Methods for measuring the potency of AADC viral vectors
Inventors
Drouin, Lauren M. • Starremans, Patrick • Horowitz, Eric D. • Ciatto, Carlo • Mombeleur, Joseph
Assignees
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Publication Number
US-12129476-B2
Publication Date
2024-10-29
Expiration Date
Abstract
The present disclosure presents improved analytical tools, systems and methods related to AADC viral vectors, including AADC potency assays for measuring and analyzing AADC expression potency (i.e. enzymatic activity) related to AADC vectors such as adeno-associated virus (AAV) AADC vectors.
Core Innovation
The disclosure describes a method for measuring aromatic L-amino acid decarboxylase (AADC) viral vector potency for an adeno-associated virus (AAV) formulation comprising an AADC viral vector. The AADC viral vector comprises a polynucleotide encoding one or more AADC polypeptides, and the method contacts the AAV formulation with a cell under conditions that result in the cell producing one or more AADC polypeptides from the polynucleotide, then lyses the cell to form a first cell lysate containing at least a portion of the AADC polypeptides.
Potency is determined by adding L-3,4 dihydroxyphenylalanine (L-DOPA) to the first cell lysate under conditions that allow at least a portion of the AADC polypeptides to react with L-DOPA to produce dopamine, and measuring the amount of dopamine produced after the reaction. The disclosure also provides an AADC viral vector reference standard for dopamine production, comprising a polynucleotide encoding one or more AADC polypeptides produced by cells transduced with the reference standard.
The amount of dopamine produced from the AADC viral vector reference standard is determined by adding L-DOPA to a second cell lysate containing at least a portion of the AADC polypeptides produced by the cells transduced with the reference standard, then measuring dopamine after the reaction. The method compares the amount of dopamine produced using the AADC viral vector to the amount produced using the AADC viral vector reference standard, such that an AADC Relative Potency is measured.
The disclosed framework also characterizes analytical performance for dopamine measurement by UHPLC-UV and extends the assay to UHPLC-ECD, including assay qualification and robustness elements such as accuracy/precision, repeatability, LOD/LOQ, calibration ranges, specificity, linearity, chromatographic peak quality criteria, and reference standard and lysate stability.
Claims Coverage
The independent claim coverage centers on 4 core inventive features: AAV vector AADC expression in cells, L-DOPA conversion to dopamine in cell lysate, reference standard normalized dopamine production, and relative potency by comparison to the reference standard.
AAV vector AADC expression in cells
Providing an adeno-associated virus (AAV) formulation comprising an AADC viral vector that comprises a polynucleotide encoding one or more AADC polypeptides; contacting the AAV formulation with a cell under conditions that result in the cell producing one or more AADC polypeptides from the polynucleotide; lysing the cell to form a first cell lysate comprising at least a portion of the AADC polypeptides.
L-DOPA conversion to dopamine in cell lysate
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; measuring the amount of dopamine in the first cell lysate after the reaction.
Reference standard normalized dopamine production
Providing an AADC viral vector reference standard for dopamine production, wherein the reference standard comprises a polynucleotide encoding one or more AADC polypeptides; adding L-DOPA to a second cell lysate comprising at least a portion of the AADC polypeptides produced by cells transduced with the reference standard; measuring the amount of dopamine produced after the reaction.
Relative potency by comparison to reference standard
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.
Overall, the claims cover measuring AADC viral vector potency by producing AADC polypeptides in cells from an AAV formulation, converting L-DOPA to dopamine in lysates, quantifying dopamine, and determining AADC Relative Potency by comparison to an AADC viral vector reference standard.
Stated Advantages
Measures AADC viral vector potency by determining AADC Relative Potency through comparison to an AADC viral vector reference standard.
Documented Applications
Producing a gene therapy product by measuring the AADC Relative Potency, comparing it to a threshold value, and aliquoting the formulation into a container if the potency meets the criterion.
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