-, di- or triglycerides (e.g. Kolliphor® EL), medium chain mono- and / or diglycerides (e.g. C6 to C12 fatty acids), ethoxylated plant oil (e.g. ethoxylated castor oil), monoglycerides of C12 to C20 saturated or unsaturated fatty acids (e.g. C14 to C18), esters of saturated or unsaturated fatty acids with diols (such as 08 to C20 fatty acids; e.g. 2-hydroxypropyl octanoate; propylene glycol monolaurate), mono- and di-esters esters of polyethylene glycol with medium chain fatty acids (e.g. 06 to C10 fatty acids), sorbitol or sorbitan esters, esters of sorbitol or sorbitan with polyethylene glycol and / or fatty acids (e.g. polysorbates), transesterified ethoxylated vegetable oils (e.g. Labrafil® M1944CS), polyethers (e.g. copolymers of polyalkylene glycols, such as Polox- amer®), salts of fatty acids, cetrimonium bromide, bis(2-ethylhexyl) sulfosuccinate and mixtures thereof.

[0130] The total amount of oily components in the composition comprises the cumulative amounts of the oily components listed above, in particular of steroids (including cholesterol and its derivatives), fatty acids, fatty alcohols, fatty amines, hydrocarbons with carbon chain lengths of at least eight carbon atoms, phospholipids, sphingolipids, ceramides, glycolipids, etherlipids, polyethers, carotenoids, and glycerides (mono-, di- and / or triglycerides) and combinations thereof, including modified mono-, di- or triglycerides and / or modified fatty acids, such as PEGylated, ethoxylated, esterified (e.g. with propylene glycol, sorbitol or sorbitan) and / or in salt form. Mono- , di- or triglycerides include vegetable oils.

[0131] In one embodiment the oily components in the agent may be up to 500 g / mol, may be up to 600 g / mol, be up to 700 g / mol, or up to 1000 g / mol. Optionally, the oily components in the agent may be from 10 g / mol to 1000 g / mol, from 50 g / mol to 700 g / mol, or from 100 g / mol to 500 g / mol.

[0132] The agent may include oily components in amounts of at least 1.0 wt.-%, at least 5.0 wt.-%, at least 10.0 wt.-%, at least 20.0 wt.-%, at least 30.0 wt.-%, at least 40.0 wt.-% or at least 50.0 wt.- %. Optionally, the amount of oily components in the composition may be limited to up to 99.0 wt.-%, up to 95.0 wt.-%, up to 90.0 wt.-%, or up to 85.0 wt.-%. Optionally, the agent may include oily components in amounts from 1.0 wt.-% to 98.0 wt.-%, from 2.0 wt.-% to 90.0 wt.-%, from 5.0 wt.-% to 80.0 wt.-%, from 10.0 wt.-% to 50.0 wt.-%, or from 20.0 wt.-% to 40.0 wt.-%.

[0133] In certain embodiments with higher amounts of oily components, such as SECs, the total amount of oily components in the agent may be at least 60.0 wt.-%, at least 70.0 wt.-%, or at least 80.0 wt.-%. Optionally, in particular in case of SECs, the total amount of oily components in the agent may be from 50 wt.-% to 99 wt.-%, from 60 wt.-% to 98 wt.-%, from 65 wt.-% to 95 wt.-%, from 70 wt.-% to 90 wt.-%, or from 75 wt.-% to 85 wt.-%.

[0134] Alternative embodiments of agents with lower amounts of oily components, such as optional types of solid dosage forms, include up to 10.0 wt.-%, up to 8.0 wt.-%, up to 6.0 wt.-%, or up to 4.0 wt.-% of oily components. Optionally, agents with lower amounts of oily components include oily components from 0.1 wt.-% to 10.0 wt.-%, from 0.2 wt.-% to 9.8 wt.-%, from 0.5 wt.-% to 9.5 wt.-%, from 1.0 wt.-% to 9.0 wt.-%, or from 2.0 wt.-% to 8.0 wt.-%.

[0135] Optional embodiments include oily components in amounts of from 1.0 wt.-% to 99.0 wt.-%, from 5.0 wt.-% to 95.0 wt.-%, or from 10.0 wt.-% to 90.0 wt.-%.

[0136] The methods and agents of the present disclosure can be used for any cell type, in one embodiment a eukaryotic cell. However, due to its improved efficiency they are specifically useful to transfect immune cells, such as lymphocytes (B-lymphocytes and T-lymphocytes). This has been shown by an excellent transfection of P388 cells, which may serve as a model-cell-line for transfection of lymphocytes. Thus, in one embodiment, the methods and agents of the present disclosure related to the transfection of immune cells, such as lymphocytes, optionally B-lym- phocytes and / or T-lymphocytes.

[0137] In a further aspect the invention includes the use of the agent in a method of treatment of a patient, comprising administering to said patient an effective amount of a nucleic acid in a composition according to this disclosure. Uses

[0138] The invention includes methods and agents as described herein for use in prevention, diagnosis and / or therapy.

[0139] In one further aspect the present disclosure relates to the use of the transfecting composition comprising the transfection enhancing agent and a nucleic acid of the present disclosure for transfecting cells. One further embodiment pertains to the use of the transfecting composition within the transfection method disclosed herein.

[0140] In yet another aspect the present disclosure relates to the transfecting composition comprising the transfection enhancing agent and a nucleic acid of the present disclosure for use in a method of treatment of a disease, wherein cells are transfected.

[0141] In one embodiment the disclosed methods and / or the transfecting composition comprising the transfection enhancing agent and nucleic acid are used to transfect a nucleic acid into a eukaryotic target cell in vivo or in vitro, for laboratory, diagnostic and / or therapeutic applications. In one embodiment the target cell which is transfected is a leukocyte cell. In some embodiments the leukocyte cell is selected from neutrophils, lymphocytes, monocytes, eosinophils and basophils, as well as any combination thereof. In certain embodiments the method and / or transfecting composition comprising the transfection enhancing agent and a nucleic acid may be used for topical delivery, parenteral delivery, inhalation delivery, intravenous delivery and / or intra-arterial delivery of a nucleic acid.

[0142] In one embodiment the methods and transfecting composition comprising the transfection enhancing agent and nucleic acid disclosed herein are suitable for in vitro and in vivo gene therapy, which is a technique used to treat or prevent diseases by modifying the genetic information of a person, such as Adenosine Deaminase Deficiency (ADA), Cystic Fibrosis (CF), Hemophilia, Severe Combined Immunodeficiency (SCID), Parkinson's disease, and / or Retinal Disorders, such as Leber's congenital amaurosis (LCA).

[0143] Since the methods and transfecting composition comprising the transfection enhancing agent and nucleic acid of the present invention are specifically efficient in transfecting lymphocytes, the methods and agents could be used in one embodiment for the treatment of lymphocyte-related diseases, such as lymphoma, a type of cancer that affects the lymphatic system, characterized by the abnormal growth of lymphocytes; leukaemia, a type of blood cancer where there is an overproduction of abnormal white blood cells, including lymphocytes; rheumatoid arthritis, an autoimmune disease in which the immune system attacks the body's own tissues, including those that contain lymphocytes; HIV / AIDS, a viral infection that primarily affects CD4+ T lymphocytes, leading to a weakened immune system; multiple sclerosis, an autoimmune disease that damages the myelin sheath around nerve fibres, which can be caused by an attack by lymphocytes; sarcoidosis, an inflammatory disease that can involve the lymphatic system, with accumulation of immune cells in various organs and tissues; lupus, another autoimmune disease in which the immune system attacks healthy tissues, including lymphocytes; mononucleosis, a viral infection caused by the Epstein-Barr virus that can affect lymphocytes and cause symptoms such as swollen lymph nodes, fatigue, and sore throat, graves' disease, an autoimmune disease that can affect the thyroid gland, and is associated with the presence of lymphocytes in the thyroid tissue and / or Hodgkin's disease, a type of lymphoma that specifically affects the lymphocytes in the lymph nodes.

[0144] In one further embodiment the transfection enhancing agent may be used to transfect CAR T- cells during the preparation of immunotherapy. “CAR T” stands for chimeric antigen receptor T- cell therapy, which is a type of immunotherapy that uses a patient's own T cells to fight cancer. CAR T therapy involves collecting a patient's T cells and modifying them in a laboratory so that they can better recognize and attack cancer cells. During the CAR T therapy process, T cells are collected from a patient's blood through a process and then genetically modified in a laboratory to produce chimeric antigen receptors (CARs) on their surface. These CARs are engineered to target specific proteins on the surface of cancer cells. This can be facilitated more efficiently by transfecting nucleic acids with the disclosed transfection enhancing agents.

[0145] In yet, another embodiment the disclosed transfection enhancing agents may be used for CRISPR / Cas9 experiments or other gene-editing experiments, i.e., in order to deliver the nucleic acid into the target cell.

[0146] In yet, another embodiment the disclosed transfection enhancing agents may be used in order to deliver a nucleic acid vaccination into a target cell.

[0147] In yet, another embodiment the disclosed composition comprises a transfection enhancing agent and a nucleic acid for transfecting the nucleic acids into a target cell, wherein the transfection enhancing agent comprises:

[0148] - a conjugate of a cell penetrating peptide (CPP) and a lipid,

[0149] - optionally, an oily component,

[0150] - optionally, a surfactant

[0151] - optionally, at least one solubilizer; wherein the CPP is selected from the group consisting of cyclic or linear penetratin, TAT (transactivator of transcription), MAP (model amphiphatic peptide), polyarginines, such as R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12, pVEC, transportan, and MPG.

[0152] In one embodiment the disclosure relates to a transfecting composition comprising a transfection enhancing agent and a nucleic acid for transfecting the nucleic acids into a target cell, wherein the transfection enhancing agent comprises:

[0153] - a conjugate of a cell penetrating peptide (CPP) and a lipid,

[0154] - optionally, an oily component,

[0155] - optionally, a surfactant

[0156] - optionally, at least one solubilizer; wherein the CPP is selected from the group consisting of cyclic or linear penetratin, TAT (transactivator of transcription), MAP (model amphiphatic peptide), polyarginines, such as R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12, pVEC, transportan, and MPG, for use in a method of treatment of a disease, wherein the cell is incubated with the transfecting composition, and the cell is transfected.

[0157] In one embodiment the disclosure relates to the method of treatment, wherein the composition is administered parenterally.

[0158] In one embodiment, the composition is administered parenterally, such as via intravenous, intramuscular, or subcutaneous injection, to ensure rapid bioavailability and precise dosing. In another embodiment, the parenteral administration is achieved through a controlled-release depot formulation to provide sustained therapeutic effects over an extended period.

[0159] In one embodiment the disclosure relates to the method of treatment, wherein the composition is administered orally. In one embodiment, the composition is formulated for oral administration in the form of tablets, capsules, or liquid suspensions, ensuring ease of use and patient compliance. In another embodiment, the oral formulation includes an enteric coating to protect the active ingredient from degradation in the acidic environment of the stomach and enhance bioavailability in the intestine.

[0160] In one embodiment the disclosure relates to the method of treatment of a disease, wherein the method comprises the transfection of a gut cell.

[0161] In one embodiment, the gut cell is transfected with mRNA or DNA encoding an antigenic protein, enabling in vivo production of a vaccine antigen to elicit a systemic and / or mucosal immune response. In another embodiment, the gut cell is transfected with nucleic acids encoding a therapeutic protein, which is expressed locally or systemically to treat diseases such as inflammatory bowel disease or enzyme deficiencies. In another embodiment, the vaccine antigen expressed by the gut cells interacts with immune tissues in the gastrointestinal tract, such as Peyer’s patches, to stimulate mucosal immunity against enteric pathogens. In one embodiment, gut cells transfected with nucleic acids produce therapeutic proteins that modulate the gut microbiota, enhance gut barrier integrity, or provide localized anti-inflammatory effects.

[0162] In one embodiment the disclosure relates to the method of treatment, wherein the method is a vaccination and / or a transient expression of a therapeutic protein.

[0163] In one embodiment, the vaccination involves the delivery of mRNA encoding an immunogenic peptide or protein into target cells, where the mRNA is translated, leading to in vivo production of the antigen and stimulation of an immune response. In another embodiment, the vaccine comprises plasmid DNA that is transfected into host cells, enabling the expression of the antigenic protein in vivo and subsequent activation of the adaptive immune system. In another embodiment, the vaccination induces both humoral and cellular immunity by generating the antigen in situ, ensuring robust and long-lasting protection against the targeted pathogen. In one embodiment, the antigen produced by the host cells is a protein or peptide that mimics a pathogenspecific structure, thereby training the immune system to recognize and neutralize the pathogen during future exposures.

[0164] In one embodiment, the therapeutic protein is expressed in vivo within a target cell following the transfection of the cell with DNA or RNA encoding the protein. In another embodiment, the therapeutic protein is produced in vivo after administration of a nucleic acid-based formulation, such as plasmid DNA or mRNA encapsulated in lipid nanoparticles, facilitating efficient delivery and cellular uptake. In one embodiment, the therapeutic protein is a monoclonal antibody engineered to specifically target and neutralize a disease-associated antigen. In one embodiment, the therapeutic protein serves as a vaccine antigen, where in vivo expression induces an immune response to protect against a specific pathogen. In another embodiment, the therapeutic protein acts as a therapeutic agent for treating non-infectious diseases, such as cancer or genetic disorders, by providing a functional protein directly within the patient’s cells.

[0165] Method of making

[0166] In an aspect, the invention includes a method of making a transfection enhancing agent, comprising the steps of a) reacting at least one CPP (cell penetrating peptide) with at least one lipid to obtain a lipid conjugate, b) optionally, using a linker structure to covalently bind the CPP to the lipid; c) optionally, purifying the lipid conjugate to obtain a purified lipid conjugate, d) optionally, reconstituting the transfection enhancing agent by incubation in water; wherein the wt / wt-ratio of the lipid conjugate to the oily component is from 0.1 to 10 and wherein the amount of lipid conjugate in the composition is from 1 to 20 mg / ml.

[0167] The method of this invention may include the step of lyophilizing the lipid conjugate to obtain a lyophilizate. Preferred lyophilizates have limited water content, of not more than 10 wt.-%, and / or less than 3 wt.-%. The water content can be determined by Karl-Fischer-titration or automated systems such as Water Content Analyzer. Compared to liquid formulations lyophilized lipid conjugates have better long-term stability.

[0168] In one embodiment the disclosure relates to a method for transfecting nucleic acid into a target cell comprising the steps of a) Providing a transfecting composition of a nucleic acid and a transfection enhancing agent, the transfection enhancing agent comprising a conjugate of a cell penetrating peptide (CPP) and a lipid; b) Bringing the transfecting composition of step a) into contact with a target cell;

[0169] In another embodiment the disclosure relates to above-mentioned method comprising before step a) the steps of: aa) Providing a transfection enhancing agent comprising a conjugate of a cell penetrating peptide (CPP) and a lipid; ab) Bringing the transfection enhancing agent and a nucleic acid into contact; ac) Obtaining a transfecting composition comprising the nucleic acid and the transfection enhancing agent.

[0170] In another embodiment the disclosure relates to above-mentioned method comprising an additional step c) comprises incubating said transfecting composition with said target cell.

[0171] In another embodiment the disclosure relates to above-mentioned method wherein the additional step(s) c) and / or d) comprise(s) obtaining a target cell transfected with the nucleic acid.

[0172] In another embodiment the disclosure relates to a method wherein the transfection efficiency of the method is at least 10% and / or wherein the transfection efficiency is increased at least 1.5- fold as compared to the transfection efficiency of the same transfecting composition without the transfection enhancing agent.

[0173] In another embodiment the disclosure relates to a method wherein a wt:wt-ratio between the conjugate and the nucleic acid is from 1 :10 to 10.000:1.

[0174] In another embodiment the disclosure relates to a method wherein the nucleic acid is selected from the group consisting of ribonucleic acid (RNA), desoxyribonucleic acid (DNA), xenonucleic acid (XNA), peptide-nucleic acids (PNA) and locked nucleic acid (LNA), and any combinations thereof.

[0175] In yet another embodiment the disclosure relates to a use of a transfecting composition comprising a transfection enhancing agent and a nucleic acid for transfecting the nucleic acids into a target cell, wherein the transfection enhancing agent comprises:

[0176] - a conjugate of a cell penetrating peptide (CPP) and a lipid,

[0177] - optionally, an oily component,

[0178] - optionally, a surfactant

[0179] - optionally, at least one solubilizer.

[0180] In another embodiment the disclosure relates to a use wherein the conjugate of a cell penetrating peptide (CPP) and a lipid has the general structure of formula I:

[0181] L - [LINKER]n- CPP (Formula I) wherein L is the lipid; wherein Linker is a linker of n repeating units; wherein n is 0 or any integer from 1 to 1000; wherein CPP is the cell-penetrating peptide.

[0182] In another embodiment the disclosure relates to a use wherein the amount of CPP-lipid conjugate in the transfection enhancing agent is from 0.05 to 100 wt.-%.

[0183] In another embodiment the disclosure relates to a use wherein the CPP comprises cationic amino acids.

[0184] In another embodiment the disclosure relates to a use wherein the CPP is selected from the group consisting of cyclic or linear penetratin, TAT (transactivator of transcription), MAP (model amphiphatic peptide), polyarginines, such as R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12, pVEC, transportan, MPG, and functional derivatives, peptide-mimetics, and any combinations thereof.

[0185] In another embodiment the disclosure relates to a use wherein the lipid is selected from the group consisting of a phospholipid, (optionally selected from phosphatidylcholines, phosphatidylethanolamines, phosphatidylinosites, phosphatidylserines, cephalines, phosphatidylglycerols, lysophospholipids), steroids (including cholesterol and its derivatives), fatty acids, fatty alcohols, fatty amines, hydrocarbons with carbon chain lengths of at least eight carbon atoms, sphingolipids, ceramides, glycolipids, etherlipids, carotenoids, and glycerides, as well as any combination thereof.

[0186] In another embodiment the disclosure relates to a use wherein the target cell is a leukocyte cell.

[0187] In one embodiment the disclosure relates to a use of the method as disclosed hereinunder for the transfection of a nucleic acid into a target cell, optionally for the transfection in a method according as disclosed hereinunder

[0188] Brief description of figures

[0189] Figure 1 - Characterization of the CPP-lipid-conjugate transfection enhancing agent (cf. examples 1-4). A. Depicted are size, PDI and zetapotential of CPP-lipid-conjugate (c = 10 mg / ml) in SEC (n = 3). The CPP-lipid-conjugate SECs showed a size of approx. 200 nm with a PDI of about 0.3. The positive zetapotential is related to the positively charged CPP-lipid-conjugate. B. Depicted is the zetapotential of CPP-lipid-conjugate SECs containing different amounts of the lipid-conjugate (n = 3). The lipid-conjugate was partly replaced by DSPE-PEG-2000. The zetapotential correlates with the amount of lipid-conjugate in the respective SEC formulation. C. Depicted is the zetapotential of CPP-lipid-conjugate SECs containing different lipoplex components with constant CPP-lipid-conjugate concentration (c = 10 mg / ml) (n = 3). The zetapotential is independent from the lipoplex component (concentration of each lipoplex was 50 pg / ml). These results demonstrate that the positive zetapotential of the SECs is caused by the lipid- conjugate. D. Depicted is the zetapotential of the lipid-conjugate SECs. It is independent from the amount of lipoplex in the formulation, demonstrating that the SECs are mainly composed of the lipid-conjugate (n = 3). Composition of SECs as described in the examples. E. Depicted is that the size and PDI of lipid-conjugate SECs depends on the lipid-conjugate concentration. Increasing amounts of lipid-conjugate lead to higher PDI (n = 3).

[0190] Figure 2 - Cytotoxicity studies of transfection enhancing agent (cf. example 5). P388 cells were exposed to different concentrations of transfection enhancing agent preparations. After for 4 h of compound exposure, 10 pl of alamarBlue reagent were added to each well for another 4 h. The abundance of the resulting resorufin was quantified by its fluorescence (excitation 530 nm; emission 590 nm). The data shows the mean + / - S.D. of four technical replicates normalized to cells treated with 0.1% of the respective compound. Composition of SECs as described in the examples.

[0191] Figure 3 - Transfection studies of different formulations of the transfection enhancing agent (cf. example 6). A. P388 cells were exposed for 4 h to green fluorescent protein (GFP)-encoding mRNA, as well as different transfection preparations of this GFP mRNA, including the disclosed transfection enhancing agents and Xfect®. As further control, cells were exposed to GFP mRNA-free SECs. 24 h after transfection, median GFP fluorescence of 30,000 cells was recorded using the MACSQuant Analyzer 10. Data shown is the mean + / - S.D. of two technical replicates. Composition of SECs and further details are described in the examples. B. LS180 cells were exposed for 4 h to green fluorescent protein-encoding (GFP) DNA vector (3.3 pg) prepared in either Fugene® or in various concentrations of the disclosed transfection enhancing agent. 24 h after transfection, median GFP fluorescence of 30,000 cells was recorded using the MACSQuant Analyzer 10. Data shown is the mean + / - S.D. of two technical replicates (arbitrary unit). Composition of micelles as described in the examples. C. Further results of the transfection efficacy of different SEC and micelle compositions and concentrations vs. xFect in P388 cells.

[0192] Figure 4 - Chemical structure of Tfp-PEG13-DSPE.

[0193] Figure 5 - Chemical structure of Mal-PEG12-DSPE.

[0194] Figure 6 - Synthesis of exemplary conjugates.

[0195] Figure 7 - Structure of a modified phospholipid used to make a conjugate.

[0196] Figure 8 - Example conjugate and modified phospholipid.

[0197] Figure 9 - Further example conjugate and modified phospholipid.

[0198] Figure 10 - Further example of a conjugated lipid (peptide part - SEQ ID NO: 11).

[0199] Figure 11 - Western blot results of eGFP-expression in P388-cells with the compositions of the present disclosure vs. standard transfection formulations .

[0200] Examples

[0201] Example 1 : Synthesis of CPP-lipid conjugates

[0202] Figures 6, 8 and 9 illustrate the synthesis of exemplary conjugates. a.) Synthesis of a first CPP-lipid conjugate

[0203] Cysteine-modified penetratin was coupled to the headgroup-modified phospholipid which is shown in Figure 7. Its chemical name is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide(polyethylene glycol)-2000], ammonium salt.

[0204] The resulting lipid conjugate is shown in Figure 6. b.) Synthesis of a second CPP-lipid conjugate

[0205] The synthesis of the CPP-lipid conjugate consists of the solid-phase peptide synthesis of lysinyl-nona-arginine utilizing the Fmoc / tBu strategy. Purity is controlled after loading of lysine on a solid phase resin, after coupling of five arginines, and after completion of the solid-phase synthesis. The peptide is cleaved from the resin with side-chain protection groups intact using HFIP / DCM and purified via HPLC. The head-to-tail cyclization of the side-chain protected peptide is per-formed in solution (ACN / DCM) using HATU / DIEA, which impedes racemization, followed by deprotection with TFA / water / anisole and precipitation with MTBE to obtain the cyclo- (Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Lys) as intermediate. Purification of the peptide intermediate is performed by HPLC on a Phenyl / Hexyl column.

[0206] The peptide intermediate is conjugated in solution (DMF / water) with 1.2 equivalents of the second intermediate (2R)-3-((((4,46-dioxo-46-(2,3,5,6-tetrafluorophenoxy)-

[0207] 7, 10, 13, 16, 19,22,25,28,31 ,34, 37,40, 43-tridecaoxa-3-azahexatetracontyl)oxy)(hydroxy)-phos- phoryl)oxy)propane-1,2-diyl distearate and purified via HPLC on a Phenyl / Hexyl column with concurrent ion exchange to acetic acid.

[0208] The resulting lipid conjugate is shown in Figure 8. c.) Synthesis of a third CPP-lipid conjugate

[0209] 3 equivalents of CPP (cyclic R9-peptides) and 10 equivalents of DI PEA were added to malei- mido-PEG(12)-distearoylphosphatidylethanolamine (Iris Biotech) or PEG(13)-dis- tearoylphosphatidylethanolamine-tetrafluorophenyl ester (Iris Biotech) dissolved in DMF in a concentration of 5 mg / ml. The reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with a 1:2 mixture of ACN / H2O and purification was performed via HPLC using a Chromolithe® Performance RP-C18e column (100 x 3 mm). Water and acetonitrile containing 0.05 % TFA were used as eluents with a flow rate of 2 ml / min.

[0210] The resulting lipid conjugate is shown in Figure 9. Example 2. Preparation of transfecting composition comprising SECs

[0211] SECs were prepared as follows:

[0212] The respective amount of RNA was dissolved in the respective amount of water. Corn oil and / or propylene glycol was added to the solution in the desired amounts. After short shaking, the CPP-Lipid-Conjugate dissolved in ethanol was added and the mixture was shaken again. Subsequently the required amount of water was added to reach the final concentration specified and the mixture shaken again. The SECs were subsequently analyzed with respect to size, size distribution and zetapotential. a.) Examples of transfecting enhancing agents and transfecting compositions comprising SECs

[0213] Basic transfection enhancing agent comprising SEC; “SEC No. 0”:

[0214] • CPP-Lipid-Conjugate = 10 mg / ml

[0215] • Cationic surfactant = Hexadecyltrimethylammoniumchloride: 50 pg / ml

[0216] • Corn Oil: 3 mg / ml

[0217] • Propylene glycol: 50 pl / 1 ml SEC

[0218] • RNA: 0.5-2.0 pg / pl or “quantum satis (q.s.)”

[0219] Transfecting composition SEC No. 1 :

[0220] CPP-Lipid-Conjugate = 3 mg / ml

[0221] Cationic surfactant = Hexadecyltrimethylammoniumchloride: 50 pg / ml

[0222] Corn Oil: 3 mg / ml

[0223] Propylene glycol: 50 pl / 1 ml SEC

[0224] RNA: 0.5 pg / pl or “quantum satis (q.s.)”

[0225] Transfecting composition SEC No. 2:

[0226] CPP-Lipid-Conjugate = 3 mg / ml

[0227] Cationic surfactant = none

[0228] Corn Oil: 3 mg / ml

[0229] Propylene glycol: 50 pl / 1 ml SEC

[0230] RNA: 0.5 pg / pl or “quantum satis (q.s.)”

[0231] Transfecting composition SEC No. 3:

[0232] CPP-Lipid-Conjugate = 0.3 mg / ml DSPE-PEG-2000 = 2.7 mg / ml Cationic surfactant = Hexadecyltrimethylammoniumchloride: 50 pg / ml Corn Oil: 3 mg / ml

[0233] Propylene glycol: 50 pl / 1 ml SEC RNA: 0.05 pg / p or “quantum satis (q.s.)

[0234] Example 3: Preparation of transfection compositions comprising micelles

[0235] The CPP-Lipid-Conjugate was dissolved in ethanol or methanol at a concentration of 100 mg / mL and added to the buffer or medium in the respective amount. Subsequently mRNA or DNA (solution in water) was added in the respective amount.

[0236] Example 4: Characterization of lipid-conjugate SECs

[0237] SECs the composition according to composition “SEC No. 0” were measured regarding size, PDI and zetapotential (n= 3). The lipid-conjugate SECs showed a size of approx. 200 nm with a PDI of about 0.3. The positive zetapotential is related to the positively charged lipid-conjugate (cf. Figure 1A).

[0238] Zetapotential of lipid-conjugate SECs according to composition “SEC No. 0” but containing different amounts of the lipid-conjugate were measured (n = 3). The lipid-conjugate was partly replaced by DSPE-PEG-2000. The zetapotential correlates with the amount of lipid-conjugate in the respective SEC formulation (cf. Figure 1B).

[0239] Zetapotential of lipid-conjugate SECs according to composition “SEC No. 0” but containing different lipoplex components with constant lipid-conjugate concentration (c = 10 mg / ml) (n = 3). The zetapotential is independent from the lipoplex component (concentration of each lipoplex was 50 pg / ml). These results demonstrate that the positive zetapotential of the SECs is caused by the lipid-conjugate (cf. Figure 1C). The zetapotential of the lipid-conjugate SECs is also independent from the amount of lipoplex in the formulation, demonstrating that the SECs are mainly composed of the lipid-conjugate (n = 3). Composition of SECs according to composition “SEC No. 0” but 1% lipoplex = 100 pg / ml, 0.5% lipoplex = 50 pg / ml, 0.25% lipoplex = 25 pg / ml and 0.1% lipoplex = 10 pg / ml (cf. Figure 1 D).

[0240] The size and PDI of lipid-conjugate SECs depend on the lipid-conjugate concentration. Increasing amounts of lipid-conjugate lead to higher PDI (n = 3) (cf. Figure 1E).

[0241] Example 5 Cytotoxicity studies of different transfecting compositions

[0242] 5 x 104cells / 100 pl per well were exposed to different concentrations of transfection compositions. After for 4 h of compound exposure, 10 pl of Alamar Blue reagent were added to each well for another 4 h. The abundance of the resulting resorufin was quantified by its fluorescence (excitation 530 nm; emission 590 nm). Data shown in Figure 2 is the mean + / - S.D. of four technical replicates normalized to cells treated with 0.1% of the respective compound. Composition of SECs according to composition “SEC No. 0” but CPP-lipid-conjugate was partly replaced by DSPE-PEG-2000 = PEG-lipid). SECs + 1 / 3 CPP-lipid-conjugate + 2 / 3 PEG-lipid means c(CPP- lipid-conjugate) = 3.33 mg / ml and c(DSPE-PEG-2000 = 6,66 mg / ml). SECs + 1 / 10 CPP-lipid- conjugate + 9 / 10 PEG-lipid means c(CPP-lipid-conjugate) = 1 mg / ml and c(DSPE-PEG-2000 = 9 mg / ml).

[0243] Example 6: Transfection studies of different transfecting compositions in different celltypes

[0244] 4 x 104P388 cells / 1 ml were exposed for 4 h to green fluorescent protein (GFP)-encoding mRNA (1 pg) or different transfection compositions of this GFP mRNA, including Xfect® (1 pg) or CPP-lipid-conjugate-SECs (0.375 pg, 0.1875 pg, 0.075 pg). As controls, cells were exposed to GFP mRNA-free CPP-lipid-conjugate-SECs.

[0245] 24 h after transfection, median GFP fluorescence of 30,000 cells was recorded using the MACSQuant Analyzer 10. Data shown is the mean + / - S.D. of two technical replicates (arbitrary unit).

[0246] In general, specified amounts of lipid-conjugate were added to medium and the mRNA was added. Finally, the mixture was added to the cells.

[0247] • 0.5% CPP-lipid-conjugate-SECs with mRNA -> 5.0 pl CPP-lipid-conjugate-SEC diluted in 1 ml suspension added to cells-> c(CPP-lipid-conjugate) = 50 pg / ml

[0248] • 0.25% CPP-lipid-conjugate-SECs with mRNA -> 2.5 pl CPP-lipid-conjugate-SEC diluted in 1 ml suspension added to cells -> c(CPP-lipid-conjugate) = 25 pg / ml

[0249] • 0.1% CPP-lipid-conjugate-SEC with mRNA -> 1.0 pl CPP-lipid-conjugate-SEC diluted in 1 ml suspension added to cells -> c(CPP-lipid-conjugate) = 10 pg / ml.

[0250] Results are shown in Figure 3A.

[0251] 4 x 104LS180 cells / 1 ml were exposed for 4 h to green fluorescent protein-encoding (GFP) DNA vector (3.3 pg) prepared in either Fugene® or in various concentrations of CPP-lipid-conju- gate-micelles. 24 h after transfection, median GFP fluorescence of 30,000 cells was recorded using the MACSQuant Analyzer 10. Data shown is the mean + / - S.D. of two technical replicates (arbitrary unit).

[0252] • 1% CPP-lipid-conjugate-micelles with mRNA -> 10 pl CPP-lipid-conjugate-Peptide (c = 100 mg / ml) -> c(CPP-lipid-conjugate) in 1 ml suspension given to cells = 1 mg / ml; other concentrations analogous.

[0253] Results are shown in Figure 3B.

[0254] 4 x 104P388 cells / 1 ml were exposed for 4 h to green fluorescent protein (GFP)-encoding mRNA (1 g) or different transfection compositions of this GFP mRNA, including Xfect® (1 pg) or CPP-lipid-conjugate-SEC (1.0 pg (HC), 0.5 pg (MC), 0.25 pg (LG)) or micelles formulations (all 1.0 pg).

[0255] • HC CPP-lipid-conjugate-SEC -> 20 pl CPP-lipid-conjugate-SEC (c = 3 mg / ml) diluted in 1 ml cell suspension -> c(CPP-lipid-conjugate) = 60 pg / ml

[0256] • MC CPP-lipid-conjugate-SEC -> 10 pl CPP-lipid-conjugate-SEC (c = 3 mg / ml) diluted in

[0257] 1 ml cell suspension -> c(CPP-lipid-conjugate) = 30 pg / ml

[0258] • LC CPP-lipid-conjugate-SEC -> 5 pl CPP-lipid-conjugate-SEC (c = 3 mg / ml) diluted in 1 ml cell suspension -> c(CPP-lipid-conjugate) = 15 pg / ml

[0259] • HC micelles -> 10 pl micelle-mix with mRNA (c = 100 mg / ml) diluted in 1 ml and given to cells -> c(CPP-lipid-conjugate) = 1 mg / ml MC micelles -> 1 pl micelles (c = 100 mg / ml) diluted in 1 ml -> c(CPP-lipid-conjugate) = 0.1 mg / ml

[0260] • LC micelles -> 0.1 pl micelle-mix with mRNA (c = 100 mg / ml) diluted in 1 ml and given to cells -> c(CPP-lipid-conjugate) = 0.01 mg / ml.

[0261] Results are shown in Figure 3C.

[0262] A usual transfection protocol utilizing the transfection enhancing agent of the disclosure, looks as follows: a). Storage & handling

[0263] • Thaw the transfection enhancing agent at room temperature just prior to use. Once thawed, store transfection enhancing agent at 4 °C for up to 12 months.

[0264] • Thaw transfection reaction buffer at room temperature (20 °C) just prior to use. Vortex after thawing. Once thawed, store transfection reaction buffer at 4 °C for up to 12 months.

[0265] • After each use make sure that the cap for the transfection enhancing agent is closed tightly and return to the supplied foil pouch containing desiccant. b). Mock transfections

[0266] Use a plasmid that does not contain your gene of interest. You should include a source of nucleic acids to assemble with the transfection enhancing agent. c.) Transfection Protocol

[0267] 1. Prepare cells for transfection

[0268] • Adherent cells: One day prior to the transfection, plate cells in 1 ml of complete growth medium so that the cells will be 50-70% sub confluent at the time of transfection.

[0269] • Suspension cells: Just prior to preparing the transfection enhancing agent (step 2), plate 5 x 105-1.25 x 106cells in 1 ml of growth medium.

[0270] 2. Thoroughly vortex transfection enhancing agent.

[0271] 3. In a microcentrifuge tube, dilute 5 pg of your nucleic acid, e.g., RNA or plasmid DNA, with transfection reaction buffer to a final volume of 100 pl. Mix well by vortexing for 5 s at high speed.

[0272] NOTES:

[0273] • Always add your plasmid to the buffer before adding transfection enhancing agent.

[0274] • At least 50 pl of the solution must be transfection reaction buffer.

[0275] • Do not use less than 2.5 pg of nucleic acid, e.g., RNA or plasmid DNA, per well of a 6- well plate. However, the first time you use transfection enhancing agent, it is recommend testing 2.5 pg, 5 pg, and 7.5 pg. Using less than 2.5 pg per well in a 6-well plate may result in a low transfection efficiency.

[0276] 4. Add 1.5 pl transfection enhancing agent to the diluted nucleic acid, e.g., RNA or plasmid DNA. Mix well by vortexing for 10 sec at high speed.

[0277] NOTE:

[0278] • Always keep the ratio of transfection enhancing agentmucleic acid the same. Use 0.3 pl of transfection enhancing agent per 1 pg of nucleic acid, e.g., RNA or plasmid DNA.

[0279] 5. Incubate for 10 min at room temperature to allow transfecting composition to form.

[0280] 6. Spin down for 1 sec to collect the contents at the bottom of the tube.

[0281] 7. Add the entire 100 pl of transfecting composition dropwise to the cell culture medium. Rock the plate gently back and forth to mix.

[0282] NOTE: It is not necessary to remove serum from your cell culture medium. It is normal for the medium to change color slightly upon addition of the transfecting composition.

[0283] 8. Incubate the plate at 37 °C for 4 h to overnight.

[0284] NOTE:

[0285] • 4 h incubation with transfecting composition of a nucleic acid and a transfection enhancing agent, the transfection enhancing agent comprising a conjugate of a cell penetrating peptide (CPP) and a lipid, is sufficient for optimal transfection.

[0286] • Incubation overnight is possible for convenience but does not generally increase transfection efficiency. If you have sensitive cells we recommend incubating for no more than 4 h.

[0287] 9. Remove nanoparticle complexes from cells by aspiration, replace with 2 ml fresh complete growth medium, and return the plate to the 37 °C incubator until time of analysis. Peak expression is typically reached 48 h post transfection.

[0288] Transfection efficiency is tested according to the protocol as published in “A method to evaluate the efficiency of transfection reagents in an adherent zebrafish cell line”, BioResearch open access, Vol. 2, No. 1, Feb. 2013 (DOI: 10.1089 / biores.2012.0287).

[0289] In short, 24 h after seeding and growth, P388 cells in 96-well plates are replenished with fresh medium and then transfected with 1 pg / pL of a GFP-containing plasmid (such as pmaxGFP vector). Two wells for each variation and transfection enhancing agent are treated identically to reduce errors. Transfection volumes are scaled down to fit 96-well plate dimensions. To measure maxGFP fluorescence in relation to cell density, transfected cells are stained with the fluorescent nuclear dye Hoechst 33342 (Invitrogen, Germany) according to manual. Fluorescence signals were measured after 4 h post transfection and after 80 min post Hoechst 3342-treat- ment from the bottom in a fluorescence plate reader (Victor X4, Perkin Elmer) using a 355-nm excitation filter and a 460-nm emission filter for measurement of the Hoechst 33342 signal and a 485-nm excitation filter and 535-nm mission filter for measurement of the maxGFP signal. Positive and negative controls are used as well. The measurement is repeated after 48 h post transfection.

[0290] Transfection efficiency is calculated as percentage of transfected cells from all viable cells by counting cells holding a maxGFP signal as well as a Hoechst-stained nuclei signal.

[0291] Results of the Hoechst 33342 fluorescence measurement are blank-corrected with background signal from cell-free wells holding only staining solution. The blank-corrected values are transformed into cells per square centimeter using a calibration curve that was established with Hoechst-stained cells seeded at various densities.

[0292] Results of the maxGFP fluorescence measurements are blank-corrected with signal from nontransfected but Hoechst-stained cells and normalized to 10,000 cells with the corresponding cell density value from the same well.

[0293] Cell viability at 2 and 4 h post transfection is calculated from cell density numbers from each well, normalized to the mean cell density calculated from three non-transfected control wells. Values of two corresponding wells with transfected cells are combined to a mean value in order to exclude pseudo-replicates and other errors.

[0294] Results are statistically analyzed to define the mean values and the standard deviation. Significant differences are accepted when p < 0.05.

[0295] Values are represented as percentage of transfected cells, maxGFP relative fluorescence (rfu) per 10 cells and / or relative transfection efficiency as compared to negative control, e.g., cells incubated with nucleic acid, but without transfection enhancing agent.

[0296] Example 7: P388-cell test a.) Cell culture

[0297] P388 cells are in culture under normal cell culture conditions. One day before, the cells were transferred to FCS-free medium. The P388 are transferred to 6-well plates (suspension cells).

[0298] P388 were diluted to 2*10x6 / ml with medium.

[0299] After the formulations were added, the samples were incubated for 6 h.

[0300] A cell pellet was made from the sample (30 sec, 10,000 G) and dissolved with RIPA buffer + protease inhibitors. Supernatant was frozen in -20°C until Western blotting. b.) Compositions

[0301] All samples are made into duplicates.

[0302] • eGFP mRNA is dissolved with 20 pL double-distilled water - cone. 1 pg / pL

[0303] • CPP-lipid conjugate are added directly to the medium in the well.

[0304] The medium is prepared in the reaction-container for the micelles. First RNA is added and then the micelles. This is mixed well and then poured onto the cells. c.) Volumes provided:

[0305] CPP-lipid conjugate #1: 200 l -> CPP-lipid conjugate (c = 5 mg / ml) with mRNA (c = 0.1 pg / pl); V=200 pl

[0306] Micelles #2: -> CPP-lipid conjugate (c = 100 mg / ml) -> 11.78 mg CPP-lipid conjugate in 117.8 pl MeOH d.) Western blot for eGFP

[0307] Western blot is performed according to usual protocols. 5% bovine serum albumin was used as blocking agent for unspecific interactions. eGFP was detected by commercially available anti- eGFP mouse-antibody, p-actin served as positive control and was detected by commercially available anti-p-actin-antibody, secondary antibody was a commercially available anti-mouse antibody. e.) eGFP detection after transfection in P388 cells:

[0308] Continuous 4%-15% precast gels from Bio-rad® were used to get a better differentiation of small proteins.

[0309] The expected band of (denaturated) eGFP was at 27 kDa.

[0310] Between 42 kDa and 26 kDa the membrane was cut to detect eGFP and p-actin on the same membrane with different methods.

[0311] - An eGFP antibody dilution of 1 : 100 was used.

[0312] - A p-actin antibody dilution of 1 :2000 was used.

[0313] Results of the western blot are depicted in figure 11. Western blot proves that eGFP-protein was formed in P388-cells, and the signals in FACS are reliable. Xfect®-control did not lead to successful transfection CPP-lipid conjugate and -micelles lead to successful transfection.

Claims

Claims1. A method for transfecting nucleic acid into a target cell comprising the steps of a) Providing a transfecting composition of a nucleic acid and a transfection enhancing agent, the transfection enhancing agent comprising a conjugate of a cell penetrating peptide (CPP) and a lipid; b) Bringing the transfecting composition of step a) into contact with a target cell. wherein the CPP is selected from the group consisting of cyclic or linear penetratin, TAT (transactivator of transcription), MAP (model amphiphatic peptide), polyarginines, such as R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12, pVEC, transportan, and MPG.

2. The method according to claim 1, comprising before step a) the steps of: aa) Providing a transfection enhancing agent comprising a conjugate of a cell penetrating peptide (CPP) and a lipid; ab) Bringing the transfection enhancing agent and a nucleic acid into contact; ac) Obtaining a transfecting composition comprising the nucleic acid and the transfection enhancing agent.

3. The method according to any one of claims 1 or 2, wherein an additional step c) comprises incubating said transfecting composition with said target cell.

4. The method according to any one of claims 1 to 3, wherein an additional step c) and / or d) comprises obtaining a target cell transfected with the nucleic acid.

5. The method according to any of the previous claims, wherein the transfection efficiency of the method is at least 10% and / or wherein the transfection efficiency is increased at least 1.5- fold as compared to the transfection efficiency of the same transfecting composition without the transfection enhancing agent.

6. The method according to any of the previous claims, wherein a wt:wt-ratio between the conjugate and the nucleic acid is from 1 :10 to 10.000:1.

7. The method according to any of the previous claims, wherein the nucleic acid is selected from the group consisting of ribonucleic acid (RNA), desoxyribonucleic acid (DNA), xenonucleic acid (XNA), peptide-nucleic acids (PNA) and locked nucleic acid (LNA), and any combinations thereof.

8. Use of a transfecting composition comprising a transfection enhancing agent and a nucleic acid for transfecting the nucleic acids into a target cell, wherein the transfection enhancing agent comprises:- a conjugate of a cell penetrating peptide (CPP) and a lipid,- optionally, an oily component,- optionally, a surfactant- optionally, at least one solubilizer; wherein the CPP is selected from the group consisting of cyclic or linear penetratin, TAT (transactivator of transcription), MAP (model amphiphatic peptide), polyarginines, such as R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12, pVEC, transportan, and MPG.

9. The use according to claim 8, wherein the conjugate of a cell penetrating peptide (CPP) and a lipid has the general structure of formula I:L - [LINKER]n- CPP (Formula I) wherein L is the lipid; wherein Linker is a linker of n repeating units; wherein n is 0 or any integer from 1 to 1000; wherein CPP is the cell-penetrating peptide.

10. The use according to any of claims 8 or 9, wherein the amount of CPP-lipid conjugate in the transfection enhancing agent is from 0.05 to 100 wt.-%.

11. The use according to any of claims 8 to 10, wherein the CPP comprises cationic amino acids.

12. The use according to any of claims 8 to 11, wherein the lipid is selected from the group consisting of a phospholipid, (optionally selected from phosphatidylcholines, phosphatidylethanolamines, phosphatidylinosites, phosphatidylserines, cephalines, phosphatidylglycerols, lysophos- pholipids), steroids (including cholesterol and its derivatives), fatty acids, fatty alcohols, fatty amines, hydrocarbons with carbon chain lengths of at least eight carbon atoms, sphingolipids, ceramides, glycolipids, etherlipids, carotenoids, and glycerides, as well as any combination thereof.

13. The use according to any of claims 8 to 12, wherein the target cell is a leukocyte cell.

14. Use of the method according to claims 1 to 7 for the transfection of a nucleic acid into a target cell, optionally for the transfection in a method according to one of claims 1 to 7.

15. A transfecting composition comprising a transfection enhancing agent and a nucleic acid for transfecting the nucleic acid into a target cell, wherein the transfection enhancing agent comprises:- a conjugate of a cell penetrating peptide (CPP) and a lipid,- optionally, an oily component,- optionally, a surfactant- optionally, at least one solubilizer; wherein the CPP is selected from the group consisting of cyclic or linear penetratin, TAT (transactivator of transcription), MAP (model amphiphatic peptide), polyarginines, such as R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12, pVEC, transportan, and MPG, for use in a method of treatment of a disease, wherein the cell is incubated with the transfecting composition, and the cell is transfected.

16. The transfecting composition according to claim 15, wherein the composition is administered parenterally.

17. The transfecting composition according to claim 15, wherein the composition is administered orally.

18. The transfecting composition according to any of claims 15 to 17, wherein the method comprises a transfection of a gut cell.

19. The transfecting composition according to any of claims 15 to 18, wherein the method of treatment comprises a vaccination and / or a transient expression of a therapeutic protein.

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