MicroRNA-regulated, systemically delivered rAAV9: a step closer to CNS-restricted transgene expression - PubMed (original) (raw)
doi: 10.1038/mt.2010.279. Epub 2010 Dec 21.
Qing Xie, Hongwei Zhang, Stefan L Ameres, Jui-Hung Hung, Qin Su, Ran He, Xin Mu, Seemin Seher Ahmed, Soyeon Park, Hiroki Kato, Chengjian Li, Christian Mueller, Craig C Mello, Zhiping Weng, Terence R Flotte, Phillip D Zamore, Guangping Gao
Affiliations
- PMID: 21179009
- PMCID: PMC3048189
- DOI: 10.1038/mt.2010.279
MicroRNA-regulated, systemically delivered rAAV9: a step closer to CNS-restricted transgene expression
Jun Xie et al. Mol Ther. 2011 Mar.
Abstract
Recombinant adeno-associated viruses (rAAVs) that can cross the blood-brain-barrier and achieve efficient and stable transvascular gene transfer to the central nervous system (CNS) hold significant promise for treating CNS disorders. However, following intravascular delivery, these vectors also target liver, heart, skeletal muscle, and other tissues, which may cause untoward effects. To circumvent this, we used tissue-specific, endogenous microRNAs (miRNAs) to repress rAAV expression outside the CNS, by engineering perfectly complementary miRNA-binding sites into the rAAV9 genome. This approach allowed simultaneous multi-tissue regulation and CNS-directed stable transgene expression without detectably perturbing the endogenous miRNA pathway. Regulation of rAAV expression by miRNA was primarily via site-specific cleavage of the transgene mRNA, generating specific 5' and 3' mRNA fragments. Our findings promise to facilitate the development of miRNA-regulated rAAV for CNS-targeted gene delivery and other applications.
Figures
Figure 1
In vitro validation of artificial miRNA-binding sites for reporter silencing. Plasmids harboring the rAAVCB_nLacZ_ genome with or without miR-1 or miR-122-binding sites were transfected into human hepatoma (HuH7) cells (a) which express miR-122 or cotransfected into 293 cells, together with a plasmid expressing either pri-miR-122 (b) or pri-miR-1 (c) at molar ratios of 1:3 (low) or 1:10 (high). 0X: no miRNA-binding site; 1X: one miRNA-binding site; 3X: three miRNA-binding sites. The cells were fixed and stained histochemically with X-gal 48 hours after transfection and blue cells counted. The percentage of nLacZ-positive cells in each transfection were compared to transfection of the control plasmid (prAAVCB_nLacZ_). CB, chicken β-actin; miR, microRNA; nLacZ, β-galactosidase reporter transgene; rAAV, recombinant adeno-associated viruses.
Figure 2
In vivo evaluation of endogenous miRNA-mediated transgene silencing in rAAV9 transduction. (a–c) Adult male C58BL/6 mice were injected intravenously with 5 × 1013 genome copies per kg (GC/kg) each of rAAV9CB_nLacZ_ (no binding site), (a) rAAVCB9_nLacZ-miR-122BS_ (one miR-122-binding site) and rAAV9CB_nLacZ-(miR-122BS)_ 3 (three miR-122-binding sites), (b) rAAV9CB_nLacZ-miR-1BS_ (one miR-1 binding site) and rAAV9CB_nLacZ-(miR-1BS)_ 3 (three miR-1-binding sites, and (c) rAAV9CB_nLacZ-miR-1BS-miR-122BS_ (1X each binding site) and rAAV9CB_nLacZ-(miR-1BS)_ 3 –(miR-122BS) 3 (three miR-1 and three miR-122-binding sites). The animals were necropsied 4 weeks after vector administration, and appropriate tissues were harvested for cryosectioning and X-gal histochemical staining. miR, microRNA; nLacZ, β-galactosidase reporter transgene; rAAV, recombinant adeno-associated viruses.
Figure 3
Analysis of expression levels of cognate miRNA, mRNA, and protein of endogenous miRNA target genes in mice transduced with rAAV9CB_nLacZ_ with or without miRNA-binding sites. Total cellular RNA or protein was prepared from (a–c) liver or (d) heart. (a) Northern blot detection of miRNAs. U6 small nuclear RNA provides a loading control. (b) Quantitative reverse-transcription PCR measuring cyclin G1 mRNA. The data are presented as relative cyclin G1 mRNA levels normalized to β-actin. (c,d) Western blot analyses of protein levels of endogenous targets of miR-122 and miR-1. Total cellular protein prepared from (c) liver or (d) heart was analyzed for cyclin G1 and calmodulin. (e) Serum cholesterol levels. Serum samples from mice that received rAAV9 with or without miRNA-binding sites were collected after 4 weeks and measured for total cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL). miR, microRNA; nLacZ, β-galactosidase reporter transgene; rAAV, recombinant adeno-associated viruses.
Figure 4
Molecular characterization of transgene mRNAs with or without miRNA-binding sites. (a) Locations of the probes and primers, the sequences of mature miR-122 and its perfectly complementary binding site in the transgene mRNA are presented. (b) Total cellular RNA from liver was analyzed either by conventional reverse-transcription PCR (RT-PCR) by using primers that span a region between the 3′ end of nLacZ and the 5′ end of poly(A) signal (c) or by quantitative RT-PCR; data are presented as relative nLacZ mRNA levels normalized to β-actin. (d) For the northern blot analysis of nLacZ mRNA, 18S RNA served as a loading control, and the blots were hybridized with either a transgene DNA (e) or RNA probe. (f) In addition, poly(A) bearing mRNA from the liver of an animal received rAAV containing three miR-1- and three miR-122-binding sites was analyzed by 5′ RACE; the PCR product was resolved on an ethidium bromide-stained agarose gel. miR, microRNA; nLacZ, β-galactosidase reporter transgene; rAAV, recombinant adeno-associated viruses.
Figure 5
Alignment of sequences spanning the miRNA-binding sites and poly(A) signal regions recovered by 5′ RACE. Poly(A)-containing mRNA was isolated from the (a) liver and (b) heart of an animal injected with rAAV9CB_nLacZ-(miR-1BS)_ 3 –(miR-122BS) 3. Twenty-one liver-derived and 22 heart-derived clones were sequenced. The putative cleavage sites in each clone are identified by arrows; the frequencies of miRNA-directed, site-specific cleavage for each miRNA-binding site are reported; triangles point to the positions of the expected miRNA-directed cleavage sites (a,b). miRNA, microRNA, nLacZ, β-galactosidase reporter transgene; rAAV, recombinant adeno-associated viruses.
Figure 6
Endogenous miRNA-repressed, CNS-directed EGFP gene transfer by systemically delivered rAAV9. Ten-week-old male C57BL/6 mice were injected intravenously with scAAV9CBEGFP or scAAV9CB_nLacZ-(miR-1BS)_ 3 –(miR-122BS) 3 at a dose of 2 × 1014 genome copies per kg (GC/kg) body weight. The animals were necropsied 3 weeks later for whole body fixation by transcardiac perfusion. (a) Brain, spinal cord, liver, heart, and muscle were harvested for cryosectioning, immunofluorescent staining for EGFP (brain and cervical spinal cord), and fluorescence microscopy to detect EGFP. Total cellular DNA and RNA were extracted from brain, liver, heart and muscle to measure the amount of persistent vector genome by qPCR and EGFP mRNA by qRT-PCR. (b) For each tissue, the relative abundance of the EGFP mRNA containing miRNA-binding sites was compared to that of the EGFP mRNA lacking miRNA-binding sites. For each sample, mRNA abundance was normalized to the amount of vector genome detected in the tissue. EGFP, enhanced green fluorescent protein; miRNA, microRNA; nLacZ, β-galactosidase reporter transgene; qRT-PCR, quantitative reverse-transcription PCR; rAAV, recombinant adeno-associated viruses.
Figure 7
A molecular model for endogenous miRNA-regulated rAAV expression. miRNA, microRNA; rAAV, recombinant adeno-associated viruses.
Similar articles
- Application of mutated miR-206 target sites enables skeletal muscle-specific silencing of transgene expression of cardiotropic AAV9 vectors.
Geisler A, Schön C, Größl T, Pinkert S, Stein EA, Kurreck J, Vetter R, Fechner H. Geisler A, et al. Mol Ther. 2013 May;21(5):924-33. doi: 10.1038/mt.2012.276. Epub 2013 Feb 26. Mol Ther. 2013. PMID: 23439498 Free PMC article. - Circumventing cellular immunity by miR142-mediated regulation sufficiently supports rAAV-delivered OVA expression without activating humoral immunity.
Xiao Y, Muhuri M, Li S, Qin W, Xu G, Luo L, Li J, Letizia AJ, Wang SK, Chan YK, Wang C, Fuchs SP, Wang D, Su Q, Nahid MA, Church GM, Farzan M, Yang L, Wei Y, Desrosiers RC, Mueller C, Tai PW, Gao G. Xiao Y, et al. JCI Insight. 2019 May 21;5(13):e99052. doi: 10.1172/jci.insight.99052. JCI Insight. 2019. PMID: 31112525 Free PMC article. - Intravenous Infusion of AAV for Widespread Gene Delivery to the Nervous System.
Gessler DJ, Tai PWL, Li J, Gao G. Gessler DJ, et al. Methods Mol Biol. 2019;1950:143-163. doi: 10.1007/978-1-4939-9139-6_8. Methods Mol Biol. 2019. PMID: 30783972 Free PMC article. - Recombinant adeno-associated viral vectors in the nervous system.
Burger C, Nash K, Mandel RJ. Burger C, et al. Hum Gene Ther. 2005 Jul;16(7):781-91. doi: 10.1089/hum.2005.16.781. Hum Gene Ther. 2005. PMID: 16000060 Review. - Progress in microRNA delivery.
Zhang Y, Wang Z, Gemeinhart RA. Zhang Y, et al. J Control Release. 2013 Dec 28;172(3):962-74. doi: 10.1016/j.jconrel.2013.09.015. Epub 2013 Sep 25. J Control Release. 2013. PMID: 24075926 Free PMC article. Review.
Cited by
- Sub-genomic flaviviral RNA elements increase the stability and abundance of recombinant AAV vector transcripts.
Meganck RM, Ogurlu R, Liu J, Moller-Tank S, Tse V, Blondel LO, Rosales A, Hall AC, Vincent HA, Moorman NJ, Marzluff WF, Asokan A. Meganck RM, et al. J Virol. 2024 Aug 20;98(8):e0009524. doi: 10.1128/jvi.00095-24. Epub 2024 Jul 31. J Virol. 2024. PMID: 39082815 Free PMC article. - Adeno-associated virus as a delivery vector for gene therapy of human diseases.
Wang JH, Gessler DJ, Zhan W, Gallagher TL, Gao G. Wang JH, et al. Signal Transduct Target Ther. 2024 Apr 3;9(1):78. doi: 10.1038/s41392-024-01780-w. Signal Transduct Target Ther. 2024. PMID: 38565561 Free PMC article. Review. - Novel AAV variants with improved tropism for human Schwann cells.
Drouyer M, Chu TH, Labit E, Haase F, Navarro RG, Nazareth D, Rosin N, Merjane J, Scott S, Cabanes-Creus M, Westhaus A, Zhu E, Midha R, Alexander IE, Biernaskie J, Ginn SL, Lisowski L. Drouyer M, et al. Mol Ther Methods Clin Dev. 2024 Mar 11;32(2):101234. doi: 10.1016/j.omtm.2024.101234. eCollection 2024 Jun 13. Mol Ther Methods Clin Dev. 2024. PMID: 38558569 Free PMC article. - Development of AAV-Mediated Gene Therapy Approaches to Treat Skeletal Diseases.
Lin C, Greenblatt MB, Gao G, Shim JH. Lin C, et al. Hum Gene Ther. 2024 May;35(9-10):317-328. doi: 10.1089/hum.2024.022. Epub 2024 Apr 8. Hum Gene Ther. 2024. PMID: 38534217 Review. - Improved gene therapy for spinal muscular atrophy in mice using codon-optimized hSMN1 transgene and hSMN1 gene-derived promotor.
Xie Q, Chen X, Ma H, Zhu Y, Ma Y, Jalinous L, Cox GF, Weaver F, Yang J, Kennedy Z, Gruntman A, Du A, Su Q, He R, Tai PW, Gao G, Xie J. Xie Q, et al. EMBO Mol Med. 2024 Apr;16(4):945-965. doi: 10.1038/s44321-024-00037-x. Epub 2024 Feb 27. EMBO Mol Med. 2024. PMID: 38413838 Free PMC article.
References
- Kaplitt MG, Feigin A, Tang C, Fitzsimons HL, Mattis P, Lawlor PA, et al. Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trial. Lancet. 2007;369:2097–2105. - PubMed
- Federici T., and, Boulis NM. Invited review: festschrift edition of neurosurgery peripheral nervous system as a conduit for delivering therapies for diabetic neuropathy, amyotrophic lateral sclerosis, and nerve regeneration. Neurosurgery. 2009;65 4 Suppl:A87–A92. - PubMed
- Bjorklund T., and, Kordower JH. Gene therapy for Parkinson's disease. Mov Disord. 2010;25 Suppl 1:S161–S173. - PubMed
- Vandenberghe LH, Wilson JM., and, Gao G. Tailoring the AAV vector capsid for gene therapy. Gene Ther. 2009;16:311–319. - PubMed
Publication types
MeSH terms
Substances
Grants and funding
- P30 DK032520/DK/NIDDK NIH HHS/United States
- P01 DK058327/DK/NIDDK NIH HHS/United States
- R01 GM065236/GM/NIGMS NIH HHS/United States
- 5P30DK 32520/DK/NIDDK NIH HHS/United States
- P01 DK 58327/DK/NIDDK NIH HHS/United States
- DK 32520/DK/NIDDK NIH HHS/United States
- P01 HL059412/HL/NHLBI NIH HHS/United States
- R37 GM062862/GM/NIGMS NIH HHS/United States
- HHMI/Howard Hughes Medical Institute/United States
LinkOut - more resources
Full Text Sources
Other Literature Sources