WO2024059688A2 - Régulation de la barrière du système nerveux central sanguin (snc sanguin) et ses utilisations - Google Patents

Régulation de la barrière du système nerveux central sanguin (snc sanguin) et ses utilisations Download PDF

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WO2024059688A2
WO2024059688A2 PCT/US2023/074158 US2023074158W WO2024059688A2 WO 2024059688 A2 WO2024059688 A2 WO 2024059688A2 US 2023074158 W US2023074158 W US 2023074158W WO 2024059688 A2 WO2024059688 A2 WO 2024059688A2
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cns
pacsin2
blood
cede
disease
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WO2024059688A9 (fr
WO2024059688A3 (fr
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Chenghua GU
Shachar Y. DAGAN
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Harvard University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]

Definitions

  • 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.
  • kits and methods for regulating Blood-Central Nervous System (blood-CNS) barrier permeability e.g., increasing or decreasing Blood-CNS barrier permeability
  • 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.
  • 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.
  • a method for increasing Blood-Central Nervous System (Blood-CNS) Barrier permeability to treat a CNS disease in a subject comprises administering the subject an inhibitor of Cede 141 at the Blood-CNS Barrier.
  • the blood-CNS barrier is the blood-brain barrier. In other embodiments, the blood-CNS barrier is the blood-retina barrier.
  • the inhibitor of Cede 141 is capable of inhibiting Cede 141 expression and/or activity.
  • the inhibitor of Cede 141 is an inhibitory nucleic acid targeting Cede 141.
  • the inhibitory nucleic acid targets the coding sequence of Cede 141 comprising SEQ ID NO: 1 or SEQ ID NO: 2.
  • the inhibitory nucleic acid targeting Cede 141 is a siRNA, a shRNA, a miRNA, an AmiRNA, an aptamer, or an ASO.
  • the inhibitory nucleic acid targeting Cede 141 is a siRNA targeting Cede 141.
  • the siRNA targeting Cede 141 comprises an antisense strand complementary to any one of SEQ ID NOs: 9-21. 1
  • the inhibitory nucleic acid targeting Cede 141 is a shRNA targeting Cede 141.
  • the shRNA comprises a guide strand complementary to any one of SEQ ID NOs: 9-21.
  • the inhibitor of Cede 141 is an antibody, an antibody variant or an antigen-binding fragment targeting Cede 141.
  • the inhibitor of Cede 141 is a small molecule.
  • the inhibitor of Cede 141 is delivered to CNS endothelial cells.
  • the injection is systemic injection comprising intravenous injection, intramuscular injection, subcutaneous injection, or intraperitoneal injection.
  • the injection is direct injection to the CNS comprising intracerebral injection, intraventricular injection, intracisternal injection, intraparenchymal injection, intrathecal injection, and any combination thereof.
  • 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.
  • the method comprising administering an isolated nucleic acid comprising a transgene encoding Pacsin2.
  • the inhibitory nucleic acid is delivered by a recombinant adeno-associated virus (rAAV).
  • rAAV recombinant adeno-associated virus
  • the capsid protein is BRI, BI30, AAV1, AAV2, AAV9, or variant thereof (e.g., AAV-PHP or AAV-PHP.B).
  • the capsid protein is BRI.
  • the capsid protein is BI30.
  • the inhibitor of Cede 141 is administered to the subject via injection.
  • the transgene further comprises a promoter operably linked to a nucleotide sequence encoding Pacsin2.
  • the isolated nucleic acid further comprises two adeno-associated virus inverted terminal repeats (ITRs) flanking the transgene encoding Pacsin2.
  • the isolated nucleic acid is administered to the subject via a recombinant adeno-associated virus (rAAV).
  • the rAAV further comprises a capsid protein.
  • the rAAV comprises a capsid protein comprising BRI, BI30, AAV1, AAV2, AAV9, or variant thereof.
  • the capsid protein is BI30.
  • the method further comprises administering a therapeutic agent for treating the CNS disease.
  • the present disclosure provides a method for delivering a therapeutic agent to the central nervous system to treat a CNS disease is described herein.
  • 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.
  • Blood-CNS Blood-Central Nervous System
  • the agent for increasing Blood-CNS barrier permeability is an inhibitor of Cede 141.
  • the agent for increasing Blood-CNS barrier permeability is an isolated nucleic acid comprising a transgene encoding Pacsin2.
  • the CNS disease is a neuromuscular disease, neurodegenerative disease, brain and nerve tumors, Neurogenetic Diseases, Cognitive disorders, Familial dystonia, Neuroinfectious disease, neuropsychiatric disorders.
  • the neuromuscular disease is Amyotrophic Lateral Sclerosis (ALS), Ataxia, Cerebral Palsy, Muscular Dystrophy.
  • 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.
  • the present disclosure provides a method for decreasing Blood-Central Nervous System (Blood-CNS) Barrier permeability to treat a CNS disease in a subject.
  • the method comprises administering the subject an inhibitor of Pacsin2 at the Blood-CNS Barrier.
  • the inhibitor of Pacsin2 is capable of inhibiting Pacsin2 expression and/or activity.
  • the inhibitor of Pacsin2 is an inhibitory nucleic acid targeting Pacsin2.
  • the inhibitory nucleic acid targets the coding sequence of Pacsin2 comprising SEQ ID NOs: 5 and 6.
  • the inhibitory nucleic acid targeting Pacsin2 is a siRNA, a shRNA, a miRNA, an AmiRNA, an aptamer, or an ASO.
  • the inhibitory nucleic acid targeting Pacsin2 is a siRNA targeting Pacsin2.
  • the siRNA targeting Pacsin2 comprises an antisense strand complementary to any one of SEQ ID NOs: 22-31, and 36-45.
  • the inhibitory nucleic acid targeting Pacsin2 is a shRNA targeting Pacsin2.
  • the shRNA targeting Pacsin2 comprises a guide strand complementary to any one of SEQ ID NOs: 22-31, and 36-45.
  • the inhibitor of Pacsin2 is an antibody, an antibody variant or an antigen-binding fragment targeting Pacsin2.
  • the inhibitor of Pacsin2 is a small molecule.
  • 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).
  • the rAAV comprises a capsid protein comprising BRI, BI30, AAV1, AAV2, AAV9, or variant thereof.
  • the capsid protein is BRI. In some embodiments, the capsid protein is BI30.
  • the inhibitor of Pacsin2 is administered to the subject via injection.
  • the injection is systemic injection comprising intravenous injection, intramuscular injection, subcutaneous injection, or intraperitoneal injection.
  • 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.
  • 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.
  • the method comprises administering an isolated nucleic acid comprising a transgene encoding Cede 141.
  • the transgene further comprises a promoter operably linked to a nucleotide sequence encoding Cede 141.
  • the isolated nucleic acid further comprises two adeno- associated virus inverted terminal repeats (ITRs) flanking the transgene encoding Cede 141.
  • the isolated nucleic acid is administered to the subject via a recombinant adeno-associated virus (rAAV).
  • the rAAV further comprises a capsid protein.
  • the capsid protein is BRI, BI30, AAV1, AAV2, AAV9, or variant thereof.
  • the capsid protein is BRI.
  • the capsid protein is BI30.
  • 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.
  • the retinal disease is diabetic retinopathy.
  • the neurodegenerative disease is Huntington’s disease, Alzheimer's disease, Parkinson's disease, motor neuron disease, Amyotrophic lateral sclerosis, spinal muscular atrophy, spinocerebral ataxia.
  • the acute injury of the CNS is stroke or head trauma.
  • the neuroinfectious disease is encephalitis, sepsis, or COVID- 19.
  • the primary cancer of the CNS is glioblastoma, meningioma, or lymphoma.
  • the metastatic cancer of the CNS is lung cancer, metastatic breast cancer, GI track cancers, or melanoma.
  • the autoimmune disease of the CNS is multiple sclerosis.
  • the neuroinflammatory condition is CNS Lupus, CNS Lyme Disease, Neurosarcoidosis, Neuromyelitis optica (NMO), or Paraneoplastic and Autoimmune Encephalitis.
  • the cognitive disorder is dementia resulting from Alzheimer’s disease, Lewy body dementia, frontotemporal dementia, encephalopathy, or post-acute COVID syndrome.
  • 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.
  • the condition is associated with aging.
  • the condition is early onset dementia.
  • the condition is stroke.
  • the condition is spinal cord injury.
  • 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. 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.
  • 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.
  • FIG. 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. 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
  • 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.
  • 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.
  • 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.
  • 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
  • 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 the Coiled-Coil Domain Containing 141 (Ccdcl41).
  • 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).
  • 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.
  • Cede 141 is linked to congenital hypogonadotropic hypogonadism in humans.
  • 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.
  • Cede 141 is expressed by CNS endothelial cells.
  • Cede 141 plays a role in regulating CNS endothelial cell function (e.g., regulating blood-CNS barrier permeability).
  • Ccdcl41 regulates blood-CNS barrier permeability via Pacsin2.
  • the presence of Cede 141 in CNS endothelial cells inhibits Pacsin2.
  • inhibition of Pacsin2 by Cede 141 results in decreased tubular-vesicle mediated transcytosis of the CNS endothelial cells.
  • 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.
  • ECs brain endothelial cells
  • overexpression of Pacsin2 in brain ECs increases blood-CNS barrier permeability.
  • overexpression of Pacsin2 in brain ECs up-regulates tubular vesicles.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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
  • 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).
  • the blood-CNS barrier includes the blood-brain barrier (BBB) and blood-retina barrier (BRB).
  • BBB blood-brain barrier
  • BRB blood-retina barrier
  • 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.
  • the endothelial cells of the CNS are brain endothelial cells.
  • the brain endothelial cells are arterial endothelial cells, venous endothelial cells, and/or capillary endothelial cells.
  • the brain capillary endothelial cells are brain microvascular endothelial cells (BMVECs).
  • BMVECs brain microvascular endothelial cells
  • the endothelial cells of the CNS are spinal cord endothelial cells.
  • the endothelial cells of the CNS are retina vasculature endothelial cells.
  • 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.
  • 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.
  • pathogens e.g., bacteria, viruses, or parasites
  • 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.
  • the blood-CNS barrier is generally very effective at excluding, e.g., pathogens, from the CNS, the blood-CNS barrier poses a daunting obstacle when a drug needs to be delivered to the CNS.
  • the present disclosure 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)).
  • 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.
  • Cede 141 is expressed by CNS endothelial cells.
  • the Cede 141 protein expressed by CNS endothelial cells regulates tubular vesicle-mediated transcytosis via inhibition of Pacsin2 in endothelial cells in the CNS.
  • 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.
  • transcytosis e.g., tubular vesicle mediated transcytosis
  • 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.
  • a disease e.g., brain and CNS tumors, ALS, etc.
  • 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).
  • 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.
  • an inhibitor e.g., inhibitory nucleic acids, small molecule inhibitors, and/or antibodies targeting Pacsin2 signaling.
  • 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).
  • 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
  • diseases associated with increased blood-CNS barrier permeability e.g., ageing related neurodegenerative conditions (e.g., early onset dementia), brain vasculature diseases (e.g.
  • a molecule e.g., a therapeutic agent
  • an inhibitor of Cede 141 or an activator of Pacsin2 e.g., Pacsin2 coding sequence
  • 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).
  • blood-CNS Blood- Central Nervous System
  • the present disclosure provides compositions and methods, kits and uses for increasing blood-CNS barrier permeability.
  • the method comprises administering to a subject an inhibitor of Cede 141 signaling.
  • the Ccdcl41 is expressed by CNS endothelial cells.
  • 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.
  • the inhibitor of Cede 141 signaling is a Cede 141 inhibitor.
  • inhibition of Cede 141 in CNS endothelial cells results in increased blood-CNS barrier permeability.
  • the Cede 141 inhibitor is capable of inhibiting Cede 141 expression and/or activity.
  • the Cede 141 inhibitor is an inhibitory nucleic acid targeting Cede 141 mRNA.
  • 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
  • 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 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 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 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.
  • 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.
  • siRNA molecules may be determined by the binding of the antisense strand of the molecule to its target RNA (e.g., Cede 141 mRNA).
  • target RNA e.g., Cede 141 mRNA
  • the siRNA molecules are 60, 65, 70, 75, 80, 85, 90, 95, 100, or more base pairs in length.
  • 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.
  • 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.
  • 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.
  • the antisense sequence of the siRNA molecule is 7, 8, 9, 10,
  • 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.
  • the sense sequence of the siRNA molecule is 7, 8, 9, 10, 11,
  • 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.
  • 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)).
  • a Cede 141 mRNA e.g., human Cede 141 mRNA (SEQ ID NO: 1) or mouse Ccdcl41 mRNA (SEQ ID NO: 2).
  • 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).
  • the target region is a region of consecutive nucleotides in a CCDC141 mRNA (e.g., human CCDC141 mRNA or mouse Cede 141 mRNA).
  • 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).
  • 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.
  • 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.
  • 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,
  • 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)).
  • 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 (SEQ ID NO: 1) or mouse Cede 141 mRNA (SEQ ID NO: 2)).
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • target RNA sequence e.g., Cede 141 mRNA; SEQ ID NO: 1 or SEQ ID NO: 2.
  • the shRNA molecules are 60, 65, 70, 75, 80, 85, 90, 95, 100 or more base pairs in length.
  • 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.
  • 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.
  • shRNA molecules that comprise a nucleotide sequence complementary to all or a portion of the target sequence i.e., an antisense sequence
  • shRNA molecules that comprise a nucleotide sequence complementary to all or a portion of the target sequence i.e., an antisense sequence
  • shRNA molecules that comprise a nucleotide sequence complementary to all or a portion of the target sequence i.e., an antisense sequence
  • 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.
  • the shRNA targeting inhibitory nucleic acid described herein comprises an antisense sequence and a sense sequence.
  • the antisense sequence of the shRNA molecule is 7, 8, 9, 10,
  • 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.
  • the sense sequence of the shRNA molecule is 7, 8, 9, 10, 11,
  • 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.
  • 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).
  • a Cede 141 mRNA e.g., human Cede 141 mRNA or mouse Cede 141 mRNA.
  • 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.
  • a Ccdcl41 mRNA e.g., human Ccdcl41 mRNA or mouse Ccdcl41 mRNA
  • 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).
  • 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).
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the Cede 141 inhibitor is an antibody, an antibody variant or an antigen-binding fragment thereof targeting Cede 141.
  • An 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.
  • an antibody is a full-length antibody.
  • 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.
  • 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.
  • 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).
  • an antibody comprises a constant domain, e.g., an Fc region.
  • An immunoglobulin constant domain refers to a heavy or light chain constant domain.
  • the heavy chain of an antibody described herein can be an alpha (a), delta (A), epsilon (E), gamma (y) or mu (p) heavy chain.
  • 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.
  • an antibody described herein comprises a human gamma 1 CHI, CH2, and/or CH3 domain.
  • 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.
  • a human constant region sequence such as any known in the art.
  • 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.
  • 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.
  • an antibody is modified, e.g., modified via glycosylation, phosphorylation, sumoylation, and/or methylation.
  • an antibody is a glycosylated antibody, which is conjugated to one or more sugar or carbohydrate molecules.
  • 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.
  • the one or more sugar or carbohydrate molecule are monosaccharides, disaccharides, oligosaccharides, or glycans.
  • the one or more sugar or carbohydrate molecule is a branched oligosaccharide or a branched glycan.
  • the one or more sugar or carbohydrate molecule includes a mannose unit, a glucose unit, an N-acetylglucosamine unit, or a phospholipid unit.
  • 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.
  • 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).
  • 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.
  • 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.
  • administering results in increased blood-CNS barrier permeability.
  • 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.
  • an agent that promotes the Pacsin2 signaling at the blood-CNS barrier is a recombinant human Pacsin2 or a fragment thereof.
  • 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).
  • an exemplary human Pacsin2 amino acid sequence is set forth in SEQ ID NO: 7 (NP_001171899.1): MSVTYDDSVGVEVSSDSFWEVGNYKRTVKRIDDGHRLCSDLMNCLHERARIEKAYAQQLTEW ARRWRQLVEKGPQYGTVEKAWMAFMSEAERVSELHLEVKASLMNDDFEKIKNWQKEAFHKQM MGGFKETKEAEDGFRKAQKPWAKKLKEVEAAKKAHHAACKEEKLAISREANSKADPSLNPEQ LKKLQDKIEKCKQDVLKTKEKYEKSLKELDQGTPQYMENMEQVFEQCQQFEEKRLRFFREVL LEVQKHLDLSNVAGYKAI YHDLEQSIRAADAVEDLRWFRANHGPGMAMNWPQFEEWSADLNR TLSRREKKKATDGVTLTGINQTGDQSLPSKPSSTLNVPSNPAQSQSSY
  • an agent that promotes the Pacsin2 signaling at the blood-CNS barrier is a recombinant mouse Pacsin2 or a fragment thereof.
  • 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).
  • an exemplary mouse Pacsin2 amino acid sequence is set forth in SEQ ID NO: 8
  • an agent that promotes the Pacsin2 signaling at the blood-CNS barrier is a nucleic acid encoding Pacsin2 (e.g., human or mouse Pacsin2).
  • the nucleic acid encoding Pacsin2 encodes a mammalian Pacsin2 (e.g., human Pacsin2, mouse Pacsin2, rat Pacsin2, non-human primate Pacsin2, etc.).
  • the nucleic acid encoding Pacsin2 encodes a human Pacsin2.
  • the nucleic acid encoding Pacsin2 encodes a mouse Pacsin2.
  • 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:
  • 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):
  • mouse Pacsin2 mRNA sequence is set forth in SEQ ID NO: 6 (NM_001159509.1).
  • 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):
  • an effective amount of an agent that increases blood-CNS barrier permeability 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.
  • 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.
  • 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.
  • 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 inhibitor e.g., Cede 141 inhibitor
  • the inhibitor described herein is a small molecule and can be taken orally.
  • an agent promoting Pacsin2 expression/activity is an rAAV encoding Pacsin2.
  • administering an inhibitor of Cede 141 signaling decreases the level and/or activity of Cede 141.
  • 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.
  • administration of an inhibitors described herein results in increasing of the permeability of the blood-CNS barrier in the subject.
  • 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.
  • administering an agent that promotes Pacsin2 expression/activity e.g., rAAV encoding Pacsin 2.
  • 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.
  • 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.
  • administering 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.
  • 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
  • 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.
  • PCT dynamic perfusion CT
  • DCEMRI dynamic contrast-enhanced magnetic resonance imaging
  • 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.
  • NSE neuron-specific enolase
  • GFAP glial fibrillary acidic protein
  • CSF cerebral spinal fluid
  • 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.
  • a family history of the condition e.g., by genetic analysis
  • 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.
  • compositions for increasing blood-CNS permeability can be administered to a subject having or diagnosed as having a disease affecting the CNS.
  • 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.
  • “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.
  • 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.
  • 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.
  • methods which comprise administering an inhibitor of Cede 141 signaling can further comprise administering a therapeutic agent to the subject.
  • 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).
  • a therapeutic agent and a composition for increasing blood-CNS permeability are administered sequentially (e.g., administration of a composition for increasing blood-CNS permeability prior to administration of the therapeutic agent).
  • a therapeutic agent and a composition for increasing blood- CNS permeability 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).
  • a therapeutic agent and a composition for increasing blood-CNS permeability 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).
  • 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.
  • 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,
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • the therapeutic agent is an agent that does not normally cross the blood-CNS barrier.
  • 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.
  • 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.
  • Delivery of an inhibitor of Cede 141 signaling at the blood-CNS barrier to a mammalian subject may be by, for example, injection to the CNS.
  • 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).
  • the injection is systemic injection (e.g., intravenous injection, intradermal injection, or subcutaneous injection).
  • the inhibitor can be administered orally.
  • the present disclosure also provides methods for decreasing blood- CNS barrier permeability (i.e., maintaining blood-CNS integrity) in a subject.
  • the method comprises administering to the subject an agent that promotes Cede 141 signaling.
  • the method comprises administering to the subject an agent that inhibits Pacsin2 signaling at the blood-CNS barrier.
  • an agent that promotes the Cede 141 signaling decreases expression and/or activity of Pacsin2.
  • an agent that promotes the Cede 141 signaling at the blood- CNS barrier is a recombinant Cede 141 or a fragment thereof.
  • an agent that promotes the Cede 141 signaling at the blood-CNS barrier is a recombinant human Cede 141 or a fragment thereof.
  • 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).
  • an exemplary human Ccdcl41 amino acid sequence is set forth in SEQ ID NO: 3 (NP_001303674.1):
  • an agent that promotes the Ccdcl41 signaling at the blood- CNS barrier is a recombinant mouse Cede 141 or a fragment thereof.
  • 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).
  • an exemplary mouse Cede 141 amino acid sequence is set forth in SEQ ID NO: 4 (NP_001020747.2):
  • an agent that promotes the Ccdcl41 signaling at the blood- CNS barrier is a nucleic acid encoding Cede 141.
  • 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).
  • the nucleic acid encoding Cede 141 encodes a human Cede 141.
  • the nucleic acid encoding Cede 141 encodes a mouse Cede 141.
  • 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:
  • 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,
  • An exemplary nucleotide sequence encoding mouse Cede 141 protein is set forth in SEQ ID NO: 2 (NM-001025576.3):
  • the present disclosure 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.
  • 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).
  • the method comprises administering to a subject an inhibitor of Pacsin2 signaling.
  • the Pacsin2 is expressed by CNS endothelial cells.
  • the method comprises administering to the subject an agent that inhibits Pacsin2 signaling at the blood-CNS barrier.
  • the inhibitor of Pacsin2 signaling is a Pacsin2 inhibitor.
  • inhibition of Pacsin2 in CNS endothelial cells results in decreased blood- CNS barrier permeability.
  • inhibition of Pacsin2 does not result in decreased blood-CNS barrier permeability.
  • the Pacsin2 inhibitor is capable of inhibiting Pacsin2 expression and/or activity.
  • the Pacsin2 inhibitor is an inhibitory nucleic acid targeting Pacsin2 mRNA.
  • 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.
  • an inhibitory nucleic acid targeting Pacsin2 is an siRNA.
  • siRNA molecules comprise a specific antisense sequence in addition to the reverse complement (sense) sequence.
  • 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).
  • the siRNA molecules are 60, 65, 70, 75, 80, 85, 90, 95, 100 or more base pairs in length.
  • 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.
  • 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.
  • 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)).
  • a Pacsin2 mRNA e.g., human Pacsin2 mRNA (SEQ ID NO: 5) or mouse Pacsin2 mRNA (SEQ ID NO: 6).
  • 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).
  • the target region is a region of consecutive nucleotides in a Pacsin2 mRNA (e.g., human Pacsin2 mRNA or Pacsin2 Cede 141 mRNA).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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)).
  • a Pacsin2 mRNA e.g., human Pacsin2 mRNA (SEQ ID NO: 5) or mouse Pacsin2 mRNA (SEQ ID NO: 6).
  • 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.
  • a Pacsin2 mRNA e.g., human Pacsin2 mRNA or mouse Pacsin2 mRNA.
  • 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.
  • 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.
  • 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.
  • Pacsin2 siRNAs target sequences are set forth below:
  • CAAAGAGGAGAAGCTGGCTATCTCA (SEQ ID NO: 26)
  • CAGCTACGAGAAGACCCAGAGCTAT (SEQ ID NO: 30)
  • 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.
  • 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
  • an inhibitory nucleic acid targeting Pacsin2 is an shRNA.
  • 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).
  • the shRNA molecules are 60, 65, 70, 75, 80, 85, 90, 95, 100 or more base pairs in length.
  • 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.
  • 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.
  • 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).
  • shRNA Short Hairpin RNA
  • 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.
  • the shRNA targeting inhibitory nucleic acid described herein comprises an antisense sequence and a sense sequence.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • a Pacsin2 mRNA e.g., human Pacsin2 mRNA or mouse Pacsin2 mRNA.
  • 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.
  • a Pacsin2 mRNA e.g., human Pacsin2 mRNA or mouse Pacsin2 mRNA
  • the target region is a region of consecutive nucleotides in a Pacsin2 mRNA (e.g., human Pacsin2 mRNA or mouse Pacsin2 mRNA).
  • 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).
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • viral vector mediated delivery e.g., AAV, retrovirus, adenovirus, or lentivirus
  • ceDNA ceDNA
  • 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.
  • administering results in decreased blood-CNS barrier permeability.
  • an effective amount of an agent that decreases blood-CNS barrier permeability 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.
  • administering may result in inhibition of Pacsin2 signaling and/or increase of Cede 141 signaling of one or more of the CNS endothelial cells.
  • 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.
  • 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).
  • a Pacsin2 inhibitor described herein is a small molecule and can be administered orally.
  • administering an inhibitor of Pacsin2 signaling at the blood- CNS Barrier decreases the level and/or activity of Pacsin2.
  • 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.
  • administration of the Pacsin2 inhibitors described herein results in decreasing of the permeability of the blood-CNS barrier in the subject.
  • 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.
  • 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.
  • PCT dynamic perfusion CT
  • DCEMRI dynamic contrast- enhanced magnetic resonance imaging
  • NSE neuron-specific enolase
  • GFAP glial fibrillary acidic protein
  • CSF cerebral spinal fluid
  • administering an agent that promotes Cede 141 signaling at the blood-CNS Barrier increases the level and/or activity of Ccdcl41.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • NSE neuron-specific enolase
  • GFAP glial fibrillary acidic protein
  • S 100P cerebral spinal fluid
  • 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).
  • 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.
  • 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.
  • Endothelial cells 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).
  • endothelial cells e.g., endothelial cells in the CNS not having a dysfunction, or at risk of developing endothelial cell dysfunction
  • dysfunctional endothelial cells e.g., endothelial cells causing abnormal vasculature permeability, hemodynamics, or neuroimmune crosstalk, et cetera.
  • “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).
  • 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).
  • 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.
  • the disease associated with CNS endothelial cell dysfunction is a CNS primary cancer, such as glioblastoma, meningioma, or lymphoma.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • PCT dynamic perfusion CT
  • DCEMRI dynamic contrast-enhanced magnetic resonance imaging
  • protein biomarkers e.g., neuron- specific enolase (NSE), glial fibrillary acidic protein (GFAP), and S 100(3) in the cerebral spinal fluid (CSF), etc.
  • 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.
  • the disease is a CNS primary cancer, such as glioblastoma, meningioma, or lymphoma.
  • the disease is a metastatic cancer to the brain such as metastatic lung cancer, metastatic breast cancer, or melanoma.
  • the disease is a neuroinflammatory disease, such as CNS Lupus, CNS Lyme Disease, Neurosarcoidosis, Neuromyelitis optica (NMO), or Paraneoplastic and Autoimmune Encephalitis.
  • 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.
  • 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.
  • 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).
  • the injection is systemic injection (e.g., intravenous injection, intradermal injection, or subcutaneous injection).
  • an agent that promotes Cede 141 signaling or inhibits Pacsin2 signaling at the blood-CNS barrier can be administered orally.
  • the present disclosure provides compositions and methods for delivering a transgene to endothelial cells throughout the CNS in a subject.
  • the disclosure provides isolated and/or engineered AAVs.
  • 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).
  • an AAV capsid protein e.g., AAV-BI30, AAV-BR1, AAV9 or variants thereof
  • 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).
  • 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 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).
  • transgene 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.
  • the AAV capsid is an important element in determining tissue-specific targeting capabilities of the virus.
  • a rAAV having a capsid appropriate for the tissue being targeted can be selected.
  • 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).
  • 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.
  • 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.
  • ITRs AAV inverted terminal repeats
  • 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.
  • the molecular weights of VP1, VP2 and VP3 are about 87 kDa, about 72 kDa, and about 62 kDa, respectively.
  • capsid proteins upon translation, form a spherical 60-mer protein shell around the viral genome.
  • the functions of the capsid proteins are to protect the viral genome, deliver the genome, and interact with the host.
  • capsid proteins deliver the viral genome to a host in a tissue-specific or cell-specific manner.
  • 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).
  • the AAV capsid protein is AAV-BI30.
  • the AAV capsid protein is AAV-BR1.
  • the capsid protein is of AAV serotype 9 (AAV9).
  • an AAV capsid protein is of a serotype derived from AAV9 (e.g., an AAV9 capsid variant).
  • the AAV9 capsid variant is AAV9.PHP.B.
  • the isolated nucleic acids of the invention may be recombinant adeno-associated virus (AAV) vectors (rAAV vectors).
  • AAV adeno-associated virus
  • 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
  • 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.
  • an rAAV encoding a Cede 141 protein comprises the nucleic acid sequence of SEQ ID NOs: 1 or 2.
  • an rAAV encoding a Pacsin2 protein comprises the nucleic acid sequence of SEQ ID NOs: 5 or 6.
  • an rAAV encoding an inhibitory nucleic acid targeting Pacsin2 comprises the nucleic acid sequence of SEQ ID NOs: 9-21.
  • an rAAV encoding an inhibitory nucleic acid targeting Cede 141 comprises the nucleic acid sequence of SEQ ID NOs: 22-31, and 36-45
  • 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.
  • a 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.
  • 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
  • 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.
  • 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.
  • 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.
  • the promoter is a constitutive promoter.
  • 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).
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • PGK phosphoglycerol kinase
  • 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.
  • 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.
  • 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.
  • MT zinc-inducible sheep metallothionine
  • Dex dexamethasone
  • MMTV mouse mammary tumor virus
  • T7 polymerase promoter system WO 98/10088
  • ecdysone insect promoter No et al., Proc. Natl. Acad. Sci. USA, 93:3346- 3351 (
  • 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.
  • 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.
  • other native expression control elements such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may also be used to mimic the native expression.
  • the promoter is a native promoter (e.g., Cede 141 and/or Pacsin2 promoter).
  • 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.
  • the regulatory sequences impart tissue-specific gene expression capabilities.
  • the tissue-specific regulatory sequences bind tissuespecific transcription factors that induce transcription in a tissue-specific manner.
  • tissue-specific regulatory sequences e.g., promoters, enhancers, etc.
  • the tissue- specific promoter is an endothelial cell-specific promoter.
  • 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).
  • a transgene e.g., transgene for expressing a Cede 141 and/or Pacsin2 protein
  • 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).
  • 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.
  • a transgene described by the disclosure comprises one or more (1, 2, 3, 4, 5, or more) artificial introns.
  • the one or more artificial introns are positioned between a promoter and a nucleotide sequence encoding the transgene.
  • the isolated nucleic acid comprises a chimeric intron.
  • 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.
  • the 3 ' UTR is Cede 141 and/or Pacsin2 gene 3 ' UTR.
  • the transgene comprises a 3 '-untranslated region (3 -UTR).
  • 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.
  • miRNA binding sites 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.
  • incorporation of one or more miRNA binding sites into a transgene allows for de-targeting of transgene expression in a cell-type specific manner.
  • 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.
  • 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.
  • agents e.g., inhibitory nucleic acid inhibiting Cede 141 or Pacsin2, or nucleic acid encoding Cede 141 or Pacsin2
  • 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.
  • the transgene of the present disclosure encodes Pacsin2 (e.g., human Pacsin2 or mouse Pacsin2).
  • 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.
  • Cede 141 e.g., human Cede 141 or mouse Ccdcl41
  • 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).
  • an “effective amount” of a rAAV is an amount sufficient to infect a sufficient number of cells of target cells in a subject.
  • the target cells of the rAAV are endothelial cell of the CNS.
  • the endothelial cells of the CNS are brain endothelial cells.
  • the brain endothelial cells are arterial endothelial cells, venous endothelial cells, and capillary endothelial cells.
  • the brain capillary endothelial cells are brain microvascular endothelial cells (BMVECs).
  • the endothelial cells of the CNS are spinal cord endothelial cells.
  • the endothelial cells of the CNS are retina vasculature endothelial cells.
  • 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.
  • 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.
  • the rAAV comprises a capsid protein of an AAV serotype of AAV-BI30.
  • the rAAV comprises a capsid protein of an AAV serotype of AAV-BR1.
  • AAV-BI30 or AAV-BR1 has tropism for endothelial cells.
  • AAV-BI30 has tropism for endothelial cells in the central nervous system (CNS).
  • the endothelial cells of the CNS are brain endothelial cells.
  • AAV-BI30 has tropism for brain arterial endothelial cells, brain venous endothelial cells, and brain capillary endothelial cells.
  • AAV- BI30 has tropism for brain microvascular endothelial cells (BMVECs).
  • BMVECs brain microvascular endothelial cells
  • AAV-BI30 has tropism for spinal cord endothelial cells.
  • 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.
  • endothelial cells in the lung e.g., lung microvasculature endothelial cells
  • endothelial cells in the aorta e.g., endothelial cells in the aorta, or endothelial cells in the
  • 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).
  • administration of the rAAV or a composition thereof described herein may result in transduction of one or more of the foregoing endothelial cells.
  • administration of the rAAV or a composition thereof described herein may result in transduction of all of the foregoing endothelial cells.
  • 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.
  • 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.
  • 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.
  • the injection is intravenous injection (i.v.).
  • single dose of a rAAV is administered.
  • multiple doses of a rAAV are administered.
  • 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.
  • 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.
  • Delivery of the rAAVs to a mammalian subject may be by, for example, injection to the CNS.
  • 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).
  • the injection is systemic injection (e.g., intravenous injection, intradermal injection, or subcutaneous injection).
  • 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.
  • 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.
  • one acceptable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline).
  • 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.
  • 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.
  • DPBS Dulbecco’s phosphate-buffered saline
  • PBS Phosphate-buffered Saline
  • buffers may comprise disodium hydrogen phosphate and sodium chloride, or potassium dihydrogen phosphate and potassium chloride.
  • 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.
  • the rAAV containing pharmaceutical composition described herein comprises one or more suitable surface- active agents, such as a surfactant.
  • a surfactant 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., TweenTM 20, 40, 60, 80, or 85) and other sorbitans (e.g., SpanTM 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.
  • 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.
  • a desired tissue e.g., endothelial cells in the CNS
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • kits may include one or more containers housing the components described herein and instructions for use.
  • kits may include one or more agents described herein, along with instructions describing the intended application and the proper use of these agents.
  • 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.
  • 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 may be provided in liquid form (e.g., in solution) or in solid form (e.g., a dry powder, a lyophilized powder).
  • 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.
  • a suitable solvent or other species for example, water, buffered solution, or a cell culture medium
  • “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
  • audiovisual e.g., videotape, DVD, et cetera
  • internet e.g., a wireless local area network
  • 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.
  • governmental agency e.g., US FDA or European Medicines Agency
  • the kit may contain any one or more of the components described herein in one or more containers.
  • 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.
  • the kit may include the active agents premixed and shipped in a syringe, vial, tube, or other container.
  • 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).
  • AAV adeno-associated virus
  • 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.
  • 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).
  • Cede 141 protein expression in CNS endothelial cells 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
  • Cede 141 was knocked down using AAV-BR1 delivered sgRNAs targeting Ccdcl41 in Cas9f/-;Tie2Cre and control (Cas9f/-) mice.
  • 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).
  • Ccdcl41 regulates BBB function by suppressing tubular vesicle trafficking
  • 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.
  • 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
  • Pacsin2 over-expression in CNS ECs is sufficient to cause BBB leakage and upregulation of tubular vesicle transcytosis
  • 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.
  • FIG. 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).
  • 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.
  • Pacsin2 is upregulated in the HSV-induced neuroinflammation
  • 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).
  • 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.
  • 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.
  • 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.

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Abstract

La présente divulgation concerne des compositions, des méthodes, des kits et des utilisations de régulation de la perméabilité de la barrière du système nerveux central sanguin (CNS sanguin) (p. ex. augmentation ou diminution de la perméabilité de la barrière du CNS sanguin) par régulation de la signalisation entre la cellule endothéliale dérivée de Ccdc 141 et la Pacsin2 exprimée dans les cellules endothéliales du SNC.
PCT/US2023/074158 2022-09-14 2023-09-14 Régulation de la barrière du système nerveux central sanguin (snc sanguin) et ses utilisations Ceased WO2024059688A2 (fr)

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