SUPPLEMENTARY RESULTS
We confirmed that androgen-induced expression also translated to the AAV8-mediated expression
of α-gal by implanting male and female mice with 21-day slow release dihydrotestosterone (DHT)
pellets 14 days prior to vector administration. Four weeks after vector administration, male
expression levels were essentially unaffected by the added DHT, while α-gal expression from
female mice was increased to male levels (Supplementary Figure S2a). However, unlike the
situation for AAV2-FIX (10), similar results were seen when DHT was given the same day as
vector administration, or even 14 days after vector administration (data not shown).
As another test of the androgen dependence of α-gal expression, male mice were surgically
castrated 4 weeks after vector administration. Supplementary Figure S2b shows that in these
castrated mice α-gal expression quickly dropped to female levels (<100 ng/ml). Eight weeks later a
21-day DHT pellet was implanted. Expression rapidly returned to near pre-castration levels before
decreasing with pellet exhaustion. (DHT levels were quantified and had returned to background in
these animals by 21 days after pellet implantation (data not shown)). These results suggest that in
male mice, liver-based expression from the AAV8-αgal vector requires the ongoing presence of
DHT.
In female mice, we similarly noted that after DHT administration expression levels were not
stable, but rather increased for a few weeks after DHT pellet exhaustion and then fell.
Supplementary Figure S2c shows the kinetics of this phenomenon, which indicates an effect of
DHT on expression that is prolonged relative to its actual presence. When α-gal serum levels
reached a nadir, we administered a second DHT pellet, and once again saw an immediate increase
in expression that persisted for about 3 weeks beyond pellet exhaustion before declining. Analysis
of vector copy number in the liver of both male and female mice throughout the experimental time
course showed that vector copies remained relatively constant throughout, stabilizing after an initial
decline in the first week (data not shown). As in the male mice, these results suggest that continued
high expression in female mice is dependent on the ongoing presence of DHT.