Dakai Mu

Heparin affinity purification of extracellular vesicles

Extracellular vesicles (EVs) are lipid membrane vesicles released by cells. They carry active biomolecules including DNA, RNA, and protein which can be transferred to recipient cells. Isolation and purification of EVs from culture cell media and biofluids is still a major challenge. The most widely used isolation method is ultracentrifugation (UC) which requires expensive equipment and only partially purifies EVs. Previously we have shown that heparin blocks EV uptake in cells, supporting a direct EV-heparin interaction. Here we show that EVs can be purified from cell culture media and human plasma using ultrafiltration (UF) followed by heparin-affinity beads. UF/heparin-purified EVs from cell culture displayed the EV marker Alix, contained a diverse RNA profile, had lower levels of protein contamination, and were functional at binding to and uptake into cells. RNA yield was similar for EVs isolated by UC. We were able to detect mRNAs in plasma samples with comparable levels to UC samples. In conclusion, we have discovered a simple, scalable, and effective method to purify EVs taking advantage of their heparin affinity.

Adenoassociated Virus Serotype 9-Mediated Gene Therapy for X-Linked Adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a devastating neurological disorder caused by mutations in the ABCD1 gene that encodes a peroxisomal ATP-binding cassette transporter (ABCD1) responsible for transport of CoA-activated very long-chain fatty acids (VLCFA) into the peroxisome for degradation. We used recombinant adenoassociated virus serotype 9 (rAAV9) vector for delivery of the human ABCD1 gene (ABCD1) to mouse central nervous system (CNS). In vitro, efficient delivery of ABCD1 gene was achieved in primary mixed brain glial cells from Abcd1-/- mice as well as X-ALD patient fibroblasts. Importantly, human ABCD1 localized to the peroxisome, and AAV-ABCD1 transduction showed a dose-dependent effect in reducing VLCFA. In vivo, AAV9-ABCD1 was delivered to Abcd1-/- mouse CNS by either stereotactic intracerebroventricular (ICV) or intravenous (IV) injections. Astrocytes, microglia and neurons were the major target cell types following ICV injection, while IV injection also delivered to microvascular endothelial cells and oligodendrocytes. IV injection also yielded high transduction of the adrenal gland. Importantly, IV injection of AAV9-ABCD1 reduced VLCFA in mouse brain and spinal cord. We conclude that AAV9-mediated ABCD1 gene transfer is able to reach target cells in the nervous system and adrenal gland as well as reduce VLCFA in culture and a mouse model of X-ALD.

Trafficking of adeno-associated virus vectors across a model of the blood–brain barrier; a comparative study of transcytosis and transduction using primary human brain endothelial cells

Developing therapies for central nervous system (CNS) diseases is exceedingly difficult because of the blood–brain barrier (BBB). Notably, emerging technologies may provide promising new options for the treatment of CNS disorders. Adeno‐associated virus serotype 9 (AAV9) has been shown to transduce cells in the CNS following intravascular administration in rodents, cats, pigs, and non‐human primates. These results suggest that AAV9 is capable of crossing the BBB. However, mechanisms that govern AAV9 transendothelial trafficking at the BBB remain unknown. Furthermore, possibilities that AAV9 may transduce brain endothelial cells or affect BBB integrity still require investigation. Using primary human brain microvascular endothelial cells as a model of the human BBB, we performed transduction and transendothelial trafficking assays comparing AAV9 to AAV2, a serotype that does not cross the BBB or transduce endothelial cells effectively in vivo. Results of our in vitro studies indicate that AAV9 penetrates brain microvascular endothelial cells barriers more effectively than AAV2, but has reduced transduction efficiency. In addition, our data suggest that (i) AAV9 penetrates endothelial barriers through an active, cell‐mediated process, and (ii) AAV9 fails to disrupt indicators of BBB integrity such as transendothelial electrical resistance, tight junction protein expression/localization, and inflammatory activation status. Overall, this report shows how human brain endothelial cells configured in BBB models can be utilized for evaluating transendothelial movement and transduction kinetics of various AAV capsids. Importantly, the use of a human in vitro BBB model can provide import insight into the possible effects that candidate AVV gene therapy vectors may have on the status of BBB integrity.

Mouse Gender Influences Brain Transduction by Intravascularly Administered AAV9

To the editor: Adeno-associated virus serotype 9 (AAV9) has shown remarkable efficiency in transducing organs in vivo including the heart, liver, and brain.1,2,3,4,5,6 Recently, AAV9 has gained renewed interest due to its ability in crossing the blood–brain barrier upon intravenous injection.7,8,9,10,11,12 In adult mice, AAV9 was shown to preferentially transduce endothelial cells, astrocytes, and neurons after intravenous injection.10 The implications of these findings are enormous: a noninvasive route of administration and widespread gene delivery to the brain. Complete characterization of the gene transfer properties of AAV9, including kinetics of expression, immunogenicity, and biodistribution in different species, will be necessary for clinical translation. Here we show that intravenous injection of single-stranded AAV9 encoding the reporter genes firefly luciferase (Fluc) and green fluorescent protein (GFP) yielded higher transgene expression in the brain of females compared with male mice. This observation was consistent in two strains of mice (nude and C57BL/6) and was correlated with an increased number of AAV genomes in the female brains. These findings stress the importance of proper matching of mice gender in different experimental groups when using systemic injection of AAV9. Age-matched (6–8 weeks) male and female athymic NU/NU nude mice and C57BL/6N mice were injected intravenously (through the tail vein) with 1.5 × 1012 gene copies (g.c.)/kg of single-stranded AAV9 vector encoding Fluc, driven by the ubiquitously active cytomegalovirus/chicken beta-actin (CBA) hybrid promoter (AAV9-Fluc). The transduction efficiency of AAV9 in the brain and abdomen was monitored 7 and 14 days later using bioluminescence imaging (BLI). We observed a higher bioluminescent signal in the head region of females compared with males in both strains of mice and at both time points (Figure 1a,b). In contrast to the bioluminescent signal in the head region, and in support of previous reports,13,14,15 the Fluc signal from the abdomen (most likely liver) was lower in female mice than in males (Supplementary Figure S1). Figure 1 Systemic injection of AAV9 yields higher transduction of the brain of female compared to male mice. (a,b) Male and female nude and C57BL/6 mice were injected via the tail vein with 1.5 × 1012 g.c./kg of AAV9-Fluc vector. Mice were injected 7 and ... In another set of mice, we performed a kinetic analysis of luciferase expression in the head and liver in both male and female nude and C57BL/6N mice over a 4-week period. We observed a stable luminescence expression in the head of nude and C57BL/6N mice, whereas the abdomen values showed more variability over time in both strains (Supplementary Figure S2). Next we confirmed the BLI results by direct analysis of Fluc enzymatic activity in the brain regions from dead animals, ex vivo. Consistent with BLI, the Fluc levels in the cortex, cerebellum, and olfactory bulbs of female mice were significantly higher (*P < 0.05) compared with the brain of male mice in both strains (Supplementary Figure S3). We next sought to determine the transduction profile of AAV9 expressing GFP under CBA promoter (AAV9-GFP) in the brains of male and female mice. C57BL/6N mice were injected intravenously with 5 × 1013 g.c./kg of AAV9-GFP. A higher dose was utilized in this experiment to allow visualization of GFP, which is a less sensitive reporter than the Fluc enzyme. At 2 weeks after injection, these mice were killed, then perfused with phosphate-buffered saline, and their brains and livers harvested. Immunostaining with an antibody specific for GFP revealed diffuse expression of almost exclusively neurons and astrocytes throughout the brain (Figure 1c and Supplementary Figure S4). A count of the GFP-positive neurons and astrocytes per square millimeter in the cortex revealed a 1.7-fold (P = 0.111) and 2.7-fold (P = 0.032) increase, respectively, in the female vs. the male brains (Figure 1d). Interestingly, the ratio of GFP-positive astrocytes to neurons was 2.1 and 1.333 for males and females, respectively, which may indicate a slight difference in AAV tropism in the brain between mice genders. To investigate whether the increase in transduction of the female brain was linked to higher transport of the vector to this organ, quantitative polymerase chain reaction for AAV genome was performed on tissue homogenates from the cortex of mice. In both strains a significantly higher number of AAV genome copies (*P < 0.05) was detected in the brain tissue of females compared with male mice (Supplementary Figure S5). This study is the first to provide a comparative analysis of transgene expression in the brain of male and female mice upon systemic injection of a single-stranded AAV9 vector. Previous studies using this vector serotype used either male or female mice exclusively11,13 or mixed littermates were used,10 with no direct comparison between different genders. Although the exact mechanism of increased transgene expression in the brain of female mice remains unknown, our findings are important for future AAV9 vector research, specifically when the systemic injection route is chosen. Of immediate application, our results suggest that equal distribution of mouse gender between groups or the use of one gender for a given study is crucial for accurate data interpretation. Additionally, this study warrants further investigation of AAV9-mediated brain transduction of genders of other species (e.g., rats, dogs, nonhuman primates) for possible translation of these findings to human gene therapy. SUPPLEMENTARY MATERIAL Supplementary Methods Figure S1. Bioluminescent signal in the abdomen region of male and female mice injected systemically with AAV9-Fluc. Figure S2. Bioluminescence kinetic analysis in the head and abdomen region of mice injected with AAV9-Fluc vector. Figure S3. Fluc activity in brain tissue homogenates in male and female mice injected intravenously with AAV9-Fluc. Figure S4. AAV9 transduces primarily neurons and astrocytes in male and female mice upon systemic injection. Figure S5. Quantitation of AAV9 genome copies in the brain of male and female mice after systemic vector injection.

Tailored transgene expression to specific cell types in the central nervous system after peripheral injection with AAV9.

The capacity of certain adeno-associated virus (AAV) vectors to cross the blood–brain barrier after intravenous delivery offers a unique opportunity for noninvasive brain delivery. However, without a well-tailored system, the use of a peripheral route injection may lead to undesirable transgene expression in nontarget cells or organs. To refine this approach, the present study characterizes the transduction profiles of new self-complementary AAV9 (scAAV9) expressing the green fluorescent protein (GFP) either under an astrocyte (glial fibrillary acidic (GFA) protein) or neuronal (Synapsin (Syn)) promoter, after intravenous injection of adult mice (2 × 1013 vg/kg). ScAAV9-GFA-GFP and scAAV9-Syn-GFP robustly transduce astrocytes (11%) and neurons (17%), respectively, without aberrant expression leakage. Interestingly, while the percentages of GFP-positive astrocytes with scAAV9-GFA-GFP are similar to the performances observed with scAAV9-CBA-GFP (broadly active promoter), significant higher percentages of neurons express GFP with scAAV9-Syn-GFP. GFP-positive excitatory as well as inhibitory neurons are observed, as well as motor neurons in the spinal cord. Additionally, both activated (GFAP-positive) and resting astrocytes (GFAP-negative) express the reporter gene after scAAV9-GFA-GFP injection. These data thoroughly characterize the gene expression specificity of AAVs fitted with neuronal and astrocyte-selective promoters after intravenous delivery, which will prove useful for central nervous system (CNS) gene therapy approaches in which peripheral expression of transgene is a concern.

CRISPR/Cas9 Mediated Disruption of the Swedish APP Allele as a Therapeutic Approach for Early-Onset Alzheimer’s Disease

The APPswe (Swedish) mutation in the amyloid precursor protein (APP) gene causes dominantly inherited Alzheimer’s disease (AD) as a result of increased β-secretase cleavage of the amyloid-β (Aβ) precursor protein. This leads to abnormally high Aβ levels, not only in brain but also in peripheral tissues of mutation carriers. Here, we selectively disrupted the human mutant APPSW allele using CRISPR. By applying CRISPR/Cas9 from Streptococcus pyogenes, we generated allele-specific deletions of either APPSW or APPWT. As measured by ELISA, conditioned media of targeted patient-derived fibroblasts displayed an approximate 60% reduction in secreted Aβ. Next, coding sequences for the APPSW-specific guide RNA (gRNA) and Cas9 were packaged into separate adeno-associated viral (AAV) vectors. Site-specific indel formation was achieved both in primary neurons isolated from APPSW transgenic mouse embryos (Tg2576) and after co-injection of these vectors into hippocampus of adult mice. Taken together, we here present proof-of-concept data that CRISPR/Cas9 can selectively disrupt the APPSW allele both ex vivo and in vivo—and thereby decrease pathogenic Aβ. Hence, this system may have the potential to be developed as a tool for gene therapy against AD caused by APPswe and other point mutations associated with increased Aβ.

Virus vector-mediated genetic modification of brain tumor stromal cells after intravenous delivery

The malignant primary brain tumor, glioblastoma (GBM) is generally incurable. New approaches are desperately needed. Adeno-associated virus (AAV) vector-mediated delivery of anti-tumor transgenes is a promising strategy, however direct injection leads to focal transgene spread in tumor and rapid tumor division dilutes out the extra-chromosomal AAV genome, limiting duration of transgene expression. Intravenous (IV) injection gives widespread distribution of AAV in normal brain, however poor transgene expression in tumor, and high expression in non-target cells which may lead to ineffective therapy and high toxicity, respectively. Delivery of transgenes encoding secreted, anti-tumor proteins to tumor stromal cells may provide a more stable and localized reservoir of therapy as they are more differentiated than fast-dividing tumor cells. Reactive astrocytes and tumor-associated macrophage/microglia (TAMs) are stromal cells that comprise a large portion of the tumor mass and are associated with tumorigenesis. In mouse models of GBM, we used IV delivery of exosome-associated AAV vectors driving green fluorescent protein expression by specific promoters (NF-κB-responsive promoter and a truncated glial fibrillary acidic protein promoter), to obtain targeted transduction of TAMs and reactive astrocytes, respectively, while avoiding transgene expression in the periphery. We used our approach to express the potent, yet toxic anti-tumor cytokine, interferon beta, in tumor stroma of a mouse model of GBM, and achieved a modest, yet significant enhancement in survival compared to controls. Noninvasive genetic modification of tumor microenvironment represents a promising approach for therapy against cancers. Additionally, the vectors described here may facilitate basic research in the study of tumor stromal cells in situ.Graphical abstract

295. Intrathecal Delivery of rAAV9-ABCD1 by Osmotic Pump in a Mouse Model of Adrenomyeloneuropathy

Adrenomyeloneuropathy (AMN) is a debilitating neurological disorder caused by mutations in the ABCD1 gene, which encodes a peroxisomal ATP-binding cassette transporter (ABCD1) responsible for transport of CoA-activated very long-chain fatty acids (VLCFA) into the peroxisome for degradation. The Abcd1-/- mouse develops a phenotype similar to AMN, manifesting spinal cord axon degeneration as well as peripheral neuropathy due to affected dorsal root ganglion neurons (DRGs). We previously reported successful transduction of central nervous system cells in vitro and in vivo using recombinant adeno-associated virus serotype 9 (rAAV9) vector for delivery of the human ABCD1 gene. Unfortunately, intravenous delivery in young mice was associated with cardiac toxicity due to transgene overexpression. We therefore set out to optimize delivery to the spinal cord while minimizing systemic leakage using an intrathecal osmotic pump. Self complementary AAV9 GFP(scAAV9GFP) and rAAV9 encoding ABCD1 (rAAV9-ABCD1) were delivered to Abcd1-/- mice intrathecally (IT) either by bolus over a 2min duration or by osmotic pump over 24h duration with PBS injection as sham control. Two weeks after injection, mice were sacrificed and perfused with 4% PFA. Tissues were then collected, sectioned and stained for immunofluorescence analysis. scAAV9-GFP delivered IT by osmotic pump led to widespread expression across CNS-relevant cell types and DRGs in a dose-dependent manner. Spinal cord and DRG had higher expression compared with brain, but GFP expression was also detected in peripheral organs (liver, heart and adrenal gland), with highest expression seen at 3X1011GC. A similar distribution pattern of ABCD1 protein was detected after rAAV9-ABCD1 intrathecal pump delivery. In general, higher doses (2X1011GC and 1X1011GC) led to more expression in CNS and peripheral organs compared with a lower dose (0.5X1011GC). However, widespread expression of ABCD1 across CNS was even detected after direct intrathecal bolus injection of 0.5X1011GC. Importantly, the same dose delivered by pump led to higher expression in brain and spinal cord far from the injection site and comparatively less leakage to peripheral organs compared with bolus injection. Preliminary experiments delivering rAAV9-ABCD1 at 0.5X1011GC by intracerebroventricular approach suggest behavioral improvement in the Abcd1-/- mouse despite localized expression in brain. We therefore anticipate even better performance at this dose using the outlined intrathecal pump delivery. We conclude that rAAV9-mediated ABCD1 gene transfer via intrathecal osmotic pump leads to more uniform and widespread gene delivery to CNS with reduced leakage into the systemic circulation compared with intrathecal bolus injection.

390. Impact of rAAV9-ABCD1 Upon Behavioral Outcome in a Mouse Model of X-ALD

X-linked adrenoleukodystrophy(X-ALD) is a devastating neurological disorder caused by mutations in the ABCD1 gene that encodes a peroxisomal ATP-binding cassette (ABC) transporter. We previously reported successful transduction of central nervous system cells in vitro and in vivo using recombinant adeno-associated virus serotype 9 (rAAV9) vector for delivery of the human ABCD1 gene (ABCD1). Here we report first results of long term experiments to evaluate the therapeutic effect of AAV9-ABCD1 in a mouse model of X-ALD that develops motor and sensory symptoms around 18 months of age.rAAV9 encoding ABCD1 (rAAV9-ABCD1) was delivered to Abcd1-/- mouse CNS by stereotactic intraventricular (ICV) injection at young (4 months) and old (12 months) age, while intravenous (IV) injection was tested at only old age (12 months). Mouse body weight was monitored every month, while mouse behavior including hind limb reflex extension (scoring system 0-4, paralysis to normal), mechanical sensitivity (von Frey testing) and motor function (rotarod testing) were recorded starting from 15 months of age. No changes in body weight occurred regardless of delivery route or age injected, suggesting no obvious toxicity of the rAAV9-ABCD1 vector. ICV injection at both young and old age showed significant improvement upon mouse hind limb reflex extension after 15 months of age. Scores of hind limb reflex extension in untreated Abcd1-/- mice dropped to 1.75 at 18 months of age (wild type mice: 3.5), whereas Abcd1–/– mice treated via ICV at an old age retained scores around 3.25 (p<0.05). ABCD1-/- mice treated via ICV at a young age had an average score of 3.5 at 15 months (compared to untreated Abcd1-/- mice: 2.4; p<0.05). IV injection at old age showed mild but not significant improvement. Mechanical sensitivity also improved after both ICV injection at young age and IV injection at old age but not ICV injection in old Abcd1-/- mice. Analysis of rotarod data is pending.We conclude that rAAV9-mediated ABCD1 gene transfer is safe and efficacious at improving behavior of a known X-ALD mouse model including mouse hind limb reflex extension and mechanical sensitivity. However timing and delivery route are crucial determinants of efficacy and need to be independently assessed.

152. Retinal Tropism of Exosome-Associated AAV Vector Via Intravitreal Delivery

Recent clinical studies have validated the use of adeno-associated viral vectors (AAV) as a safe and efficient gene delivery vehicle for the treatment of retinal disorders. Different studies targeting the retina using AAV vectors have been achieved transduction of retinal cells via one of two delivery routes: subretinal or intravitreal injections, with subretinal delivery by far the most efficient method. Current treatments for age-related macular degeneration (AMD) heavily rely on protein drug delivery via regular and repeated intravitreal injections making the current standard of care clearly not optimal. One alternative for AMD treatment would be to use an AAV-based gene delivery and indeed preclinical studies in both mouse and non-human primates have shown that intravitreal delivery of AAV2 can mediate sustained expression of transgenes in the vitreous cavity following a single injection. Since the DNA is encapsulated in the AAV capsid, it is the capsid that determines the majority of the pharmacological interactions with the host, including cellular specificity, binding and uptake, immune activation and stability. However intravitreal injections using AAV vectors are inefficient regarding expression levels and poses higher inflammatory risk to vector or transgene antigens.In the current study we evaluated the retinal tropism of exosome-associated AAV9 vector (vexosomes). Exosomes are naturally secreted membrane nanovesicles involved in intercellular communication. We have previously shown that vexosomes highly outperforms regular AAV in efficacy in vitro and in vivo, and are resistant to neutralizing antibodies due to shielding of the AAV by the exosomal membrane.AAV9-vexosomes were isolated from AAV-producer cell (293T) media by ultracentrifugation. AAV vexosomes were injected into C57bl/6 wild type mice and transgene expression was evaluated at 2 and 4 weeks post injections via in vivo fundus imaging. Eyes were collected at 4 weeks post injections and retinal cell tropism was analyzed. Comparison of transgene expression levels between the different vexosomes and traditionally formulated AAV vectors was done using real time PCR.Intravitreal delivery of AAV9-vexosomes showed increased level of retinal cells transduction compared to regular AAV9 vectors which show very poor transduction. Apparently, vexosomes were able to penetrate through the ILM and transduce the retina in patches, as assessed by fundus imaging. Due to the immune issues surrounding gene delivery in the eye via an intravitreal route, the results presented here indicate a promising alternative gene delivery platform using vexosomes that could circumvent the inflammatory risks expected by intravitreal injection. Further studies in assessing the extent vexosomes evade neutralization in the vitreous will be extremely important to complement the results presented here.