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. 2021 Feb 24;7(9):eabd0321.
doi: 10.1126/sciadv.abd0321. Print 2021 Feb.

Enhancement of liver-directed transgene expression at initial and repeat doses of AAV vectors admixed with ImmTOR nanoparticles

Petr O Ilyinskii  1 , Alicia M Michaud  1 , Christopher J Roy  1 , Gina L Rizzo  1 , Stephanie L Elkins  1 , Teresa Capela  1 , Aparajita C Chowdhury  1 , Sheldon S Leung  1 , Takashi K Kishimoto  2
Affiliations

Enhancement of liver-directed transgene expression at initial and repeat doses of AAV vectors admixed with ImmTOR nanoparticles

Petr O Ilyinskii et al. Sci Adv. .

Abstract

Systemic AAV (adeno-associated virus) gene therapy is a promising approach for the treatment of inborn errors of metabolism, but questions remain regarding its potency and durability. Tolerogenic ImmTOR nanoparticles encapsulating rapamycin have been shown to block the formation of neutralizing anti-capsid antibodies, thereby enabling vector re-administration. Here, we further demonstrate that ImmTOR admixed with AAV vectors also enhances hepatic transgene expression at the initial dose of AAV vector, independent of its effects on adaptive immunity. ImmTOR enhances AAV trafficking to the liver, resulting in increased hepatic vector copy numbers and transgene mRNA expression. Enhanced transgene expression occurs through a mechanism independent of the AAV receptor and cannot be replicated in vivo with free rapamycin or empty nanoparticles. The multipronged mechanism of ImmTOR action makes it an attractive candidate to enable more efficient transgene expression at first dose while simultaneously inhibiting adaptive responses against AAV to enable repeat dosing.

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Figures

Fig. 1
Fig. 1. ImmTOR suppresses induction of IgG against AAV and elevates transgene expression after initial and repeat co-administrations with AAV vector.
(A and B) C57BL/6 mice (n = 6) were injected on days 0 and 56 with AAV8-SEAP (5 × 1011 vg/kg) alone or admixed with 100 μg of ImmTOR. OD, optical density; RLU, relative luminescence unit. Anti-AAV IgG (A) and SEAP activity (B) were measured at times indicated. SEAP activity in the AAV8-SEAP + ImmTOR–treated group is shown as fold increase over the group receiving AAV8-SEAP alone (top line), and its ratio after AAV re-administration (red arrow) versus that before redosing (d46) is also shown (bottom line; untreated, nonbold; ImmTOR-treated, bold). (C) ImmTOR, but not free rapamycin or NP-Empty, enhances transgene expression after co-administration with AAV vector. BALB/c males (n = 6) were injected with AAV8-SEAP (5 × 1011 vg/kg) alone or combined with ImmTOR (200 μg), equivalent mass of NP-Empty, or empty NP combined with 200 μg of free rapamycin. SEAP activity in the ImmTOR-treated group is shown as fold increase over that group receiving AAV8-SEAP alone. AAV IgG and SEAP levels between the test groups (A and B) or between group treated with ImmTOR and all test groups (C) were different at every time point (P < 0.01, multiple t test).
Fig. 2
Fig. 2. ImmTOR admixed to AAV provides higher transduction and mRNA and protein expression but similar inhibition of IgG response.
(A to D) Groups of C57BL/6 female mice (n = 20) were injected with AAV8-SEAP (5 × 1011 vg/kg) alone or combined with 100 μg of ImmTOR, administered either as an admix or sequentially (non-admix) as shown in experimental scheme (A). Livers were harvested from five mice per group at each time point and analyzed for vector DNA (B) and SEAP mRNA (C). Sera from the same animals were analyzed for SEAP activity (D). Vector genome copies (vg per cell) and SEAP mRNA fold increase [normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] over naïve mice. (E) C57BL/6 female mice (n = 10 per group) were injected with AAV8-SEAP (5 × 1011 vg/kg) alone or combined with 100 μg of ImmTOR, either as an admix or administered sequentially. Hepatic vector DNA and SEAP mRNA and serum SEAP levels in the group receiving AAV8-SEAP admixed to ImmTOR versus the other two groups were statistically different at every time point (P < 0.01, except for the 3-day and 7-day time points for vector DNA at P < 0.05, multiple t test), as were anti-AAV IgG levels in groups treated with AAV8-SEAP combined with ImmTOR versus the group receiving AAV8-SEAP alone (P < 0.01, multiple t test).
Fig. 3
Fig. 3. Anc80 and ImmTOR admix leads to increased virion trafficking to hepatocytes, which results in emergence of a new transgene-positive cell population.
A488-labeled ImmTOR and A647-labeled Anc80 virions (A) or Cy5-labeled ImmTOR and eGFP-expressing Anc80 (B and C) were injected (intravenously, RO, retro-orbital) alone or admixed. Livers were taken at 48 hours (A) or 7 days (B and C), processed to single-cell suspension, stained with antibodies to markers indicated, and analyzed by FACS. Fractions of A647-positive (A) or GFP-positive (B and C) hepatocytes (identified as LRP-1+F4/80−CD11b−) are shown (% of total). Background fluorescence in naïve mice is shown by dotted line (A). GFP-positive hepatocytes in (C) are divided into GFP+Cy5− (red) and GFP+Cy5+ (green). Statistical difference in the size of A647-positive (A) or GFP-positive (B) hepatocyte fractions is shown (*P < 0.05, **P < 0.01, ***P < 0.001, Mann-Whitney test).
Fig. 4
Fig. 4. ImmTOR restores AAV8-Luc transgene expression after DsiRNA knockdown of AAVR in vitro and enhances AAV4 transduction in vivo.
(A) Huh-7 cells were left untreated or transfected with scrambled DsiRNA (Scr) or AAVR-specific DsiRNA and analyzed for AAVR expression at 72 hours (Western blot, quantified by densitometry). Lane 1, untreated; lane 2, Scr; lanes 3 to 5, 6 to 8, and 9 to 11, treated with 20, 10, and 1 nM AAVR DsiRNAs 1, 2, and 3, respectively. (B) Huh-7 cells were left untreated or transfected with Scr or AAVR-specific DsiRNA 3 (20 nM, 72 hours), then transduced with Anc80-luciferase alone or admixed with ImmTOR, and, 24 hours later, assayed for luciferase activity (12 to 18 replicates per group in each individual study), normalized to the average activity in controls (no DsiRNA) (****P < 0.0001, one-way ordinary ANOVA). Experiment was repeated twice with similar outcomes, and the representative result is shown. (C) Mice were injected (intravenously) with AAV4-GFP (6.25 × 1012 vg/kg) alone or admixed to ImmTOR (300 μg), and 9 days later, livers were analyzed for GFP fluorescence flow cytometry or vector genome copies (*P < 0.05, **P < 0.01, unpaired t test). Dotted green line indicates the level of background autofluorescence in naïve mice.
Fig. 5
Fig. 5. Partially neutralizing mouse anti-AAV sera can be overcome by admixing to ImmTOR.
(A) AAV8-SEAP was incubated in vitro with anti-AAV8–positive (1 and 2), normal (3 and 4), or no (5 and 6) serum, then either left untreated (1, 3, and 5) or admixed with 50 μg of ImmTOR (2, 4, and 6), and injected into groups of mice (n = 4). (B) AAV8-SEAP was admixed to 50 μg of ImmTOR (2, 4, and 6) or left untreated (1, 3, and 5), then incubated in vitro with anti-AAV8–positive (1 and 2), normal (3 and 4), or no (5 and 6) serum, and injected into groups of mice (n = 4). (C) Mice were passively immunized with 0.2 μl of anti-AAV8–positive (titer = 1:100,000) serum (1 to 3) or mock-treated (4) and then injected with AAV8-SEAP either alone (1 and 4) or with 50 μg of non-admixed (2) or admixed (3) ImmTOR. Serum SEAP was measured on day 33 (A and B) or 35 (C) and is shown as the percentage of the untreated control; readings from groups receiving ImmTOR are in bold (A and B). Experiments were repeated two to three times with similar outcomes, and representative results are shown. Statistical difference between groups admixed or not admixed to ImmTOR is shown (*P < 0.05; ns, not significant; Mann-Whitney test).
Fig. 6
Fig. 6. Partially neutralizing human anti-AAV sera can be overcome by admixing of AAV vector to ImmTOR.
(A) Correlation of neutralizing activity and anti-AAV IgG antibody levels in human donor sera. Huh-7 cells were pretreated with individual human donor sera and then transduced with AAV8-luciferase (AAV8-Luc). Luciferase activity (bars) and anti-AAV8 IgG (line) are shown on left and right y axes, respectively. (B) Mice were passively immunized with the same human sera used in (A) and then treated with AAV-SEAP alone or admixed with ImmTOR at 100 μg. Serum SEAP activity was assessed at 12 days. Serum SEAP activity is shown as a percentage of that seen in control mice treated with AAV8-SEAP alone in the absence of serum (no serum group; activity level shown by dotted line). Statistical difference in SEAP levels between otherwise identically treated groups admixed or not admixed to ImmTOR (B) is shown (*P < 0.05; ns, not significant; Mann-Whitney test).
Fig. 7
Fig. 7. ImmTOR admixed with AAV leads to elevated transgene expression and permits dose sparing and multiple repeat dosing.
(A) Anc80-SEAP was administered three times, either twice at 5 × 1011 vg/kg (days 0 and 70) followed by a third injection of 25 × 1011 vg/kg (day 167) or three times at 25 × 1011 vg/kg (days 0, 70, and 167; indicated by arrows) as shown in experimental scheme. The lower dose vector was administered either alone or admixed with ImmTOR (100 μg). (B) SEAP expression levels in groups treated with low-dose AAV + ImmTOR (bold) or with high-dose AAV alone (italics) are shown as the fold increase over the group treated with low-dose virus alone. Baseline expression levels before redosing (day 61) are shown as lines (black dotted, low-dose vector; green dashed: low-dose vector admixed with ImmTOR; blue dash-dotted, high-dose vector). Statistical difference in SEAP levels between the low-dose AAV group treated with ImmTOR and the high-dose AAV group not receiving ImmTOR is shown (*P < 0.05 and **P < 0.01; ns, not significant; multiple t test). SEAP levels in low-dose virus group not receiving ImmTOR were different from both other groups at all time points with P < 0.01. (C) Anti-Anc80 IgG levels were measured by enzyme-linked immunosorbent assay (ELISA).
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