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

Patent metadata

Field	Value
Publication number	US-9314535-B2
Application number	US-201414195864-A
Country	US
Kind code	B2
Filing date	Mar 4, 2014
Priority date	Sep 3, 2009
Publication date	Apr 19, 2016
Grant date	Apr 19, 2016

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 to methods of preparation of this inventive polymeric carrier molecule and of the inventive polymeric carrier cargo complex. The present invention also provides methods of application and use of this inventive polymeric carrier molecule 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 of such diseases.

First claim

The invention claimed is:

1. A method of treating a subject comprising administering a polymeric carrier cargo complex formed of a nucleic acid and a polymeric carrier molecule according to formula (I):
L-P 1 —S—[S—P 2 —S] n —S—P 3 -L

wherein,
P 1 and P 3 are different or identical to each other and represent a linear or branched hydrophilic polymer chain independently selected from the group consisting of polyethylene glycol (PEG), poly-N-(2-hydroxypropyl)methacrylamide, poly-2- (methacryloyloxy)ethyl phosphorylcholines, poly(hydroxyalkyl L-asparagine), poly(2-(methacryloyloxy)ethyl phosphorylcholine), hydroxyethylstarch and poly(hydroxyalkyl L-glutamine), wherein the hydrophilic polymer chain exhibits a molecular weight of 1 kDa to 100 kDa,
P 2 is a cationic or polycationic polypeptide having a length of 3 to 100 amino acids, and comprising at least two cysteine residues;
—S—S— is a reversible disulfide bond, wherein one of the sulfur positions of each of the disulfide bonds in provided by the at least two cysteine residues of P 2 ;
L is a ligand, which may be present or not, independently selected from the group consisting of 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, TAT, a ligand of a receptor, a cytokine, a hormone, a growth factor, a small molecule, a carbohydrate, mannose, galactose, a synthetic ligand, a small molecule agonist, an inhibitor or antagonist of a receptor, and a RGD peptidomimetic analogue; and
n is an integer selected from a range of 1 to 50.

2. The method according to claim 1 , wherein the polymeric carrier molecule additionally contains an amino acid component (AA) x , wherein x is an integer selected from a range of 1 to 100, and wherein the amino acid component is linked to the polymeric carrier molecule by disulfide bonds to at least two cysteine residues in the (AA) x component.

3. The method according to claim 2 , wherein the amino acid component (AA) x comprises an aromatic amino acid component, a hydrophilic amino acid component, a lipophilic amino acid component, a weak basic amino acid component, a signal peptide, a localization signal or sequence, a nuclear localization signal or sequence, a cell penetrating peptide, a therapeutically active polypeptide, an antigen, an antigenic epitope, a tumour antigen, a pathogenic antigen, an autoimmune antigen, an allergen, an antibody, an immunostimulatory polypeptide, an antigen-specific T-cell receptor, or a polypeptide suitable for a therapeutic application.

4. The method according to claim 2 , wherein the 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 1 to 30, each (AA) x component being linked by disulfide bonds to at least two cysteine residues in each (AA) x component.

5. The method according to claim 1 , wherein formula (I) is modified according to 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 before and
a+b=n, wherein
n is as defined before;
a is an integer, selected independent from integer b from a range of 1 to 50, and
b is an integer, selected independent from integer a from a range of 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 }.

6. The method according to claim 1 , wherein component P 2 is a cationic or polycationic polypeptide selected from the group consisting of protamine, nucleoline, spermine, spermidine, poly-L-lysine (PLL), basic polypeptides, poly-arginine, cell penetrating peptides (CPPs), chimeric CPPs, Transportan, MPG peptides, HIV-binding peptides, Tat, HIV-1 Tat, Tat-derived peptides, oligoarginines, members of the penetratin family, Penetratin, Antennapedia-derived peptides, pAntp, pIsl, antimicrobial-derived CPPs, 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, histones, VP22 peptides, HSV, VP22 (Herpes simplex), MAP, KALA, protein transduction domains (PTDs), PpT620, prolin-rich peptides, lysine-rich peptides, Pep-1, L-oligomers, and Calcitonin peptide(s).

7. The method according to claim 1 , wherein component P 2 comprises a cationic peptide of formula (IIb):
Cys{(Arg) l ;(Lys) m ;(His) n ;(Orn) o ;(Xaa) x }Cys,
wherein
l+m+n+o+x=8-16, and l, m, n or o are independently 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 cationic peptide; and
Xaa is any amino acid selected from native or non-native amino acids except Arg, Lys, His or Orn; and x is 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 cationic peptide.

8. The method according to claim 1 , wherein the nucleic acid is provided in a molar ratio of 5 to 10000 of polymeric carrier molecule : nucleic acid.

9. The method according to claim 1 , wherein the nucleic acid is a DNA, a RNA, a coding nucleic acid, a coding DNA, a coding RNA, a coding mRNA, an siRNA, an immunostimulatory nucleic acid, an immunostimulatory CpG nucleic acid, or an immunostimulatory RNA (isRNA).

10. The method according to claim 1 , wherein the nucleic acid encodes a therapeutically active polypeptide, an antigen, a tumor antigen, a pathogenic antigen, an animal antigen, a viral antigen, a protozoal antigen, a bacterial antigen, an allergic antigen, an autoimmune antigen, an allergen, an antibody, an immunostimulatory polypeptide, or an antigen-specific T-cell receptor.

11. The method of treating a subject according to claim 1 , wherein the subject has a disease selected from the group consisting of cancer or tumour diseases, infectious diseases, autoimmune diseases, allergies or allergic diseases, monogenetic diseases, genetic diseases, cardiovascular diseases, neuronal diseases, diseases of the respiratory system, diseases of the digestive system, diseases of the skin, musculoskeletal disorders, disorders of the connective tissue, neoplasms, immune deficiencies, endocrine, nutritional and metabolic diseases, eye diseases and ear diseases.

12. The method according to claim 7 , wherein component P2 is a cationic peptide of formula CysHis 6 Arg 4 His 6 Cys (SEQ ID NO: 83).

13. The method of claim 1 , wherein one of the sulfur positions of each of the disulfide bonds of formula (I) is provided by a cysteine residue at the N- or C-terminus of the polypeptide of P 2 .

14. The method of claim 13 , wherein one of the sulfur positions of the disulfide bonds of formula (I) is provided by a cysteine residue at the N-terminus of the polypeptide of P 2 and one of the sulfur positions of the disulfide bonds of formula (I) is provided by a cysteine residue at the C-terminus of the polypeptide of P 2 .

15. The method of claim 1 , wherein L is present and comprises the KALA cell penetrating peptide.

Assignees

• Curevac Gmbh
• Curevac Ag

Inventors

• Baumhof Patrick
• Schlake Thomas

Classifications

• A61P37/08

  Antiallergic agents (antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14) · CPC title

• A61P5/00

  Drugs for disorders of the endocrine system · CPC title

• A61P37/04

  Immunostimulants · CPC title

• A61P37/02

  Immunomodulators · CPC title

• A61P9/00

  Drugs for disorders of the cardiovascular system · CPC title