Research ArticleNIEMANN-PICK TYPE A DISEASE

Adeno-associated viral vector serotype 9–based gene therapy for Niemann-Pick disease type A

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Science Translational Medicine  21 Aug 2019:
Vol. 11, Issue 506, eaat3738
DOI: 10.1126/scitranslmed.aat3738
  • Fig. 1 CM injection of AAV9-hASM elicits broad transgene distribution in NHP brain and spinal cord with no neurological adverse effects or signs of histopathology.

    (A and B) Representative images of ASM protein expression in the cortex and cerebellum of NHP after CM delivery of 6 ml of 2.3 × 1013 VG/ml AAV9-hASM throughout CM injection at 1-month (A) and 3-month (B) survival time (n = 1 animal per group). Insets depict the intracellular lysosomal/endosomal distribution pattern of the hASM protein in the cells indicated by black arrowheads. (C) ASM protein expression in the cortex and cerebellum of control NHP not injected with AAV9-hASM. (D) Immunofluorescent colocalization of ASM and the lysosomal marker Lamp1 in Purkinje cells of the cerebellum. (E) Graphs showing the mean ± SEM of ASM-associated infrared signal per area in the indicated brain regions of noninjected NHP (white bars), 1-month survival NHP (black bars), and 3-month survival NHP (gray bars) after CM AAV9-hASM injection (n = 1 animal per group). (F) Graph showing mean ± SEM ASM-associated infrared signal per area in the thalamus of noninjected NHP (white bar), 1-month survival NHP (black bar), and 3-month survival NHP (gray bar) after CM AAV9-hASM injection (n = 1 animal per group). (G) Graph showing ASM amount in liver homogenates determined by ELISA in noninjected NHP (white bar), 1-month survival NHP (black bar), and 3-month survival NHP (gray bar) after CM AAV9-hASM injection (n = 1 animal per group). (H) Graphs showing ASM amount determined by ELISA in representative regions (cervical, thoracic, and lumbar) of the spinal cord of noninjected NHP (white bars) or in NHP 1 month (black bars) and NHP 3 months (gray bars) after CM AAV9-hASM injection (n = 1 animal per group). (I) Representative images of H&E staining in cortex and cerebellum of NHP after CM delivery of AAV9-hASM throughout CM injection at 1- and 3-month survival time and in a noninjected control NHP. (J) Representative images of Purkinje cells within the cerebellum stained against calbindin in NHP after CM delivery of AAV9-hASM at 1- and 3-month survival time and in a noninjected control NHP. Scale bars, 50 μm for (A), (B), and (C) insets; 500 μm for (A) and (B) (top left); 200 μm for (A) and (B) (bottom left and top right), (C), (I), and (J); 100 μm for (A) and (B) (bottom right); 10 μm for (D). N, nucleus; a.u., arbitrary units. In (H), the symbol ζ in the control animal denotes levels of ASM lower than the detection limit of the ELISA kit. Statistical analyses were performed using two-way ANOVA followed by Tukey’s post hoc test for multiple comparisons correction. *P < 0.05, **P < 0.01, and ***P < 0.001. N/A, not available.

  • Fig. 2 CM injection of a therapeutic dose of AAV9-hASM in ASM-KO mice results in motor improvement similar to iCb injection.

    (A) Mean ± SEM body weight gain in percentage respect to baseline (before treatment) in WT and ASM-KO mice during the 8 weeks after CM injection of 10 μl of aCSF or of AAV9-hASM at different concentrations (2.3 × 109, 2.3 × 1011, and 2.3 × 1013 VG/ml). (B) Mean ± SEM time spent on the rod in the rotarod performance test in WT and ASM-KO mice CM injected with aCSF or AAV9-hASM at the different doses (n = 4 to 9 animals per group). Two-way ANOVA followed by Bonferroni post hoc. (C) Mean ± SEM ASM activity in the CSF of WT and ASM-KO mice 8 weeks after receiving 10 μl of aCSF or of AAV9-hASM (2.3 × 1013 VG/ml) by CM injection (n = 3 to 4 animals per group). Two-way ANOVA followed by LSD post hoc. (D) Mean ± SEM body weight gain in percentage with respect to baseline (before treatment) in WT and ASM-KO mice during 8 weeks after iCb (left) or CM (right) injection of 10 μl of aCSF or of AAV9-hASM (2.3 × 1013 VG/ml). (E) Mean ± SEM time spent on the rod in the rotarod performance test in WT and ASM-KO mice after iCb or CM injection of 10 μl of aCSF or of AAV9-hASM (2.3 × 1013 VG/ml) (n = 4 to 7 animals per group). Two-way ANOVA followed by Games-Howell (iCb) and Bonferroni (CM) post hoc; *P < 0.05, **P < 0.005, and ***P < 0.001. n.s., not significant.

  • Fig. 3 iCb and CM injections of AAV9-hASM promote ASM expression and prevent molecular and cellular pathology in the cerebellum of ASM-KO mice.

    (A) Immunofluorescence images of hASM expression in Purkinje cells (indicated by asterisks) after iCb and CM injection of AAV9-hASM. ML, molecular layer; PCL, Purkinje cell layer; and GL, granular layer. (B) Phenotypic analysis of AAV9-hASM transduced cells in the cerebellum of iCb and CM AAV9-hASM–injected ASM-KO mice. Brain sections were costained in green against hASM and in red against neuronal (MAP2), astrocytic (GFAP), or microglia (Iba1) markers. Asterisks depict examples of cells coexpressing hASM and the cell type–specific marker. (C) Western blot analysis of hASM expression in cerebellar homogenates from ASM-KO mice injected with aCSF and iCb or CM AAV9-hASM. Graph shows mean ± SEM cerebellar hASM amount normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (n = 4 per group). One-way ANOVA followed by Bonferroni post hoc. (D) Mean ± SEM SM concentration in cerebellar homogenates from WT and ASM-KO mice after receiving iCb or CM injection of aCSF or AAV9-hASM (n = 3 to 6 per group). Two-way ANOVA followed by Bonferroni (iCb) and Games-Howell (CM) post hoc. (E) Immunofluorescence staining against lysosomal Lamp1 in Purkinje cells (depicted by white dot lines) after aCSF or AAV9-hASM in iCb or CM injection in WT and ASM-KO mice. Graphs show mean ± SEM size of lysosomes in Purkinje cells after aCSF or AAV9-hASM iCb or CM injection in WT and ASM-KO mice (n = 3 to 6 per group). Two-way ANOVA followed by Bonferroni post hoc. (F) Purkinje cell density analysis by immunofluorescent staining of calbindin in the posterior lobe of the cerebellum after aCSF or AAV9-hASM iCb or CM injections in WT and ASM-KO mice. Graphs show mean ± SEM number of Purkinje cells after aCSF or AAV9-hASM iCb or CM injections in WT and ASM-KO mice (n = 3 to 6 per group). Two-way ANOVA followed by Bonferroni (iCb) and Games-Howell (CM) post hoc. DAPI (4′,6-diamidino-2-phenylindole) staining in blue identifies the cell nuclei in the different panels. Scale bars, 50 μm for (A) and (B); 10 μm for (E); and 100 μm for (F). *P < 0.05, **P < 0.005, and ***P < 0.001.

  • Fig. 4 iCb, but not CM, injection of AAV9-hASM promotes inflammation in the cerebellum of ASM-KO mice.

    (A) Analysis of cerebellar astrocytes by immunofluorescent staining of GFAP in aCSF- and AAV9-hASM iCb- or CM-injected WT and ASM-KO mice. Graphs show mean ± SEM GFAP-associated intensity in aCSF or AAV9-hASM iCb- or CM-injected WT and ASM-KO mice (n = 4 per group). Two-way ANOVA followed by Bonferroni post hoc. (B) Cerebellar microglia analysis by immunofluorescent staining of Iba1 in aCSF or AAV9-hASM iCb- or CM-injected WT and ASM-KO mice. Graphs show mean ± SEM iba1+ cell density in aCSF or AAV9-hASM iCb- or CM-injected WT and ASM-KO mice (n = 4 per group). Two-way ANOVA followed by Bonferroni post hoc. DAPI staining in blue identifies the cell nuclei in the different panels. Scale bars, 100 μm for (A) and 50 μm for (B). *P < 0.05, **P < 0.005, and ***P < 0.001.

  • Fig. 5 CM, but not iCb, injection of AAV9-hASM affects the cortex and hippocampus in ASM-KO mice.

    (A) Cortex and hippocampus from ASM-KO mice stained for hASM and the neuronal marker NeuN after CM injection of AAV9-hASM. Asterisks indicate hASM-positive cells. DAPI staining in blue identifies the cell nuclei. (B) Mean ± SEM SM concentration in cortical homogenates from WT and ASM-KO mice iCb or CM injected with aCSF or AAV9-hASM (n = 4 to 6 per group). Two-way ANOVA followed by Bonferroni post hoc. (C) Mean ± SEM SM concentration in hippocampal homogenates from WT and ASM-KO mice iCb or CM injected with aCSF or AAV9-hASM (n = 3 to 6 per group). Two-way ANOVA followed by Bonferroni post hoc. (D) Mean ± SEM time spent in the novel arm of the Y maze spontaneous alternation test in WT and ASM-KO mice iCb or CM injected with aCSF or AAV9-hASM (n = 3 to 7 per group). Two-way ANOVA followed by Bonferroni post hoc. Scale bars, 25 μm. *P < 0.05 and **P < 0.005.

  • Fig. 6 CM injection of AAV9-hASM increases ASM activity in plasma and improves liver pathology in ASM-KO mice.

    (A) Mean ± SEM SM concentration in the liver of WT and ASM-KO mice CM injected with aCSF or AAV9-hASM (n = 5 to 8 per group). Two-way ANOVA followed by Bonferroni post hoc. (B) Liver from WT and ASM-KO mice injected into CM with aCSF or AAV9-hASM stained for the specific macrophage marker F4/80. Graph shows mean ± SEM area of macrophages (n = 3 to 4 per group). (C) Fold increase in ASM activity in plasma of ASM-KO mice injected into CM with AAV9-hASM over the basal values in aCSF-injected ASM-KO mice (n = 7 per group). Mann-Whitney U test. (D) Survival curve showing the percentage of WT and ASM-KO mice, CM injected with aCSF or AAV9-hASM, at the indicated time in weeks after birth (n = 6 per group). Mantel-Cox test. Scale bars, 25 μm. *P < 0.05, **P < 0.005, and ***P < 0.001.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/506/eaat3738/DC1

    Fig. S1. ASM colocalizes with the lysosomal marker Lamp1.

    Fig. S2. AAV9-hASM CM injection does not promote immune response in brain tissue of NHP.

    Fig. S3. Expression of hASM does not differ among cerebellar lobes in AAV9-hASM iCb- and CM-injected ASM-KO mice.

    Fig. S4. Purkinje cell death is more evident in cerebellar lobes of AAV9-hASM iCb- compared to CM-injected ASM-KO mice.

    Fig. S5. Astrocytosis and microgliosis are higher in cerebellar lobes of AAV9-hASM iCb- compared to CM-injected ASM-KO mice.

    Fig. S6. AAV9-hASM iCb-injected ASM-KO mice show higher ceramide levels than AAV9-hASM iCM-injected ASM-KO mice.

    Fig. S7. ASM-KO mice noninjected and CM injected with AAV9 empty vector or aCSF show similar behavioral, cellular, and molecular pathology.

    Fig. S8. Statistical comparison of the effects of cerebellar iCb and CM injection in ASM-KO mice.

    Fig. S9. CM injection of AAV9-hASM promotes hASM expression along the rostrocaudal axis.

    Fig. S10. hASM mRNA is not detected in the liver of AAV9-hASM CM-injected ASM-KO mice by RT-PCR.

    Fig. S11. ASM activity in plasma of aCSF-injected WT is higher than in ASM-KO AAV9-hASM CM-injected mice.

    Fig. S12. CM injection results in a vast transduction of different components of the ventricular system and surrounding areas.

    Fig. S13. AAV9-hASM infects primary cortical neurons of ASM-KO mice and elicits secretion of hASM.

    Table S1. AAV9-hASM CM injection does not affect general health, appearance, and behavior of NHP as determined by monthly average of daily observations.

    Data file S1. Primary data.

  • The PDF file includes:

    • Fig. S1. ASM colocalizes with the lysosomal marker Lamp1.
    • Fig. S2. AAV9-hASM CM injection does not promote immune response in brain tissue of NHP.
    • Fig. S3. Expression of hASM does not differ among cerebellar lobes in AAV9-hASM iCb- and CM-injected ASM-KO mice.
    • Fig. S4. Purkinje cell death is more evident in cerebellar lobes of AAV9-hASM iCb- compared to CM-injected ASM-KO mice.
    • Fig. S5. Astrocytosis and microgliosis are higher in cerebellar lobes of AAV9-hASM iCb- compared to CM-injected ASM-KO mice.
    • Fig. S6. AAV9-hASM iCb-injected ASM-KO mice show higher ceramide levels than AAV9-hASM iCM-injected ASM-KO mice.
    • Fig. S7. ASM-KO mice noninjected and CM injected with AAV9 empty vector or aCSF show similar behavioral, cellular, and molecular pathology.
    • Fig. S8. Statistical comparison of the effects of cerebellar iCb and CM injection in ASM-KO mice.
    • Fig. S9. CM injection of AAV9-hASM promotes hASM expression along the rostrocaudal axis.
    • Fig. S10. hASM mRNA is not detected in the liver of AAV9-hASM CM-injected ASM-KO mice by RT-PCR.
    • Fig. S11. ASM activity in plasma of aCSF-injected WT is higher than in ASM-KO AAV9-hASM CM-injected mice.
    • Fig. S12. CM injection results in a vast transduction of different components of the ventricular system and surrounding areas.
    • Fig. S13. AAV9-hASM infects primary cortical neurons of ASM-KO mice and elicits secretion of hASM.
    • Table S1. AAV9-hASM CM injection does not affect general health, appearance, and behavior of NHP as determined by monthly average of daily observations.
    • Legend for data file S1

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    Other Supplementary Material for this manuscript includes the following:

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