Complexation of nucleic acids with disulfide-crosslinked cationic components for transfection and immunostimulation

Abstract

The present invention is directed to a polymeric carrier cargo complex, comprising as a cargo at least one nucleic acid (molecule) and disulfide-crosslinked cationic components as a (preferably non-toxic and non-immunogenic) polymeric carrier. The inventive polymeric carrier cargo complex allows for both efficient transfection of nucleic acids into cells in vivo and in vitro and/or for induction of an (innate and/or adaptive) immune response, preferably dependent on the nucleic acid to be transported as a cargo. The present invention also provides, pharmaceutical compositions, particularly vaccines and adjuvants, comprising the inventive polymeric carrier cargo complex and optionally an antigen, as well as the use of such the inventive polymeric carrier cargo complex and optionally an antigen for transfecting a cell, a tissue or an organism, for (gene-)therapeutic purposes as disclosed herein, and/or as an immunostimulating agent or adjuvant, e.g. for eliciting an immune response for the treatment or prophylaxis of diseases as mentioned above. Finally, the invention relates to kits containing the inventive polymeric carrier cargo complex and/or the inventive pharmaceutical composition, adjuvant or vaccine in one or more parts of the kit.

1 . A polymeric carrier cargo complex, comprising:
 a) as a carrier a polymeric carrier formed by disulfide-crosslinked cationic polypeptide components, said carrier comprising at least a first and a second cationic polypeptide each comprising 3 to 100 amino acids and at least 50% cationic amino acids, wherein each of said first and second cationic polypeptides comprise at least one cysteine residue and wherein said first and said second cationic polypeptides are linked by a disulphide bond between said cysteine residue in the first and second cationic polypeptides, and   b) as a cargo at least one RNA molecule.   
     
     
         2 . (canceled) 
     
     
         3 . The polymeric carrier cargo complex of  claim 1 , wherein the at least one RNA is an immunostimulatory RNA. 
     
     
         4 . The polymeric carrier cargo complex of  claim 1 , wherein the at least one RNA is an mRNA. 
     
     
         5 . A polymeric carrier cargo complex of  claim 1 , wherein the nitrogen/phosphate (N/P) ratio of the cationic polypeptide components to the at least one nucleic acid molecule is in the range of 0.1-20, or in the range of 0.1-5, or in the range of 0.1-1. 
     
     
         6 . A polymeric carrier cargo complex of  claim 1 , wherein the polymeric carrier comprises a functional polypeptide additionally to the cationic polypeptide components. 
     
     
         7 . A polymeric carrier cargo complex of  claim 1 , wherein the functional polypeptide is an antigen or antigen epitope. 
     
     
         8 . A polymeric carrier cargo complex of  claim 1 , wherein the polymeric carrier additionally comprises a ligand. 
     
     
         9 . A polymeric carrier cargo complex of  claim 1 , wherein the ligand is mannose. 
     
     
         10 - 12 . (canceled) 
     
     
         13 . A polymeric carrier cargo complex of  claim 1 , wherein the at least one cysteine residue is located at the terminal end of the cationic polypeptide. 
     
     
         14 - 15 . (canceled) 
     
     
         16 . A polymeric carrier cargo complex of  claim 1 , wherein said first and second cationic polypeptides each comprise at least two cysteine residues. 
     
     
         17 . A polymeric carrier cargo complex of  claim 1 , wherein said first and second cationic polypeptides each comprise 3 to 25 amino acids and comprises at least 50% cationic amino acids. 
     
     
         18 . A polymeric carrier cargo complex of  claim 1 , wherein said first and second cationic polypeptides each comprise 5 to 20 amino acids and comprises at least 50% cationic amino acids. 
     
     
         19 . A polymeric carrier cargo complex of  claim 1 , wherein said first and second cationic polypeptides each comprise at least 5 cationic amino acid residues selected from the group consisting of Arginine, Lysine, Histidine and Ornithin. 
     
     
         20 . A polymeric carrier cargo complex of  claim 4 , wherein the mRNA encodes an antigen. 
     
     
         21 . A polymeric carrier cargo complex of  claim 4 , wherein the antigen is a tumor antigen. 
     
     
         22 . A polymeric carrier cargo complex of  claim 4 , wherein the mRNA encodes an antibody or a fragment thereof. 
     
     
         23 . A polymeric carrier cargo complex of  claim 1 , wherein the RNA comprises at least one guanosine, uridine, adenosine or cytidine nucleotide position that is substituted with an analogue of said nucleotide. 
     
     
         24 . A pharmaceutical formulation comprising a polymeric carrier cargo complex according to  claim 1 . 
     
     
         25 . A method treating a subject comprising administering an effective amount of a polymeric carrier cargo complex according to  claim 1  to the subject. 
     
     
         26 . The method of  claim 25 , further defined as a method expressing a polypeptide or stimulating an immune response in the subject.