Methods for the evaluation of neutralizing antibodies for adeno-associated virus
This disclosure describes a method that includes using dorsomorphin to increase the infectivity of adeno-associated virus (AAV). The AAV may be of any AAV serotype. In some embodiments, dorsomorphin may be used in combination with IL-6 or TNFα or both. This disclosure further describes methods for using dorsomorphin-treated cells to determine neutralizing antibody (NAb) titers.
1 . A method comprising:
providing an HEK293 cell responsive to an AMP-activated protein kinase (AMPK) inhibitor; and
infecting the cell with adeno-associated virus (AAV) in the presence of dorsomorphin.
2 . The method of claim 1 , wherein the AAV comprises AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV Anc80L65, or AAVKLK.
3 . The method of claim 1 , wherein the AAV is an AAV vector.
4 . The method of claim 3 , wherein the AAV vector comprises a reporter gene.
5 . The method of claim 4 , wherein the reporter gene encodes luciferase.
6 . The method of claim 1 , wherein dorsomorphin is added up to 30 minutes, up to 40 minutes, up to 50 minutes, up to 60 minutes, up to 90 minutes, up to 120 minutes, or up to 180 minutes prior to the addition of AAV.
7 . The method of claim 1 , wherein dorsomorphin is added to the cell at a concentration of up to 4 μM, up to 6 μM, up to 8 μM, up to 10 μM, up to 15 μM, up to 20 μM, or up to 30 μM.
8 . The method of claim 1 , wherein the method further comprises infecting the cell with AAV in the presence of IL-6 or TNFα or both.
9 . The method of claim 1 , wherein the method further comprises adding a composition to the dorsomorphin-treated cell at the time of infecting the cell with AAV, wherein the composition comprises:
a composition known to comprise a neutralizing antibody (NAb) to AAV; or
a composition being tested for the presence of a NAb to AAV.
10 . The method of claim 9 , wherein the AAV is an AAV vector comprising a reporter gene.
11 . The method of claim 9 , wherein the composition comprises serum.
12 . The method of claim 11 , wherein the serum is serum from a human or an animal.
13 . The method of claim 9 , wherein adding a composition to the dorsomorphin-treated cell comprises:
adding a first dilution of the composition to a first sample of dorsomorphin-treated cells; and
adding a second dilution of the composition to a second sample of dorsomorphin-treated cells.
14 . The method of claim 9 , wherein the method comprises determining an NAb titer within 72 hours of infecting the cell with AAV.
15 . The method of claim 10 , wherein the method comprises measuring the level of the reporter gene expressed by the cell.
16 . The method of claim 10 , wherein the reporter gene comprises luciferase.
17 . The method of claim 15 , wherein adding a composition to the dorsomorphin-treated cell comprises:
providing a plurality of samples of dorsomorphin-treated cells;
adding a plurality of serial dilutions of the composition across the plurality of samples, one serial dilution per sample; and
determining the serial dilution at which 50% or greater inhibition of the reporter gene is observed.