Adeno Associated Virus 9-Based Gene Therapy Delivers a Functional Monocarboxylate Transporter 8, Improving Thyroid Hormone Availability to the Brain of Mct8-Deficient Mice

Abstract

Background: MCT8 gene mutations produce thyroid hormone (TH) deficiency in the brain, causing severe neuropsychomotor abnormalities not correctable by treatment with TH. This proof-of-concept study examined whether transfer of human MCT8 (hMCT8) cDNA using adeno-associated virus 9 (AAV9) could correct the brain defects of Mct8 knockout mice (Mct8KO).

Methods: AAV9 vectors delivering long and/or short hMCT8 protein isoforms or an empty vector were injected intravenously (IV) and/or intracerebroventricularly (ICV) into postnatal day 1 Mct8KO and wild type (Wt) mice. Triiodothyronine (T3) was given daily for four days before postnatal day 28, at which time brains were collected after perfusion to assess increase in T3 content and effect on the T3-responsive transcription factor, Hairless.

Results: Increased pup mortality was observed after IV injection of the AAV9-long hMCT8 isoform, but not after injection of AAV9-short hMCT8 isoform. Compared to IV, ICV delivery produced more hMCT8 mRNA and protein relative to the viral dose, which was present in various brain regions and localized to the cell membranes. Despite production of abundant hMCT8 mRNA and protein with ICV delivery, only IV delivered AAV9-hMCT8 targeted the choroid plexus and significantly increased brain T3 content and expression of Hairless.

Conclusions: These results indicate that MCT8 delivery to brain barriers by IV but not ICV injection is crucial for its proper function. MCT8 has no constitutive activity but acts through an increase in T3 entering the brain tissue. Increasing MCT8 expression in brain cell membranes, including neurons, is insufficient to produce an effect without an increase in brain T3 content. The correct hMCT8 isoform along with an optimized delivery method are critical for an effective gene therapy to provide functional MCT8 in the brain of patients with MCT8 mutations.

FIG. 1.

Survival of mice up to nine days after administration of AAV9-ShMCT8 and AAV9-LhMCT8 at postnatal day 1 (P1). (A) Between 85% and 92% of the pups given AAV9-ShMCT8 survived, irrespective of viral dose or route of administration. This was not significantly different from survival after injection of empty vector (EV). (B) Survival of mice injected with AAV9-LhMCT8 decreased with increased viral dose. Only 9% of the mice injected intravenously (IV) with a high dose were alive at P10. For IV injections, low, middle, and high doses were 8 × 10¹⁰, 2 × 10¹¹, and 4 × 10¹¹ viral particles (vp)/mouse, respectively. For mice injected intracerebroventricularly (ICV), low and high doses were 5 × 10⁹ and 3 × 10¹⁰ vp/mouse, respectively

FIG. 2.

hMCT8 mRNA and protein in cerebrum of mice injected with AAV9-ShMCT8 and AAV9-LhMCT8. (A) The MCT8 cDNA contained in the AAV9 transcribed in a dose-dependent manner. Note that the ICV injection induced 10–40 times more mRNA than IV injection did using doses one order of magnitude lower than those used for IV injections. Doses were as follows: for IV administration, low = 8 × 10¹⁰, middle = 2 × 10¹¹, and high = 4 × 10¹¹ vp per mouse; for ICV administration, low = 5 × 10⁹ and high = 3 × 10¹⁰ vp/mouse. Results are given as mean ± standard error of the mean (SEM). Number (#) of animals per group is indicated. (B) Western blot analysis after SDS-PAGE of cerebrum homogenates developed with an antibody against hMCT8. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is used as a loading control. In agreement with the mRNA data, vectors injected ICV induced more protein than those injected IV did (lanes 5 and 6 compared with lanes 3 and 4). The expected molecular weight of hMCT8 is 60 kDa. ShMCT8 produced a monomer as well as a dimer (lanes 3, 5, and 6). In contrast, LhMCH8 produced predominantly a monomer (lanes 7 and 8). (C) Quantitative analysis of the MCT8 in the bands shown in (B), corrected for the GAPDH loading control. Note that the amount of MCT8 in the brain of the Wt mouse given EV was not calculated because of the unknown level of cross-reactivity.

FIG. 3.

Localization of the expressed hMCT8 protein in brains of Mct8KO mice given high doses of AAV9-ShMCT8 IV (4 × 10¹¹ vp/mouse) and ICV (3 × 10¹⁰ vp/mouse) compared with Wt and KO animals injected IV with EV. Representative images showing hMCT8 expression (in brown) detected with a specific antibody by immunohistochemistry counterstained with hematoxylin in the somatosensory region of the cerebral cortex (A, D, G, J), the cerebellar lobule 4 (B, E, H, K), and choroid plexus (C, F, I, L) of Wt (A–C) and Mct8KO (D–F) mice injected with EV. Mct8KO mice were injected with AAV9-ShMCT8 IV (G–I) or ICV (J–L). In accordance with results from Western blots, hMCT8 was present in larger quantities in the cerebral cortex of mice injected with the virus ICV (J) than IV (G). However, much of the immunoreactivity was present in aggregates (white arrowheads). Some Purkinje cells (white arrows) were observed in both IV- and ICV-injected cerebellum (H and K) with remarkable hMCT8 expression at the dendritic arborizations (black arrowheads). The control, Wt mice injected with EV, did not show the positive signal of dendritic arborizations (B). hMCT8 was abundantly present at the choroid plexus of IV-injected KO mice (I) and only spottily expressed in mice given the virus ICV (L). The scale bar for each brain region is in the lower right corner of the left photograph and equals 50 μm. Color images available online at www.liebertpub.com/thy

FIG. 4.

Membrane localization of hMCT8 demonstrated by confocal microscopy in Mct8KO mice injected with AAV9-ShMCT8 IV and ICV (for dose, see legend to Fig. 3). Representative images showing immunopositive signal for hMCT8 (in red) and nuclei stained with DAPI (in blue) in the choroid plexus (A, B), cerebral cortex (C, D) and CA1 region of the hippocampus (E, F) of Mct8KO mice injected with AAV9-ShMCT8 IV (A, C, E) or ICV (B, D, F). hMCT8 was neatly localized at the apical membrane of the choroid plexus of IV-injected mice (A). A similar localization was observed in only few cells of ICV injected animals (B). hMCT8 was localized in the membranes (see white arrows) of cortical and hippocampal neurons of animals injected either IV or ICV (C–F). However, a greater amount of hMCT8 was present in the cytoplasm of animals injected ICV. (A, B) Scale bar = 20 μm; (C–F) scale bar = 10 μm. Color images available online at www.liebertpub.com/thy

FIG. 5.

Effect of AAV9-ShMCT8 on triiodothyronine (T3) content and induction of Hairless (Hr) expression in the cerebrum of mice injected with the virus IV, ICV, and combined IV + ICV. (A) Cerebrum T3 content. (B) Hr gene expression. Relative to Mct8KO mice injected with EV, a significant increase in brain T3 content (A) and Hr gene expression (B) was observed only in mice injected with the virus IV. Middle and high doses of AAV9-ShMCT8 injected IV were 2 × 10¹¹ and 4 × 10¹¹ vp/mouse, respectively. For ICV injections, low and high dose were 5 × 10⁹ and 3 × 10¹⁰ vp/mouse, respectively. The combined IV + ICV injections contained the high doses of virus. Data from EV given IV and ICV were combined in the Wt and Mct8KO mice, as the EV did not produce any effect compared with non-injected animals of both genotypes. Data are presented as mean ± SEM with the number (#) of animals per group as indicated. Results from Wt animals given EV are adjusted to 100% in Hr expression. Statistical differences for Mct8KO animals given ShMCT8 and compared to the same genotype given EV are indicated above the bars. *p < 0.05; **p < 0.01; ***p < 0.001.