Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids

Abstract

The present invention is directed to an inventive polymeric carrier molecule according to generic formula (I) and variations thereof, which allows for efficient transfection of nucleic acids into cells in vivo and in vitro, a polymeric carrier cargo complex formed by a nucleic acid and the inventive polymeric carrier molecule, but also methods of preparation of this inventive polymeric carrier molecule and of the inventive polymeric carrier complex. The present invention also provides methods of application and use of this inventive polymeric carrier and the inventive polymeric carrier cargo complex as a medicament, for the treatment of various diseases, and in the preparation of a pharmaceutical composition for the treatment such diseases.

1 . Polymeric carrier molecule according to generic formula (I):
   L-P 1 —S—[S—P 2 —S] n —S—P 3 -L
   wherein,   P 1  and P 3  represent a linear or branched hydrophilic polymer chain, each P 1  and P 3  exhibiting at least one —SH-moiety, capable to form a disulfide linkage upon condensation with component P 2 , the linear or branched hydrophilic polymer chain selected independent from each other from polyethylene glycol (PEG), poly-N-(2-hydroxypropyl)methacrylamide, poly-2-(methacryloyloxy)ethyl phosphorylcholines, poly(hydroxyalkyl L-asparagine) or poly(hydroxyalkyl L-glutamine), wherein the hydrophilic polymer chain exhibits a molecular weight of about 1 kDa to about 100 kDa;   P 2  is a cationic or polycationic peptide or protein, having a length of about 3 to 100 amino acids, or
 is a cationic or polycationic polymer, having a molecular weight of about 0.5 kDa to about 100 kDa, 
 each P 2  exhibiting at least two —SH-moieties, capable to form a disulfide linkage upon condensation with component(s) P 1  and/or P 3 ; 
   —S—S— is a (reversible) disulfide bond;   L is an optional ligand, selected independent from the other from RGD, Transferrin, Folate, a signal peptide or signal sequence, a localization signal or sequence, a nuclear localization signal or sequence (NLS), an antibody, a cell penetrating peptide, or TAT;   n is an integer, selected from a range of about 1 to 50.   
     
     
         2 . The polymeric carrier molecule according to  claim 1 , wherein the polymeric carrier molecule additionally contains a repetitive amino acid component (AA) x , wherein x is an integer selected from a range of about 1 to 10, and wherein AA is an amino acid selected from aromatic amino acids Trp, Tyr, or Phe, or from hydrophilic, non charged polar amino acids Thr, Ser, Asn or Gln, or from lipophilic amino acids Leu, Val, Ile, Ala, or Met, or is selected from weak basic amino acids histidine or aspartate (aspartic acid). 
     
     
         3 . The polymeric carrier molecule according to  claim 1 , wherein repetitive amino acid component (AA) x  occurs as a mixed repetitive amino acid component [(AA) x ] z , wherein z is an integer selected from a range of about 1 to 30. 
     
     
         4 . The polymeric carrier molecule according to  claim 1 , wherein formula (I) is modified with mixed repetitive amino acid component [(AA) x ] z  according to following formula (Ia)
   L-P 1 —S-{[S—P 2 —S] a [S-(AA) x -S] b }—S—P 3 -L,
   wherein x, z, S, L, AA, P 1 , P 2  and P 3  are as defined herein and   a+b=n, wherein
 n is as defined herein 
 a is an integer, selected independent from integer b from a range of about 1 to 50, and 
 b is an integer, selected independent from integer a from a range of about 0 to 50, 
   and wherein the single components [S—P 2 —S] and [S-(AA) x -S] occur in any order in the subformula {[S—P 2 —S] a [S-(AA) x -S] b }.   
     
     
         5 . The polymeric carrier molecule according to  claim 1 , formula (I) or formula (Ia) are further modified with repetitive amino acid component (AA) x  or with mixed repetitive amino acid component [(AA) x ] z  according to any of following subformulae
   L-P 1 —S—S-(AA) x -S-{[S—P 2 —S] a [S-(AA) x -S] b }—S-(AA) x -S—S—P 3 -L, or
     L-P 1 —S—[S-(AA) x -S] z —{[S—P 2 —S] a [S-(AA) x -S] b }-[S-(AA) x -S] z —S—P 3 -L, or
     L-(AA) x -P 1 —S-{[S—P 2 —S] a [S-(AA) x -S] b }S—P 3 -(AA) x -L, or
     L-[(AA) x ] z P 1 —S-{[S—P 2 —S] a [S-(AA)) x —S] b }—S—P 3 -[(AA) x ] z -L, or
     L-(AA) x -S—S—P 1 —S-{[S—P 2 —S] a [S-(AA) x -S] b }—S—P 3 —S—S-(AA) x -S—S-L, or
     L-S—S-(AA) x -S—S—P 1 —S-{[S—P 2 —S] a [S-(AA) x -S] b }—S—P 3 —S—S-(AA) x -S—S-L, or
     L-S—[S-(AA) x -S] z -S—P 1 —S-{[S—P 2 —S] a [S-(AA)-S] b }—S—P 3 —S—[S-(AA) x -S] z -S-L, or
     (AA) x -P 1 —S-{[S—P 2 —S] a [S-(AA) x -S] b }—S—P 8 -(AA) x , or
     [(AA) x ] z -P 1 —S-{[S—P 2 —S] a [S-(AA) x -S] b }—S—P 3 —[(AA) x ] z , or
     (AA) x -S—S—P 1 —S-{[S—P 2 —S] a [S-(AA) x -S] b }—S—P 3 —S—S-(AA) x , or
     H—[S-(AA) x -S] z —S—P 1 —S-{[S—P 2 —S] a [S-(AA) x -S] b }—S—P 3 —S—[S-(AA) x -S] z —H,
   wherein a, b, n, x, z, S, L, AA, P 1 , P 2  and P 3  are as defined herein.   
     
     
         6 . The polymeric carrier molecule according to  claim 1 , wherein component P 2  is a cationic or polycationic peptide selected from protamine, nucleoline, spermine or spermidine, poly-L-lysine (PLL), basic polypeptides, poly-arginine, cell penetrating peptides (CPPs), chimeric CPPs, such as Transportan, or MPG peptides, HIV-binding peptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, oligoarginines, members of the penetratin family, e.g. Penetratin, Antennapedia-derived peptides (particularly from  Drosophila antennapedia ), pAntp, pIsl, etc., antimicrobial-derived CPPs e.g. Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, MAP, KALA, PpTG20, Proline-rich peptides, Loligomere, Arginine-rich peptides, Calcitonin-peptides, FGF, Lactoferrin, poly-L-Lysine, poly-Arginine, histones, VP22 derived or analog peptides, HSV, VP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs, PpT620, prolin-rich peptides, arginine-rich peptides, lysine-rich peptides, Pep-1, L-oligomers, or from Calcitonin peptide(s). 
     
     
         7 . The polymeric carrier molecule according to  claim 1 , wherein the —SH-moiety in component(s) in P 1 , P 2  and P 3  is provided by a cysteine. 
     
     
         8 . The polymeric carrier molecule according to  claim 1 , wherein component P 2  is selected from a peptide comprising a cationic peptide of formula (IIa):
   Cys((Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x )Cys,   wherein   l+m+n+o+x=8−15, and l, m, n or o independently of each other may be any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, provided that the overall content of Arg, Lys, His and Orn represents at least 10% of all amino acids of the oligopeptide; and Xaa may be any amino acid selected from native (=naturally occurring) or non-native amino acids except of Arg, Lys, His or Orn; and x may be any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, provided, that the overall content of Xaa does not exceed 90% of all amino acids of the oligopeptide.   
     
     
         9 . Polymeric carrier cargo complex formed of a polymeric carrier molecule according to  claim 1  and a nucleic acid. 
     
     
         10 . The polymeric carrier cargo complex of a polymeric carrier molecule according to  claim 9 , wherein the nucleic acid is provided in a molar ratio of about 10 to 10000 of inventive polymeric carrier molecule: nucleic acid. 
     
     
         11 . The polymeric carrier cargo complex of a polymeric carrier molecule according to  claim 9 , wherein the nucleic acid is a DNA or a RNA. 
     
     
         12 . The polymeric carrier cargo complex of a polymeric carrier molecule according to  claim 9 , wherein the nucleic acid is a coding RNA selected from an mRNA, or an siRNA. 
     
     
         13 . The polymeric carrier cargo complex of a polymeric carrier molecule according to  claim 9 , wherein the nucleic acid encodes a therapeutically active protein or peptide, an antigen, including tumor antigens, pathogenic antigens, animal antigens, viral antigens, protozoal antigens, bacterial antigens, allergic antigens, autoimmune antigens, allergens, antibodies, immunostimulatory proteins or peptides, or antigen-specific T-cell receptors. 
     
     
         14 . Method of preparing the polymeric carrier according to  claim 1  comprising following steps:
 a. providing at least one cationic or polycationic protein or peptide and/or at least one cationic or polycationic polymer as component P 2  as defined according to  claim 1  and optionally a repetitive amino acid component (AA) x  or a mixed repetitive amino acid component [(AA) x ] z  as defined according to  claim 1 , in the ratios indicated by formulae (I) or (Ia), mixing these components for a time of at least about 5 hours and thereby condensing and thus polymerizing these components with each other via disulfide bonds in a polymerization condensation or polycondensation to obtain a repetitive component H—[S—P 2 —S] n —H or H{[S—P 2 —S] a [S-(AA) x -S] b }H, as defined according to  claim 1 ; 
 b. providing a hydrophilic polymer P 1  and/or P 3  as defined according to  claim 1 , optionally modified with a ligand L and/or a repetitive amino acid component (AA) x  as defined according to  claim 1 ; 
 c. mixing the hydrophilic polymer P 1  and/or P 3  according to step b) with the repetitive component H—[S—P 2 —S] n —H or H{[S—P 2 —S] a [S-(AA) x -S] b }H obtained according to step a) in a ratio of about 2:1, and thereby terminating the polymerization condensation or polycondensation reaction and obtaining the inventive polmeric carrier according to formula (I) or (Ia); 
 d. optionally purifying the inventive polymeric carrier obtained according to step c); 
 e. optionally adding a nucleic acid as defined herein to the polymeric carrier obtained according to step c) or d) and complexing the nucleic acid with the polymeric carrier obtained according to step c) or d) to obtain a polymeric carrier cargo complex as defined according to  claim 9 . 
 
     
     
         15 . Pharmaceutical composition, comprising the polymeric carrier cargo complex according to  claim 9  and optionally a pharmaceutically acceptable carrier and/or vehicle. 
     
     
         16 . Use of the polymeric carrier molecule according to  claim 1  and a nucleic acid as defined according to  claim 9  or use of the polymeric carrier cargo complex according to  claim 9  as a medicament. 
     
     
         17 . Use of the polymeric carrier molecule according to any of  claims 1  to  8  and a nucleic acid as defined according to  claim 11 , or of the polymeric carrier cargo complex according to  claim 9 , for the preparation of a pharmaceutical composition for the prophylaxis, treatment and/or amelioration of diseases as defined herein, selected from cancer or tumor diseases, infectious diseases, including viral, bacterial or protozoological infectious diseases, autoimmune diseases, allergies or allergic diseases, monogenetic diseases, including hereditary diseases or genetic diseases, diseases which have a genetic inherited background and which are typically caused by a single gene defect and are inherited according to Mendel's laws, cardiovascular diseases, and neuronal diseases. 
     
     
         18 . Kit, including kit of parts, comprising as components at least one polymeric carrier molecule according to  claim 1  and at least one nucleic acid according to  claim 10 , provided as a polymeric carrier cargo complex according to  claim 10 , optionally with information on the administration and dosage of the polymeric carrier complex.