Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN

Journal name:
Nature Biotechnology
Volume:
28,
Pages:
271–274
Year published:
DOI:
doi:10.1038/nbt.1610
Received
Accepted
Published online

Spinal muscular atrophy (SMA), the most common autosomal recessive neurodegenerative disease affecting children, results in impaired motor neuron function1. Despite knowledge of the pathogenic role of decreased survival motor neuron (SMN) protein levels, efforts to increase SMN have not resulted in a treatment for patients. We recently demonstrated that self-complementary adeno-associated virus 9 (scAAV9) can infect ~60% of motor neurons when injected intravenously into neonatal mice2, 3, 4. Here we use scAAV9-mediated postnatal day 1 vascular gene delivery to replace SMN in SMA pups and rescue motor function, neuromuscular physiology and life span. Treatment on postnatal day 5 results in partial correction, whereas postnatal day 10 treatment has little effect, suggesting a developmental period in which scAAV9 therapy has maximal benefit. Notably, we also show extensive scAAV9-mediated motor neuron transduction after injection into a newborn cynomolgus macaque. This demonstration that scAAV9 traverses the blood-brain barrier in a nonhuman primate emphasizes the clinical potential of scAAV9 gene therapy for SMA.

At a glance

Figures

  1. Phenotypic correction of SMA mice injected on P1.
    Figure 1: Phenotypic correction of SMA mice injected on P1.

    (a) Injection of scAAV9-GFP in SMA animals results in GFP expression (green) within dorsal root ganglia and motor neurons (ChAT staining in red) in the lumbar spinal cord 10-d post-injection. (b) Western blots from tissues of control, scAAV9-SMN–treated and untreated SMA animals show elevated levels of SMN expression in SMN-treated animals compared to control animals, although levels are still lower than those of control littermates. Quantifications of western blots are available in the Supplementary Figures 1 and 7. (c) Righting ability shows that SMN-treated animals can right themselves similarly to control animals by P13. (d) SMA animals treated with scAAV9-SMN are larger than GFP-treated animals. (e) scAAV9-SMN treatment of SMA animals results in greatly extended survival over GFP treatment. (f) Body weight assessments show an increase in animals treated with scAAV9-SMN versus those treated with GFP. Scale bars, 200 μm (a); 50 μm (a inset).

  2. Effects of SMN treatment at P1 on NMJs of adult SMA mice.
    Figure 2: Effects of SMN treatment at P1 on NMJs of adult SMA mice.

    Untreated SMA mice do not survive to adulthood. (a) scAAV9-SMN treatment restores endplate currents (EPC) in ~90-d-old SMA animals. In control mice, the mean EPC amplitude was 82.6 ± 3.5 nA, and in treated SMA mice, it was 83.4 ± 4.1 nA (P = 0.89, n = 4 mice for each group). (b) Affected animals treated with scAAV9-SMN had an increase in miniature endplate currents. (c) Both control and treated SMA endplate currents had a similar degree of depression during 50 Hz nerve stimulation. (di) Representative sections from the transverse abdominis (TVA), a proximal muscle with innervation abnormalities in SMA mice2, shows normal innervation in both wild-type (df) and SMN-treated (gi) animals. Scale bars, 10 μm.

  3. Systemic injection of scAAV9-GFP into SMA mice of varying ages.
    Figure 3: Systemic injection of scAAV9-GFP into SMA mice of varying ages.

    (ac) Animals injected on P2 have a transduction pattern identical to P1-injected animals, with motoneuron transduction in lumbar spinal cord. (df) P5-injected animals have more glial transduction and less motoneuron (f inset, arrow) transduction than younger animals in lumbar spinal cord analysis. (gi) The pattern of increasing glial transduction continues in P10-injected animals. GFP (green), ChAT (red, a motoneuron marker) and merged (yellow). (jk) scAAV9-SMN injection on P2 in SMA animals rescues life span and increases body weight (n = 6), whereas P5 scAAV9-SMN delivery in SMA animals only partially rescues life span and body weight (n = 4) compared with control scAAV9-GFP–treated (n = 10). No increase in life span or body weight was seen in mice treated with scAAV9-SMN on P10 (n = 4). (lq) Systemic injection of scAAV9-GFP into a cynomolgus macaque on P1 results in a similar transduction pattern within the spinal cord as previously shown in P1-injected mice. GFP (l,o), ChAT (m,p) and merged (n,q) images from thoracic spinal cord demonstrate motor neuron transduction. A representative longitudinal section is shown in (ln), indicating transduction along the neuraxis. Transverse sections (oq) mimic the pattern of dorsal root ganglia and motor neuron transduction seen in P1-injected mice. Inset scale bars, 50 μm; c,f,i, 100 μm; n,q, 200 μm.

References

  1. Burghes, A.H. & Beattie, C.E. Spinal muscular atrophy: why do low levels of survival motor neuron protein make motor neurons sick? Nat. Rev. Neurosci. 10, 597609 (2009).
  2. Foust, K.D. et al. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat. Biotechnol. 27, 5965 (2009).
  3. Gao, G. et al. Clades of adeno-associated viruses are widely disseminated in human tissues. J. Virol. 78, 63816388 (2004).
  4. McCarty, D.M. et al. Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo . Gene Ther. 10, 21122118 (2003).
  5. Lefebvre, S. et al. Identification and characterization of a spinal muscular atrophy-determining gene. Cell 80, 155165 (1995).
  6. McGovern, V.L., Gavrilina, T.O., Beattie, C.E. & Burghes, A.H. Embryonic motor axon development in the severe SMA mouse. Hum. Mol. Genet. 17, 29002909 (2008).
  7. MacKenzie, A.E. & Gendron, N.H. Tudor reign. Nat. Struct. Biol. 8, 1315 (2001).
  8. Gavrilina, T.O. et al. Neuronal SMN expression corrects spinal muscular atrophy in severe SMA mice while muscle-specific SMN expression has no phenotypic effect. Hum. Mol. Genet. 17, 10631075 (2008).
  9. Azzouz, M. et al. Lentivector-mediated SMN replacement in a mouse model of spinal muscular atrophy. J. Clin. Invest. 114, 17261731 (2004).
  10. Avila, A.M. et al. Trichostatin A increases SMN expression and survival in a mouse model of spinal muscular atrophy. J. Clin. Invest. 117, 659671 (2007).
  11. Hastings, M.L. et al. Tetracyclines that promote SMN2 exon 7 splicing as therapeutics for spinal muscular atrophy. Sci. Transl. Med 1, 514 (2009).
  12. Duque, S. et al. Intravenous administration of self-complementary AAV9 enables transgene delivery to adult motor neurons. Mol. Ther. 17, 11871196 (2009).
  13. Le, T.T. et al. SMNDelta7, the major product of the centromeric survival motor neuron (SMN2) gene, extends survival in mice with spinal muscular atrophy and associates with full-length SMN. Hum. Mol. Genet. 14, 845857 (2005).
  14. Butchbach, M.E., Edwards, J.D. & Burghes, A.H. Abnormal motor phenotype in the SMNDelta7 mouse model of spinal muscular atrophy. Neurobiol. Dis. 27, 207219 (2007).
  15. Kong, L. et al. Impaired synaptic vesicle release and immaturity of neuromuscular junctions in spinal muscular atrophy mice. J. Neurosci. 29, 842851 (2009).
  16. Wang, X. et al. Decreased synaptic activity shifts the calcium dependence of release at the mammalian neuromuscular junction in vivo. J. Neurosci. 24, 1068710692 (2004).
  17. Cifuentes-Diaz, C. et al. Neurofilament accumulation at the motor endplate and lack of axonal sprouting in a spinal muscular atrophy mouse model. Hum. Mol. Genet. 11, 14391447 (2002).
  18. Kariya, S. et al. Reduced SMN protein impairs maturation of the neuromuscular junctions in mouse models of spinal muscular atrophy. Hum. Mol. Genet. 17, 25522569 (2008).
  19. Murray, L.M. et al. Selective vulnerability of motor neurons and dissociation of pre- and post-synaptic pathology at the neuromuscular junction in mouse models of spinal muscular atrophy. Hum. Mol. Genet. 17, 949962 (2008).
  20. Narver, H.L. et al. Sustained improvement of spinal muscular atrophy mice treated with trichostatin A plus nutrition. Ann. Neurol. 64, 465470 (2008).
  21. Clark, K.R. et al. Gene transfer into the CNS using recombinant adeno-associated virus: analysis of vector DNA forms resulting in sustained expression. J. Drug Target. 7, 269283 (1999).
  22. Duan, D. et al. Circular intermediates of recombinant adeno-associated virus have defined structural characteristics responsible for long-term episomal persistence in muscle tissue. J. Virol. 72, 85688577 (1998).
  23. Nakai, H. et al. Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo. J. Virol. 75, 69696976 (2001).
  24. Saunders, N.R., Joakim Ek, C. & Dziegielewska, K.M. The neonatal blood-brain barrier is functionally effective, and immaturity does not explain differential targeting of AAV9. Nat. Biotechnol. 27, 804805, author reply 805 (2009).
  25. Kota, J. et al. Follistatin gene delivery enhances muscle growth and strength in nonhuman primates. Sci. Transl. Med 1, 615 (2009).
  26. Koerber, J.T. et al. Molecular evolution of adeno-associated virus for enhanced glial gene delivery. Mol. Ther. 17, 20882095 (2009).
  27. Maheshri, N., Koerber, J.T., Kaspar, B.K. & Schaffer, D.V. Directed evolution of adeno-associated virus yields enhanced gene delivery vectors. Nat. Biotechnol. 24, 198204 (2006).
  28. Asokan, A. et al. Reengineering a receptor footprint of adeno-associated virus enables selective and systemic gene transfer to muscle. Nat. Biotechnol. 28, 7982 (2010).
  29. Pyatt, R.E., Mihal, D.C. & Prior, T.W. Assessment of liquid microbead arrays for the screening of newborns for spinal muscular atrophy. Clin. Chem. 53, 18791885 (2007).
  30. Monani, U.R. et al. The human centromeric survival motor neuron gene (SMN2) rescues embryonic lethality in Smn −/− mice and results in a mouse with spinal muscular atrophy. Hum. Mol. Genet. 9, 333339 (2000).

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Author information

  1. These authors contributed equally to this work.

    • Xueyong Wang &
    • Vicki L McGovern

Affiliations

  1. Center for Gene Therapy, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio, USA.

    • Kevin D Foust,
    • Lyndsey Braun,
    • Adam K Bevan,
    • Amanda M Haidet &
    • Brian K Kaspar
  2. Wright State University, Dayton, Ohio, USA.

    • Xueyong Wang &
    • Mark M Rich
  3. Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio, USA.

    • Vicki L McGovern,
    • Thanh T Le,
    • Arthur H M Burghes &
    • Brian K Kaspar
  4. Integrated Biomedical Graduate Program, The Ohio State University, Columbus, Ohio, USA.

    • Adam K Bevan,
    • Amanda M Haidet,
    • Arthur H M Burghes &
    • Brian K Kaspar
  5. The Mannheimer Foundation, Inc., Homestead, Florida, USA.

    • Pablo R Morales

Contributions

K.D.F., M.M.R., A.H.M.B. and B.K.K. designed and executed experiments and wrote the manuscript. V.L.M., X.W, L.B., A.M.H., A.K.B., P.R.M. and T.T.L. contributed to experiments.

Competing financial interests

The authors declare no competing financial interests.

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