Restrictive inverted terminal repeats for viral vectors
View Patent ↗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 polynucleotide comprising at least one parvovirus inverted terminal repeat (ITR), wherein said ITR comprises:
(a) a first structural element that functionally interacts with a large Rep protein from a first adeno-associated virus (AAV) but does not functionally interact with a large Rep protein from a second AAV; and
(b) a second structural element that functionally interacts with the large Rep protein from the second AAV but does not functionally interact with the large Rep protein from the first AAV;
wherein the ITR functionally interacts with a synthetic AAV large Rep protein; and
wherein one of the structural elements is a nicking stem.
2. The polynucleotide of claim 1 , wherein said ITR does not functionally interact with any wild-type large Rep protein.
3. The polynucleotide of claim 1 , wherein said structural elements are selected from the group consisting of a nicking stem, a Rep binding element (RBE), and an extended RBE.
4. The polynucleotide of claim 1 , wherein said first structural element is a nicking stem.
5. The polynucleotide of claim 1 , wherein said second structural element is a spacer, a RBE or an extended RBE.
6. The polynucleotide of claim 1 , wherein said ITR further comprises a third structural element that functionally interacts with a large Rep protein from an AAV that is the same as or different from the first and/or second AAV.
7. The polynucleotide of claim 1 , further comprising a heterologous nucleic acid.
8. A viral vector comprising the polynucleotide of claim 1 .
9. A recombinant parvovirus particle comprising the polynucleotide of claim 1 .
10. The polynucleotide of claim 1 , wherein said parvovirus is an adeno-associated virus (AAV).
11. The polynucleotide of claim 1 , wherein said first and/or second structural element has a modified sequence as compared to the wild-type sequence of the ITR.
12. The polynucleotide of claim 11 , wherein said modified sequence is a wild-type sequence from a different ITR.
13. The polynucleotide of claim 11 , wherein said modified sequence is a synthetic sequence.
14. The polynucleotide of claim 11 , wherein said first structural element is a nicking stem and said nicking stem comprises a wild-type AAV2 sequence.
15. The polynucleotide of claim 11 , wherein said first structural element is a modified nicking stem comprising a change in height as compared to a wild-type sequence.
16. The polynucleotide of claim 11 , wherein said first structural element is a modified nicking stem comprising a modified sequence as compared to a wild-type sequence.
17. The polynucleotide of claim 16 , wherein said modified sequence is a modified terminal resolution site (trs) sequence.
18. The polynucleotide of claim 11 , wherein said second structural element is a RBE and said RBE comprises a wild-type AAV5 sequence.
19. The polynucleotide of claim 11 , wherein said second structural element is a RBE comprising a change in length or sequence relative to a wild-type sequence.
20. The viral vector of claim 8 , which is an AAV vector.
21. The recombinant parvovirus particle of claim 9 , which is an AAV particle.