AAV vector and assay for anti-AAV (adeno-associated virus) neutralizing antibodies

A method for quantifying neutralizing antibodies that bind to AAV, comprising:

• (a) providing infectious recombinant AAV particles that encapsidate a recombinant vector, wherein (i) the vector comprises a reporter transgene, wherein the reporter transgene encodes a protein that provides an enzymatic, colorimetric, fluorescent, luminescent, chemiluminescent, or electrochemical signal, (ii) the reporter transgene comprises a self-complementary genome, and (iii) the reporter transgene is operably linked to one or more expression regulatory elements and flanked by one or more flanking elements;
• (b) providing a biological sample from a subject for quantifying neutralizing antibodies that bind to AAV;
• (c) providing human embryonic kidney (HEK) cells with a passage number up to 25 passages after initial thawing;
• (d) contacting or incubating the infectious recombinant AAV particles of (a) with the biological sample of (b) thereby producing a resulting mixture (M);
• (e) contacting the mammalian cells of (c) with resulting mixture (M) under conditions in which the infectious recombinant AAV particles of (a) can infect and express the reporter transgene in said cells;
• (f) measuring expression of the reporter transgene;
• and
• (g) comparing said reporter transgene expression of (f) to reporter transgene of a negative (−) control lacking neutralizing antibodies that bind to AAV.