Abstract
<jats:p>A companion paper reported that stochastic genome loading constrains the authentic single-genome fraction of recombinant adeno-associated virus (rAAV) to approximately 36.8%. This paper considers a theoretical structural constraint: occlusion of the five-fold genome-entry channels by intraluminal N-terminal extensions of VP1 and VP2, which are absent from VP3. Each capsid has twelve such channels. Consequently, any penton having one or more VP1 or VP2 subunits occludes its channel, so only all-VP3 pentons are packaging competent. Under stochastic incorporation of VP1, VP2, and VP3, as shown by mass spectrometry, the probability that a penton is composed entirely of VP3 is fVP35, and the number of open channels per capsid follows B(12, fVP35). At the canonical 1:1:10 ratio(fVP3 ≈ 0.83), approximately 40% of channels are open, and approximately 99.8% ofcapsids keep at least one. Because a single open channel suffices for genome entry,obstruction has negligible effect at the canonical ratio and does not reduce the stochastic packaging ceiling. At a matched (1:1) genome supply, obstruction instead decreases the multi-occupancy (Head-Full) fraction while increasing the empty-capsid fraction, showing that the two mechanisms partially oppose rather than compound one another. Channel obstruction becomes the dominant source of empty capsids only below a threshold nearfVP3 ≈ 0.75; at a 1:1:2 ratio, only approximately 32% of capsids are predicted to be packaging competent. These findings show that VP1:VP2:VP3 stoichiometry is a threshold-dependent critical “quality attribute” and recast a VP3-only capsid as a strategy to improve particle homogeneity, robustness, stoichiometric drift, and potency.</jats:p>