Efficient retinal ganglion cells transduction by retro-orbital venous sinus injection of AAV-PHP.eB in mature mice.

Gene therapy is one of the strategies that may reduce or reverse progressive neurodegeneration in retinal neurodegenerative diseases. However, efficiently delivering transgenes to retinal ganglion cells (RGCs) remains hard to achieve. In this study, we innovatively investigated transduction efficiency of adeno-associated virus (AAV)-PHP.eB in murine RGCs by retro-orbital venous sinus injection. Five doses of AAV-PHP.eB-EGFP were retro-orbitally injected in venous sinus in adult C57/BL6J mice. Two weeks after administration, RGCs transduction efficiency was quantified by retinal flat-mounts and frozen section co-labeling with RGCs marker Rbpms. In addition, safety of this method was evaluated by RGCs survival rate and retinal morphology. To conform efficacy of this new method, AAV-PHP.eB-CNTF was administrated into mature mice through single retro-orbital venous injection after optic nerve crush injury to evaluate axonal elongation. Results indicated that AAV- PHP.eB readily crossed the blood-retina barrier and was able to transduce more than 90% of RGCs when total dose of virus reached 5 × 1010 vector genomes (vg). Moreover, this technique did not affect RGCs survival rate and retinal morphology. Furthermore, retro-orbital venous delivery of AAV-PHP.eB-CNTF effectively transduced RGCs, robustly promoted axonal regeneration after optic nerve crush injury. Thus, novel AAV-PHP.eB retro-orbital injection provides a minimally invasive and efficient route for transgene delivery in treatment of retinal neurodegenerative diseases.

Group ICA of wide-field calcium imaging data reveals the retrosplenial cortex as a major contributor to cortical activity during anesthesia.

Large-scale cortical dynamics play a crucial role in many cognitive functions such as goal-directed behaviors, motor learning and sensory processing. It is well established that brain states including wakefulness, sleep, and anesthesia modulate neuronal firing and synchronization both within and across different brain regions. However, how the brain state affects cortical activity at the mesoscale level is less understood. This work aimed to identify the cortical regions engaged in different brain states. To this end, we employed group ICA (Independent Component Analysis) to wide-field imaging recordings of cortical activity in mice during different anesthesia levels and the awake state. Thanks to this approach we identified independent components (ICs) representing elements of the cortical networks that are common across subjects under decreasing levels of anesthesia toward the awake state. We found that ICs related to the retrosplenial cortices exhibited a pronounced dependence on brain state, being most prevalent in deeper anesthesia levels and diminishing during the transition to the awake state. Analyzing the occurrence of the ICs we found that activity in deeper anesthesia states was characterized by a strong correlation between the retrosplenial components and this correlation decreases when transitioning toward wakefulness. Overall these results indicate that during deeper anesthesia states coactivation of the posterior-medial cortices is predominant over other connectivity patterns, whereas a richer repertoire of dynamics is expressed in lighter anesthesia levels and the awake state.

Improving cell-specific recombination using AAV vectors in the murine CNS by capsid and expression cassette optimization.

The production of cell-type- and age-specific genetically modified mice is a powerful approach for unraveling unknown gene functions. Here, we present a simple and timesaving method that enables adeno-associated virus (AAV)-mediated cell-type- and age-specific recombination in floxed mice. To achieve astrocyte-specific recombination in floxed Ai14 reporter mice, we intravenously injected blood-brain barrier-penetrating AAV-PHP.eB vectors expressing Cre recombinase (Cre) using the astrocyte-specific mouse glial fibrillary acidic protein (mGfaABC1D) promoter. However, we observed nonspecific neuron-predominant transduction despite the use of an astrocyte-specific promoter. We speculated that subtle but continuous Cre expression in nonastrocytic cells triggers recombination, and that excess production of Cre in astrocytes inhibits recombination by forming Cre-DNA aggregates. Here, we resolved this paradoxical event by dividing a single AAV into two mGfaABC1D-promoter-driven AAV vectors, one expressing codon-optimized flippase (FlpO) and another expressing flippase recognition target-flanked rapidly degrading Cre (dCre), together with switching the neuron-tropic PHP.eB capsid to astrocyte-tropic AAV-F. Moreover, we found that the FlpO-dCre system with a target cell-tropic capsid can also function in neuron-targeting recombination in floxed mice.

Non-invasive systemic viral delivery of human alpha-synuclein mimics selective and progressive neuropathology of Parkinson's disease in rodent brains.

BACKGROUND: Alpha-synuclein (α-syn) aggregation into proteinaceous intraneuronal inclusions, called Lewy bodies (LBs), is the neuropathological hallmark of Parkinson's disease (PD) and related synucleinopathies. However, the exact role of α-syn inclusions in PD pathogenesis remains elusive. This lack of knowledge is mainly due to the absence of optimal α-syn-based animal models that recapitulate the different stages of neurodegeneration. METHODS: Here we describe a novel approach for a systemic delivery of viral particles carrying human α-syn allowing for a large-scale overexpression of this protein in the mouse brain. This approach is based on the use of a new generation of adeno-associated virus (AAV), AAV-PHP.eB, with an increased capacity to cross the blood-brain barrier, thus offering a viable tool for a non-invasive and large-scale gene delivery in the central nervous system. RESULTS: Using this model, we report that widespread overexpression of human α-syn induced selective degeneration of dopaminergic (DA) neurons, an exacerbated neuroinflammatory response in the substantia nigra and a progressive manifestation of PD-like motor impairments. Interestingly, biochemical analysis revealed the presence of insoluble α-syn oligomers in the midbrain. Together, our data demonstrate that a single non-invasive systemic delivery of viral particles overexpressing α-syn prompted selective and progressive neuropathology resembling the early stages of PD. CONCLUSIONS: Our new in vivo model represents a valuable tool to study the role of α-syn in PD pathogenesis and in the selective vulnerability of nigral DA neurons; and offers the opportunity to test new strategies targeting α-syn toxicity for the development of disease-modifying therapies for PD and related disorders.

Deletion of Gtf2i via Systemic Administration of AAV-PHP.eB Virus Increases Social Behavior in a Mouse Model of a Neurodevelopmental Disorder.

Williams syndrome (WS) is a neurodevelopmental disorder characterized by distinctive cognitive and personality profiles which also impacts various physiological systems. The syndrome arises from the deletion of about 25 genes located on chromosome 7q11.23, including Gtf2i. Prior research indicated a strong association between pre-natal Gtf2i deletion, and the hyper-social phenotypes observed in WS, as well as myelination deficits. As most studies addressed pre-natal Gtf2i deletion in mouse models, post-natal neuronal roles of Gtf2i were unknown. To investigate the impact of post-natal deletion of neuronal Gtf2i on hyper-sociability, we intravenously injected an AAV-PHP.eB virus expressing Cre-recombinase under the control of αCaMKII, a promoter in a mouse model with floxed Gtf2i. This targeted deletion was performed in young mice, allowing for precise and efficient brain-wide infection leading to the exclusive removal of Gtf2i from excitatory neurons. As a result of such gene deletion, the mice displayed hyper-sociability, increased anxiety, impaired cognition, and hyper-mobility, relative to controls. These findings highlight the potential of systemic viral manipulation as a gene-editing technique to modulate behavior-regulating genes during the post-natal stage, thus presenting novel therapeutic approaches for addressing neurodevelopmental dysfunction.

A blood-brain barrier-penetrating AAV2 mutant created by a brain microvasculature endothelial cell-targeted AAV2 variant.

Upon systemic administration, adeno-associated virus serotype 9 (AAV9) and the capsid variant PHP.eB show distinct tropism for the central nervous system (CNS), whereas AAV2 and the capsid variant BR1 transduce brain microvascular endothelial cells (BMVECs) with little transcytosis. Here, we show that a single amino acid substitution (from Q to N) in the BR1 capsid at position 587 (designated BR1N) confers a significantly higher blood-brain barrier (BBB) penetration capacity to BR1. Intravenously infused BR1N showed significantly higher CNS tropism than BR1 and AAV9. BR1 and BR1N likely use the same receptor for entry into BMVECs; however, the single amino acid substitution has profound consequences on tropism. This suggests that receptor binding alone does not determine the final outcome in vivo and that further improvements of capsids within predetermined receptor usage are feasible.

Selective transduction of cerebellar Purkinje and granule neurons using delivery of AAV-PHP.eB and AAVrh10 vectors at axonal terminal locations.

Adeno-associated virus (AAV)-based brain gene therapies require precision without off-targeting of unaffected neurons to avoid side effects. The cerebellum and its cell populations, including granule and Purkinje cells, are vulnerable to neurodegeneration; hence, conditions to deliver the therapy to specific cell populations selectively remain challenging. We have investigated a system consisting of the AAV serotypes, targeted injections, and transduction modes (direct or retrograde) for targeted delivery of AAV to cerebellar cell populations. We selected the AAV-PHP.eB and AAVrh10 serotypes valued for their retrograde features, and we thoroughly examined their cerebellar transduction pattern when injected into lobules and deep cerebellar nuclei. We found that AAVrh10 is suitable for the transduction of neurons in the mode highly dependent on placing the virus at axonal terminals. The strategy secures selective transduction for granule cells. The AAV-PHP.eB can transduce Purkinje cells and is very selective for the cell type when injected into the DCN at axonal PC terminals. Therefore, both serotypes can be used in a retrograde mode for selective transduction of major neuronal types in the cerebellum. Moreover, our in vivo transduction strategies are suitable for pre-clinical protocol development for gene delivery to granule cells by AAVrh10 and Purkinje cells by AAV-PHPeB.

Structural basis of receptor usage by the engineered capsid AAV-PHP.eB.

Adeno-associated virus serotype 9 (AAV9) is a promising gene therapy vector for treating neurodegenerative diseases due to its ability to penetrate the blood-brain barrier. PHP.eB was engineered from AAV9 by insertion of a 7-amino acid peptide and point mutation of neighboring residues, thereby enhancing potency in the central nervous system. Here, we report a 2.24-Å resolution cryo-electron microscopy structure of PHP.eB, revealing conformational differences from other 7-mer insertion capsid variants. In PHP.eB, the 7-mer loop adopts a bent conformation, mediated by an interaction between engineered lysine and aspartate residues. Further, we identify PKD2 as the main AAV receptor (AAVR) domain recognizing both AAV9 and PHP.eB and find that the PHP.eB 7-mer partially destabilizes this interaction. Analysis of previously reported AAV structures together with our pull-down data demonstrate that the 7-mer topology determined by the lysine-aspartate interaction dictates AAVR binding strength. Our results suggest that PHP.eB's altered tropism may arise from both an additional interaction with LY6A and weakening of its AAVR interaction. Changing the insertion length, but not sequence, modifies PKD2 binding affinity, suggesting that a steric clash impedes AAVR binding. This research suggests improved library designs for future AAV selections to identify non-LY6A-dependent vectors and modulate AAVR interaction strength.

AAV-PHP.eB transduces both the inner and outer retina with high efficacy in mice.

Recombinant adeno-associated virus (AAV) vectors are one of the main gene delivery vehicles used in retinal gene therapy approaches; however, there is a need to further improve the efficacy, tropism, and safety of these vectors. In this study, using a CMV-EGFP expression cassette, we characterize the retinal utility of AAV-PHP.eB, a serotype recently developed by in vivo directed evolution, which can cross the blood-brain barrier and target neurons with high efficacy in mice. Systemic and intravitreal delivery of AAV-PHP.eB resulted in the high transduction efficacy of retinal ganglion and horizontal cells, with systemic delivery providing pan-retinal coverage of the mouse retina. Subretinal delivery transduced photoreceptors and retinal pigment epithelium cells robustly. EGFP expression (number of transduced cells and mRNA levels) were similar when the retinas were transduced systemically or intravitreally with AAV-PHP.eB or intravitreally with AAV2/2. Notably, in photoreceptors, EGFP fluorescence intensities and mRNA levels were 50-70 times higher, when subretinal injections with AAV-PHP.eB were compared to AAV2/8. Our results demonstrate the pan-retinal transduction of ganglion cells and extremely efficient transduction of photoreceptor and retinal pigment epithelium cells as the most valuable features of AAV-PHP.eB in the mouse retina.

Brain-wide TVA compensation allows rabies virus to retrograde target cell-type-specific projection neurons.

Retrograde tracers based on viral vectors are powerful tools for the imaging and manipulation of upstream neural networks projecting to a specific brain region, and they play important roles in structural and functional studies of neural circuits. However, currently reported retrograde viral tracers have many limitations, such as brain area selectivity or the inability to retrograde label genetically defined brain-wide projection neurons. To overcome these limitations, a new retrograde tracing method, AAV-PHP.eB assisted retrograde tracing systems (PARTS) based on rabies virus, was established through brain-wide TVA-dependent targeting using an AAV-PHP.eB that efficiently crosses the blood-brain barrier in C57BL/6 J mice, and complementation of EnvA-pseudotyped defective rabies virus that specifically recognizes the TVA receptor. Furthermore, combined with Cre transgenic mice, cell-type-specific PARTS (cPARTS) was developed, which can retrograde label genetically defined brain-wide projection neurons. Our research provides new tools and technical support for the analysis of neural circuits.

Central nervous system-targeted adeno-associated virus gene therapy in methylmalonic acidemia.

Methylmalonic acidemia (MMA) is a severe metabolic disorder most commonly caused by a mutation in the methylmalonyl-CoA mutase (MMUT) gene. Patients with MMA experience multisystemic disease manifestations and remain at risk for neurological disease progression, even after liver transplantation. Therefore, delivery of MMUT to the central nervous system (CNS) may provide patients with neuroprotection and, perhaps, therapeutic benefits. To specifically target the brain, we developed a neurotropic PHP.eB vector that used a CaMKII neuro-specific promoter to restrict the expression of the MMUT transgene in the neuraxis and delivered the adeno-associated virus (AAV) to mice with MMA. The PHP.eB vector transduced cells in multiple brain regions, including the striatum, and enabled high levels of expression of MMUT in the basal ganglia. Following the CNS-specific correction of MMUT expression, disease-related metabolites methylmalonic acid and 2-methylcitrate were significantly (p < 0.02) decreased in serum of treated MMA mice. Our results show that targeting MMUT expression to the CNS using a neurotropic capsid can decrease the circulating metabolite load in MMA and further highlight the benefit of extrahepatic correction for disorders of organic acid metabolism.

CRISPR-mediated rapid generation of neural cell-specific knockout mice facilitates research in neurophysiology and pathology.

Inducible conditional knockout mice are important tools for studying gene function and disease therapy, but their generation is costly and time-consuming. We introduced clustered regularly interspaced short palindromic repeats (CRISPR) and Cre into an LSL-Cas9 transgene-carrying mouse line by using adeno-associated virus (AAV)-PHP.eB to rapidly knockout gene(s) specifically in central nervous system (CNS) cells of adult mice. NeuN in neurons and GFAP in astrocytes were knocked out 2 weeks after an intravenous injection of vector, with an efficiency comparable to that of inducible Cre-loxP conditional knockout. For functional testing, we generated astrocyte-specific Act1 knockout mice, which exhibited a phenotype similar to mice with Cre-loxP-mediated Act1 knockout, in an animal model of multiple sclerosis (MS), an autoimmune disorder of the CNS. With this novel technique, neural cell-specific knockout can be induced rapidly (few weeks) and cost-effectively. Our study provides a new approach to building inducible conditional knockout mice, which would greatly facilitate research on CNS biology and disease.

Apolipoprotein E, low-density lipoprotein receptor, and immune cells control blood-brain barrier penetration by AAV-PHP.eB in mice.

Rationale: The blood-brain barrier (BBB) prevents the effective delivery of therapeutic molecules to the central nervous system (CNS). A recently generated adeno-associated virus (AAV)-based vector, AAV-PHP.eB, has been found to penetrate the BBB more efficiently than other vectors including AAV-PHP.B. However, little is known about the mechanisms. In this study, we investigated how AAV-PHP.eB penetrates the BBB in mice. Methods: We injected AAV-PHP.eB into the bloodstream of wild-type C57BL/6 and BALB/c mice as well as mouse strains carrying genetic mutation in apolipoprotein E gene (Apoe) or low-density lipoprotein receptor gene (Ldlr), or lacking various components of the immune system. Then, we evaluated AAV-PHP.eB transduction to the brain and spinal cord in these mice. Results: We found that the transduction to the CNS of intravenous AAV-PHP.eB was more efficient in C57BL/6 than BALB/c mice, and significantly reduced in Apoe or Ldlr knockout C57BL/6 mice compared to wild-type C57BL/6 mice. Moreover, poor CNS transduction in BALB/c mice was dramatically increased by B-cell or natural killer-cell depletion. Conclusions: Our findings demonstrate that the ApoE-LDLR pathway underlies the CNS tropism of AAV-PHP.eB and that the immune system contributes to the strain specificity of AAV-PHP.eB.

Widespread labeling and genomic editing of the fetal central nervous system by in utero CRISPR AAV9-PHP.eB administration.

Efficient genetic manipulation in the developing central nervous system is crucial for investigating mechanisms of neurodevelopmental disorders and the development of promising therapeutics. Common approaches including transgenic mice and in utero electroporation, although powerful in many aspects, have their own limitations. In this study, we delivered vectors based on the AAV9.PHP.eB pseudo-type to the fetal mouse brain, and achieved widespread and extensive transduction of neural cells. When AAV9.PHP.eB-coding gRNA targeting PogZ or Depdc5 was delivered to Cas9 transgenic mice, widespread gene knockout was also achieved at the whole brain level. Our studies provide a useful platform for studying brain development and devising genetic intervention for severe developmental diseases.

CNS Transduction Benefits of AAV-PHP.eB over AAV9 Are Dependent on Administration Route and Mouse Strain.

Adeno-associated viral (AAV) vectors are attractive tools for central nervous system (CNS) gene therapy because some vectors can cross the blood-brain barrier (BBB), allowing them to be used as minimally invasive treatments. A novel AAV vector recently evolved in vivo, AAV-PHP.eB, has been reported to cross the BBB more effectively than the existing gold standard AAV9, but not under all conditions. Here, we compared the efficacy of single-stranded AAV-PHP.eB and AAV9 in targeting mouse CNS and peripheral tissues after administration via various routes, in two different mouse strains (C57BL/6J and B6C3), and after packaging AAV-PHP.eB with a self-complementary genome. We found that AAV-PHP.eB produced higher CNS transduction than AAV9 after intravenous injection, but only in C57BL/6J and not in B6C3 mice. AAV-PHP.eB and AAV9 produced similar CNS transduction when the administration route did not require the vectors to cross the BBB. Packaging AAV-PHP.eB with a self-complementary genome increased overall CNS transduction, but at the expense of strong neuronal tropism. AAV-PHP.eB resulted in less transduction of liver tissue than AAV9 under all conditions. Taken together, these results suggest the potential for AAV-PHP.eB as a vector for CNS gene therapy applications, but consideration will be required for translation beyond mouse models.

Efficient whole brain transduction by systemic infusion of minimally purified AAV-PHP.eB.

BACKGROUND: Adeno-associated virus (AAV) vectors have excellent properties as gene transfer vehicles. The recent development of AAV-PHP.eB, highly BBB-permeable capsid variant of AAV serotype 9, has opened up systemic application for whole brain transduction. To attain high transduction efficacy, much efforts have been paid to purify AAV vectors using gradient centrifugation or column chromatography. These methods are time-consuming, cost substantially and require expensive equipment. NEW METHOD: We propose a simple purification method for the production of systemically applicable AAV-PHP.eB targeting the brain. The new method, which we named minimal purification (MP) method, requires only 2 steps: removal of cell debris using a syringe filter and concentration using a disposable ultrafiltration device. RESULTS: The MP method yielded 2 times more AAV-PHP.eB than the standard ultracentrifuge purification (UCP) method. Intravenous injection of AAV-PHP.eB prepared using the MP method caused robust whole brain transduction without overt toxicity on the liver and kidney. Moreover, we found almost no difference in cellular density and morphology of brain microglia between control mice and mice treated systemically with the MP viral solution, suggesting no influence of the viral injection on brain immunity. COMPARISON WITH EXISTING METHODS: The new method, which requires only a benchtop centrifuge and takes only 2-4 h to obtain a ready-to-use viral solution, is much less expensive than the existing UCP method, and can avoid cumbersome and time-consuming purification processes. CONCLUSIONS: This simplified method further expands the use of AAV vectors in the neuroscience community.

Bioluminescence imaging in mice with synthetic luciferin analogues.

Luciferase enzymes from bioluminescent organisms can be expressed in mice, enabling these rodents to glow when treated with a corresponding luciferin substrate. Light emission occurs where the expression of the genetically-encoded luciferase overlaps with the biodistribution of the administered small molecule luciferin. Here we discuss differences between firefly luciferin analogues for bioluminescence imaging, focusing on transgenic and adeno-associated virus (AAV)-transduced mice.

Focused ultrasound as a novel strategy for noninvasive gene delivery to retinal Müller glia.

Müller glia are specialized retinal cells with stem cell properties in fish and frogs but not in mammals. Current efforts to develop gene therapies to activate mammalian Müller glia for retinal repair will require safe and effective delivery strategies for recombinant adeno-associated viruses (AAVs), vectors of choice for clinical translation. Intravitreal and subretinal injections are currently used for AAV gene delivery in the eye, but less invasive methods efficiently targeting Müller glia have yet to be developed. Methods: As gene delivery strategies have been more extensively studied in the brain, to validate our vectors, we initially compared the glial tropism of AAV-PHP.eB, an AAV9 that crosses the blood-brain and blood-retinal barriers, for its ability to drive fluorescent protein expression in glial cells in both the brain and retina. We then tested the glial transduction of AAV2/8-GFAP-mCherry, a virus that does not cross blood-brain and blood-retinal barriers, for its effectiveness in transducing Müller glia in murine retinal explants ex vivo. For in vivo assays we used larger rat eyes, performing invasive intravitreal injections, and non-invasive intravenous delivery using focused ultrasound (FUS) (pressure amplitude: 0.360 - 0.84 MPa) and microbubbles (Definity, 0.2 ml/kg). Results: We showed that AAV-PHP.eB carrying a ubiquitous promoter (CAG) and green fluorescent protein (GFP) reporter, readily crossed the blood-brain and blood-retinal barriers after intravenous delivery in mice. However, murine Müller glia did not express GFP, suggesting that they were not transduced by AAV-PHP.eB. We thus tested an AAV2/8 variant, which was selected based on its safety record in multiple clinical trials, adding a glial fibrillary acidic protein (GFAP) promoter and mCherry (red fluorescent protein) reporter. We confirmed the glial specificity of AAV2/8-GFAP-mCherry, showing effective expression of mCherry in astrocytes after intracranial injection in the mouse brain, and of Müller glia in murine retinal explants. For in vivo experiments we switched to rats because of their larger size, injecting AAV2/8-GFAP-mCherry intravitreally, an invasive procedure, demonstrating passage across the inner limiting membrane, leading to Müller glia transduction. We then tested an alternative non-invasive delivery approach targeting a different barrier - the inner blood-retinal-barrier, applying focused ultrasound (FUS) to the retina after intravenous injection of AAV2/8 and microbubbles in rats, using magnetic resonance imaging (MRI) for FUS targeting. FUS permeabilized the rat blood-retinal-barrier and allowed the passage of macromolecules to the retina (Evans blue, IgG, IgM), with minimal extravasation of platelets and red blood cells. Intravenous injection of microbubbles and AAV2/8-GFAP-mCherry followed by FUS resulted in mCherry expression in rat Müller glia. However, systemic delivery of AAV2/8 also had off-target effects, transducing several murine peripheral organs, particularly the liver. Conclusions: Retinal permeabilisation via FUS in the presence of microbubbles is effective for delivering AAV2/8 across the inner blood-retinal-barrier, targeting Müller glia, which is less invasive than intravitreal injections that bypass the inner limiting membrane. However, implementing FUS in the clinic will require a comprehensive consideration of any off-target tropism of the AAV in peripheral organs, combined ideally, with the development of Müller glia-specific promoters.

The GPI-Linked Protein LY6A Drives AAV-PHP.B Transport across the Blood-Brain Barrier.

Efficient delivery of gene therapy vectors across the blood-brain barrier (BBB) is the holy grail of neurological disease therapies. A variant of the neurotropic vector adeno-associated virus (AAV) serotype 9, called AAV-PHP.B, was shown to very efficiently deliver transgenes across the BBB in C57BL/6J mice. Based on our recent observation that this phenotype is mouse strain dependent, we used whole-exome sequencing-based genetics to map this phenotype to a specific haplotype of lymphocyte antigen 6 complex, locus A (Ly6a) (stem cell antigen-1 [Sca-1]), which encodes a glycosylphosphatidylinositol (GPI)-anchored protein whose function had been thought to be limited to the biology of hematopoiesis. Additional biochemical and genetic studies definitively linked high BBB transport to the binding of AAV-PHP.B with LY6A (SCA-1). These studies identify, for the first time, a ligand for this GPI-anchored protein and suggest a role for it in BBB transport that could be hijacked by viruses in natural infections or by gene therapy vectors to treat neurological diseases.