Relative potency assay for viral vector encoding isomerohydrolases

A method for measuring isomerohydrolase activity, wherein the method is linear from about 1×104 to about 2×106 AAV vector genomes per cell, comprising:

• (a) contacting cells expressing Lecithin Retinol Acyltransferase (LRAT) with an adeno-associated viral (AAV) vector genomes comprising a transgene encoding an isomerohydrolase protein under conditions allowing cell transduction;
• (b) incubating transduced cells under conditions allowing expression of the encoded isomerohydrolase protein;
• (c) collecting and lysing the transduced cells to produce an extract comprising the encoded isomerohydrolase protein;
• (d) incubating said extract with a substrate for a period of time and under conditions allowing conversion of the substrate by the isomerohydrolase protein to a 11-cis-retinol reaction product;
• (e) subjecting said 11-cis-retinol reaction product to column chromatography thereby producing a column chromatography purified 11-cis-retinol reaction product;
• and
• (f) subjecting said column chromatography purified 11-cis-retinol reaction product to mass spectrometry thereby quantifying the 11-cis-retinol reaction product, wherein the amount of the reaction product reflects isomerohydrolase activity thereby measuring isomerohydrolase activity, wherein the method is linear from about 1×104 to about 2×106 AAV vector genomes per cell.