Materials and methods for intracellular transport and their uses

Abstract

Coupled polypeptides and fusion polypeptides for intracellular transport and their preparation and use, include (i) an aminoacid sequence with the transport function of herpesviral VP22 protein (or a homologue, e.g. from VZV, BHV or MDV) and (ii) another protein sequence selected from (a) proteins for cell cycle control; (b) suicide proteins; (c) antigenic sequences or antigenic proteins from microbial and viral antigens and tumour antigens; (d) immunomodulating proteins; and (e) therapeutic proteins. The coupled proteins can be used for intracellular delivery of protein sequences (ii), to exert the corresponding effector function in the target cell, and the fusion polypeptides can be expressed from corresponding polynucleotides, vectors and host cells.

1 . Coupled polypeptides and fusion polypeptides comprising (i) an aminoacid sequence with the transport function of herpesviral VP22 protein and (ii) another protein sequence selected from (a) proteins for cell cycle control; (b) suicide proteins (proteins that are conditionally cytotoxic or lethal upon administration, to a cell containing them, of a corresponding (pro)drug or activator compound): (c) antigenic sequences or antigenic proteins (e.g. of greater than 12 aminoacid residues in length) from microbial and viral antigens and tumour antigens; (d) immunomodulating proteins; and (e) therapeutic proteins.  
     
     
         2 . A polypeptide according to  claim 1  where said other protein sequence is from a mammalian (e.g. human) cell cycle control protein.  
     
     
         3 . A polypeptide according to  claim 2  where said other protein sequence is from a mammalian (e.g. human) protein for increasing or inducing cell apoptosis or for conferring on 2 cell the ability to undergo apoptosis.  
     
     
         4 . A polypeptide according to  claim 2  where said other protein sequence is from a mammalian (e.g. human) cell cycle control protein selected from p53 protein, cyclin dependent kinase inhibitors, and proteins oil the bcl2 and bax families.  
     
     
         5 . A polypeptide according to  claim 4 , which is a fusion polypeptides and comprises a sequence from a p53 protein.  
     
     
         6 . A fusion polypeptide according to  claim 5 , comprising substantially a full length VP22 sequence and substantially a full length p53 sequence.  
     
     
         7 . A polypeptide according to  claim 1  where said other protein sequence is from a suicide protein.  
     
     
         8 . A polypeptide according to  claim 7  where said suicide protein is selected from thymidine kinase and nitroreductase.  
     
     
         9 . A polypeptide according to  claim 7 , comprising substantially the full length VP22 sequence.  
     
     
         10 . A polypeptide according to  claim 1 , which is a fusion polypeptide, and comprises a cleavage-inducing linker sequence located between the VP22 sequence and said other protein sequence.  
     
     
         11 . A polypeptide according to  claim 1 , comprising a sub-sequence of HSV VP22 starting from about aa 159 and extending to about aa 301, and having (relative to the full VP22 sequence) at least one deletion of at least part of the VP22 sequence extending for example from the N-terminal to the sequence of about aa 1-158.  
     
     
         12 . A polypeptide according to  claim 1 , which comprises a sequence corresponding to aminoacids 60-301 or 159-301 of the full HSV VP22 sequence.