Adeno-associated-virus rep sequences, vectors and viruses

Abstract

The invention provides adeno-associated virus (AAV) replication (Rep) sequences. In one embodiment, the invention provides nucleotide sequences encoding a chimeric protein, wherein the encoded chimeric protein contains a wild type AAV Rep inhibitory amino acid sequence, and wherein the nucleotide sequences contain a scrambled and/or deoptimized polynucleotide sequence encoding the wild type AAV Rep inhibitory amino acid sequence. The invention provides vectors, cells, and viruses containing the invention's sequences. Also provided are methods for detecting portions of the AAV Rep inhibitory amino acid sequence, which reduce replication and/or infection and/or productive infection by viruses. The invention's compositions and methods are useful for site-specific integration and/or expression of heterologous sequences by recombinant adeno-associated virus (rAAV) vectors and by rAAV virus particles, such as hybrid viruses (e.g., Ad-AAV) comprising such vectors. The invention's compositions and methods find application in, for example, gene therapy and/or vaccines.

1 - 42 . (canceled) 
     
     
         43 . A recombinant nucleotide sequence encoding a chimeric protein,
 a) wherein the encoded chimeric protein
 i) comprises wild type AAV Rep inhibitory amino acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:25, and SEQ ID NO:28, and 
 ii) has Rep-mediated nuclease activity, and 
   b) wherein the recombinant nucleotide sequence comprises a scrambled polynucleotide sequence encoding said wild type AAV Rep inhibitory amino acid sequence.   
     
     
         44 . The recombinant nucleotide sequence of  claim 43 , wherein
 i) said scrambled polynucleotide sequence encoding said wild type SEQ ID NO:22 comprises SEQ ID NO:23,   ii) said scrambled polynucleotide sequence encoding said wild type SEQ ID NO:25 comprises SEQ ID NO:26, and   iii) said scrambled polynucleotide sequence encoding said wild type SEQ ID NO:28 comprises SEQ ID NO:29.   
     
     
         45 . The recombinant nucleotide sequence of  claim 43 , wherein said scrambled polynucleotide sequence comprises a deoptimized AAV Rep inhibitory nucleotide sequence. 
     
     
         46 . An expression vector comprising the recombinant nucleotide sequence of  claim 43 . 
     
     
         47 . A recombinant adeno-associated virus (rAAV) comprising the recombinant nucleotide sequence of  claim 43 . 
     
     
         48 . The recombinant adeno-associated virus (rAAV) of  claim 47 , wherein the rAAV is infectious. 
     
     
         49 . The recombinant adeno-associated virus (rAAV) of  claim 48 , wherein the infectious rAAV is replication competent. 
     
     
         50 . The recombinant adeno-associated virus (rAAV) of  claim 49 , wherein the replication competent rAAV is productive. 
     
     
         51 . The recombinant adeno-associated virus (rAAV) of  claim 47 , wherein the rAAV is produced by a permissive cell at substantially the same copy number as the copy number of a control AAV that lacks expression of AAV Rep protein. 
     
     
         52 . The recombinant adeno-associated virus (rAAV) of  claim 47 , wherein the rAAV is characterized by site-specific integration into adeno-associated virus integration site 1 (AAVS1) sequence. 
     
     
         53 . The recombinant adeno-associated virus (rAAV) of  claim 47 , wherein the rAAV expresses Rep78 protein SEQ ID NO:04 at a reduced level compared to the level expressed by a control hybrid virus that comprises wild type amino acid sequence SEQ ID NO:20 that is encoded by the wild type AAV Rep inhibitory nucleotide sequence listed as SEQ ID NO:17. 
     
     
         54 . The recombinant adeno-associated virus (rAAV) of  claim 47 , wherein the rAAV is a hybrid virus that comprises at least a portion of a heterologous virus genome sequence. 
     
     
         55 . The recombinant adeno-associated virus (rAAV) of  claim 54 , wherein the heterologous virus is selected from the group consisting of adenovirus, herpes simplex virus, retrovirus, lentivirus, and baculovirus. 
     
     
         56 - 60 . (canceled) 
     
     
         61 . A method for producing a recombinant adeno-associated virus (rAAV) particle, comprising
 a) providing an expression vector comprising the recombinant nucleotide sequence of  claim 43 ,   b) providing an adeno-associated virus (AAV) packaging cell, and   c) transfecting the packaging cell with the expression vector to produce a recombinant adeno-associated virus (rAAV).   
     
     
         62 - 65 . (canceled) 
     
     
         66 . A method for reducing one or more symptoms of disease in a mammalian subject, comprising administering a therapeutically effective amount of the vector of  claim 46  to a mammalian subject in need of the therapy. 
     
     
         67 - 71 . (canceled)