Lentivector-mediated SMN replacement in a mouse model of spinal muscular atrophy

Abstract

Spinal muscular atrophy (SMA) is a frequent recessive autosomal disorder. It is caused by mutations or deletion of the telomeric copy of the survival motor neuron (SMN) gene, leading to depletion in SMN protein levels. The treatment rationale for SMA is to halt or delay the degeneration of motor neurons, but to date there are no effective drug treatments for this disease. We have previously demonstrated that pseudotyping of the nonprimate equine infectious anemia virus (using the lentivector gene transfer system) with the glycoprotein of the Evelyn-Rokitnicki-Abelseth strain of the rabies virus confers retrograde axonal transport on these vectors. Here, we report that lentivector expressing human SMN was successfully used to restore SMN protein levels in SMA type 1 fibroblasts. Multiple single injections of a lentiviral vector expressing SMN in various muscles of SMA mice restored SMN to motor neurons, reduced motor neuron death, and increased the life expectancy by an average of 3 and 5 days (20% and 38%) compared with LacZ and untreated animals, respectively. Further extension of survival by SMN expression constructs will likely require a knowledge of when and/or where high levels of SMN are needed.

Figure 1

EIAV-based lentivector-mediated gene transfer in vivo. Transverse sections of lumbar spinal cord (A), brain stem (B and C), and muscle (D) showing transduction of both muscle and MNs after injection of 30 μl rabies G pseudotyped lentivector-LacZ vector in the gastrocnemius (A) and facial muscles (B and C) of postnatal P2 FVB mice. (E) Cell counts of total transduced MNs in the lumbar spinal cord, facial nucleus, and motor trigeminal nucleus (MTN) after intramuscular injections of lentivector-LacZ into 2-day-old SMA mice. Data are means ± SEM. Expression of β-gal (green) (F) colocalizes with the immunofluorescence of CGRP (red) in spinal MNs, producing yellow staining (G). Scale bars: 400 μm (A and B), 200 μm (C and D), 100 μm (F and G).

Figure 2

Lentivector-mediated restoration of SMN protein expression in vitro. (A) Schematic representation of Lentivector encoding for human SMN gene. CMV, cytomegalovirus; cPPT, central polypurine tract; ΔΔ env, double-deleted envelope leaving only rev response element; WPRE, Woodchuck hepatitis virus posttranscriptional regulation element; SIN, self-inactivating; LTR, long-terminal repeat. (B) Lentivector-mediated expression of SMN in fibroblasts from type I SMA patients. Lentivector-SMN restores SMN expression in gems (arrows). No restoration of gems was observed in fibroblasts incubated with lentivector-LacZ (C). Expression of SMN in human fibroblasts (green) (D) colocalizes with the red immunofluorescence of gemin2 (E), producing yellow staining (F). Arrow indicates colocalization of SMN with gemin2 in gems. Lentivector-LacZ–treated fibroblasts stained with SMN Abs (G) and gemin2 (H). (I) Merged image from G and H. (J) Gem counts in lentivector-SMN–treated and control fibroblast cells. (K) Immunoblot confirming lentivector-mediated SMN expression in human fibroblasts using Abs against SMN. Scale bars: 50 μm (B and C), 100 μm (D–F), 200 μm (G–I).

Figure 3

Retrograde lentivector delivery of SMN at the onset of disease extends survival and delays the phenotype in SMA mice. (A) Weight measurements of animals treated with lentivector-SMN or lentivector-LacZ. (B) Survival analysis in SMA mice injected at 2 days of age with lentivector-LacZ or lentivector-SMN. Mortality was significantly delayed in mice treated with lentivector-SMN compared with the control LacZ group. (C) Image showing lentivector-mediated expression of SMN in spinal MN at the end stage of disease as monitored using Abs against the HA tag. Arrow indicates gems. (D) Double labeling using HA (green, arrow indicates gems) and CGRP Abs (red) in spinal sections. No HA staining was detected in LacZ-injected mice (E). (F and G) SMN expression in muscles from SMN and LacZ-treated mice, respectively. Arrows in F indicate lentivector-mediated SMN expression. (H) Western blot analysis of ventral spinal cord in LacZ, SMN, and WT animals. Scale bars: 50 μm (C–E), 100 μm (F and G).

Figure 4

SMN gene replacement protects spinal and brain stem MNs in SMA mice. ChAT immunoreactivity in lentivector-SMN–treated (A, insert) and lentivector–Lac-Z–treated (B, insert) mice. Immunohistochemistry showing CGRP-positive MNs in lumbar spinal cord of lentivector-SMN–injected (C) and lentivector-LacZ–injected (D) animals. (E) Cell counts of surviving lumbar spinal cord MNs in WT (control), lentivector–Lac-Z–, and lentivector-SMN–treated SMA mice at end stage of disease. (F) Quantification of facial nucleus MNs in control, lentivector-LacZ–, and lentivector-SMN–injected animals at end stage of disease. Scale bars: 100 μm.

Figure 5

Immune response in SMA mice 1 week after intramuscular lentivector-SMN delivery. Abs used to detect components of the immune response in the injected area of muscle were (A) P7/7: MHC class II; (B) CD3: T cells; and (C) FA11: macrophage. All animals exhibited a minor infiltration at site of injections (arrows). Scale bars: 100 μm.