Adeno-associated viruses and uses thereof
AssigneeUniversity of Iowa Research Foundation
InventorsJohn F. Engelhardt, Dongsheng Duan, Ziying Yan
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Abstract
The invention provides an isolated and purified DNA molecule comprising at least one DNA segment, a biologically active subunit or variant thereof, of a circular intermediate of adeno-associated virus, which DNA segment confers increased episomal stability, persistence or abundance of the isolated DNA molecule in a host cell. The invention also provides a composition comprising at least two adeno-associated virus vectors.
Claims21
A composition comprising at least two recombinant adeno-associated viruses (rAAV), comprising:
- a) a first rAAV comprising a first recombinant DNA molecule comprising linked:
- i) a first DNA segment comprising a 5′-inverted terminal repeat of AAV;
- ii) a second DNA segment which comprises at least one heterologous enhancer and a heterologous promoter;
- and
- iii) a third DNA segment comprising a 3′-inverted terminal repeat of AAV;
- and
- b) a second rAAV comprising a second recombinant DNA molecule comprising linked:
- i) a first DNA segment comprising a 5′-inverted terminal repeat of AAV;
- ii) a second DNA segment which comprises an entire open reading frame for a therapeutic gene product;
- and
- iii) a third DNA segment comprising a 3′-inverted terminal repeat of AAV,
- wherein the recombinant DNA molecules of the two rAAVs, when contacted with a host cell, become linked so that the heterologous promoter initiates transcriptional expression of the gene product encoded by the entire open reading frame, and wherein the second rAAV does not comprise a heterologous promoter 5′ to the open reading frame.
A first rAAV vector which comprises a cis-acting heterologous transcriptional regulatory including a heterologous enhancer and a heterologous promoter positioned in the vector so that the heterologous promoter is capable of regulating, in a host cell, transcriptional expression of a therapeutic gene product encoded by an entire open reading frame in a second rAAV vector, after sequences in the first and second rAAV vectors become linked in the host cell, and wherein the second rAAV does not comprise a heterologous promoter 5′ to the open reading frame.
A host cell contacted with at least two rAAV, wherein a first rAAV comprises a first recombinant DNA molecule comprising linked:
- i) a first DNA segment comprising a 5′-inverted terminal repeat of AAV;
- ii) a second DNA segment which comprises a heterologous enhancer and a heterologous promoter;
- and
- iii) a third DNA segment comprising a 3′-inverted terminal repeat of AAV;
- and
- wherein a second rAAV comprises a second recombinant DNA molecule comprising linked:
- i) a first DNA segment comprising a 5′-inverted terminal repeat of AAV;
- ii) a second DNA segment which comprises an entire open reading frame for a therapeutic gene product;
- and
- iii) a third DNA segment comprising a 3′-inverted terminal repeat of
- AAV, wherein the heterologous promoter initiates transcriptional expression of the gene product encoded by the open reading frame in the second rAAV in the host cell, and wherein the second rAAV does not comprise a heterologous promoter 5′ to the open reading frame.
A method to transfer recombinant DNAs to a host cell, comprising:
- contacting the host cell with at least two rAAV,
- wherein a first rAAV comprises a first recombinant DNA molecule comprising linked:
- i) a first DNA segment comprising a 5′-inverted terminal repeat of AAV;
- ii) a second DNA segment which comprises a heterologous enhancer and a heterologous promoter;
- and
- iii) a third DNA segment comprising a 3′-inverted terminal repeat of AAV;
- and
- wherein a second rAAV comprises a second recombinant DNA molecule comprising linked:
- i) a first DNA segment comprising a 5′-inverted terminal repeat of AAV;
- ii) a second DNA segment which comprises an entire open reading frame for a therapeutic gene product;
- and
- iii) a third DNA segment comprising a 3′-inverted terminal repeat of AAV, wherein the heterologous promoter initiates transcriptional expression of the gene product encoded by the open reading frame in the second rAAV in the host cell, and wherein the second rAAV does not comprise a heterologous promoter 5′ to the open reading frame.
A first rAAV comprising a first recombinant DNA molecule comprising linked:
- a first DNA segment comprising a 5′-inverted terminal repeat of AAV;
- a second DNA segment which comprises a heterologous enhancer and a heterologous promoter;
- and a third DNA segment comprising a 3′-inverted terminal repeat of AAV;
- and
- a second rAAV comprising a second recombinant DNA molecule comprising linked:
- a first DNA segment comprising a 5′-inverted terminal repeat of AAV;
- a second DNA segment which comprises an entire open reading frame for a therapeutic gene product;
- and a third DNA segment comprising a 3′-inverted terminal repeat of AAV,
- wherein the heterologous promoter in the first rAAV initiates transcriptional expression of the gene product encoded by the open reading frame in the second rAAV in a host cell contacted with the first and second rAAVs and wherein the second rAAV does not comprise a heterologous promoter 5′ to the open reading frame.
Dependencies are inferred from the claim text.