Preexisting immunity and low expression in primates highlight translational challenges for liver-directed AAV8-mediated gene therapy

Abstract

Liver-directed gene therapy with adeno-associated virus (AAV) vectors effectively treats mouse models of lysosomal storage diseases (LSDs). We asked whether these results were likely to translate to patients. To understand to what extent preexisting anti-AAV8 antibodies could impede AAV8-mediated liver transduction in primates, commonly preexposed to AAV, we quantified the effects of preexisting antibodies on liver transduction and subsequent transgene expression in mouse and nonhuman primate (NHP) models. Using the highest viral dose previously reported in a clinical trial, passive transfer of NHP sera containing relatively low anti-AAV8 titers into mice blocked liver transduction, which could be partially overcome by increasing vector dose tenfold. Based on this and a survey of anti-AAV8 titers in 112 humans, we predict that high-dose systemic gene therapy would successfully transduce liver in >50% of human patients. However, although high-dose AAV8 administration to mice and monkeys with equivalent anti-AAV8 titers led to comparable liver vector copy numbers, the resulting transgene expression in primates was ~1.5-logs lower than mice. This suggests vector fate differs in these species and that strategies focused solely on overcoming preexisting vector-specific antibodies may be insufficient to achieve clinically meaningful expression levels of LSD genes using a liver-directed gene therapy approach in patients.

Figure 1

Primary mouse and primate hepatocytes show equivalent αgal expression and secretion in response to vector treatment. Mouse and cynomolgus macaque primary hepatocytes (light gray and black bars, respectively) were transduced with Ad2, AAV8, or AAV2 vectors carrying the DC190-αgal cassette at a multiplicity of infection of 75 for Ad2, and 1,000 for AAV2 and AAV8. Untreated primary hepatocytes of both species were included as controls. For each treatment N = 3 and error bars indicate the SEM. P values were determined by unpaired two-tailed Student's t-test. (a) Vector copy numbers per cell in mouse and primate primary hepatocytes are shown 48 hours after treatment with vector. All vector-treated samples differed significantly in vector copies per cell relative to untreated controls with a P value ≤1 × 10⁻⁶. # indicates a statistically significant difference in vector copies per cell between mouse and cynomolgus macaque primary hepatocytes with a P value ≤0.05 by unpaired two-tailed t-test; ‡ indicates a P value of ≤0.001. (b) 18S-normalized mRNA levels of the αgal transgene at 48 hours post-treatment are shown (arbitrary units). All vector-treated samples differed significantly in transgene-specific mRNA relative to untreated controls with a P value ≤0.05. ‡ indicates a statistically significant difference in transgene mRNA between mouse and cynomolgus macaque primary hepatocytes with a P value of 0.008. (c) Levels of secreted αgal in cell culture media 48 hours post-treatment. All Ad2-αgal treated samples differed significantly from untreated controls in levels of secreted αgal with a P value ≤0.001. Apart from detectable secreted αgal in the media of mouse primary hepatocyte cultures treated with AAV8-αgal, neither AAV8 nor AAV2-αgal treatment resulted in secreted αgal levels significantly above that of untreated controls. # indicates a statistically significant difference in secreted αgal between mouse and cynomolgus macaque primary hepatocytes treated with Ad2-αgal with a P value 0.03. (d) Vector-normalized αgal transgene expression indicates DC190 promoter efficiency in mouse and primate primary hepatocytes. Transgene-specific mRNA levels at 48 hours post-treatment are shown after normalization to vector copies per cell. # indicates a statistically significant difference in vector-normalized transgene mRNA between mouse and cynomolgus macaque primary hepatocytes treated with AAV2-αgal with a P value 0.02. (e) For Ad2-αgal treatment (the only group with secreted αgal levels at 48 hours above background for both mouse and primate samples) αgal levels are shown after normalization to transgene-specific mRNA levels. No statistically significant difference was observed between mouse and primate groups suggesting equivalent αgal translation and secretion efficiency in mouse and primate primary hepatocytes. (f) Secreted αgal levels after normalization to vector copies per cell are shown 48 hours after treatment with Ad2-αgal. No statistically significant difference was observed between mouse and primate groups. AAV, adeno-associated virus; Ad2, adenovirus-2; αgal, α-galactosidase A.

Figure 2

Sustained serum αgal levels are substantially lower in primates than in mice. Serum human αgal levels are shown in mice for 16 weeks after treatment with AAV8-αgal at 5 × 10¹² drp/kg (light gray diamonds, N = 10 to week 11 and N = 5 thereafter) and rhesus macaques (R-6 to R-8, Table 1) for 17 weeks after treatment with the same vector at four times the dose (2 × 10¹³ drp/kg, black squares, N = 3). Error bars indicate the SEM. After expression stabilization (after 14 days), average serum αgal levels were ~260 ng/ml in mice and ~20 ng/ml in macaques. AAV, adeno-associated virus; αgal, α-galactosidase A.

Figure 3

Passive transfer of low anti-AAV8 antibody titer nonhuman primate (NHP) serum reduces mouse liver transduction by AAV8-αgal. (a) Passive transfer of naive mouse serum or diluted low-titer NHP serum has no effect on expression levels; high-titer NHP serum abrogates expression. Mice were pretreated with naive mouse serum, serum from five different NHPs (R-23 to R-27, Supplementary Table S1) to an anti-AAV8 total antibody titer in the mouse of ≤10, or serum from an NHP with a high anti-AAV8 total titer (R-28, Supplementary Table S1) to an in vivo titer of 10,240, followed 1 hour later by intravenous administration of the AAV8-αgal vector at 2 × 10¹² drp/kg or saline vehicle without vector (Untreated). Mean serum αgal levels 1 week post-treatment are shown. For each group N = 4 and error bars indicate SEM. Compared to mice pretreated with naive mouse serum, no statistically significant reduction in serum αgal levels was observed for mice pretreated with low-titer NHP serum to in vivo anti-AAV8 total titers of ≤10. However, for mice pretreated with high-titer NHP serum to an in vivo titer of 10,240, ‡ indicates a statistically significant reduction in serum αgal relative to controls pretreated with naive mouse serum (P value ≤0.001 by unpaired two-tailed t-test). In b and c, high-titer inhibitory NHP serum (R-28, Supplementary Table S1) was serially diluted into NHP serum with no detectable anti-AAV8 titer (R-23, Supplementary Table S1) and passively transferred into mice to obtain a range of in vivo anti-AAV8 total antibody titers. Pretreatment with sera or a saline control was followed 1 hour later by intravenous administration of AAV8-αgal at 2 × 10¹² drp/kg or saline vehicle as indicated. Error bars indicate SEM and 4–5 animals were included in each group. (b) Pretreatment to in vivo anti-AAV8 titers of ≥160 blocks gene transfer after low-dose AAV8-αgal. Mean serum αgal levels are shown 1 (checked light gray bars) and 2 weeks (solid light gray bars) after vector administration. # indicates a statistically significant difference in serum αgal levels at that time point relative to mice administered vector after saline pretreatment with a P value ≤0.05 by unpaired two-tailed t-test; ‡ indicates a P value of ≤0.001. (c) Decreased serum αgal correlates with reduced vector copies per liver cell. Mean vector copies per liver cell are shown 2 weeks after treatment with vehicle (white bar) or the AAV8-αgal vector at 2 × 10¹² drp/kg (light gray bars). No vector was detected in control animals treated with vehicle alone (arbitrarily assigned a vector copy per cell value of 0.0001 for log-scale display). # indicates a statistically significant difference in vector copies/cell relative to mice administered vector after saline pretreatment with a P value ≤0.05 by unpaired two-tailed t-test; ‡ indicates a P value of ≤0.001. αgal, α-galactosidase A.

Figure 4

Increasing vector dose tenfold partially overcomes preexisiting antibodies to AAV8. To assess the potential of higher vector dose to overwhelm existing anti-AAV8 antibodies, 2 × 10¹³ drp/kg (high dose) AAV8-αgal was administered to mice pretreated with saline or serially diluted inhibitory nonhuman primate (NHP) sera to obtain a range of in vivo anti-AAV8 total antibody titers by passive transfer as shown. Mice injected with vector-free vehicle without pretreatment (Untreated) were included for comparison. To ensure inhibitory activity was comparable to Figure 3, additional control groups were treated with vector at 2 × 10¹² drp/kg after pretreatment with inhibitory NHP serum, diluted in either saline or naive NHP serum, to the minimum in vivo inhibitory titer of 40 as determined for that dose in Figure 3b. Error bars indicate SEM and 4–5 animals were included in each group. P values were determined by unpaired two-tailed Student's t-test. (a) Mean serum αgal levels are shown at a vector dose of 2 × 10¹² (gray bars) and 2 × 10¹³ (black bars) at 1 (striped bars) and 2 weeks (solid bars) after administration. For low-dose controls pretreated to a titer of 40, serum αgal levels are similar to those of Figure 3b. # indicates a statistically significant difference in serum αgal levels at that time point relative to mice administered 2 × 10¹³ drp/kg vector after saline pretreatment with a P value ≤0.05 by unpaired two-tailed t-test; ‡ indicates a P value of ≤0.001. (b) Mean vector copies per cell are shown 2 weeks after treatment with saline only (white bar) or the AAV8-αgal vector at a dose of 2 × 10¹² drp/kg (gray bars) or 2 × 10¹³ drp/kg (black bars) after passive transfer of inhibitory serum to the in vivo titer indicated. No vector was detected in vehicle-treated control animals, which were arbitrarily assigned a vector copy per cell value of 0.0001 for log-scale display. Roughly 25 vector copies/cell were detected in animals dosed with vector at 2 × 10¹³ drp/kg after pretreatment with saline or permissive NHP serum without detectable anti-AAV8 antibodies (R-23, Supplementary Table S1). † indicates a statistically significant difference in vector copies/cell relative to mice administered 2 × 10¹³ drp/kg vector after saline pretreatment with a P value ≤0.01; ‡ indicates a P value of ≤0.001. No statistically significant difference in vector copies/cell was seen for controls treated with low-dose vector (2 × 10¹² drp/kg) after pretreatment with NHP serum to an in vivo anti-AAV8 titer of 40 (gray bars) compared to the similarly treated groups in Figure 3c. (c) Mean vector copies per cell 2 weeks after administration the AAV8-αgal vector at a drp/kg dose of 2 × 10¹² (open diamonds with dashed connecting line) or 2 × 10¹³ (filled squares with solid connecting line) are shown as a function of the anti-AAV8 total titer in the mouse passive transfer model (Figures 3 and 4). In c and d, saline pretreatment was arbitrarily assigned an anti-AAV8 titer of 0.1, and permissive NHP serum, without detectable anti-AAV8 antibodies, a titer of 1 for the purpose of log-scale display. (d) Mean serum αgal levels in Figures 3 and 4 are shown as a function of in vivo anti-AAV8 total antibody titers in the mouse passive transfer model. For the purposes of log-scale display, αgal levels below the limit of detection were arbitrarily assigned a value of 0.1 ng/ml. Squares with solid connecting lines show serum αgal levels at 1 (open squares) and 2 weeks (filled squares) after treatment with a vector dose of 2 × 10¹³ drp/kg. Diamonds with dashed connecting lines show serum αgal levels at 1 (open diamonds) and 2 weeks (filled diamonds) after treatment with a vector dose of 2 × 10¹² drp/kg at the indicated titer. AAV, adeno-associated virus; αgal, α-galactosidase A.

Figure 5

Serum αgal levels correlate with AAV8 vector copy number per cell in mice but not in nonhuman primates (NHPs). Mean serum αgal levels as a function of vector copies per liver cell are compared between the mouse passive transfer and NHP models. In mice, αgal expression and vector copy number in Figures 3 and 4 were positively correlated (R² = 0.93). Vector copies above 0.8/cell led to detectable αgal expression, which increased linearly according to the equation y = 18.0x + 11.2. In NHPs (Table 1), however, no correlation was observed. Linear trend lines are shown for mouse (solid line) and NHP (dashed line) data. Error bars indicate SEM. AAV, adeno-associated virus; αgal, α-galactosidase A.

Figure 6

Anti-AAV8 titers in human subjects. Anti-AAV8 total antibody titers were determined for 112 human volunteers. Percent of individuals with each titer are shown. Extrapolating from the passive transfer model, a vector dose of 2 × 10¹³ drp/kg would be predicted to overcome preexisting antibodies to AAV8, allowing systemic vector-mediated transgene delivery to the liver at >5 vector copies/cell in humans with titers ≤640 (white bars), or ~52% of individuals. An anti-AAV8 titer of 800 (gray bar) would be predicted to be borderline permissive to liver transduction at high vector dose (2 × 10¹³). Individuals with titers >800 (black bars) would be predicted to be nonpermissive to liver transduction (even at high dose) based both on the passive transfer and NHP data (Table 1). AAV, adeno-associated virus.

Figure 7

Anti-AAV8 total titers are a more sensitive indicator of prior virus exposure in humans than neutralizing titers. (a) Total versus neutralizing anti-AAV8 antibody titers in human subjects. Neutralizing antibody and total anti-AAV8 antibody titers were measured in 70 human subjects (Supplementary Table S3). Although all 70 had measurable anti-AAV8 total antibody titers, only 33 had a detectable neutralizing titer (titer ≥25). In individuals where both total and neutralizing titers could be determined, total titer and neutralizing titer correlated linearly (dashed line) with an R² value of 0.70, as shown. Limits of detection for total and neutralizing antibody assays are indicated by dashed lines. (b) Distribution of anti-AAV8 total antibody titers in 70 human subjects for which neutralizing titers were also determined. The distribution of anti-AAV8 total antibody titers is shown for subjects with neutralizing antibody titers above (≥25, black bars) and below (<25, gray bars) the limit of detection for the neutralizing titer assay. AAV, adeno-associated virus.