RNA formulations suitable for therapy
The present invention relates to compositions comprising RNA, preferably messenger RNA (mRNA), more preferably self-amplifying RNA (saRNA), and polymers, in particular cationic polymers, such as polyethylenimine (PEI), poly-L-Lysin (PEL), polyvinylamine (PVA) or polyallylamine (PAA), where individual RNA molecules are present in solution. In the formulations, the RNA is preferentially present in the form of monomers, dimers, timers or oligomers, but not as aggregates comprising a large number of RNA molecules per aggregate, in particular large polyplex nanoparticles. The formulations display improved transfection efficacy and they can be used for delivery of RNA to a subject, where they have an improved dose response relationship in comparison to formulations where large aggregates in the form of polyplex nanoparticles are present.
1 . A composition comprising RNA and a cationic or polycationic polymer in an aqueous phase, wherein the ratio of the number of cationic or polycationic polymer nitrogen groups to phosphate groups of RNA (N/P) is at least 48 and wherein the cationic or polycationic polymer has an average molecular weight between 10000 Da and 50000 Da, wherein the predominant fraction of the RNA molecules comprises individual RNA molecules associated with the cationic or polycationic polymer in solution, wherein the predominant fraction of the RNA molecules is the fraction which contains the most RNA on a mass basis, wherein the RNA is mRNA or saRNA, and wherein the cationic or polycationic polymer is or comprises poly(ethyleneimine).
2 . The composition of claim 1 , wherein the majority of the RNA molecules associated with the polymer are present as RNA monomolecular species.
3 . The composition of 1 , wherein the poly(ethyleneimine) is a linear cationic or polycationic polymer, or wherein the poly(ethyleneimine) is a branched cationic or polycationic polymer.
4 . The composition of claim 1 , wherein the poly(ethyleneimine) has a mean molar mass between 15000 Da and 30000 Da, between 20000 Da and 25000 Da, or of about 22500 Da.
5 . The composition of claim 1 , which additionally comprises a buffering substance selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), 2-(N-morpholino) ethanesulfonic acid (MES), Bis-tris buffering systems, carboxylic acid buffering systems, phosphatic acid buffering systems, or citric acid buffering systems, where the pH is in the range from 4 to 8; or wherein the composition has an ionic strength of 50 mM or less; or wherein the concentration of free positively charged divalent cationic ions is 20 uM or less.
6 . The composition of claim 1 , wherein the RNA predominantly consists of individual cationic or polycationic polymer-associated RNA molecules.
7 . The composition of claim 1 , wherein the mass fraction of RNA present as RNA monomers or RNA oligomers having four or less RNA copies per oligomer unit is higher than 60%, higher than 70%, higher than 80%, higher than 90%, higher than 95%, higher than 96%, higher than 97%, higher than 98%, or higher than 99% of the total amount of RNA.
8 . The composition of claim 1 , wherein the composition does not comprise RNA aggregates.
9 . The composition of claim 1 , wherein the composition does not comprise viral RNA particles.
10 . The composition of claim 1 , formulated for use in non-viral gene delivery.
11 . The composition of claim 1 , wherein the ratio of the number of cationic or polycationic polymer nitrogen groups to phosphate groups of RNA (N/P) ranges from 48 to 300.
12 . The composition of claim 1 , wherein the ratio of the number of cationic or polycationic polymer nitrogen groups to phosphate groups of RNA (N/P) is at least 72.
13 . The composition of claim 11 , wherein the ratio of the number of cationic or polycationic polymer nitrogen groups to phosphate groups of RNA (N/P) ranges from 60 to 200, 80 to 150, or 70 to 120.
14 . A method for forming a composition comprising RNA and a cationic or polycationic polymer in an aqueous phase, wherein the predominant fraction of the RNA molecules comprises individual RNA molecules associated with the cationic or polycationic polymer in solution, wherein the predominant fraction of the RNA molecules is the fraction which contains the most RNA on a mass basis, the method comprising mixing RNA and an excess of cationic or polycationic polymer, or mixing a cationic or polycationic polymer containing solution and a RNA containing solution, and wherein the ratio of the number of the cationic or polycationic polymer nitrogen groups to phosphate groups of RNA (N/P) is at least 48 and wherein the cationic or polycationic polymer has an average molecular weight between 10000 Da and 50000 Da, wherein the RNA is mRNA or saRNA, and wherein the cationic or polycationic polymer is or comprises poly(ethyleneimine).
15 . The method of claim 14 , wherein the ratio of the number of cationic or polycationic polymer nitrogen groups to phosphate groups of RNA (N/P) ranges from 48 to 300.
16 . The method of claim 15 , wherein the ratio of the number of cationic or polycationic polymer nitrogen groups to phosphate groups of RNA (N/P) ranges from 60 to 200, 80 to 150, or 70 to 120.
17 . The method of claim 14 , wherein the mixing of RNA and cationic or polycationic polymer comprises mixing a solution of the RNA and a solution of the cationic or polycationic polymer, or mixing equal volumes of a solution of the RNA and a solution of the cationic or polycationic polymer, or dissolving dehydrated RNA in a solution of the cationic or polycationic polymer.