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. 2022 Mar 16:25:170-189.
doi: 10.1016/j.omtm.2022.03.011. eCollection 2022 Jun 9.

Therapeutic advantages of combined gene/cell therapy strategies in a murine model of GM2 gangliosidosis

Davide Sala  1 , Francesca Ornaghi  1 , Francesco Morena  2 , Chiara Argentati  2 , Manuela Valsecchi  3 , Valeria Alberizzi  4 , Roberta Di Guardo  4 , Alessandra Bolino  4 , Massimo Aureli  3 , Sabata Martino  2 , Angela Gritti  1
Affiliations

Therapeutic advantages of combined gene/cell therapy strategies in a murine model of GM2 gangliosidosis

Davide Sala et al. Mol Ther Methods Clin Dev. .

Abstract

Genetic deficiency of β-N-acetylhexosaminidase (Hex) functionality leads to accumulation of GM2 ganglioside in Tay-Sachs disease and Sandhoff disease (SD), which presently lack approved therapies. Current experimental gene therapy (GT) approaches with adeno-associated viral vectors (AAVs) still pose safety and efficacy issues, supporting the search for alternative therapeutic strategies. Here we leveraged the lentiviral vector (LV)-mediated intracerebral (IC) GT platform to deliver Hex genes to the CNS and combined this strategy with bone marrow transplantation (BMT) to provide a timely, pervasive, and long-lasting source of the Hex enzyme in the CNS and periphery of SD mice. Combined therapy outperformed individual treatments in terms of lifespan extension and normalization of the neuroinflammatory/neurodegenerative phenotypes of SD mice. These benefits correlated with a time-dependent increase in Hex activity and a remarkable reduction in GM2 storage in brain tissues that single treatments failed to achieve. Our results highlight the synergic mode of action of LV-mediated IC GT and BMT, clarify the contribution of treatments to the therapeutic outcome, and inform on the realistic threshold of corrective enzymatic activity. These results have important implications for interpretation of ongoing experimental therapies and for design of more effective treatment strategies for GM2 gangliosidosis.

Keywords: CNS; GM2 gangliosidosis; Sandhoff disease; bone marrow transplantation; cell therapy; gene therapy; hexosaminidase; lentiviral vectors; lysosomal storage disorders.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Experimental plan LVs (LV.hexA+ LV.hexB) were co-injected bilaterally into the EC of neonatal mice at 2 days. Transplantation of total BM isolated from tgGFP WT donors was performed at 60 days of age after busulfan treatment (myeloablation). Untreated (UT) and/or treated SD mice (Hexb−/−) were analyzed during disease progression (30 days, 60 days, asymptomatic; 120 days, terminal stage of disease) and after treatment (120 days; 240 days, end of the study). Intracerebral (IC) gene therapy (GT), BM transplantation (BMT), and combined (IC GT + BMT) treatments were applied to SD mice, WT (Hexb+/+) and heterozygous (Het; Hexb+/−) littermates.
Figure 2
Figure 2
Synergy of treatments in increasing the lifespan and preserving the body weight of SD mice (A) Kaplan-Meier survival curves showing the percentage survival of treated and UT SD mice. SD UT, n = 93; SD IC GT = 6; SD BMT, n = 13; SD IC GT + BMT, n = 12. Data were analyzed by log rank (Mantel-Cox) test: ∗∗p < 0.005, ∗∗∗∗p < 0.0001. (B) Body weight of treated SD mice and UT littermates was monitored from 50 days of age. BMT-treated WT/Het mice were used as controls to evaluate the effect of busulfan conditioning on body weight. The higher body weight of IC GT-treated mice reflected the absence of the myeloablative regimen in this treatment group. Data represent the mean ± SEM; n = 5–19 mice/group.
Figure 3
Figure 3
Transduction of neural cells upon IC GT and brain myeloid cell reconstitution upon BMT in SD mice (A) Schematic of LV.hexA+ LV.hexBinjection and immunofluorescence (IF) confocal image showing clearance of GM2 storage and reduction in CD68+ cells in a region of SD brain tissue (box) close to the LV injection site. Top panel: GM2, red. Bottom panel: CD68, red; IBA1, blue. Nuclei are counterstained with Hoechst (white). Scale bar, 25 μm. (B and C) Percentages of donor-derived GFP+ cells in the peripheral blood (PB; (B) and bone marrow (BM; (C) of BMT- and IC GT + BMT-treated SD and WT mice evaluated 30 days (PB) and 60 days (BM) after BMT. The percentage of GFP+ cells in the BM of tgGFP mice (donors) is shown for comparison. (D) Cellular composition of the BM analyzed 60 days after transplantation into BMT- and IC GT + BMT-treated SD mice and age-matched UT controls (WT, SD, and tgGFP mice). CD19, marker for B cells; CD11b, marker for monocytes, neutrophils, and natural killer (NK) cells; CD3, marker of mature T cells. Data represent the mean ± SEM; n = 3–13 mice/group. (E) IF image showing the distribution of GFP+ cells in a whole brain sagittal section of an IC GT + BMT-treated SD mouse at 120 days. Boxes highlight brain regions in (F) at higher magnification. (F) IF pictures showing GFP+ cells engrafted into the olfactory bulb (OB), hippocampus (Hip), thalamus (Thal), and pons/medulla (Pons) of IC GT + BMT-treated SD mice at 120 days and 240 days. (E) and (F) Direct GFP fluorescence, green; nuclei counterstained with Hoechst, gray. Scale bars, 3,000 μm (E) and 500 μm (F).
Figure 4
Figure 4
Hex enzymatic activity in CNS tissues, PNS, and periphery of treated SD mice and controls (A) Enzymatic activity measured in the telencephalon (TEL), cerebellum (CB), and spinal cord (SC) tissues of IC GT-, BMT-, and IC GT + BMT-treated SD mice and age-matched UT SD mice at 120 days and 240 days. Enzymatic activity was measured as degradation of the artificial substrates MUG (left graph) and MUGS (right graph) and expressed as the percentage of age-matched UT WT control data. (B) Enzymatic activity (MUG, MUGS; expressed as nanomoles per milligram per hour) in the TEL, CB, and SC tissues of WT and Het mice at 120 days and 240 days. ∗p < 0.01, ∗∗p < 0.005, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (C and D) Enzymatic activity (MUG, MUGS; expressed as the percentage of age-matched UT WT controls) measured in the cerebrospinal fluid (CSF; (C) and BM (D) of treated SD mice (IC GT, BMT, and IC GT + BMT) and UT controls (Het and SD) at 120 days and 240 days. ∗p < 0.05 (C); ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001 SD IC GT 120 days versus all other treatments (D). Combined treatment versus WT UT, not significant. (E and F) Confocal IF images showing donor-derived GFP+ cells (green) expressing the microglial and macrophagic markers Iba1 (red) and CD68 (blue), respectively, in the liver (E) and spleen (F) of BMT-treated SD mice at 120 days. Scale bar, 50 μm. (G–I) Enzymatic activity measured in the liver (G), spleen (H), and sciatic nerve (I) of treated SD mice (IC GT, BMT, and IC GT + BMT) and UT controls (Het and SD) at 120 days and 240 days. Enzymatic activity was measured as degradation of MUG and MUGS and expressed as the percentage of age-matched UT WT controls. ∗∗p < 0.01, ∗∗∗∗p < 0.0001 SD IC GT 120 days versus all other treatments (G and H). ∗∗∗∗p < 0.0001, ∗∗p < 0.01 SD IC GT + BMT 240 days versus all other treatments (I). Combined treatment versus Het (liver) and WT (spleen), p > 0.05. Data in (A)–(D) and (G)–(I) are expressed as the mean ± SEM; n = 3–9 mice/group. Data were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons test.
Figure 5
Figure 5
Rescue of LAMP1 expression and GM2 storage in treated SD mice (A) Representative western blot analyses and relative quantification showing LAMP1 protein expression in whole-brain lysate (TEL and CB) of treated mice (IC GT, BMT, and IC GT + BMT) at 120 days and 240 days and age-matched UT controls (WT, Het, and SD). Data are expressed as the mean ± SEM; n = 3–11 mice/group; one-way ANOVA followed by Kruskal-Wallis multiple comparisons test, ∗∗∗p < 0.001. (B and C) Representative HPTLC and relative quantification of the aqueous phase (GM2, (B) and organic phase (GA2, (C) of total lipids obtained from whole-brain lysate (TEL and CB) of BMT- and IC GT + BMT-treated SD mice (120 days and 240 days) and age-matched UT controls (WT and SD). Quantifications are expressed as the percentage of total gangliosides (GM2) and percentage of total neutral sphingolipids (GA2). Data represent the mean ± SEM; n = 4–8 mice/group. One-way ANOVA followed by Dunnett’s multiple comparison test, ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (D) Representative IF pictures showing GM2 expression (anti-GM2 antibody, blue) and relative mask selection of immunopositive signal (black) in sagittal brain sections taken from IC GT + BMT-treated SD mice and UT controls (SD and WT) at 120 days. Magnification 10×; scale bars, 1,000 μm. (E) Correlation of Hex enzymatic activity (y axis; MUG, nanomoles per milligram per hour) and GM2 or GA2 levels (x axis, values expressed as the percentage of total gangliosides or total neutral sphingolipids, respectively) in the TEL of treated SD mice at 120 days and 240 days and age-matched UT controls (SD, WT, and Het). Each dot represents one animal. Treatment groups are shown in the legend.
Figure 6
Figure 6
Reduction of neuroinflammatory markers in the CNS of treated SD mice (A) Relative mRNA expression of neuroinflammatory cytokines (Ccl3 and Ccl5), macrophage (Cd68), and astrocytic (Gfap) markers in whole-brain lysate (TEL and CB) of treated mice (IC GT, BMT, and IC GT + BMT) at 120 days and 240 days and age-matched UT controls (WT, Het, and SD). Data are expressed as fold change with respect to the WT (set as 1) after normalization to Gapdh expression. (B) Representative western blot and relative quantification showing the expression of GFAP protein in treated mice (IC GT, BMT, and IC GT + BMT) at 120 days and 240 days and age-matched UT controls (WT, Het, and SD). Data are expressed as fold change with respect to the WT (set as 1) after normalization to calnexin (CNX) expression. Data in (A) and (B) represent the mean ± SEM; n = 3–11 animals/group. One-way ANOVA followed by Kruskal-Wallis multiple comparisons test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (C) 3D projections of z stack images showing the presence of donor-derived GFP+ cells (green) and resident cells expressing Iba1 (microglia, red) and CD68 (macrophages, blue) in the TEL of IC GT + BMT-treated SD mice at 120 days and 240 days and UT controls (WT and SD). Nuclei counterstained with Hoechst, gray. Images were acquired at 40× magnification.
Figure 7
Figure 7
Effect of treatments on cerebellar pathology of SD mice (A) Representative confocal pictures showing GM2 storage in the cerebellar molecular layer, Purkinje cell (PC) layer, and granular layer of UT WT and SD mice at 120 days. GM2, green; calbindin (Calb), red; nuclei counterstained with Hoechst, white. Scale bar, 25 μm. (B) Representative confocal images showing calbindin+ PCs (red) in UT mice of different ages (WT, 120 days; SD, 30 days, 60 days, and 120 days) and IC GT + BMT-treated SD mice at 120 days and 240 days. Calbindin, red. Nuclei counterstained with Hoechst, gray. Scale bar, 50 μm. (C) Quantification of the number of PCs in UT SD and WT mice at 30 days, 60 days, and 120 days. Data represent the number of PCs per millimeter and are expressed as the mean ± SEM; n = 1–10 mice/group. two-way ANOVA followed by Bonferroni’s multiple comparisons test; ∗∗p < 0.01. (D) Quantification of the number of PCs in treated mice (IC GT, BMT, and IC GT + BMT) at 120 days and 240 days and age-matched UT controls (WT and SD). Data represent the number of PCs per millimeter in all cerebellar lobules of each brain section (3–4 sagittal sections per mouse). Data are expressed as mean ± SEM; n = 4–6 mice/group; one-way ANOVA followed by Kruskal-Wallis multiple comparisons test, ∗∗p < 0.01.
Figure 8
Figure 8
Analyses of the myelin compartment in treated SD mice (A) Quantification of myelinated fibers and percentage of demyelinated/degenerated (aberrant) fibers among total fibers in SC sections from treated mice (IC GT, BMT, and IC GT + BMT) at 120 days and 240 days and age-matched UT controls (WT and SD). Data are expressed as mean ± SEM; n = 3–4 mice/group; nonparametric one-way ANOVA followed by Dunn’s multiple comparisons test, ∗p < 0.05. (B) Semithin section analysis of lumbar SC of IC GT + BMT-treated SD mice at 120 days and 240 days and UT controls (WT and SD). Red arrows indicate aberrant myelin. Scale bar, 10 μm. (C) Electron microscopy images of optic nerves in treated SD and UT controls at 120 days and 240 days. Red arrows indicate aberrant myelin, and asterisks mark macrophages. Scale bar, 2 μm. (D) Representative western blot analyses and relative quantification showing MBP and MAG protein expression in whole-brain lysate (TEL and CB) of treated mice (IC GT, BMT, and IC GT + BMT) at 120 days and 240 days and age-matched UT controls (WT, Het, and SD). Data are expressed as fold change with respect to the WT (set as 1) after normalization to β-actin expression. Data represent the mean ± SEM; n = 3–10 mice/group. One-way ANOVA followed by Kruskal-Wallis multiple comparisons test, ∗p < 0.05. (E) Expression of myelin-related genes (Mbp, Mag, Plp1, and Ugt8a) in whole-brain lysate (TEL and CB) of treated mice (IC GT, BMT, and IC GT + BMT) at 120 days and 240 days and age-matched UT controls (WT, Het, and SD). Data are expressed as fold change with respect to the WT (set as 1) after normalization to Gapdh. Data represent the mean ± SEM; n = 2–12 mice/group. One-way ANOVA followed by Kruskal-Wallis multiple comparisons test, ∗∗p < 0.01. No significant differences are found between UT SD and UT WT mice or between SD-treated mice and SD UT counterparts.
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