Systemic administration of AAV8-α-galactosidase A induces humoral tolerance in nonhuman primates despite low hepatic expression

Abstract

In mice, liver-restricted expression of lysosomal enzymes from adeno-associated viral serotype 8 (AAV8) vectors results in reduced antibodies to the expressed proteins. To ask whether this result might translate to patients, nonhuman primates (NHPs) were injected systemically with AAV8 encoding α-galactosidase A (α-gal). As in mice, sustained expression in monkeys attenuated antibody responses to α-gal. However, this effect was not robust, and sustained α-gal levels were 1-2 logs lower than those achieved in male mice at the same vector dose. Because our mouse studies had shown that antibody levels were directly related to expression levels, several strategies were evaluated to increase expression in monkeys. Unlike mice, expression in monkeys did not respond to androgens. Local delivery to the liver, immune suppression, a self-complementary vector and pharmacologic approaches similarly failed to increase expression. While equivalent vector copies reached mouse and primate liver and there were no apparent differences in vector form, methylation or deamination, transgene expression was limited at the mRNA level in monkeys. These results suggest that compared to mice, transcription from an AAV8 vector in monkeys can be significantly reduced. They also suggest some current limits on achieving clinically useful antibody reduction and therapeutic benefit for lysosomal storage diseases using a systemic AAV8-based approach.

Figure 1

Despite the relatively low expression levels generated in primates, humoral immune tolerance to human α-galactosidase can be achieved after prolonged expression. Two juvenile male rhesus macaques (R-15, R-18) were injected intravenously with 2 × 10¹³ drp/kg AAV8-αgal; one juvenile male rhesus macaque (R-22) was a naive (non-transduced) control. (a) Serum α-galactosidase A (α-gal) and (b) anti-α-gal titers were determined over time. Day 0 levels represent background for each animal, i.e., before vector administration. All three monkeys were challenged with α-gal in complete Freund's adjuvant (CFA; arrows): R-22 at day 0, R-18 at day 84, and R-15 at day 112. Monkey R-15 was rechallenged with α-gal in incomplete Freund's adjuvant (IFA; arrows) at day 200. Three juvenile male cynomolgus macaques (C-1, C-2, C-3) were injected intravenously with 2 × 10¹³ drp/kg AAV8-αgal. (c) Serum α-gal and (d) anti-α-gal titers were determined over time. Day 0 levels represent background for each animal, i.e., before vector administration. One monkey (C-2) was challenged with α-gal in CFA at day 28, and the remaining two were challenged at day 84 (arrows).

Figure 2

Sustained α-galactosidase A (α-gal) serum levels are significantly lower in nonhuman primates (NHPs) than in mice. Juvenile male rhesus monkeys (R-9, R-10, R-11) and both male and female C57BL/6J mice were dosed intravenously with 2 × 10¹³ drp/kg AAV8-αgal. Serum samples were analyzed for α-gal over time. As expected, the sustained expression levels in male mice were significantly greater than those seen in female mice. Sustained levels in female mice were significantly greater than those seen in the juvenile male primates.

Figure 3

Expression in monkeys does not respond to androgen. Two approaches were taken to explore the potential effects of androgen on expression from male nonhuman primates (NHPs). To increase serum androgen levels a 21-day release dihydrotestosterone (DHT) pellet (25 mg) was implanted in a juvenile male rhesus macaque (R-20) 3 days before the animal received virus. Expression over time was comparable to that of a juvenile male rhesus (R-18) in the absence of added DHT. In a second approach, expression was compared from administration of vector (2 × 10¹³ drp/kg AAV8-αgal) to a juvenile (C-1) and an adult (C-3) male cynomolgus monkey.

Figure 4

Low sustained expression levels in nonhuman primates are unaffected by multiple pharmacologic and delivery strategies. Several strategies were used in an attempt to increase the long-term, sustained expression levels generated from liver-based expression in rhesus macaques. AAV8-αgal vector (2 × 10¹³ drp/kg) was delivered to juvenile male rhesus macaques that were: (i) naive to treatment (AAV8; R-9, R-10, R-11), (ii) immune suppressed and dosed either systemically (IS-Sys; R-6, R-7, R-8) or locally (IS-Local; R-1, R-2, R-3, R-4, R-5), (iii) treated with an anti-inflammatory (Anti-Infl; R-12, R-13, R-14), or (iv) treated with inhibitors of DNA silencing (Dep/Dac; R-21). Two juvenile male monkeys also received 2 × 10¹³ drp/kg of a self-complementary AAV8-αgal vector (scAAV8; R-15, R-16). Sustained expression levels (after day 15) were essentially indistinguishable for all groups. N values for each group are shown in parentheses.

Figure 5

Vector copy numbers in liver are similar for male mouse and monkey but mRNA levels are significantly lower in nonhuman primates (NHPs). (a) Adeno-associated viral (AAV) vector copies were determined by real time PCR using a primer/probe set spanning the vector-specific α-gal/BGH poly A junction. Day 3 copy numbers are essentially the same in both mice and monkey (R-17); day 28 copy numbers decline slightly but remain essentially equivalent in male mice and monkeys (R-9 to 11). (b) Vector-specific α-gal mRNA copy numbers were determined for the same mice and monkeys shown in a. mRNA copies in monkeys were 1–2 logs lower than those seen in male mice at both time points.

Figure 6

Vector methylation and deamination do not differ between mouse and primate. (a) Schematic of the AAV8-αgal vector. Shown are the locations of 29 CpG dinucleotides in three vector regions that were analyzed for methylation by quantitative bisulfite pyrosequencing. In all panels, error bars indicate SEM. (b) Percent methylation for each CpG at day 28 in nonhuman primates (NHPs) (R-9 to 13) and four male mice is compared. For specific CpGs in each region, significantly more methylation is observed in mice than NHPs. (c) Percent methylation for 27 CpG in two locations near the 5′ inverted terminal repeat (ITR) and within the hybrid intron at day 28 in mice from b is compared with four male mice and NHP R-17 at day 3, and two day 145 NHPs (C-1, C-3). At specific CpGs, significantly greater percent methylation is observed in day 28 mice than either day 3 mice or NHPs at either time point. (d) Summary of methylation for NHPs and mice across all analyzed CpG sites. In panels (e–h), portions of the (e) hybrid intron region and (f) near ITR region were analyzed for possible vector deoxycytidine (dC) deamination by pyrosequencing. Percent dC to deoxythymidine (dT) sequence conversion, indicative of dC deamination to uracil, is shown for sites meeting analysis criteria. Very low levels of dC to dT conversion consistent with possible deamination were observed in both regions without significant differences between mice and primate groups at any time point. For the (g) hybrid intron and (h) near ITR regions, the single existing CCA site of each was specifically assessed for dC to dT conversion consistent with vector deamination in animals from panels b and c. No significant difference is observed between species at either CCA site. As single vector dC signals in a single animal were analyzed, measurement error could not be determined for the day 3 NHP data in panels g and h.

Figure 7

Vector form does not differ between mouse and primate. Vector molecular form as a function of time was assessed in liver samples from mice and primates by Southern blot. Assignments of vector form are tentative as they are interpreted from molecular weights and published studies. (a) Vector form in five mice at days 3 and 28. Relative molecular weights are provided and the source animal for each sample listed. Liver DNA from a naive mouse was included as a control for probe specificity. At each time point, the molecular weight and relative prevalence of vector forms are consistent across animals. (b) Vector forms in mice at days 3 (M-130) and 28 (M-138) and NHPs at days 3 (R-17), 28 (R-9 to 14) and 145 (C-1 to 3) were compared. Vector copies per liver genome as determined for each sample by quantitative PCR are provided. C-2 was included as a control for probe specificity in nonhuman primates. In this animal, liver transduction failed due to its acquisition of high anti-AAV8 titer prior to vector administration.