Extracellular vesicles-associated AAVs for the treatment of Machado-Joseph disease

Carina Henriques; Patrícia Albuquerque; David Rufino-Ramos; Miguel M Lopes; Kevin Leandro; Catarina O Miranda; Rita Almeida; Guilherme Lopes-Gabriel; João de Sousa-Lourenço; Sara M Lopes; Laetitia S Gaspar; Diana D Lobo; Ana Carolina Silva; Teresa M Ribeiro-Rodrigues; Henrique Girão; Célia M Gomes; Rafael Baganha; Sónia Duarte; Casey A Maguire; Magda M Santana; Luís Pereira de Almeida; Rui Jorge Nobre

doi:10.1016/j.ymthe.2025.10.022

. 2025 Oct 11;34(1):161–178. doi: 10.1016/j.ymthe.2025.10.022

Extracellular vesicles-associated AAVs for the treatment of Machado-Joseph disease

Carina Henriques ^1,^2,^3,^4,^5,¹⁴, Patrícia Albuquerque ^1,^2,^5,¹⁴, David Rufino-Ramos ^1,^2,^5,^6,^7,⁸, Miguel M Lopes ^1,^2,^3,^4,⁹, Kevin Leandro ^1,^2,^3,⁵, Catarina O Miranda ^1,^2,^3,⁹, Rita Almeida ^1,^2,^3,⁴, Guilherme Lopes-Gabriel ^1,^2,^3,^4,⁵, João de Sousa-Lourenço ^1,^2,^3,⁴, Sara M Lopes ^1,^2,^3,⁹, Laetitia S Gaspar ^1,^2,^3,⁹, Diana D Lobo ^1,^2,^3,⁹, Ana Carolina Silva ^1,^2,^3,⁹, Teresa M Ribeiro-Rodrigues ^2,^10,^11,¹², Henrique Girão ^2,^10,^11,¹², Célia M Gomes ^2,^10,^11,¹², Rafael Baganha ^1,^2,^3,^4,⁹, Sónia Duarte ^1,^2,^3,⁹, Casey A Maguire ¹³, Magda M Santana ^1,^2,^3,⁹, Luís Pereira de Almeida ^1,^2,^3,^4,^5,^15,^∗, Rui Jorge Nobre ^1,^2,^3,^4,^9,^15,^∗∗

PMCID: PMC12925786 PMID: 41077785

Abstract

Machado-Joseph disease (MJD) is the most common dominant autosomal inherited ataxia worldwide, caused by the overrepetition of the trinucleotide CAG in the ATXN3 gene. This leads to the accumulation of mutant ataxin-3 protein and neurodegeneration. Currently, treatment remains symptomatic, although gene therapy has emerged as a promising approach. However, efficient and minimally invasive gene delivery to the brain remains a challenge. Extracellular vesicle-associated adeno-associated virus (EV-AAV) vectors are a novel delivery system, combining the ability of AAV vectors to deliver genes with the capacity of extracellular vesicles to bypass the immune system and cross the blood-brain barrier (BBB). Previous studies, however, have only combined AAV serotypes known to efficiently cross the BBB with EVs as a non-invasive delivery system to the brain. Thus, the ability of EV-AAVs to cross the BBB remained inconclusive. In this study, we evaluated whether AAV1/2 serotype, combined with rabies virus glycoprotein (RVg)-coated EVs, could effectively target the brain. Two isolation methods, differential ultracentrifugation and size-exclusion chromatography (SEC) were compared, with SEC yielding higher EV recovery. Moreover, RVg-EV-AAV1/2 successfully crossed the BBB and transduced mouse brains, leading to motor and neuropathologic improvements in an MJD mouse model. This study demonstrates that RVg-EV-AAVs are promising non-invasive delivery systems for MJD gene therapy.

Keywords: adeno-associated virus vectors, extracellular vesicles, Machado-Joseph disease, spinocerebellar ataxia type 3, gene therapy, RNA interference

Graphical abstract

This study demonstrates that rabies virus glycoprotein-coated EV-AAV1/2 successfully cross the blood-brain barrier, delivering genes non-invasively and improving motor and neuropathological outcomes in a mouse model of Machado-Joseph disease, showcasing their potential for gene therapy applications in neurodegenerative disorders.

Introduction

Machado-Joseph disease (MJD), or spinocerebellar ataxia type 3, is a neurodegenerative polyglutamine disorder caused by an unstable repetition of the trinucleotide cytosine-adenine-guanine (CAG) in exon 10 of the ATXN3 gene.¹^,²^,³^,⁴^,⁵^,⁶ Despite being the most common autosomal dominantly inherited ataxia worldwide,⁷^,⁸ to date, there is no cure or treatment for MJD, and the management of affected individuals remains limited to supportive care.⁹^,¹⁰ Different strategies have been explored to treat MJD in pre-clinical studies, with gene therapy pointed to as a strong candidate.¹¹^,¹²^,¹³^,¹⁴^,¹⁵^,¹⁶^,¹⁷^,¹⁸^,¹⁹^,²⁰^,²¹^,²²^,²³^,²⁴^,²⁵^,²⁶^,²⁷ However, there is still a need for delivery systems capable of effectively transporting genetic material to the central nervous system (CNS) with the minimal invasiveness.

In recent years, the vectors of adeno-associated virus (AAV) have emerged as the preferred platform for delivering gene products to the human body, especially to the brain.²⁸ Wild-type (WT) AAVs are small viruses that have a large repertoire of serotypes with different tissue tropisms.²⁹^,³⁰ Currently, seven AAV-based products have been approved for the treatment of human diseases in Europe and the United States.³¹^,³²^,³³^,³⁴^,³⁵^,³⁶^,³⁷ However, difficulties have emerged with the clinical translation and increasing use of these vectors.³⁸ A major challenge is the high prevalence of preexisting neutralizing antibodies against AAV capsids in humans, related to previous natural AAV infection and to the cross-reactivity between different AAV serotypes.³⁹^,⁴⁰ In addition, high doses of AAV vectors are required to reach the CNS, when systemically delivered, even when using AAV serotypes known to cross the blood-brain barrier (BBB) (e.g., AAV8, AAV9). Moreover, upon intravenous (IV) administration, most AAV serotypes exhibit tropism for peripheral organs, leading to undesired off-target expression and toxicity.⁴¹ To circumvent these limitations, significant effort has been made toward the development of new AAV capsids capable of avoiding neutralization, efficiently crossing the BBB, and transducing specific regions or cell populations of the CNS, while simultaneously maintaining low transduction of peripheral organs.³⁸^,⁴²

During the production of AAVs, a fraction of these vectors was found to be secreted into the culture medium in association with extracellular vesicles (EVs), the so-called EV-AAVs.⁴² Several reports have demonstrated that EV-AAVs not only achieve higher levels of cell transduction compared to AAV vectors⁴³^,⁴⁴^,⁴⁵^,⁴⁶^,⁴⁷^,⁴⁸ but also circumvent antibody neutralization⁴²^,⁴³^,⁴⁵^,⁴⁷ and cross biological barriers, including the BBB.⁴³^,⁴⁹ However, to our knowledge, only AAV serotypes with a known ability to cross the BBB have been used in previous studies, where EV-AAVs were developed to target the CNS through IV administration. Thus, it remained unclear whether AAV serotypes lacking the natural capacity to cross the BBB, such as AAV1 and AAV2,⁵⁰^,⁵¹^,⁵²^,⁵³ could reach the brain upon IV injection when associated with EVs.

The ability of EV-AAVs to cross the BBB, independently of the AAV serotype, could open new opportunities for the treatment of neurodegenerative diseases, such as MJD, enabling the systemic delivery of therapeutic AAVs, encapsulated in EVs, with higher tropisms for specific brain regions or cell populations. Therefore, the present work aimed to develop a minimally invasive therapeutic strategy for the treatment of MJD using EV-AAVs to deliver a microRNA (miRNA)-based silencing sequence. For that, we first compared two methods for the isolation of EV-AAVs: differential ultracentrifugation (UC) and size-exclusion chromatography (SEC). We then investigated whether a typically non-BBB-penetrating AAV serotype, the mosaic AAV1/2, can target the brain when associated with brain-targeting EVs. Finally, we assessed whether EV-AAVs could be used as minimally invasive vehicles to deliver therapeutic silencing artificial miRNAs for MJD treatment.

EV-AAVs isolated by UC and SEC showed functional cargo delivery in vitro and in vivo. Furthermore, the ability of rabies virus glycoprotein (RVg)-coated EV-AAV1/2 to cross the BBB and deliver their cargo into the brain upon IV injection was evaluated in WT and MJD transgenic mice. Remarkably, we found that RVg-EV-AAVs crossed the BBB, transduced neuronal cells, and ameliorated neuropathology and motor impairments in a severely impaired mouse model of MJD.

Results

SEC and UC can isolate intact RVg-EV-AAVs

To evaluate whether AAV vectors were secreted in association with EVs during standard AAV vector production, we performed two different isolation protocols, UC and SEC, that separate particles based on their sedimentation rate or size, respectively. For that, we generated AAV1/2 vectors encoding the enhanced green fluorescent protein (EGFP) using a transfection protocol previously optimized in our group (Figure 1A).⁵⁴ The mosaic AAV1/2 vector was chosen due to the neurotropic features of AAV serotypes 1 and 2 and the low ability to cross the BBB.⁵⁴^,⁵⁵ Importantly, an additional plasmid encoding the platelet-derived growth factor receptor transmembrane domain fused with RVg peptide (pRVg), which has been successfully used for the delivery of both EVs and EV-AAVs to the brain,²⁶^,⁴³ was co-transfected into the AAV-producer cells to enhance in vivo brain targeting. The ability of pRVg to increase brain targeting has been previously described by us¹⁷^,²⁶ and others.⁴³^,⁵⁶^,⁵⁷^,⁵⁸ AAV-producer cell conditioned medium was harvested 48–72 h post-transfection and pre-cleared by low-speed centrifugations.

Isolation and characterization of EV-AAVs isolated by UC and SEC

(A) Schematic representation of the protocol of production of extracellular vesicle-associated adeno-associated virus (EV-AAV). Briefly, HEK293T cells were transfected with five plasmids: one plasmid encoding the transgene of interest between the ITRs (pITRs), two plasmids containing the wild-type AAV1 and AAV2 rep and cap open reading frames (pAAVs), one helper plasmid (pHelper), and one plasmid encoding for the rabies virus glycoprotein peptide (pRVg). On the following day, the culture medium was replaced with fresh medium supplemented with 2% fetal bovine serum free of EVs. Conditioned medium was collected at 48–72 h post-transfection. (B) Schematic representation of the EV-AAV isolation protocol by ultracentrifugation (UC). AAV production medium was submitted to two steps of UC: 1 h at 20,000 × g (UC-20K) followed by 1 h 30 min at 100,000 × g (UC-100K). In both steps, pelleted material was resuspended in PBS for subsequent analysis. (C) Western blot (WB) showing the presence of EV protein marker flotillin-1 (FLOT-1), AAV capsid proteins (VP1, VP2, and VP3), and cellular contamination marker calnexin (CNX). Commercial HEK293T EVs isolated by UC, AAV vectors purified by FPLC, and HEK293T cell lysates were used as control samples. (D) WB immunoreactivity quantification of UC-100K fraction relative to UC-20K fraction. FLOT-1 and AAV capsid proteins were present in UC-20K and UC-100K fractions. CNX was mostly present on the UC-20K fraction. Results were normalized to the UC-20K fraction. (E) Transmission electron microscopy (TEM) of UC-20K and UC-100K fractions. EVs were present in both fractions. Scale bar, 50 nm. (F) The ratio between the number of particles and protein (n°/μg) showed no significant differences between UC-20K and UC-100K fractions. Particle number was determined by nanoparticle tracking analysis (NTA), and the protein content was quantified by microBCA. (G) Viral genome quantification was performed by quantitative polymerase chain reaction (qPCR). The number of viral genomes (vg) per nanoparticle showed a tendency to be slightly increased in the UC-100K fraction. Vg copies were normalized to the initial medium volume (mL). (D, F, and G) Results are shown as mean ± SEM for UC-20K and UC-100K (n = 3–6 per group). Statistical analysis was performed using the two-tailed unpaired t test (∗p < 0.05; ∗∗p < 0.01). (H) Schematic representation of the EV-AAV isolation protocol by size-exclusion chromatography (SEC). Briefly, concentrated conditioned medium was loaded into an SEC column, and 30 fractions of 0.5 mL were collected for further analysis. (I) Representative WB. Ponceau staining shows protein distribution in the WB membrane. Blocked membranes were horizontally cut into two pieces. The top piece was immunoblotted for CNX and AAV and the bottom piece for FLOT-1 and AAV. EV-marker FLOT-1, AAV capsid proteins (VP1, VP2, and VP3) and cell-contaminating protein CNX are labeled. Control AAV vectors purified by FPLC and HEK293T cell lysates were used as controls. (J) Immunoreactivity quantification of FLOT-1 (orange) and AAV capsid proteins (green) from F7 to F13. FLOT-1 distribution showed a peak at F9, while proteins from AAV capsid were more abundant in F13. Data were normalized to maximum optical density (OD). (K) TEM images obtained from SEC F7–F12 showed the presence of EVs in all fractions. Scale bar, 50 nm. (L) The ratio between the number of particles and protein levels (n°/μg) was assessed in F7–F13. Particle number was determined by NTA and protein amount by microBCA. The number of nanoparticles per protein decreased across fractions. (M) Viral yield (in vg) of F7–F13 was quantified by qPCR and normalized to the initial medium volume. (J, L, and M) Results are shown as mean ± SEM for F7–F13 (n = 3–12 per group).

For EVs isolation by UC, two steps of UC were performed, one at 20,000 × g (UC-20K), to collect larger vesicles and a second UC at 100,000 × g (UC-100K), for the recovery of smaller EVs (Figure 1B).⁵⁹ On each UC step, the pellets were collected for further analysis.

Western blot (WB) analysis confirmed the presence of the EV marker flotillin-1 (FLOT-1) in both UC fractions, with no significant differences observed between UC-20K and UC-100K (Figures 1C, 1D, and S1).⁶⁰^,⁶¹ Moreover, nanoparticles were visualized by negative stain transmission electron microscopy (TEM) and size was determined by nanoparticle tracking analysis (NTA), confirming EV-like shapes (Figure 1E) and sizes (Figure S3A). Nevertheless, calnexin (CNX), an endoplasmic reticulum marker, was detected on UC-20K, suggesting cellular contamination in this fraction (Figures 1C and 1D). We also compared the ratio of nanoparticles per microgram of protein in those fractions, as previously described by Webber and Clayton,⁶² and, despite the presence of CNX in UC-20K, no significant differences in this ratio were observed between UC-20K and UC-100K (Figure 1F).

After confirming EV isolation by UC, we verified whether AAV vectors were present on the same UC fractions. WB analysis revealed the presence of AAV capsids (VP1, VP2, and VP3) in both UC-20K and UC-100K fractions, with a significant increase in the UC-100K fraction (Figures 1C and 1D). Viral genomes (vg) were also quantified by qPCR. A tendency for an increased number of vg was observed at UC-100K as compared to UC-20K (Figure 1G).

Next, EVs were isolated by SEC, following protocols previously developed by our group.⁶³^,⁶⁴^,⁶⁵ For this, pre-cleared conditioned medium was concentrated using ultrafiltration with a molecular weight cutoff (MWCO) of 10 kDa to reduce the EV loss⁶⁶ and then applied into a Sepharose-based SEC column. Thirty individual fractions (F) of 0.5 mL were collected for posterior analysis (Figure 1H).

FLOT-1 marker was used to investigate in which SEC fraction(s) EVs were eluted. As shown in Figures 1I and 1J (see also Figure S2), FLOT-1 was first detected on F7, reaching higher concentrations on F9. CNX was not detected in FLOT-1-enriched fractions, indicating the absence of cellular contamination. Negative stain TEM analysis confirmed the presence of intact EVs (Figure 1K), with different sizes, morphology, and staining features. Electron-dense material in EVs surface was also visualized, but due to the low resolution of TEM images and the small size of AAVs (25 nm) it was not possible to confirm whether these were solo AAV vectors. NTA analysis confirmed EV sizes ranging from 70 to 130 nm in F7–F13 (Figure S3B). Additionally, between F7 and F13 there was a decrease in the ratio of nanoparticles per microgram of protein (Figure 1L).

The AAV elution profile was also evaluated by WB in the collected fractions. AAV capsid proteins were first detected on F8 and showed a gradual increase in F9–F13 (Figures 1I and 1J). The same was observed by quantification of vg using qPCR (Figure 1M).

A pool of SEC-isolated EVs containing F8–F11 was used in the following experiments. F7 was rejected from the pool due to the low amount of AAV vectors present in that fraction, while F12 and F13 were rejected due to the potential isolation of solo AAV vectors in these last fractions.

Next, we performed a side-by-side comparison of UC-100K and SEC F8–F11 samples regarding number and size of EVs, quantity of AAV vectors, and total protein content. For this, after cell debris removal, the conditioned medium was divided into two equal parts: half was used for UC-100K and the other half for SEC (Figure 2A). No significant differences were observed in the total protein amount (Figure 2B) nor the size of the RVg-EV-AAVs isolated by the two methods (Figures 2C and S3C). Nevertheless, the yield of nanoparticles was drastically higher (6.6-fold) by SEC (Figures 2D and S3D), suggesting a probable loss of EVs during UC. A higher ratio of nanoparticles per protein was also attained by SEC (Figure 2E). However, the viral vector yield was 3.8 times higher in RVg-EV-AAVs isolated by UC (Figure 2F), which may indicate that UC simultaneously isolates solo AAV vectors, EVs, and EV-AAVs. As a result, the heterogeneous mixture isolated by UC presents a higher vg-to-nanoparticle ratio (SEC: 5.023 ± 1.879; UC-100K: 94.77 ± 10.38), when compared to RVg-EV-AAVs isolated by SEC (Figure 2G). Overall, these data suggest that SEC and UC isolation methods can isolate different populations of intact RVg-EV-AAVs.

Different populations of RVg-EV-AAVs are obtained depending on the isolation method

(A) Schematic representation of the experimental design. EV-AAVs from the AAV vector production medium were purified by UC and SEC in parallel. A pool of SEC-isolated EV-AAVs was created containing F8–F11. (B) Protein content was measured by microBCA (μg). UC-100K showed a tendency for higher protein amounts when compared to pooled SEC F8–F11. (C) Nanoparticle size distribution (nm) was determined by NTA. Both isolation methods allowed the recovery of particles with similar size modes. (D) NTA showed higher total nanoparticle number (n°) in the pooled SEC fractions. (E) The ratio between the number of particles and protein (n°/μg) was higher in the pooled SEC fractions (F8–F11) compared to UC-100K. (F) Viral genome (vg) copies, quantified by qPCR, were 3.8-fold higher in the UC-100K fraction compared to pooled SEC fractions (F8–F11). (G) The vg per nanoparticle ratio (vg/nanoparticle) was higher in the UC-100K fraction when compared to the pooled SEC fraction. (B–G) Results are shown as mean ± SEM for UC-100K and SEC F8–F11 (n = 3 per group). Statistical analysis was performed using the two-tailed unpaired t test (∗p ≤ 0.05; ∗∗p < 0.01).

RVg-EV-AAVs deliver functional cargo to neuronal cells in vitro

To test the efficiency of transgene expression of the most promising SEC fractions containing AAV vectors in association with EVs, mouse neural crest-derived neuroblast (Neuro-2a [N2a]) cells were transduced with each fraction from F7 to F13 at an MOI of 10,000 vg/cell (Figure 3A). Transduction was qualitatively and quantitatively evaluated through the analysis of GFP expression by immunocytochemistry (ICC) (Figure 3B) and flow cytometry (FC) (Figures 3C and 3D), respectively. All conditions incubated with F7–F13 were positive for GFP expression, with F8-incubated cells showing a peak of transduction with 23.08% ± 1.69% GFP⁺ cells (Figure 3D). In contrast, cells incubated with F12 and F13 showed the lowest transduction efficiency. These results suggest that the most biologically active population of EV-AAV1/2 coated with RVg is collected in SEC F7–F11.

RVg-EV-AAVs efficiently deliver functional cargo to neuronal cells *in vitro*

(A) Schematic representation of the transduction assay. N2a cells were individually transduced at an MOI of 10,000 vg/cell with SEC fractions F7–F13 (B–D) or UC-20K, UC-100K, SEC F8–F11, and solo AAV1/2 (E–G). The medium was changed 48 h post-infection (pi) and immunocytochemistry (ICC) and flow cytometry (FC) were performed. (B) GFP expression was evaluated 48 h post-transduction by ICC. The presence of GFP⁺ cells was observed in all conditions (F7–F13). Non-transduced cells were used as negative control (CTRL). Anti-GFP, green; DAPI, blue. Scale bar, 200 μm. (C) FC was performed to quantify GFP expression levels (green) in all conditions. Non-transduced cells were used as negative controls for GFP (gray peaks). (D) FC quantification of GFP⁺ cells showing the highest transduction efficiency in conditions transduced with F8. Results are shown as mean ± SEM for F7–F13 and CTRL (n = 4 per group). (E) Neuronal cells transduced with UC-20K, UC-100K, SEC F8–F11, and solo AAV1/2 particles expressing GFP. Anti-GFP, green; DAPI, blue. Scale bar, 200 μm. Images are representative and may show field-to-field variability; quantitative comparison was based on FC and is shown in (F) and (G). (F) FC was performed to quantify GFP expression levels (green peaks) in transduced N2a cells. Cells incubated with PBS were used as controls (gray peaks). (G) FC quantification of GFP⁺ cells showed that SEC F8–F11 has the highest transduction efficiency, followed by UC-100K. Results are shown as mean ± SEM for UC-20K, UC-100K, F8–F11 (n = 6 per group), and AAV1/2 (n = 3). (D and G) Statistical analysis was performed using a two-tailed unpaired t test for comparisons between conditions (#p ≤ 0.05; ##p < 0.01), and a one-way ANOVA with Dunnett’s post hoc test for comparisons with the control group, respectively (∗p ≤ 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001).

Subsequently, we evaluated the neuronal transduction efficiency of RVg-EV-AAVs isolated by UC-20K, UC-100K, SEC (pooled F8–F11), and solo AAV1/2. N2a cells were incubated with 10,000 vg/cell, and the percentage of cells expressing GFP was assessed either by ICC (Figure 3E) or FC (Figures 3F and 3G) 48 h post-transduction. Although both ICC and FC analysis showed the presence of GFP⁺ cells in all conditions, the quantification of GFP⁺ cells revealed that SEC F8–F11 and UC-100K are the most effective conditions to transduce neuronal cells in vitro, with a percentage of 8.9 ± 0.75 and 7.5 ± 0.37, respectively (Figures 3F and 3G). Interestingly, RVg-EV-AAVs isolated by either UC or SEC showed 3.0 to 3.6 times higher transduction efficiency, respectively, in N2a cells when compared to solo AAV1/2 (Figures 3F and 3G).

RVg-EV-AAVs can cross the BBB and deliver functional cargo to neurons upon IV administration in vivo

Given the efficacy of RVg-EV-AAVs to deliver functional cargoes in vitro, we next evaluated the possibility of using these particles as a non-invasive delivery system to the brain. For this purpose, WT C57BL/6 mice were injected with RVg-EV-AAVs carrying an EGFP transgene in the facial vein (2 μL containing 1 × 10¹⁰ vg of RVg-EV-AAVs purified by SEC, F8–F11) at postnatal day 1 (P1) (Figure 4A). Mice were sacrificed at P100, and sagittal brain sections were collected to evaluate brain transduction of RVg-EV-AAVs by immunohistochemistry (IHC), using an antibody specific for GFP.

RVg-EV-AAVs can cross the blood-brain barrier and deliver functional cargo to the brain *in vivo*

(A) Schematic representation of the study design. Wild-type C57BL/6 mice were intravenously (IV) injected at postnatal day 1 (P1) with 1 × 10¹⁰ vg of RVg-EV-AAVs encoding EGFP (SEC pool from F8 to F11). Mice were sacrificed at P100, and brains were processed for immunohistochemical analysis (IHC). (B) Representative brain sections of P100 mice labeled with anti-GFP antibody. GFP labeling shows the preferential tropism of RVg-EV-AAVs to transduce the cerebellum (lobule IX and X are depicted), brainstem, and choroid plexus in neonatal WT mice injected with RVg-EV-AAVs (n = 4). A non-injected mouse was used as a staining control (CTRL). Cerebellum and brainstem scale bars, 250 μm; choroid plexus scale bar, 500 μm. (C) Higher magnification of representative images of the cerebellum, brainstem, lateral ventricle, and fourth ventricle from IHC-processed sections, with anti-GFP staining from the brains of P100 mice. Cerebellum, brainstem, lateral ventricle, and fourth ventricle scale bars, 50 μm. (D) Schematic representation of the IV injection of EV-AAVs into adult mice. Six-week-old WT C57BL/6 mice were IV injected with 2.5 × 10¹⁰ vg of uncoated EV-AAVs (unc-EV-AAVs) or RVg-coated EV-AAVs (RVg-EV-AAVs) encoding the firefly luciferase (FLuc) transgene. FLuc expression was assessed at 3, 6, 20, 23, and 30 days post-injection to monitor brain transduction in living mice. (E) Bioluminescence activity was observed in injected animals, with a significantly higher signal being measured in mice injected with RVg-EV-AAVs. This expression was stable over time, showing that EV-AAVs can stably transduce the cells in the brain. (F) Quantification of average radiance in the brain of unc-EV-AAVs and RVg-EV-AAVs-injected animals across all time points. Results are shown as mean ± SEM for PBS-injected control animals (n = 5), RVg-EV-AAVs (n = 4), and unc-EV-AAVs injected animals (n = 4). Statistical analysis was performed using the two-tailed unpaired t test (∗p < 0.05).

GFP expression was mostly observed in the cerebellum, particularly in Purkinje cells at lobule X, brainstem, and ependymal cells of the choroid plexus (lateral and fourth ventricles) (Figures 4B and 4C). These data suggest that RVg-EV-AAVs effectively deliver their cargo to the brain of mice, when injected at P1, resulting in long-term expression of the EGFP transgene.

Next, we aimed to further evaluate whether RVg-EV-AAVs could reach the brains of adult WT mice upon a single IV administration and to understand the impact of RVg peptide in EV-AAVs on brain tropism. For that, two groups of 6-week-old female mice were administered, in the tail vein, with either RVg-coated or uncoated EV-AAVs, encoding the firefly luciferase (FLuc) transgene (Figure 4D). FLuc is a bioluminescence reporter that emits light in the presence of luciferin substrate, allowing gene expression monitoring in living animals over time. A third group of mice was injected with phosphate buffered saline (PBS), to serve as experimental control. In this experiment, EV-AAVs were obtained by UC-100K since a higher concentration of vg was required. Bioluminescence analysis was used to monitor brain transduction in living animals at days 3, 6, 20, 23, and 30 post-injection (Figure 4D). A significant increase in bioluminescence signal was observed in the heads of RVg-EV-AAV-injected mice compared to uncoated EV-AAVs (Figures 4E and 4F), suggesting a superior brain transduction profile in the RVg-EV-AAV group. Transgene expression persisted 30 days post-injection, further supporting efficient brain delivery of EV-AAV1/2. In peripheral organs, RVg-EV-AAV-treated mice also showed a significant increase in bioluminescence signal in the lungs, along with a trend toward higher signal in the liver and heart compared to uncoated EV-AAVs (Figure S4).

IV administration of RVg-EV-AAVs encoding miR-ATXN3 alleviates neuropathological and motor impairments in MJD transgenic mice

Having demonstrated the efficacy of RVg-EV-AAVs to deliver therapeutic cargo to the brains of mice, we sought to evaluate the capability of these vectors to deliver an engineered miRNA-based silencing sequence to specifically target mutant ataxin-3 mRNA for the treatment of MJD.²⁶^,⁶⁷ We used a severe MJD transgenic mouse that presents motor impairments and neuropathologic features since the 3^rd week of age.⁶⁸ Cerebellar atrophy is specifically accentuated in this mouse model, in which the cerebellar volume is ∼40% smaller than in WT animals.⁶⁹

In our study, 9-week-old transgenic female MJD mice were IV injected in the tail vein with 1 × 10¹¹ vg of RVg-EV-AAVs encoding a miR-control or an miR-ATXN3 (Figure 5A).²⁶^,⁶⁷ We started by evaluating the expression levels of mutant ataxin-3 protein in the cerebellum of injected mice by WB. Despite non-significant statistical differences, there was a trend toward reduction in mutant ataxin-3 protein (hemagglutinin [HA]-tag) in MJD mice treated with miR-ATXN3 (Figures 5B and 5C). The same trend was observed by immunofluorescence (IF), where there was a tendency toward reduction in the number of ataxin-3 aggregates in the cerebellum, particularly in lobule X (Figures 5D and 5E). Moreover, treatment with miR-ATXN3 led to a significant preservation of the interlobular thickness region between lobules VII and VIII (Figures 5F and 5G), indicating the rescue of the typical cerebellar cortex atrophy observed in this model.

IV administration of RVg-targeted EV-AAVs encoding miR-ATXN3 alleviates motor and neuropathologic impairments in MJD transgenic mice

(A) Schematic representation of the study design. Nine-week-old transgenic MJD mice were IV injected on the tail vein with 1 × 10¹¹ vg of RVg-targeted EV-AAVs containing either miR-control or miR-ATXN3. Motor assessment was performed every 3 weeks for a total of 12 weeks after IV injection. Mice were sacrificed at 21 weeks of age (12 weeks after IV injection). Brains were divided into two halves for subsequent neuropathological analysis: left hemispheres were post-fixed and sliced for histological analysis, while right hemispheres were used for biochemical analysis. (B) WB evaluation of mutant ataxin-3 protein levels in the cerebellum (HA-tag antibody). (C) WB immunoreactivity quantification of HA-tag relative to miR-control showed a trend toward reduction of mutant ataxin-3 protein levels in miR-ATXN3-treated mice. Protein levels were normalized with β-actin immunoreactivity. (D) Representative images of immunofluorescence labeling of mutant ataxin-3 aggregates (anti-HA staining) in the cerebellum. Scale bars, 50 μm. (E) Quantification of the number of mutant ataxin-3 aggregates per mm² in lobule X of the cerebellum. miR-ATXN3-treated mice (red) showed a tendency for the reduction in the number of mutant ataxin-3 aggregates when compared with the group injected with miR-control (blue). (F) Representative bright-field microscopy image displaying the thickness of granular (GL), Purkinje cells (PC), and molecular (ML) layers after cresyl violet staining. Scale bars, 50 μm. (G) Quantification of the interlobular thickness (μm) across different lobules of the cerebellum showed an increase in lobules VII–VIII in miR-ATXN3-treated mice (red). (H and I) The motor phenotype was analyzed by measuring the mean latency time to fall (in seconds) at (H) constant rotarod test (5 rpm) and at (I) accelerated rotarod test (4–40 rpm). Mice treated with miR-ATXN3 had a higher latency to fall (red) when compared to miR-control animals (blue). (J) Gait performance by hindbase width in the footprint test. miR-ATXN3-treated mice (red) presented a reduced hindbase width when compared to miR-control animals (blue). (C and E–J) Results are shown as mean ± SEM for miR-control and miR-ATXN3 (n = 6–7 per group). Statistical analysis was performed using a two-tailed t test (∗p ≤ 0.05).

To determine whether the observed amelioration in biochemical and neuropathological markers of MJD would impact the phenotype of MJD mice, we conducted motor assessments using rotarod and footprint tests (Figures 5H–5J and S5). Behavioral assessments were performed every 3 weeks, up to 12 weeks after treatment. MJD mice treated with miR-ATXN3 endured for a longer period both in stationary (Figure 5H) and accelerated rotarod (Figure 5I) compared with miR-control. Moreover, MJD mice treated with miR-ATXN3 showed a statistically significant reduced hindbase width compared to the controls (Figure 5J), suggesting both motor and gait performance improvement.

Overall, these results suggest that targeting mutant ataxin-3 mRNA with miRNA-based therapy, delivered with RVg-EV-AAVs to 9-week-old mice, mitigates biochemical, neuropathological, and behavioral manifestations of MJD.

Discussion

The present study demonstrates that: (1) EV-AAVs can be isolated by different methods, namely UC and SEC; (2) RVg-EV-AAVs can efficiently deliver functional cargo to neuronal cells in vitro; (3) RVg-EV-AAVs can cross the BBB and deliver the functional cargo of AAV vectors to the brain and sustain stable transduction; and (4) IV administration of RVg-EV-AAVs encoding miR-ATXN3 alleviates motor behavior and neuropathological impairments in MJD transgenic mice.

AAV vectors are one of the preferred vehicles for gene therapy targeting the CNS³⁰ due to their ability to infect dividing and non-dividing cells, including neurons. Additionally, they exhibit lower cytotoxicity and immunogenicity in comparison with other viral vectors such as adenovirus, allowing high efficiency and sustained expression of transgenes.⁷⁰^,⁷¹^,⁷² However, the use of AAV vectors presents some limitations, such as the small transgene size capacity, the inability of certain serotypes to cross biological barriers like the BBB, and preexisting neutralizing antibodies that can eliminate incoming vectors and prevent therapeutic delivery.³⁹^,⁴⁰ To overcome these challenges, different strategies have been explored, including the engineering of AAV capsids and genomes and the use of immunosuppressants.³⁸^,⁴⁰

In 2012, it was demonstrated that during normal production of AAV vectors in human embryonic kidney 293 cells stably expressing the SV40 large T antigen (HEK293T), AAV vectors were also secreted in association with EVs.⁴² EVs have been widely studied due to their unique characteristics and possible application as gene delivery vectors.⁷³^,⁷⁴^,⁷⁵ They are naturally formed vesicles, with a lipid membrane identical to the cell membrane, involved in intercellular communication.⁷³^,⁷⁶ These small vesicles can circulate through the bloodstream, cross biological barriers such as the BBB, and deliver their cargo to tissues distant from their progenitor cells.⁵⁷^,⁷⁷^,⁷⁸ Similar to EVs, EV-AAVs can cross the BBB upon systemic injection and transduce neuronal cells.⁴²^,⁴³^,⁴⁴^,⁴⁵^,⁴⁶^,⁴⁷^,⁴⁸^,⁵⁹^,⁷⁹^,⁸⁰^,⁸¹^,⁸² Despite the relevance of this discovery, to date, only AAV serotypes able to cross the BBB (e.g., AAV9, AAV8) have been employed in EV-AAV systems to target the CNS in vivo, which does not eliminate the possible contribution of AAV vectors (either bound to the surface of EVs or present as free vectors) to these tropism observations.⁴³^,⁴⁴^,⁴⁹

In this context, to determine whether EV-AAVs could reach the brain without the contribution of the AAV serotype, we chose AAV1/2 mosaic vectors to generate EV-AAVs. AAV1/2 mosaic vectors combine the properties of both parental AAVs, including neurotropic features; however, they are still unable to efficiently cross the BBB.⁵¹^,⁵²^,⁵³ To direct EV-AAVs to the brain, the pRVg was engineered on the surface of EVs, as previously demonstrated by György and colleagues.⁴³ RVg peptides bind to acetylcholine receptors and selectively target neuronal cells and brain endothelial cells, enabling nanoparticles to cross the BBB with mild immunogenicity.¹⁷^,²⁶^,⁵⁶^,⁵⁷^,⁸³

Although UC has been the most commonly used protocol for the isolation of EV-AAVs, UC is considered a method with limited capabilities regarding reproducibility, scalability, and purity of the isolated product.⁸⁴ Moreover, it remains unclear whether the isolation of EV-AAVs by UC leads to the formation of EV-AAV associations in addition to the endogenously produced EV-AAVs.⁴³ For this reason, before moving to in vivo studies, we characterized the population of EV-AAVs obtained by UC and by an SEC protocol previously optimized in our laboratory.⁶³^,⁶⁴^,⁶⁵ It was possible to obtain EV-AAVs from the medium of a standard AAV vector production by both methods. Namely, in the fractions UC-100K and SEC F7–F13, no cell contamination was observed. Due to the low number of vg on SEC F7 and the potential isolation of solo AAV vectors on SEC F12 and F13, a pool of SEC-isolated EV-AAVs was created containing fractions F8–F11.

Overall, both isolation methods retrieved EVs with a similar size; however, SEC resulted in a 6.6-fold higher EV yield compared to UC, suggesting that SEC is more efficient in the recovery of EVs from the culture medium. However, the viral yield was 3.8-fold higher on EV-AAVs isolated by UC, which might be explained by the co-isolation of solo AAV vectors with EV-AAVs by UC. In fact, the centrifugation speed used (100,000 × g) is close to the sedimentation coefficient of AAVs.⁸⁵ Based on this, the differences observed in the vg-to-nanoparticle ratio in SEC and UC may be attributed not only to a higher number of EVs without AAV vectors obtained when isolated by SEC, but also to the co-isolation of solo AAV vectors with EVs during UC. Immunoaffinity-based methodologies can be further used in conjunction with SEC to specifically isolate an EV-AAV subpopulation, further increasing the homogeneity of the sample.⁸⁶

Despite the aforementioned differences observed in EV-AAVs obtained by SEC and UC, no significant differences were observed in the ability of EV-AAVs generated by the two methods to transduce neuronal cells in vitro. Nevertheless, EV-AAVs isolated by UC and SEC mediated higher neuronal transduction than solo AAV vectors. This difference may be due to the presence of pRVg⁴³^,⁵⁶ or other proteins on the surface of EVs, which facilitates internalization into neuronal cells.

Having demonstrated the efficiency of RVg-EV-AAV1/2 in vitro, we aimed to evaluate the possibility of using these vectors as a non-invasive delivery system to target the CNS. Following an IV injection in neonatal WT mice, we showed that RVg-EV-AAV1/2 can reach the mouse brain and mediate an efficient and sustained transgene expression at P100, with GFP being detected in the cerebellum and lateral ventricles. To delve deeper into the capacity of RVg-EV-AAV1/2 to transduce adult mouse brains upon IV injection, we performed a tail vein injection in a group of 6-week-old mice. In this experiment, we also aimed to assess the impact of RVg coating in EV-AAVs. Despite that both uncoated and RVg-coated EV-AAVs were able to transduce mouse brains, RVg-EV-AAV1/2-FLuc achieved superior transduction levels, with transgene expression persisting in adult WT mice for at least 30 days post-injection. Of note, since solo AAV1/2 are unlikely to cross the BBB and target the brain,⁵³ their potential co-isolation during UC should have a negligible impact on the observed results. Furthermore, co-isolated EVs are unlikely to mediate long-term expression. This suggests that RVg-coated EV-AAVs are the most predominant nanoparticles delivering cargo to the brain over time, in a sustained manner, in both neonatal and adult mice.

In addition, systemic administration of EV-AAVs, with or without RVg, led to transgene expression in peripheral organs, most notably the liver. This raises important safety considerations, since strong hepatic expression may increase the risk of toxicity. Potential strategies to mitigate this include the incorporation of liver-specific miRNA target sites (e.g., miR-122) in the transgene cassette to suppress hepatic expression⁸⁷ or glycan remodeling of EV surfaces to reduce asialoglycoprotein receptor-mediated uptake.⁸⁸^,⁸⁹ These approaches are expected to improve the brain specificity and overall safety of RVg-EV-AAVs in future developments. Addressing these safety issues will be essential to maximize the translational potential of EV-AAVs.

To our knowledge, this is the first time that EV-AAVs incorporating non-brain-targeting AAV serotypes, such as AAV1/2, were used in association with RVg-EVs to specifically target the brain. This demonstrates that EV-AAVs are naturally formed, and RVg-EV-AAVs can effectively access the brain parenchyma through IV administration, independently of the tropism of the AAV serotype. It should be noted that the systemic dose of EV-AAVs in this study was low (∼5 × 10¹² vg/kg) compared to what is used for brain tropic AAV9 vectors, which is typically 20-fold higher (∼1 × 10¹⁴ vg/kg).⁹⁰^,⁹¹ Thus, once we are able to scale up production of EV-AAVs using combined methods such as suspension neuronal or red blood cells, tangential flow filtration, and SEC, we can compare RVg-EV-AAVs to conventional AAV vectors at higher doses to fully assess its potential.

Due to the nature of these particles, we anticipate that a higher therapeutic dose compared to single AAV vectors will be well tolerated, with minimal immunogenicity. Future experimental settings will be needed to evaluate the maximal dose possible with EV-AAV treatment without apparent toxicity or other side effects.

Finally, we investigated whether RVg-EV-AAVs could be used for the delivery of an artificial miRNA (miR-ATXN3), previously used in our group to efficiently and selectively silence mutant ataxin-3.²⁶^,⁶⁷ It is highly relevant to mention that we used a severe MJD mouse model, characterized by a pronounced phenotype, including weight differences immediately after birth, as well as cerebellar atrophy and an ataxic phenotype since 3 weeks of age.⁶⁸ The transgenic MJD mice were treated at 9 weeks old, corresponding to an advanced disease stage and a late therapeutic intervention. Remarkably, IV injection of RVg-EV-AAV-miR-ATXN3 led to a tendency toward a decrease in ataxin-3 aggregates and the preservation of interlobular thickness in the cerebellum and the improvement of motor performance, as assessed through rotarod and footprint tests. With further improvements in the scalability of EV-AAV production, in the future we will be able to assess the effect of higher doses of RVg-EV-AAV-miR-ATXN3, which will likely enhance therapeutic benefits.

Overall, our work demonstrates that RVg-EV-AAVs represent promising minimally invasive gene delivery vectors for the treatment of neurodegenerative disorders, including MJD.

Materials and methods

In vitro and in vivo experiments

All work involving cells and viral vectors was performed in appropriate biosafety cabinets and incubators, separated from those used for maintaining cell lines. All equipment and reagents in contact with cells in culture and viruses were sterilized, and after usage, virally contaminated materials were properly sterilized or disposed of, according to Good Laboratory Practice.

Cell lines

HEK293T and N2a cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) high glucose (Thermo Fisher Scientific), supplemented with 10% fetal bovine serum (FBS; Biowest) and 1% penicillin-streptomycin (P/S; Life Technologies). Cells were maintained at 37°C under a humidified atmosphere containing 5% CO₂, unless otherwise mentioned. Sterile PBS (pH 7.4) and 0.05% trypsin (Life Technologies) were used for cell passage.

Production of AAV vectors and EV-AAVs

Twenty-four hours before transfection, 1.05 × 10⁷ HEK293T cells were plated into 15-cm dishes and cultured in DMEM supplemented with 10% FBS, 1% P/S, and 1 M hydroxyethyl-piperazine ethane sulfonic acid (HEPES; Life Technologies) buffer. AAV vectors were produced according to the standard transfection method.⁵⁴^,⁹² To make mosaic AAV1/2 capsids, during transfection in HEK293T cells, instead of a single rep/cap plasmid, we used a 1:1 ratio of rep1/cap1 plasmid (AAV1) and rep2/cap2 plasmid (AAV2). This strategy has been reported previously.⁵⁴^,⁹³^,⁹⁴ Briefly, four plasmids were used: (1) a plasmid encoding the EGFP gene, FLuc,⁴³ artificial miR-control or miR-ATXN3²⁶^,⁶⁷, under the control of chicken β-actin promoter, and placed between the inverted terminal repeat sequences (pITRs); (2, 3) two plasmids containing the WT rep and cap genome sequences of serotypes AAV1 and AAV2; and (4) a plasmid encoding the adenovirus proteins (E1A, E1B, E4, and E2A) along with the adenovirus’ RNAs necessary for helper functions (pHelper). A fifth plasmid encoding for the pRVg-platelet-derived growth factor receptor transmembrane domain was also transfected for the production of RVg-EV-AAVs.⁴³ For an efficient transfection, polyethylenimine (linear, MW 40,000 Da) (Polysciences, Inc.) was used as the transfection reagent.

Medium was replaced 16 h post-transfection by fresh DMEM, supplemented with 2% EV-free FBS (to minimize bovine EVs contamination), 1% P/S, and 1% HEPES. Conditioned medium was collected at 48 h and 72 h post-transfection and centrifuged for 10 min at 300–500 × g to remove cells, followed by another centrifugation at 1,000–2,000 × g for 10–15 min to remove cell debris. Medium was stored at −80°C for future isolation of EV-AAVs, while cells were harvested for AAV vector purification.

Isolation of EV and EV-AAVs by differential centrifugation

Differential centrifugation was performed using a 70-Ti rotor in an Optima XE-100 ultracentrifuge (Beckman Coulter). Conditioned medium was centrifuged for a minimum of 1 h at 20,000 × g. The pellet was discarded, and the supernatant was centrifuged at 100,000 × g for at least 1 h. The final pellet containing EV-AAVs was resuspended in PBS.

Isolation of EV-AAVs by SEC

Conditioned medium was concentrated, depending on the initial volume, using centrifugal concentrators (Vivaspin 20/Vivacell 70, Sartorius) or pressure concentrators (Vivacell 250, Sartorius) with an MWCO of 10 kDa. For EV-AAV isolation, commercial 70-nm agarose-based size exclusion columns (qEVoriginal, Izon Science) were used as follows: 1 mL concentrated medium was loaded on qEVoriginal columns, followed by PBS.⁶³^,⁶⁴^,⁶⁵ Fractions of 0.5 mL were collected and immediately used or stored at −80°C for no more than 3 months before further analysis. For in vivo experiments, after SEC, an additional concentration step was performed using centrifugal concentrators (Vivacell 20, Sartorius).

Isolation of AAV vectors

AAV vectors were purified in our core facility ViraVector (CNC-UC) with an AKTA Pure 25 L system (GE Healthcare, Life Sciences), using a protocol based on fast protein liquid chromatography (FPLC), previously optimized in our group.⁵⁴ At the end, purified AAV vectors were concentrated using centrifugal concentrators (Amicon Ultra-15 and Ultra-0.5, Merck) with an MWCO of 100 kDa and supplemented with 0.001% Pluronic F-68 100X (Life Technologies) to avoid the further loss of AAV vectors that tend to adhere to the surface of materials.

Protein quantification

Total protein was quantified in samples isolated by UC and SEC through (1) bicinchoninic acid (BCA) (Thermo Scientific), in the case of lysed samples (lysis described below), and (2) microBCA (Thermo Scientific) assays, when using non-lysed samples. For the in vivo experiments, the protein concentration was estimated through the Bradford protein assay (Bio-Rad).

WB

All samples used for WB analysis were lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris base [Thermo Scientific], 150 mM sodium chloride [Acros Organics], 5 mM ethylene glycol tetraacetic acid [Thermo Scientific], 1% Triton X-100 [Thermo Scientific], 0.5% sodium deoxycholate [Sigma-Aldrich], and 0.1% sodium dodecyl sulfate [SDS; Thermo Scientific], pH 7.5), supplemented with a protease inhibitor cocktail (Roche) and 200 μM phenylmethylsulfonyl fluoride (Sigma-Aldrich), 1 mM dithiothreitol (DTT; Thermo Scientific), 1 mM activated sodium orthovanadate (Sigma-Aldrich), and 5 mM sodium fluoride (Acros Organics), except for solo AAV vectors purified by FPLC. Samples were then denatured with 6× sample buffer (9.3% DTT, 10% SDS, 30% glycerol [Thermo Scientific], and 0.012% bromophenol blue [Sigma-Aldrich]) in 0.5 M Tris-HCl (Thermo Scientific)/0.4% SDS (pH 6.8), for 5 min at 95 °C. For protein extracts from in vitro experiments, the same volume was used for each sample, and protein extracts from HEK293T cells were used as reference. For in vivo protein extracts, samples were sonicated by two series of 5-s ultrasound pulses (1 pulse/s), and equal amounts of protein (40 μg) from each condition were used.

Protein extracts were resolved in SDS-polyacrylamide gels (4% stacking and 10% running, Bio-Rad) and transferred onto polyvinylidene difluoride membranes (Immobilon-P, EMD Millipore), according to standard protocols. After protein electrotransfer, membranes were stained with Ponceau S (Thermo Scientific) to visualize total protein and subsequently washed with 0.1 M sodium hydroxide (Thermo Scientific). Then, membranes were blocked by incubation in 5% non-fat milk powder in 0.1% Tween 20 (VWR Chemicals) in Tris-buffered saline, for 1 h at room temperature (RT). Immunoblotting was performed overnight at 4°C with the following primary antibodies, diluted in the blocking solution: anti-CNX H70 (rabbit polyclonal, Santa Cruz Biotechnology, 1:1,000), anti-FLOT-1 clone 18 (mouse monoclonal, BD Transduction Laboratories, 1:400), anti-AAV (VP1/VP2/VP3) clone B1 (mouse monoclonal, American Research Products, 1:1,000), HA-11-Tag (mouse monoclonal, BioLegend, 1:1,000), calbindin D-28K (rabbit polyclonal, Millipore, 1:1,000), and β-actin (mouse monoclonal, Sigma-Aldrich, 1:10,000), followed by incubation with the corresponding alkaline phosphatase-coupled secondary antibodies (goat anti-rabbit polyclonal antibody, Thermo Scientific, 1:10,000, or goat anti-mouse polyclonal antibody, Invitrogen, 1:10,000), diluted in the blocking solution. For in vitro experiments, after blocking, membranes were horizontally cut into two pieces, between 63 and 48 kDa, using the MW ladder as a guide. The top piece was incubated with the antibodies anti-CNX H70, followed by anti-AAV clone B1, and the bottom piece with the antibodies anti-FLOT-1 clone 18, followed by anti-AAV clone B1 (Figure S2). Alternatively, if membranes were done in duplicate, the top piece was incubated with anti-CNX H70, the bottom piece with the anti-FLOT-1 clone 18, and the second membrane was incubated with anti-AAV clone B1 (Figure S1). The presence of antigens of interest was observed with enhanced chemifluorescence substrate (GE Healthcare) and chemifluorescence imaging (ChemiDoc Imaging System, Bio-Rad). For in vivo experiments, the specific optical density was normalized to the β-actin amount in the corresponding lane of the same membrane.

Quantification of AAV vectors by real-time PCR

Absolute quantification of AAV titer (vg/μL) was performed by quantitative real-time PCR using the AAVpro Titration Kit (for real-time PCR) version 2 (Takara Bio), according to the manufacturer’s instructions. Briefly, following DNase I treatment, AAV capsids were lysed, samples were diluted, and qPCR was performed using primers targeting the ITRs of AAVs. A standard curve was used for absolute quantification.

NTA

Measurements of nanoparticle size and concentration in RVg-EV-AAVs preparations were performed by NTA. A NanoSight NS300 instrument (Marvell Panalytical), with a 488-nm laser and sCMOS camera module (Malvern Panalytical) was used, following the manufacturer’s instructions. Five videos of 30 s were recorded for each sample using a syringe pump speed of 40. Measurements were performed using NTA 3.2 software (Malvern).

TEM

TEM was performed according to Théry and collaborators.⁹⁵ Briefly, RVg-EV-AAVs were fixed with 2% paraformaldehyde (PFA, Acros Organics) and deposited on Formvar carbon-coated grids (TAAB Laboratories Equipment) for 20 min. Grids were washed with PBS and fixed for 5 min with 1% glutaraldehyde. Following a cycle of washes using distilled water, grids were contrasted with a uranyl-oxalate solution (pH 7) for 5 min and transferred to methyl-cellulose-uranyl acetate for 10 min on ice. Images were obtained using a Tecnai G2 Spirit BioTWIN electron microscope (FEI Company) at 80 kV.

In vitro transduction assay

To evaluate the transduction efficiency of RVg-EV-AAVs and AAV vectors, N2a cells were seeded in either 24-well plates (5 × 10⁴ cells/well) for FC or 48-well plates (2 × 10⁴ cells/well) for ICC analysis. On the following day, half of the medium was collected, and cells were infected, with different samples equally diluted in PBS, at 10,000 vg/cell. Medium was replaced 16 h post-vector addition. Transgene expression was evaluated 48 h post-infection.

FC

Two days post-infection, infected N2a cells were processed for FC analysis. Briefly, cells were harvested, washed with cold PBS, collected by centrifugation, and resuspended in PBS in conic tubes (BD Biosciences). Samples were kept on ice before being analyzed for GFP expression in an FC Calibur flow cytometer (BD Biosciences). A total of 2 × 10⁴ cells were scanned during each acquisition, and GFP fluorescence was evaluated in the FL-1 channel. Data analysis was performed using FlowJo software (Tree Star).

ICC

Two days post-infection, infected N2a cells were washed with PBS and fixed in 4% PFA in PBS for 15 min at RT. After fixation, cells were washed and permeabilized with 0.1% Triton X-100 in PBS for 5 min at RT. Blockage of non-specific staining was performed with 10% bovine serum albumin (BSA, Acros Organics) in PBS for 30 min at 37°C, and cells were then incubated overnight at 4°C with the rabbit anti-GFP primary antibody (1:1,000, Invitrogen) diluted in 3% BSA in PBS. Subsequently, cells were washed and incubated for 45 min at 37°C with the goat anti-rabbit secondary antibody (1:250, Thermo Scientific), also diluted in 3% BSA in PBS. Cells were washed with PBS, incubated for 7 min with DAPI (1:5,000, Sigma-Aldrich), rinsed, and maintained in PBS at 4°C until visualization. Images were obtained using the CELENA S digital cell imaging system (Logos Biosystems) equipped with a 10× objective.

Animals

Animals were housed in a temperature-controlled room maintained on a 12-h light/12-h dark cycle. Food and water were provided ad libitum. All experiments involving animals were carried out in accordance with the European Union Community directive (2010/63/EU) for the care and use of laboratory animals, transposed into Portuguese law in 2013 (Decree Law 113/2013). Additionally, experiments involving mice were approved by the Responsible Organization for the Animal Welfare (ref. 66_2015/22062015) of the Faculty of Medicine and Center for Neuroscience and Cell Biology of the University of Coimbra licensed animal facility (Coimbra, Portugal). The involved researchers received adequate training (Federation of European Laboratory Animal Science Associations-certified course), and certification to perform animal experiments from the Portuguese authorities (Directorate-General for Food and Veterinary Medicine, Lisbon, Portugal; ref. 0421/000/000/2015).

Neonatal IV injections

C57BL/6 pregnant mice were housed and monitored daily from embryonic days 17 to 21, with minimal disturbance, to ensure that newborn pups could be dosed with vectors at P1.

Newborn mice were initially rested on a bed of ice for approximately 1 min for anesthetization. RVg-EV-AAV-EGFP samples isolated by SEC, containing a total of 1 × 10¹⁰ vg (5.9 × 10¹² vg/kg) diluted in 50 μL PBS, were manually injected into the facial vein of seven newborn mice, using a 100-μL Hamilton syringe connected to a 30G beveled tip needle (Hamilton). A correct injection was verified by noting the blanching of the vein. After injection, pups were identified by toe tattooing, carefully cleaned, rubbed with their original bedding to prevent maternal rejection, and then returned to their original cage. Animals were sacrificed at P100 (n = 4) for histologic analysis of the brain.

Adult IV injections

To ascertain the brain-targeting potential of RVg-coated EV-AAV1/2 by bioluminescence, 6-week-old WT C57BL/6 female mice were injected in the tail vein with 2.5 × 10¹⁰ vg of either uncoated EV-AAVs (unc-EV-AAVs, n = 4) or RVg-EV-AAVs (n = 4) expressing FLuc isolated by UC. Animals injected with PBS were used as controls (n = 5).

To assess the therapeutic potential of EV-AAVs, 9-week-old female MJD transgenic mice were used (C57BL/6 background). This animal model expresses a truncated form of human ataxin-3 with 69 glutamine repeats (Tg-ATXN3-69Q), together with an N-terminal HA epitope, driven by the L7 promoter.⁶⁸ Mice were injected in the tail vein with 1 × 10¹¹ vg (4.3 × 10¹² vg/kg) of either RVg-EV-AAV-miR-control (n = 6) or RVg-EV-AAV-miR-ATXN3 (n = 7) isolated by UC. Briefly, mice were placed into a restrainer (BioSeb), their tails were warmed with a heat lamp to dilate the veins, and 0.15 mL vector was slowly injected into the lateral tail vein. To stop the bleeding, gentle finger clamping was applied at the injection site.

Animals were sacrificed 30 days or 12 weeks post-injection (EV-AAV-FLuc and EV-AAV-miR experiments, respectively). For therapeutic evaluation, brains were divided into two halves: one hemisphere was stored directly at −80°C and the other hemisphere was post-fixed in PFA for histological analysis, as detailed below.

Bioluminescence imaging

Bioluminescence imaging was performed using an IVIS Lumina XR imaging system (PerkinElmer). In vivo imaging was performed in anesthetized mice that were previously shaved around the head. Mice were then injected intraperitoneally (IP) with 100 μL d-luciferin (Promega) resuspended in Dulbecco’s (d)PBS (30 mg/mL final concentration) and imaged 5 min later for luciferase activity using the auto-acquisition mode. Mice were imaged at 3, 6, 20, 23, and 30 days after the single IV injection on day 0. Mice were sacrificed at day 30 with an overdose of xylazine/ketamine and transcardially perfused with d-luciferin (Promega) resuspended in dPBS (240 μg/mL final concentration). Peripheral organs were collected and bioluminescence acquired in IVIS Lumina XR imaging system (PerkinElmer).

The data analysis for signal intensities and image comparisons was performed using Living Image software (version 4.10, PerkinElmer).

Rotarod performance test

To evaluate motor coordination and balance, stationary and accelerating rotarod tests were performed using a rotarod apparatus (Panlab Harvard Apparatus). This test was performed before the injection with either RVg-EV-AAV-miR-control or RVg-EV-AAV-miR-ATXN3 vectors and every 3 weeks until 12 weeks after injection. On the stationary rotarod test, mice were placed on the rotarod apparatus at a constant speed of 5 rpm for a maximum of 300 s (5 min). On the accelerating rotarod test, mice were placed on the apparatus at an accelerated speed (from 4 to 40 rpm) over a period of 300 s (5 min). The total time during which mice remained running in the rotation roll was recorded (latency to fall).

Footprint analysis

To evaluate the gait profile, footprint tests were performed every 3 weeks post-RVg-EV-AAV injection for a total of 4 time points. Briefly, mice paws were coated with non-toxic paints and animals were allowed to walk on a 100-cm-long white sheet of paper (10-cm-wide runway with 15-cm-high walls). To assess mice gait, three parameters were analyzed: (1) hindbase width, the distance between the left and right hind footprints; (2) overlap length, the distance between the center of the hind footprint and the center of the preceding front footprint; and (3) stride length, determined by measuring the perpendicular distance of a given step and proceeding steps. A sequence of six consecutive steps was used for evaluation, excluding the footprints at the beginning and end of the run.

Tissue collection and preparation

Mice were terminally anesthetized through the IP route and transcardially perfused with cold PBS (pH 7.4). For biochemistry, brains were immediately stored at −80°C for posterior analysis. For immunostaining, excised brains were post-fixed with 4% PFA for 48 h at 4°C, transferred to 20% sucrose in PBS, and frozen at −80°C upon sinking. Sagittal brain sections with 30 μm thickness were subsequently obtained by using a cryostat, with the temperature set at −20°C. Sections were collected as free-floating sections in PBS supplemented with 5% sodium azide (Sigma-Aldrich) in 48-well trays and stored at 4°C until further processing.

Cresyl violet staining

Brain sections were mounted on gelatin-coated slides, hydrated with ultrapure water, and dehydrated in an ascending ethanol sequence (95% and 100%, Thermo Scientific), cleared with xylene solution (Thermo Scientific), hydrated with ethanol 75% and ultrapure water, stained with cresyl violet (Sigma-Aldrich) for 5 min and rehydrated with ultrapure water, dehydrated in an ascending ethanol sequence (75%, 95%, and 100%), cleared with xylene solution, and finally coverslipped with Eukitt (O. Kindler GmbH).⁹⁶ Images were acquired using a Zeiss Axio Imager Z2 microscope (Carl Zeiss Microscopy GmbH) equipped with a high-resolution color camera and with a Plan-Apochromat 20×/0.8 M27 objective. Interlobular thickness was manually measured using ZEN 2 software (Blue Edition, Carl Zeiss Microscopy).

Immunostaining

For IHC, sections were incubated in 0.1% phenylhydrazine (Merck) in PBS for 30 min at 37°C to block endogenous peroxidases. For both IHC and IF, simultaneous blocking and permeabilization were performed for 1 h at RT in blocking solution (0.1% Triton X-100 containing 10% normal goat serum [Gibco] in PBS). Brain slices were then incubated overnight at 4°C with the rabbit anti-GFP primary antibody (1:1,000, Invitrogen) or mouse anti-HA-11-Tag antibody (BioLegend, 1:1,000), diluted in the blocking solution. Following three washing steps in PBS, free-floating sections were incubated for 2 h at RT with the anti-rabbit biotinylated secondary antibody (Vector Laboratories) or Alexa Fluor 568 anti-mouse antibody (Invitrogen), diluted in blocking solution (1:250). Subsequently, free-floating sections were rinsed with PBS. For IHC, bound antibodies were visualized by the avidin-biotin complex amplification system (Vectastain ABC Kit, Vector Laboratories) using 3,3′-diaminobenzidine tetrahydrochloride (DAB Substrate Kit, Vector Laboratories) as substrate. The reaction was stopped by washing sections in PBS after achieving optimal staining. For IF, sections were incubated for 7 min with DAPI (1: 5,000, Sigma-Aldrich) and rinsed in PBS.

Sections were mounted on gelatin-coated slides. For IHC, sections were hydrated with ultrapure water and dehydrated in an ascending ethanol sequence (70%, 95%, and 100%). Slides were then cleared with xylene solution and finally coverslipped with Eukitt (O. Kindler GmbH). For IF, slides were coverslipped with fluorescence mounting medium (DAKO).

Images were acquired using a Zeiss Axio Imager Z2 microscope (Carl Zeiss Microscopy GmbH), equipped with a high-resolution color camera. Images of the cerebellum, the brainstem, and the choroid plexus were obtained with a Plan-Apochromat 20×/0.8 M27 or 63×/1.4 objective. Ataxin-3 inclusions (HA⁺) were manually counted using Fiji software.

Statistical analysis

All statistical analyses were performed using GraphPad Prism software (version 9.0.0). Data are presented as mean ± standard error of the mean (SEM). Unpaired Student’s t tests and one-way ANOVA tests were performed when applicable. Significance was determined according to the following criteria: ∗p ≤ 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Data and code availability

Data will be available on reasonable request to the corresponding authors.

Acknowledgments

This work was funded by the European Regional Development Fund (ERDF) through the Centro 2020 Regional Operational Programme, the Operational Programme for Competitiveness and Internationalisation (COMPETE 2020), and by Portuguese national funds via Fundação para a Ciência e a Tecnologia (FCT), under the projects UIDB/04539/2025, UIDP/04539/2025, LA/P/0058/2020 and 2022.06118.PTDC. The work also received funding from the projects SpreadSilencing (POCI-01-0145-FEDER029716), ViraVector (CENTRO-01-0145-FEDER-022095) and Neurodiet (JPND/0001/2022). Additional support was provided by CinTech under the Recovery and Resilience Plan (RRP); by Accelerating Research & Development for Advanced Therapies (ARDAT), under the IMI2 JU grant agreement no. 945473 (co-funded by the European Union and the European Federation of Pharmaceutical Industries and Associations); and Capacity 2023 (ID: 101145599), GeneT (ID: 101059981), European Rare Diseases Research Alliance (ERDERA) (ID: 101156595), GeneH (ID: 101186939) and GCure (ID: 101186929), under the European Union’s Horizon Europe program. Further funding was received from the American Portuguese Biomedical Research Fund, the European Advanced Translational Research Infrastructure for Neurosciences (NeurATRIS), and the Richard Chin and Lily Lock MJD Research Fund. C.H. was supported by 2021.06939.BD; D.R.-R. is supported by Friedreich’s Ataxia Research Alliance (FARA) and FARA Australia; G.G. was supported by 2024.04513.BD; K.L. was supported by SFRH/BD/09513/2020; M.M.L. was supported by 2021.05776.BD; and P.A. was supported by SFRH/BD/90730/2012. We thank M. Zuzarte (Faculty of Medicine, University of Coimbra) for electron microscopy imaging. We thank Luisa Cortes, Tatiana Catarino, Margarida Caldeira, and the CNC Microscopy Imaging Center of Coimbra (MICC) team for assistance with microscopy imaging. We thank all members of the L.P.d.A. lab for all the support, discussion, and comments.

Author contributions

C.H., P.A., M.M.L., M.M.S., L.P.d.A., and R.J.N. conceived and designed the experiments. C.H., P.A., D.R.-R., M.M.L., K.L., C.O.M., R.A., G.G., J.d.S.-L., S.M.L., L.S.G., T.M.R.-R., and M.M.S. performed the experiments. C.H., P.A., K.L., and G.G. analyzed the data. D.R.-R., M.M.L., K.L., D.D.L., A.C.S., H.G., C.M.G., R.B., S.D., and C.A.M. shared knowledge. C.H., P.A., and D.R.-R. wrote the first draft of the paper. All the authors reviewed and edited the paper.

Declaration of interests

R.J.N. and L.P.d.A. are the inventors listed on patent application WO2020144611A1, which is related to the subject of this article.

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.ymthe.2025.10.022.

Contributor Information

Luís Pereira de Almeida, Email: luispa@cnc.uc.pt.

Rui Jorge Nobre, Email: rui.nobre@cnc.uc.pt.

Supplemental information

Document S1. Figures S1–S5

mmc1.pdf^{(473.8KB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(26MB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S5

mmc1.pdf^{(473.8KB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(26MB, pdf)}

Data Availability Statement

Data will be available on reasonable request to the corresponding authors.

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PERMALINK

Extracellular vesicles-associated AAVs for the treatment of Machado-Joseph disease

Carina Henriques

Patrícia Albuquerque

David Rufino-Ramos

Miguel M Lopes

Kevin Leandro

Catarina O Miranda

Rita Almeida

Guilherme Lopes-Gabriel

João de Sousa-Lourenço

Sara M Lopes

Laetitia S Gaspar

Diana D Lobo

Ana Carolina Silva

Teresa M Ribeiro-Rodrigues

Henrique Girão

Célia M Gomes

Rafael Baganha

Sónia Duarte

Casey A Maguire

Magda M Santana

Luís Pereira de Almeida

Rui Jorge Nobre

Abstract

Graphical abstract

Introduction

Results

SEC and UC can isolate intact RVg-EV-AAVs

Figure 1.

Figure 2.

RVg-EV-AAVs deliver functional cargo to neuronal cells in vitro

Figure 3.

RVg-EV-AAVs can cross the BBB and deliver functional cargo to neurons upon IV administration in vivo

Figure 4.

IV administration of RVg-EV-AAVs encoding miR-ATXN3 alleviates neuropathological and motor impairments in MJD transgenic mice

Figure 5.

Discussion

Materials and methods

In vitro and in vivo experiments

Cell lines

Production of AAV vectors and EV-AAVs

Isolation of EV and EV-AAVs by differential centrifugation

Isolation of EV-AAVs by SEC

Isolation of AAV vectors

Protein quantification

WB

Quantification of AAV vectors by real-time PCR

NTA

TEM

In vitro transduction assay

FC

ICC

Animals

Neonatal IV injections

Adult IV injections

Bioluminescence imaging

Rotarod performance test

Footprint analysis

Tissue collection and preparation

Cresyl violet staining

Immunostaining

Statistical analysis

Data and code availability

Acknowledgments

Author contributions

Declaration of interests

Footnotes

Contributor Information

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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