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. 2011 Mar;19(3):526-35.
doi: 10.1038/mt.2010.279. Epub 2010 Dec 21.

MicroRNA-regulated, systemically delivered rAAV9: a step closer to CNS-restricted transgene expression

Jun Xie  1 , 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

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.

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Figures

Figure 1
Figure 1
In vitro validation of artificial miRNA-binding sites for reporter silencing. Plasmids harboring the rAAVCBnLacZ 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 (prAAVCBnLacZ). CB, chicken β-actin; miR, microRNA; nLacZ, β-galactosidase reporter transgene; rAAV, recombinant adeno-associated viruses.
Figure 2
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 rAAV9CBnLacZ (no binding site), (a) rAAVCB9nLacZ-miR-122BS (one miR-122-binding site) and rAAV9CBnLacZ-(miR-122BS)3 (three miR-122-binding sites), (b) rAAV9CBnLacZ-miR-1BS (one miR-1 binding site) and rAAV9CBnLacZ-(miR-1BS)3 (three miR-1-binding sites, and (c) rAAV9CBnLacZ-miR-1BS-miR-122BS (1X each binding site) and rAAV9CBnLacZ-(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
Figure 3
Analysis of expression levels of cognate miRNA, mRNA, and protein of endogenous miRNA target genes in mice transduced with rAAV9CBnLacZ 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
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
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 rAAV9CBnLacZ-(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
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 scAAV9CBnLacZ-(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
Figure 7
A molecular model for endogenous miRNA-regulated rAAV expression. miRNA, microRNA; rAAV, recombinant adeno-associated viruses.
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