Expression levels of VP3 is thought to be controlled by the Kozak sequence where A in the -3 position and G in the +4 position, assuming A in the initiation codon AUG is +1, is considered to be the optimal and heavily favoured sequence . For AAV6 and AAV8 respectively, the ATG initiation codon at M203 and M204 has this optimal sequence while the second ATG initiation codon at M211 and M212, respectively, has C in the -3 position and G in the +4 position, resulting in the population of (Ac)VP3 being significantly greater than the population of the (Ac)VP3 Variant, as observed in this study and others (Table ) (24). AAV9 does not contain an M211 amino acid as it does not contain the second ATG initiation codon in its DNA sequence at that position, explaining why no (Ac)VP3 Variant is detected in the AAV9 samples.