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. 2021 Jul 16:22:279-292.
doi: 10.1016/j.omtm.2021.06.015. eCollection 2021 Sep 10.

ImmTOR nanoparticles enhance AAV transgene expression after initial and repeat dosing in a mouse model of methylmalonic acidemia

Petr O Ilyinskii  1 , Alicia M Michaud  1 , Gina L Rizzo  1 , Christopher J Roy  1 , Sheldon S Leung  1 , Stephanie L Elkins  1 , Teresa Capela  1 , Aparajita Chowdhury  1 , Lina Li  2 , Randy J Chandler  2 , Irini Manoli  2 , Eva Andres-Mateos  3 , Lloyd P M Johnston  1 , Luk H Vandenberghe  3 , Charles P Venditti  2 , Takashi Kei Kishimoto  1
Affiliations

ImmTOR nanoparticles enhance AAV transgene expression after initial and repeat dosing in a mouse model of methylmalonic acidemia

Petr O Ilyinskii et al. Mol Ther Methods Clin Dev. .

Abstract

A major barrier to adeno-associated virus (AAV) gene therapy is the inability to re-dose patients due to formation of vector-induced neutralizing antibodies (Nabs). Tolerogenic nanoparticles encapsulating rapamycin (ImmTOR) provide long-term and specific suppression of adaptive immune responses, allowing for vector re-dosing. Moreover, co-administration of hepatotropic AAV vectors and ImmTOR leads to an increase of transgene expression even after the first dose. ImmTOR and AAV Anc80 encoding the methylmalonyl-coenzyme A (CoA) mutase (MMUT) combination was tested in a mouse model of methylmalonic acidemia, a disease caused by mutations in the MMUT gene. Repeated co-administration of Anc80 and ImmTOR was well tolerated and led to nearly complete inhibition of immunoglobulin (Ig)G antibodies to the Anc80 capsid. A more profound decrease of plasma levels of the key toxic metabolite, plasma methylmalonic acid (pMMA), and disease biomarker, fibroblast growth factor 21 (FGF21), was observed after treatment with the ImmTOR and Anc80-MMUT combination. In addition, there were higher numbers of viral genomes per cell (vg/cell) and increased transgene expression when ImmTOR was co-administered with Anc80-MMUT. These effects were dose-dependent, with the higher doses of ImmTOR providing higher vg/cell and mRNA levels, and an improved biomarker response. Combining of ImmTOR and AAV can not only block the IgG response against capsid, but it also appears to potentiate transduction and enhance therapeutic transgene expression in the mouse model.

Keywords: ImmTOR rapamycin-encapsulated nanoparticles; gene therapy; immunogenicity mitigation; re-dosing.

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Conflict of interest statement

P.O.I., A.M.M., G.L.R., C.J.R., S.S.L., S.L.E., T.C., A.C., L.P.M.J., and T.K.K. are employees and shareholders of Selecta Biosciences. E.A.-M is an employee and holds stocks of Akouos, Inc. L.H.V. received consulting fees and research funding from Selecta Biosciences and holds equity in and serves on the Scientific Advisory Board of Akouos. He is an inventor of Anc80L65, licensed to biopharmaceutical companies, including Selecta Biosciences, from which he receives royalties. C.P.V. received research funding from Selecta Biosciences. R.J.C., L.L., I.M., and C.P.V. are co-inventors on patents and patent applications filed by the NIH on their behalf.

Figures

None
Graphical abstract
Figure 1
Figure 1
Weight, biomarker, and anti-vector IgG dynamics in Mmut–/–;TgINS-MCK-Mmut mice after initial treatment with Anc80-MMUT at 2.5 × 1012 vg/kg combined with ImmTOR (A) Weight gains (in % increase versus pre-injection weight) after initial treatment either with Anc80-MMUT alone or combined with 100 or 300 μg of ImmTOR. Mice were 24–28 days of age at treatment initiation (day 0, indicated by arrow in B and C). (B) Methylmalonic acid concentration in plasma after initial treatment. Relative levels (versus pre-treatment levels as 100%) are also shown for each group at every time point (levels in normal mice were <20 μM). Time points with statistically significant (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001) differences versus group not receiving ImmTOR are indicated. Number of mice per each group is shown in parentheses (six mice/group were terminated at day 30 for tissue analysis). (C) Dynamics of serum FGF21 levels after initial treatment (levels in normal mice were 207 ± 110 μM). (D) Dynamics of serum IgG antibody response to Anc80-MMUT. Anti-Anc80 IgG is presented as top OD. Averages with SD are shown. Statistical difference between the group treated with Anc80-MUT alone versus those treated with Anc80 combined with ImmTOR is shown for all time points (∗∗∗∗p < 0.0001).
Figure 2
Figure 2
AAV-driven transduction of liver cells of Mmut–/–;TgINS-MCK-Mmut mice after initial treatment with Anc80-MMUT combined with ImmTOR Efficacy of Anc80-MMUT transduction of Mmut–/–;TgINS-MCK-Mmut mouse livers with or without ImmTOR (100 or 300 μg) measured as number of viral genome copies per cell (vg/cell) on day 30 after a single vector injection (∗p < 0.05).
Figure 3
Figure 3
Weight gains in Mmut–/–;TgINS-MCK-Mmut mice after repeat treatment with Anc80-MMUT at 2.5 × 1012 vg/kg combined with ImmTOR Gains (in % increase versus pre-re-dosing weight) in mice co-treated either with 100 or 300 μg of ImmTOR are shown versus those in mice treated only with Anc80-MMUT. The number of mice in each group is shown in parentheses (two mice treated with Anc80-MMUT alone were taken from the study into breeding 30 days after re-dosing). Time points with statistically significant (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001) weight differences versus group not receiving ImmTOR are indicated (gray and black symbols stand for 100 and 300 μg of ImmTOR, respectively). Dashed line indicates the interval during which collective weights in mice co-treated with ImmTOR (100 and 300 μg) show statistical difference (p < 0.01–0.05) versus those in mice treated only with Anc80-MMUT. Averages with SD are shown.
Figure 4
Figure 4
Therapeutic benefit of repeat dosing with Anc80-MMUT combined with ImmTOR in Mmut–/–;TgINS-MCK-Mmut mice (A and B) Dynamics of absolute (A) (in μM) and relative (B) (versus those prior to initial day 0 treatment as 100%) plasma levels of methylmalonic acid before and after therapeutic Anc80-MMUT re-dosing on day 56 (indicated by arrow) with statistical significance indicated (∗p < 0.05, ∗∗p < 0.01). The number of mice per each group at each time point is shown in parentheses. (C) Dynamics of plasma FGF21 levels after repeat Anc80-MMUT injection (day 56, indicated by an arrow). Relative levels (versus pre-treatment day 0 levels as 100%) are also shown for each group at every time point with statistical significance indicated (∗p < 0.05). Averages with SD are shown in all graphs. Data for mice treated with Anc80-MMUT alone are shown in normal font, those treated with Anc80-MMUT combined with 100 μg of ImmTOR are in bold, and those treated with Anc80-MMUT combined with 300 μg of ImmTOR are in bold and italicized. (D) Long-term AAV-driven transduction of liver cells of Mmut–/–;TgINS-MCK-Mmut mice after repeat dosing with Anc80-MMUT combined with ImmTOR (100 or 300 μg) measured as vg/cell at approximately 250 days after therapeutic re-dosing (300–310 days after initial treatment) with Anc80-MMUT either alone or combined with 100 or 300 μg of ImmTOR. Statistical significance (∗p < 0.05) is shown with p values between selected groups also indicated. Averages with SD are shown in all graphs.
Figure 5
Figure 5
Therapeutic effect of repeat Anc80-MMUT dosing correlates with long-term liver cell transduction and MMUT mRNA expression Two groups of mice were treated twice (days 0 and 56, indicated by arrows) with Anc80-MMUT alone or combined with 300 μg of ImmTOR and followed up for 300 days. A single fatality (shown in A and B) occurred in the group treated with Anc80-MMUT alone on day 88 after initial inoculation. Both groups received 10 mg/mL dexamethasone concurrently with Anc80 inoculation. (A) Dynamics of pMMA levels. Relative pMMA levels (versus pre-treatment levels as 100%) are also shown for each group at every time point. Time points with statistically significant (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001) differences between the groups indicated. Number of mice per group at each time point is shown in parentheses. (B) Dynamics of plasma FGF21 levels. Time points with statistically significant (∗p < 0.05, ∗∗p < 0.01) differences between the groups are indicated. (C) Efficacy of Anc80-MMUT transduction of Mmut–/–;TgINS-MCK-Mmut mouse livers (in vg/cell) at approximately 300 days after therapeutic re-dosing with Anc80-MMUT either alone or combined with 300 μg of ImmTOR (340–360 days after initial treatment). Statistical significance (∗p < 0.05) is shown. (D) Efficacy of MMUT mRNA transcription in Mmut–/–;TgINS-MCK-Mmut mouse livers (as measured by fold increase over the baseline, GAPDH normalized) at the same time point as in (C). Statistical significance (∗∗p < 0.01) is shown. Averages with SD are shown in all graphs. (E) Inverse correlation of AAV long-term transduction efficacy and pMMA levels. Summary of two individual studies conducted using similar protocols and identical Anc80 doses and injection schedule (with or without dexamethasone). Data presented in Figures 4A, 4C, 5A, and 5C are used for analysis with no samples omitted. The p value of correlation is shown.
Figure 6
Figure 6
Long-term alleviation of hepatic pathology by Anc80-MMUT and ImmTOR Representative photomicrographs of liver sections from heterozygous MMUT+/− (A) and Anc80-MMUT-treated Mmut–/–;TgINS-MCK-Mmut mice (B–F) are shown (original magnification, ×400). Anc80-MMUT (days 0 and 56) was used alone (B) or combined with 100 μg (C) or 300 μg (D) of ImmTOR, combined with dexamethasone (E) or dexamethasone and 300 μg of ImmTOR (F). Hematoxylin and eosin staining of liver sections from 1-year-old mice are shown. Scale bars, 20 μm. In heterozygous control (A), hepatocytes (H) lack vacuoles and intracytoplasmic inclusions; pale-staining cytoplasm shows the presence of glycogen, which is within normal limits. Sinusoids containing erythrocytes (blue arrows) between hepatocyte cords and a small central vein (C) are indicated. In mice treated with Anc80-MMUT without ImmTOR (B and E), numerous hepatocytes have hyalinized with moderately intense eosinophilic pink inclusions (black arrowheads) within their cytoplasm or small round non-staining vacuoles (blue arrowhead). A sinusoid containing erythrocytes (blue arrow) is indicated in (E). Mice co-treated with Anc80-MMUT combined with low dose ImmTOR (C) possess hepatocytes that contain only rare eosinophilic inclusions and more numerous non-stained vacuoles, while in those co-treated with high-dose ImmTOR (D and F) histologic findings are minimal to absent. (G) Mean histopathology scores with statistical significance shown (∗p < 0.05, ∗∗∗p < 0.001); group size is 10–13 mice (except for mock-treated control, 1 mouse); mice receiving the same amounts of ImmTOR (0, 100, or 300 μg) in both studies are unified in a single group.
Figure 7
Figure 7
ImmTOR suppresses the induction of IgG against Anc80 vector after initial and repeat co-administrations Groups of mice (six to nine per group at all of the following time points) were injected twice with an 8-week interval with 2.5 × 1012 vg/kg of Anc80-MMUT alone or combined with ImmTOR (at 100 or 300 μg of rapamycin; see Figures 1, 2, 3, and 4) and Anc80 IgG in serum measured at times indicated and presented as EC50. Averages with SD are shown. Statistical difference between the group treated with Anc80-MUT alone versus those treated with Anc80 combined with ImmTOR is shown for all time points (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).
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