Detailed Method for Intrathecal Delivery of Gene Therapeutics by Direct Lumbar Puncture in Mice

  • Kelsey R. Pflepsen
  • Cristina D. Peterson
  • Kelley F. Kitto
  • Lucy Vulchanova
  • George L. Wilcox
  • Carolyn A. FairbanksEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1937)


Delivery of viral vectors directly into the central nervous system (CNS) has emerged as an important tool for the refinement of gene therapy. Intrathecal delivery by direct lumbar puncture in conscious rodents offers a minimally invasive approach that avoids tissue damage and/or destruction. Here we describe delivery of small quantities of viral vector product to the intrathecal space of rodents via direct lumbar puncture aided by a catheter.

Key words

Intrathecal Gene therapy AAV gene therapy Intrathecal gene delivery Direct lumbar puncture 



This work was supported by NIDA grant R01DA035931-01, DoD grant W81XWH-15-1-0494, T32 training grant T32DA07234 to K.P., and T32 training grant T32DA007097 to C.P.


  1. 1.
    Hylden JLK, Wilcox GL (1980) Intrathecal morphine in mice: a new technique. Eur J Pharmacol 67:313–316CrossRefGoogle Scholar
  2. 2.
    Mestre C, Pelissier T, Fialip J, Wilcox G, Eschalier A (1994) A method to perform direct transcutaneous intrathecal injection in rats. J Pharmacol Toxicol Methods 32(4):197–200CrossRefGoogle Scholar
  3. 3.
    Fairbanks CA (2003) Spinal delivery of analgesics in experimental models of pain and analgesia. Adv Drug Deliv Rev 55(8):1007–1041CrossRefGoogle Scholar
  4. 4.
    Vulchanova L, Schuster DJ, Belur LR, Riedl MS, Podetz-Pedersen KM, Kitto KF, Wilcox GL, McIvor RS, Fairbanks CA (2010) Differential adeno-associated virus mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture. Mol Pain 6:31. Scholar
  5. 5.
    Schuster DJ, Dykstra JA, Riedl MS, Kitto KF, Honda CN, McIvor RS, Fairbanks CA, Vulchanova L (2013) Visualization of spinal afferent innervation in the mouse colon by AAV8-mediated GFP expression. Neurogastroenterol Motil 25(2):e89–e100. Scholar
  6. 6.
    Schuster DJ, Dykstra JA, Riedl MS, Kitto KF, Belur LR, McIvor RS, Elde RP, Fairbanks CA, Vulchanova L (2014) Biodistribution of adeno-associated virus serotype 9 (AAV9) vector after intrathecal and intravenous delivery in mouse. Front Neuroanat 8:42. Scholar
  7. 7.
    Schuster DJ, Belur LR, Riedl MS, Schnell SA, Podetz-Pedersen KM, Kitto KF, McIvor RS, Vulchanova L, Fairbanks CA (2014) Supraspinal gene transfer by intrathecal adeno-associated virus serotype 5. Front Neuroanat 8:66. Scholar
  8. 8.
    Hardcastle N, Boulis NM, Federici T (2018) AAV gene delivery to the spinal cord: serotypes, methods, candidate diseases, and clinical trials. Expert Opin Biol Ther 18(3):293–307. Scholar
  9. 9.
    Dirren E, Aebischer J, Rochat C, Towne C, Schneider BL, Aebischer P (2015) SOD1 silencing in motoneurons or glia rescues neuromuscular function in ALS mice. Ann Clin Transl Neurol 2(2):167–184. Scholar
  10. 10.
    Hirai T, Enomoto M, Kaburagi H, Sotome S, Yoshida-Tanaka K, Ukegawa M, Kuwahara H, Yamamoto M, Tajiri M, Miyata H, Hirai Y, Tominaga M, Shinomiya K, Mizusawa H, Okawa A, Yokota T (2014) Intrathecal AAV serotype 9-mediated delivery of shRNA against TRPV1 attenuates thermal hyperalgesia in a mouse model of peripheral nerve injury. Mol Ther 22(2):409–419. Scholar
  11. 11.
    Shyng C, Nelvagal HR, Dearborn JT, Tyynela J, Schmidt RE, Sands MS, Cooper JD (2017) Synergistic effects of treating the spinal cord and brain in CLN1 disease. Proc Natl Acad Sci U S A 114(29):E5920–E5929. Scholar
  12. 12.
    Watson G, Bastacky J, Belichenko P, Buddhikot M, Jungles S, Vellard M, Mobley WC, Kakkis E (2006) Intrathecal administration of AAV vectors for the treatment of lysosomal storage in the brains of MPS I mice. Gene Ther 13(11):917–925. Scholar
  13. 13.
    Liu CC, Huang ZX, Li X, Shen KF, Liu M, Ouyang HD, Zhang SB, Ruan YT, Zhang XL, Wu SL, Xin WJ, Ma C (2018) Upregulation of NLRP3 via STAT3-dependent histone acetylation contributes to painful neuropathy induced by bortezomib. Exp Neurol 302:104–111. Scholar
  14. 14.
    Li YY, Li H, Liu ZL, Li Q, Qiu HW, Zeng LJ, Yang W, Zhang XZ, Li ZY (2017) Activation of STAT3-mediated CXCL12 up-regulation in the dorsal root ganglion contributes to oxaliplatin-induced chronic pain. Mol Pain 13:1744806917747425. Scholar
  15. 15.
    Zhang XS, Li X, Luo HJ, Huang ZX, Liu CC, Wan Q, Xu SW, Wu SL, Ke SJ, Ma C (2017) Activation of the RAGE/STAT3 pathway in the dorsal root ganglion contributes to the persistent pain hypersensitivity induced by lumbar disc herniation. Pain Physician 20(5):419–427PubMedGoogle Scholar
  16. 16.
    Morioka N, Zhang FF, Nakamura Y, Kitamura T, Hisaoka-Nakashima K, Nakata Y (2015) Tumor necrosis factor-mediated downregulation of spinal astrocytic connexin43 leads to increased glutamatergic neurotransmission and neuropathic pain in mice. Brain Behav Immun 49:293–310. Scholar
  17. 17.
    Morioka N, Fujii S, Kondo S, Zhang FF, Miyauchi K, Nakamura Y, Hisaoka-Nakashima K, Nakata Y (2018) Downregulation of spinal astrocytic connexin43 leads to upregulation of interleukin-6 and cyclooxygenase-2 and mechanical hypersensitivity in mice. Glia 66(2):428–444. Scholar
  18. 18.
    Towne C, Pertin M, Beggah AT, Aebischer P, Decosterd I (2009) Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery. Mol Pain 5:52. Scholar
  19. 19.
    Lu Z, Xu J, Rossi GC, Majumdar S, Pasternak GW, Pan YX (2015) Mediation of opioid analgesia by a truncated 6-transmembrane GPCR. J Clin Invest 125(7):2626–2630. Scholar
  20. 20.
    Yang X, Liu J, Liu ZJ, Xia QJ, He M, Liu R, Liu W, Wang W, Liu J, Zhou XF, Zhang YH, Wang TH (2014) Reversal of bone cancer pain by HSV-1-mediated silencing of CNTF in an afferent area of the spinal cord associated with AKT-ERK signal inhibition. Curr Gene Ther 14(5):377–388CrossRefGoogle Scholar
  21. 21.
    Zou W, Guo Q, Chen C, Yang Y, Wang E (2011) Intrathecal herpes simplex virus type 1 amplicon vector-mediated human proenkephalin reduces chronic constriction injury-induced neuropathic pain in rats. Mol Med Rep 4(3):529–533. Scholar
  22. 22.
    Hu C, Lu Y, Cheng X, Cui Y, Wu Z, Zhang Q (2017) Gene therapy for neuropathic pain induced by spared nerve injury with naked plasmid encoding hepatocyte growth factor. J Gene Med 19:12. Scholar
  23. 23.
    Vanderwall AG, Noor S, Sun MS, Sanchez JE, Yang XO, Jantzie LL, Mellios N, Milligan ED (2018) Effects of spinal non-viral interleukin-10 gene therapy formulated with d-mannose in neuropathic interleukin-10 deficient mice: Behavioral characterization, mRNA and protein analysis in pain relevant tissues. Brain Behav Immun 69:91–112. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Kelsey R. Pflepsen
    • 1
  • Cristina D. Peterson
    • 2
  • Kelley F. Kitto
    • 2
  • Lucy Vulchanova
    • 2
  • George L. Wilcox
    • 2
    • 3
    • 4
  • Carolyn A. Fairbanks
    • 1
    • 2
    • 3
    Email author
  1. 1.Department of PharmaceuticsUniversity of MinnesotaMinneapolisUSA
  2. 2.Department of NeuroscienceUniversity of MinnesotaMinneapolisUSA
  3. 3.Department of PharmacologyUniversity of MinnesotaMinneapolisUSA
  4. 4.Department of DermatologyUniversity of MinnesotaMinneapolisUSA

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