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. Author manuscript; available in PMC: 2016 Jan 1.
Published in final edited form as: Gene Ther. 2014 Nov 6;22(1):104–110. doi: 10.1038/gt.2014.91

Non-invasive, neuron-specific gene therapy can be facilitated by focused ultrasound and recombinant adeno-associated virus

Shutao Wang 1, Oluyemi O Olumolade 1, Tao Sun 1, Gesthimani Samiotaki 1, Elisa E Konofagou 1,2
PMCID: PMC4294560  NIHMSID: NIHMS650171  PMID: 25354683

Abstract

Recombinant adeno-associated virus (rAAV) has shown great promise as a potential cure for neurodegenerative diseases. The existence of the blood-brain barrier (BBB), however, hinders efficient delivery of the viral vectors. Direct infusion through craniotomy is the most commonly used approach to achieve rAAV delivery, which carries increased risks of infection and other complications. Here we report a focused ultrasound (FUS) facilitated, non-invasive rAAV delivery paradigm that is capable of producing targeted and neuron-specific transductions. Oscillating ultrasound contrast agents (i.e. microbubbles), driven by focused ultrasound waves, temporarily “unlocking” the BBB, allowing the systemically administrated rAAVs to enter the brain parenchyma, while maintaining their bioactivity and selectivity. Taking the advantage of the neuron-specific promoter-synapsin, rAAV gene expression was triggered almost exclusively (95%) in neurons of the targeted (i.e. caudate-putamen) region. Both behavioral assessment and histological examination revealed no significant long term adverse effects (in the brain and several other critical organs) for this combined treatment paradigm. Results from this study demonstrated the feasibility and safety for the non-invasive, targeted rAAV delivery technique, which might have provided a new arena for gene therapy in both pre-clinical and clinical settings.

Introduction

The pathology of Parkinson’s Disease (PD) is characterized by the relatively selective death of neuronal subtypes, namely the nigro-striatal dopaminergic (DA) neurons1. Although the exact pathogenesis of PD is still under investigation, the discovery of the dopamine precursor L-dopa2, which can be taken up by the remaining DA neurons and converted to dopamine, alleviated symptoms for PD patients. Unfortunately, with ongoing loss of DA neurons, the uptake of extracellular L-dopa gradually declines. Alternatively, gene therapy was proposed as a means for achieving localized and prolonged transgene expression3. Until now, several clinical trials designed for PD patients were performed using recombinant adeno-associated virus serotype 2 (rAAV2) carrying various genomes: human aromatic acid decarboxylase (hAADC)4; glutamic acid decarboxylase (GAD)5; and more recently neurturin (NTN)6. Although all of these trials demonstrated the tolerability of rAAV2 vectors in PD patients, the insignificant therapeutic effects might have been results from limited injection sites and lack of an effective transduction volume. To overcome these limitations, multiple injections were employed. However, the increased number of injections poses much higher risks in targeting accuracy, mechanical failure, infection and other complications for PD patients5,7. Moreover, given the relatively large molecular size of rAAV (approximately 20 nm) and the high intracranial pressure8, the vectors are expected to remain in the proximity of the catheter tip9, leading to a suboptimal transduction volume.

Alternatively, rAAV could be administrated systemically and by adapting the design of viral vectors, cell-specific transduction may be achieved. Nonetheless, the presence of the blood-brain barrier (BBB) prevents almost all large molecules (including rAAV) from entering the brain parenchyma10. Recent advances in therapeutic ultrasound and its application in BBB disruption provide a promising alternative for circumventing these obstacles11,12. The ultrasound beam can be focused at the targeted region (on the order of millimeters), causing oscillations of the systemically administrated microbubbles. Focused ultrasound (FUS) in combination with microbubbles has been shown to be capable of non-invasively inducing reversible BBB opening in rodents as well as non-human primates13,14. Molecules of various sizes were successfully delivered to the brain: neurotropic factors15, antibodies16, as well as chemotherapeutic agents17. Several efforts have been reported towards non-invasively delivering rAAV across the BBB for the purpose of gene therapy18–20. Among the reported studies, the neuron transduction rate (18%-40%) was either significantly below the effective level or not quantified. In addition, no safety profile of the proposed treatment paradigm was adequately established and documented. Therefore, the goal of this study is to demonstrate the feasibility of FUS facilitated, neuron-specific rAAV transduction and establish safety profiles for this technique.

Results and Discussion

The caudate-putamen (CPu) was chosen as the primary target for FUS-induced BBB opening because it is directly linked to motor functions of the animal model used in this study. The acoustic beam was tightly focused (with a −6dB pressure volume of 1 × 1 × 7 mm3) through a coupling medium on to the intended target. A mixture of rAAV-synapsin-GFP (either serotype 1 or 2) and in-house manufactured microbubbles (mean diameter of 0.9 μm) was administrated via the tail vein immediately before sonication. The oscillation of microbubbles (driven by the ultrasound field) was monitored with a passive cavitation detector and the cavitation dose was quantified (Fig. 1a) for each group as an indicator for bubble-blood vessel wall interactions. The cavitation doses of the BBB opened groups were significantly higher (P<0.001 for rAAV1 and P<0.01 for rAAV2) than that of the control. The temporary opening of the BBB was confirmed with T1-weighted contrast-enhanced magnetic resonance imaging (MRI). The contrast agent gadodiamide (MW 573.66 Da) does not cross the BBB under normal, physiologic conditions. However, the BBB opened region (namely, left CPu) was highlighted by the diffusion of gadodiamide (Fig. 1b) indicating localized BBB opening. The diffusion of the MR contrast agent was clearly revealed as the bright region from the reconstructed 3D MR image (Fig. 1c).

Figure 1.

Figure 1

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Targeted BBB disruption is achieved by FUS and microbubbles, and the transgene is primarily expressed in neurons. a, Increased cavitation doses, indicating microbubble-blood vessel interaction, were found in FUS treated groups (red: rAAV1; black: rAAV2). b, Contrast-enhanced T1-weighted MR images revealed targeted BBB disruption (highlighted region). c, The bright region from the 3D reconstructed mouse brain image revealed the volume of BBB opening. d, Transgene expression was found only in the FUS treated region. The co-localization of GFP-positive and NeuN-positive cells indicates neuron-specific transduction. Scale bars 20 μm. e, Transgene expression was almost exclusively restricted in neurons and only approximately 5% of transduced cells were astrocytes and unidentified cells. f, Examples of transgene expression in a neuron and an astrocyte. Scale bars 20 μm. **P < 0.01, ***P < 0.001 by unpaired t-test. Error bars indicate s.d.

Mice were sacrificed four weeks following the initial sonication. The coronally sectioned brain slices were stained for neurons and astrocytes with anti-NeuN and anti-GFAP antibodies, respectively. By comparing the FUS-treated side to the contralateral side (which served as a rAAV only control), as shown in Fig. 1d, significant transgene expression (GFP) was observed on the BBB opened side, while the contralateral side was almost free of rAAV transduction. We also identified the types of transduced cells using a customized image processing algorithm based on the fluorescent images. It was found that for both types of rAAVs, approximately 95% (96.3±1.9% for rAAV1 and 93.7±2.4% for rAAV2) of the transduced cells were neurons (Fig. 1e). In addition, transductions of astrocytes and unidentified cells were also observed, although to a lesser degree (i.e. altogether ~5%). Magnification of transgene-expressing neurons and astrocytes is shown in Fig. 1f. Such neuron-specific transduction was achieved primarily by the viral promoter synapsin for both rAAV1 and rAAV2 vectors. Kügler et al.21 reported that the transgene expression from adenoviral vectors (directly infused into the rat brain) can be restricted exclusively to neurons by using a small fragment of the human synapsin 1 gene. In this study, we demonstrated that systemically administrated rAAV1 and rAAV2 with synapsin promoter also exhibited neuron-specific transduction once across the BBB.

We quantified the distribution of the rAAV transduction in the targeted CPu region. As shown in Fig. 2a, GFP was stained in brown color using the ABC-DAB method. The rAAV1 (Fig. 2a left column) transduced cells (dark brown) can be clearly observed on the left side (FUS treated) of the brain sections (corresponding to sections 2, 4, 6 in Fig. 2b), while rAAV2 presented much lower transduction efficiency (Fig. 2a right column, corresponding to sections 4, 6, 8 in Fig. 2c). The contralateral side (untreated, right bottom corner of each image in Fig. 2a) showed minimal or no rAAV transduction. The total number of transgene-expressing neurons was quantified with a custom-written program, which was verified with manual counting (discrepancy was within 15%). For each brain, eight sections (thickness of 40 μm), which had an inter-sectional space of 200 μm, were stained and quantified. The number of rAAV transduced neurons for each section was normalized against the maximum neuron count of each brain. As shown in Figs. 2b&2c, the transduced neurons followed a Gaussian distribution for both rAAV1- and rAAV2-treated groups (R2 = 0.7594 and 0.6504, respectively). This was caused by the circular shape of the FUS beam in the radial dimension. In other words, the acoustic pressure field also exhibited a Gaussian distribution across the radial direction, intrinsically resulting from the focusing geometry of the FUS beam. As shown in Fig. 2d, the total number of rAAV1 transduced neurons in the CPu region, with a single sonication, was estimated to be 4,581±2005, which is significantly higher than the contralateral side (P = 0.012). The number of rAAV2 transduced neurons on the treated side, although significantly higher (P = 0.028) than the contralateral side, is much less effective than that of the rAAV1 treated groups (P = 0.014). A magnified observation of the bright field images is given in Figs. 2f&2g, where the soma and dendrites of the transduced neurons are clearly marked with dark brown color.

Figure 2.

Figure 2

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FUS facilitated rAAV delivery is localized and rAAV1 exhibits more efficient transduction than rAAV2. a, Examples of rAAV1&2 transgene expression (dark brown) in the targeted region (left CPu). The inter-section distance was 440 μm. The contralateral side of the brain is shown in the black square at the bottom right corner of each image. Scale bars 500 μm. b&c, Normalized numbers of rAAV1 (b) and rAAV2 (c) transduced cells revealed Gaussian distributions, corresponding to the profile of the FUS pressure field in the radial direction. d&e, The FUS treated side showed significant higher number of transduced cells than the contralateral side for both rAAV1 (d) and rAAV2 (e). f&g, Magnified bright field images revealing rAAV transgene expression. Scale bars 50 μm (f) and 20 μm (g).

We have investigated the long term safety aspects of the proposed treatment paradigm with both histological examinations and behavioral tests. One concern for the systemically administrated rAAV vectors was transduction in unintended organs. To address this concern, four critical organs (heart, lung, liver and kidney) from each mouse were harvested upon sacrificing. The organs were then sectioned at 20 μm and imaged for transgene expression (GFP). As shown in Fig. 3a, no increased GFP signal was observed from the rAAV treated groups when compared with the sham group. Histological examinations were performed using mice treated with FUS only, since rAAV vectors have been shown to be safe and tolerable in clinical trials6,22,23. After initial treatment, these mice were sacrificed following a survival time of four weeks (to be consistent with the rAAV + FUS treated groups). Based on the H&E staining and Nissl staining results, no long term brain damage was observed in either the FUS treated region or the contralateral side (Fig. 3b&3c).

Figure 3.

Figure 3

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The combined FUS and rAAV treatment showed no significant adverse effect in vivo. a, Fluorescent images of the heart, lung, kidney and liver showed no rAAV transduction when compared with sham. Scae bars 100 μm. b, H&E staining and c, Nissl staining of the FUS treated brains showed no long term cellular damage. Scale bars 60 μm. d&e, No significant change was observed across the four-week survival period from the vertical poll test (d) and the rotarod performance test (e).

Since the FUS targeted region was the CPu, which is closely linked to motor functions, the rotarod performance test and the vertical pole test were selected to the monitor potential changes in behavior24. The tests were repeated on a weekly basis for rAAV1 + FUS treated, rAAV2 + FUS treated, and sham groups until the end of the survival period. No significant change was observed between these groups from the vertical pole test, shown in Fig. 3d. There was, however, statistically significant difference (P < 0.05) between week 0 and week 3 within the rAAV2-treated group. This may be attributed to the learning curve of the animals, as the same trend can be found in the other two groups (although no significant difference). Furthermore, the rotarod test (shown in Fig. 3e) showed no significant difference between the rAAV + FUS treated groups and the sham group from each week. These observations demonstrated, from both behavioral and histological perspectives, that the combined rAAV and FUS-induced BBB opening paradigm is safe for the non-invasive and targeted gene therapy.

Gene therapy has been proposed for the treatment of neurodegenerative diseases for its persistent transgene expression. Among all gene therapeutic vehicles, rAAV showed great potential, especially its safety and tolerability profile from clinical trials. Conventionally, these vectors are infused through a cannula directly into the targeted brain region and convection-enhanced delivery systems were then developed to increase the diffusion volume9. More recently, novel cannulae were proposed to further enhance the delivery efficiency. For instance, a micro-fabricated catheter with MR compatibility was evaluated in vivo for intraparenchymal delivery25; a radially branched cannula was designed for efficient delivery at the scale of the human brain26. Nevertheless, all cannula-based approaches require invasive procedures and may cause various complications. Here, we propose a non-invasive, targeted and neuron-specific gene therapy paradigm using focused ultrasound and rAAV vectors with the -synapsin promoter. Our approach, unlike the cannula-based delivery systems, is capable of achieving completely non-invasive rAAV delivery to the brain. More importantly, this technique allows flexible implementation of multi-site delivery by simply re-positioning the FUS transducer.

One limitation of this study is that the FUS sonication and rAAV administration were carried out in mice at a relatively young age (10 weeks old). Nevertheless, successful repeated FUS-induced BBB openings have been reported in non-human primate up to 26 weeks27. Compared to cannula-based approaches, our technique required a higher dose in that viral vectors were administrated intravenously and the optimal dose needs to be determined in future studies. By selecting appropriate promoters, non-specific transduction can be minimized and no long term adverse effects were observed from behavioral tests and histological examinations. The results presented here demonstrated the feasibility and safety of a non-invasive and neuron-specific gene therapy approach.

Materials and Methods

Animal preparation

All experimental procedures involving animals were approved by the Columbia University Institutional Animal Care and Use Committee. A total of 18 mice (age 10 weeks) were used in this study and were divided into four groups: FUS + rAAV1 (n=5), FUS + rAAV2 (n=5), FUS only (n=5), and sham (n=3). Mice were anesthetized with a mixture of oxygen and 1–2% isoflurane (SurgiVet, Smiths Medical PM, Inc., WI) and placed prone with its head immobilized by a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). The scalp hair was removed by an electrical trimmer and depilatory cream to minimize acoustic impedance mismatch.

Microbubble and rAAV vectors

Microbubbles were manufactured in-house according to previously published protocol28. Briefly, the 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and polyethylene Glycol 2000 (PEG2000) were mixed at a 9:1 ratio. Two milligrams of the mixture was dissolved in a 2 ml solution consisted of filtered PBS/glycerol (10% volume)/propylene glycol (10% volume) using a sonicator (Model 1510, Branson Ultrasonics, Danbury, CT, USA) and stored in a 5 ml vial at 4°C. The empty space of the vial was filled with decafluorobutane (C4F10) gas and activated via mechanical agitation using VialMix™ (Lantheus Medical Imaging, N. Billerica, MA) shaker for a pre-set time of 45 s. The concentration and size distribution of each microbubble vial were measured with a Coulter Counter Multisizer (Beckman Coulter Inc., Fullerton, CA).

The rAAV vectors used in this study were purchased from SignaGen Laboratories (Rockville, MD). Both rAAV1 and rAAV2 expressing eGFP under the control of -synapsin promoter were employed. The titer of the viral vectors were provided by the manufacturer (quantified with real-time PCR) and diluted in PBS to 1.1×1012 GC/ml. For each mouse (average body weight of 25 g), a total of 100 μl of diluted rAAV was mixed with approximately 2.5×107 microbubbles and injected via the tail vein.

Focused ultrasound

The FUS sonications were carried out with a single element focused transducer (focal length: 60 mm and radius: 30 mm, Imasonic, France), which has a center frequency of 1.5 MHz. The −6dB pressure focal zone of the FUS transducer was measured by a needle hydrophone (Precision Acoustics Ltd., Dorchester, UK) in degassed water to be 7.5 × 1 × 1 mm3. A confocally mounted pulse-echo transducer (radius: 11.2 mm, focal length: 60 mm, and center frequency: 10 MHz, Olympus NDT, Waltham, MA) was used for targeting and receiving cavitation signal during the treatments29. The pulse-echo transducer was driven by a pulser-receiver (Olympus, Waltham, MA), which was connected to a digitizer (Gage Applied technologies, Inc., Lachine, QC, Canada) for data acquisition. The transducer cone was filled with de-ionized and degassed water and sealed with a piece of polyurethane membrane (Trojan; Church & Dwight Co., Inc., Princeton, NJ). The FUS transducer was connected to a matching circuit and driven by a computer controlled function generator (Agilent, Palo Alto, CA) and a 50 dB power amplifier (ENI Inc., Rochester, NY). The transducer system was then mounted onto a computer controlled three-dimensional positioner (Velmex Inc., Lachine, QC, Canada).

The intended target in this study was the caudate-putamen and the targeting was achieved using a metallic grid approach as described in previous reports29,30. Briefly, a metallic grid was placed on top of lambda and the center of the grid was found from the reflected ultrasound signal. The FUS transducer was then moved according to the following coordinates: AP +6 mm and ML −2 mm. The center of the FUS focus was placed 3 mm below the skull and 30% attenuation was accounted for acoustic pressure loss through the skull. An estimated in situ peak rarefactional pressure (PRP) of 0.45 MPa was used for all sonications with a pulsing sequence of pulse repetition frequency (PRF) 5 Hz, pulse length 20 ms, and a total duration of 300 s. The rAAV and microbubble mixture was administrated as a bolus via the tail vein immediately before the sonication. The cavitation signals were recorded by the pulse-echo transducer and the cavitation dose was quantified from the Fast Fourier Transformed spectra (4 – 12 MHz) of each pulse with a custom written program (MATLAB R2011a, MathWorks, Inc., Natick, MA). The net cavitation emission from the contrast microbubbles was determined by subtracting the background signal measured using the identical ultrasound exposure prior to microbubble administration.

Magnetic resonance imaging

MRI was performed for the FUS only group upon completion of the FUS treatment. The BBB opening was confirmed with a 9.4T MRI system (Bruker Medical, Boston, MA). The mice were placed in a birdcage coil (diameter 3 cm), while being anesthetized with 1 – 2% isoflurane and vital sign monitored throughout the imaging sessions. A bolus of 0.3 ml of gadodiamide (GD-DTPA) (Omniscan®, GE Healthcare, Princeton, NJ) was injected intraperitoneally in each mouse. Approximately 50 min post the injection, MR images were collected using a contrast-enhanced T1-weighted 2D FLASH sequence31.

Behavioral testing

To assess the safety of the BBB opening and rAAV systemic delivery, two types of behavior testing were performed: rotarod performance test and vertical pole test. The rotarod apparatus (Med Associates, St. Albans, VT) is composed a plastic rod (32 mm in diameter) and a motion senor underneath the rod. Each mouse was placed on the accelerating rod (initially at rest, acceleration 4 revolution/min2) and allowed to stay on the rod for a maximum of 3 min. Falling off the rotarod, as well as latching on to the rotarod (rotating with the rod) for two consecutive revolutions was considered the end of a trial. The vertical pole test was carried out using a rough surfaced pole (length 80 cm). Mice were positioned upward on the pole and the time for the mice to climb down was recorded. For both tests, three trials were conducted for each mouse, and the average value was used for analysis. The rAAV1, rAAV2 and sham groups underwent initial training for both tests (week 0) before the beginning of the FUS treatment. The testing was repeated on a weekly basis for each mouse until the end point of the survival period (week 3).

Immunohistochemistry and histology

Mice were sacrificed and transcardially perfused with 30 mL PBS and followed by 60 mL 4% paraformaldehyde. The brains as well as several critical organs (i.e. heart, lung, liver, and kidney) were harvested and soaked in paraformaldehyde. The skull was removed from the brains 24 hours later and the brains were fixed for one additional day. All tissues were cryo-protected with gradient sucrose solution (10% for 1 hr, 20% for 2 hrs, and 30% overnight), frozen on dry ice, and kept in a −80°C freezer. Brain samples were sectioned coronally at 40 μm and the entire CPu region was collected. For immunohistochemistry staining, every sixth section was treated with anti-GFP antibody (dilution 1:5000, Novus Biologicals, Littleton, CO) and stained using the ABC-DAB method. Samples were then imaged with a bright field microscope (Olympus, Center Valley, PA). In order to identify AAV transduced cell type, immunofluorescent stainings were also carried out. Brain sections were incubated with primary antibodies: mouse anti-NeuN (dilution 1:200, Millipore, Temecula, CA) and rabbit anti-GFAP (dilution 1:200, Invitrogen, Camarillo, CA). Samples were then stained with fluorescently labeled secondary antibodies goat anti-mouse conjugated with Cy3 (dilution 1:200, Millipore, Temecula, CA) and goat anti-rabbit conjugated with Cy5 (dilution 1:200, Invitrogen, Camarillo, CA). Fluorescent images were obtained with a confocal laser scanning microscope (Leica, Buffalo Grove, IL). Paraffin-embedded brains (from the FUS only group) were sectioned at 7 μm and used for Hematoxylin and Eosin (H&E) staining. The organs were sectioned at 20 μm in thickness and stained with DAPI upon imaging. The images were taken with a Nikon confocal microscopy (Nikon Instruments Inc., Melville, NY).

Image analysis

All image analyses were done using custom-written programs in MATLAB (R2011a, MathWorks, Inc., Natick, MA). To identify the rAAV transduced cell types, ten Z-stacked fluorescent images (of GFP, NeuN, and GFAP channels) were taken from each brain. The maximum projected GFP channel images were loaded into the program and used as the base for locating GFP positive cells. Once a GFP positive cell is identified by thresholding the grayscale images, the locations of its pixels were recorded. The intensity of these pixels from the NeuN channel and GFAP channel were quantified and determined to be NeuN positive or GFAP positive if 70% of the pixels were above the threshold. The total number of neurons for each brain was quantified based on the DAB stained sections (8 sections per brain with inter-sectional space of 200 μm). The bright field images were first converted into grayscale format and the transduced cells were segmented. The results (from three different brains) from automatic counting program were compared with manual counting results, and the discrepancy was within 15%.

Statistics

Comparison between two groups was made using two-tailed unpaired Student’s t-test and a p-value less than 0.05 was considered to be statistically significant. Comparisons between groups in behavioral testing were performed using one-way ANOVA followed by the Bohferroni’s post-hoc test.

Acknowledgments

This work was supported in part by NIH R01EB009041, NIH R01AG038961, and the Kinetics Foundation. We thank C. Chen and H. Chen for insightful discussion and comments. The authors also appreciate the assistance of Y. Han and C. Acosta in generating MRI and fluorescent images.

Footnotes

Conflict of interest

The authors declare no conflict of interest.

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