Long-term comparative analysis of AAV9-mediated gene replacement therapies for spinal muscular atrophy in mice

Figures

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  Fig. 1. Therapeutic efficacy comparison in SMA mice. a Benchmark (BMK) and 2nd-generation (2nd-G) rAAV vectors. b Schematic of experimental design in the SMA (SMN∆7) mice. Mice were treated at P0 with 1.1E + 13 vg/kg by ICV injection. Non-treated SMA mice (SMA) and healthy carriers (HC) were used as controls. c Kaplan-Meier survival curves for the SMA mice receiving the BMK or 2nd-G vector in a 75-day study period, Log-rank (Mantel-Cox) test. d, e Surface righting reflex and grid test of treated mice (n = 10 in SMA, n = 10 in HC, n = 20 in BMK, n = 20 in 2nd-G). f Rotarod testing of the treated mice on days 30 and 60 (on day 30: n = 13 in BMK, n = 20 in 2nd-G, n = 10 in HC; on day 60: n = 5 in BMK, n = 10 in 2nd-G, n = 9 in HC). g Representative images of hind-limb and tail necrosis and frequencies of occurrence in each group. h Immunostaining with ChAT in the spinal cord (SC) on day 60. The L1 and L5 segments are shown in the top and bottom panels. Four mice per group were used for quantitative analysis. To calculate soma size, ~85 and 40 motor neurons (MN) were used for L1 MNs and L5 MMNs, respectively, in each mouse. LMC lateral motor column, MMC medial motor column. Medial, M;…

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  Fig. 2. Comparison of long-term efficacy in SMA mice. a Kaplan-Meier survival curve of the SMA mice and HC in a 12-month study period. All SMA mice were treated at P0 at a dose of 3.3E + 13 vg/kg by ICV injection. b Western blot analysis of pan-SMN protein expression in the SC of SMA mice treated for three and 12 months, n = 3. c Immunostaining with SMN and SmB in ChAT+ motor neurons in the L5 lumbar spinal cord segment from the SMA mice post-injection for three months. d Immunostaining of SMN, PV and SmB in the L5 DRGs from the SMA mice post-injection for three months. Four mice in each group, PV+ neurons were used to quantify the SMN aggregates (n = 309 in HC, n = 244 in BMK, n = 279 in 2nd-G). e Immunostaining of MNs in the L1 and L5 segments from the SMA mice same as in (a). Four mice in each group. MN numbers in L1 MNs (n = 303 in HC, n = 267 in BMK, n = 290 in 2nd-G) and L5 MMNs (n = 58 in HC, n = 50 in BMK, n = 62 in 2nd-G). L1 MN soma size (n = 256 in HC, n = 189 in BMK, n = 224 in 2nd-G), L5 MMN soma size (n = 105 in HC, n = 81 in BMK, n = 129 in 2nd-G). The violin plots show the median (solid line) and interquartile range (dotted lines). f Rotarod test of the SMA mice three and…

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  Fig. 3. Supraphysiological SMN protein induces cardiac thrombosis in both SMA and WT mice. a H&E staining of mouse hearts from SMA mice treated with the benchmark vector at 3.3E + 13 vg/kg for 12 months. Occurrences by percentage are shown at the right. b SMN protein expression in the left atrium of SMA mice treated for one month by Western blot, three animals in each group. c Gross image of heart from FVB WT mice two weeks post-injection of a 1.0E + 14 vg/kg dose delivered via ICV administration at P0-P1. The frequency of left atrial thrombosis (LAT) is shown at the bottom. The CB-opti construct is a re-engineered version of the benchmark vector, created by replacing the native SMN1 coding sequence with codon-optimized hSMN1 to maximize SMN expression. NT, non-treated FVB mice. d H&E staining of the FVB mouse hearts two weeks after treatment. e SMN protein expression in the left atrium of FVB WT mice after receiving the AAV treatment for three days by ICV injection at 1.0E + 14 vg/kg at P0. Three animals in each group. f Immunostaining of the whole heart with anti-SMN antibody from FVB WT mice on day 7 post-injection. Bar plots in (b, e) represent mean ± SEM, and the statistical tests…

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  Fig. 4. The 2nd-G vector impaired the motor function of WT mice. a Kaplan-Meier survival curve of C57BL/6 mice in a 20-month study period. All C57BL/6 mice were treated at P0 with 1.0E + 14 vg/kg via ICV injection. Non-treated (NT) healthy littermates were used for comparison. b Rotarod test at five and twenty months after treatment. Animals in 5 months: n = 5 in NT and BMK, n = 7 in 2nd-G. In 20 months: n = 6 in NT, n = 10 in BMK, n = 11 in 2nd-G. c Hind-limb clasping phenotype observed in C57BL/6 mice in 2nd-G vector-treated mice. d SMN protein expression in the spinal cord (SC) and brain of C57BL/6 mice after receiving the AAV treatment for 20 months. Quantification of SMN is shown at the bottom. Three animals in each group. e Immunostaining of ChAT+ motor neurons in the L1 and L5 segments from the C57BL/6 mice post-injection for twenty months. Four animals were included in each group in comparison. MN numbers were quantified in L1 MNs (n = 299 in NT, n = 257 in BMK, n = 264 in 2nd-G) and L5 MMNs (n = 58 in NT, n = 59 in BMK, n = 54 in 2nd-G). L1 MN soma size (n = 407 in NT, n = 290 in BMK, n = 311 in 2nd-G), L5 MMN soma size (n = 97 in NT, n = 124 in BMK, n = 110 in 2nd-G). f ChAT,…

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  Fig. 5. Benchmark vector integrates into Rtl1-Rian locus and induces hepatocellular carcinoma. a, b Gross images of the liver and lung from the C57BL/6 mice 20 months after receiving AAV treatment. All mice were treated at P0 by ICV injection at a dose of 1.0E + 14 vg/kg. Non-treated mice were used as controls. The percentage of animals with tumors is shown on the right. c H&E staining and immunohistochemistry (IHC) staining with Alpha Fetoprotein (AFP), epithelial cellular adhesion molecule (EpCAM), and Kiel 67 antibodies in the liver of mice that received AAV treatment for 20 months. Four mice per group were analyzed, and a representative image is shown. d Transcriptome comparison between the liver of the 2nd-G vector group vs. non-treated C57BL/6 mice, the BMK vector group vs non-treated C57BL/6 mice, and the liver tumor from the BMK vector group versus its adjacent liver or the non-treated group. FC, Fold Change, adjusted P values (P_adj) were calculated using the Benjamini–Hochberg (BH) method, and P_adj <0.05 was considered statistically significant, indicated by dashed lines in the graphs. e Rian, Mirg, and Rtl1 expression by qRT-ddPCR in the liver and liver tumor tissues, n = 5…