REGULATION OF BLOOD-CENTRAL NERVOUS SYSTEM (BLOOD-CNS) BARRIER AND USES THEREOF

RELATED APPLICATIONS rooou This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application, U.S.S.N. 63/406,463, filed September 14, 2022, which is incorporated herein by reference.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[00021 The contents of the electronic sequence listing (H082470416WO00-SEQ-LJG.xml; Size: 64,042 bytes; and Date of Creation: September 11, 2023) is incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

[00031 This invention was made with government support under grant numbers NS 116820 and HL153261 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

BACKGROUND

[00041 The CNS requires an optimal and tightly regulated microenvironment for efficient synaptic transmission. This is achieved by blood-CNS barriers that regulate substance flux to maintain tissue homeostasis. Barrier properties of CNS endothelial cells require induction and maintenance from brain parenchymal cells. However, the signal(s) that facilitate endothelial cells to maintain barrier integrity remain elusive.

SUMMARY

[00051 The present disclosure, at least in part, provides compositions and the uses thereof, kits and methods for regulating Blood-Central Nervous System (blood-CNS) barrier permeability (e.g., increasing or decreasing Blood-CNS barrier permeability) by regulating signaling between Cede 141 and Pacsin2. In some embodiments, the methods described herein comprises regulating (e.g., increase) blood-CNS barrier permeability by administering to a subject an inhibitor of Cede 141, and/or an agent that increases Pacsin2 expression and/or activity. In some embodiments, the methods described herein comprises regulating (e.g., decrease) blood-CNS barrier permeability by administering to a subject an inhibitor of Pacsin2, and/or an agent that increases Cede 141 expression and/or activity.

[00061 According to some aspects, a method for increasing Blood-Central Nervous System (Blood-CNS) Barrier permeability to treat a CNS disease in a subject is provided herein. In some embodiments, the method comprises administering the subject an inhibitor of Cede 141 at the Blood-CNS Barrier.

[00071 In some embodiments, the blood-CNS barrier is the blood-brain barrier. In other embodiments, the blood-CNS barrier is the blood-retina barrier.

[00081 In some embodiments, the inhibitor of Cede 141 is capable of inhibiting Cede 141 expression and/or activity. In some embodiments, the inhibitor of Cede 141 is an inhibitory nucleic acid targeting Cede 141. In some embodiments, the inhibitory nucleic acid targets the coding sequence of Cede 141 comprising SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the inhibitory nucleic acid targeting Cede 141 is a siRNA, a shRNA, a miRNA, an AmiRNA, an aptamer, or an ASO.

[00091 In some embodiments, the inhibitory nucleic acid targeting Cede 141 is a siRNA targeting Cede 141. In some embodiments, the siRNA targeting Cede 141 comprises an antisense strand complementary to any one of SEQ ID NOs: 9-21. 1

[00101 In some embodiments, the inhibitory nucleic acid targeting Cede 141 is a shRNA targeting Cede 141. In some embodiments, the shRNA comprises a guide strand complementary to any one of SEQ ID NOs: 9-21.

[00111 In some embodiments, the inhibitor of Cede 141 is an antibody, an antibody variant or an antigen-binding fragment targeting Cede 141.

[00121 In some embodiments, the inhibitor of Cede 141 is a small molecule.

[00131 In some embodiments, the inhibitor of Cede 141 is delivered to CNS endothelial cells.

[00141 In some embodiments, the injection is systemic injection comprising intravenous injection, intramuscular injection, subcutaneous injection, or intraperitoneal injection.

[00151 In some embodiment, the injection is direct injection to the CNS comprising intracerebral injection, intraventricular injection, intracisternal injection, intraparenchymal injection, intrathecal injection, and any combination thereof.

[00161 According to some aspects, the present disclosure provides a method for increasing Blood-Central Nervous System (Blood-CNS) Barrier permeability to treat a CNS disease in a subject is provided herein. In some embodiments, the method comprising administering an isolated nucleic acid comprising a transgene encoding Pacsin2. [00171 In some embodiments, the inhibitory nucleic acid is delivered by a recombinant adeno-associated virus (rAAV). In some embodiments, the capsid protein is BRI, BI30, AAV1, AAV2, AAV9, or variant thereof (e.g., AAV-PHP or AAV-PHP.B). In some embodiments, the capsid protein is BRI. In some embodiments, the capsid protein is BI30. In some embodiments, the inhibitor of Cede 141 is administered to the subject via injection.

[00181 In some embodiment, the transgene further comprises a promoter operably linked to a nucleotide sequence encoding Pacsin2. In some embodiments, the isolated nucleic acid further comprises two adeno-associated virus inverted terminal repeats (ITRs) flanking the transgene encoding Pacsin2. In some embodiments, the isolated nucleic acid is administered to the subject via a recombinant adeno-associated virus (rAAV). In some embodiments, the rAAV further comprises a capsid protein. In some embodiments, the rAAV comprises a capsid protein comprising BRI, BI30, AAV1, AAV2, AAV9, or variant thereof. In some embodiments, the capsid protein BRI. In some embodiments, the capsid protein is BI30. [00191 In some embodiments, the method further comprises administering a therapeutic agent for treating the CNS disease.

[00201 According to some aspects, the present disclosure provides a method for delivering a therapeutic agent to the central nervous system to treat a CNS disease is described herein. In some embodiments, the method comprises administering a subject, a therapeutic agent for treating the disease; and an agent for increasing Blood-Central Nervous System (Blood-CNS) Barrier.

[00211 In some embodiments, the agent for increasing Blood-CNS barrier permeability is an inhibitor of Cede 141.

[00221 In some embodiments, the agent for increasing Blood-CNS barrier permeability is an isolated nucleic acid comprising a transgene encoding Pacsin2.

[00231 In some embodiments, the CNS disease is a neuromuscular disease, neurodegenerative disease, brain and nerve tumors, Neurogenetic Diseases, Cognitive disorders, Familial dystonia, Neuroinfectious disease, neuropsychiatric disorders. In some embodiments, the neuromuscular disease is Amyotrophic Lateral Sclerosis (ALS), Ataxia, Cerebral Palsy, Muscular Dystrophy.

[00241 In some embodiments, the therapeutic agent comprises antibiotics, antibodies, anticonvulsants (e.g., gabapentin), chemotherapeutic s, anti-inflammatories, neurotransmitters, pain medication (e.g., morphine), peptides, nucleic acids (e.g. RNAi-based therapies), or psychiatric drugs. [00251 According to some aspects, the present disclosure provides a method for decreasing Blood-Central Nervous System (Blood-CNS) Barrier permeability to treat a CNS disease in a subject. In some embodiments, the method comprises administering the subject an inhibitor of Pacsin2 at the Blood-CNS Barrier.

[00261 In some embodiments, the inhibitor of Pacsin2 is capable of inhibiting Pacsin2 expression and/or activity. In some embodiments, the inhibitor of Pacsin2 is an inhibitory nucleic acid targeting Pacsin2. In some embodiments, the inhibitory nucleic acid targets the coding sequence of Pacsin2 comprising SEQ ID NOs: 5 and 6. In some embodiments, the inhibitory nucleic acid targeting Pacsin2 is a siRNA, a shRNA, a miRNA, an AmiRNA, an aptamer, or an ASO. In some embodiments, the inhibitory nucleic acid targeting Pacsin2 is a siRNA targeting Pacsin2. In some embodiments, the siRNA targeting Pacsin2 comprises an antisense strand complementary to any one of SEQ ID NOs: 22-31, and 36-45.

[00271 In some embodiments, the inhibitory nucleic acid targeting Pacsin2 is a shRNA targeting Pacsin2. In some embodiments, the shRNA targeting Pacsin2 comprises a guide strand complementary to any one of SEQ ID NOs: 22-31, and 36-45.

[00281 In some embodiments, the inhibitor of Pacsin2 is an antibody, an antibody variant or an antigen-binding fragment targeting Pacsin2.

[00291 In some embodiments, the inhibitor of Pacsin2 is a small molecule.

[00301 In some embodiments, the inhibitor of Pacsin2 is delivered to CNS endothelial cells. [00311 In some embodiments, the inhibitor of Pacsin2 is delivered by a recombinant adeno- associated virus (rAAV). In some embodiments, the rAAV comprises a capsid protein comprising BRI, BI30, AAV1, AAV2, AAV9, or variant thereof. In some embodiments, the capsid protein is BRI. In some embodiments, the capsid protein is BI30.

[00321 In some embodiments, the inhibitor of Pacsin2 is administered to the subject via injection. In some embodiments, the injection is systemic injection comprising intravenous injection, intramuscular injection, subcutaneous injection, or intraperitoneal injection. In some embodiments, the injection is direct injection to the CNS comprising intracerebral injection, intraventricular injection, intracisternal injection, intraparenchymal injection, intrathecal injection, intravitreal injection, and any combination thereof.

[00331 According to some aspects, the present disclosure provides methods for decreasing Blood-Central Nervous System (Blood-CNS) Barrier permeability to treat a CNS disease in a subject are provided herein. In some embodiments, the method comprises administering an isolated nucleic acid comprising a transgene encoding Cede 141. [00341 In some embodiments, the transgene further comprises a promoter operably linked to a nucleotide sequence encoding Cede 141.

[00351 In some embodiments, the isolated nucleic acid further comprises two adeno- associated virus inverted terminal repeats (ITRs) flanking the transgene encoding Cede 141. In some embodiments, the isolated nucleic acid is administered to the subject via a recombinant adeno-associated virus (rAAV). In some embodiments, the rAAV further comprises a capsid protein. In some embodiments, the capsid protein is BRI, BI30, AAV1, AAV2, AAV9, or variant thereof. In some embodiments, the capsid protein is BRI. In some embodiments, the capsid protein is BI30.

[00361 In some embodiments, the CNS disease is retinal disease, neurodegenerative disease, acute injury of the CNS, neuroinfectious disease, primary and metastatic cancers f the CNS, autoimmune disease of the CNS, neuroinflammatory conditions, or cognitive disorder. In some embodiments, the retinal disease is diabetic retinopathy. In some embodiments, the neurodegenerative disease is Huntington’s disease, Alzheimer's disease, Parkinson's disease, motor neuron disease, Amyotrophic lateral sclerosis, spinal muscular atrophy, spinocerebral ataxia. In some embodiments, the acute injury of the CNS is stroke or head trauma. In some embodiments, the neuroinfectious disease is encephalitis, sepsis, or COVID- 19. In some embodiments, the primary cancer of the CNS is glioblastoma, meningioma, or lymphoma. In some embodiments, the metastatic cancer of the CNS is lung cancer, metastatic breast cancer, GI track cancers, or melanoma. In some embodiments, the autoimmune disease of the CNS is multiple sclerosis. In some embodiments, the neuroinflammatory condition is CNS Lupus, CNS Lyme Disease, Neurosarcoidosis, Neuromyelitis optica (NMO), or Paraneoplastic and Autoimmune Encephalitis.

[00371 In some embodiments, the cognitive disorder is dementia resulting from Alzheimer’s disease, Lewy body dementia, frontotemporal dementia, encephalopathy, or post-acute COVID syndrome.

[00381 In some aspects, the present disclosure provides composition and method for treating a condition associated with increased blood-CNS barrier permeability, the method comprising administering to a subject a Pacsin2 inhibitor or an agent promoting Cede 141 expression/activity. In some embodiments, the condition is associated with aging. In some embodiments, the condition is early onset dementia. In some embodiments, the condition is stroke. In some embodiments, the condition is spinal cord injury.

[00391 The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawing and detailed description of certain embodiments and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[00401 The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the Detailed Description of Specific Embodiments presented herein.

[00411 FIGs. 1A-1H show the delivery of gene-specific single-guide RNA (sgRNAs) using adeno-associated virus (AAV) that specifically infects brain microvessels called AAV-BR1 and results in increased blood-brain barrier leakage. FIG. 1A shows a genetic construct containing sgRNA incorporated into AAV-BR1 and injected into mice to achieve CNS- specific CRISPR/Cas9 gene knockout. FIG. IB shows expression of AAV-BR1 (left panel) in CNS endothelial cells (right paten). FIG. 1C shows the elimination of Glutl expression in CNS endothelial cells infected with AAV-BR1 sgRNA:S7c7a. FIG. ID shows that elimination of Glutl through AAV-Glutl injection results in blood-brain barrier leakage. FIG. IE shows increased brain leakage in the brain of mice injected with BR 1 -sgRNA:.S7c2a/ relative to controls. FIG IF shows increased brain leakage in coronal sections of a mouse brain injected with BR l -sgRNA:.S7c2a/ relative to controls. FIG. 1G shows increased leakage area outside of Glutl ^KD vessels in the brains of mice injected with BRl-sgRNA:S7c2a7 relative to controls. FIG. 1H shows quantifications of increased tracer leakage outside of vessels in Glutl ^KD vs controls.

[00421 FIGs. 2A-2D show the enrichment of Ccdcl41 to the vessels of the CNS. FIG. 2A shows the vessels, nuclei and, Cede 141 mRNA 1 in the brain and lungs. FIG. 2B shows the mRNA density of Ccdcl41 (dots/area) in the brain and lungs. FIG. 2C shows Ccdcl41 protein expression in purified endothelial prep from brain and lung. FIG. 2D shows Cede 141 protein quantification using western-blot of purified endothelial prep from brain and lung. This analysis showed 4-fold higher Cede 141 protein in brain ECs compared to lung ECs. [00431 FIGs. 3A-3G show effects of knocking down Ccdcl41 expression in CNS endothelial cells. FIG. 3A shows Ccdcl41 ablation using AAV-BR1 delivered sgRNAs targeting Cede 141. FIG. 3B shows no apparent tracer extravasation in the brains of control mice. FIG. 3C shows increased tracer extravasation in the brains of Cdcl41 ^KD mice. FIG. 3D shows increased leakage area outside of Ccdcl41 ^KD vessels in the brain of mice injected with BR1- sgRNA:Cc<7c747 relative to controls. FIG. 3E shows the correlation of virus infection in vessels to leakage sites as measured by increased NHS-biotin concentrations surrounding vessels in sgCcdcl41 injected mice versus controls. FIG. 3F shows quantifications of increased tracer leakage outside of vessels in Ccdcl41 ^KD vs controls. FIG. 3G Shows increased endogenous IgG leakage outside of Ccdcl41KD vessels in the brain of mice injected with BR 1 -sgRNA:.S7c2a/ relative to controls.

100441 FIGs 4A-4B show the effects of endothelial- specific Ccdcl41 ^KD on endothelial cells and neurons of the brain. FIG. 4A shows that acute endothelial specific ablation of Cede 141 results in neuronal cell death. FIG. 4B shows that acute endothelial specific ablation of Cede 141 does not affect endothelial cell viability.

100451 FIGs. 5A-5B show the effects of Ccdcl41 ^KD on vessel area in the CNS. FIG. 5A shows the percent vessel area in the CNS in Ccdcl41 ^KD and control mice. FIG. 5B shows confocal micrographs of control and Ccdcl41 ^KD mice.

100461 FIGs. 6A-6E show the effects of Cede 141 on BBB function through suppression of tubular vesicle trafficking. FIG. 6A shows an increase in intracellular tubular transport vesicles in Ccdcl41 ^KD CNS endothelial cells vs controls. FIG. 6B shows increases in total vesicle density and tubular vesicle density in Ccdcl41 ^KD mice relative to control mice. There was no change in round vesicle density between Ccdcl41KD mice and control mice. FIG. 6C shows magnified electron microscopy images showing the tubular structure of the transport vesicles following Cede 141 knockdown. FIG. 6D shows that Ccdcl41 ^KD has no effect on caveolae-mediated transcytosis at the BBB in CNS endothelial cells. FIG. 6E shows the quantification of Cavl vesicle density in Ccdcl41 ^KD and control mouse brains.

[00471 FIGs. 7A-7D show the effects of Ccdcl41 ^KD on tight-junctions in the BBB endothelial cells of the CNS. FIG. 7A shows that Ccdcl41 ^KD does not induce tracer transfer between adjacent endothelial cells, therefore exhibiting functional tight junctions. FIG. 7B shows that Wnt/p-catenin signaling is unchanged after Cede 141 ablation in brain endothelial cells. FIG. 7C shows that Cldn5 expression in CNS endothelial cells was unaffected following Ccdcl41 ^KD relative to controls. FIG. 7D shows that the expression of tight-j unction protein ZO-1 was unaffected following Ccdcl41 ^KD relative to controls.

[00481 FIGs. 8A-8F show that Pacsin2 expression is increased following Ccdcl41 ^KD . FIG. 8A shows the relative expression of Pacsin2 mRNA in the brain and lungs of mice. FIG. 8B shows Pacsin2 protein expression in purified endothelial prep from brain and lung FIG. 8C shows Pacsin2 protein quantification using western blot of purified endothelial prep from brain and lung. FIG. 8D shows increased Pacsin2 expression in Ccdcl41 ^KD vessels relative to controls. FIG. 8E shows a graph demonstrating the increased percentage of Pacsin2 mRNA positive vessels following Ccdcl41 ^KD relative to controls. FIG. 8F shows that ablation of Cede 141 in CNS capillaries results in upregulation of Pacsin2.

100491 FIGs. 9A-9J show that Pacsin2 overexpression in CNS endothelial cells increases BBB leakage and tubular vesicle transcytosis. FIG. 9A shows increased Pacsin2 expression in brain endothelial cells following injection with AAV-BR1 expressing Pacsin2 in adult mice. FIG. 9B shows a graph demonstrating the increased percentage of Pacsin2 positive vessels following Pacsin2 overexpression relative to controls. FIG. 9C shows Pacsin2 protein upregulation following Pacsin2 overexpression compared to controls. FIG. 9D shows that Pacsin2 overexpression increases brain leakiness as measured by increased permeation of sulfo-NHS -biotin into adult mouse brain tissues. FIG. 9E shows confocal micrographs of leakage of sulfo-NHS -biotin into brain regions surrounding the vessels of adult mice overexpressing Pacsin2. FIG. 9F shows a graph quantifying the leakage of sulfo-NHS -biotin into adult mouse brain tissues surrounding vessels of endothelial cells overexpressing Pacsin2. FIG. 9G contains electron microscopy images showing that Pacsin2 overexpression results in increased tubular vesicles in CNS endothelial cells relative to controls. FIG. 9H shows increase in tubular vesicle density following Pacsin2 overexpression relative to controls. FIG. 91 shows that Pacsin2 overexpression results in the leakage of mouse endogenous IgG (mlgG). FIG. 9J shows a graph quantifying the leakage of endogenous mouse IgG into adult mouse brain parenchyma.

[00501 FIGs. 10A-10B show that Cede 141 suppressing tubular vesicle mediated transcytosis and BBB leakiness by inhibiting Pacsin2 expression. FIG. 10A shows that double knockdown of Pacsin2 and Cede 141 in adult mouse brains rescues leakiness phenotype observed in Ccdcl41 ^KD animals. FIG. 10B shows a graph quantifying the percentage of leakage area outside of the vessels in Cdcl41 ^KD , Pacsin2 ^KD , and Ccdcl41 ^KD +Pacsin ^KD adult mouse brains. 100511 FIG. 11 shows Pacsin2 protein (middle panel) in the vessels (represented by Glutl expression in the left panel) of the post-mortem temporal cortex of the aged human individual (78 years old). The right panel shows a merged image of Glutl and Pacsin2 expression. 100521 FIG. 12 shows that Pacsin2 is up-regulated in the endothelial cells in HSV-induced neuroinflammation. The immunostaining of mouse brain stem sections shows increased Pacsin2 protein levels (middle panel in infected region compared to middle panel in uninfected region) in the vessels (represented by Icam2 expression in the left panel of the uninfected region and the infected region) of the HSV infected mice compared to uninfected mice. DETAILED DESCRIPTION

[00531 The present disclosure, at least in part, provides compositions and uses thereof, kits and uses thereof, and methods for regulating Blood-Central Nervous System (blood-CNS) barrier permeability (e.g., increasing or decreasing blood-CNS barrier permeability) by regulating the Coiled-Coil Domain Containing 141 (Ccdcl41). In some aspects, the present disclosure provides compositions and uses thereof, kits and uses thereof, and methods for regulating Blood-Central Nervous System (blood-CNS) barrier permeability (e.g., increasing or decreasing blood-CNS barrier permeability) by regulating protein kinase C and casein kinase substrate in neurons protein 2 (Pacsin2). In some embodiments, the present disclosre provides composition and methods for regulating Blood-Central Nervous System (blood- CNS) barrier permeability (e.g., increasing or decreasing blood-CNS barrier permeability) by regulating Cede 141 and Pacsin2.

[00541 Cede 141 is linked to congenital hypogonadotropic hypogonadism in humans. During embryonic development, gonadotropin releasing hormone (GnRH) neurons originating in the nasal placode migrate into the dorsal region of the olfactory bulb (OB) and then caudally towards the hypothalamus (Hutchins et al., CCDC141 mutation identified in anosmic hypogonadotropic hypogonadism (Kallmann syndrome) alters GnRH neuronal migration. Endocrinology, 157(5), 1956-1966 (2016)) Genetic screenings identified families with congenital hypogonadotropic hypogonadism carry novel mutations in Cede 141 and hypothesized a role in neuronal migration. The present disclosure, at least in part, is based on the discovery that Cede 141 is expressed by CNS endothelial cells. In some embodiments, Cede 141 plays a role in regulating CNS endothelial cell function (e.g., regulating blood-CNS barrier permeability). In some embodiments, Ccdcl41 regulates blood-CNS barrier permeability via Pacsin2. In some embodiments, the presence of Cede 141 in CNS endothelial cells inhibits Pacsin2. In some embodiments, inhibition of Pacsin2 by Cede 141 results in decreased tubular-vesicle mediated transcytosis of the CNS endothelial cells.

[00551 Pacsin2 colocalizes with amyloid-P particles, suggesting that Pacsin2 may have a protective role in the aging brain by providing clearance of amyloid-P particles. Pacsin2 is normally expressed at low levels in brain endothelial cells (ECs) compared to periphery ECs but is upregulated in EC-specific knockdown of Cede 141 mutant mice. In some embodiments, overexpression of Pacsin2 in brain ECs increases blood-CNS barrier permeability. In some embodiments, overexpression of Pacsin2 in brain ECs up-regulates tubular vesicles. In some embodiments, knocking down Pacsin2 in brain ECs decreases blood-CNS barrier permeability (e.g., rescued the leakage phenotype observed in brain EC- specific Cede 141 knockdown). The present disclosure enables specific manipulation of blood-CNS barrier permeability to improve the delivery of therapeutics, or to ameliorate ailments associated with blood-CNS leakage.

100561 Cede 141 inhibits tubular vesicle transcytosis that is mediated by Pacsin2. Cede 141 gene ablation or Pacsin2 gene overexpression in brain endothelial cells in mice leads to an accumulation of tracer- filled tubular vesicles (e.g., tubular vesicles are involved in transcytosis (z.e., vesicular trafficking) of molecules across the blood-brain barrier endothelial cell layer, from blood into the brain tissue). Analogous tracer-filled tubular vesicles have been observed in the disrupted blood-brain barrier vasculature in the brain and spinal cord, under several neuropathological conditions. Specifically, tubular vesicles have been observed in the brain endothelial cells after traumatic brain injuries suggesting Pacsin2 overexpression in these conditions (see, e.g., Lossinsky et al., New ultrastructural evidence for a protein transport system in endothelial cells of gerbil brains. Acta Neuropathol. 47, 105-110; Lossinsky et al., Ultracytochemical studies of vesicular and canalicular transport structures in the injured mammalian blood-brain barrier. Acta Neuropathol. 61, 239-245; Lossinsky et al., Ultracytochemical evidence for endothelial channel-lysosome connections in mouse brain following blood-brain barrier changes. Acta Neuropathol. 53, 197-202; Lossinskyet al., A comparative ultrastructural study of endothelial cell tubular structures from injured mouse blood-brain barrier and normal hepatic sinusoids demonstrated after perfusion fixation with osmium tetroxide. Microvasc Res 31, 333-344) and stroke (see, e.g., Tagami, M. et al., Increased transendothelial channel transport of cerebral capillary endothelium in stroke-prone SHR. Stroke 14, 591-596) and in the spinal cord endothelial cells in the experimental autoimmune encephalomyelitis(EAE) mouse and rat models for multiple sclerosis (see, e.g., Claudio et al., Increased vesicular transport and decreased mitochondrial content in bloodbrain barrier endothelial cells during experimental autoimmune encephalomyelitis. Am J Pathol 135, 1157-1168 (1989); Lossinsky et al., Sites of egress of inflammatory cells and horseradish peroxidase transport across the blood-brain barrier in a murine model of chronic relapsing experimental allergic encephalomyelitis. Acta Neuropathol 78, 359-371. In other embodiments, Pacsin2 is overexpressed in ageing brain where blood-CNS barrier permeability has been shown to be increased (e.g. , early onset dementia such as frontotemporal dementia (see, Gerrits et al., Neurovascular dysfunction in GRN-associated frontotemporal dementia identified by single-nucleus RNA sequencing of human cerebral cortex, Nat. Neurosci. 2022 Aug;25(8): 1034-1048). In some embodiments, Ccdcl41 is decreased in conditions where blood-CNS barrier permeability have been shown to be increased (e.g., stoke (see, Garcia-Bonilla et al., Brain and blood single-cell transcriptomics in acute and subacute phases after experimental stroke; bioRxiv. Preprint. 2023 Apr 3), spinal cord injury (see, Cao et al., Single-cell RNA sequencing for traumatic spinal cord injury, The FASEB Journal. 2022; 36:e22656), ageing (see, Ximerakis et al., Single-cell transcriptomic profiling of the aging mouse brain, Nature Neuroscience, volume 22, pages 1696-1708 (2019)). Accordingly, in some embodiments, the present disclosure contemplates treating diseases and conditions associated with increased blood-CNS permeability (e.g., ageing, early onset dementia, stroke, or traumatic spinal cord injury, Multiple sclerosis, epilepsy, cerebral edema) by administering a subject in need thereof an agent that inhibits Pacsin2 expression/activity and/or promotes Cede 141 expression/activity. In some embodiments, the present disclosure provides methods of inhibiting transcytosis at the blood-CNS barrier by administering to a subject in need thereof an agent that inhibits Pacsin2 expression/activity and/or promotes Cede 141 expression/activity. The blood-CNS barrier is a structure that separates circulating blood from the central nervous system (CNS). In some embodiments, the blood-CNS barrier includes the blood-brain barrier (BBB) and blood-retina barrier (BRB). The blood-CNS barrier lines the capillaries associated with the CNS and is comprised of endothelial cells and the tight junctions between them. The blood-brain barrier is formed by endothelial cells of the blood vessel (e.g., capillary) wall, astrocyte end-feet ensheathing the capillary, and pericytes embedded in the blood vessel (e.g., capillary) basement membrane. In some embodiments, the endothelial cells of the CNS are brain endothelial cells. In some embodiments, the brain endothelial cells are arterial endothelial cells, venous endothelial cells, and/or capillary endothelial cells. In some embodiments, the brain capillary endothelial cells are brain microvascular endothelial cells (BMVECs). In some embodiments, the endothelial cells of the CNS are spinal cord endothelial cells. In some embodiments, the endothelial cells of the CNS are retina vasculature endothelial cells. In some embodiments, the retina vasculature endothelial cells are superficial plexus arterial endothelial cells, superficial plexus venous endothelial cells, intermediate plexus endothelial cells, or deep plexus endothelial cells.

100571 The blood-CNS barrier generally excludes large hydrophilic molecules and pathogens (e.g., bacteria, viruses, or parasites) from entering the CNS while allowing the passage of small hydrophobic molecules, such as lipids and oxygen. Other molecules are actively transported across the blood-CNS barrier, e.g., glucose. The restrictive permeability of CNS endothelial cells that constitute these barriers is a result of specialized tight junctions and low rates of transcytosis, which limit substance exchange between blood and CNS tissue. While the blood-CNS barrier is generally very effective at excluding, e.g., pathogens, from the CNS, the blood-CNS barrier poses a formidable obstacle when a drug needs to be delivered to the CNS. For example, antibodies and most antibiotics will not cross the blood-CNS barrier. The degradation of the blood-CNS barrier is a feature of many neurodegenerative diseases, e.g., multiple sclerosis. Accordingly, methods and compositions for modulating the permeability of the blood-CNS barrier, both by increasing or decreasing the permeability of the barrier, have a role in the treatment of a wide variety of diseases that impact the CNS. (See, e.g., Kadry et al., A blood-brain barrier overview on structure, function, impairment, and biomarkers of integrity, Fluids and Barriers of the CNS, volume 17, Article number: 69 (2020)).

[00581 The present disclosure, at least in part, is based on the theory that the barrier properties of the CNS endothelial cells are not intrinsic to those cells. Rather, the CNS endothelial cells require active induction and maintenance from the CNS environment to induce and maintain the barrier properties (see, e.g., P. A. Stewart et al., Developing nervous tissue induces formation of blood-brain barrier characteristics in invading endothelial cells: A study using quail-chick transplantation chimeras. Dev. Biol. 84, 183-192 (1981)). In some aspects, the present disclosure is based on the discovery that the scaffolding protein coiled- coil domain 141 (Ccdcl41) inhibits expression of protein kinase C and casein kinase substrate in neurons 2 (Pacsin2) protein in CNS endothelial cells. In some embodiments, Cede 141 is expressed by CNS endothelial cells. In some embodiments, the Cede 141 protein expressed by CNS endothelial cells regulates tubular vesicle-mediated transcytosis via inhibition of Pacsin2 in endothelial cells in the CNS. In some embodiments, inhibition of Cede 141 increases Pacsin2 expression in CNS endothelial cells and increases blood-CNS barrier permeability by increasing transcytosis in CNS endothelial cells. In some embodiments, inhibition of Pacsin2 in CNS endothelial cells decreases blood-CNS barrier permeability by regulating transcytosis (e.g., tubular vesicle mediated transcytosis) in CNS endothelial cells.

100591 In some aspects, the present disclosure provides compositions and methods for increasing Blood-Central Nervous System (blood-CNS) barrier permeability to treat a disease (e.g., brain and CNS tumors, ALS, etc.) in a subject, the method comprising administering to the subject an inhibitor (e.g., inhibitory nucleic acids, small molecule inhibitors, and/or antibodies targeting Cede 141) of Cede 141 at the blood-CNS barrier. In some aspects, the present disclosure also provides methods for increasing blood-CNS barrier permeability, the method comprising administering to a subject an activator of Pacsin2 (e.g., coding sequence of Pacsin2). In some aspects, the present disclosure provides compositions and methods for decreasing blood-CNS barrier permeability for treating or preventing diseases associated with increased blood-CNS barrier permeability (e.g., ageing related neurodegenerative conditions (e.g., early onset dementia), brain vasculature diseases (e.g., stroke, spinal cord injury), chemical exposures, and/or bacterial and viral infections) in a subject, the method comprising administering to a subject an inhibitor (e.g., inhibitory nucleic acids, small molecule inhibitors, and/or antibodies targeting Pacsin2) of Pacsin2 signaling. In some aspects, the present disclosure also provides compositions and methods of decreasing blood-CNS permeability for treating or preventing diseases associated with increased blood-CNS barrier permeability (e.g., ageing related neurodegenerative conditions (e.g., early onset dementia), brain vasculature diseases (e.g., stroke, spinal cord injury), chemical exposures, and/or bacterial and viral infections) in a subject, the method comprising increasing Cede 141 expression and/or activity in the CNS endothelial cells (e.g., overexpressing Ccdcl41). [00601 Other aspects of the present disclosure relate to co-delivering a molecule (e.g., a therapeutic agent) with an inhibitor of Cede 141 or an activator of Pacsin2 (e.g., Pacsin2 coding sequence) at the blood-CNS Barrier to facilitate the CNS delivery of the molecule (e.g., a therapeutic agent).

100611 In some aspects, the present disclosure provides methods for decreasing Blood- Central Nervous System (blood-CNS) barrier permeability for treating a disease in a subject, the method comprising administering to the subject an agent that inhibits Pacsin2 or increases Cede 141 expression and/or activity in the central nervous system (CNS).

I. Increasing Blood-Central Nervous System (blood-CNS) Barrier Permeability 100621 In some aspects, the present disclosure provides compositions and methods, kits and uses for increasing blood-CNS barrier permeability. In some embodiments, the method comprises administering to a subject an inhibitor of Cede 141 signaling. In some embodiments, the Ccdcl41 is expressed by CNS endothelial cells. In some embodiments, the method comprises administering to the subject an agent that promotes Pacsin2 expression and/or activity (e.g., an isolated nucleic acid encoding Pacsin2) at the blood-CNS barrier. 100631 In some embodiments, the inhibitor of Cede 141 signaling is a Cede 141 inhibitor. In some embodiments, inhibition of Cede 141 in CNS endothelial cells results in increased blood-CNS barrier permeability.

100641 In some embodiments, the Cede 141 inhibitor is capable of inhibiting Cede 141 expression and/or activity. In some embodiments, the Cede 141 inhibitor is an inhibitory nucleic acid targeting Cede 141 mRNA. As described herein, an inhibitory nucleic acid, refers to nucleic acids capable of inhibiting expression or activity of a target gene (e.g., DNA, RNA, or protein of the target gene), for example, CCDC141. Non- limiting examples of inhibitory nucleic acids include e.g., dsRNA, siRNA, shRNA, miRNA, amiRNA, antisense oligonucleotides (ASOs), DNA or RNA aptamers, et cetera An ASO is a small chain of nucleotides, generally 18-30 nucleotides long, that targets messenger RNA (mRNA) and is capable of altering mRNA expression through a variety of mechanisms, including ribonuclease H mediated decay of the pre-mRNA, direct steric blockage, and exon content modulation through splicing site binding on pre-mRNA. A small interfering RNA (siRNA), also known as short interfering RNA or silencing RNA, is a double-stranded non-coding RNA molecules, typically 20-27 base pairs in length, which interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation. A short hairpin RNA or small hairpin RNA (shRNA) is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). A microRNA (abbreviated miRNA) is a small single- stranded non-coding RNA molecule (containing about 22 nucleotides) that functions in RNA silencing and post-transcriptional regulation of gene expression via base-pairing with complementary sequences within mRNA molecules. An amiRNA is an artificial miRNA. A mixmer is an oligomer consisting of alternating short stretch of LNA and DNA. An LNA, or Locked Nucleic Acid, also known as bridged nucleic acid (BNA), or inaccessible RNA, is a modified RNA nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon. Aptamers are short sequences of artificial DNA or RNA that bind a specific target molecule.

100651 In some embodiments, an inhibitory nucleic acid targeting Cede 141 is an siRNA. siRNA molecules comprise a specific antisense sequence in addition to the reverse complement (sense) sequence.

100661 The specificity of siRNA molecules may be determined by the binding of the antisense strand of the molecule to its target RNA (e.g., Cede 141 mRNA). In some embodiments, the siRNA molecules are 60, 65, 70, 75, 80, 85, 90, 95, 100, or more base pairs in length. In some embodiments, the antisense sequence of the siRNA molecules is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more base pairs in length. In some embodiments, the antisense sequence of the siRNA molecules are 8 to 30 base pairs in length, 10 to 15 base pairs in length, 10 to 20 base pairs in length, 15 to 25 base pairs in length, 19 to 21 base pairs in length, or 21 to 23 base pairs in length.

[00671 Following selection of an appropriate target RNA sequence, siRNA molecules that comprise a nucleotide sequence complementary to all or a portion of the target sequence, i.e., an antisense sequence, can be designed and prepared using methods known in the art.

[00681 In some embodiments, the antisense sequence of the siRNA molecule is 7, 8, 9, 10,

11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more nucleotides in length. In some embodiments, the antisense sequence is 8 to 50 nucleotides in length, 8 to 40 nucleotides in length, 8 to 30 nucleotides in length, 10 to 15 nucleotides in length, 10 to 20 nucleotides in length, 15 to 25 nucleotides in length, 19 to 21 nucleotides in length, or 21 to 23 nucleotides in lengths.

[00691 In some embodiments, the sense sequence of the siRNA molecule is 7, 8, 9, 10, 11,

12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more nucleotides in length. In some embodiments, the sense sequence is 8 to 50 nucleotides in length, 8 to 40 nucleotides in length, 8 to 30 nucleotides in length, 10 to 15 nucleotides in length, 10 to 20 nucleotides in length, 15 to 25 nucleotides in length, 19 to 21 nucleotides in length, or 21 to 23 nucleotides in lengths.

[00701 In some embodiments, siRNA molecules comprise an antisense sequence comprising a region of complementarity to a target region in a Cede 141 mRNA (e.g., human Cede 141 mRNA (SEQ ID NO: 1) or mouse Ccdcl41 mRNA (SEQ ID NO: 2)). In some embodiments, the region of complementarity is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to a target region in a Cede 141 mRNA (e.g., human Cede 141 mRNA or mouse Cede 141 mRNA). In some embodiments, the target region is a region of consecutive nucleotides in a CCDC141 mRNA (e.g., human CCDC141 mRNA or mouse Cede 141 mRNA). In some embodiments, a complementary nucleotide sequence need not be 100% complementary to that of its target to be specifically hybridizable or specific for a Cede 141 mRNA (e.g., human Cede 141 mRNA or mouse Cede 141 mRNA). Exemplary human CCDC141 mRNA and mouse Cede 141 mRNA sequences are set forth in SEQ ID NOs: 1 and 2.

[00711 In some embodiments, siRNA molecules comprise an antisense sequence that comprises a region of complementarity to in a Ccdcl41 mRNA (e.g., human Ccdcl41 mRNA or mouse Cede 141 mRNA) sequence and the region of complementarity is in the range of 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 nucleotides in length. In some embodiments, the region of complementarity is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,

44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the region of complementarity is complementary to at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of a Cede 141 mRNA (e.g., human Cede 141 mRNA (SEQ ID NO: 1) or mouse Cede 141 mRNA (SEQ ID NO: 2)). In some embodiments, the region of complementarity comprises a nucleotide sequence that contains no more than 1, 2, 3, 4, or 5 base mismatches compared to the complementary portion of a Cede 141 mRNA (e.g., human

Cede 141 mRNA or mouse Cede 141 mRNA). In some embodiments, the region of complementarity comprises a nucleotide sequence that has up to 3 mismatches over 15 bases, up to 2 mismatches over 10 bases, or up to 1 mismatch over 5 bases.

[00721 Exemplary Cede 141 target sequences are set forth below:

GGTAAGAATGGCATTAAATGGATCA (SEQ ID NO: 9)

GAATGGCATTAAATGGATCACACCT (SEQ ID NO: 10)

TGGCATTAAATGGATCACACCTTCT (SEQ ID NO: 11)

GGCATTAAATGGATCACACCTTCTG (SEQ ID NO: 12)

CATTAAATGGATCACACCTTCTGCA (SEQ ID NO: 13)

TCACACCTTCTGCAGGGATACTCAT (SEQ ID NO: 14)

ACACCTTCTGCAGGGATACTCATCA (SEQ ID NO: 15)

CACCTTCTGCAGGGATACTCATCAA (SEQ ID NO: 16)

CCTTCTGCAGGGATACTCATCAAAG (SEQ ID NO: 17)

TCTGCAGGGATACTCATCAAAGTCA (SEQ ID NO: 18)

GGTAAGAATGGCATTAAATGG (SEQ ID NO: 19)

GGCATTAAATGGATCACACCT (SEQ ID NO: 20)

GCAGGGATACTCATCAAAGTC (SEQ ID NO: 21)

[00731 In some embodiments, siRNA molecules targeting Cede 141 comprise an antisense strand which comprises a region of complementarity that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the Cede 141 target sequences as set forth in any one of SEQ ID NOs: 9- 21. In some embodiments, siRNA molecules targeting Cede 141 comprise an antisense strand which comprises a region of complementarity that is complementary to at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides sequences as set forth in any one of SEQ ID NOs: 9-21.

[00741 In some embodiments, siRNA molecules targeting Cede 141 comprise a sense strand which comprises a nucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the sequences as set forth in SEQ ID NOs: 9-18. In some embodiments, siRNA molecules targeting Cede 141 comprise a sense strand at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of the sequence as set forth in SEQ ID NOs: 9-21.

[00751 In some embodiments, an inhibitory nucleic acid targeting Cede 141 is an shRNA. shRNA molecules comprise a specific antisense sequence in addition to the reverse complement (sense) sequence, typically separated by a spacer or loop sequence. Cleavage of the spacer or loop provides a single-stranded RNA molecule and its reverse complement, such that they may anneal to form a dsRNA molecule (optionally with additional processing steps that may result in the addition or removal of one, two, three, or more nucleotides from the 3' end and/or (e.g., and) the 5' end of either or both strands). A spacer can be of a sufficient length to permit the antisense and sense sequences to anneal and form a doublestranded structure (or stem) prior to cleavage of the spacer (and, optionally, subsequent processing steps that may result in the addition or removal of one, two, three, four, or more nucleotides from the 3' end and/or (e.g., and) the 5' end of either or both strands). A spacer sequence may be an unrelated nucleotide sequence that is situated between two complementary nucleotide sequence regions which, when annealed into a double-stranded nucleic acid, comprise a shRNA.

[00761 The specificity of shRNA molecules may be determined by the binding of the antisense strand of the molecule to its target RNA sequence (e.g., Cede 141 mRNA; SEQ ID NO: 1 or SEQ ID NO: 2). In some embodiments, the shRNA molecules are 60, 65, 70, 75, 80, 85, 90, 95, 100 or more base pairs in length. In some embodiments, the antisense sequence of the shRNA molecules is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more base pairs in length. In some embodiments, the antisense sequence of the shRNA molecules are 8 to 30 base pairs in length, 10 to 15 base pairs in length, 10 to 20 base pairs in length, 15 to 25 base pairs in length, 19 to 21 base pairs in length, or 21 to 23 base pairs in length. [00771 Following selection of an appropriate target RNA sequence, shRNA molecules that comprise a nucleotide sequence complementary to all or a portion of the target sequence, i.e., an antisense sequence, can be designed and prepared using methods known in the art (see, e.g., Moore et al., Short Hairpin RNA (shRNA): Design, Delivery, and Assessment of Gene Knockdown, Methods Mol Biol. 2010; 629: 141-158).

[00781 The shRNA molecules can comprise a hairpin i.e., when two regions of the same strand, usually complementary in nucleotide sequence when read in opposite directions, basepair to form a double helix that ends in an unpaired loop), or asymmetric hairpin (i.e., hairpin with a strand overhang) secondary structure, having self-complementary sense and antisense strands. In some embodiments, the shRNA targeting inhibitory nucleic acid described herein comprises an antisense sequence and a sense sequence.

[00791 In some embodiments, the antisense sequence of the shRNA molecule is 7, 8, 9, 10,

11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more nucleotides in length. In some embodiments, the antisense sequence is 8 to 50 nucleotides in length, 8 to 40 nucleotides in length, 8 to 30 nucleotides in length, 10 to 15 nucleotides in length, 10 to 20 nucleotides in length, 15 to 25 nucleotides in length, 19 to 21 nucleotides in length, or 21 to 23 nucleotides in lengths.

[00801 In some embodiments, the sense sequence of the shRNA molecule is 7, 8, 9, 10, 11,

12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more nucleotides in length. In some embodiments, the sense sequence is 8 to 50 nucleotides in length, 8 to 40 nucleotides in length, 8 to 30 nucleotides in length, 10 to 15 nucleotides in length, 10 to 20 nucleotides in length, 15 to 25 nucleotides in length, 19 to 21 nucleotides in length, or 21 to 23 nucleotides in lengths.

[00811 In some embodiments, shRNA molecules comprise an antisense sequence comprising a region of complementarity to a target region in a Cede 141 mRNA (e.g., human Cede 141 mRNA or mouse Cede 141 mRNA). In some embodiments, the region of complementarity is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target region in a Ccdcl41 mRNA (e.g., human Ccdcl41 mRNA or mouse Ccdcl41 mRNA) of SEQ ID NOs: 9-21. In some embodiments, the target region is a region of consecutive nucleotides in a Cede 141 mRNA (e.g., human Cede 141 mRNA or mouse Cede 141 mRNA). In some embodiments, a complementary nucleotide sequence need not be 100% complementary to that of its target to be specifically hybridizable or specific for a Ccdcl41 mRNA (e.g., human Ccdcl41 mRNA or mouse Cede 141 mRNA).

100821 In some embodiments, shRNA molecules comprise an antisense sequence that comprises a region of complementarity in a Cede 141 mRNA (e.g., human Cede 141 mRNA or mouse Cede 141 mRNA) sequence and the region of complementarity is in the range of 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 nucleotides in length. In some embodiments, the region of complementarity is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the region of complementarity is complementary to at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of a Cede 141 mRNA (e.g., human Cede 141 mRNA or mouse Cede 141 mRNA). In some embodiments, the region of complementarity comprises a nucleotide sequence that contains no more than 1, 2, 3, 4, or 5 base mismatches compared to the complementary portion of a Cede 141 mRNA (e.g., human Cede 141 mRNA or mouse Cede 141 mRNA). In some embodiments, the region of complementarity comprises a nucleotide sequence that has up to 3 mismatches over 15 bases, up to 2 mismatches over 10 bases, or up to 1 mismatch over 5 bases.

[00831 In some embodiments, shRNA molecules targeting Cede 141 comprises an antisense strand comprising a region of complementarity that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% complement to the sequences as set forth in any one of SEQ ID NOs: 9-21. In some embodiments, shRNA molecules targeting Cede 141 comprise an antisense strand comprising a region of complementarity that is complementary to at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of the sequences as set forth in any one of SEQ ID NOs: 9-21. 100841 In some embodiments, the inhibitory nucleic acids targeting Cede 141 can be administered to the subject using any suitable known method, for example, but not limited to, direct injection, viral vector mediated delivery (e.g., AAV, retrovirus, adeno virus, or lentivirus), or ceDNA. In some embodiments, the Cede 141 inhibitor is an antibody, an antibody variant or an antigen-binding fragment thereof targeting Cede 141. An antibody: As used herein, the term “antibody” refers to a polypeptide that includes at least one immunoglobulin variable domain or at least one antigenic determinant, e.g. , paratope that specifically binds to an antigen. In some embodiments, an antibody is a full-length antibody. In some embodiments, an antibody is a chimeric antibody. In some embodiments, an antibody is a humanized antibody. However, in some embodiments, an antibody is a Fab fragment, a F(ab')2 fragment, a Fv fragment or a scFv fragment. In some embodiments, an antibody is a nanobody derived from a camelid antibody or a nanobody derived from shark antibody. In some embodiments, an antibody is a diabody. In some embodiments, an antibody comprises a framework having a human germline sequence. In another embodiment, an antibody comprises a heavy chain constant domain selected from the group consisting of IgG, IgGl, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgAl, IgA2, IgD, IgM, and IgE constant domains. In some embodiments, an antibody comprises a heavy (H) chain variable region (abbreviated herein as VH), and/or a light (L) chain variable region (abbreviated herein as VL). In some embodiments, an antibody comprises a constant domain, e.g., an Fc region. An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences and their functional variations are known. With respect to the heavy chain, in some embodiments, the heavy chain of an antibody described herein can be an alpha (a), delta (A), epsilon (E), gamma (y) or mu (p) heavy chain. In some embodiments, the heavy chain of an antibody described herein can comprise a human alpha (a), delta (A), epsilon (E), gamma (y) or mu (p) heavy chain. In a particular embodiment, an antibody described herein comprises a human gamma 1 CHI, CH2, and/or CH3 domain. In some embodiments, the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (y) heavy chain constant region, such as any known in the art. Non-limiting examples of human constant region sequences have been described in the art, e.g., see U.S. Pat. No. 5,693,780 and Kabat E A et al., (1991) NIH publication no. 91-3242. In some embodiments, the VH domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein. In some embodiments, an antibody is modified, e.g., modified via glycosylation, phosphorylation, sumoylation, and/or methylation. In some embodiments, an antibody is a glycosylated antibody, which is conjugated to one or more sugar or carbohydrate molecules. In some embodiments, the one or more sugar or carbohydrate molecule are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and/or phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecule are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecule is a branched oligosaccharide or a branched glycan. In some embodiments, the one or more sugar or carbohydrate molecule includes a mannose unit, a glucose unit, an N-acetylglucosamine unit, or a phospholipid unit. In some embodiments, an antibody is a construct that comprises a polypeptide comprising one or more antigen binding fragments of the disclosure linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Examples of linker polypeptides have been reported (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123). Still further, an antibody may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal poly histidine tag to make bivalent and biotinylated scFv molecules, Mol. Immunol. 31: 1047-1058). Antibodies, antibody variants and antigen-binding fragments targeting Ccdcl41 have been previously described, see, e.g., PA5-21169, PA5-116237 from Invitrogen, DPABH- 17228, DPABH-24009, DPAB-L20267 from Creative Diagnostics, 6399 from ProSci Inc. Non-limiting examples of anti-Ccdcl41 antibody include LS-B9522 from LS Bio; CAT#: AP50767PU-N, CAT#: TA320162, CAT#: TA334850 from Origene.

100851 In some embodiments, administration of an effective amount of any one of the Cede 141 inhibitors described herein or a combination thereof results in increased blood-CNS barrier permeability.

100861 In some embodiments, an agent that promotes Pacsin2 expression and/or activity at the blood-CNS barrier is an isolated nucleic acid encoding Pacsin2. In some embodiments, promoting expression/activity in CNS endothelial cells results in increased blood-CNS barrier permeability.

100871 In some embodiments, an agent that promotes the Pacsin2 signaling at the blood-CNS barrier is a recombinant human Pacsin2 or a fragment thereof. In some embodiments, the present disclosure provides a recombinant human Pacsin2 comprises an amino acid sequence. An exemplary human Pacsin2 amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the amino acid sequence as set forth in SEQ ID NO: 7 (NP_001171899.1). In some embodiments, an exemplary human Pacsin2 amino acid sequence is set forth in SEQ ID NO: 7 (NP_001171899.1): MSVTYDDSVGVEVSSDSFWEVGNYKRTVKRIDDGHRLCSDLMNCLHERARIEKAYAQQLT EW ARRWRQLVEKGPQYGTVEKAWMAFMSEAERVSELHLEVKASLMNDDFEKIKNWQKEAFHK QM MGGFKETKEAEDGFRKAQKPWAKKLKEVEAAKKAHHAACKEEKLAISREANSKADPSLNP EQ LKKLQDKIEKCKQDVLKTKEKYEKSLKELDQGTPQYMENMEQVFEQCQQFEEKRLRFFRE VL LEVQKHLDLSNVAGYKAI YHDLEQSIRAADAVEDLRWFRANHGPGMAMNWPQFEEWSADLNR TLSRREKKKATDGVTLTGINQTGDQSLPSKPSSTLNVPSNPAQSAQSQSSYNPFEDEDDT GS TVSEKDDTKAKNVSSYEKTQSYPTDWSDDESNNPFSSTDANGDSNPFDDDATSGTEVRVR AL YDYEGQEHDELSFKAGDELTKMEDEDEQGWCKGRLDNGQVGLYPANYVEAIQ

100881 In some embodiments, an agent that promotes the Pacsin2 signaling at the blood-CNS barrier is a recombinant mouse Pacsin2 or a fragment thereof. In some embodiments, the recombinant mouse Pacsin2 comprises an amino acid sequence. An exemplary mouse Pacsin2 amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the amino acid sequence as set forth in SEQ ID NO: 8 (NP_001152981.1). In some embodiments, an exemplary mouse Pacsin2 amino acid sequence is set forth in SEQ ID NO: 8

(NP_001152981.1):

MSVTYDDSVGVEVSSDSFWEVGNYKRTVKRIDDGHRLCGDLMNCLHERARIEKAYAQ QLTEW ARRWRQLVEKGPQYGTVEKAWIAVMSEAERVSELHLEVKASLMNEDFEKIKNWQKEAFHK QM MGGFKETKEAEDGFRKAQKPWAKKLKEVEAAKKAHHTACKEEKLAISREANSKADPSLNP EQ LKKLQDKIEKCKQDVLKTKDKYEKSLKELDQTTPQYMENMEQVFEQCQQFEEKRLRFFRE VL LEVQKHLDLSNVASYKTI YRELEQSIKAADAVEDLRWFRANHGPGMAMNWPQFEEWSADLNR TLSRREKKKAVDGVTLTGINQTGDQSGQNKPGSNLSVPSNPAQSTQLQSSYNPFEDEDDT GS SISEKEDIKAKNVSSYEKTQTYPTDWSDDESNNPFSSTDANGDSNPFDEDTTSGTEVRVR AL YDYEGQEHDELSFKAGDELTKIEDEDEQGWCKGRLDSGQVGLYPANYVEAIQ

100891 In some embodiments, an agent that promotes the Pacsin2 signaling at the blood-CNS barrier is a nucleic acid encoding Pacsin2 (e.g., human or mouse Pacsin2). In some embodiments, the nucleic acid encoding Pacsin2 encodes a mammalian Pacsin2 (e.g., human Pacsin2, mouse Pacsin2, rat Pacsin2, non-human primate Pacsin2, etc.). In some embodiments, the nucleic acid encoding Pacsin2 encodes a human Pacsin2. In some embodiments, the nucleic acid encoding Pacsin2 encodes a mouse Pacsin2.

100901 Human Pacsin2 mRNA sequence is set forth in SEQ ID NO: 5 (NM_001184970.3). In some embodiments, the nucleic acid encoding human Pacsin2 comprises a nucleic acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the nucleotide sequence of SEQ ID NO:

5. The nucleotide sequence encoding human Pacsin2 includes NM_001184970.3. An exemplary nucleotide sequence encoding human Pacsin2 protein is set forth in SEQ ID NO: 5 (NM_001184970.3):

AGAAGCGGCCGCAGGGGTCTGGGCAGGGCTGGGCAGTGCTGCCGGAGCAAAAGCGGT AGCGG GAGCCCGGCCGGAGCTGGGTCTGGAGACGCCGTGGCAGCCTGAACGGAGTGTGCGACGGA TT GGGAGGTTTGTCTACAGATTTTGAGCGTTCGAAGTTGACCCCTGACTAAGTATACTTTGC TG CTCCCTCAGCCTTTGAAAAAATGTCTGTCACATATGATGATTCCGTTGGAGTAGAAGTGT CC AGCGACAGCTTCTGGGAGGTCGGGAACTACAAGCGGACTGTGAAGCGGATCGACGATGGC CA CCGCCTGTGCAGCGACCTCATGAACTGCCTGCATGAGCGGGCGCGCATCGAGAAGGCGTA TG CGCAGCAGCTCACTGAGTGGGCCCGGCGCTGGAGGCAGCTCGTGGAGAAAGGGCCCCAGT AC GGGACCGTGGAGAAGGCCTGGATGGCCTTCATGTCCGAGGCAGAGAGGGTGAGCGAGCTG CA CCTCGAGGTGAAGGCCTCACTGATGAACGATGACTTCGAGAAGATCAAGAACTGGCAGAA GG AAGCCTTTCACAAGCAGATGATGGGCGGCTTCAAGGAGACCAAGGAAGCTGAGGACGGCT TT CGGAAGGCACAGAAGCCCTGGGCCAAGAAGCTGAAAGAGGTAGAAGCAGCAAAGAAAGCC CA CCATGCAGCGTGCAAAGAGGAGAAGCTGGCTATCTCACGAGAAGCCAACAGCAAGGCAGA CC CATCCCTCAACCCTGAACAGCTCAAGAAATTGCAAGACAAAATAGAAAAGTGCAAGCAAG AT GTTCTTAAGACCAAAGAGAAGTATGAGAAGTCCCTGAAGGAACTCGACCAGGGCACACCC CA GTACATGGAGAACATGGAGCAGGTGTTTGAGCAGTGCCAGCAGTTCGAGGAGAAACGCCT TC GCTTCTTCCGGGAGGTTCTGCTGGAGGTTCAGAAGCACCTAGACCTGTCCAATGTGGCTG GC TACAAAGCCATTTACCATGACCTGGAGCAGAGCATCAGAGCAGCTGATGCAGTGGAGGAC CT GAGGTGGTTCCGAGCCAATCACGGGCCGGGCATGGCCATGAACTGGCCGCAGTTTGAGGA GT GGTCCGCAGACCTGAATCGAACCCTCAGCCGGAGAGAGAAGAAGAAGGCCACTGACGGCG TC ACCCTGACGGGCATCAACCAGACAGGCGACCAGTCTCTGCCGAGTAAGCCCAGCAGCACC CT TAATGTCCCGAGCAACCCCGCCCAGTCTGCGCAGTCACAGTCCAGCTACAACCCCTTCGA GG ATGAGGACGACACGGGCAGCACCGTCAGTGAGAAGGACGACACTAAGGCCAAAAATGTGA GC AGCTACGAGAAGACCCAGAGCTATCCCACCGACTGGTCAGACGATGAGTCTAACAACCCC TT CTCCTCCACGGATGCCAATGGGGACTCGAATCCATTCGACGACGACGCCACCTCGGGGAC GG AAGTGCGAGTCCGGGCCCTGTATGACTATGAGGGGCAGGAGCATGATGAGCTGAGCTTCA AG GCTGGGGATGAGCTGACCAAGATGGAGGACGAGGATGAGCAGGGCTGGTGCAAGGGACGC TT GGACAACGGGCAAGTTGGCCTATACCCGGCAAATTATGTGGAGGCGATCCAGTGATGAGT CG GGGACAGGCCAGCGGGGGGACGGAGGCGGCGGGCCCAGGAGCCTCAGCCAGCCACGTGGG CA TCCACTCCTTTTCCTGCAAGAGATGATGGTTCCATTGCTCTTGGCTTCATGGTGTTCCTG GA AGGCAGATGAGCTGGTCATTTCGCCTGGGACTCGGCACCTTTCCGAGTGCAGCTGGAGGG AT CTGAGCGCAGGAAGACGCAGAACAACAGAAATAGCCGCCCCTCCCCGCCCACTGTGCCTG TT GGCCTATCATAGATCTCTATGTTCTTGACTTTGTCTCTCCTTTCCGAGTCAATGGTGGGT TA CACTGATCTTGTTCCACTGATTACTCTCTCTGACGAGTCCATCACCTGCAACTTAAATGA AC AAGCTTACATCCCATTTTGAGTGAAGATTTTGAGGTTTTTAATTTAAAGGCTGTGTACAG TT ATACTTTTTTATACACCTGTTCATTTCTACTTAAATTATGGCACAGATTGATGCGCACCA GT CTTGAGGAAACGATCTCCCTATTCCCTTACCCTGTTACTCAGCCACGCCGTGTGTAGGCT TA GCCTCAGGTGGCAGATGTTTGAGGAAAGGAATTATGCCAGGAAGGTGGGACCGGGTTATG GT CGGGTTTCTATTGGGAATGCTCTTTGTGCTTTTGGGCATCTGAATGGAAGCTTTACATAG AA CCTTAGGTAGAACTCCCCCAAATCGCCATATTTAAAAATTATTTTCACTCTATTCTTGCT TA AAAC TGTACTCTTTTG C AAAT TAACAATTTTATCACTGATTCAGAGT T AAAAAG AAG AC T AA CTTTTCAAGCAAATGCATCTGTAAAGATGCTTTAGATTAGACTGTCATGTCTCAGTGTCT AT CTGTATATATTATTTGATATT C AG AG AAT C T AAAG CACTCGTCTACTGTTTTAATGAGATTT AACAGCTTTTAACAGTGAGTTTCGTTTGTAAACTGCTTGAAGTCTGTGGCATTCAGGCAC AC GTCTGGCTGGCCGGCTGGGTCTCCTCCCGGGCTCAGTGGGCCTGGGGCCTCTCTGACGTG GT GCCTGCTGGAGGGAGGCTCGTCGTCACCAGCTGACTGCTGGTCCGGCTTCTGACCGGCCT TT GTCCTGGCTCCGTAGCAGAACACTGTAAAAGTGCCCGCGTCTTTGCAGTAGTTGCAGATT TC AGTCGTCGTGTTACTTGTGCACAAACAGAAGCTGGGTCTTACCCGCAGCACGAGTGTCTC GG GCTGCCCGGAGTCGCCCGGGAGCAGGTGCTGCAGCCAGAGTTACGCGGGGGCCACGCGGG CC GGCGGGGGTGGGGGGAACGTGGGGGAACCTGTGTTTCACGTGACTCAGCAGTGCCCGCCG CC GTCACCAGCTATGCATTCACTCCGTTTCCAGTGAGCAGATGTCTTGCTTGGAAAGTGGAC CT GTGTCTGTGTCTGTCCTGAGAACTTACCAGCAGAAATCCTCATTTCTGTGCTACGGATTT AC CAAAAATTGTCAAGTCTTTTTCAGTTTAACAGTTCCTTTACATGTGTAGTATTTGAGGAA AA AAAT C AAT AAAC AG T T G AT C T C G T G C A

[00911 Mouse Pacsin2 mRNA sequence is set forth in SEQ ID NO: 6 (NM_001159509.1). In some embodiments, the nucleic acid encoding mouse Pacsin2 comprises a nucleic acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the nucleotide sequence of SEQ ID NO: 6. An exemplary nucleotide sequence encoding mouse Pacsin2 protein is set forth in SEQ ID NO: 6 (NM_001159509.1):

GCGGCGCGGTCGCGGCTGGCGGCAGAAGCGGCTGCGGCGGGTCTGGGCAGGGCCGGG GAGCT CGGTTGGAGCGGAACGGGAAGGGGTCGGAGTCGCAGCCCGGCCGGGGCGGCGTGCTCAGT GG GACCCGAGGCGAACGGCGGCCGAGGTCTGTCTGCAGGTTTTACACATCTGACCCCTGAAC TG GAGTGTCTGCTGCCACCCCCTCGTCCTCTGCAGAAATGTCTGTCACCTACGATGACTCTG TG GGAGTGGAAGTGTCCAGCGACAGCTTCTGGGAGGTTGGGAACTACAAACGGACTGTGAAG CG GATTGACGATGGCCACCGCCTGTGTGGTGACCTCATGAACTGTCTGCATGAGCGGGCACG CA TCGAGAAGGCGTATGCACAGCAGCTCACTGAGTGGGCCCGACGCTGGAGGCAGCTGGTAG AG AAGGGACCACAGTATGGGACCGTGGAGAAGGCCTGGATAGCTGTCATGTCTGAAGCAGAG AG GGTGAGTGAACTGCACCTGGAAGTGAAGGCATCACTGATGAATGAAGACTTTGAGAAGAT CA AGAACTGGCAGAAGGAAGCCTTTCACAAGCAGATGATGGGAGGCTTCAAGGAGACCAAAG AA GCAGAGGATGGCTTTCGGAAGGCCCAGAAGCCCTGGGCCAAGAAGCTGAAAGAGGTGGAA GC GGCAAAGAAGGCGCACCACACAGCGTGCAAAGAGGAGAAGCTGGCCATCTCCCGGGAAGC CA ACAGCAAGGCAGATCCATCCCTCAACCCTGAGCAGCTGAAGAAACTGCAAGACAAGATAG AA AAATGCAAACAGGACGTTCTAAAGACCAAGGACAAGTATGAGAAGTCCCTGAAGGAGCTT GA TCAGACCACACCCCAGTACATGGAGAACATGGAGCAGGTGTTCGAGCAGTGCCAGCAGTT TG AAGAGAAGCGCCTGCGCTTCTTCCGGGAGGTTCTGCTGGAGGTTCAGAAGCACTTGGATC TG TCCAATGTGGCTAGCTATAAAACCATTTACCGGGAGCTGGAGCAGAGCATCAAAGCAGCA GA TGCGGTAGAGGACCTGAGGTGGTTCCGGGCTAACCATGGGCCAGGCATGGCTATGAACTG GC CACAGTTTGAGGAGTGGTCTGCAGATCTGAATCGAACTCTCAGCCGGAGAGAGAAGAAGA AG GCTGTTGACGGTGTCACCCTAACAGGGATCAACCAGACAGGTGACCAGTCTGGACAGAAC AA GCCTGGCAGCAACCTTAGTGTCCCGAGCAACCCCGCCCAGTCCACGCAGTTACAGTCCAG CT ACAACCCCTTCGAGGACGAGGACGACACGGGCAGCAGCATCAGTGAGAAGGAGGACATTA AG GCCAAAAATGTCAGCAGCTATGAGAAGACTCAGACTTACCCCACTGACTGGTCTGATGAT GA GTCTAACAACCCTTTCTCCTCCACGGATGCCAACGGGGATTCGAACCCATTTGATGAGGA CA CGACCTCAGGAACAGAAGTGCGAGTTCGGGCCCTCTATGACTATGAGGGGCAGGAACATG AT GAGCTGAGCTTCAAGGCTGGGGATGAACTGACCAAGATAGAGGATGAAGATGAACAGGGT TG GTGCAAGGGACGTTTAGACAGCGGCCAGGTTGGCCTATACCCAGCCAACTATGTCGAGGC TA TCCAGTGACAGCCCATGGGCAGGCTGGCGGAGAGACGGAAATGGGCAGTTCAGGAGCTCC GT TAGCCTTGGCCTGGGCAGTGACACCTCTAGTGCCCCCAGCAGCCATGTAGGCATCCACTC CA CCTGCAAAAGACGATGGCTCTGTTGTTCTTGGCTTCCTGGTGTGCTTTGAAGGCAGATGA GC TGGTGATTTCATTGGGCACTTGGCCCTTTTCCAAGCACATCTGGGCAGATATAGACACAG GA AGATAGGGTCCAACAGCGAGAGCCAGGCCCCTCCCCACCCCCACCAGCTCTCTCTATCAT GG ATCTGCACCTTCTCGCCCTTGTCTCTCCTGAGTCATGACGGGTCATACTGATTCTTGTTC CA CTGATGATTTTCTCTGATGAGGTCCTATCTGCAAGGTCAATGAGCAGACTTACATGCCAT CT TCTGAGTAAAGAGTTTGAGGTTTTAATTTAAAGGCAATGTACAGCTATACTTTTTTATAT GC TCTTCCAGTCAGTTAAATTATGGCCTACACTGATCTGAGATGTTCTCCACGTGAGCTGTC TT CATTTCTCTGTGCTATGTCCAGATGTGGGGTTGCTGCAGCCGGGGTTCTATGGCAAGTGC CA GTTGCAGGGCTAACCTTGTGCAACGTTCCCCAACACTTCCACATACAGAAATTATTTTCA CT CTATCCCTGCTTCAGTTTTTGCAGATTAACAGTTCTATTAGTGATTTGGAAAGTTAACAG TA AGAAGACTAACTTTTCAAAACAGTTGCATCTGTAGATTAAGATGCTTTACATTAGACCGT TG TGTCTCGATGTATATCTGTATATATTATTTGATAAT C AG AAAAT CTATAGAGTTCACCCACT GTTGAATGAGAGCTGGTGGCTTCTGACAGCAGATCTGGTCAACTGCTTGAAGCCCATGGC AT TGAAGCACAGGCACGGCTGGTTAACGGTGCCCACCCAGTTAGGATGTGGCTCTGGCCTCT GA GTGGAGCTGCTGGGAAGACTGATTCTCATTGGCCTGGGCTCCAAGCTCATGACCGAGCAC TG GAAAAGCTCCTAGGACTTGGTAGTAATCGTAGACTTCACAGTCCCTGTGTCACTCACTGG AG AGCTAGAGGGAGGGGTTCGACACCCTCCACCACACACACACACACACACACACACACACA CA CAAGTTCCTCCAGTTGCCCTTGTCCTCAGGTGCAGTGGGACTGTTGTGAGCCCCAGGGAT GG GCACAAAGAGGACTTTTATTTTGTTAGCTCGGACAGTGCAGTGGTGCACATCAGCAACTT GT ATTTCTTCGGTGTTTGGCACGAGCACTGTCTCGCTGTGGCTGTGTGTCATGAGAACTTAC CA GCAGAAATCCTTGTTCCTAAGCTACAGAATGACCAAAAGCTGTCAAGTCCTTAATGTTTA GA AAC T C C T T AAAAT GTATAGTATTT T AG AAC AAC AAC AAC AAAAC T C AAT AAAC AG T T G AT C T TGTGTGTTTGACAGTCCCTTTGCCTTGCATCCACCCTCATGTTCACACAGCTGATAGACC TG TCTCCTTGGGGGTGTACCAGCTTTGAAACTGGGCAAGATCCAGTTCATAGGATCCCTTCT CG GGACCATGTCTCAGTTCCTGCACCAGAGGCTGGGGACTTGGGGCACCTTGGGCATCCCTG AG GAGGGCATCCCTGGTGCCACCCTCTCTCCCCTGACCAACAACCGCTCCTCCCCAAACCTG CG TGTGTTGACCTTTATGTGCCACTGTTCTGTTTTCTCAAGCTGATGATCTCTGGGTGTAAT GT GATCAATGCAGGTCCCAGCTGGTCCTGCTCGCACAAAGAACCTCCAAGGATCTTGCCTCC TA TTGGGATCAGCTAAGGAGATAAAGCCAGTGCGGTAGCCCTGACCAGTCCCGGTGAACGGA CT GCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCT GC TCTTCAACACTAGTAAAGCTTCTCTCTGGCCTTCC

[00921 An effective amount of an agent that increases blood-CNS barrier permeability (e.g., e.g., Cede 141 inhibitor or an agent that promotes Pacsin2 expression/activity) may be an amount sufficient to have a therapeutic benefit in a subject, e.g., to extend the lifespan of a subject, to improve and/or reverse in the subject one or more symptoms of disease, or to slow disease progression. The effective amount will depend on a variety of factors such as, for example, the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among subject and tissue. An effective amount may also depend on the inhibitor used.

[00931 In some embodiments, administration of the inhibitors described herein may result in inhibition of Cede 141 signaling of one or more of the CNS endothelial cells. In some embodiments, administration of the inhibitors described herein may result in inhibition of Cede 141 signaling in all of the CNS endothelial cells.

[00941 In some embodiments, administration of an agent promoting Pacsin2 expression/activity described herein may result in increasing of Pacsin2 signaling in one or more of the CNS endothelial cells. In some embodiments, administration of an agent promoting Pacsin2 expression/activity described herein may result in increasing of Pacsin2 signaling in all of the CNS endothelial cells.

100951 An effective amount may also depend on the mode of administration. For example, targeting endothelial cells in the CNS by intravenous administration or subcutaneous injection may require different (e.g., higher, or lower) doses, in some cases, than targeting endothelial cells in the CNS by another method (e.g., local injection to the CNS). In some embodiments, the inhibitor (e.g., Cede 141 inhibitor) described herein is a small molecule and can be taken orally. In some embodiments, an agent promoting Pacsin2 expression/activity is an rAAV encoding Pacsin2.

100961 In some embodiments, administering an inhibitor of Cede 141 signaling decreases the level and/or activity of Cede 141. In some embodiments, administration of an Ccdcl41inhibitor described herein decreases the expression and/or activity of Cede 141 by at least 10% or more, e.g., by 10% or more, 50% or more, 100% or more, 200% or more, 500% or more, or 1000% or more. In some embodiments, administration of an inhibitors described herein results in increasing of the permeability of the blood-CNS barrier in the subject. In some embodiments, administration of an inhibitor described herein results in increasing the permeability of the blood-CNS barrier in the subject by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500% or more compared the permeability of the blood-CNS barrier of the subject prior to administration of the inhibitor.

100971 In some embodiments, administering an agent that promotes Pacsin2 expression/activity (e.g., rAAV encoding Pacsin2). In some embodiments, administration of an agent that promotes Pacsin2 expression/activity (e.g., rAAV encoding Pacsin2) by at least 10% or more, e.g., by 10% or more, 50% or more, 100% or more, 200% or more, 500% or more, or 1000% or more. In some embodiments, administration of an agent that promotes Pacsin2 expression/activity (e.g., rAAV encoding Pacsin2) described herein results in increasing of the permeability of the blood-CNS barrier in the subject. In some embodiments, administration of an agent that promotes Pacsin2 expression/activity (e.g., rAAV encoding Pacsin2) described herein results in increasing the permeability of the blood-CNS barrier in the subject by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, or more compared the permeability of the blood-CNS barrier of the subject prior to administration of the inhibitor. [00981 The permeability of blood-CNS barrier can be measured using any suitable technique or method known in the art, e.g., neuroimaging techniques including dynamic perfusion CT (PCT) and dynamic contrast-enhanced magnetic resonance imaging (DCEMRI), quantification of protein biomarkers (e.g., neuron- specific enolase (NSE), glial fibrillary acidic protein (GFAP), and S 100(3) in the cerebral spinal fluid (CSF), et cetera.

100991 In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having a disease affecting the CNS, e.g., a neurological disease or a condition treated by delivering therapeutic agents suitable for treating the suspected or diagnosed disease to the CNS. Subjects having a disease affecting the CNS can be identified by a physician using current methods of diagnosing such conditions. Symptoms and/or complications of such conditions which characterize these conditions and aid in diagnosis are known in the art and include, but are not limited to, loss of neural function (e.g., lack of coordination, lack of sensation, altered behaviors, inflammation of the CNS, headaches, et cetera). Tests that may aid in a diagnosis of such conditions can include, but are not limited to, CT scan, MRI scan, spinal tap, brain biopsy, nerve biopsy, electroencephalogram (EEG), lumbar puncture, physical examination, nerve conduction studies, and/or blood tests. For some conditions, a family history of the condition (e.g., by genetic analysis), or exposure to risk factors for the condition can also aid in determining if a subject is likely to have the condition or in making a diagnosis.

(01001 In some embodiments, compositions for increasing blood-CNS permeability (e.g., Cede 141 inhibitors or rAAV encoding Pacsin2) described herein can be administered to a subject having or diagnosed as having a disease affecting the CNS. In some embodiments, the methods described herein comprise administering an effective amount of a composition for increasing blood-CNS permeability (e.g., Cede 141 inhibitors or rAAV encoding Pacsin2) described herein or a composition thereof, to a subject in order to alleviate a symptom of a disease affecting the CNS. As used herein, “alleviating a symptom” is ameliorating any condition or symptom associated with the disease affecting the CNS. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique.

(01011 In some embodiments, a composition for increasing blood-CNS permeability (e.g., an inhibitor of Cede 141 signaling or rAAV encoding Pacsin2) can be administered to a subject in need thereof in combination with a therapeutic agent to the central nervous system. In some embodiments, the administration of a composition for increasing blood-CNS permeability (e.g., an inhibitor of Cede 141 signaling or rAAV encoding Pacsin2) at the blood-CNS barrier increases blood-CNS barrier permeability thereby facilitating the delivery of the therapeutic agent to the CNS. The therapeutic agent can be any agent for the treatment of any disease, provided that it is desired that the therapeutic agent reaches the central nervous system. In some embodiments, methods which comprise administering an inhibitor of Cede 141 signaling can further comprise administering a therapeutic agent to the subject. In some embodiments, the present disclosure provides a methods for delivering a therapeutic agent to the CNS, the method comprising administering to the subject (i) a therapeutic agent, and (ii) a composition for increasing blood-CNS permeability (e.g., an inhibitor of Cede 141 signaling or rAAV encoding Pacsin2). In some embodiments, a therapeutic agent and a composition for increasing blood-CNS permeability (e.g., an inhibitor of Cede 141 signaling or rAAV encoding Pacsin2) are administered sequentially (e.g., administration of a composition for increasing blood-CNS permeability prior to administration of the therapeutic agent). In some embodiments, a therapeutic agent and a composition for increasing blood- CNS permeability (e.g., an inhibitor of Cede 141 signaling or rAAV encoding Pacsin2) are administered concurrently (e.g., administration of a composition for increasing blood-CNS permeability and the therapeutic agent at the same time in one formulation or separate formulations). In some embodiments, a therapeutic agent and a composition for increasing blood-CNS permeability (e.g., an inhibitor of Cede 141 signaling or rAAV encoding Pacsin2) are administered at different frequencies (e.g., administration of a composition for increasing blood-CNS permeability once followed by administration of the therapeutic agent multiple times). Non-limiting examples of such therapeutic agents can include, antibiotics, antibodies, anticonvulsant (e.g., gabapentin), chemotherapeutics, anti-inflammatories, neurotransmitters, pain medication (e.g., morphine), peptides, nucleic acids (e.g., RNAi-based therapies), or psychiatric drugs. In some embodiments, a subject in need of increased permeability of the blood-brain barrier is in need of treatment for a condition selected from the group consisting of neuromuscular diseases (e.g., Amyotrophic Lateral Sclerosis (ALS), Ataxia, Cerebral Palsy, Muscular Dystrophy), neurodegenerative disease (e.g., Alzheimer’s Disease, Parkinson's disease, motor neuron disease, Amyotrophic lateral sclerosis, spinal muscular atrophy, spinocerebral ataxia.), brain and nerve tumors (e.g., Chordomas, Craniopharyngiomas, Gangliocytomas, Glomus jugulare, Meningiomas, Pineocytomas, Pituitary adenomas, Schwannomas, Gliomas, Astrocytomas, Ependymomas, Glioblastoma multiforme (GBM), Medulloblastomas, Oligodendrogliomas, Hemangioblastomas, Rhabdoid tumors, medulloblastomas, low-grade astrocytomas (pilocytic), ependymomas, craniopharyngiomas and brainstem gliomas, et cetera), Neurogenetic Diseases (e.g., Ataxia including spinocerebellar ataxias, olivopontocerebellar atrophies, and multiple system degeneration, CAD AS IL (Cerebral Autosomal Dominant Arteriopathy with Sub-cortical Infarcts and Leukoencephalopathy), Charcot-Marie-Tooth disease (hereditary neuropathy), Cognitive disorders (e.g., familial Alzheimer's disease and other familial dementias including frontotemporal dementia, familial Pick’s disease, familial Creutzfeldt- Jakob disease, Familial amyotrophic lateral sclerosis (familial ALS also known as Lou Gehrig's disease), Familial dystonia including Dopa-responsive dystonia, Fragile X and Fragile X associated Tremor Ataxia Syndrome (FXTAS), Hereditary Spastic Paraplegia, Huntington's disease, Leukodystrophy including adrenomyeloneuropathy and cerebro tendinous xanthomatosis, Lysosomal storage disorders including Gaucher, Niemann-Pick, and Fabry diseases, Mitochondrial encephalomyopathies (MELAS syndrome), Mucopolysaccharidoses, Neurofibromatosis, Primary Lateral Sclerosis, Tourette's syndrome, Tuberous sclerosis, Von- Hippel-Lindau disease, Wilsons disease), or neuropsychiatric disorders (e.g., schizophrenia (SZ), bipolar disorder (BD), major depressive disorder (MDD) and attention deficit hyperactivity disorder (ADHD), et cetera). The identity of such CNS therapeutic agents is known in the art and described, e.g., in Ghose et al., J Comb Chem 1999 1:55-68 and Pardridge. NeuroRx 2005 2:3-14; each of which is incorporated by reference herein in its entirety. In some embodiments, a central nervous system therapeutic agent can inhibit the activity and/or expression of a therapeutic target gene associated with a central nervous system disease (e.g., examples of such genes are described below herein), e.g., it can be an inhibitory nucleic acid or an inhibitory antibody reagent.

101021 In some embodiments, the central nervous system therapeutic agent is less than about 1000 kDa in size. In some embodiments, the central nervous system therapeutic agent is less than about 500 kDa in size. In some embodiments, the central nervous system therapeutic reagent is less than about 300 kDa in size. In some embodiments, the central nervous system therapeutic reagent is less than about 200 kDa in size. In some embodiments, the central nervous system therapeutic reagent is less than about 70 kDa in size. In some embodiments, the central nervous system therapeutic reagent is less than about 50 kDa in size. In some embodiments, the central nervous system therapeutic reagent is less than about 20 kDa in size. In some embodiments, the central nervous system therapeutic reagent is less than about 10 kDa in size. In some embodiments, the central nervous system therapeutic reagent is less than about 1 kDa in size. In some embodiments, a CNS therapeutic agent is between 1 and 1000 kDa in size, between 5 and 1000 kDa in size, between 10 and 1000 kDa in size, between 20 and 1000 kDa in size, between 50 and 1000 kDa in size, between 100 and 1000 kDa in size, between 150 and 1000 kDa in size, between 200 and 1000 kDa in size, between 300 and 1000 kDa in size, between 400 and 1000 kDa in size, between 500 and 1000 kDa in size, between 600 and 1000 kDa in size, between 700 and 1000 kDa in size, between 800 and 1000 kDa in size, between 900 and 1000 kDa in size, between 1 and 750 kDa in size, between 5 and 750 kDa in size, between 10 and 750 kDa in size, between 20 and 750 kDa in size, between 50 and 750 kDa in size, between 100 and 750 kDa in size, between 150 and 750 kDa in size, between 200 and 750 kDa in size, between 300 and 750 kDa in size, between 400 and 750 kDa in size, between 500 and 750 kDa in size, between 600 and 750 kDa in size, between 700 and 750 kDa in size, between 1 and 500 kDa in size, between 5 and 500 kDa in size, between 10 and 500 kDa in size, between 20 and 500 kDa in size, between 50 and 500 kDa in size, between 100 and 500 kDa in size, between 150 and 500 kDa in size, between 200 and 500 kDa in size, between 300 and 500 kDa in size, between 400 and 500 kDa in size, between 1 and 250 kDa in size, between 5 and 250 kDa in size, between 10 and 250 kDa in size, between 20 and 250 kDa in size, between 50 and 250 kDa in size, between 100 and 250 kDa in size, between 150 and 250 kDa in size, between 200 and 250 kDa in size, between 300 and 250 kDa in size, between 400 and 250 kDa in size, between 1 and 200 kDa in size, between 5 and 200 kDa in size, between 10 and 200 kDa in size, between 20 and 200 kDa in size, between 50 and 200 kDa in size, between 100 and 200 kDa in size, between 150 and 200 kDa in size, between 5 and 180 kDa in size, between 5 and 160 kDa in size, between 5 and 150 kDa in size, between 5 and 125 kDa in size, between 5 and 120 kDa in size, between 5 and 100 kDa in size, between 5 and 90 kDa in size, between 5 and 80 kDa in size, between 5 and 75 kDa in size, between 5 and 60 kDa in size, between 5 and 50 kDa in size, between 5 and 45 kDa in size, between 5 and 40 kDa in size, between 5 and 30 kDa in size, between 5 and 20 kDa in size, between 5 and 10 kDa in size, between 10 and 200 kDa, between 10 and 180 kDa in size, between 10 and 160 kDa in size, between 10 and 150 kDa in size, between 10 and 125 kDa in size, between 10 and 120 kDa in size, between 10 and 100 kDa in size, between 10 and 90 kDa in size, between 10 and 80 kDa in size, between 10 and 75 kDa in size, between 10 and 60 kDa in size, between 10 and 50 kDa in size, between 10 and 45 kDa in size, between 10 and 40 kDa in size, between 10 and 30 kDa in size, between 10 and 20 kDa in size, between 20 and 200 kDa, between 20 and 180 kDa in size, between 20 and 160 kDa in size, between 20 and 150 kDa in size, between 20 and 125 kDa in size, between 20 and 120 kDa in size, between 20 and 100 kDa in size, between 20 and 90 kDa in size, between 20 and 80 kDa in size, between 20 and 75 kDa in size, between 20 and 60 kDa in size, between 20 and 50 kDa in size, between 20 and 45 kDa in size, between 20 and 40 kDa in size, between 20 and 30 kDa in size, between 30 and 200 kDa, between 30 and 180 kDa in size, between 30 and 160 kDa in size, between 30 and 150 kDa in size, between 30 and 125 kDa in size, between 30 and 120 kDa in size, between 30 and 100 kDa in size, between 30 and 90 kDa in size, between 30 and 80 kDa in size, between 30 and 75 kDa in size, between 30 and 60 kDa in size, between 30 and 50 kDa in size, between 30 and 45 kDa in size, between 30 and 40 kDa in size, between 40 and 200 kDa, between 40 and 180 kDa in size, between 40 and 160 kDa in size, between 40 and 150 kDa in size, between 40 and 125 kDa in size, between 40 and 120 kDa in size, between 40 and 100 kDa in size, between 40 and 90 kDa in size, between 40 and 80 kDa in size, between 40 and 75 kDa in size, between 40 and 60 kDa in size, between 40 and 50 kDa in size, between 40 and 45 kDa in size, between 50 and 200 kDa in size, between 50 and 180 kDa in size, between 50 and 160 kDa in size, between 50 and 150 kDa in size, between 50 and 125 kDa in size, between 50 and 120 kDa in size, between 50 and 100 kDa in size, between 50 and 90 kDa in size, between 50 and 80 kDa in size, between 50 and 75 kDa in size, between 50 and 60 kDa in size, between 75 and 200 kDa in size, between 75 and 180 kDa in size, between 75 and 160 kDa in size, between 75 and 150 kDa in size, between 75 and 125 kDa in size, between 75 and 120 kDa in size, between 75 and 100 kDa in size, between 75 and 90 kDa in size, between 75 and 80 kDa in size, between 100 and 200 kDa in size, between 100 and 120 kDa, between 100 and 125 kDa in size, between 100 and 150 kDa in size, between 100 and 175 kDa in size, between 125 and 200 kDa in size, between 150 and 175 kDa in size, or between 100 and 175 kDa in size.

101031 . In some embodiments, the central nervous system therapeutic agent can be, e.g., a biologic agent (e.g., an enzyme, an antibody, a polypeptide), a sugar, and/or a small molecule. In some embodiments, the CNS therapeutic agent is a therapeutic antibody or fragment thereof. Any suitable therapeutic antibody can be delivered to the CNS using the methods described herein.

101041 In some embodiments, the therapeutic agent is an agent that does not normally cross the blood-CNS barrier. In some embodiments, the CNS therapeutic agent is an agent that inefficiently crosses the blood-CNS barrier, e.g., a therapeutically effective dose of the agent is unable to cross the blood-CNS barrier when administered systemically. In some embodiments, the CNS therapeutic agent is an agent that does efficiently cross the blood- CNS barrier, e.g., a therapeutically effective dose of the agent is able to cross the blood-CNS barrier when administered systemically. Administration of an inhibitor of Cede 141 signaling at the blood-CNS barrier can increase the permeability of the blood-CNS barrier such that, e.g., a therapeutically effective dose of the CNS therapeutic agent is able to reach the CNS, or the necessary dose of the CNS therapeutic agent is lowered.

[01051 Delivery of an inhibitor of Cede 141 signaling at the blood-CNS barrier to a mammalian subject (e.g., human) may be by, for example, injection to the CNS. In some embodiments, the injection is direct injection to the CNS (e.g., intracerebral injection, intraventricular injection, intracisternal injection, intraparenchymal injection, intrathecal injection, and any combination of the foregoing). In some embodiments, the injection is systemic injection (e.g., intravenous injection, intradermal injection, or subcutaneous injection). In some embodiments, the inhibitor can be administered orally.

II. Decreasing blood-CNS Barrier Permeability

101061 In some aspects, the present disclosure also provides methods for decreasing blood- CNS barrier permeability (i.e., maintaining blood-CNS integrity) in a subject. In some embodiments, the method comprises administering to the subject an agent that promotes Cede 141 signaling. In some embodiments, the method comprises administering to the subject an agent that inhibits Pacsin2 signaling at the blood-CNS barrier. In some embodiments, an agent that promotes the Cede 141 signaling decreases expression and/or activity of Pacsin2. 101071 In some embodiments, an agent that promotes the Cede 141 signaling at the blood- CNS barrier is a recombinant Cede 141 or a fragment thereof. In some embodiments, an agent that promotes the Cede 141 signaling at the blood-CNS barrier is a recombinant human Cede 141 or a fragment thereof. In some embodiments, the recombinant human Cede 141 comprises an amino acid sequence. The human Cede 141 amino acid sequence has been described in NM_001316745. An exemplary human Cede 141 amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the amino acid sequence as set forth in SEQ ID NO: 3 (NP_001303674.1). In some embodiments, an exemplary human Ccdcl41 amino acid sequence is set forth in SEQ ID NO: 3 (NP_001303674.1):

MSSQGSPSVALSTTTVSSVAVQAGDSKIVIAVIKCGKWVQLQLAESQPNLLEIGSSQ DETKK LLHDHELLLAKLKALEDRVWELLQEADKTAEENKDQSQVYDAMAETLGEAWAALVSMLER RT ELLRLTSEFFENALEFAIKIDQAEDFLQNTHEFESAESLKSLLQLHEHHTKELLERSLAL LN KSQQLTDFIEKFKCEGPNVNPELTQGAHSSCLKVDRLLELLQDRRRQLDKYLKQQWQELS QV LQICQWDQQENQVTCWFQKTIRNLQEQSLGSSLSDNEDRIHKQEELI IKAKEWNSAVEKLKS EALRILLSKDYVEKEHLQLSHQKLSQLQEEFGQLMVERNTWLKKANEFFNSANKAFDVLG RV EAYLKLLKSEGLSLAVLAVRHEELHRKIKDCTTDALQKGQTLISQVDSCSSQVSGIHEMM GC IKRRVDHLTEQCSAHKEYALKKQQLTASVEGYLRKVEMSIQKISPVLSNAMDVGSTRSES EK ILNKYLELDIQAKETSHELEAAAKTMMEKNEFVSDEMVSLSSKARWLAEELNLFGQSIDY RS QVLQTYVAFLKSSEEVEMQFQSLKEFYETEIPQKEQDDAKAKHCSDSAEKQWQLFLKKSF IT QDLGLEFLNLINMVRMALNGSHLLQGYSSKSK

[01081 In some embodiments, an agent that promotes the Ccdcl41 signaling at the blood- CNS barrier is a recombinant mouse Cede 141 or a fragment thereof. In some embodiments, the recombinant mouse Cede 141 comprises an amino acid sequence An exemplary mouse Cdcl41 amino acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the amino acid sequence as set forth in SEQ ID NO: 4 (NP_001020747.2). In some embodiments, an exemplary mouse Cede 141 amino acid sequence is set forth in SEQ ID NO: 4 (NP_001020747.2):

MSCKESPHVGASTTTVSSVAVHAGDSKIVIAWKCGKWVRLQLAESQPNLLEIGSSQD ETKK LLHDHELLLAKLKALEDRVWELLREADRTAEANKAQSQVYDAMAQTLGEAWATLVSMLER RR ELLGLTSEFFQSALEFAIKIDQAEAFLQNPHEFESTEALQSLLLLHDRHAKELLERSLDL LN KSQQLTDFIEKFKCEGSTMNSELIQGAQSSCLKIDSLLELLQDRRRQLDKYLQQQRQELS QV LQLCLWDQQENQVSSWFQKAIRDLQEQSLGASLSDNRELICKHEDLIVKAKEWDSAVEKL KS QALGILLSKDLAGKEHLQLSNQKLNRLQEEFGRLMVERKAWLSMANDFFTSANKSFDVLG KV EAYLKLLKSEGLSLPVLAAKHEELHREIKDSTATALQKGRTLISQVDSCRSRVTGIHEMM GY IQNRVDCLTEQCTAHEEFARKRQQLATSVDDYLRKVEMSIQEIRPILATTLDVASSPSES EK ILNKYLELDIQVKETAHALEAAAKIMTEKNELELNEVALLPLKVKWLEEELSTLGRSISC RS RILQTYVAFRKSSEEAEEQLQSLKEFYLTEIPWKDEDDAWKCQSNSAERKWQLFLKKSFL T QDLSLEFLNLINMAKENEILNVKNEMHIMENIMEKQTNGREELSHLRVAWYLKAIEGKPA RE QWEMFKEKLTKTTHSVKLLHEVLMPVSALDLGGSLQSTSDLRRRWIAMKPQLQQLHEDVQ QI TKEWEVLSSQGAPLKEKAEQLKDLVHLHRRQRERIQEYEEILYKTVQFHQVKEELVHLIK PR ELELLAQPMELASSEEVQMQLGRSQERRAHVDHLHQLALTLGVDI ISSVQQPNCSNISAKNL QQQLEALELESRSWSAQAKEHERVLSCSLEYCTARDEISELKESFKDIKKKFNNLKFNYS KK NEKSRNLKTLQYQIQQVDTYAEKIQALRKKMEKVNNKTSDSFLSYPSNKANMLSEAMEDL KK NVDDFDKWTDYKMNLDLTEHLQEVIEECNFWYEDASATWRVGKYSMECQTREAVDILHRQ FNKFITPSVPQQEERIQEVIDLAQRLYGLEEGQKYAEKIVTRHKEILESITELCGSLVEL KE KLMQGEVPKMNSDLEDFHDNCIDLLKGPGKDDQKTFSEERNEGQVQGADVLAVNGTREDG LP MDLRRTSSDKEDSAQGLILPEDTLSGEESECISSDDISLPPLPVSPESPLAPSDMEVEEL TS SSALALHISGYRMHPGTGGLGKAQESALPSPIAFADGGHSKKDTFTSHFERPYPQLKAES SL ASRGSAEMSTKLHINVKCPASMPHEVHDKALQQCSQARESTLEMQEKVHADSNVTKTQDR LH AALDVSPGLGSQPDTSESHQRRVGPQGNKKNSSAENSWSLAGQAPHFSRLLSNVTVMEGS P VTLEVEVTGFPEPTLTWFKKGQKLCADGHLQVLHKDTKHSVFIPKVCEADAGLYVAQAQN SS GTLSSKAILHVTGNHGPPITRLNWIMLCI IYVSVSVIYWLLTR

[01091 In some embodiments, an agent that promotes the Ccdcl41 signaling at the blood- CNS barrier is a nucleic acid encoding Cede 141. In some embodiments, the nucleic acid encoding Cede 141 encodes a mammalian Cede 141 (e.g., human Cede 141, mouse Cede 141, rat Cede 141, non-human primate Cede 141, et cetera). In some embodiments, the nucleic acid encoding Cede 141 encodes a human Cede 141. In some embodiments, the nucleic acid encoding Cede 141 encodes a mouse Cede 141.

[01101 Human Ccdcl41 mRNA sequence is set forth in SEQ ID NO: 1 (NM_000638). In some embodiments, the nucleic acid encoding human Cede 141 comprises a nucleic acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the nucleotide sequence of SEQ ID NO: 1. The nucleotide sequence encoding human Cede 141 includes NM_001316745. An exemplary nucleotide sequence encoding human Ccdcl41 protein is set forth in SEQ ID NO:

1 (NM_001316745.2):

GCTCCTTTCTCTCTCACGATAAACTCAATGGGGTTGTCTATTTTTAACCTCCTGTTC TTTCC TTTTGGCAGAGTAATCAGTGTGAGCTGAGCTCTTTCCCTGAATGAATCCAAGTAACCCTG GC CTTCTGAAATGACCAGCAGACACACAACTGTCCAGAGGCTGCCCAAAGTATAAACTCTGG TT CTAAAGTACCATGTCCAGCCAAGGAAGTCCTAGTGTTGCGCTTTCTACGACGACAGTCAG TT CAGTTGCTGTGCAGGCTGGGGACTCCAAAATCGTTATAGCTGTCATAAAGTGTGGCAAAT GG GTACAACTTCAACTGGCTGAATCACAGCCCAATCTTCTAGAAATTGGCAGCAGTCAAGAT GA AACCAAAAAACTTCTTCATGATCATGAACTTCTTTTGGCCAAGCTCAAGGCTTTGGAAGA TC GGGTATGGGAACTCTTGCAGGAAGCAGACAAGACAGCTGAAGAGAACAAGGATCAGAGTC AG GTCTATGATGCCATGGCCGAGACTCTGGGTGAAGCATGGGCAGCTCTGGTGTCCATGCTT GA AAGAAGAACAGAGCTCCTTAGGTTGACTTCTGAATTTTTTGAAAATGCCTTAGAGTTTGC TA TTAAAATAGACCAAGCTGAAGATTTCCTCCAGAATACTCATGAGTTTGAGAGTGCTGAGT CC TTAAAATCACTTCTTCAGCTTCATGAACATCATACTAAAGAACTCTTGGAACGGTCTTTA GC C C T T T T AAAC AAAAG TCAACAACTCACTGACTT C AT AG AAAAAT T C AAG T G T G AAG G AC C T A ATGTGAATCCTGAGTTGACTCAGGGAGCTCATAGCAGCTGTCTGAAGGTTGACCGCCTTC TT GAACTTCTACAAGACAGGAGAAGACAACTAGACAAGTACTTGAAGCAACAGTGGCAAGAA TT GAGTCAAGTTCTGCAGATATGTCAGTGGGACCAACAAGAAAACCAGGTTACTTGTTGGTT TC AGAAAACTATAAGAAATTTACAGGAACAAAGTCTAGGTTCATCACTTTCAGACAATGAGG AT CGAATTCATAAGCAAGAGGAACTGATAATAAAAGCAAAGGAATGGAATTCTGCTGTTGAG AA GCTGAAGAGTGAGGCACTGAGAATTCTGCTGTCAAAGGACTACGTGGAGAAAGAACACCT CC AGCTCTCTCACCAGAAACTCAGTCAGCTTCAAGAAGAATTTGGTCAACTCATGGTGGAAA GG AATACCTGGTTAAAGAAGGCGAATGAATTTTTTAACAGTGCTAACAAGGCATTTGATGTA CT TGGAAGAGTTGAAGCTTACCTTAAGCTCCTTAAATCAGAGGGTTTAAGTCTGGCTGTTTT GG CAGTGAGGCATGAGGAATTACACAGAAAAATTAAAGACTGCACAACTGATGCTTTGCAAA AG GGACAAACCTTAATCAGCCAAGTAGACTCCTGCAGCTCTCAGGTGTCCGGCATCCATGAG AT

GATGGGGTGCATTAAGAGACGAGTGGATCATCTGACCGAACAGTGTTCAGCGCACAA GGAAT ATGCTCTTAAGAAACAACAACTAACAGCCTCAGTGGAGGGTTACCTACGGAAGGTGGAAA TG TCAATTCAGAAAATCAGTCCAGTACTTTCTAATGCAATGGATGTTGGTTCTACCCGTTCT GA AT C AG AG AAG AT T T T G AAT AAAT AT C T G G AAC TAGATATCCAAGCTAAGGAGACATCACATG AATTAGAAGCAGCTGCAAAAACCATGATGGAGAAAAATGAATTTGTATCTGATGAAATGG TA TCACTTTCCTCTAAAGCTAGATGGCTAGCAGAAGAATTAAACCTATTTGGCCAAAGCATT GA CTATAGATCGCAAGTCCTGCAAACTTACGTGGCATTTCTGAAGTCATCAGAGGAGGTAGA GA TGCAGTTTCAGAGCTTAAAAGAATTTTATGAAACCGAAATCCCTCAGAAGGAGCAGGATG AT GCTAAAGCCAAGCATTGTTCTGACTCGGCTGAGAAGCAGTGGCAGCTATTTTTAAAGAAG AG TTTTATAACACAAGATCTAGGGCTTGAGTTCCTTAATTTAATAAATATGGTAAGAATGGC AT TAAATGGATCACACCTTCTGCAGGGATACTCATCAAAGTCAAAATAATAATTTCAAAGGT TC AGCTGTCTATTAATCAGCAGTTCTCTCCTAGGGAGTTCTCTTTAGCTTCATTCAACCTAG AC CAGTCCTGAAGCCCAACATTTAAATGCTTGCTTGGCCTAGAACTTTGGTTAGTTTATAAC AG GAAGGCTGTTTTAACTATTAAGAAGAAAGTGACTGTAATCCAAATGCAACATTGTTATTA TT AAATAGAGTCCTCAGAAGTCCACCCAAAAGTTCTGTGTTATTTTGAAAGCATGCAACAAG GT CCTTTCAAAATTTCTAGGGGCTAAAGACAACTTCTAAGATGAATCTTCTTTTGAAGAGTT TC TTACCATAGCAGATCTCTTTGTAGT G AAG AT CCATTGCATT T AG AAAAAT AC CCTAACCTTT AACTGTCTT G AAT TATATCTG T AAAAT AT T T T AG AAT CCTTATATCCACCCATAACCCATAT AAAATTCCCTGTAGGATACTTAAAAGATAGTCCACTTCTCTTTCAATACCTCAAGTATTT TC CTAATGAAACTGCTCATTTGATTGTTGGAGAATGCCAGCTGCTAGAAACCTTCTTTTTTT AA TGTTAAATATATCTGTTTCCGTATCTACTCCTAATCATCTGTAGCTACACAGGGACAAAA AG TATTCATATTTAATTTCTCTTACACAT G AAAG TCCTTCCAACAT T AAAAAC AG TTGCTATGT CTGAGTTTTTTCTGCAGCATTAAAAATCCTTTCCATCATTTATGAAGGAGCTTTATTTTT CT TTCTATGCTGATCCCCCCTTCTCAGGATTTTCTATGGTTTGTCAAGATCATTCATTAAAT GT AACCAAAAAGTGAACTACAGGTGCAACACAGAGTACAGTAAACTGGACATTATTATTCAC TA ATGAGACCTACAATTATGCTAACATTTTTAGCAGTAGTTTCACTTTGCTGGTACATATTA AG CTTGTGATAGAACAAAACTCTCATAATATTTTTATTGCAGCTTCTGTCAAACCATGTCTC TC CCATATTATACCTGTGTCAGCCATTTACCTTTTTTATACAAATGTGGGCCTTTATATTTT TT CTTATTTAATTTTATTTTGTTAGTCTTCACACATGATCCCATCTTGCTAAACTAATTTGA AC CTACCTAGTATATTAGCCATCTGTCTTGGGTTTAATTCTGTAGATATGACAAGTAAGCTT TC TATGGCAGCAATTAGTTCTTAAATATAAATGCCTAAAGGGCAGATTGGCATTCCAGTTCA TG AAGCAGTCTATTGATTAAACCAGTTTTTAAAATAATCATGAATGCTCCCAATAGTACTTC AT TCAGCCGCTACTTCATAATCTTTGTATTAAGTCTATAATGGGAGACTTTCTCAAATAAAC TG C T C AAAAG AAG GTTTACCTACCTAGTATCTTATCAATGACATAGACAATTT G AC AT AAAT C A GGTTGGCTCCTAGCGATCAAGGCATTCTTTCCAAAATGCTCACAAAGTGCTTGATAAGTA AT GAACTTATTGCACTGATTTTCAGTGTGAAGCTTACCAATCTGTAAATTGTGTAGTCCATC CT TTCCTCCTTTTGAAAATCAGGGCATTTTCTGTTCTCTAATTTTATGGCTCTTCTAGTTTC TG TAGATTTTCAAAGATAACTGACAGCTGCAATTTTCTCTGTACCTGGGATGTGATTAATCT CA GTCTGGAGCCTGAATTCATTTGGAAGAGTTAGGTTCTAACTACCCTCATCTACTTGGAGC TG CAATTTTCTCTTTATCTTGTTTCAACTTTTGTTGTTTGAAGACCATTCTTGATAAACCTA CT T G G AAAT AAAAT AT GCATTAGCTGATTCTCATTTATTCAACATGATTCTTAAGCAC T AAAG A AAACTTGCTAAGCAATTTGCCCATGTTCCTATGTGATCTAGGTGCTTTACTGCAAAAAAA AA GAAAAGTTATTTTTCGTTTAGAACATTTAGAACATGTATTATCTATCTATCTATCTCTGT CT CTCTCTCACACTTCTACGTGGTTCAATCAGTCTTCTCCCATCTGTTGATGATGTATGTTT CT GATTTACCTAAGGGAATTCAAATACTTTCAAACTGCGTAATTTTGTCTACCTTTTTATTA CT AACTTCATTTCAGAAATTTTAGTTCTGGAATCTACAGTCGGTACTTTTATTTATTAAATT AC TGATGAAATTCTGTTTCTTGTCTGCTCTTTTCCCATCTTTCCAATACTTTCTTGAACTAT AT TAACCCTAGGCACTTAAAGTGTTCATCAGCTAGCTACAACATTTGTACCTGGAGCTCTTT CT TTCTTTCTTTCCTTTCTTTTCTTTTTTTTTTAGATGGAGTCTCGCTCTGTCACCCAGACT GG AGTGCAGTGGTGCGATCTCAGCTCCCTTTAACCTCCAGCTCCTGGGTTCAAGCAATTCTC AT GCCTCAGTCCCCCGATTAGCTGGAATTACAGGTGCCTGCCACCATGCCCGGCTAATACTT GT _{ATTTTTAGTAGAGACGAGGTTTCACCATGTTGACCAGGCTGGTCTCAAACTCCTG ACTTCAA} GTGATCTGCCCACCTCTGCCTCCCAAAGTGCTGGGATTACAGGCATGAACCACAGCGCCC GG CCAATCTGGAGCTGTTTCTGTTGTCTAATTTTTCTCTTGGTTTCCAGTCCTGTAGTCCTG TT TCTTGGCAGAACTAGCGAATATTTATCAAATGCCAGACATTATGTATAAAAAATTGGAGG AT CAGCAGATGGTGTTTTCTTCTTCTAGAAAGGGTCACCCTTCTTTCTCCAGGCAGGCATTG GT GGGCAATCACTCAAGCCAATCAGAAGCTGGCAGAGTTGTGTTTGCAGTGTAGTCCTGTCC CC TCAGGCTCCCAGAGTTTTGTATTGAGAGCCAAGTGCTTTCTCTGACGTCTCTGCCCCTGG CA GGTCACAAACTCCAATCTTTGTTTGGGTAGTCTGCTGAAAAACTCCACTCTGCTTTTCAG AG GCCTTTTGCTTAGGTTTTTTAATCCACTGCCCGATATACCCCTGGTATTTTGTCAACGTC TT TAGAAGATCATTGTGCTTGAAGCCCTCCAAGTGTCTGCCAAAATCTCTGGTGTTTTCTCG GC TCCAGGGAAGTCTCGATTATCAGCTTCCTGTCTGCACAGAATTGGGAAATCCCCCTGGGA TG ATAGGTGAGCTCACCCCTGTCAGGTTCTTCCCTCTCTGGAGTCAGCCTCTCTAGTTCCGG TT GTCCGACCAGTTCTCTGCTTCCTTCAAATGGATTCCTTCTGCATTTTACCCAGCTTTTCT AG GTATTCTAAGCAAAAGCACTGCTCTGCCACTGGCTACCCCATTCTACTCAGAAGTCCTTT GT TACTTATTTAGAATGTTTAAGATTGTACAGTCAGATTTTTGTTTTTAAAGATCTGCAACA AT AACTTATTC C AAAT TATATTTTGTATCTATATC T AAAT AAAC TTAGGTTATCATAATATTCA ATGCCCACTTAGATCTCCCAGAAATAGCATATATTTTACTACTGTGCTTTGGTGTTCTTT TA TCTTGATGTCATAAACACGGTCACTCTACCCTAAGGTCTTTACCACTATCATATGATAAA TA AGTTCCTCCTTATTGATAAAATTAAAGCCCACGTACTAAGGAAATTTATTGGTTAGCAAC AT TTGGAGAGATGAAATTGTTGGTAAAGTAAGTAAAACGATTTTTTTTATTCTTATATCTTT TG GACCACTAGGCTTTTAAACAAACATTATTGTTGAATCATGGTGACTAGTTTGCTTGCTAA GA AGGCAGAGGCCAGCTGGTATATTCAGTTGAGTGTTCTGTTCTTTATTGGGTTGTCGTGGC TC ATATTTTCAAGC C AG AAAT AG AAAAAAT TTCAGTTCATC T AAAAAT C AT T AAG AT C AG AG AA ACTGATTCAGGCCCAAATTATGGCAGACATCTTGATGAAAGTGCTAATTTATTAATTTAT AA TGGCCAATACCAAGAATGCTATCTGGGATACAGCCTTCCTATTATCAAGGTATCTGAAAA GC AAGACATCAGAAAACCATTCATGGTGCATGAATATAAACGAAAAATGAATGCTTTAGTGG CC TGTTTAGACTGGATTATGACAAACATTTTCCATGTCCTACACAGAAGTAAAATGAGTACT GT GTTTACTAATTAGACCTCAGGCAAATTATTCAATCTTCTTGAGTGGCCAAAATATTCGTC TG C C AAAT AC AT GATCACTTTTCACAGATCCTACTAGCTAGACATC T AC AAAT CCTACTTTAGC TCTCCTTTCTCTAGACCTCACTCAGACCACCATATCCTTCTGATTAAAGTTGTCATTGGC AT GCTGCAGTTTCAAAATTTATATTAAATTATATAATACATTACATTCTGGTTAATCATTTC TT TAAAATATGTGAAAGATAATTTTATTTATGATAAAAGTATAATTTGGGGTGAATTCTGCT GT AGTTGTCTAATATCTTTCATAATTTATTATGTATCATTATTAATAATGAGGGAATATAAC GT TTTATTACTAGATTCTTAAGTTCATGGGGGAAATCTCATTTAGGACCTACAGATATATAT TT TTTTGGAGTTGCCACTTTATAATGTGTAAAATAACATAAAAATATAAGTGTGCAACATCA AT AT CAT T T AAAAT AAT T T T AAAAT AAAAT GGCCTATCTT T AAAAT AT CCTGTTAAGTATTTAT GATACTGTCAATTGCTGTCATTGAGATGATAGAAATTTGAAGATTTATTGAGAAAGCCTG GC TATTAAAGTAAAAAACATAAGAATTGTTTCGCCAAATGAATGTTCCTGTGCATTTTTTAA AT G T T T C T AC AG AAAT AAAG CACGCATAGTCTTA Olll] Mouse Ccdcl41 mRNA sequence is set forth in SEQ ID NO: 2 (NM_001025576.3).

In some embodiments, the nucleic acid encoding mouse Cede 141 comprises a nucleic acid sequence at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to the nucleotide sequence of SEQ ID NO: 2. Nucleotide sequences encoding mouse Ccdcl41 protein include NM_001025576,

XM_6 19767, XM_886279, XM_896648, XM_896657, XM_896667, XM_896676, XM_896697, XM_896711, XM_896724, XM_912240, XM_922274, XM_922277, XM_922280, XM_922286, XM_922295, XM_922299, XM_922303, XM_922309. An exemplary nucleotide sequence encoding mouse Cede 141 protein is set forth in SEQ ID NO: 2 (NM-001025576.3):

TGTTCTTTCCTTTTGGCCGGGCAATCAGTGTGAACAGAGGCGCGTTCCCTGAAAGAG TCCCG GTAGCCTCGTGGAAGCCTTCTGACATGACCAGCAGACAGCATCACTACCAAGGCTGCTCG GG GTTTAAGCCCTGCTTCTAAAGTGCCATGTCCTGCAAAGAAAGTCCCCATGTGGGTGCTTC CA CCACCACTGTCAGCTCTGTAGCCGTGCACGCTGGGGACTCCAAGATCGTGATCGCCGTTG TC AAGTGTGGCAAATGGGTACGACTCCAACTGGCTGAATCGCAGCCCAATCTCCTCGAAATT GG GAGCAGTCAAGATGAAACCAAAAAACTCCTTCACGATCATGAGCTGCTTCTGGCCAAGCT TA AGGCCTTGGAAGATCGTGTGTGGGAACTCTTACGGGAAGCAGACCGGACAGCTGAAGCAA AC AAGGCTCAAAGTCAGGTGTATGACGCCATGGCCCAGACGCTGGGTGAGGCATGGGCAACC CT GGTCTCCATGCTTGAAAGAAGAAGGGAGCTCCTTGGGCTGACGTCCGAGTTCTTTCAAAG TG CTTTGGAGTTTGCTATAAAAATAGACCAAGCTGAAGCTTTTCTGCAGAATCCTCATGAGT TT GAGAGCACTGAAGCCTTACAGTCACTTCTTCTGCTTCATGACCGACATGCCAAAGAACTC TT AG AAAG AT CCCTAGACCTTT T AAAC AAAAG TCAACAACTGACTGACTT C AT AG AAAAAT T C A AGTGTGAAGGATCTACTATGAATTCTGAGCTGATCCAGGGAGCTCAGAGCAGCTGTCTGA AG ATCGACAGCCTCCTTGAACTTCTGCAAGACAGGAGAAGGCAGCTCGACAAGTACTTGCAG CA ACAGCGGCAGGAATTGTCTCAGGTTCTGCAGTTGTGTCTGTGGGACCAACAAGAAAACCA GG TTTCTTCTTGGTTTCAGAAAGCAATAAGAGATCTGCAGGAACAGAGTCTGGGCGCATCAC TT TCAGACAACAGAGAGCTAATCTGCAAGCACGAGGACCTGATAGTCAAAGCAAAGGAATGG GA TTCGGCCGTAGAGAAGCTGAAAAGCCAGGCTTTGGGGATTCTATTGTCCAAGGACTTGGC AG GAAAGGAACATCTACAGCTCTCTAACCAGAAACTGAATCGGCTTCAAGAAGAATTTGGAA GA CTCATGGTGGAAAGGAAAGCCTGGTTATCGATGGCAAATGACTTTTTTACCAGTGCTAAC AA GTCATTTGATGTACTTGGGAAAGTTGAAGCTTACCTTAAGCTCCTTAAATCAGAGGGTTT AA GCCTGCCTGTCTTGGCAGCGAAGCATGAGGAATTACACAGAGAAATTAAAGACTCTACAG CC ACTGCTCTGCAAAAGGGACGAACCTTAATCAGCCAAGTAGACTCCTGCCGCTCTCGGGTG AC AGGAATCCACGAGATGATGGGATACATTCAGAACCGAGTGGATTGTCTCACTGAACAGTG TA CAGCGCATGAGGAATTTGCCCGCAAGAGACAGCAATTAGCAACATCAGTGGACGACTACC TA AGGAAGGTGGAAATGTCAATTCAGGAAATCAGGCCCATTCTTGCTACCACATTGGATGTT GC TTCCAGCCCTTCT G AAT C AG AG AAG AT T T T AAAT AAAT AT C T G G AAT TGGATATCCAAGTCA AGGAGACAGCACATGCATTAGAAGCAGCTGCAAAAATCATGACAGAGAAAAATGAACTTG AA CTCAATGAAGTGGCATTGCTTCCTCTTAAAGTGAAATGGCTAGAGGAAGAATTAAGCACG CT TGGTCGAAGCATCAGCTGCAGGTCACGGATCCTGCAGACGTACGTGGCATTTCGAAAGTC AT CCGAGGAGGCAGAGGAGCAGCTTCAGAGCCTAAAGGAATTTTATCTGACTGAAATTCCTT GG AAAGATGAAGACGATGCTGTGGTCAAGTGCCAGTCTAACTCCGCTGAGAGGAAATGGCAG CT GTTTTTAAAGAAGAGTTTTTTGACCCAGGACTTGAGCCTCGAGTTTCTGAACTTAATAAA CA TGGCAAAAGAGAATGAGATATTAAATGTTAAAAATGAAATGCACATTATGGAGAACATTA TG GAAAAGCAAACAAATGGAAGGGAAGAGCTCAGCCATCTCCGAGTGGCGTGGTACCTCAAA GC TATCGAAGGCAAGCCTGCGAGAGAGCAGTGGGAAATGTTCAAAGAGAAACTTACAAAGAC TA CTCACAGTGTAAAACTTCTCCACGAAGTTCTCATGCCTGTCTCTGCGCTTGACCTTGGGG GG AGCCTCCAGAGCACGTCAGATCTGCGGAGGAGGTGGATCGCAATGAAGCCTCAGCTCCAG CA ACTGCATGAGGATGTTCAGCAAATCACGAAAGAGTGGGAAGTGTTAAGCAGTCAGGGAGC TC CTCTGAAGGAGAAGGCTGAACAGCTGAAGGACCTTGTCCATCTCCACCGGAGGCAGCGAG AG CGAATCCAGGAATATGAGGAAATCCTATACAAGACAGTCCAGTTCCACCAGGTCAAGGAA GA GCTGGTCCATCTCATCAAACCAAGAGAACTGGAACTTCTAGCACAGCCCATGGAACTGGC AA GTTCTGAGGAGGTCCAGATGCAACTGGGCCGTTCGCAAGAAAGGCGGGCACATGTAGACC AT CTCCATCAGCTGGCCCTGACCTTAGGAGTGGACATCATTTCATCCGTGCAACAACCTAAT TG CTCTAATATTTCTGCAAAGAACCTGCAGCAGCAGCTGGAGGCCCTTGAACTGGAGAGCAG GA GTTGGAGTGCCCAAGCCAAAGAACATGAGCGCGTCCTGTCCTGCAGCTTGGAGTACTGCA CT GCACGTGATGAGATCAGTGAGCTCAAAGAGTCATTCAAAGACATAAAAAAGAAATTCAAC AA T C T G AAG TTTAATTACTC C AAG AAAAAT GAG AAAT C T C G AAAT T T AAAG AC TCTTCAGTATC AAATCCAACAAGTGGATACATATGCTGAAAAAATTCAGGCTCTAAGGAAGAAAATGGAAA AA GTTAATAATAAGACATCTGATTCATTCTTAAGTTATCCAAGTAATAAAGCTAACATGCTT TC GGAAGCCATGGAGGATTTGAAAAAGAACGTGGATGACTTTGACAAAGTTGTGACAGATTA CA AAATGAATTTGGACCTGACTGAGCATCTTCAGGAAGTAATAGAAGAGTGTAACTTTTGGT AT GAAGATGCAAGTGCCACCGTTGTGAGAGTTGGGAAGTATTCCATGGAGTGCCAAACAAGG GA AGCTGTGGACATCCTCCACAGGCAGTTCAATAAGTTTATTACTCCCTCGGTGCCCCAGCA AG AAGAAAGGATTCAGGAGGTCATTGACCTTGCTCAGCGCTTATATGGTTTGGAAGAGGGGC AG AAAT AT G C AG AG AAG AT TGTCACAAGACACAAGGAGATTCTT G AAT CTATTACT G AAT T AT G TGGGTCTCTCGTGGAACTTAAAGAAAAACTGATGCAGGGAGAGGTTCCAAAGATGAATTC AG ATTTGGAAGATTTCCATGACAATTGCATTGATCTGCTAAAGGGGCCAGGGAAGGATGACC AG AAAACATTCAGTGAAGAAAGGAATGAGGGGCAGGTGCAGGGAGCAGATGTATTGGCCGTC AA TGGAACAAGAGAAGATGGGCTTCCCATGGACTTGAGGCGGACCTCCTCTGACAAAGAGGA CA GCGCCCAGGGTCTGATCCTTCCAGAAGACACACTATCTGGAGAAGAATCTGAGTGTATCT CA TCCGATGACATCTCTTTGCCTCCACTGCCAGTGAGCCCCGAGTCCCCTCTGGCACCATCA GA CATGGAGGTAGAAGAACTTACCAGCTCCTCTGCCCTCGCCCTGCACATCAGCGGCTACAG GA TGCATCCAGGGACAGGTGGTCTAGGAAAGGCCCAGGAGTCTGCTCTTCCCTCACCCATTG CC TTTGCGGATGGGGGCCATAGTAAGAAAGACACATTTACAAGTCATTTTGAGAGGCCCTAT CC ACAGTTGAAAGCTGAATCCTCATTAGCCTCCAGAGGATCAGCAGAGATGAGTACCAAGTT GC ACATCAATGTGAAATGTCCAGCGAGCATGCCCCATGAAGTGCACGACAAAGCTTTACAGC AA TGCTCCCAGGCTCGGGAGAGCACACTAGAAATGCAGGAAAAAGTGCATGCTGATTCTAAC GT CACTAAAACCCAAGACAGGCTGCATGCTGCCCTTGATGTGTCCCCTGGCCTCGGCTCTCA AC CAGACACCAGCGAGAGCCATCAGAGGCGAGTGGGTCCCCAAGGAAACAAGAAAAACTCAT CC GCAGAAAACAGCGTGGTCAGCCTAGCTGGCCAGGCACCTCATTTCTCCAGGCTGCTGTCC AA TGTAACTGTCATGGAAGGTTCCCCAGTGACTTTGGAAGTCGAAGTAACAGGATTCCCAGA GC CTACACTGACATGGTTCAAGAAGGGCCAGAAACTGTGTGCAGATGGACACTTACAGGTCT TG CACAAGGACACAAAGCATTCGGTGTTCATTCCGAAGGTATGTGAGGCAGATGCTGGCCTC TA TGTGGCTCAAGCCCAAAACTCTAGTGGCACCCTGTCTTCCAAAGCCATCCTCCATGTGAC AG GTAACCATGGGCCACCAATTACAAGACTAAACTGGATTATGCTGTGTATCATCTATGTCA GT GTATCAGTAATATACTGGCTACTTACACGATAGTGACATGTTGCCATCAATGGACATTGT TA T G AG C C T AAAAG AC AAT AT GATTGTTGATTTTCTCATACAT C AC AC AAAG CACTCCCCCAAC ATACACACACCAATTTATCTCCACTTTGACTAATCCCACTGCTAGGTGAAATAGTTGTAT TT TCTCTACGATTTAACATACTTGGGAAACACGGGAAATTATGTACAGCGGGTAATGATACA TT TCATACTCTAGGAAGAGAAAGATTCTGTCTAATAAGCTGATCTCTTGTTTAAAGGCATAT CA ATGCGCACATAGTTCACAAGGATCATAGGGTTTTGTCCTAATTTTCAAATCCAAATGTTT TT CATTTCTCCTTCACTGTGCTCATAACTATCTCTTTGTAGGGCATTGCATCAAACTGAGGT TG TGATTCTCAATGTTGTATTAGCTTCCATTAACTGTGCTTTTCACATTTTGATCTTGTTTA CC AAACAAACTTGACCTATGCTGTCTCAACCTACTCTACATTCCTACGTCGGAGGATGTCAC TA TCAATAACCATTCCTCATGATGGTCTCTTGGGCTTGATGTTGGAGAACAGACGTGCCATC AG CTTCAAAATGGAGTCAACCTTTCTATTAGCTCTGAATGTCATATGTCAAAATGACATACA GG CTGCAGGAGGGGACCAGTTTTCCCCCTGTCCTTCCACTGCTGTGCATTGACCATCTGTGC AT AACTATAGGCAGGTCCTACTGCATCTGCTGCTGCTGCTGTTGCTGCTGAACACTGTGTAG CC CTTTACAAGGCATCTCAGTGCCCCCCCTTTCCCATCTGGAAGAGGGGCTCATCTACCTCT TG ACAACTGAGTTTTTATTTTATGTCCTGTGGTGATCTGGCCAATGGAAAAATACTCAGAGA AG ACTCATTACAATGCTTGGTGTGATTTGCCATCATGCTACTCATTACCCATTAATTCCCTT TC AGATTCTGATCTTT TAG AAT AT T AAAT G AAAAG AG TGCTGTTAATATGTAATTTCTAACTAC TCCCTTGGGGGAGGGTAAGACTGATGTTCTATGAATTAGAGTTTAAATTCCTTTCTAACT TC TTACAACTACATCCTTTTCAAAGGCTCTGGACGGTAACCTCTGGAGGATATAGTTAGCGT GT TTTAGAGGGAGTCTGGGTAAGATAGAAGAAGACAATTCTAGCTTTCTTTTGTGCAATTAT CT CTGTAATATTGAAGAATGTTGTATGGCTTCCTAATACGAATTATTAAAAAAAATAAGAGA GG TGTTTGTTTTCATATTTTAAACTTTGTCATGCTAGGGTAAAATCAAGTTGAAAGAATAAT TT ATTACAGATTTTTGATGAAGGGAAGAAATTAATTATGTTGCTGAAAGACTGTCTACAAAT AA TCAAAATATTTAAATAGAATATATTTGATGTGATATTAAAAAGCTACTGAATGTTGTGGA AG ATAAGAAATGTCTAGTCTAGTAAAAAATTGGCCTATTTTTTCTTGTCCTGTGTATACAAT TT AAT T G AAAAAT T G T AAC T G T T T T C T T T C AAG T T T T G AC AG AAAG C T AT T AAAAT C T T AT AT A TACAC [01121 In some embodiments, the nucleic acids encoding a Cede 141 can be administered to the subject using any suitable known method, for example, but not limited to, direct injection, viral vector mediated delivery (e.g., AAV, retrovirus, adeno virus, or lentivirus), or ceDNA. In some embodiments, an agent that promotes Cede 141 expression/activity is an rAAV encoding Cede 141.

[01131 The present disclosure, at least in part, is based on the surprising discovery that inhibition of Pacsin2 in CNS endothelial cells results in an increase in blood-CNS barrier permeability (z.e., maintaining blood-CNS integrity) through decreased tubular vesicle- mediated transcytosis. In some aspects, the method comprises administering to the subject a Pacsin2 inhibitor. Pacsin2 protein is involved in linking the actin cytoskeleton with vesicle formation by regulating tubulin polymerization and mediates vesicle or tubule formation for endocytosis across brain endothelial cells (Ritter et al., PACSIN 2, a novel member of the PACSIN family of cytoplasmic adapter proteins, FEBS Leters (1999) 454(9): 356-362; Leite et al., The Role of BAR Proteins and the Glycocalyx in Brain Endothelium Transcytosis, Cells (2020), 9(12): 2685).

[01141 In some embodiments, the method comprises administering to a subject an inhibitor of Pacsin2 signaling. In some embodiments, the Pacsin2 is expressed by CNS endothelial cells. In some embodiments, the method comprises administering to the subject an agent that inhibits Pacsin2 signaling at the blood-CNS barrier.

[01151 In some embodiments, the inhibitor of Pacsin2 signaling is a Pacsin2 inhibitor. In some embodiments, inhibition of Pacsin2 in CNS endothelial cells results in decreased blood- CNS barrier permeability. In some embodiments, inhibition of Pacsin2 does not result in decreased blood-CNS barrier permeability.

[01161 In some embodiments, the Pacsin2 inhibitor is capable of inhibiting Pacsin2 expression and/or activity. In some embodiments, the Pacsin2 inhibitor is an inhibitory nucleic acid targeting Pacsin2 mRNA. As described herein, an inhibitory nucleic acid, refers to nucleic acids capable of inhibiting expression or activity of a target gene (e.g., DNA, RNA, or protein of the target gene), for example, PACSIN2. Non-limiting examples of inhibitory nucleic acids include e.g., dsRNA, siRNA, shRNA, miRNA, amiRNA, antisense oligonucleotides (ASOs), DNA or RNA aptamers, etc.

[01171 In some embodiments, an inhibitory nucleic acid targeting Pacsin2 is an siRNA. siRNA molecules comprise a specific antisense sequence in addition to the reverse complement (sense) sequence. [01181 The specificity of siRNA molecules may be determined by the binding of the antisense strand of the molecule to its target RNA (e.g., Pacsin2 mRNA). In some embodiments, the siRNA molecules are 60, 65, 70, 75, 80, 85, 90, 95, 100 or more base pairs in length. In some embodiments, the antisense sequence of the siRNA molecules is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more base pairs in length. In some embodiments, the antisense sequence of the siRNA molecules are 8 to 30 base pairs in length, 10 to 15 base pairs in length, 10 to 20 base pairs in length, 15 to 25 base pairs in length, 19 to 21 base pairs in length, or 21 to 23 base pairs in length.

[01191 In some embodiments, siRNA molecules comprise an antisense sequence comprising a region of complementarity to a target region in a Pacsin2 mRNA (e.g., human Pacsin2 mRNA (SEQ ID NO: 5) or mouse Pacsin2 mRNA (SEQ ID NO: 6)). In some embodiments, the region of complementarity is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to a target region in a Pacsin2 mRNA (e.g., human Pacsin2 mRNA or mouse Pacsin2 mRNA). In some embodiments, the target region is a region of consecutive nucleotides in a Pacsin2 mRNA (e.g., human Pacsin2 mRNA or Pacsin2 Cede 141 mRNA). In some embodiments, a complementary nucleotide sequence need not be 100% complementary to that of its target to be specifically hybridizable or specific for a Pacsin2 mRNA (e.g., human Pacsin2 mRNA or mouse Pacsin2 mRNA). Exemplary human Pacsin2 mRNA and mouse Pacsin2 mRNA sequence is set forth in SEQ ID NO: 5 and SEQ ID NO: 6.

[01201 In some embodiments, siRNA molecules comprise an antisense sequence that comprises a region of complementarity to in a Pacsin2 mRNA (e.g., human Pacsin2 mRNA or mouse Pacsin2 mRNA) sequence and the region of complementarity is in the range of 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 nucleotides in length. In some embodiments, the region of complementarity is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the region of complementarity is complementary to at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of a Pacsin2 mRNA (e.g., human Pacsin2 mRNA (SEQ ID NO: 5) or mouse Pacsin2 mRNA (SEQ ID NO: 6)). In some embodiments, the region of complementarity comprises a nucleotide sequence that contains no more than 1, 2, 3, 4, or 5 base mismatches compared to the complementary portion of a Pacsin2 mRNA (e.g., human Pacsin2 mRNA or mouse Pacsin2 mRNA). In some embodiments, the region of complementarity comprises a nucleotide sequence that has up to 3 mismatches over 15 bases, up to 2 mismatches over 10 bases, or up to 1 mismatch over 5 bases.

101211 In some embodiments, siRNA molecules targeting Pacsin2 comprise an antisense strand which comprises a region of complementarity that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the sequences as set forth in any one of SEQ ID NOs: 22-31, and 36-45. In some embodiments, siRNA molecules targeting Pacsin2 comprise an antisense strand which comprises a region of complementarity that is complementary to at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of the sequences as set forth in any one of SEQ ID NOs: 22-31, and 36-45.

Exemplary Pacsin2 siRNAs target sequences are set forth below:

101221 Pacsin2 target sequences:

CACATATGATGATTCCGTTGGAGTA (SEQ ID NO: 22)

TCGGGAACTACAAGCGGACTGTGAA (SEQ ID NO: 23)

CAAGAACTGGCAGAAGGAAGCCTTT (SEQ ID NO: 24)

GAAAGAGGTAGAAGCAGCAAAGAAA (SEQ ID NO: 25)

CAAAGAGGAGAAGCTGGCTATCTCA (SEQ ID NO: 26)

CAAGCAAGATGTTCTTAAGACCAAA (SEQ ID NO: 27)

TAGACCTGTCCAATGTGGCTGGCTA (SEQ ID NO: 28)

CCTGTCCAATGTGGCTGGCTACAAA (SEQ ID NO: 29)

CAGCTACGAGAAGACCCAGAGCTAT (SEQ ID NO: 30)

AGTTGGCCTATACCCGGCAAATTAT (SEQ ID NO: 31)

GGCCTCACTGATGAACGATGA (SEQ ID NO: 36)

GGTAGAAGCAGCAAAGAAAGC (SEQ ID NO: 37)

GCAAGCAAGATGTTCTTAAGA (SEQ ID NO: 38)

GCACCTAGACCTGTCCAATGT (SEQ ID NO: 39)

GGCTGGCTACAAAGCCATTTA (SEQ ID NO: 40)

GCTGGCTACAAAGCCATTTAC (SEQ ID NO: 41)

GCTACAAAGCCATTTACCATG (SEQ ID NO: 42) GGTCAGACGATGAGTCTAACA (SEQ ID NO: 43)

GGCCTATACCCGGCAAATTAT (SEQ ID NO: 44)

GCCTATACCCGGCAAATTATG (SEQ ID NO: 45)

[01231 In some embodiments, the present disclosure also contemplates delivering inhibitory nucleic acids targeting Pacsin2 mRNA that are known in the art, for example, CAAAUUAUGUGGAGGCGAU (SEQ ID NO: 32), CCCUUAAUGUCCCGAGCAA (SEQ ID NO: 33), CCUCACUGAUGAACGAUGA (SEQ ID NO: 34), or CUGAGGUGGUUCCGAGCCA (SEQ ID NO: 35) as described by Hansen et al. (Pacsin2 is recruited to caveolae and functions in caveolar biogenesis, J Cell Sci (2011) 124 (16): 2777- 2785), or sc-36174(m), and sc-36173(h) from Santa Cruz Biotechnology.

[01241 In some embodiments, an inhibitory nucleic acid targeting Pacsin2 is an shRNA. [01251 The specificity of shRNA molecules may be determined by the binding of the antisense strand of the molecule to its target RNA sequence (e.g., Pacsin2 mRNA; SEQ ID NO: 5 or EQ ID NO: 6). In some embodiments, the shRNA molecules are 60, 65, 70, 75, 80, 85, 90, 95, 100 or more base pairs in length. In some embodiments, the antisense sequence of the shRNA molecules is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more base pairs in length. In some embodiments, the antisense sequence of the shRNA molecules are 8 to 30 base pairs in length, 10 to 15 base pairs in length, 10 to 20 base pairs in length, 15 to 25 base pairs in length, 19 to 21 base pairs in length, or 21 to 23 base pairs in length.

[01261 Following selection of an appropriate target RNA sequence, shRNA molecules that comprise a nucleotide sequence complementary to all or a portion of the target sequence, i.e., an antisense sequence, can be designed and prepared using methods known in the art (see, e.g., Moore et al., Short Hairpin RNA (shRNA): Design, Delivery, and Assessment of Gene Knockdown, Methods Mol Biol. 2010; 629: 141-158).

[01271 The shRNA molecules can comprise a hairpin i.e., when two regions of the same strand, usually complementary in nucleotide sequence when read in opposite directions, basepair to form a double helix that ends in an unpaired loop), or asymmetric hairpin (i.e., hairpin with a strand overhang) secondary structure, having self-complementary sense and antisense strands. In some embodiments, the shRNA targeting inhibitory nucleic acid described herein comprises an antisense sequence and a sense sequence.

[01281 In some embodiments, the antisense sequence of the shRNA molecule is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more nucleotides in length. In some embodiments, the antisense sequence is 8 to 50 nucleotides in length, 8 to 40 nucleotides in length, 8 to 30 nucleotides in length, 10 to 15 nucleotides in length, 10 to 20 nucleotides in length, 15 to 25 nucleotides in length, 19 to 21 nucleotides in length, or 21 to 23 nucleotides in lengths.

101291 In some embodiments, the sense sequence of the shRNA molecule is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more nucleotides in length. In some embodiments, the sense sequence is 8 to 50 nucleotides in length, 8 to 40 nucleotides in length, 8 to 30 nucleotides in length, 10 to 15 nucleotides in length, 10 to 20 nucleotides in length, 15 to 25 nucleotides in length, 19 to 21 nucleotides in length, or 21 to 23 nucleotides in lengths.

[01301 In some embodiments, shRNA molecules comprise an antisense sequence comprising a region of complementarity to a target region in a Pacsin2 mRNA (e.g., human Pacsin2 mRNA or mouse Pacsin2 mRNA). In some embodiments, the region of complementarity is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target region in a Pacsin2 mRNA (e.g., human Pacsin2 mRNA or mouse Pacsin2 mRNA) of SEQ ID NOs: 22-31 and 36-45. In some embodiments, the target region is a region of consecutive nucleotides in a Pacsin2 mRNA (e.g., human Pacsin2 mRNA or mouse Pacsin2 mRNA). In some embodiments, a complementary nucleotide sequence need not be 100% complementary to that of its target to be specifically hybridizable or specific for a Pacsin2 mRNA (e.g., human Pacsin2 mRNA or mouse Pacsin2 mRNA).

101311 In some embodiments, shRNA molecules comprise an antisense sequence that comprises a region of complementarity in a Pacsin2 mRNA (e.g., human Pacsin2 mRNA or mouse Pacsin2 mRNA) sequence and the region of complementarity is in the range of 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 nucleotides in length. In some embodiments, the region of complementarity is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the region of complementarity is complementary to at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of a Pacsin2 mRNA (e.g., human Pacsin2 mRNA or mouse Pacsin2 mRNA). In some embodiments, the region of complementarity comprises a nucleotide sequence that contains no more than 1, 2, 3, 4, or 5 base mismatches compared to the complementary portion of a Pacsin2 mRNA (e.g., human Pacsin2 mRNA or mouse Pacsin2 mRNA). In some embodiments, the region of complementarity comprises a nucleotide sequence that has up to 3 mismatches over 15 bases, up to 2 mismatches over 10 bases, or up to 1 mismatch over 5 bases.

101321 In some embodiments, shRNA molecules target a Pacsin2 sequence comprises an antisense strand that comprises a region of complementarity that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the sequences as set forth in any one of SEQ ID NOs: 22-31, and 36-45. In some embodiments, shRNA molecules targeting Pacsin2 comprise an antisense strand that is complementary to at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of the sequence as set forth in any one of SEQ ID NOs: 22-31 and 36-45.

101331 In some embodiments, the inhibitory nucleic acids targeting Pacsin2 can be administered to the subject using any suitable known method, for example, but not limited to, direct injection, viral vector mediated delivery (e.g., AAV, retrovirus, adenovirus, or lentivirus), or ceDNA.

101341 In some embodiments, the Pacsin2 inhibitor is an antibody, an antibody variant or an antigen-binding fragment thereof targeting Pacsin2. Antibodies, antibody variants and antigen-binding fragments targeting Pacsin2 have been previously described, see, e.g., ab228589, ab262841 from Abeam, aa400-450 from CSBio, PA5-83983 , PA5-84299, MAS- 37727, PA5-99031, PA5-118137 or PAI-41564 from Invitrogen.

101351 In some embodiments, administration of an effective amount of any one of the Pacsin2 inhibitors or an agent that promotes Cede 141 expression/activity described herein or a combination thereof at the blood-CNS barrier results in decreased blood-CNS barrier permeability.

101361 An effective amount of an agent that decreases blood-CNS barrier permeability (e.g., a Pacsin2 inhibitor or an agent that promotes Cede 141 expression/activity) described herein may be an amount sufficient to have a therapeutic benefit in a subject, e.g., to extend the lifespan of a subject, to improve and/or reverse in the subject one or more symptoms of disease, or to slow disease progression. The effective amount will depend on a variety of factors such as, for example, the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among subject and tissue. An effective amount may also depend on the inhibitor used. [01371 In some embodiments, administration of an agent that decreases blood-CNS barrier permeability (e.g., a Pacsin2 inhibitor or an agent that promotes Ccdcl41 expression/activity) described herein may result in inhibition of Pacsin2 signaling and/or increase of Cede 141 signaling of one or more of the CNS endothelial cells. In some embodiments, administration of the inhibitors described herein may result in inhibition of Pacsin2 signaling and/or Cede 141 signaling in all of the foregoing endothelial cells.

[01381 An effective amount may also depend on the mode of administration. For example, targeting endothelial cells in the CNS by intravenous administration or subcutaneous injection may require different (e.g., higher, or lower) doses, in some cases, than targeting endothelial cells in the CNS by another method (e.g., local injection to the CNS). In some embodiments, a Pacsin2 inhibitor described herein is a small molecule and can be administered orally.

[01391 In some embodiments, administering an inhibitor of Pacsin2 signaling at the blood- CNS Barrier decreases the level and/or activity of Pacsin2. As used herein, administration of the inhibitor described herein decreases the expression and/or activity of Pacsin2 by at least 10% or more, e.g., by 10% or more, 50% or more, 100% or more, 200% or more, 500% or more, or 1000% or more. In some embodiments, administration of the Pacsin2 inhibitors described herein results in decreasing of the permeability of the blood-CNS barrier in the subject. In some embodiments, administration of the Pacsin2 inhibitor described herein results in decreasing the permeability of the blood-CNS barrier in the subject by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500% or more compared the permeability of the blood-CNS barrier of the subject prior to administration of the inhibitor. The permeability of blood-CNS barrier can be measured using any suitable technique or method known in the art, e.g., neuroimaging techniques including dynamic perfusion CT (PCT) and dynamic contrast- enhanced magnetic resonance imaging (DCEMRI), quantification of protein biomarkers (e.g., neuron- specific enolase (NSE), glial fibrillary acidic protein (GFAP), and S 100(3) in the cerebral spinal fluid (CSF), etc.

[01401 In some embodiments, administering an agent that promotes Cede 141 signaling at the blood-CNS Barrier increases the level and/or activity of Ccdcl41. As used herein, administration of an agent that promotes Cede 141 signaling described herein increases the expression and/or activity of Ccdcl41 by at least 10% or more, e.g., by 10% or more, 50% or more, 100% or more, 200% or more, 500% or more, or 1000% or more. In some embodiments, administration of an agent that promotes Cede 141 signaling described herein results in decreasing of the permeability of the blood-CNS barrier in the subject. In some embodiments, administration of an agent that promotes Cede 141 signaling described herein results in decreasing the permeability of the blood-CNS barrier in the subject by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 150%, at least 200%, at least

300%, at least 400%, at least 500% or more compared the permeability of the blood-CNS barrier of the subject prior to administration of the inhibitor. The permeability of blood-CNS barrier can be measured using any suitable technique or method known in the art, e.g., neuroimaging techniques including dynamic perfusion CT (PCT) and dynamic contrast- enhanced magnetic resonance imaging (DCEMRI), quantification of protein biomarkers (e.g., neuron- specific enolase (NSE), glial fibrillary acidic protein (GFAP), and S 100P) in the cerebral spinal fluid (CSF), etc.

101411 Without wishing to be bound by any particular theory, efficient delivery of an agent that promotes Cede 141 signaling or inhibits Pacsin2 signaling at the blood-CNS barrier may be useful for the treatment of a subject having a disease associated with endothelial cell dysfunction (e.g., head trauma, stroke, et cetera). In certain embodiments, the endothelial cells are endothelial cells in the CNS. Accordingly, methods and compositions for treating diseases associated with endothelial cell dysfunction are also provided herein. In some aspects, the disclosure provides a method for treating a disease associated with endothelial cell dysfunction, the method comprising: administering to a subject having or suspected of having a disease associated with endothelial cell dysfunction an effective amount of an agent that promotes Cede 141 signaling or inhibits Pacsin2 signaling at the blood-CNS barrier. 101421 Endothelial cells (e.g., endothelial cells in the CNS) can be healthy endothelial cells (e.g., endothelial cells in the CNS not having a dysfunction, or at risk of developing endothelial cell dysfunction), or dysfunctional endothelial cells (e.g., endothelial cells causing abnormal vasculature permeability, hemodynamics, or neuroimmune crosstalk, et cetera). As used herein, “or at risk of developing endothelial cell dysfunction” refers to a subject having an increased probability of developing endothelial cell dysfunction than the general population due to the presence of a risk factor. Exemplary categories of risk factors for developing endothelial cell dysfunction include, but are not limited to, genetics, head trauma, vascular disease, prior brain surgery, disease, age, race, and family history (e.g., positive family history of vascular disease, high cholesterol, high blood pressure, or diabetes). As used herein, an “disease associated with endothelial cell dysfunction” is a disease or condition that results from the dysfunction of endothelial cells (e.g., endothelial cells in the CNS). In some embodiments, a disease associated with CNS endothelial cell dysfunction includes but is not limited to, retinal disease (e.g., diabetic retinopathy), neurodegenerative disease (e.g., Huntington’s disease, dementia), acute injury of the CNS (e.g., stroke and head trauma), Neuroinfectious disease (e.g., encephalitis, sepsis, COVID- 19), primary and metastatic cancers of the CNS, autoimmune disease of the CNS (e.g., multiple sclerosis), and other neuroinflammatory conditions. In some embodiments, the disease associated with CNS endothelial cell dysfunction is a CNS primary cancer, such as glioblastoma, meningioma, or lymphoma. In some embodiments, the disease associated with CNS endothelial cell dysfunction is a metastatic cancer to the brain such as metastatic lung cancer, metastatic breast cancer, or melanoma. In some embodiments, the disease associated with CNS endothelial cell dysfunction is a neuroinflammatory disease, such as CNS Lupus, CNS Lyme Disease, Neurosarcoidosis, Neuromyelitis optica (NMO), or Paraneoplastic and Autoimmune Encephalitis. In some embodiments, the disease associated with CNS endothelial cell dysfunction is a dementia and/or cognitive disorder, such as dementia resulting from Alzheimer’s disease, Lewy body dementia, frontotemporal dementia, encephalopathy, or post-acute COVID syndrome.

101431 Cede 141 inhibits tubular vesicle transcytosis that is mediated by Pacsin2. Cede 141 gene ablation or Pacsin2 gene overexpression in the brain endothelial cells in mice leads to an accumulation of tracer- filled tubular vesicles (e.g., tubular vesicles are involved in transcytosis (z.e., vesicular trafficking) of molecules across the blood-brain barrier endothelial cell layer, from blood into the brain tissue). Analogous tracer-filled tubular vesicles have been observed in the disrupted blood-brain barrier vasculature in the brain and spinal cord, under several neuropathological conditions. Specifically, tubular vesicles have been observed in the brain endothelial cells after traumatic brain injuries suggesting Pacsin2 overexpression in these conditions (see., e.g., Lossinsky et al., New ultrastructural evidence for a protein transport system in endothelial cells of gerbil brains. Acta Neuropathol 47, 105-110; Lossinsky et al., Ultracytochemical studies of vesicular and canalicular transport structures in the injured mammalian blood-brain barrier. Acta Neuropathol 61, 239-245; Lossinsky et al., Ultracytochemical evidence for endothelial channel-lysosome connections in mouse brain following blood-brain barrier changes. Acta Neuropathol 53, 197-202; Lossinskyet al., A comparative ultrastructural study of endothelial cell tubular structures from injured mouse blood-brain barrier and normal hepatic sinusoids demonstrated after perfusion fixation with osmium tetroxide. Microvasc Res 31, 333-344, doi: 10.1016/0026-2862(86)90022-l (1986)) and stroke (see, e.g., Tagami, M. et al. Increased transendothelial channel transport of cerebral capillary endothelium in stroke-prone SHR. Strokel4, 591-596, doi: 10.1161/01. str.14.4.591 (1983)) and in the spinal cord endothelial cells in the EAE mouse and rat models for multiple sclerosis (see, e.g., Claudio et al., Increased vesicular transport and decreased mitochondrial content in blood-brain barrier endothelial cells during experimental autoimmune encephalomyelitis. Am J Pathol 135, 1157-1168 (1989); Lossinsky et al., Sites of egress of inflammatory cells and horseradish peroxidase transport across the blood-brain barrier in a murine model of chronic relapsing experimental allergic encephalomyelitis. Acta Neuropathol 78, 359-371. In other embodiments, Pacsin2 is overexpressed in ageing brain where blood-CNS barrier permeability has been shown to be increased (e.g., early onset dementia such as frontotemporal dementia (see, Gerrits et al., Neurovascular dysfunction in GRN-associated frontotemporal dementia identified by singlenucleus RNA sequencing of human cerebral cortex, Nat Neurosci. 2022 Aug;25(8): 1034- 1048). In some embodiments, Cede 141 is decreased in conditions where blood-CNS barrier permeability have been shown to be increased (e.g, stoke (see, Garcia-Bonilla et al., Brain and blood single-cell transcriptomics in acute and subacute phases after experimental stroke; bioRxiv. Preprint. 2023 Apr 3), spinal cord injury (see, Cao et al., Single-cell RNA sequencing for traumatic spinal cord injury, The FASEB Journal. 2022; 36:e22656), ageing (see, Ximerakis et al., Single-cell transcriptomic profiling of the aging mouse brain, Nature Neuroscience volume 22, pagesl696-1708 (2019)). Accordingly, in some embodiments, the present disclosure contemplates treating diseases and conditions associated with increased blood-CNS permeability (e.g., ageing, early onset dementia, stroke, or traumatic spinal cord injury) by administering a subject in need thereof an agent that inhibits Pacsin2 expression/activity and/or promotes Cede 141 expression/activity. In some embodiments, the present disclosure provides methods of inhibiting transcytosis at the blood-CNS barrier by administering a subject in need thereof an agent that inhibits Pacsin2 expression/activity and/or promotes Cede 141 expression/activity.

101441 In some embodiments, an agent that promotes Cede 141 signaling or inhibits Pacsin2 signaling at the blood-CNS can be administered to a subject in need of improved integrity (e.g., decreased permeability) of the blood-CNS barrier. In some embodiments, the subject in need of improved quality of vesicle trafficking in the blood-CNS barrier can be a subject who has been diagnosed with or determined to have abnormally high permeability of the blood- brain barrier, e.g., repeated infections of the CNS, or in which abnormal levels of a systemically administered tracer molecule reach the CNS. In some embodiments, the subject having abnormally high blood-CNS barrier permeability described herein has an increasing of the permeability of the blood-CNS barrier compared to a healthy subject. In some embodiments, the subject having abnormally high blood-CNS barrier permeability described herein has an increasing of the permeability of the blood-CNS barrier by at least 10%, at least

15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least

50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 85%, at least

90%, at least 95%, at least 99%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500% or more compared the permeability of the blood-CNS barrier of a healthy subject. The permeability of blood-CNS barrier can be measured using any suitable technique or method known in the art, e.g., neuroimaging techniques including dynamic perfusion CT (PCT) and dynamic contrast-enhanced magnetic resonance imaging (DCEMRI), quantification of protein biomarkers (e.g., neuron- specific enolase (NSE), glial fibrillary acidic protein (GFAP), and S 100(3) in the cerebral spinal fluid (CSF), etc.

101451 In some embodiments, the subject in need of improved quality of vesicle trafficking of the blood-brain barrier can be a subject in need of treatment e.g. having, diagnosed as having, or at risk of developing) a condition selected from the group consisting of dementia, encephalitis, sepsis, COVID-19, primary and metastatic cancers of the CNS, autoimmune disease of the CNS (e.g., multiple sclerosis), and other neuroinflammatory conditions. In some embodiments, the disease is a CNS primary cancer, such as glioblastoma, meningioma, or lymphoma. In some embodiments, the disease is a metastatic cancer to the brain such as metastatic lung cancer, metastatic breast cancer, or melanoma. In some embodiments, the disease is a neuroinflammatory disease, such as CNS Lupus, CNS Lyme Disease, Neurosarcoidosis, Neuromyelitis optica (NMO), or Paraneoplastic and Autoimmune Encephalitis. In some embodiments, the disease is a dementia and/or cognitive disorder, such as dementia resulting from Alzheimer’ s disease, Lewy body dementia, frontotemporal dementia, encephalopathy, or post-acute COVID syndrome.

[0105] In some embodiments, administration of an agent that promotes Cede 141 signaling or inhibits Pacsin2 signaling at the blood-CNS barrier can slow or halt the progression of a neurodegenerative disease. In some embodiments, administration of an agent that promotes the Cede 141 signaling at the blood-CNS barrier can slow or prevent the development of at least some signs or symptoms of any of the diseases described herein. [01061 Delivery of an agent that promotes Cede 141 signaling at the blood-CNS barrier or inhibits Pacsin2 signaling in a mammalian subject may be by, for example, injection to the CNS. In some embodiments, the injection is direct injection to the CNS (e.g., intracerebral injection, intraventricular injection, intracisternal injection, intraparenchymal injection, intrathecal injection, and any combination of the foregoing). In some embodiments, the injection is systemic injection (e.g., intravenous injection, intradermal injection, or subcutaneous injection). In some embodiments, an agent that promotes Cede 141 signaling or inhibits Pacsin2 signaling at the blood-CNS barrier can be administered orally.

III. Delivery of nucleic acids using rAAV

101071 The present disclosure provides compositions and methods for delivering a transgene to endothelial cells throughout the CNS in a subject. In some aspects, the disclosure provides isolated and/or engineered AAVs. In some aspects, the present disclosure provides a recombinant adeno-associated virus (rAAV), wherein the rAAV comprises: (i) an AAV capsid protein (e.g., AAV-BI30, AAV-BR1, AAV9 or variants thereof), and (ii) an isolated nucleic acid encoding an agent for modulating blood-CNS barrier as described herein (e.g., an agent increasing or decreasing Cede 141 and/or Pacsin2 expression/activity).

101081 As used herein with respect to AAVs, the term “isolated” refers to an AAV that has been artificially produced or obtained. Isolated AAVs may be produced using recombinant methods. Such AAVs are referred to herein as “recombinant AAVs”. Recombinant AAVs (rAAVs) preferably have tissue-specific targeting capabilities, such that a transgene (e.g., a transgene encoding Cede 141 and/or Pacsin2 or an inhibitory nucleic acid targeting Cede 141 and/or Pacsin2) of the rAAV will be delivered specifically to one or more predetermined tissue(s) or cell(s) (e.g., endothelial cells in the CNS).

101091 The term “transgene,” as used herein, refers to a gene that has been transferred from one organism to another by any known suitable genetic engineering techniques. The introduction of a transgene has the potential to change the phenotype of an organism. Transgene describes a segment of DNA containing a gene sequence that has been isolated from a first organism and is introduced into a second organism. This non-native segment of DNA may either retain the ability to produce RNA or protein in the transgenic organism or alter the normal function of the transgenic organism’s genetic code. In some embodiments, the transgene encodes a Cede 141 and/or Pacsin2 protein. In some embodiments, the transgene encodes an inhibitory nucleic acid targeting Cede 141 and/or Pacsin2. IOHOI The AAV capsid is an important element in determining tissue-specific targeting capabilities of the virus. Thus, a rAAV having a capsid appropriate for the tissue being targeted can be selected. In some embodiments, the target tissue/cells of the present disclosure are endothelial cells in the CNS (e.g., brain arterial endothelial cells, brain venous endothelial cells, brain capillary endothelial cells, endothelial cells of the spinal cord, or endothelial cells of the retina). romi Methods for obtaining recombinant AAVs having a desired capsid protein are well known in the art. See, for example, US Patent Application Publication, US-2003-0138772, the contents of which are incorporated herein by reference. Typically the methods involve culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; a recombinant AAV vector comprising AAV inverted terminal repeats (ITRs) and an isolated nucleic acid comprising a transgene (e.g., a transgene for expressing a Cede 141 and/or Pacsin2 protein or an inhibitory nucleic acid targeting Cede 141 and/or Pacsin2); and sufficient helper functions to permit packaging of the recombinant AAV vector into the AAV capsid.

101121 In some embodiments, capsid proteins are structural proteins encoded by the cap gene of an AAV. AAVs comprise three capsid proteins, virion proteins 1 to 3 (named VP1, VP2 and VP3), all of which are transcribed from a single cap gene via alternative splicing. In some embodiments, the molecular weights of VP1, VP2 and VP3 are about 87 kDa, about 72 kDa, and about 62 kDa, respectively. In some embodiments, upon translation, capsid proteins form a spherical 60-mer protein shell around the viral genome. In some embodiments, the functions of the capsid proteins are to protect the viral genome, deliver the genome, and interact with the host. In some aspects, capsid proteins deliver the viral genome to a host in a tissue-specific or cell-specific manner. In some embodiments, an AAV capsid protein is of an AAV serotype selected from the group consisting of AAV-BI30 (see, e.g., W02023004367), AAV-9, AAV9.PHP.B, AAV9.PHP.eB, or AAV-BR1 (see, e.g., WO 2015158749). In some embodiments, the AAV capsid protein is AAV-BI30. In some embodiments, the AAV capsid protein is AAV-BR1. In some embodiments, the capsid protein is of AAV serotype 9 (AAV9). In some embodiments, an AAV capsid protein is of a serotype derived from AAV9 (e.g., an AAV9 capsid variant). In some embodiments, the AAV9 capsid variant is AAV9.PHP.B.

101131 The isolated nucleic acids of the invention may be recombinant adeno-associated virus (AAV) vectors (rAAV vectors). In some embodiments, an isolated nucleic acid as described by the disclosure comprises two adeno-associated virus (AAV) inverted terminal repeats (ITR) flanking the transgene. The isolated nucleic acid (e.g., the recombinant AAV vector) may be packaged into a capsid protein and administered to a subject and/or delivered to a selected target cell. “Recombinant AAV (rAAV) vectors” are typically composed of, at a minimum, a transgene and its regulatory sequences (e.g., a promoter), and 5' and 3' AAV inverted terminal repeats (ITRs). The transgene may comprise, as disclosed elsewhere herein, a nucleotide sequence encoding a Cede 141 and/or Pacsin2 protein or an inhibitory nucleic acid targeting Cede 141 and/or Pacsin2. In some embodiments, an rAAV encoding a Cede 141 protein comprises the nucleic acid sequence of SEQ ID NOs: 1 or 2. In some embodiments, an rAAV encoding a Pacsin2 protein comprises the nucleic acid sequence of SEQ ID NOs: 5 or 6. In some embodiments, an rAAV encoding an inhibitory nucleic acid targeting Pacsin2 comprises the nucleic acid sequence of SEQ ID NOs: 9-21. In some embodiments, an rAAV encoding an inhibitory nucleic acid targeting Cede 141 comprises the nucleic acid sequence of SEQ ID NOs: 22-31, and 36-45

101141 In some embodiments, the transgene (e.g., a transgene encoding a Cede 141 and/or Pacsin2 protein or an inhibitory nucleic acid targeting Cede 141 and/or Pacsin2) may further comprise a promoter operably linked to the coding sequence (e.g., nucleotide sequence encoding the Cede 141 and/or Pacsin2 protein or the inhibitory nucleic acid targeting Cede 141 and/or Pacsin2). A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases “operatively linked,” “under control,” or “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. A promoter may be a constitutive promoter, inducible promoter, or a tissue- specific promoter.

101151 As used herein, a nucleotide sequence (e.g., a nucleotide sequence encoding Cede 141 and/or Pacsin2 protein or an inhibitory nucleic acid targeting Cede 141 and/or Pacsin2) and regulatory sequences are said to be operably linked when they are covalently linked in such a way as to place the expression or transcription of the nucleic acid sequence encoding Cede 141 and/or Pacsin2 protein or an inhibitory nucleic acid targeting Cede 141 and/or Pacsin2 under the influence or control of the regulatory sequences. If it is desired that the nucleic acid sequences be translated into a functional protein, two DNA sequences are said to be operably linked if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence, and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame- shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably linked to a nucleic acid sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.

[01161 In some embodiments, the promoter is a constitutive promoter. Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the P-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFl-a promoter (Invitrogen). In some embodiments, the promoter is hybrid cytomegalovirus (CMV) immediate-early /Chicken betaactin promoter (CAG promoter). In some embodiments, the promoter is a chicken beta-actin (CBA) promoter. In some embodiments, the promoter is a minimal promoter. A minimal promoter is a part of a promoter located between -35 to +35 region with respect to the transcription start site. It has one or more of 3 conservative sequences, i.e., Tata box, initiator region, binding site for RNA polymerase, and downstream promoter element. Exemplary minimal promoters can be less than 400, 400, 200, 195, 190, 185, 180, or less nucleotides in length. In some embodiments, the promoter us a U6 promoter.

101171 Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech, and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346- 3351 (1996)), the tetracycline-repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-inducible system (Gossen et al., Science, 268: 1766- 1769 (1995), see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), the RU486- inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)) and the rapamycin-inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Still other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only.

101181 In another embodiment, the native promoter for the transgene is used. The native promoter may be preferred when native expression of the transgene is desired. The native promoter may be used when expression of the transgene must be regulated temporally or developmentally, or in a tissue- specific manner, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may also be used to mimic the native expression. In some embodiments, the promoter is a native promoter (e.g., Cede 141 and/or Pacsin2 promoter). In some examples, the promoter can drive the transgene expression (e.g., Cede 141 and/or Pacsin2 protein or inhibitory nucleic acid targeting Cede 141 and/or Pacsin2) in endothelial cells in the CNS.

101191 In some embodiments, the regulatory sequences impart tissue- specific gene expression capabilities. In some cases, the tissue-specific regulatory sequences bind tissuespecific transcription factors that induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. In some embodiments, the tissue- specific promoter is an endothelial cell-specific promoter.

101201 The 5' untranslated region (5' UTR) (also known as a leader sequence or leader RNA) is the region of an mRNA that is directly upstream from the initiation codon. The 5' UTR plays important roles in both transcriptional and translational regulation of the downstream gene (e.g., the Cede 141 and/or Pacsin2 gene). In some embodiments, a transgene (e.g., transgene for expressing a Cede 141 and/or Pacsin2 protein) comprises a nucleotide encoding a 5' UTR.

101211 The presence of an intron or intervening sequence in mRNA was first described, in vitro, to be important for mRNA processing and increased transgene expression (e.g., Huang ZM, Yen TS. Role of the hepatitis B virus posttranscriptional regulatory element in export of intron less transcripts. Mol Cell Biol. 1995;15(7):3864-3869; Niwa M, Rose SD, Berget SM. In vitro polyadenylation is stimulated by the presence of an upstream intron. Genes Dev. 1990;4(9): 1552-1559, which are incorporated herein by reference). In some embodiments, an isolated nucleic acid described herein may also contain an artificial intron, desirably located between the promoter/enhancer sequence and the nucleotide sequence encoding a Cede 141 and/or Pacsin2 protein or an inhibitory nucleic acid targeting Cede 141 and/or Pacsin2). In some embodiments, an intron is a synthetic or artificial (e.g., heterologous) intron. Examples of synthetic introns include an intron sequence derived from SV-40 (referred to as the SV-40 T intron sequence) and intron sequences derived from the chicken beta-actin gene. In some embodiments, a transgene described by the disclosure comprises one or more (1, 2, 3, 4, 5, or more) artificial introns. In some embodiments, the one or more artificial introns are positioned between a promoter and a nucleotide sequence encoding the transgene. In some embodiments, the isolated nucleic acid comprises a chimeric intron.

101221 In some embodiments, the transgene (e.g., the transgene for expressing a Cede 141 and/or Pacsin2 protein or an inhibitory nucleic acid targeting Cede 141 and/or Pacsin2) expression cassette further comprises a nucleotide sequence encoding a 3 ' UTR located 3' of the nucleotide sequence encoding the Cede 141 and/or Pacsin2 protein or an inhibitory nucleic acid targeting Cede 141 and/or Pacsin2. In some embodiments, the 3 ' UTR is Cede 141 and/or Pacsin2 gene 3 ' UTR.

101231 In some embodiments, the transgene comprises a 3 '-untranslated region (3 -UTR). In some embodiments, the disclosure relates to isolated nucleic acids comprising a transgene encoding a Cede 141 and/or Pacsin2 protein or an inhibitory nucleic acid targeting Cede 141 and/or Pacsin2, and one or more miRNA binding sites. Without wishing to be bound by any particular theory, incorporation of miRNA binding sites into gene expression constructs allows for regulation of transgene expression (e.g., inhibition of transgene expression) in cells and tissues where the corresponding miRNA is expressed. In some embodiments, incorporation of one or more miRNA binding sites into a transgene allows for de-targeting of transgene expression in a cell-type specific manner. In some embodiments, one or more miRNA binding sites are positioned in the 3 ' untranslated region (3 ' UTR) of a transgene, for example between the last codon of a nucleic acid sequence encoding a Cede 141 and/or Pacsin2 protein or an inhibitory nucleic acid targeting Cede 141 and/or Pacsin2, and a poly A sequence.

101241 The methods typically involve administering to a subject an effective amount of a rAAV encoding an agent (e.g., inhibitory nucleic acid inhibiting Cede 141 or Pacsin2, or nucleic acid encoding Cede 141 or Pacsin2) for expressing a transgene in the subject to increase or decrease BBB permeability. Additional embodiments involve administering to a subject an effective amount of a rAAV comprising a nucleic acid encoding a transgene in the subject to increase or decrease BBB permeability. In some embodiments, the transgene of the present disclosure encodes Pacsin2 (e.g., human Pacsin2 or mouse Pacsin2). In some embodiments, the transgene of the present disclosure encodes Cede 141 (e.g., human Cede 141 or mouse Ccdcl41). In some embodiments, the transgene of the present disclosure encodes an inhibitory nucleic acid targeting Cede 141 (e.g., siRNA or shRNA targeting Cede 141 as described herein). In some embodiments, the transgene of the present disclosure encodes an antibody targeting Cede 141. In some embodiments, the transgene of the present disclosure encodes an inhibitory nucleic acid targeting Pacsin2 (e.g., siRNA or shRNA targeting Pacsin2 as described herein). In some embodiments, the transgene of the present disclosure encodes an antibody targeting Pacsin2. An “effective amount” of a rAAV is an amount sufficient to infect a sufficient number of cells of target cells in a subject. In some embodiments, the target cells of the rAAV are endothelial cell of the CNS. In some embodiments, the endothelial cells of the CNS are brain endothelial cells. In some embodiments, the brain endothelial cells are arterial endothelial cells, venous endothelial cells, and capillary endothelial cells. In some embodiments, the brain capillary endothelial cells are brain microvascular endothelial cells (BMVECs). In some embodiments, the endothelial cells of the CNS are spinal cord endothelial cells. In some embodiments, the endothelial cells of the CNS are retina vasculature endothelial cells. In some embodiments, the retina vasculature endothelial cells are superficial plexus arterial endothelial cells, superficial plexus venous endothelial cells, intermediate plexus endothelial cells, or deep plexus endothelial cells. An effective amount of a rAAV may be an amount sufficient to have a therapeutic benefit in a subject, e.g., to extend the lifespan of a subject, to improve and/or reverse in the subject one or more symptoms of disease, or to slow disease progression. The effective amount will depend on a variety of factors such as, for example, the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among subject and tissue.

101251 An effective amount may also depend on the rAAV used. The invention is based, in part on the recognition that rAAV comprising capsid proteins having a particular serotype (e.g., an AAV9 capsid protein variant such as AAV-BI30 or an AAV2 capsid protein variant such as AAV-BR1) mediate more efficient transduction of endothelial cells throughout the CNS than a rAAV comprising capsid proteins having a different serotype. In some embodiments, the rAAV comprises a capsid protein of an AAV serotype of AAV-BI30. In some embodiments, the rAAV comprises a capsid protein of an AAV serotype of AAV-BR1. 101261 In some embodiments, AAV-BI30 or AAV-BR1 has tropism for endothelial cells. In some embodiments, AAV-BI30 has tropism for endothelial cells in the central nervous system (CNS). In some embodiments, the endothelial cells of the CNS are brain endothelial cells. In some embodiments, AAV-BI30 has tropism for brain arterial endothelial cells, brain venous endothelial cells, and brain capillary endothelial cells. In some embodiments, AAV- BI30 has tropism for brain microvascular endothelial cells (BMVECs). In some embodiments, AAV-BI30 has tropism for spinal cord endothelial cells. In some embodiments AAV-BI30 has tropism for retina vasculature endothelial cells. In some embodiments, AAV- BI30 has tropism for retina endothelial cells such as superficial plexus arterial endothelial cells, superficial plexus venous endothelial cells, intermediate plexus endothelial cells, or deep plexus endothelial cells. In some embodiments, AAV-BI30 has tropism for peripheral endothelial cells, such as endothelial cells in the lung (e.g., lung microvasculature endothelial cells), endothelial cells in the aorta, or endothelial cells in the interlobular vessels of the kidney. In some embodiments, AAV-BI30 shows stronger tropism toward endothelial cells in the CNS than endothelial cells in peripheral tissues as measured by any suitable method in the art (e.g., transduce higher percentage of endothelial cells in the CNS and/or with higher transduction efficiency compared to endothelial cells in peripheral tissues). In some embodiments, administration of the rAAV or a composition thereof described herein may result in transduction of one or more of the foregoing endothelial cells. In some embodiments, administration of the rAAV or a composition thereof described herein may result in transduction of all of the foregoing endothelial cells. In some embodiments, administration of the rAAV or a composition thereof described herein may result in transduction of the endothelial cells throughout the CNS, but not the endothelial cells in peripheral tissues.

[0110] In certain embodiments, the effective amount of rAAV is IO ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , or 10 ¹⁴ genome copies per kg. In certain embodiments, the effective amount of rAAV is IO ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , or 10 ¹⁵ genome copies per subject.

[0111] An effective amount may also depend on the mode of administration. For example, targeting endothelial cells in the CNS by intravenous administration or subcutaneous injection may require different (e.g., higher or lower) doses, in some cases, than targeting endothelial cells in the CNS by another method (e.g., local injection to the CNS). The disclosure is based, in part, on the recognition that intravenous injection (i.v.) of rAAV having certain serotypes (e.g., AAV-BI30) mediates efficient transduction of endothelial cells in the CNS. Thus, in some embodiments, the injection is intravenous injection (i.v.). In some embodiments, single dose of a rAAV is administered. In some cases, multiple doses of a rAAV are administered.

[0112] Without wishing to be bound by any particular theory, efficient transduction of endothelial cells in the CNS by rAAV described herein may be useful for the treatment of a subject having a disease associated with endothelial cell dysfunction (e.g., head trauma). Accordingly, methods and compositions for treating disease associated with endothelial cell dysfunction are also provided herein. In some aspects, the disclosure provides a method for treating a disease associated with endothelial cell dysfunction, the method comprising: administering to a subject having or suspected of having a disease associated with endothelial cell dysfunction an effective amount of rAAV, wherein the rAAV comprises (i) an AAV- BI30 capsid protein or an AAV-BR1 capsid protein; and (ii) a nucleic acid comprising a promoter operably linked to a transgene.

[01131 Delivery of the rAAVs to a mammalian subject may be by, for example, injection to the CNS. In some embodiments, the injection is direct injection to the CNS (e.g., intracerebral injection, intraventricular injection, intracisternal injection, intraparenchymal injection, intrathecal injection, and any combination of the foregoing). In some embodiments, the injection is systemic injection (e.g., intravenous injection, intradermal injection, or subcutaneous injection).

[01141 In some embodiments, a composition further comprises a pharmaceutically acceptable carrier. Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the rAAV is directed. “Acceptable” means that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated.

Pharmaceutically acceptable excipients (carriers) including buffers, which are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover. For example, one acceptable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present disclosure.

[01151 The rAAV containing pharmaceutical composition disclosed herein may further comprise a suitable buffer agent, include, but are not limited to, HEPES (4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic acid) buffer, Dulbecco’s phosphate-buffered saline (DPBS) buffer, or Phosphate-buffered Saline (PBS) buffer. Such buffers may comprise disodium hydrogen phosphate and sodium chloride, or potassium dihydrogen phosphate and potassium chloride.

[01161 Optionally, the compositions of the disclosure may contain, in addition to the rAAV and carrier(s), other pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

101171 The rAAV containing pharmaceutical composition described herein comprises one or more suitable surface- active agents, such as a surfactant. Surfactants are compounds that lower the surface tension (or interfacial tension) between two liquids, between a gas and a liquid, or between a liquid and a solid. Surfactants may act as detergents, wetting agents, emulsifiers, foaming agents, and dispersants. Suitable surfactants include, in particular, nonionic agents, such as polyoxyethylenesorbitans (e.g., Tween™ 20, 40, 60, 80, or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80, or 85). Compositions with a surface-active agent will conveniently comprise between 0.05 and 5% surface-active agent, and can be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added, for example, mannitol or other pharmaceutically acceptable vehicles, if necessary.

101181 The rAAVs are administered in sufficient amounts to transduce the cells of a desired tissue (e.g., endothelial cells in the CNS) and to provide sufficient levels of gene transfer and expression without undue adverse effects. Examples of pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ (e.g., CNS), intravenous, intramuscular, subcutaneous, intradermal, and other parental routes of administration. Routes of administration may be combined, if desired.

101191 The dose of rAAV virions required to achieve a particular “therapeutic effect,” e.g., the units of dose in genome copies/per kilogram of body weight (GC/kg), will vary based on several factors including, but not limited to: the route of rAAV virion administration, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. One of skill in the art can readily determine a rAAV virion dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors.

101201 In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar day (e.g., a 24-hour period). In some embodiments, a dose of rAAV is administered to a subject no more than once per 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar week (e.g., 7 calendar days). In some embodiments, a dose of rAAV is administered to a subject no more than bi-weekly (e.g., once in a two-calendar week period). In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar month (e.g., once in 30 calendar days). In some embodiments, a dose of rAAV is administered to a subject no more than once per six calendar months. In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar year (e.g., 365 days or 366 days in a leap year). In some embodiments, a dose of rAAV is administered to a subject once.

IV. Kits and Related Composition

101211 The agents described herein may, in some embodiments, be assembled into pharmaceutical or research kits to facilitate their use in therapeutic, or research applications. A kit may include one or more containers housing the components described herein and instructions for use. Specifically, such kits may include one or more agents described herein, along with instructions describing the intended application and the proper use of these agents. In certain embodiments agents in a kit may be in a pharmaceutical formulation and dosage suitable for a particular application and for a method of administration of the agents. Kits for research purposes may contain the components in appropriate concentrations or quantities for running various experiments.

101221 The kit may be designed to facilitate use of the methods described herein by researchers and can take many forms. Each of the compositions of the kit, where applicable, may be provided in liquid form (e.g., in solution) or in solid form (e.g., a dry powder, a lyophilized powder). In certain cases, some of the compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water, buffered solution, or a cell culture medium), which may or may not be provided with the kit. As used herein, “instructions” can define a component of instruction and/or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, et cetera), internet, and/or web-based communications, et cetera The written instructions may be in a form prescribed by a governmental agency (e.g., US FDA or European Medicines Agency) regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions can also reflect approval by the agency of manufacture, use, or sale for animal administration and/or human use.

101231 The kit may contain any one or more of the components described herein in one or more containers. As an example, in one embodiment, the kit may include instructions for mixing one or more components of the kit and/or isolating and mixing a sample and applying to a subject. The kit may include a container housing agents described herein. The agents may be in the form of a liquid, gel, or solid (e.g., powder). The agents may be prepared sterilely, packaged in syringe, and shipped refrigerated. Alternatively, it may be housed in a vial or other container for storage. A second container may have other agents prepared sterilely. Alternatively, the kit may include the active agents premixed and shipped in a syringe, vial, tube, or other container.

101241 Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

VI. General techniques

101251 The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture-. Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis, et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995) [01261 Without further elaboration, it is believed that one skilled in the art can, based on the present disclosure, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

[01271 Exemplary embodiments of the invention will be described in more detail by the following examples. These embodiments are exemplary of the invention, which one skilled in the art will recognize is not limited to the exemplary embodiments.

[01281 The aspects described herein are not limited to specific embodiments, systems, compositions, methods, or configurations, and as such can, of course, vary. The terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting.

EXAMPLES

Example 1: Regulation of blood-CNS Barrier Permeability by Manipulating Ccdcl41- Pacsin2 Interaction

A platform to rapidly knockdown genes in a brain EC in adult mice

[01291 A brain endothelial- specific viral delivery platform approach was developed to acutely knock out candidate genes in adult mice. Specifically, a mouse line that expresses Cas9 in ECs was generated by crossing the Cre-dependent Cas9 mouse with Tie2:Cre +/- mice. Gene-specific singe-guide RNAs (sgRNAs) were delivered to central nervous system (CNS) endothelial cells in mice using adeno-associated virus (AAV) that specifically infect brain microvessels named AAV-BR1 which allowed acute knock out of any gene of interest in a CRISPR/Cas9 background (FIG. 1A). Injection of 5*el l AAV-BR1 viral particles via tail-vein infected the majority (78%) of blood microvessels in the cortex of adult mice (FIG. IB). This approach enhanced the ability to rapidly identify novel determinants of blood-brain barrier (BBB) integrity in vivo. The system was validated through selective ablation of the glucose transporter 1 (Glutl) in brain endothelial cells in adult mice. Glutl, encoded by Slc2al . is expressed in brain ECs and is essential for neuronal survival and proper brain function and ablation of endothelial Glutl in adult mice, results in a reduction of tight- junction proteins, reduced pericyte coverage and BBB breakdown. Examination of Glutl expression using immunohistochemistry revealed that AAV-BR1- sgRNA:.S7c2a/ successfully eliminated Glutl expression specifically in infected cells (FIG. 1C) of Cre- dependent Cas9 mouse with Tie2:Cre +/- mice. To assess barrier function, mice were intravenously injected with sulfo-NHS -biotin, a small 556 daltons tracer prior to tissue harvesting. Examination of brain tissue revealed robust tracer extravasation throughout the brain of AAV-BR 1 -sgRNA:.S7c2a/ injected mice while no leakage was observed in control mice (FIGs. 1D-1G). These results show that combining CRISPR/Cas9 with AAV-BR1 enable targeted loss-of-function genetic studies in brain endothelial to identify their roles in BBB maintenance in adult mice.

Transcriptome analysis of CNS and periphery ECs identified Ccdcl41, which is highly enriched in CNS ECs

[01301 A comparative analysis of CNS and peripheral endothelial cells from published transcriptome datasets was performed to identify genes enriched at the BBB. Cede 141 was identified as highly enriched in BBB endothelial cells compared to peripheral endothelial cells in all published datasets, including in newly formed blood-retina-barrier ECs. To confirm Cede 141 enrichment in CNS vasculature, Cede 141 mRNA localization in brain and lung was analyzed using commercially available RNAscope probes for Cede 141 (FIGs. 2A- 2B). Cede 141 mRNA probes were specifically localized to ECs in the cortex and showed ~ 14-fold higher density compared to lung ECs (FIG. 2C). To validate Cede 141 protein expression in CNS endothelial cells, a western-blot of purified endothelial prep from brain and lung tissues was performed. Cede 141 protein concentrations in brain endothelial cells were four-times greater than lung endothelial cells (FIG. 2D). Together, these results confirm that Cede 141 is expressed in CNS endothelial cells.

Ccdcl41 is required for BBB maintenance but not for normal vessel patterning 101311 To examine whether Cede 141 is important for BBB maintenance, Cede 141 was knocked down using AAV-BR1 delivered sgRNAs targeting Ccdcl41 in Cas9f/-;Tie2Cre and control (Cas9f/-) mice. Three weeks post viral injection, an 85% reduction in Ccdcl41 protein level in brain capillary lysates from Ccdcl41KD (Ccdcl41 knockdown) mice (AAV- BRl-sg Ccdcl41; Cas9f/-;Tie2Cre) was observed relative to control animals (AAV-BRl-sg Cede 141; Cas9f/-) (FIGs. 3A-3C). To determine the effects of endothelial- specific Cede 141 reduction on blood-brain barrier function, sulfo-NHS -biotin was injected into Ccdcl41KD mice prior to tissue harvesting. Ablation of Cede 141 resulted in tracer extravasation in multiple brain areas including the cortex, thalamus, hippocampus, and olfactory bulb while in control animals the tracer was confined to blood vessels (FIG. 3D). Increased tracer leakage was observed specifically surrounding vessels of Ccdcl41KD mice (FIGs. 3E). Tracer extravasation in the cortex permeated -23% of the parenchyma in sgCcdcl41,Cre+ animals compared to less than 1% in control animals (FIG. 3F). Leakage was not limited to small tracers, as tracer extravasation was observed also using 10 kD dextran and 44 kD horseradish peroxidase (HRP) and >100 kD endogenous IgG (FIG. 3G). Cede 141 ablation in endothelial cells did not affect viability or other vascular functions. Cede 141 ablation in neurons altered neuronal cell morphology and caused cell death without impacting vessel morphology (FIGs. 4A-4B). Vessel morphology and density was unchanged in Ccdcl41KD mice relative to controls (FIG. 5A-5B). These data demonstrate that Ccdcl41 is specifically required for BBB integrity but not for vessel patterning and health, indicating a direct role of Cede 141 in barrier function.

Ccdcl41 regulates BBB function by suppressing tubular vesicle trafficking

[01321 To understand the underlying subcellular cause for BBB leakage in sgCcdcl41 KD animals, brain sections of HRP-iv injected animals were examined using electron microscopy (EM). Surprisingly, the HRP-filled vesicles were tubular in shape, in contrast to previously observed caveolae vesicles. There was a ~3-fold increase in tracer-filled vesicles in endothelial cells of sgCcdcl41 injected animals compared to control animals (FIGs. 6A-6C). Caveolin-1 protein was not located in increased tracer- filled tubular vesicles observed in Ccdcl41KD mice, as determined by immune-electron microscopy using caveolin-1 antibodies. As a control, caveolin-1 gold particles labeled robustly the caveolae vesicles in the arteriolar endothelial and neighboring smooth muscle cells (FIG. 6D-6E). Therefore, it was determined that Cede 141 regulates tubular vesicle transcytosis is caveolae-independent. Cede 141 KD and control mice exhibit functional tight junctions with 44kDa HRP-and 1.9kDa microperoxidase tracers successfully halted between endothelial cells. Expression of tight junction proteins including Cldn5, Zol (FIGs. 7C-7D) and Cadh5 were indistinguishable between sgCcdcl41 RNA-treated and control mice.

Ccdcl41 inhibits Pacsin2 expression, a known tubular vesicle trafficking regulator, in CNS ECs

[01331 The family of BAR-domain containing proteins previously described as effectors in tubular shaped vesicle endocytosis were examined in response to Cede 141 inhibition. Pacsin2 (protein kinase C and casein kinase substrate in neurons protein 2, also known as Syndapin- 2), which are normally expressed at high levels in periphery endothelial cells and low levels in brain endothelial cells, was upregulated in the brain endothelial cells of Cede 141 KD mice (FIGs. 8A-8F) indicating that a reduction Cede 141 induces upregulation in Pacsin2 and subsequent formation of tubular transcytotic vesicles.

Pacsin2 over-expression in CNS ECs is sufficient to cause BBB leakage and upregulation of tubular vesicle transcytosis

The effects of Pacsin2 over-expression were examined in the brain. AAV-BR1 expressing either Pacsin2 or mRuby3 fluorescent protein were iv injected into adult mice and sulfo- NHS-biotin leakage assays were performed after 3 weeks of the viral injection. Pacsin2 expression was increased in brain endothelial cells following injection with AAV-BR1 expressing Pacsin2, but not with mRuby3 (FIG. 9A-9C). Mice injected with AAV- Pacsin2 but not AAV-mRuby3 exhibited leakage in the brain (FIGs. 9D-9F) an increase in tubular vesicles in brain ECs in AAV-Pacsin2 injected mice but not in AAV-mRuby3 injected mice (FIG. 9G) demonstrating that upregulation of Pacsin2 in brain ECs was sufficient to increase BBB permeability and tubular vesicle-mediated transcytosis. The analysis of electron microscopy images of these brains showed that Pacsin2 overexpression results in increased tubular vesicles in CNS endothelial cells relative to controls (FIG. 9H). Further, leakage was not limited to small tracers, and endogenous IgG leakage was also observed in AAV-Pacsin2 injected mice but not in AAV-mRuby3 injected mice (FIGs. 9I-9J). Ccdcl41 regulates BBB by inhibiting Pacsin2 expression thus suppressing tubular vesicle mediated transcytosis Genetic rescue experiments were performed to determine if induced tubular vesicle transcytosis and BBB leakage were due to increased Pacsin2 expression in brain endothelial cells resulting from Cede 141 knockdown. AAVs harboring the sgRNAs targeting Pacsin2 (sgPacsin2); Tie2Cre or its Cre- control mice were iv injected to Ccdcl41 KD and control mice. sgCcdcl41 injected animals exhibit upregulation of Pacsin2 in brain endothelial cells; however, sgPacsin2 and double infected animals did not exhibit elevated Pacsin2 immunoreactivity. BBB permeability was evaluated using sulfo-NHS -biotin tracer localization in the brain after intravenous injection. sgCcdcl41 animals demonstrated increased tracer leakage, while an intact barrier was observed in sgPacsin2 injected mice. Mice that were infected with both sgCcdcl41 and sgPacsin2 exhibited only mild leakage, wherein about 6% of the parenchyma was positive for the sulfo-NHS -biotin tracer (FIGs. 10A-10B). These findings suggest that upregulation in Pacsin2 is a crucial component in the leakage observed after Cede 141 ablation in brain endothelial cells. Pacsin2 is expressed in aged human brain [01341 Cognitive impairment in aging and neurodegenerative disorders has been linked to a dysfunctional blood-brain barrier. The immunohistological examination of an aging human brain (78 years old) showed the expression of Pacsin2 in the blood vessels (FIG. 11).

Pacsin2 is upregulated in the HSV-induced neuroinflammation

[01351 Neuroinflammatory pathways are critical components in the pathogenesis of neurodegenerative diseases (e.g., ageing, early onset dementia). Pacsin2 protein levels were studied in mouse brains in a HSV-induced neuroinflammation model. Immunostained mouse cortical 1 sections show that Pacsin2 protein was upregulated in endothelial cells of HSV- infected mice compared to uninfected mice (FIG. 12).

References

[01361 Ritter, B., Modregger, J., Paulsson, M., & Plomann, M. (1999). PACSIN 2, a novel member of the PACSIN family of cytoplasmic adapter proteins. FEBS letters, 454(3), 356- 362.

[01371 Leite, D. M., Matias, D., & Battaglia, G. (2020). The role of BAR proteins and the glycocalyx in brain endothelium transcytosis. Cells, 9(V2), 2685.

[01381 Hansen, C. G., Howard, G., & Nichols, B. J. (2011). Pacsin 2 is recruited to caveolae and functions in caveolar biogenesis. Journal of Cell Science, 124( 6), 2777-2785.

[01391 Hutchins, B. I., Kotan, L. D., Taylor-Burds, C., Ozkan, Y., Cheng, P. J., Gurbuz, F., ... & Wray, S. (2016). CCDC141 mutation identified in anosmic hypogonadotropic hypogonadism (Kallmann syndrome) alters GnRH neuronal migration. Endocrinology , 157(5), 1956-1966.

OTHER EMBODIMENTS

[01401 All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[01411 From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims. INCORPORATION BY REFERENCE

[01421 The present application refers to various issued patent, published patent applications, scientific journal articles, and other publications, all of which are incorporated herein by reference. The details of one or more embodiments of the invention are set forth herein. Other features, objects, and advantages of the invention will be apparent from the Detailed Description, the Figures, the Examples, and the Claims.

EQUIVALENTS AND SCOPE

[01431 In the articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Embodiments or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[01441 Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claims that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

101451 This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the embodiments. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any embodiment, for any reason, whether or not related to the existence of prior art.

101461 Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended embodiments. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.