Isolation of Biologically Active Extracellular Vesicles-Associated AAVs for Gene Delivery to the Brain by Size Exclusion Chromatography

doi:10.21203/rs.3.rs-3220758/v1

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Isolation of Biologically Active Extracellular Vesicles-Associated AAVs for Gene Delivery to the Brain by Size Exclusion Chromatography

https://doi.org/10.21203/rs.3.rs-3220758/v1

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Extracellular vesicles-associated adeno-associated viral vectors (EV-AAVs) emerged as a new opportunity for non-invasive gene therapy targeting the central nervous system (CNS). However, in previous reports, only AAV serotypes with known ability to cross the blood-brain barrier (BBB) have been used for EV-AAV production and testing through non-invasive strategies. In this work, we aimed at optimizing a size exclusion chromatography (SEC) protocol for the production and isolation of natural and biologically active brain-targeting EV-AAVs, that could be applied to any AAV serotype and further used for non-invasive gene delivery to the CNS. We performed a comparison between SEC and differential ultracentrifugation (UC) isolation protocols in terms of yield, contaminants, and transgene expression efficiency. We found that SEC allows a higher recovery of EV-AAVs, free of cell contaminating proteins and with less solo AAVs than UC. Remarkably, SEC-purified EV-AAVs also showed to be more potent at transgene expression than solo AAVs in neuronal cell lines. Brain-targeting EV-AAVs exhibited the ability to reach be brain upon intravenous administration. In conclusion, SEC-purified brain-targeting EV-AAVs show to be a promising gene delivery vector for therapy of brain disorders.

Biological sciences/Biotechnology/Gene delivery

Biological sciences/Biological techniques/Isolation, separation and purification

Extracellular vesicles

Adeno-associated virus

Size exclusion chromatography

Gene therapy

Brain

In the last few years, adeno-associated viral vectors (AAVs) have been the vector of choice for the delivery of gene products to the human body, specially to the brain¹. Wild-type AAVs are small viruses, with a big repertory of serotypes with different tissue tropisms^2,3. Currently, six AAV-based products have been approved for the treatment of human diseases in Europe and the USA^4–9. However, new difficulties have emerged with clinical translation and use of these vectors¹⁰. A major challenge is the high prevalence of pre-existing neutralizing antibodies against AAV capsids in humans, related to previous AAV infection or cross-reactivity between different AAV serotypes¹¹. In addition, for central nervous system (CNS) disorders, high doses of systemically delivered AAVs need to be used to reach the brain, even when AAV serotypes known to cross the blood brain barrier (BBB) are used (e.g. AAV8 and AAV9). Moreover, upon intravenous (IV) administration, most AAV serotypes show tropism to peripheral tissues (e.g. liver) causing the risk of undesired expression in off-target tissues and toxicity¹². To circumvent these limitations, a significant effort has been made to develop new AAV capsids able to avoid neutralization, efficiently cross the BBB and transduce specific regions or cell populations of the CNS, while simultaneously maintaining a low transduction of peripheral organs^10,13.

During the production of AAVs, a portion of these vectors were found to be secreted into the media in association with extracellular vesicles (EVs), the so-called EV-AAVs¹³. Several reports have demonstrated that EV-AAVs are not only able to achieve a higher cell transduction than AAVs^14–19, but also to circumvent antibody neutralization^{13–16, 19} and biological barriers such as the BBB^14,20. However, only AAV serotypes with already known ability to cross the BBB upon IV injection were used for the development of EV-AAVs targeting the CNS. It remains unclear whether AAV serotypes without natural capacity to cross the BBB upon IV injection, such as AAV1 and AAV2^21–24, can reach the brain in association with EVs. Moreover, there is still the need for an isolation protocol that allows the recovery of pure and biologically formed EV-AAVs with potential for scale-up and clinical translation.

The present work aimed to optimize and validate a protocol for the isolation of purer and biologically active EV-AAVs, able to reach the brain upon IV injection and transduce the CNS (Graphical Abstract).

2.1. Size exclusion chromatography allows the isolation of pure and biologically active EV-AAVs

Size exclusion chromatography (SEC) has been shown to be a simple and fast strategy to isolate highly pure, intact and functional EVs, with potential application in clinical setting^25–27. In the present work, we aimed at optimizing a SEC protocol for the isolation of EV-AAVs, based on a protocol previously developed by our group to isolate EVs from plasma²⁸.To achieve that, we took advantage of mosaic AAV vectors carrying capsid proteins from AAV1 and AAV2, thus combining the properties of both AAV serotypes that, despite their neurotropic features, are not able to efficiently^21–24. In our protocol, HEK293T cells were transfected with the four plasmids for AAV1/2 production: (I) a plasmid encoding the enhanced green fluorescent reporter gene (GFP), under the action of chicken beta-actin (CBA) promoter placed between the ITRs sequences (pITR), (II, III) two plasmids containing the wild-type (WT) AAV genome rep and serotype 1 and 2 cap ORFs and (IV) a helper plasmid²⁹. A fifth plasmid encoding for Rabies virus glycoprotein peptide (RVg) was also used to target EV-AAVs to the brain¹⁴. The conditioned medium from AAV-producer cells was then collected at 48 h and 72 h post-transfection (Fig. 1.A), pre-cleared by low-speed centrifugations and concentrated using ultrafiltration (UF) with a molecular weight cut-off (MWCO) of 10 000 kDa to reduce EVs loss³⁰ (Fig. 1.B). The filtered concentrated medium was then applied into an agarose-based SEC column and 30 fractions (F) of 0.5 mL were collected individually for posterior analysis (Fig. 1.C).

To identify the SEC fractions where most proteins eluted, we performed a western blot on the 30 collected fractions and assessed total protein by staining membranes with Ponceau S solution. Proteins were mainly detected between F12 and F26, with a peak at F18 (Supplementary Fig. 1.A and Supplementary Fig. 1.B). The same profile was obtained when proteins were quantified using the bicinchoninic acid assay (BCA) (Supplementary Fig. 1.C).

To find out in which SEC fraction(s) EVs were eluted, we used the marker flotillin 1 (FLOT-1). This marker is expressed in both small and medium/large EVs allowing us to identify the range of fractions containing both entities. As shown in Fig. 2.A and Fig. 2.B, FLOT-1 was firstly detected on F7, and showed a peak between F8 and F11. Calnexin (CNX), a typical cellular marker, was not detected between F7 to F13, indicating the absence of cell contamination. Nanoparticle analysis by NTA confirmed the presence of EVs sized from 70 nm to 130 nm in F7-13. In these fractions, we also found a gradual decrease in size mode and in the nanoparticle/protein ratio (Fig. 2.C-E). Negative stain Transmission Electron Microscopy (TEM) also confirmed the presence of intact EVs between F7 and F12 (Supplementary Fig. 3).

The AAV elution profile was also evaluated by western blot. AAV capsid proteins (VP1, VP2, and VP3, that assemble in a 1:1:10 ratio) were firstly detected on F8 and showed a gradual increase in F9-F13 (Fig. 2.A-B and Supplementary Fig. 2). Viral genome quantification by qPCR also showed a gradual increase in viral titer along these fractions (Fig. 2.F). While the viral genome/protein ratio oscillated between F9-13, the ratio between viral genomes and nanoparticles (viral genome/nanoparticle) gradually increased between F7 and F12 (Fig. 2.G-H).

To test the transgene expression efficiency of the most promising SEC fractions regarding the presence of EV-AAVs, Neuro 2A (N2a) cells were independently transduced with F7 to F13 at a MOI of 10 000 viral genomes/cell (Fig. 3.A). Transduction was qualitatively evaluated through the analysis of GFP expression by immunocytochemistry (ICC) and quantitatively by Fluorescence-Activated Cell Sorting (FACS). F7 to F11 showed to be the most potent fractions compared to control cells, with F8 being the most potent (23.08% ± 1.69% GFP-positive cells) (Fig. 3.B-D).

Taking these results into account, we infer that the purest and biologically active population of EV-AAV1/2 coated with RVg (referred in our work as EV-AAV) is collected on SEC fractions 7 to 11. However, because of the low viral titer and EV amount observed in fraction F7 (Fig. 2.A-C and Fig. 2-F), we chose to exclude this fraction and proceed only with fractions F8 to F11 in the following experiments.

2.2. SEC allows a better purification of EV-AAVs compared to UC

Once the SEC protocol was optimized, we performed a side-by-side comparison of SEC and the standard protocol of differential centrifugation (UC)³¹ (see supplementary Fig. 4). Cell medium was collected at 48h and 72h post-transfection of HEK293T cells with the same five plasmids for AAV1/2 production and RVg expression mentioned above. Cell debris were removed by low-speed centrifugations. Half of the medium was then purified by UC (UC-100k) and the other half by our SEC protocol (Fig. 4.A). EV-AAVs isolated from the medium by UC and SEC were then characterized using microBCA, NTA and qPCR.

No significant differences were observed in the total protein amount, nor in the size of the EV-AAVs isolated by the two methods (Fig. 4.B and 4.D, respectively). Nevertheless, the yield of nanoparticles was drastically higher (6.6-fold) by SEC (Fig. 4.C), suggesting a probable loss of EVs during UC. Nanoparticles per protein ratio was higher by SEC (Fig. 4.E).

On the other hand, the viral titer was 3.8 times higher in EV-AAVs isolated by UC, which may indicate that UC simultaneously isolate AAVs, EVs and EV-AAVs. As result, the heterogeneous mixture of EV-AAVs isolated by UC present a higher viral genome to nanoparticle ratio (SEC: 5.023 ± 1.879; UC-100k: 94.77 ± 10.38) when compared to EV-AAVs isolated by SEC (Fig. 4.G).

To compare the in vitro transduction of EV-AAVs isolated by UC and SEC, N2a cells were incubated with 10 000 viral genomes/cell. At 48 h post-infection, the percentage of cells expressing GFP was determined by FACS (Fig. 5). The highest percentage of GFP-positive cells was detected in cells treated with EV-AAVs isolated by SEC (8.9 ± 0.75) (Fig. 5.B-D). Interestingly, EV-AAVs isolated by SEC or UC showed higher N2a cells transduction than solo AAVs (Fig. 5.B-D).

Since EV-AAVs isolated by SEC contained higher yields of nanoparticles, with less co-isolated contaminant proteins and solo AAVs, we proceeded to in vivo experiments with EV-AAVs isolated by SEC (F8-F11).

2.3. EV-AAVs transduce C57BL/6 mice brains upon intravenous injection

Given the in vitro efficacy of EV-AAVs purified by SEC, we next evaluated the possibility of using EV-AAVs as a non-invasive delivery system for the treatment of CNS diseases. For this purpose, WT C57BL/6 neonatal mice (P1) were intravenously injected in the facial vein with 1x10¹⁰ vg of EV-AAVs expressing GFP (Fig. 6.A). Mice were sacrificed at P100 and immunohistochemistry against GFP was performed in sagittal brain sections to evaluate neuronal transduction.

GFP expression was mostly observed in the cerebellum (particularly in Purkinje cells at lobule 10), brainstem and ependymal cells of the choroid plexus (lateral and fourth ventricles) (Fig. 6.B-C). GFP expression was similar between animals sacrificed at P100. These results show that biologically formed EV-AAVs coated with RVg are able to reach the brain and transduce the CNS.

Adeno-associated viral (AAV) vectors are one of the preferential vehicles for gene therapy targeting the central nervous system (CNS)³. AAVs can infect both dividing and non-dividing cells, including neurons. Additionally, they exhibit no cytotoxicity and, in comparation with other viral vectors, have mild immunogenicity, allowing high efficiency and sustained expression of transgenes^32–34. However, the use of AAVs has some limitations, such as the small transgene size capacity, the presence of biological barriers that limit gene delivery, such as the BBB, or the induction of an immune response due to capsid similarities with wild-type AAVs. To overcome these challenges, AAV capsids and genomes have been extensively engineered¹⁰.

In 2012, it was demonstrated that, during a normal production of AAVs in HEK293T cells, AAVs are also secreted in association with EVs¹³. EVs have been largely studied due to their unique characteristics and possible application, for example, as gene delivery vectors³⁵. EVs are naturally formed vesicles, with a lipid membrane identical to the cell membrane, involved in intercellular communication. These small vesicles can travel through circulation, cross biological barriers, such as BBB and deliver their cargo to deep tissues^36–38. Similar to EVs, EV-AAVs are also able to cross the BBB upon systemic injection and transduce neuronal cells, with the same tropism as standard AAVs, but with higher efficiency at lower vector doses^13,18. Moreover, it was observed that EV-AAVs transduce cells more efficiently than solo AAVs both in vitro and in vivo^{14–19, 31,39–42}. Despite the relevance of this discovery, to date, only AAV serotypes able to cross the BBB (e.g. AAV9 and AAV8) have been used to target the CNS in vivo, which does not eliminate the possible contribution of solo AAVs to these observations^14,18,20. Moreover, in these studies the purification method used for the isolation of EV-AAVs was based on UC^13,18. Although UC has been the most used protocol for the isolation of EV and EV-AAVs, solo AAVs can be co-isolated with EV-AAVs upon UC isolation¹⁴. Moreover, UC is limited by the lack of reproducibility, purity, and scalability which hampers its translation into the clinical setting. In this context, there was a need to optimize a reproducible and scalable protocol for the isolation of pure and biologically formed EV-AAVs, translatable for future clinical applications.

Previously, our group optimized a protocol for the isolation of EVs from human plasma by SEC, a method that renders highly pure EVs, without compromising their integrity and functionality^25,26. Here, we adapted this protocol to isolate EVs from conditioned media of AAV-producing HEK293T cells. To understand in which SEC F(s) EV-AAVs were isolated, we characterized the first 30 fractions of 500 uL eluted from the column. A large amount of proteins was obtained between F12 and F26, which we considered to be “free protein”⁴³. Pure EVs were present mostly between F8 and F11, as shown by the presence of flotillin 1 (EVs marker) and absence of calnexin (cellular marker). Markers of AAV capsids (VP1-3) were also detected in these fractions. Since the pore size of the chosen SEC column is 70 nm, solo AAVs (~ 25 nm) are not expected to elute in the same fractions of EVs. This indicates that AAVs might be eluted in association with EVs. Indeed, the fractions containing EV-AAVs (F7 to F11) showed to be more biologically active than those containing solo AAVs (i.e. F12, F13), as shown by the higher expression of GFP in F7 to F11 upon infection. Our results suggest that it is possible to isolate potent and naturally formed EV-AAVs, with less contaminant proteins and solo AAVs, by SEC.

Following the optimization of the SEC protocol, we next compared EV-AAVs isolated by UC-100k and SEC. Both methods isolated EVs with a similar size, however SEC allowed a yield 6.6-fold higher compared to UC, suggesting that SEC is more efficient in the recovery of EVs from the media. On the other hand, viral titer was 3.8-fold higher on EV-AAVs isolated by UC, which might be explained due to higher co-isolation of solo AAVs with EV-AAVs by UC. In fact, the centrifugation speed used is close to the sedimentation coefficient of AAVs⁴⁴.

Based on that, the differences observed in the viral genome/nanoparticle ratio in SEC and UC may be attributed to not only a higher number of EVs without AAVs isolated by SEC, but also to the co-isolation of solo AAVs 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 sample⁴⁵. Additionally, EV-AAVs isolated by UC were 10.88-fold less pure than those isolated by SEC, according to the Nanoparticles/Protein ratio previously described by Webber and Clayton⁴⁶.

In any case, EV-AAVs isolated by UC and SEC were more potent at transduction than solo AAVs in vitro, possibly due to the higher neuronal targeting capacity of EV-AAVs, as compared to solo AAVs, due to the presence of RVg peptide on their surface^14,47.

Having demonstrated the efficiency and advantages of isolating EV-AAVs by SEC in vitro, we aimed at evaluating the possibility of using EV-AAVs as a non-invasive delivery system targeting the CNS. To determine whether EV-AAVs could reach the brain without the contribution of AAV serotype, we chose mosaic AAV1/2 vectors for the generation of EV-AAVs. Mosaic AAV1/2 vectors combine the properties of both parental AAVs, including neurotropic features; however, they are still unable to efficiently cross the BBB on their own^21–24. To direct EV-AAVs to the brain, the RVg peptide was engineered on the surface of EVs, as previously demonstrated by György and colleagues¹⁴. RVg peptides bind to acetylcholine receptors and selectively target neuronal cells and brain endothelial cells, enabling EVs to cross the BBB^47–49.

Following an intravenous injection of SEC EV-AAVs, we showed that SEC purified EV-AAV1/2 can cross reach the mouse brain upon IV injection. To our knowledge, this is the first time that EV-AAVs comprising AAV serotypes without natural ability to cross the BBB were used. This demonstrates that EV-AAVs are naturally formed and can effectively access the brain parenchyma through IV administration, independent of AAV serotype involvement.

Interestingly, the high rate of transduction of ependymal cells upon IV administration makes EV-AAVs strong candidates to gene delivery through the cerebrospinal fluid, allowing a long-term supply of transgenic product⁵⁰. On the other hand, the capacity to target the cerebellum in mice highlights the potential of EV-AAVs as a non-invasive gene delivery platform for diseases such as spinocerebellar ataxias that mainly affect the cerebellum⁵¹.

Finally, it is important to note that in the present work, we used 10 to 38-fold fewer viral genomes for IV injection (1x10¹⁰ vg/animal) compared to what has been used with solo AAV9 (1x10¹¹ − 3.8x10¹¹ vg/animal)^3,52–55. Based on this, we can speculate that using a higher amount of EV-AAVs, but still smaller than solo AAV9, it would be possible to achieve an higher CNS transduction.

In both studies investigating the transduction of EV-AAVs in N2a cells, it was not possible to determine the specific impact of cDNA inside vesicles, but not packaged within the AAV capsid. To assess the individual contribution of the cDNA to transduction, it would be necessary to repeat the EV production protocol without using the "AAV rep and cap genes" plasmid. Earlier work has suggested that transgene DNA outside of the capsid is unlikely to play a role in transduction¹³. Furthermore, plasmid DNA loaded into EVs is not efficient at transgene expression⁵⁶. However, it is evident that the presence of AAVs associated with EVs is the primary factor affecting transduction efficiency, as demonstrated by the observed brain transduction in rodents 100 days after the injection of EV-AAVs.

In conclusion, SEC allows the recovery of pure and biologically formed EV-AAVs that can be scaled up for future clinical applications. Given their outstanding features, not only at evading the immune system and achieving higher cell transduction but also at crossing biological barriers, EV-AAVs may be a promising approach for gene therapy of CNS disorders.

In vitro experiments

All work involving cells and viral vectors was performed in dedicated biosafety cabinets and incubators separated from those used for maintaining cell lines. The equipment and reagents in contact with cells in culture and viruses were sterilized and materials virally contaminated were properly rinsed or disposed, according to good laboratory practices.

6.1. Cell lines

Human Embryonic Kidney cells 293T (HEK293T) and Mouse Neural Crest-derived cell line (Neuro 2A cells) were obtained from the American Type Culture Collection cell biology bank (CRL-11268 and CCL-131, respectively). Both cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin (P/S). Cells were maintained in 5% CO2 at 37°C, unless mentioned otherwise. Sterile Phosphate Buffered Saline (PBS), pH = 7.4, and 0.05% trypsin were used for cell passage.

6.2. Production of AAVs and EV-AAVs

Twenty four hours before transfection, 1.05 x 10⁷ HEK293T cells were plated into 15-cm dishes and cultured in DMEM supplemented with 10% FBS, 1% P/S and 1M hydroxyethyl-piperazine ethane sulfonic acid (HEPES) buffer. AAVs were produced according to the standard transfection method²⁹. Briefly, four plasmids were used: i) a plasmid encoding the enhanced green fluorescent reporter gene (GFP) under the chicken beta-actin (CBA) promoter placed between the ITRs (pITR) sequences, ii) two plasmids containing the wild-type (WT) AAV genome rep and cap ORFs (AAV1 and AAV2) and iii) 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 Rabies virus glycoprotein peptide-platelet-derived growth factor receptor (RVg-PDGFR) transmembrane domain was also transfected¹⁴.For an efficient transfection, Polyethylenimine (PEI) linear MW 40000 (Polysciences, Inc) was used as the transfection reagent.

Medium was replaced by fresh DMEM 2% EV-free FBS, 1% P/S and 1% HEPES 16h post-transfection to reduce bovine EVs.

Conditioned medium was collected at 48 h and 72 h post-transfection and centrifuged for 10 min at 500 g followed by another centrifugation at 1 000 g for 15 min to remove cell debris. Medium was stored at -80ºC. Cells were harvested for AAV purification.

6.3. 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 60 min at 20 000 g and supernatant was centrifuged once again at 100 000 g for 90 min. Resulting pellets were resuspended in PBS.

6.4. Isolation of EV-AAVs by Size Exclusion Chromatography

Conditioned medium was concentrated using centrifugal concentrators (Vivaspin 20/Vivacell 70, Sartorius) or pressure concentrators (Vivacell 250, Sartorius) with a MWCO of 10 000 kDa. For EV-AAV purification, commercial agarose-based size exclusion columns (qEVoriginal, Izon Science) were used. For each assay, 1 mL of concentrated medium was loaded on qEVoriginal columns followed by PBS. Fractions of 0.5 mL were collected and immediately used or stored at − 80ºC before further analysis.

For in vivo experiments, after SEC, an additional concentration step was performed using centrifugal concentrators (Vivacell20, Sartorius).

6.5. Isolation of AAVs

AAVs were purified based on a Fast Protein Liquid Chromatography (FPLC) in our core facility ViralVector (CNC), using the AKTA pure 25 system (GE Healthcare, Life sciences). Purified rAAVs were subsequently concentrated using centrifugal concentrators (Amicon Ultra-15 and Ultra-0.5, Merck) with a molecular weight cut-off (MWCO) of 100 000 kDa and supplemented with 0.001% Pluronic F-68 100X (Life Technologies) to avoid aggregation.

6.6. Protein quantification

Total protein was quantified in samples isolated by UC and SEC through bicinchoninic acid (BCA) (Thermo Scientific), using lysed (described below), and MicroBCA (Thermo Scientific) assays, using non-lysed samples.

6.7. Western Blot

All samples used for western blot analysis, were lysed with RIPA buffer [150 mM NaCl, 50 mM Tris-Cl, 5 mM ethylene glycol tetraacetic acid (EGTA), 1% Triton X-100, 0.5% deoxycholic acid (DOC), 0.1% sodium doceylsulphate (SDS) pH = 7.5) supplemented with a protease inhibitor cocktail (Roche) and 200 mM phenylmethylsulphonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 1 mM activated sodium orthovanadate and 10 mM sodium fluoride (NaF)], with the exception of solo AAVs purified by FPLC. Protein extracts from HEK293T cells were also used as reference. Protein extracts were resolved in SDS-polyacrylamide gels (4% stacking and 10% running) and transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P, EMD Millipore), according to standard protocols. After protein eletrotransfer, membranes were stained with Ponceau S to visualize total protein and subsequently washed with 0.1 M NaOH. Then, membranes were blocked by incubation in 5% non-fat milk powder in 0.1% Tween 20 in Tris buffered saline (TBS-T), for 1 h at room temperature (RT). Immunoblotting was performed overnight at 4 ºC with the following primary antibodies: anti-calnexin H70 (rabbit polyclonal, Santa Cruz Biotechnology, 1:1000), anti-flotilin-1 clone 18 (mouse monoclonal, BD transduction laboratories, 1:400), anti-AAV (VP1/VP2/VP3) clone B1 (mouse monoclonal, American Research Products, Inc, 1:1000), followed by incubation with corresponding alkaline phosphatase-coupled secondary antibodies (goat anti-rabbit polyclonal antibody, Thermo Scientific, 1:10000; or goat anti-mouse polyclonal antibody, Invitrogen, 1:10000). The presence of the antigens of interest was observed with Enhanced Chemifluorescence substrate (ECF, Ge Healthcare) and chemifluorescence imaging (ChemiDoc™ Imaging System, BioRad).

6.8. Quantification of AAVs by Real-Time PCR

Absolute quantification of rAAV titer (vg/µL) was performed by quantitative real-time polymerase chain reaction (PCR) using the AAVpro™ Titration Kit (for Real Time PCR) Ver.2 (Takara Bio Inc), according to the manufacturer’s instructions. In resume, after DNaseI treatment, AAV capsids were lysed, samples were diluted and a qPCR with primers for the inverted terminal repeats (ITRs) of AAVs was performed. A standard curve was used for absolute quantification.

6.9. Nanoparticle Tracking Analysis (NTA)

Measurements of nanoparticle size and concentration in EV-AAVs preparations were performed by Nanoparticle Tracking Analysis (NTA). A NanoSight NS300 instrument (Marvell Panalytical, Malvern) with a 488 nm laser and sCMOS camera module (Malvern Panalytical, Malvern, United Kingdom) 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).

6.10. Transmission Electron Microscopy (TEM)

Transmission electron microscopy was performed according to ^{57 57}. Briefly, EV-AAVs were fixed with 2% paraformaldehyde (PFA) and deposited on Formvar-carbon coated grids (TAAB Laboratories Equipment) for 20 min. Grids were washed with PBS and fixed for 5min 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) at 80 kV.

6.11. In vitro transduction Assay

To evaluate the transduction efficiency of EV-AAVs and EV + AAV, Neuro 2A (N2a) cells were seeded in either 24-well plates (5x10⁴ cells/well) or 48-well plates (2x10⁴ cells/well), for fluorescence-activated cell sorting (FACS) and immunocytochemistry (ICC) analysis, respectively. On the following day, half of the medium was collected and cells were infected at 10 000 vg/cell, with different samples equally diluted in PBS. Sixteen-hours post-vector addition, the medium was replaced. Transgene expression was evaluated 48 hours post-infection.

6.12. Fluorescence-Activated Cell Sorting (FACS)

Two days post-infection, infected N2a cells were processed for flow cytometry 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 a FACS Calibur flow cytometer (BD, Biosciences). A total of 2x10⁴ 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 Inc., Ashland, USA).

6.13. Immunocytochemistry

Two days post-infection, infected N2a cells were washed with PBS and fixed in 4% paraformaldehyde in PBS, for 15 min at RT. After fixation, cells were washed and permeabilized with 0.1% Triton X-100/PBS, for 5 min at RT. Blockage of non-specific staining was performed with 10% bovine serum albumin (BSA) in PBS, for 30 min at 37 ºC, and cells were then incubated overnight at 4 ºC with the rabbit anti-GFP primary antibody (1:1000, Invitrogen) diluted in 3% BSA in PBS. Subsequently, cells were washed and incubated for 45 min at 37 ºC with the goat anti-rabbit secondary antibody (1:250, Thermo Scientific), also diluted in 3% BSA in PBS. Cells were washed with PBS and incubated for 7 min with DAPI (1: 5 000, Sigma).

Images were obtained using CELENA® S digital cell imaging system (Logos Biosystems) with a 10X objective.

In vivo Experiments

6.14. Animals

Neonatal (P1) WT (N = 7) pups (C57BL/6 background) were used to evaluate the transduction efficiency of the EV-AAV purified by SEC, upon a minimally invasive intravenous injection.

Animals were housed in a temperature-controlled room maintained on a 12-hour light / 12-hour dark cycle. Food and water were provided ad libitum. Experiments involving mice were previously approved by the Responsible Organization for the Animals Welfare (ORBEA) of the Faculty of Medicine, Directorate-general for Food and Veterinary Medicine (DGAV) and the Center for Neuroscience and Cell Biology of the University of Coimbra (FMUC/CNC, Coimbra, Portugal). All experiments were carried out in accordance with the European Union Community directive (2010/63/EU) for the care and use of laboratory animals, transposed into the Portuguese law in 2013 (Decree Law 113/2013). The involved researchers received adequate training (Felasa-certified course), and certification to perform animal experiments from the Portuguese authorities (Direcção Geral de Alimentação e Veterinária, Lisbon, Portugal). All efforts were made to minimize animal suffering.

6.15. Neonatal Intravenous Injections

C57BL/6 pregnant mice were housed and monitored daily from embryonic day 17 to 21, with the least possible disturbance, to ensure that newborn pups could be dosed with vectors on P1.

Newborn mice were initially rested on a bed of ice for approximately 1 min for anesthetization. EV-AAV samples, isolated by SEC, containing a total of 1x10¹⁰ vg, diluted in 50 µL of PBS, were manually injected into the facial vein of seven newborn mice using a 100 µL Hamilton syringe connected to a 30-gauge beveled tip needle (Hamilton). A correct injection was verified by noting blanching of the vein. After injection, pups were identified by toe tattooing, carefully cleaned, rubbed with their original bedding to prevent rejection by the mother, and then returned to their original cage. Animals were sacrificed at 100 (N = 4) days post-injection, for histological analysis of the brain.

Histological Processing

6.16. Tissue Collection and Preparation

Mice were terminally anesthetized through the intraperitoneal route and transcardially perfused with cold PBS (pH = 7.4). Brains were excised and post-fixed with 4% PFA for 48 h at 4°C, and then transferred to 20% sucrose in PBS for cryoprotection. Brains were frozen and stored at − 80°C upon sinking (approximately 48h later).

Sagittal sections were subsequently cut at 30 µm thickness, using a cryostat (CRYOSTAR NX50, Thermo Scientific) at -20°C. Sections were stored in 48-well trays as free-floating sections in PBS supplemented with 5% sodium azide. Trays were stored at 4°C until further processing.

6.17. Bright-field immunohistochemistry

After being rinsed in PBS, free-floating sections were incubated in 0.1% phenylhydrazine in PBS (Merck) for 30 min at 37°C, to block endogenous peroxidases. Tissue blocking and permeabilization was subsequently performed in blocking solution (0.1% Triton X-100 containing 10% Normal Goat Serum (NGS, Gibco) in PBS), for 1 h at RT. Brain slices were then incubated overnight at 4 ºC with the rabbit anti-GFP primary antibody (1:1000, Invitrogen) diluted in blocking solution. Following three washing steps in PBS, free-floating sections were incubated for 2h at RT with the anti-rabbit biotinylated secondary antibody (Vector Laboratories) diluted in blocking solution (1:250). Subsequently, free-floating sections were rinsed and bound antibodies were visualized by the Avidin-Biotin complex (ABC) 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.

Sections were mounted on gelatin-coated slides, 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 Eukit (O. Kindler GmbH & CO). Images were acquired using a Zeiss Axio Imager Z2 microscope (Carl Zeiss Microscopy GmbH), equipped with a High Resolution Colour Camera. Images of cerebellum, brainstem and choroid plexus were obtained with a Plan-Apochromat 20X/0.8 M27 or 63x/1.4 objective.

6.18. Statistical Analysis

All statistical analyses were performed using the GraphPad Prism software (version 9.0.0). Data are presented as mean ± standard error of 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, and **** p < 0.0001.

Acknowledgments

This work was funded by the European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Program; through the COMPETE 2020 – Operational Programme for Competitiveness and Internationalization, and Portuguese national funds via FCT under the projects: UIDB/04539/2020, UIDP/04539/2020, LA/P/0058/2020, SpreadSilencing (POCI-01-0145-FEDER029716), ViraVector (CENTRO-01-0145-FEDER-022095), Fighting Sars-CoV-2 (CENTRO-01-01D2- FEDER-000002), BDforMJD (CENTRO-01-0145-FEDER-181240), ModelPolyQ2.0 (CENTRO-01- 0145-FEDER-181258), and MJDEDIT (CENTRO-01-0145-FEDER-181266); ARDAT under the IMI2 JU grant agreement no. 945473 supported by the European Union's H2020 programme and EFPIA; by the American Portuguese Biomedical Research Fund (APBRF) and the Richard Chin and Lily Lock Machado-Joseph Disease Research. C.H. was supported by 2021.06939.BD; M.M.L. was supported by 2021.05776.BD; D.R.R. was supported by SFRH/BD/132618/2017; D.L. was supported by 2020.09668.BD; K.L. was supported by SFRH/BD/09513/2020; and A.C.S. was supported by 2020.07721.BD. We thank M. Zuzarte (Faculty of Medicine, University of Coimbra) for electron microscopy imaging. We thank Luisa Cortes, Tatiana Catarino and Margarida Caldeira, the CNC 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., M.M.L., M.S., L.P.d.A. and R.J.N. conceived and designed the experiments. C.A.M. shared knowledge and material. C.H., M.M.L., P.A., D.R.R., L.S.G., D.L., K.L., A.C.S., R.B., S.D. and M.S. performed the experiments. C.H., M.S., R.J.N. analyzed the data. C.H., M.S., R.J.N. wrote the first draft of the paper. All the authors reviewed and edited the paper.

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There is NO Competing Interest.

GraphicalAbstract.png
Graphical Abstract During the production of AAV vectors, a small percentage of AAVs is secreted in association with extracellular vesicles, named “EV-AAVs”. EV-AAVs can be efficiently isolated by size exclusion chromatography (SEC). When intravenously injected in mice, brain targeting EV-AAVs can cross the blood brain barrier (BBB) and transduce neuronal cells.
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Isolation of Biologically Active Extracellular Vesicles-Associated AAVs for Gene Delivery to the Brain by Size Exclusion Chromatography

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Abstract

Figures

1. Introduction

2. Results

2.1. Size exclusion chromatography allows the isolation of pure and biologically active EV-AAVs

2.2. SEC allows a better purification of EV-AAVs compared to UC

2.3. EV-AAVs transduce C57BL/6 mice brains upon intravenous injection

3. Discussion

4. Limitations

5. Conclusions

6. Materials and Methods

6.1. Cell lines

6.2. Production of AAVs and EV-AAVs

6.3. Isolation of EV and EV-AAVs by Differential Centrifugation

6.4. Isolation of EV-AAVs by Size Exclusion Chromatography

6.5. Isolation of AAVs

6.6. Protein quantification

6.7. Western Blot

6.8. Quantification of AAVs by Real-Time PCR

6.9. Nanoparticle Tracking Analysis (NTA)

6.10. Transmission Electron Microscopy (TEM)

6.11. In vitro transduction Assay

6.12. Fluorescence-Activated Cell Sorting (FACS)

6.13. Immunocytochemistry

6.14. Animals

6.15. Neonatal Intravenous Injections

6.16. Tissue Collection and Preparation

6.17. Bright-field immunohistochemistry

6.18. Statistical Analysis

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