Formulation for administration of rna

Abstract

The present invention relates to compositions comprising polyplex formulations for delivery of RNA to a target organ or a target cell after parenteral administration, in particular after intramuscular administration. More precisely, the present invention relates to formulations for administration of RNA such as self-replicating RNA, in particular by intramuscular injection. In more detail, the formulations comprise polyplex particles from single stranded RNA and a polyalkyleneimine. The RNA may encode a protein of interest, such as a pharmaceutically active protein. Furthermore, the present invention relates to pharmaceutical products, comprising said RNA polyplex formulations for parenteral application to humans or to animals. The present invention relates as well to manufacturing of such pharmaceutical products, comprising, optionally, steps of sterile filtration, freezing and dehydration.

1 .- 51 . (canceled) 
     
     
         52 . A composition comprising:
 (a) single stranded RNA; and   (b) polyethyleneimine   
       wherein the single stranded RNA and the polyethyleneimine are present in polyplex particles and the molar ratio of the number of nitrogen atoms (N) in the polyethyleneimine to the number of phosphor atoms (P) in the single stranded RNA (N:P ratio) is 2.0 to 15.0, 
       wherein the ionic strength of the composition is 50 mM or less. 
     
     
         53 . The composition according to  claim 52 , wherein the N:P ratio is 6.0 to 12.0. 
     
     
         54 . The composition according to  claim 52 , wherein the concentration of monovalent cationic ions is 25 mM or less and the concentration of divalent cationic ions is 20 μM or less. 
     
     
         55 . The composition according to  claim 52 , wherein the pH of the composition is between 4 and 8; between 5.5 and 8, between 6 and 7.5, between 6.5 and 7.1, between 6.5 and 7, or between 6.5 and 6.9; or between 5 and 7.5. 
     
     
         56 . The composition according to  claim 52 , wherein the polyethyleneimine comprises the following general formula (I): 
       
         
           
           
               
               
           
         
         wherein
 R is H; 
 n is 2; and 
 p is an integer, wherein p is such that the average molecular weight of the polymer is 1.5·10 2  to 10 7  Da, 5000 to 105 Da, 10000 to 40000 Da, 15000 to 30000 Da, or 20000 to 25000 Da. 
 
       
     
     
         57 . The composition according to  claim 52 , wherein at least 92% of the N atoms in the polyethyleneimine are protonatable. 
     
     
         58 . The composition according to  claim 52 , further comprising one or more additives. 
     
     
         59 . The composition according to  claim 58 , wherein the one or more additives are selected from the group consisting of buffering substances, saccharides, stabilizers, cryoprotectants, lyoprotectants, and chelating agents. 
     
     
         60 . The composition according to  claim 59 , wherein
 (i) the buffering substances comprise at least one selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), acetic acid, acetate buffers and analogues, phosphoric acid and phosphate buffers, and citric acid and citrate buffers; and/or   (ii) the saccharides comprise at least one selected from the group consisting of monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, glucose, trehalose, and saccharose; and/or   (iii) the cryoprotectants comprise at least one selected from the group consisting of glycols, ethylene glycol, propylene glycol, and glycerol; and/or   (iv) the chelating agent comprises EDTA.   
     
     
         61 . The composition according to  claim 52 , wherein the composition comprises HEPES buffered glucose (HBG), HEPES buffered trehalose (HBT), or MES-buffered glucose (MBG). 
     
     
         62 . The composition according to  claim 61 , wherein the HBG comprises 5% glucose (w/v) and 10 mM HEPES, pH 7.1, the HBT comprises 10% trehalose (w/v) and 10 mM HEPES, pH 7.1, or the MBG comprises 5% glucose (w/v) and 10 mM MES, pH 6.1. 
     
     
         63 . The composition according to  claim 52 , wherein:
 (i) the z-average as derived from dynamic light scattering measurements of the particles is less than 200 nm, less than 150 nm, or less than 100 nm; or the z-average size of the particles is between 50 nm and 200 nm; and/or   (ii) the polydispersity index as derived from dynamic light scattering measurements of the particles is less than 0.5, less than 0.3, or less than 0.2; and/or   (iii) the Zeta-potential of the particles is 20 mV or more, 20 to 40 mV, 20 to 25 mV or 25 to 40 mV; and/or   (iv) the particles are neutral or positively charged at physiological pH or at a pH between 4.5 and 7.5.   
     
     
         64 . The composition according to  claim 52 , wherein the single stranded RNA is a molecule of 6000 to 15000 bases or of 9000 to 12000 bases, and/or wherein the single stranded RNA:
 (i) encodes at least one protein of interest; and/or   (ii) is a replicon, a self-replicating, or a self-amplifying RNA; and/or   (iii) comprises an open reading frame encoding at least one peptide or protein of interest.   
     
     
         65 . The composition according to  claim 64 , wherein the replicon, the self-replicating or the self-amplifying RNA is derived from or comprises elements derived from an alphavirus and/or wherein the replicon can be replicated by a replicase from an alphavirus and/or wherein the replicon comprises a 5′ replication recognition sequence from an alphavirus and a 3′ replication recognition sequence from an alphavirus. 
     
     
         66 . The composition according to  claim 65 , wherein the alphavirus is Venezuelan equine encephalitis virus (VEEV). 
     
     
         67 . The composition according to  claim 64 , wherein the peptide or protein of interest is a pharmaceutically active peptide or protein 
     
     
         68 . The composition according to  claim 52 , wherein the composition is an aqueous composition. 
     
     
         69 . The composition according to  claim 52 , wherein
 (i) the composition is a pharmaceutical composition; and/or   (ii) the composition is for use in therapy; and/or   (iii) the composition is a vaccine composition; and/or   (iv) the composition is for introducing RNA into a cell; and/or   (v) the composition is for expressing RNA in a cell; and/or   (vi) the composition is for intramuscular administration of the RNA.   
     
     
         70 . A method for introducing RNA into a cell of a patient in need thereof, said method comprising administering the composition according to  claim 52  to the patient. 
     
     
         71 . The method according to  claim 70 , wherein the cell is a muscle cell. 
     
     
         72 . A method of intramuscular administration of RNA comprising the step of intramuscularly administering the composition according to  claim 52 . 
     
     
         73 . A method for treating a cancer associated with a cancer-antigen in a subject, comprising administering the composition according to  claim 52 , wherein the single stranded RNA encodes the cancer-antigen. 
     
     
         74 . The composition according to  claim 52 , wherein the composition is a frozen, lyophilized or spray dried composition,
 wherein the composition comprises a cryoprotectant and/or lyoprotectant.   
     
     
         75 . The composition according to  claim 74 , wherein:
 (i) the lyoprotectant is a disaccharide or a polysaccharide; and/or   (ii) the composition further comprises a chelating agent; and/or   (iii) the composition comprises trehalose, saccharose, lactose, or maltose; and/or   (iv) the composition is prepared from an aqueous composition comprising a disaccharide at 5-20% (w/v).   
     
     
         76 . The composition according to  claim 75 , wherein:
 (i) the disaccharide is trehalose or the polysaccharide is dextran; and/or   (ii) the chelating agent is EDTA; and/or   (iii) the chelating agent is at 80 μM to 10 mM; and/or   (iv) the aqueous composition comprises trehalose, HEPES, and EDTA.   
     
     
         77 . The composition according to  claim 76 , wherein the aqueous composition comprises 10% trehalose (w/v), 2.8 mM HEPES, 80 μM EDTA, pH 7.1. 
     
     
         78 . An aqueous composition obtainable by thawing the frozen composition, or reconstituting the lyophilized or spray dried composition according to  claim 74 . 
     
     
         79 . A method of preparing a frozen, lyophilized or spray dried composition comprising:
 (i) preparing an aqueous composition according to  claim 68  comprising a cryoprotectant and/or lyoprotectant and   (ii) freezing, lyophilizing or spray drying the composition.   
     
     
         80 . The method according to  claim 79 , wherein:
 (i) the lyoprotectant is a disaccharide or a polysaccharide; and/or   (ii) the aqueous composition further comprises a chelating agent; and/or   (iii) the aqueous composition comprises a disaccharide at 5-20% (w/v).   
     
     
         81 . The method according to  claim 80 , wherein:
 (i) the disaccharide is trehalose or the polysaccharide is dextran, and/or   (ii) the chelating agent is EDTA; and/or   (iii) the chelating agent is at 80 μM to 10 mM; and/or   (iv) the aqueous composition comprises trehalose, HEPES, and EDTA.   
     
     
         82 . The method according to  claim 80 , wherein the aqueous composition comprises 10% trehalose (w/v), 2.8 mM HEPES, 80 μM EDTA, pH 7.1.