Manufacturing and use of recombinant AAV vectors

1. A transfection system for the production of AAV vectors, the system comprising;

a closed, circular DNA plasmid consisting of a polynucleotide sequence of SEQ ID NO:2 as a Rep/Helper plasmid; and

a Payload/Cap plasmid consisting of a polynucleotide sequence of SEQ ID NO: 11, a polynucleotide sequence encoding a Cap gene and a polynucleotide sequence encoding a transgene,

wherein the polynucleotide sequence encoding the Cap gene is inserted before position 2025 of SEQ ID NO:11, and

wherein the polynucleotide sequence encoding the transgene is inserted after position 2663 of SEQ ID NO: 11.

2. The transfection system of claim 1 , wherein the plasmid ratio of the Rep/Helper plasmid to the Payload/Cap plasmid is 1.5:1 up to 10:1.

3. The transfection system of claim 2 , wherein the transfection system comprises no plasmid other than the Rep/Helper plasmid and the Payload/Cap plasmid.

4. The transfection system of claim 1 , wherein the transfection system comprises no plasmid other than the Rep/Helper plasmid and the Payload/Cap plasmid.

5. A method of producing an AAV vector in a cell line, the method comprising the step of transfecting the cell line with only two unique plasmids in the presence of a transfection reagent, the two plasmids consisting of:

(a) a closed, circular DNA plasmid consisting of a polynucleotide sequence of SEQ ID NO:2 as a first plasmid, and

(b) a second Payload/Cap plasmid consisting of a polynucleotide sequence of SEQ ID NO:11, a polynucleotide sequence encoding a Cap gene, and a polynucleotide sequence encoding a transgene,

wherein the polynucleotide sequence encoding the Cap gene is inserted before positions 2025 of SEQ ID NO:11, and

wherein the polynucleotide sequence encoding the transgene is inserted after position 2663 of SEQ ID NO:11

wherein the transfected cell line expresses the plasmids to produce the AAV vector.

6. The method of claim 5 , wherein the cell line is a HEK293 cell line.

7. The method of claim 5 , wherein the transfection reagent is a cationic polymer reagent.

8. The method of claim 5 , wherein the method comprises transfecting the cell line with the first plasmid and the second plasmid with a plasmid ratio of about 1.5:1 to 10:1.

9. The method of claim 8 , wherein the method comprises transfecting the cell line with the first plasmid and the second plasmid with a plasmid ratio of about 1.5:1.

10. The method of claim 8 , wherein

(a) the cell line is a HEK293 cell line; and

(b) the Transfection Reagent is a cationic polymer reagent.

11. The method of claim 10 , wherein the method comprises transfecting the cell line with the first plasmid and the second plasmid with a plasmid ratio of about 1.5:1.

12. The method of claim 10 , wherein the cell line is HEK293F.

13. A method of expressing producing an AAV vector in a HEK293 cell line, the method comprising the step of transfecting the cell line with only two unique plasmids in the presence of a transfection reagent, the two plasmids consisting of:

(a) a closed, circular DNA plasmid consisting of a polynucleotide sequence of SEQ ID NO:2 as a Rep/Helper plasmid, and

(b) a Payload/Cap plasmid consisting of a polynucleotide sequence of SEQ ID NO:11, a polynucleotide sequence encoding a Cap gene, and a polynucleotide sequence encoding a transgene,

wherein the polynucleotide sequence encoding the Cap gene is inserted before position 2025 of SEQ ID NO:11, and

wherein the polynucleotide sequence encoding the transgene is inserted after position 2663 of SEQ ID NO:11; and wherein the HEK293 cell line is transfected with the Rep/Helper plasmid and the Payload/Cap plasmid with a plasmid ratio of about 1.5:1 to 10:1 and in the absence of any other Rep/helper or Payload/Cap Plasmid,

wherein the transfected HEK293 cell line expresses the plasmids to produce the AAV vector.