Methods of raav packaging

The method of any one of the preceding claim, wherein the host cell is a mammalian cell.

The method of any one of the preceding claim, wherein the host cell is a human cell, optionally a HEK 293T cell.

The method of any one of the preceding claims, wherein the cis-element nucleic acid, the helper nucleic acid, and/or the packaging nucleic acid is a plasmid.

The method of any one of the preceding claims, wherein the cis-element nucleic acid comprises a transgene flanked by two inverted terminal repeats (ITRs).

The method of any one of the preceding claims, wherein the cis-element nucleic acid is a self-complementary nucleic acid comprising at least one AITR or mTR.

The method of any one of the preceding claims, wherein the transgene encodes a protein, optionally a therapeutic protein.

The method of any one of the preceding claims, wherein the transgene is cytotoxic or comprises one or more physiochemical characteristics that are detrimental to the fitness of the host cell (e.g., the transgene encodes a protein that forms a secondary structure with high thermal stabilities).

The method of any one of the preceding claims, wherein the adenoviral helper genes comprise E4, E2a and/or VA genes.

The method of any one of the preceding claims, wherein the concentration of the cis-element nucleic acid that is introduced to the host cell is 10-fold to 100-fold, 10-fold to 90-fold, 10-fold to 80-fold, 10-fold to 70-fold, 10-fold to 60-fold, 10-fold to 50-fold, 10-fold to 40-fold, 10-fold to 30-fold, or 10-fold to 20-fold less than the concentration of the helper nucleic acid and/or the packaging nucleic acid that is introduced to the host cell.

The method of any one of the preceding claims, wherein the ratio of the concentration of (i) relative to (ii) is between 0.01:1 and 0.1:1, optionally between 0.01:1 and 0.05:1 or between 0.01:1 and 0.025:1.

The method of any one of the preceding claims, wherein the ratio of the concentration of (i) relative to (iii) is between 0.01:1 and 0.1:1, optionally between 0.01:1 and 0.05:1 or between 0.01:1 and 0.025:1.

The method of any one of the preceding claims, wherein the cis-element nucleic acid, the helper nucleic acid, and/or the packaging nucleic acid are introduced into the host cell simultaneously.

The method of any one of the preceding claims, wherein a composition comprising the cis-element nucleic acid, the helper nucleic acid, and/or the packaging nucleic acid is introduced into the host cell.

The method of any one of the preceding claims, wherein the helper nucleic acid, and/or the packaging nucleic acid are introduced into the host cell using electroporation or transfection techniques.

The method of any one of the preceding claims, wherein less than 5% of the produced rAAV produced comprise read-through rAAV or reverse packaging rAAV.

The method of any one of the preceding claims, wherein the method produces a lower relative amount of read-through rAAV or reverse packaging rAAV compared to a traditional triple-transfection method, optionally wherein the traditional triple-transfection method produces rAAVs using a 1:1 ratio for (i):

• (ii) and/or (i):
• (iii).