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Open Access 01-12-2013 | Research

Intraperitoneal administration of AAV9-shRNA inhibits target gene expression in the dorsal root ganglia of neonatal mice

Authors: Akira Machida, Hiroya Kuwahara, Azat Mayra, Takayuki Kubodera, Takashi Hirai, Fumiko Sunaga, Mio Tajiri, Yukihiko Hirai, Takashi Shimada, Hidehiro Mizusawa, Takanori Yokota

Published in: Molecular Pain | Issue 1/2013

Abstract

Background

There is considerable interest in inducing RNA interference (RNAi) in neurons to study gene function and identify new targets for disease intervention. Although short interfering RNAs (siRNAs) have been used to silence genes in neurons, in vivo delivery of RNAi remains a major challenge, especially by systemic administration. We have developed a highly efficient method for in vivo gene silencing in dorsal root ganglia (DRG) by using short hairpin RNA–expressing single-stranded adeno-associated virus 9 (ssAAV9-shRNA).

Results

Intraperitoneal administration of ssAAV9-shRNA to neonatal mice resulted in highly effective and specific silencing of a target gene in DRG. We observed an approximately 80% reduction in target mRNA in the DRG, and 74.7% suppression of the protein was confirmed by Western blot analysis. There were no major side effects, and the suppression effect lasted for more than three months after the injection of ssAAV9-shRNA.

Conclusions

Although we previously showed substantial inhibition of target gene expression in DRG via intrathecal ssAAV9-shRNA administration, here we succeeded in inhibiting target gene expression in DRG neurons via intraperitoneal injection of ssAAV9-shRNA. AAV9-mediated delivery of shRNA will pave the way for creating animal models for investigating the molecular biology of the mechanisms of pain and sensory ganglionopathies.

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Chen Y, Willcockson HH, Valtschanoff JG: Increased expression of CGRP in sensory afferents of arthritic mice–effect of genetic deletion of the vanilloid receptor TRPV1. Neuropeptides 2008, 42: 551–556. 10.1016/j.npep.2008.08.001PubMedCentralCrossRefPubMed

Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC: Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998, 391: 806–811. 10.1038/35888CrossRefPubMed

Dorn G, Patel S, Wotherspoon G, Hemmings-Mieszczak M, Barclay J, Natt FJ, Martin P, Bevan S, Fox A, Ganju P, et al.: siRNA relieves chronic neuropathic pain. Nucleic Acids Res 2004, 32: e49. 10.1093/nar/gnh044PubMedCentralCrossRefPubMed

Mikami M, Yang J: Short hairpin RNA-mediated selective knockdown of NaV1.8 tetrodotoxin-resistant voltage-gated sodium channel in dorsal root ganglion neurons. Anesthesiology 2005, 103: 828–836. 10.1097/00000542-200510000-00022CrossRefPubMed

Li G, Li D, Xie Q, Shi Y, Jiang S, Jin Y: RNA interfering connective tissue growth factor prevents rat hepatic stellate cell activation and extracellular matrix production. J Gene Med 2008, 10: 1039–1047. 10.1002/jgm.1223CrossRefPubMed

Brummelkamp TR, Bernards R, Agami R: A system for stable expression of short interfering RNAs in mammalian cells. Sci (New York, NY) 2002, 296: 550–553. 10.1126/science.1068999CrossRef

McCown TJ: Adeno-associated virus (AAV) vectors in the CNS. Curr Gene Ther 2005, 5: 333–338. 10.2174/1566523054064995CrossRefPubMed

Terzi D, Zachariou V: Adeno-associated virus-mediated gene delivery approaches for the treatment of CNS disorders. Biotechnol J 2008, 3: 1555–1563. 10.1002/biot.200800284CrossRefPubMed

Van Vliet K, Mohiuddin Y, McClung S, Blouin V, Rolling F, Moullier P, Agbandje-McKenna M, Snyder RO: Adeno-associated virus capsid serotype identification: analytical methods development and application. J Virol Meth 2009, 159: 167–177. 10.1016/j.jviromet.2009.03.020CrossRef

10.

Vandenberghe LH, Wilson JM, Gao G: Tailoring the AAV vector capsid for gene therapy. Gene Ther 2009, 16: 311–319. 10.1038/gt.2008.170CrossRefPubMed

11.

Xu Q, Chou B, Fitzsimmons B, Miyanohara A, Shubayev V, Santucci C, Hefferan M, Marsala M, Hua XY: In vivo gene knockdown in rat dorsal root ganglia mediated by self-complementary adeno-associated virus serotype 5 following intrathecal delivery. PLoS One 2012, 7: e32581. 10.1371/journal.pone.0032581PubMedCentralCrossRefPubMed

12.

Hirai T, Enomoto M, Machida A, Yamamoto M, Kuwahara H, Tajiri M, Hirai Y, Sotome S, Mizusawa H, Shinomiya K, et al.: Intrathecal shRNA-AAV9 inhibits target protein expression in the spinal cord and dorsal root ganglia of adult mice. Hum Gene Ther Part B Meth 2012, 23: 119–127. 10.1089/hgtb.2012.035CrossRef

13.

Zheng H, Qiao C, Wang CH, Li J, Li J, Yuan Z, Zhang C, Xiao X: Efficient retrograde transport of adeno-associated virus type 8 to spinal cord and dorsal root ganglion after vector delivery in muscle. Hum Gene Ther 2010, 21: 87–97. 10.1089/hum.2009.131PubMedCentralCrossRefPubMed

14.

Foust KD, Nurre E, Montgomery CL, Hernandez A, Chan CM, Kaspar BK: Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol 2009, 27: 59–65. 10.1038/nbt.1515PubMedCentralCrossRefPubMed

15.

Miyake N, Miyake K, Yamamoto M, Hirai Y, Shimada T: Global gene transfer into the CNS across the BBB after neonatal systemic delivery of single-stranded AAV vectors. Brain Res 2011, 1389: 19–26.CrossRefPubMed

16.

Wang DB, Dayton RD, Henning PP, Cain CD, Zhao LR, Schrott LM, Orchard EA, Knight DS, Klein RL: Expansive gene transfer in the rat CNS rapidly produces amyotrophic lateral sclerosis relevant sequelae when TDP-43 is overexpressed. Mol Ther J Am Soc Gene Ther 2010, 18: 2064–2074. 10.1038/mt.2010.191CrossRef

17.

Reaume AG, Elliott JL, Hoffman EK, Kowall NW, Ferrante RJ, Siwek DF, Wilcox HM, Flood DG, Beal MF, Brown RH Jr, et al.: Motor neurons in Cu/Zn superoxide dismutase-deficient mice develop normally but exhibit enhanced cell death after axonal injury. Nat Genet 1996, 13: 43–47. 10.1038/ng0596-43CrossRefPubMed

18.

Kawase M, Murakami K, Fujimura M, Morita-Fujimura Y, Gasche Y, Kondo T, Scott RW, Chan PH: Exacerbation of delayed cell injury after transient global ischemia in mutant mice with CuZn superoxide dismutase deficiency. Stroke J Cereb Circ 1999, 30: 1962–1968. 10.1161/01.STR.30.9.1962CrossRef

19.

Mayra A, Tomimitsu H, Kubodera T, Kobayashi M, Piao W, Sunaga F, Hirai Y, Shimada T, Mizusawa H, Yokota T: Intraperitoneal AAV9-shRNA inhibits target expression in neonatal skeletal and cardiac muscles. Biochem Biophys Res Commun 2011, 405: 204–209. 10.1016/j.bbrc.2011.01.009CrossRefPubMed

20.

Storek B, Reinhardt M, Wang C, Janssen WG, Harder NM, Banck MS, Morrison JH, Beutler AS: Sensory neuron targeting by self-complementary AAV8 via lumbar puncture for chronic pain. Proc Natl Acad Sci USA 2008, 105: 1055–1060. 10.1073/pnas.0708003105PubMedCentralCrossRefPubMed

21.

Foust KD, Wang X, McGovern VL, Braun L, Bevan AK, Haidet AM, Le TT, Morales PR, Rich MM, Burghes AH, Kaspar BK: Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN. Nat Biotechnol 2010, 28: 271–274. 10.1038/nbt.1610PubMedCentralCrossRefPubMed

22.

Ehlert EM, Eggers R, Niclou SP, Verhaagen J: Cellular toxicity following application of adeno-associated viral vector-mediated RNA interference in the nervous system. BMC Neurosci 2010, 11: 20. 10.1186/1471-2202-11-20PubMedCentralCrossRefPubMed

23.

Anesti AM, Peeters PJ, Royaux I, Coffin RS: Efficient delivery of RNA Interference to peripheral neurons in vivo using herpes simplex virus. Nucleic Acids Res 2008, 36: e86. 10.1093/nar/gkn371PubMedCentralCrossRefPubMed

24.

Flotte TR, Conlon TJ, Poirier A, Campbell-Thompson M, Byrne BJ: Preclinical characterization of a recombinant adeno-associated virus type 1-pseudotyped vector demonstrates dose-dependent injection site inflammation and dissemination of vector genomes to distant sites. Hum Gene Ther 2007, 18: 245–256. 10.1089/hum.2006.113CrossRefPubMed

25.

Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey K, Davis CR, Marion P, Salazar F, Kay MA: Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 2006, 441: 537–541. 10.1038/nature04791CrossRefPubMed

26.

Wang Z, Zhu T, Qiao C, Zhou L, Wang B, Zhang J, Chen C, Li J, Xiao X: Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart. Nat Biotechnol 2005, 23: 321–328. 10.1038/nbt1073CrossRefPubMed

27.

Bostick B, Ghosh A, Yue Y, Long C, Duan D: Systemic AAV-9 transduction in mice is influenced by animal age but not by the route of administration. Gene Ther 2007, 14: 1605–1609. 10.1038/sj.gt.3303029CrossRefPubMed

28.

Abbott NJ, Patabendige AA, Dolman DE, Yusof SR, Begley DJ: Structure and function of the blood-brain barrier. Neurobiol Dis 2010, 37: 13–25. 10.1016/j.nbd.2009.07.030CrossRefPubMed

29.

Persidsky Y, Ramirez SH, Haorah J, Kanmogne GD: Blood-brain barrier: structural components and function under physiologic and pathologic conditions. J Neuroimmune Pharmacol Offic J Soc NeuroImmune Pharmacol 2006, 1: 223–236. 10.1007/s11481-006-9025-3CrossRef

30.

Hirakawa H, Okajima S, Nagaoka T, Kubo T, Takamatsu T, Oyamada M: Regional differences in blood-nerve barrier function and tight-junction protein expression within the rat dorsal root ganglion. Neuroreport 2004, 15: 405–408. 10.1097/00001756-200403010-00004CrossRefPubMed

31.

Yaksh TL: Spinal Drug Delivery. The Netherlands: Elsevier Health Science; 2009.

32.

Foust KD, Poirier A, Pacak CA, Mandel RJ, Flotte TR: Neonatal intraperitoneal or intravenous injections of recombinant adeno-associated virus type 8 transduce dorsal root ganglia and lower motor neurons. Hum Gene Ther 2008, 19: 61–70. 10.1089/hum.2007.093CrossRefPubMed

33.

Luo MC, Zhang DQ, Ma SW, Huang YY, Shuster SJ, Porreca F, Lai J: An efficient intrathecal delivery of small interfering RNA to the spinal cord and peripheral neurons. Mol Pain 2005, 1: 29. 10.1186/1744-8069-1-29PubMedCentralCrossRefPubMed

34.

Stucky CL, Dubin AE, Jeske NA, Malin SA, McKemy DD, Story GM: Roles of transient receptor potential channels in pain. Brain Res Rev 2009, 60: 2–23. 10.1016/j.brainresrev.2008.12.018PubMedCentralCrossRefPubMed

35.

Garraway SM, Xu Q, Inturrisi CE: siRNA-mediated knockdown of the NR1 subunit gene of the NMDA receptor attenuates formalin-induced pain behaviors in adult rats. J Pain Offic J Am Pain Soc 2009, 10: 380–390. 10.1016/j.jpain.2008.09.013CrossRef

36.

Tan PH, Yang LC, Shih HC, Lan KC, Cheng JT: Gene knockdown with intrathecal siRNA of NMDA receptor NR2B subunit reduces formalin-induced nociception in the rat. Gene Ther 2005, 12: 59–66. 10.1038/sj.gt.3302376CrossRefPubMed

37.

Manno CS, Pierce GF, Arruda VR, Glader B, Ragni M, Rasko JJ, Ozelo MC, Hoots K, Blatt P, Konkle B, et al.: Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med 2006, 12: 342–347. 10.1038/nm1358CrossRefPubMed

38.

Saito Y, Yokota T, Mitani T, Ito K, Anzai M, Miyagishi M, Taira K, Mizusawa H: Transgenic small interfering RNA halts amyotrophic lateral sclerosis in a mouse model. J Biol Chem 2005, 280: 42826–42830. 10.1074/jbc.M507685200CrossRefPubMed

39.

Yokota T, Miyagishi M, Hino T, Matsumura R, Tasinato A, Urushitani M, Rao RV, Takahashi R, Bredesen DE, Taira K, Mizusawa H: siRNA-based inhibition specific for mutant SOD1 with single nucleotide alternation in familial ALS, compared with ribozyme and DNA enzyme. Biochem Biophys Res Commun 2004, 314: 283–291. 10.1016/j.bbrc.2003.12.098CrossRefPubMed

40.

Hermens WT, ter Brake O, Dijkhuizen PA, Sonnemans MA, Grimm D, Kleinschmidt JA, Verhaagen J: Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system. Hum Gene Ther 1999, 10: 1885–1891. 10.1089/10430349950017563CrossRefPubMed

41.

Taymans JM, Vandenberghe LH, Haute CV, Thiry I, Deroose CM, Mortelmans L, Wilson JM, Debyser Z, Baekelandt V: Comparative analysis of adeno-associated viral vector serotypes 1, 2, 5, 7, and 8 in mouse brain. Hum Gene Ther 2007, 18: 195–206. 10.1089/hum.2006.178CrossRefPubMed

42.

Hargreaves K, Dubner R, Brown F, Flores C, Joris J: A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 1988, 32: 77–88. 10.1016/0304-3959(88)90026-7CrossRefPubMed

Title: Intraperitoneal administration of AAV9-shRNA inhibits target gene expression in the dorsal root ganglia of neonatal mice
Authors: Akira Machida
Hiroya Kuwahara
Azat Mayra
Takayuki Kubodera
Takashi Hirai
Fumiko Sunaga
Mio Tajiri
Yukihiko Hirai
Takashi Shimada
Hidehiro Mizusawa
Takanori Yokota
Publication date: 01-12-2013
Publisher: BioMed Central
Published in: Molecular Pain / Issue 1/2013
Electronic ISSN: 1744-8069
DOI: https://doi.org/10.1186/1744-8069-9-36

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Intraperitoneal administration of AAV9-shRNA inhibits target gene expression in the dorsal root ganglia of neonatal mice

Abstract

Background

Results

Conclusions

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Background

Results

Conclusions

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