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


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.


rAAV AAV Amyotrophic lateral sclerosis Pain SMA Gene therapy 



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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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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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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  1. 1.
    Ray MK, Fagan SP, Brunicardi FC (2000) The Cre-loxP system: a versatile tool for targeting genes in a cell- and stage-specific manner. Cell Transplant 9(6):805–815PubMedGoogle Scholar
  2. 2.
    Zinn E, Vandenberghe LH (2014) Adeno-associated virus: fit to serve. Curr Opin Virol 8:90–97CrossRefPubMedGoogle Scholar
  3. 3.
    Snyder BR, Gray SJ, Quach ET, Huang JW, Leung CH, Samulski RJ, Boulis NM, Federici T (2011) Comparison of adeno-associated viral vector serotypes for spinal cord and motor neuron gene delivery. Hum Gene Ther 22(9):1129–1135CrossRefPubMedGoogle Scholar
  4. 4.
    Federici T, Taub JS, Baum GR, Gray SJ, Grieger JC, Matthews KA, Handy CR, Passini MA et al. (2012) Robust spinal motor neuron transduction following intrathecal delivery of AAV9 in pigs. Gene Ther 19(8):852–859CrossRefPubMedGoogle Scholar
  5. 5.
    Bevan AK, Duque S, Foust KD, Morales PR, Braun L, Schmelzer L, Chan CM, McCrate M et al. (2011) Systemic gene delivery in large species for targeting spinal cord, brain, and peripheral tissues for pediatric disorders. Mol Ther 19(11):1971–1980. doi: 10.1038/mt.2011.157 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Samaranch L, Salegio EA, San Sebastian W, Kells AP, Foust KD, Bringas JR, Lamarre C, Forsayeth J et al. (2012) Adeno-associated virus serotype 9 transduction in the central nervous system of nonhuman primates. Hum Gene Ther 23(4):382–389CrossRefPubMedGoogle Scholar
  7. 7.
    Gray SJ, Nagabhushan Kalburgi S, McCown TJ, Jude Samulski R (2013) Global CNS gene delivery and evasion of anti-AAV-neutralizing antibodies by intrathecal AAV administration in non-human primates. Gene Ther 20:450–459CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Wang H, Yang B, Qiu L, Yang C, Kramer J, Su Q, Guo Y, Brown RH Jr et al. (2014) Widespread spinal cord transduction by intrathecal injection of rAAV delivers efficacious RNAi therapy for amyotrophic lateral sclerosis. Hum Mol Genet 23(3):668–681CrossRefPubMedGoogle Scholar
  9. 9.
    Hordeaux J, Dubreil L, Deniaud J, Iacobelli F, Moreau S, Ledevin M, Le Guiner C, Blouin V et al. (2015) Efficient central nervous system AAVrh10-mediated intrathecal gene transfer in adult and neonate rats. Gene TherGoogle Scholar
  10. 10.
    Schuster DJ, Dykstra JA, Riedl MS, Kitto KF, Belur LR, McIvor RS, Elde RP, Fairbanks CA et al. (2014) Biodistribution of adeno-associated virus serotype 9 (AAV9) vector after intrathecal and intravenous delivery in mouse. Front Neuroanat 8:42PubMedPubMedCentralGoogle Scholar
  11. 11.
    Zhang H, Yang B, Mu X, Ahmed SS, Su Q, He R, Wang H, Mueller C et al. (2011) Several rAAV vectors efficiently cross the blood–brain barrier and transduce neurons and astrocytes in the neonatal mouse central nervous system. Mol Ther 19(8):1440–1448. doi: 10.1038/mt.2011.98 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Fairbanks CA (2003) Spinal delivery of analgesics in experimental models of pain and analgesia. Adv Drug Deliv Rev 55(8):1007–1041CrossRefPubMedGoogle Scholar
  13. 13.
    Ciesielska A, Hadaczek P, Mittermeyer G, Zhou S, Wright JF, Bankiewicz KS, Forsayeth J (2013) Cerebral infusion of AAV9 vector-encoding non-self proteins can elicit cell-mediated immune responses. Mol Ther 21(1):158–166CrossRefPubMedGoogle Scholar
  14. 14.
    Klein RL, Dayton RD, Leidenheimer NJ, Jansen K, Golde TE, Zweig RM (2006) Efficient neuronal gene transfer with AAV8 leads to neurotoxic levels of tau or green fluorescent proteins. Mol Ther 13(3):517–527CrossRefPubMedGoogle Scholar
  15. 15.
    Rennels ML, Gregory TF, Blaumanis OR, Fujimoto K, Grady PA (1985) Evidence for a ‘paravascular’ fluid circulation in the mammalian central nervous system, provided by the rapid distribution of tracer protein throughout the brain from the subarachnoid space. Brain Res 326(1):47–63CrossRefPubMedGoogle Scholar
  16. 16.
    Iliff JJ, Wang M, Liao Y, Plogg BA, Peng W, Gundersen GA, Benveniste H, Vates GE et al. (2012) A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid beta. Sci Transl Med 4(147):147ra111CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Duque S, Joussemet B, Riviere C, Marais T, Dubreil L, Douar AM, Fyfe J, Moullier P et al. (2009) Intravenous administration of self-complementary AAV9 enables transgene delivery to adult motor neurons. Mol Ther 17(7):1187–1196. doi: 10.1038/mt.2009.71 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Foust KD, Nurre E, Montgomery CL, Hernandez A, Chan CM, Kaspar BK (2009) Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol 27(1):59–65. doi: 10.1038/nbt.1515 CrossRefPubMedGoogle Scholar
  19. 19.
    Yang B, Li S, Wang H, Guo Y, Gessler DJ, Cao C, Su Q, Kramer J et al. (2014) Global CNS transduction of adult mice by intravenously delivered rAAVrh.8 and rAAVrh.10 and nonhuman primates by rAAVrh.10. Mol Ther 22(7):1299–1309CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Rennels ML, Blaumanis OR, Grady PA (1990) Rapid solute transport throughout the brain via paravascular fluid pathways. Adv Neurol 52:431–439PubMedGoogle Scholar
  21. 21.
    Aird RB (1984) A study of intrathecal, cerebrospinal fluid-to-brain exchange. Exp Neurol 86(2):342–358CrossRefPubMedGoogle Scholar
  22. 22.
    Crawley JN, Fiske SM, Durieux C, Derrien M, Roques BP (1991) Centrally administered cholecystokinin suppresses feeding through a peripheral-type receptor mechanism. J Pharmacol Exp Ther 257(3):1076–1080PubMedGoogle Scholar
  23. 23.
    Bozanovic-Sosic R, Mollanji R, Johnston MG (2001) Spinal and cranial contributions to total cerebrospinal fluid transport. Am J Physiol Regul Integr Comp Physiol 281(3):R909–R916PubMedGoogle Scholar
  24. 24.
    Johnston M, Papaiconomou C (2002) Cerebrospinal fluid transport: a lymphatic perspective. News Physiol Sci 17:227–230PubMedGoogle Scholar
  25. 25.
    Pollay M (2010) The function and structure of the cerebrospinal fluid outflow system. Cerebrospinal Fluid Res 7:9CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Nonnenmacher M, Weber T (2012) Intracellular transport of recombinant adeno-associated virus vectors. Gene Ther 19(6):649–658CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Liu B, Paton JF, Kasparov S (2008) Viral vectors based on bidirectional cell-specific mammalian promoters and transcriptional amplification strategy for use in vitro and in vivo. BMC Biotechnol 8:49CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    von Jonquieres G, Mersmann N, Klugmann CB, Harasta AE, Lutz B, Teahan O, Housley GD, Frohlich D et al. (2013) Glial promoter selectivity following AAV-delivery to the immature brain. PLoS One 8(6):e65646CrossRefGoogle Scholar

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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