Restrictive inverted terminal repeats for viral vectors
AssigneeThe University of North Carolina at Chapel Hill
InventorsCurtis Hewitt, Richard Jude Samulski
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Abstract
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.
Claims23
1-28. (canceled)
A synthetic large Rep protein comprising a first portion that functionally interacts with a first structural element of a parvovirus ITR and a second portion that functionally interacts with a second structural element of a parvovirus ITR, wherein said first structural element functionally interacts with a large Rep protein from a first AAV but does not functionally interact with a large Rep protein from a second AAV and said second structural element functionally interacts with a large Rep protein from a second AAV but does not functionally interact with a large Rep protein from the first AAV.
A method of producing a recombinant parvovirus particle, comprising providing to a cell permissive for parvovirus replication:
- (a) a recombinant parvovirus template comprising (i) a heterologous nucleic acid, and (ii) a parvovirus terminal repeat sequence, wherein said terminal repeat sequence 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 a 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;
- (b) a polynucleotide encoding a synthetic large Rep protein comprising a first portion that functionally interacts with a first structural element of a parvovirus ITR and a second portion that functionally interacts with a second structural element of a parvovirus ITR, wherein said first structural element functionally interacts with a large Rep protein from a first AAV but does not functionally interact with a large Rep protein from a second AAV and said second structural element functionally interacts with a large Rep protein from a second AAV but does not functionally interact with a large Rep protein from the first AAV;
- under conditions sufficient for the replication and packaging of the recombinant parvovirus template;
- whereby recombinant parvovirus particles are produced in the cell.
48-54. (canceled)
A method of delivering a nucleic acid to a cell, comprising introducing into a cell a recombinant parvovirus particle comprising 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.
56-77. (canceled)
Dependencies are inferred from the claim text.