AAV hamartin gene therapy in a stochastic, cerebral mouse model of tuberous sclerosis type 1

Abstract

Tuberous sclerosis complex (TSC) is a dominantly inherited disease in which most individuals are born with one defective allele encoding for either hamartin (TSC1) or tuberin (TSC2), with a somatic loss of the other allele leading to abnormal neurodevelopment and upregulation of cell growth in susceptible tissues. Ninety percent of affected individuals have brain involvement, including epilepsy, cognitive impairment, autism, and/or sleep disorders. In the stochastic, cerebral mouse model of Tsc1, loss of function of hamartin is induced in the CNS by injection of an adeno-associated virus (AAV) vector encoding Cre recombinase into the cerebral ventricles of homozygous Tsc1^flox/flox mice at birth. In the brain, Tsc1 loss leads to increased proliferation of subventricular zone cells, disrupted neuronal migration and cortical cytoarchitecture, dysmyelination, and microglia-mediated inflammation, ultimately resulting in early mortality. Systemic administration of an AAV9 vector encoding human hamartin at postnatal day 21 significantly ameliorated these abnormalities at 3 and 6 weeks post-injection and markedly extended survival in this TSC1 mouse model. This work reveals the ability of hamartin replacement therapy to reverse some of the brain abnormalities caused by its loss in different cell types and provides support for the potential use of gene replacement therapy in the treatment of TSC1 patients.

Keywords: AAV9; TSC1; brain; gene replacement; microglia activation; myelination; tuberous sclerosis complex.

Graphical abstract

Figure 1

Minimum effective dose determination and expression of Cre recombinase and human hamartin in Tsc1-deficient mice (A) Schematic representation of the experimental setup. Tsc1-floxed mouse pups received i.c.v. injections of AAV1-Cre (2 × 10¹⁰ vg) into both cerebral lateral ventricles at P0 to induce Tsc1 deletion. At P21, mice were randomly assigned to five groups for RO injections at the medial canthus with PBS (group 1, n = 18) or escalating doses of AAV9-hamartin (group 2: 1 × 10¹² vg/kg, n = 18; group 3: 5 × 10¹² vg/kg, n = 21; group 4: 1 × 10¹³ vg/kg, n = 22; group 5: 5 × 10¹³ vg/kg, n = 20). Survival was monitored up to P120, at which point animals were euthanized. (B) RT-qPCR analysis of Cre recombinase transcript levels in cortex, hippocampus, and cerebellum from WT mice (not shown), mice treated with AAV1-Cre or AAV1-Cre + AAV9-hamartin (5 × 10¹³ vg/kg, n = 3 per group). Ct values are shown (inverted y axis); lower Ct indicates higher transcript abundance. Cre expression was detected in all regions, with no significant differences between cohorts across brain regions (p = 0.1, p = 0.2, p > 0.09). Data are presented as mean ± SD. Statistical analysis were made using two-tailed unpaired Mann-Whitney U test. DL, detection level. (C) RT-qPCR detection of hamartin transcript in the same brain regions. Signal was detected exclusively in AAV9-hamartin-treated mice, confirming transgene expression (∗∗p = 0.009, ∗∗p = 0.004, ∗∗∗p = 0.0004). Data are presented as mean ± SD. Statistical comparisons were made using unpaired t test with Welch’s correction. ND, not detected. (D) Kaplan-Meier survival analysis revealed dose-dependent increases in survival. Median survival times were 46, 52, 63, 73, and >120 days, for groups 1 through 5, respectively. Statistical significance was determined using the log rank Mantel-Cox test (∗∗p = 0.0012, ∗∗∗∗p < 0.0001).

Figure 2

Widespread neuronal Cre expression induces cortical and hippocampal disorganization (A) Representative β-gal-stained coronal brain sections from AAV1-Cre-injected mice at P42 showed Cre recombinase activity in blue. Insets highlight specific ROI, including the hippocampus (left inset), cortex (left and right insets), and lateral ventricles (right inset). Scale bars, 1,000 μm and 100 μm (insets). (B) Immunofluorescent analysis of Cre recombinase (magenta) in the cortex colocalized with cell-type-specific markers: NeuN (neurons, green), GFAP (astrocytes, green), Olig2 (oligodendrocytes, green), and Iba1 (microglia, green). Arrowheads indicate colocalization. DAPI (blue) was used for nuclear staining. Scale bars, 100 μm. (C) Representative sections stained for NeuN (magenta) from WT, AAV1-Cre, and AAV9-hamartin-treated Tsc1-floxed mice at P42. Images were acquired across four anatomically matched regions: (1) cortex + hippocampus, (2) somatosensory cortex, (3) hippocampal CA1, and (4) layer 5 of the primary somatosensory cortex (S1L5). AAV1-Cre-injected mice and AA9-hamartin-treated mice displayed abnormal neuronal migration, uneven dispersion and cortical cytoarchitecture disruption (white and yellow arrowheads in 1), neuronal clusters and aggregates (white arrowheads in 2), attenuated CA1 sector of hippocampal formation (white and yellow arrowheads in 2 and 3). Dashed lines delineate cortical and hippocampal boundaries in 1, 2, and 3; S1L5 in 4. Brain images are annotated based on the Allen Mouse Brain Atlas. CA1 (cornu ammonis area 1), S1L5 (primary somatosensory cortex layer V). Scale bars, 100 μm.

Figure 3

Ki67 immunostaining shows that cell proliferation in the dorsolateral SVZ of Tsc1-deficient mice is reduced by AAV9-hamartin treatment (A) Representative sections showing Ki67 (red) and DAPI (blue) staining in the SVZ of the lateral ventricle in WT, AAV1-Cre, and AAV1-Cre + AAV9-hamartin-treated mice at P42. Arrowheads indicate Ki67-positive cells within the dorsolateral SVZ. AAV1-Cre-injected animals (ii) exhibited a marked increase in Ki67+ cells compared with WT (i), indicating elevated cell proliferation. This phenotype was attenuated in AAV1-Cre + AAV9-hamartin-treated mice (iii), suggesting rescue. Scale bars, 100 μm. (B) Quantification of Ki67+ cells in the dorsolateral SVZ (n = 3 mice per group). AAV1-Cre-injected mice showed significant increase in SVZ proliferation compared with WT, which was normalized by hamartin treatment. Brain image was adapted from the Allen Mouse Brain Atlas. Data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA with Bonferroni’s post hoc test (∗p = 0.0171, ∗p = 0.0339).

Figure 4

MRI analysis reveals AAV9-hamartin treatment restores ventricular volume and partially reduces T2-hyperintense lesion burden in Tsc1-deficient mice (A) Representative T2-weighted MRI scans showing enlarged lateral ventricles in AAV1-Cre-injected mice (middle panel) compared with WT controls (left), with normalization of ventricular volume following AAV9-hamartin treatment (right; red arrows) at P42. (B) Quantification of lateral ventricular volume (mm³) showed a significant reduction in AAV1-Cre-injected mice (n = 5) compared with WT (n = 3), and restoration to near-WT levels in AAV1-Cre + AAV9-hamartin-treated mice (n = 6). Data are log₁₀ transformed, shown as mean ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s post hoc test (∗p = 0.0348, ∗p = 0.0411). (C) Representative MRI scans highlighting increased T2-hyperintense lesions representing dysmyelination in AAV1-Cre-injected and AAV1-Cre + AAV9-hamartin-treated mice (red arrows, middle and right) compared with WT (left) at P42. (D) Quantification of mean total lesions size per mouse revealed a significant increase in AAV1-Cre-injected mice (n = 5) compared with WT (n = 3), with a trend toward reduction in AAV9-hamartin-treated animals (n = 6). Data are log₁₀ transformed, shown as mean ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s post hoc test (∗p = 0.0156, p = 0.1192).

Figure 5

AAV9-hamartin treatment rescues myelination deficits in Tsc1-deficient mice over time (A) Representative brain sections showing immunostaining for MBP (red) at P21 (i), P42 (ii), and P63 (iii). Images are shown for WT, AAV1-Cre-injected, and AAV1-Cre + AAV9-hamartin treatment groups. The thresholded MBP channel used for quantification is shown in the far-right column. Dashed lines outline MBP level in the cortex. Scale bars, 1,000 μm. (B) Quantification of percent MBP-positive area in the dorsal forebrain (cortex + corpus callosum) across treatment groups and time points (n = 3 per group). At P21 and P42, AAV1-Cre-injected mice exhibited significantly reduced MBP signal compared with WT controls (∗p = 0.03 for both time points). At P42, AAV9-hamartin treatment resulted in partial recovery that did not reach statistical significance (p = 0.19). By P63, MBP levels in treated mice were comparable with WT (p > 0.99). Data are log₁₀ transformed, shown as mean ± SD. Statistical significance was determined using two-way ANOVA with Sidak’s post hoc test. D, death. (C) Quantification of MBP-positive area in the hippocampus across treatment groups and time points (n = 3 per group). AAV1-Cre-injected mice showed significantly reduced MBP signal compared with WT at P21 and P42 (∗p = 0.02 for both). AAV9-hamartin treatment showed a trend toward recovery at P42 but did not reach significance (p = 0.14). By P63, MBP levels in treated mice remained statistically comparable with WT (p = 0.99). Data are shown as mean ± SD. Statistical significance was determined using two-way ANOVA with Sidak’s post hoc test. D, death.

Figure 6

AAV9-hamartin treatment potentially induces a shift toward an activated, reparative microglial phenotype (A) Representative brain section showing immunostaining for Iba1 (microglial marker, red) and CD68 (activation marker, green) in the brain hemi-section of AAV1-Cre-injected and AAV9-hamartin (5 × 10¹⁰ vg/kg)-treated mice at P63. The merged panel demonstrates regional overlap of CD68 with Iba1-positive microglia (white arrowheads), indicating activated microglial populations in cortex, corpus callosum, and internal capsule. Brain image was adapted from the Allen Mouse Brain Atlas. Scale bar, 1,000 μm. C, cortex; CC, corpus callosum; IC, internal capsule. (B) Quantification of CD68 integrated density in the hemisphere of WT, AAV1-Cre-injected, and AAV1-Cre + AAV9-hamartin-treated mice (n = 3 per group) at P21, P42, and P63. AAV1-Cre-injected mice displayed significantly increased CD68 expression at P21 and P42 compared with WT (∗∗p = 0.0092). AAV9-hamartin treatment partially attenuated CD68 expression at P42 (p = 0.42). At P63, both WT and AAV9-hamartin-treated mice showed increased CD68 signal compared with P42 (∗∗p = 0.001), but the difference between treated and WT animals was not statistically significant (p = 0.67). Data are log₁₀ transformed, shown as mean ± SD. Statistical significance was determined using two-way ANOVA with Sidak’s post hoc test. (C) Immunostaining in the cortex, corpus callosum, and internal capsule of WT and AAV9-hamartin-treated mice at P63 showed CD68 colocalization with different microglial markers only in the treated mice. Left panel (cortex): CD68 (green) colocalized with CD206 (red). Right panel (internal capsule): CD68 (green) colocalized with IL-10 (red). Bottom panel: CD68 (green) colocalized with ARG1 (red). Merged panels and insets highlight areas of signal overlap. Scale bars, 100 μm and 10 μm (insets). D, death.