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.
Claims29
1-77. (canceled)
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) 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
- 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.
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) at least one parvovirus ITR, wherein said ITR comprises:
- a) a first structural element that functionally interacts with a large Rep protein from one or more of AAV1, AAV2, AAV3, AAV4, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13 but does not functionally interact with a large Rep protein from AAV5;
- and
- b) a second structural element that functionally interacts with the large Rep protein from AAV5 but does not functionally interact with the large Rep protein from one or more of AAV1, AAV2, AAV3, AAV4, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, and AAV13;
- wherein the ITR functionally interacts with a synthetic AAV large Rep protein comprising an amino acid sequence selected from SEQ ID NO:
- 79, 81, and 83;
- and
- 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.
Dependencies are inferred from the claim text.