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Molecular Neurobiology

, Volume 53, Issue 5, pp 3235–3248 | Cite as

A Single Injection of Recombinant Adeno-Associated Virus into the Lumbar Cistern Delivers Transgene Expression Throughout the Whole Spinal Cord

  • Yansu Guo
  • Dan Wang
  • Tao Qiao
  • Chunxing Yang
  • Qin Su
  • Guangping Gao
  • Zuoshang Xu
Article

Abstract

The lack of methods to deliver transgene expression in spinal cord has hampered investigation of gene function and therapeutic targets for spinal cord diseases. Here, we report that a single intrathecal injection of recombinant adeno-associated virus rhesus-10 (rAAVrh10) into the lumbar cistern led to transgene expression in 60 to 90 % of the cells in the spinal cord. The transgene was expressed in all cell types, including neurons, glia, ependymal cells, and endothelial cells. Additionally, the transgene was expressed in some brain areas up to the frontal cortex and the olfactory bulb. The rAAV was distributed predominantly in the spinal cord, where its genome copy was over ten times that of the peripheral organs. Compared with intravenous injection, another method for rAAV delivery to the broad central nervous system (CNS), the intrathecal injection reduced the dosage of rAAV required to achieve similar or higher levels of transgene expression in the CNS by ~100-fold. Finally, the transduced areas were co-localized with the perivascular spaces of Virchow-Robin, from which the rAAV spreads further into the CNS parenchyma, thus suggesting that rAAV penetrated the CNS parenchyma through this pathway. Taken together, we have defined a fast and efficient method to deliver widespread transgene expression in mature spinal cord in mice. This method can be applied to stably overexpress or silence gene expression in the spinal cord to investigate gene functions in mammalian CNS. Additionally, this method can be applied to validate therapeutic targets for spinal cord diseases.

Keywords

rAAV AAV Amyotrophic lateral sclerosis Pain SMA Gene therapy 

Notes

Acknowledgments

We thank Dr. Hongyan Wang for the advice and Ms Karen Tran for editing the manuscript. This work was supported by grants from the ALS Association to Z.X. and from Jacob’s Cure, NTSAD Foundation, and Canavan Foundation and National Institutes of Health R01 grant (1R01NS076991) to G.G., and partially supported by a grant from National High Technology Research and Development Program (“863” Program) of China (2012AA020810) to G.G., and from China scholarship council and Chinese society of neurology to Y.G..

Conflict of Interest

G. Gao is a founder of Voyager Therapeutics and holds equity in the company. G. Gao is an inventor on patents on many rAAV serotypes including rAAVrh10 with potential royalties licensed to Voyager Therapeutics and other biopharmaceutical companies.

Supplementary material

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Figure S1

Solutes spread quickly and widely in the CNS after injection into the lumbar cistern. Ten min after injecting 8 μl PBS containing 0.4% trypan blue, brain and spinal cord were dissected and photographed from the dorsal (a) and ventral (b) sides. Notice that the more dye was distributed on the ventral side than the dorsal side. This was likely a result of injection from the dorsal side and of the animal posture, which led to the spinal sinking to the ventral side of the spinal canal by gravity, leaving more CSF volume on the dorsal side for the dye to distribute than the ventral side. UI = uninjected, 0-5 = weakness scores ranging from the poorest without observable weakness at score 0 to the most severe with paralysis in the four limbs at score 5 (see Methods for details). (GIF 23 kb)

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Figure S2

rAAVrh10 injected into the lumbar cistern transduced neurons in DRG and Schwann cells along the lumbar ventral and dorsal roots. Bar = 50 μm. (GIF 42 kb)

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Figure S3

rAAVrh10 injected into the lumbar cistern transduced cells in the lower medulla oblongata. The section was immunostained for GFP. Positive cells are shown in (A) dorsal motor nucleus of vagus, (B) hypoglossal nucleus, (C) lateral reticular nucleus, (D) pyramidal tract, (E) external cuneate nucleus, (F) nucleus of solitary tract, (G) from left to right: inferior cerebellar peduncle, spinal trigeminal tract and spinal V nucleus, (H) rostroventrolateral reticular nucleus, (I) inferior olive, (J) interpolar part of spinal V nucleus. Bar = 50 μm. (GIF 196 kb)

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Figure S4

rAAVrh10 injected into the lumbar cistern transduced cells in the rostral medulla oblongata. The section was immunostained for GFP. Positive cells are shown in (A) solitary tract, (B) spinal vestibular nucleus, (C) medial vestibular nucleus, (D) prepositus nucleus, (E) inferior cerebellar peduncle, (F) spinal trigeminal tract, (G) interpolar part of spinal V nucleus, (H) pyramidal tract, (I) ventral gigantocellular reticular nucleus, (J) lateral paragigantocellular nucleus, (K) Botzinger complex. Arrows in the central panel point to patches of transduction that appeared to project from the edge into the parenchyma of the medulla oblongata (see text). Bar = 50 μm. (GIF 391 kb)

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High resolution image (TIFF 5880 kb)
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Figure S5

rAAVrh10 injected into the lumbar cistern transduced cells in the pons and the cerebellum. The section was immunostained for GFP. Positive cells are shown in (A) facial nucleus, (B) pyramidal tract, (C) gigantocellular reticular nucleus, alpha, (D) spinal 5 nucleus, (E) spinal trigeminal tract, (F) medial vestibular nucleus, (G) lateral vestibular nucleus, (H) prepositus nucleus, (I) molecular and Pukinje layers of 1st cerebellar lobule, (J) granular layer of 1st cerebellar lobule, (K) white matter of cerebellum, (L) cerebellar nuclei, (M) Pukinje and granular layers of 3rd cerebellar lobule, (N) molecular layer of 3rd cerebellar lobule, (O) molecular and Pukinje layers of 4&5th cerebellar lobules, (P) cortex and white matter of simple lobule. Arrows in the central panel point to patches of transduction that appeared to project from the edge into the parenchyma of the pons (see text). Bar = 50 μm. (GIF 150 kb)

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High resolution image (TIFF 2320 kb)
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Figure S6

rAAVrh10 injected into the lumbar cistern transduced cells in the lower midbrain. The section was immunostained for GFP. Positive cells are shown in (A) oculomotor nucleus, (B) interpeduncular nucleus, (C) ventral tegmental area, (D) brachium pontis, (E) deep gray layer of superior colliculus, (F) Superficial gray layer of superior colliculus, (G), (I), (J) substantia nigra, (H) periaqueductal gray, (K) subbrachial nucleus, (L) intermediate gray layer of superior colliculus. Bar = 50 μm. (GIF 359 kb)

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High resolution image (TIFF 5232 kb)
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Figure S7

rAAVrh10 injected into the lumbar cistern transduced cells in the frontal cortex. The section was immunostained for GFP. Positive cells are shown in (A), (B) cingulate cortex, (C) indusium griseum, (D) genu of corpus callosum, (E) dorsal peduncular cortex, (F) lateral ventricle, (G) caudate putamen, (H) medial septal nucleus, (I) ventral pallidum, (J) anterior commissure, (K) accumbens nucleus, (L) nucleus of vert limb diagonal band. (M)-(P) cortex. Bar = 50 μm. (GIF 372 kb)

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Figure S8

rAAVrh10 injected into the lumbar cistern transduced cells in the olfactory bulb. The section was immunostained for GFP. Some areas are enlarged (A-E) showing that cells with neuronal and glial morphology were transduced. Bar = 50 μm. (GIF 602 kb)

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Figure S9

rAAVrh10 injected into the lumbar cistern transduced neurons, astrocytes, oligodendrocytes and microglia in the brain. The sections were doubly stained for GFP and various cell markers: NeuN for neurons, GFAP for astrocytes, APC for oligodenrocytes and Iba1 for microglia. Bar = 100 μm. (GIF 450 kb)

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Figure S10

rAAVrh10 injected into the lumbar cistern did not cause inflammation. Immunostained sections from cortex (CTX), cervical (CSC) and lumbar spinal cord (LSC) detected no evidence for microgliosis, astrogliosis or neuronal loss. VH = ventral horn, DH = dorsal horn, UI = uninjected, IT = intrathecally injected. Bar = 100 μm. (GIF 518 kb)

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Figure S11

The transduction in liver is correlated with the injection quality as illustrated by the weakness score. The highest transduction levels are correlated with the best injections with the highest weakness scores. Mice scored zero showed no GFP signal in the liver. UI = uninjected. Bar = 50 μm. (GIF 493 kb)

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of NeurologyThe Second Hospital of Hebei Medical UniversityShijiazhuangChina
  2. 2.Department of Biochemistry and Molecular PharmacologyUniversity of Massachusetts Medical School WorcesterWorcesterUSA
  3. 3.Gene Therapy CenterUniversity of Massachusetts Medical School WorcesterWorcesterUSA
  4. 4.Viral Vector CoreUniversity of Massachusetts Medical School WorcesterWorcesterUSA
  5. 5.Microbiology and Physiology SystemsUniversity of Massachusetts Medical School WorcesterWorcesterUSA
  6. 6.Department of Cell BiologyUniversity of Massachusetts Medical School WorcesterWorcesterUSA
  7. 7.Neuroscience ProgramUniversity of Massachusetts Medical School WorcesterWorcesterUSA

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