Adeno-associated-virus Rep sequences, vectors and viruses

A method for detecting a portion of wild type AAV Rep inhibitory nucleotide sequence that reduces replication by a hybrid Adenovirus that contains said portion, comprising

• a) providing
• i) a first hybrid Adenovirus vector comprising a first nucleotide sequence comprising a scrambled polynucleotide sequence encoding a portion of wild type AAV Rep inhibitory amino acid sequence listed as SEQ ID NO:20,
• ii) a second hybrid Adenovirus vector comprising a second nucleotide sequence, wherein the second nucleotide sequence is produced by substituting a portion of the scrambled polynucleotide sequence with a corresponding portion of wild type AAV Rep inhibitory nucleotide sequence listed as SEQ ID NO:17, and
• iii) a host cell that is permissive for the hybrid Adenovirus,
• b) transfecting
• i) the first hybrid Adenovirus vector into the permissive cell under conditions to produce a first hybrid Adenovirus that comprises a first amino acid sequence encoded by the first nucleotide sequence, and
• ii) the second hybrid Adenovirus vector into the permissive cell under conditions to produce a second hybrid Adenovirus that comprises a second amino acid sequence encoded by the second nucleotide sequence, and
• c) determining the level of replication of the first hybrid Adenovirus and of the second hybrid Adenovirus in the transfected permissive cell, wherein a reduced level of replication of the second hybrid Adenovirus compared to the first hybrid Adenovirus identifies the portion of wild type AAV Rep inhibitory nucleotide sequence as reducing replication by the hybrid Adenovirus.