US 12,129,476 B2
Methods for measuring the potency of AADC viral vectors
Lauren M. Drouin, Cambridge, MA (US); Patrick Starremans, Cambridge, MA (US); Eric D. Horowitz, Norwell, MA (US); Carlo Ciatto, Brookline, MA (US); and Joseph Mombeleur, Bridgewater, MA (US)
Assigned to Voyager Therapeutics, Inc., Cambridge, MA (US)
Appl. No. 17/050,703
Filed by VOYAGER THERAPEUTICS, INC., Cambridge, MA (US)
PCT Filed Apr. 26, 2019, PCT No. PCT/US2019/029297
§ 371(c)(1), (2) Date Oct. 26, 2020,
PCT Pub. No. WO2019/210137, PCT Pub. Date Oct. 31, 2019.
Claims priority of provisional application 62/663,958, filed on Apr. 27, 2018.
Claims priority of provisional application 62/703,590, filed on Jul. 26, 2018.
Claims priority of provisional application 62/741,463, filed on Oct. 4, 2018.
Prior Publication US 2021/0238630 A1, Aug. 5, 2021
Int. Cl. C12N 15/86 (2006.01); A61K 48/00 (2006.01); G01N 30/02 (2006.01)
CPC C12N 15/86 (2013.01) [A61K 48/005 (2013.01); C12N 2750/14143 (2013.01); G01N 2030/027 (2013.01)] 20 Claims
 
1. A method for measuring aromatic L-amino acid decarboxylase (AADC) viral vector potency, comprising:
(a) providing an adeno-associated virus (AAV) formulation comprising an AADC viral vector, wherein the AADC viral vector is an AAV that comprises a polynucleotide encoding one or more AADC polypeptides;
(b) contacting the AAV formulation with a cell under conditions that result in the cell producing one or more AADC polypeptides from the polynucleotide;
(c) lysing the cell to form a first cell lysate comprising at least a portion of the AADC polypeptides;
(d) determining the potency of the AADC viral vector by:
(i) adding L-3,4 dihydroxyphenylalanine (L-DOPA) to the first cell lysate under conditions that allow at least a portion of the AADC polypeptides in the first cell lysate to react with at least a portion of the L-DOPA to produce dopamine;
(ii) measuring the amount of dopamine in the first cell lysate after the reaction between the AADC polypeptides and the L-DOPA;
(iii) providing an AADC viral vector reference standard for dopamine production, wherein the AADC viral vector reference standard comprises a polynucleotide encoding one or more AADC polypeptides, and wherein the amount of dopamine produced by the AADC viral vector reference standard is determined by:
adding L-DOPA to a second cell lysate that comprises at least a portion of the AADC polypeptides, wherein the AADC polypeptides in the second cell lysate are produced by cells transduced with the AADC viral vector reference standard; and
measuring the amount of dopamine produced in the second cell lysate after the reaction between the AADC polypeptides and the L-DOPA; and
(iv) comparing the amount of dopamine produced using the AADC viral vector to the AADC viral vector reference standard, such that an AADC Relative Potency of the AADC viral vector is measured.