Here we describe the delivery of microRNA using a recombinant adeno-associated virus serotype 9 in a mouse model of a neuromuscular disease. A single peripheral administration in mice resulted in sustained miRNA overexpression in muscle and motor neurons, providing an opportunity to study miRNA function and therapeutic potential in vivo.
RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno-associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice.
MiRNAs are non-coding RNAs, 21-23 nucleotides in length, that play an important role in the regulation of gene expression and control of diverse cellular and metabolic pathways.1 Gene expression is mainly regulated by inducing post-transcriptional gene silencing or mRNA degradation.2 MiRNAs are usually complementary to the 3' untranslated region (UTR) of coding genes, although binding to the 5'UTR and coding regions of the target mRNA has also been described.3
As the current understanding of the roles of miRNA in the pathogenesis of human diseases expands, pharmacological modulation of individual miRNAs or miRNA families is increasingly becoming a viable therapeutic option. Compared to other RNA inhibition strategies, miRNAs have many advantages: miRNAs are less toxic and less immunogenic and can be easily delivered into cells because of their small size.4,5,6 MiRNAs typically have many targets within cellular networks, therefore potential off-target effects and safety concerns need to be taken into account, together with efficient delivery to target tissues.
Neuromuscular diseases are acquired or inherited conditions that affect muscle and motor neurons. The targeting of drugs to skeletal muscle is an emerging area of research, where the main challenge is to achieve widespread distribution within the therapeutic window.7 Motor neurons are more difficult to target, mainly because drug access is precluded by the blood brain barrier.
Cell culture and mouse models of spinal and bulbar muscular atrophy (SBMA) were used in this study. SBMA is a neuromuscular disease caused by a toxic gain of function mechanism, in which both muscle and motor neurons are affected.8,9 SBMA (Kennedy's disease; OMIM #313200) is an X-linked disease, characterized by muscle weakness and atrophy, caused by CAG repeat expansion in the AR gene, which encodes an extended polyglutamine tract in the AR protein.10 No disease-modifying treatment is currently available for this disorder. The transgenic mouse model used in this study recapitulates the features of the disease, including the gender specificity, motor-neuron pathology, and progressive muscle atrophy.11
In this study, our efforts focused on identifying a miRNA that directly downregulates expression of the mutant AR transgene and on designing a safe and efficient mode of delivery of the miRNA to the spinal cord and skeletal muscle of our disease mouse model.
Here we identified a relatively uncharacterized miRNA, miRNA-298 (accession number MIMAT0004901),12 to directly reduce mutant AR expression in SBMA models. In order to achieve delivery of miR-298 to target tissues, we used a viral strategy, with recombinant adeno-associated virus serotype 9 (rAAV9). rAAV9 is capable of crossing the blood brain barrier and mediating long-term gene expression in non-dividing cells, including neurons.13 A single systemic administration of AAV9-miR-298 resulted in sustained expression of the miRNA, efficient transduction of muscle and motor neurons, down-regulation of AR expression, and amelioration of the disease phenotype in SBMA mice.14 This methodology can be used to provide miRNA or antagomirs overexpression in vivo.
All procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th ed., National Academies Press, Revised 2011), and have been approved by the NINDS Animal Care Committee.
1. AAV9-miRNA design strategy and miRNA selection
2. Tail Vein Injection of AAV-miRNA Plasmid
NOTE: This step needs adjustment according to the target gene and the age and weight of the mice. Use the Institutional and Animal Care and Use Committee (IACUC) guidelines to determine the dose range, volume and best route of administration based on the age and weight of the mice. GFP fluorescence signal and miRNA expression levels in target tissues are expected to peak starting 2 weeks after the injection. The following protocol refers to male mice in early adulthood. AAV should be handled as a biohazard under Biosafety Level 1 guidelines.
3. Behavioral Assays
NOTE: All experiments were conducted blindly by a third party with concealment of treatments by uniquely coded vials. Order of treatments was randomized. When performing the tests described below, experimental conditions (type of room and time of the day) should be controlled to reduce variation. This test should be performed on mice older than four weeks to attain reliable results. Mouse underwent behavioral assessment once before viral injection at 5 weeks of age to obtain baseline of normal performance. After viral injection, mice were assessed once a week starting 48 h after injection, up to 40 weeks of age.
4. Euthanasia and Tissue Harvest
A viral load of 1011 vg of AAV9-miR-298 was injected through a single tail-vein injection into 5 week old SBMA mice. These mice carry the human AR transgene with abnormally expanded polyglutamine tract in the AR (AR97Q) and develop signs of neuromuscular disease by 10 weeks of age (weight loss, hunched back, and muscle atrophy).11 Lumbar spinal cord and quadriceps muscle were harvested at 2, 4, 8 and 12 weeks after administration for miRNA quantification, biochemical assay and immunohistochemistry (Figure 1). Administration of the treatment and the subsequent analyses were performed by blinded investigators.
qRT-PCR analysis showed miR-298 expression in the skeletal muscle and the spinal cord two weeks and four weeks after treatment respectively, with peak expression levels at 8 weeks in the skeletal muscle and 12 weeks in the spinal cord after the injection (Figure 2). Using a microscope (Axiovert 100 M), green fluorescence signal was detected in muscle tissue and in spinal motor neurons by co-localization of GFP and the motor neuron marker choline acetyltransferase (ChAT) 10 weeks after treatment, when the mice start to show disease manifestations (Figure 2).
Using the same dose regimen, a cohort of SBMA mice was randomized to receive either miR-298 or mock at 7 weeks of age via tail vein injection for biochemical analyses and functional characterization. Injection was followed by weekly weight and behavioral assessment up to 40 weeks of age. qRT-PCR analysis showed that miR-298 treatment reduces the levels of mutant AR in affected tissues (Figure 3), and increased body weight and improved motor performance (Figure 4) starting at 10 weeks after the injection.
Figure 1: Schematic of study design. To increase expression levels of miR-298 in vivo, we injected SBMA mice with (A) the dual promoter AAV vector plasmid expressing GFP and either miR-298 or mock. (B) Mice were injected via a single tail-vain injection at 7 weeks of age (pre-symptomatic stage). Spinal cord and quadriceps muscle were collected from a cohort of SBMA mice at different time points for tissue distribution analysis (green). A cohort of SBMA mice was treated and sacrificed at 16 weeks of age for biochemical analyses (red). Weight and behavioral assays were performed weekly up to 40 weeks of age for functional characterization (blue). Please click here to view a larger version of this figure.
Figure 2: AAV9-miR-298 delivery in mice. Using the method described here, mice received either AAV9-miR-298-GFP or AAV9-mock-GFP through intravenous injection. Total miRNA was collected from lumbar spinal cord and quadriceps muscle at 2,4,8 and 12 weeks after injection. qRT-PCR was performed to estimate expression level of miR-298 in (A) quadriceps muscle (n = 5) (B) lumbar spinal cord (n = 5, P <0.01). All data are reported as means ± standard error mean. The widespread transduction of the AAV vector in tissues harvested at 10 weeks after treatment was confirmed by localization of staining for GFP in the (C) quadriceps muscle (original magnification, 10X. Scale bar = 100 µm) and (D) motor neurons in lumbar spinal cord (original magnification, 40X. Scale bar = 10 µm. GFP (green), ChAT (red) and DAPI (blue). The figure has been modified from doi: 10.1038/mt.2016.13.14 Please click here to view a larger version of this figure.
Figure 3: MiRNA-298 over-expression downregulates mutant AR in spinal cord and quadriceps muscle in mice. qRT-PCR was performed to estimate expression levels of AR mRNA in (A) lumbar spinal cord and (B) quadriceps muscle treated with AAV9-miR-298-GFP or AAV9-mock-GFP. Transcript levels were normalized to snoRNA202 (n = 5 per treatment). *P <0.05, **P <0.01. All data are reported as means ± standard errors. The figure has been modified from doi: 10.1038/mt.2016.13.14
Figure 4: MiR-298 over-expression improves motor function and reduces weight loss. Behavioral assessment was performed once a week (w), between week 5 to 40. Body weight (left) and hanging wire (right) performance of mice (n = 15 per group). All data are reported as means ± standard errors. The figure has been modified from doi: 10.1038/mt.2016.13.14
Here we demonstrate a highly efficient and accessible methodology to select and deliver via tail injection a miRNA using rAAV9 as a viral vector to target skeletal muscle and motor neurons in mice.14 Compared to other RNAi strategies, miRNAs are less toxic and less immunogenic.18 In addition, their small size make them well suited to the limited packaging capacity of viral vectors.2 Computational algorithms and prediction tools allow the identification of putative miRNA-mRNA targets. Once identified, the effects of miRNA on target gene expression must be verified. A common approach is to overexpress a given miRNA in vitro and detect target protein expression levels using western analysis.14,19
Various studies have used viral and non-viral strategies for delivering miRNAs.13 Here we used adeno-associated virus (AAV) as a gene delivery tool. Compared to other viral vectors, AAV elicits low immunogenicity, allows long-term gene transfer, and has a broad spectrum of tropism in dividing and non-dividing cells.18 This delivery method can also circumvent the need for chemical modification that may affect functionality and specificity of the RNA molecule. There are several serotypes of AAV, which are mostly determined by the composition of the capsid proteins. These serotypes differ in their tropism and transduce different cell types. It is necessary to select the correct serotype when considering the target tissue. We selected rAAV9, due to its high transduction efficiency in the central nervous system and skeletal muscle following peripheral administration19,20. This approach has shown greater transduction efficacy in neonatal animals compared to adult animals, likely due to differences in extracellular matrix composition, neuron-to-glia ratio, and maturity of the blood-brain-barrier.21,22
An important step in this protocol is the design of the expression vector. Compared with bicistronic vectors, which are hampered by lower expression of the second gene compared with the first gene next to the promoter, the dual promoter vector allows a back-to-back configuration yielding high expression of both the miRNA and EGFP, thus allowing localization of the miRNA in mouse tissue by immunofluorescence.
Intravenous injection of AAV9 resulted in high efficiency and homogenous transduction in the target tissues, skeletal muscle, and motor neurons. This route of injection allows the administered dose to reach the systemic circulation and cross the brain blood barrier, which is important for therapies targeting the central nervous system. Furthermore, it is a non-invasive method for delivery to the CNS. MiR-298 expression increased in the spinal cord and muscle after 2-4 weeks with a single peripheral injection at 5 weeks of age. When using single-stranded genome AAV vectors, the de novo synthesis of the second DNA strand may account for the delayed transduction.23
Levels of human miR-298 were undetectable in treated mice 20 weeks after a single administration, suggesting that for chronic diseases, multiple injections may be required to reach a therapeutic benefit. However, miRNA degradation over time can be limiting when a lifelong repeated administration is required. In addition, adaptive immunity to AAV vectors can form another barrier to a successful gene delivery.24 Thus generation of AAV vectors that are immunologically inert is crucial for repeated administration and achieving long-term effects with this promising delivery system.25
A limitation on the use of miRNA as a therapeutic strategy is the risk of off-target effects. MiRNAs may interact through incomplementary base pairing with other gene transcripts, which poses a safety risk with this approach. Improved designing of the RNAi sequences, including use of non-canonical miRNAs, such as mirtrons, which bypass the micro-processor complex and extensive long-term safety and tolerability studies are critical before translating this strategy into a safe and effective therapy.26,27,28
The method described here was originally developed for miRNA overexpression, but it can also be utilized for miRNA inhibition therapy using antagomirs.
The authors have nothing to disclose.
The authors declare no conflict of interest. We thank SignaGen Laboratories (Rockvile, MD, USA) for the AAV9 virus production. This research was supported by the Intramural Research Program of the NINDS, NIH. Philip R. Lee was supported by funds from the Division of Intramural Research of NICHD. Carlo Rinaldi was supported by a fellowship from the Association Française contre les Myopathies (AFM).
QIAzol Lysis Reagent | Qiagen | 79306 | Lysis of fatty and standard tissues before RNA isolation |
miRNeasy Mini Kit | Qiagen | 217004 | Purification of miRNA and total RNA |
TaqMan MicroRNA Reverse Transcription kit | ThermoFisher Scientific | 4366596 | A highly specific kit that quantitates only mature miRNAs |
snoRNA202 Primer | ThermoFisher Scientific | 1232 | Taqman miRNA control assay in mouse |
miR-298 Primer | ThermoFisher Scientific | 2190 | Taqman miRNA-298 assay in mouse |
Anti-GFP antibody | abcam | ab290 | Rabbit polyclonal to GFP – ChIP Grade |
Red ink pad | Dovecraft Essentials | ||
Blue ink pad | Dovecraft Essentials | ||
AAV9-GFP vector | SignaGen Laboratories | SL100840 | Large scale AAV plasmid construction, packaging and purification |
mmu-miR-298-5p | ThermoFisher Scientific | MC12525 | mirVana miRNA mimic |
AAV9-EF1a-has-mir-298-GFP | Vector Biosystems Inc |