Restrictive inverted terminal repeats for viral vectors
This invention relates to modified parvovirus inverted terminal repeats (ITRs) that do not functionally interact with wild-type large Rep proteins, synthetic Rep proteins that functionally interact with the modified ITRs, and methods of using the same for delivery of nucleic acids to a cell or a subject. The modifications provide a novel Rep-ITR interaction that limits vector mobilization, increasing the safety of viral vectors.
1. A synthetic large Rep protein comprising a first portion from a first adeno-associated virus (AAV) and a second portion from a second AAV;
wherein one of the first AAV and the second AAV is AAV5 and the other is an AAV other than AAV5;
wherein the synthetic large Rep protein functionally interacts with a synthetic inverted terminal repeat (ITR) comprising a first structural element from the first AAV and a second structural element from the second AAV; and
wherein the synthetic large Rep protein does not functionally interact with a wild-type ITR from the first AAV or with a wild-type ITR from the second AAV; and
wherein said first and second structural element is a nicking stem, a spacer or an RBE.
2. The protein of claim 1 , wherein said protein comprises a third portion and wherein the synthetic large Rep protein functionally interacts with a synthetic ITR comprising the first structural element, the second structural element, and a third structural element.
3. The protein of claim 1 , wherein said first portion comprises an amino acid sequence from about residue 97 to about residues 146-151 of a wild-type AAV5 Rep sequence.
4. The protein of claim 1 , wherein said second portion comprises an amino acid sequence from about residue 149 to about residue 187 of a wild-type AAV2 Rep sequence.
5. The protein of claim 1 , wherein said first portion comprises an amino acid sequence from about residue 97 to about residues 146-151 of a wild-type AAV5 Rep sequence and said second portion comprises an amino acid sequence from about residue 149 to about residue 187 of a wild-type AAV2 Rep sequence.
6. The protein of claim 1 , wherein said first portion comprises an amino acid sequence from about residue 1 to about residues 146-151 of a wild-type AAV5 Rep sequence and said second portion comprises an amino acid sequence from about residue 149 to about residue 621 of a wild-type AAV2 Rep sequence.
7. The synthetic large Rep protein of claim 1 , wherein the AAV other than AAV5 is selected from the group consisting of AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13.
8. The synthetic large Rep protein of claim 1 , wherein the first AAV is AAV5.
9. The synthetic large Rep protein of claim 8 , wherein the second AAV is AAV2.
10. The synthetic large Rep protein of claim 1 , wherein the first AAV is AAV2.
11. The synthetic large Rep protein of claim 10 , wherein the second AAV is AAV5.