Integrin-targeting vectors having transfection activity

Abstract

A complex that comprises (i) a nucleic acid, (ii) an integrin-binding component, for example, an intergrin-binding peptide, (iii) a polycationic nucleic acid-binding component, for example, oligolysine, and (iv) a lipid component, for example, a cationic liposome, has transfection activity.

1 . A complex that comprises 
 (i) a nucleic acid,    (ii) an integrin-binding component,    (iii) a polycationic nucleic acid-binding component, and    (iv) a lipid component    
     
     
         2 . A complex as claimed in  claim 1 , wherein the integrin-binding component is an integrin-binding peptide  
     
     
         3 . A complex as claimed in  claim 2 , wherein the peptide consists of or comprises all or part of the integrin-binding domain of a naturally-occurring integrin ligand.  
     
     
         4 . A complex as claimed in  claim 3 , wherein the integrin-binding peptide comprises the conserved amino acid sequence arginine-glycine-aspartic acid (RGD).  
     
     
         5 . A complex as claimed in  claim 4 , wherein a peptide comprising the sequence RGD has a cyclic region in which the conformational freedom of the RGD sequence is restricted.  
     
     
         6 . A complex as claimed in  claim 5 , wherein a cyclic peptide has two or more cysteine residues that form one or more disulphide bond(s).  
     
     
         7 . A complex as claimed in  claim 6 , wherein the peptide consists of or comprises the sequence CRGDMFGC [SEQ.ID.NO.:5].  
     
     
         8 . A complex as claimed in  claim 7 , wherein the peptide consists of or comprises the sequence GGCRGDMFGC [SEQ.ID.NO.:6], GGCRGDMFGCG (SEQ.ID.NO.7], GGCRGDMFGCA [SEQ.ID.NO.:8] or GACRGDMFGCA [SEQ.ID.NO.:9].  
     
     
         9 . A peptide as claimed in  claim 6 , which peptide consists of or comprises the sequence GACDCRGDCFCA [SEQ.ID.NO.:10].  
     
     
         10 . A peptide as claimed in  claim 2 , which peptide consists of or comprises the sequence CRRETAWAC [SEQ.ID.NO.:13].  
     
     
         11 . A peptide as claimed in  claim 10 , which consists of or comprises the sequence GACRRETAWACA [SEQ.ID.NO.:11] or GACRRETAWACG [SEQ.ID.NO.:12].  
     
     
         12 . A peptide as claimed in  claim 2 , which consists of or comprises the sequence GAGPEILDVPST [SEQ.ID.NO.:14], GACQIDSPCA [SEQ.ID.NO.:15] or GACRRETAWACGKGACRRETAWACG [SEQ.ID.NO.:16].  
     
     
         13 . A complex as claimed in any one of  claims 1  to  12 , wherein the nucleic acid component is or relates to a gene that is the target for gene therapy, gene vaccination or anti-sense therapy.  
     
     
         14 . A complex as claimed in any one of  claims 1  to  13 , wherein transcriptional and/or translational control elements for the nucleic acid are provided and the nucleic acid is optionally packed in a phage or vector.  
     
     
         15 . A complex as claimed in any one of  claims 1  to  14 , wherein the nucleic acid component is DNA.  
     
     
         16 . A complex as claimed in any one of  claims 1  to  14 , wherein the nucleic acid component is RNA.  
     
     
         17 . A complex as claimed in any one of  claims 1  to  16 , wherein the nucleic acid-binding component has from 3 to 100 cationic monomers.  
     
     
         18 . A complex as claimed in any one of  claims 1  to  17 , wherein the polycationic nucleic acid-binding component is an oligolysine.  
     
     
         19 . A complex as claimed in  claim 18 , wherein the oligolysine has from 10 to 20, especially 16 lysine residues.  
     
     
         20 . A complex as claimed in any one of  claims 1  to  19 , wherein the lipid component is or is capable of forming a cationic liposome.  
     
     
         21 . A complex as claimed in any one of  claims 1  to  20 , wherein the lipid component is or comprises one or more lipids selected from cationic lipids and lipids having membrane destabilising or fusogenic properties.  
     
     
         22 . A complex as claimed in  claim 21 , wherein the lipid component is or comprises the neutral lipid dioleyl phosphatidyl-ethanolamine (DOPE) or a lipid having similar membrane destabilising or fusogenic properties.  
     
     
         23 . A complex as claimed in  claim 21  or  claim 22 , wherein the lipid component is or comprises the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) or a lipid having similar cationic properties.  
     
     
         24 . A complex as claimed in  claim 23 , wherein the lipid component is or comprises a mixture of DOPE and DOTMA, especially an equimolar mixture thereof.  
     
     
         25 . A complex as claimed in  claim 24 , which comprises an equimolar mixture of DOPE and DOTMA as the lipid component, an integrin-binding peptide as the integrin-binding component, and [K] 16  as the polycationic nucleic acid-binding component.  
     
     
         26 . A complex as claimed in  claim 24  or  claim 25 , wherein the ratio lipid component:integrin-binding/polycationic nucleic acid-binding component: nucleic acid is 0.75:4:1 by weight or 0.5 nmol:1.25 nmol:0.25 nmol on a molar basis.  
     
     
         27 . A complex as claimed in any one of  claims 1  to  24 , wherein the lipid component is or comprises 2,3-dioleyloxy-N-[2-(spermidinecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium-trifluoridoacetate (DOSPA) or a lipid having similar properties to those of DOSPA.  
     
     
         28 . A complex as claimed in  claim 27 , wherein the lipid component is or comprises a mixture of DOPE and DOSPA, especially a mixture of one part by weight DOPE to 3 parts by weight DOSPA.  
     
     
         29 . A complex as claimed in  claim 28 , which comprises a mixture of DOPE and DOSPA as the lipid component, an integrin-binding peptide as the integrin-binding component, and [K] 16  as the polycationic nucleic acid-binding component.  
     
     
         30 . A complex as claimed in  claim 29 , wherein the ratio lipid component:polycationic nucleic acid-binding component: nucleic acid is 12:4:1 by weight.  
     
     
         31 . A process for the production of a complex as claimed in any one of  claims 1  to  30 , which comprises admixing components (i), (ii), (iii) and (iv).  
     
     
         32 . A process as claimed in  claim 31 , wherein the components are admixed in the following order: lipid component, integrin-binding component/polycationic nucleic acid-binding component, nucleic acid.  
     
     
         33 . A complex as claimed in any one of  claims 1  to  30 , obtainable by a process as claimed in  claim 31  or  claim 32 .  
     
     
         34 . A mixture comprising an integrin-binding component, a polycationic nucleic acid-binding component, and a lipid component.  
     
     
         35 . A mixture as claimed in  claim 34 , wherein the integrin-binding component is as defined in any one of  claims 2  to  12 .  
     
     
         36 . A mixture as claimed in  claim 34  or  claim 35 , wherein the polycationic nucleic acid-binding component is as defined in any one of  claims 17  to  19 .  
     
     
         37 . A mixture as claimed in any one of  claims 34  to  36 , wherein the lipid component is as defined in any one of  claims 20  to  24 ,  27  and  28 .  
     
     
         38 . A mixture as claimed in  claim 34 , which comprises an equimolar mixture of DOPE and DCTMA as the lipid component, an integrin-binding peptide as the integrin-binding component, and [K] 16  as the polycationic component nucleic acid-binding component.  
     
     
         39 . A mixture as claimed in  claim 38 , wherein the ratio lipid component:combined integrin-binding/polycationic nucleic acid-binding component is 0.75:4 by weight.  
     
     
         40 . A process for producing a complex as claimed in  claim 1 , which comprises incorporating a nucleic acid with a mixture as claimed in any one of  claims 34  to  39 .  
     
     
         41 . A method of transfecting a cell with a nucleic acid, which comprises contacting the cell in vitro or in vivo with a complex as claimed in any one of  claims 1  to  30  or  claim 33 .  
     
     
         42 . A pharmaceutical composition which comprises a complex as claimed in any one of  claims 1  to  30   claim 33 , in admixture or conjunction with a pharmaceutically suitable carrier.  
     
     
         43 . A method for the treatment or prophylaxis of a condition caused in human or or in a non-human animal by a defect and/or a deficiency in a gene, which comprises administering a complex as claimed in any one of  claims 1  to  30  or  claim 33  to the human or to the non-human animal.  
     
     
         44 . A method for therapeutic or prophylactic immunisation of a human or of a non-human animal, which comprises administering a complex as claimed in any one of  claims 1  to  30  or  claim 33  to the human or to the non-human animal.  
     
     
         45 . A method of anti-sense therapy, which comprises administering a complex as claimed in any one of  claims 1  to  30  or  claim 33  to a human or to a non-human animal.  
     
     
         46 . A complex as claimed in any one of  claims 1  to  30  or  claim 33  for use as a medicament or a vaccine.  
     
     
         47 . Use of a complex as claimed in any one of  claims 1  to  30  or  claim 33  for the manufacture of a medicament for the prophylaxis of a condition caused in a human or a non-human animal by a defect and/or a deficiency in a gene, or for therapeutic or prophylactic immunisation, or for anti-sense therapy.  
     
     
         48 . A kit that comprises (i) an integrin-binding component, 
 (ii) a polycationic nucleic acid-binding component, and    (iii) a lipid component.    
     
     
         49 . A kit as claimed in  claim 48 , which also comprises (a) a plasmid or vector suitable for the expression of a nucleic acid, the plasmid or vector being either empty or comprising the nucleic acid, or (b) a nucleic acid.  
     
     
         50 . A kit as claimed in  claim 48  or  claim 49 , wherein components (i) to (iii) are as defined in any one of  claims 2  to  29 .  
     
     
         51 . Use of a lipid component as defined in any one of  claims 20  to  24 ,  27  and  28  for the manufacture of a medicament comprising (i) a nucleic acid, (ii) an integrin-binding component, (iii) a polycationic nucleic acid-binding component and (iv) the lipid component.  
     
     
         52 . A method for transfecting a cell using (i) a nucleic acid, (ii) an integrin-binding component, and (iii) a polycationic nucleic acid-binding component, characterised in that a lipid component is used in addition to components (i) to (iii).  
     
     
         53 . Use as claimed in  claim 51  or a method as claimed in claim  
     
     
         52 , wherein the lipid component is as defined in any one of  claims 20  to  24 ,  27  and  28 .  
     
     
         54 . A method for expressing a nucleic acid in a host cell, which comprises bringing the cell into contact with a complex as claimed in any one of  claims 1  to  30  or  claim 33 .  
     
     
         55 . A method for producing a protein, which comprises transfecting a cell in vitro with a complex as claimed in any one of  claims 1  to  30  or  claim 33 , wherein the nucleic acid component of the complex encodes the protein, allowing the cell to express the protein, and obtaining the protein.