Primate carbonic anhydrase IV binding peptide and AAV

Abstract

Disclosed herein, including novel BBB spanning receptors on the blood brain barrier (BBB) interface, targeting peptides and derivatives thereof capable of binding to these novel receptors, and related methods of using these receptors to increase permeability of BBB and deliver agents to the nervous system (e.g., CNS).

Description

[0001] Statement on federally funded research and development This invention was carried out with government support under license number NS111369 granted by the National Institutes of Health. The government holds certain rights in this invention. Technical Field

[0002] This disclosure generally relates to the field of gene delivery. More specifically, methods and compositions for crossing the blood-brain barrier are disclosed.

[0003] Reference to sequence list This application is filed together with an electronic sequence list. The sequence list is provided as a file named RECE-004-01WO.xml, which is 3,502 kilobytes in size and was created on May 15, 2024. The information in the electronic sequence list is incorporated herein by reference in its entirety. Background Technology

[0004] Targeting therapeutic and research molecules to the desired tissue and cell types, while keeping them away from those mediating potentially dangerous side effects, is a fundamental challenge in drug development. This is especially true for molecules targeting the brain. The blood-brain barrier (BBB) ​​severely limits the properties of therapeutic and research molecules that, after systemic administration into the peripheral bloodstream, may enter the central nervous system (CNS). The BBB is a fundamental bottleneck in developing effective CNS research tools and therapies. This structure primarily comprises brain endothelial cells, necessitating the delivery of macromolecules via invasive intracranial injection, technically challenging focused ultrasound, or receptor-mediated transcytosis. For a long time, an incomplete understanding of the mechanisms involved in transcytosis has hindered the rational design of macromolecules across the BBB, with only a few targets, such as the transferrin receptor, validated for research and therapy.

[0005] Specifically, the transport of therapeutic and diagnostic materials (drugs, genes, antibodies, oligonucleotides) across the blood-brain barrier (BBB) ​​to the central nervous system (CNS) and across the blood-retinal barrier to the eye poses a significant obstacle to addressing brain and eye diseases. (Challis, RC, Ravindra Kumar, S., Chen, X., Goertsen, D., Coughlin, GM, Hori, AM, Chuapoco, MR, Otis, TS, Miles, TF, and Gradinaru, V. (2022). Adeno-Associated Virus Toolkit to Target Diverse Brain Cells. *Annu Rev Neurosci*) 45 , 447-469.10.1146 / annurev-neuro-111020-100834. Summary of the Invention

[0006] The need to transport therapeutic and diagnostic materials (drugs, genes, antibodies, oligonucleotides) across the blood-brain barrier (BBB) ​​to the central nervous system (CNS) and across the blood-retinal barrier to the eye poses a significant obstacle to addressing brain and eye diseases. Conventional methods require intracranial injection of molecules to deliver therapeutic targets to cells.

[0007] Therefore, in some aspects, the present invention provides a method for increasing the permeability of the blood-brain barrier. In some embodiments, the method comprises providing a CA4-binding peptide (also referred to as a “targeting peptide”) capable of binding to primate and / or human carbonic anhydrase 4 (CA4 or CA-IV), thereby increasing the permeability of the blood-brain barrier.

[0008] In some respects, CA4-binding peptides are listed in Tables 1, 3, 4, and 5. In some embodiments, the targeting peptides of the present invention increase the permeability of the blood-brain barrier by at least 25%, 50%, 75%, 100%, or more compared to the absence of a targeting peptide.

[0009] The present invention further provides a method for delivering a payload to the nervous system of a subject. In some embodiments, the method comprises: providing a targeting peptide or a derivative thereof capable of binding to human CA4, primate CA4, or a derivative thereof, wherein the targeting peptide is part of a delivery system and wherein the delivery system comprises a payload to be delivered to the nervous system; and administering the delivery system to the subject.

[0010] In some embodiments, the delivery system comprises nanoparticles, nanotubes, nanowires, dendritic macromolecules, liposomes, liposomes and aqueous liposomes, polymer vesicles and nonionic surfactant vesicles, foams, hydrogels, cubes, quantum dots, exosomes, macrophages, and any combination thereof. In some embodiments, the delivery system comprises a viral vector or a non-viral vector.

[0011] In some embodiments, the CA4-binding peptide enhances the binding affinity of the viral vector or the non-viral vector to human CA4 or primate CA4. In some embodiments, the viral vector comprises an AAV vector. In some embodiments, the targeting peptide is part of the capsid protein of the AAV vector. In some embodiments, the AAV vector is a vector selected from the group consisting of: AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-DJ, human isolate hu.31, human isolate hu.32, rhesus monkey isolate rh.8, rhesus monkey isolate rh.10, and variants thereof. In some embodiments, the non-viral vector comprises lipid-based nanoparticles, polymer nanoparticles, inorganic nanoparticles, surfactant-based emulsions, nanowires, silica nanoparticles, peptide- or protein-based particles, lipid-polymer particles, nanolipoprotein particles, and combinations thereof.

[0012] In some embodiments, the payload or therapeutic cargo to be delivered to the nervous system is a biomolecule, a non-biological molecule, or a combination thereof. In some embodiments, the biomolecule is selected from the group consisting of nucleic acid sequences, proteins, peptides, lipids, polysaccharides, and any combination thereof. In some embodiments, the payload is a therapeutic molecule. In some embodiments, the nucleic acid sequence to be delivered to the nervous system comprises one or more of the following: a) a sequence encoding a nutrient factor, growth factor, or other soluble factor that may be released from the transduced cell and affect the survival or function of the cell and / or surrounding cells; b) DNA for restoring protein function in a human or animal carrying a gene mutation; c) DNA encoding a protein capable of controlling or altering the activity or state of a cell; d) DNA encoding a protein or nucleic acid for assessing the state of a cell; e) DNA and / or associated guide RNA for genome engineering; f) a sequence for genome editing via homologous recombination; g) a DNA sequence encoding therapeutic RNA; h) shRNA or artificial miRNA delivery systems; or i) a DNA sequence affecting the splicing of endogenous genes.

[0013] The present invention has the advantageous recognition that systemic delivery of adeno-associated virus (AAV) provides a more widely distributed, non-invasive method compared to direct brain or eye injection, which is particularly beneficial for diffuse neurological disorders.

[0014] Therefore, in some aspects, the present invention provides methods for increasing the permeability of the blood-brain barrier to deliver therapeutic cargo across the BBB to the central nervous system and / or the eyes. In some embodiments, the present invention provides AAV bound to rhesus monkey CA4 (rhCA4) or human CA4 (huCA4) for CNS therapeutic cargo delivery in both preclinical and clinical applications in these species. In other advantageous aspects of the invention, CA4 is also highly concentrated in the brain, eyes, and intestines of primates compared to other reported transport proteins with broader expression patterns, such as transferrin receptors. Therefore, the methods for delivering therapeutic cargo across the CNS provided by the present invention may reduce off-target organ delivery.

[0015] Mouse CA4 is typically used to screen and develop AAVs to assess the BBB permeability of capsid-containing AAVs. However, there are differences in the AAV binding pockets between mouse CA4 and primate CA4. Specifically, engineered msCar4-binding AAVs, such as 9P31 or 9P36, which do not bind to rhCA4 or huCA4, have been identified in mice via directed evolution without mechanistic insights. Therefore, there is a need to identify AAVs capable of binding to primate or human CA4. This invention recognizes that such AAVs would be able to deliver therapeutic cargo across the CNS of primates and / or humans.

[0016] Therefore, in some aspects, the present invention provides CA4-binding peptides that bind to human CA4 and / or primate CA4. Unless otherwise stated, the CA4-binding peptides mentioned herein are peptides that bind to human CA4 and / or primate CA4. In some embodiments, the CA4-binding peptide is inserted into the capsid protein of an AAV. In some preferred embodiments, the CA4-binding peptide is inserted between two adjacent amino acids of AA587-594 in SEQ ID NO: 1 of the AAV9 vector, or between functional equivalents of AA587-594 in an amino acid sequence that is at least 80% identical to that of SEQ ID NO: 1. In some embodiments, the targeting peptide is inserted between AA588-589 in SEQ ID NO: 1 of the AAV9 vector, or between functional equivalents of AA588-589 in an amino acid sequence that is at least 80% identical to that of SEQ ID NO: 1. In some embodiments, the CA4-binding peptide is listed in Tables 1, 3, 4, and 5.

[0017] In some aspects, the present invention provides AAV capsid proteins that bind to primate CA4 and / or human CA4. In some embodiments, these AAV capsid proteins include an insertion of a CA4-binding peptide. In some embodiments, these AAV capsid proteins or portions thereof that bind to primate and / or human CA4 are listed in Tables 1, 3, 4, and 5.

[0018] In some aspects, the present invention provides AAVs comprising an AAV capsid protein that binds to primate CA4 and / or human CA4. In some embodiments, these AAV capsid proteins comprise an insertion of a CA4-binding peptide. In some embodiments, these AAV capsid proteins or portions thereof that bind to primate and / or human CA4 are listed in Tables 1, 3, 4, and 5.

[0019] In some aspects, the present invention provides a recombinant adeno-associated virus (rAAV). In some embodiments, the rAAV comprises any of the AAV capsid proteins disclosed herein. In some embodiments, the rAAV comprises an AAV capsid protein containing a CA4-binding peptide having binding specificity to human CA4 and / or primate CA4, wherein the amino acid sequence of the targeting peptide is inserted between two adjacent amino acids or their functional equivalents in AA587-594 of the AAV9 capsid protein. In some embodiments, the two adjacent amino acids are AA588 and AA589. In some embodiments, the rAAV exhibits enhanced tropism for this nervous system compared to rAAV without the targeting peptide. In some embodiments, the rAAV is capable of transducing this nervous system at a transduction efficiency at least twice that of rAAV without the targeting peptide.

[0020] In some embodiments, the AAV or rAAV of the present invention is used to cross the BBB and efficiently transduce the CNS of primates using a variety of non-invasive administration routes, including systemic administration.

[0021] In some embodiments, the methods of the present invention enable these AAVs to be more readily applicable in preclinical testing and clinical intervention for neurological diseases. Additionally, in some embodiments, peptide modifications of these AAVs are further recruited for non-viral delivery, such as for antibodies, antibody-drug conjugates, oligonucleotides, enzymes, proteins, larger synthetic molecules, exosomes, nanoparticles, and contrast agents.

[0022] In some aspects, the present invention provides carbonic anhydrase IV (CA4) binding peptides listed in Tables 1, 3, 4, and 5. In some aspects, the present invention provides AAV listed in Tables 1, 3, 4, and 5. These CA4 binding peptides and AAVs listed in Tables 1, 3, 4, and 5 provide genetic pathways for the central nervous system of primates after noninvasive systemic delivery. In some embodiments, the present invention provides 9-amino acid peptides listed in Tables 4 and 5.

[0023] In some embodiments, the present invention provides the AAV provided in Tables 4 and 5. In some embodiments, the AAV comprises a 7-amino acid peptide insertion at position 588 / 589 of AAV9 and an AQ or DG at positions 587 to 588.

[0024] In some aspects, the present invention provides huCA4 binding AAV Alpha 43, 45, 48, and 49. In some embodiments, huCA4 binding AAV-binding peptides are listed in Table 1. In some preferred embodiments, the present invention provides huCA40 binding AAV peptides: DGVVHETVR; DGVVGVNIR; DGEVGLTVR; DGIVGSTIR.

[0025] In some preferred aspects, the present invention provides a method for treating or diagnosing conditions related to the CNS and / or the eye by administering a composition comprising the peptides or AAVs provided herein. In some embodiments, the peptides and / or AAVs of the present invention allow for the delivery across the BBB of therapeutic goods for treating the CNS and / or the eye. In some embodiments, the present invention provides a method for treating brain conditions. In some embodiments, the brain conditions treated by the compositions of the present invention include brain cancer, neurodegeneration, and glioblastoma. In some embodiments, the eye-related condition is glaucoma.

[0026] In some embodiments, the present invention provides compositions for delivering a pharmaceutical agent to the nervous system of a subject in need. In some embodiments, the composition comprises an AAV comprising (1) the AAV capsid protein disclosed herein and (2) a pharmaceutical agent to be delivered to the nervous system of the subject; optionally wherein the nervous system is the central nervous system (CNS), the peripheral nervous system (PNS), or a combination thereof. In some embodiments, the nervous system is brain endothelial cells, neurons, capillaries in the brain, small arteries in the brain, arteries in the brain, or a combination thereof. In some embodiments, the composition is a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers. In some embodiments, the pharmaceutical agent to be delivered comprises nucleic acids, peptides, small molecules, aptamers, or a combination thereof.

[0027] In some aspects, the present invention provides a delivery system for delivering peptides in the CNS. In some embodiments, the delivery system comprises (1) a CA4-binding peptide specific for primate CA4 and / or human CA4; and (2) a pharmaceutical agent. In some embodiments, the targeting peptide (1) is displayed on the surface of the delivery system; or (2) is partially embedded in the delivery system. In some embodiments, the delivery system may be selected from the group consisting of nanoparticles, nanotubes, nanowires, dendritic macromolecules, liposomes, liposomes and aqueous liposomes, polymer vesicles and nonionic surfactant vesicles, foams, hydrogels, cubes, quantum dots, exosomes, macrophages, and combinations thereof. In some embodiments, the delivery system comprises a viral vector or a nonviral vector. In some embodiments, the delivery system comprises nanoparticles selected from the group consisting of lipid-based nanoparticles, polymer nanoparticles, inorganic nanoparticles, surfactant-based emulsions, nanowires, silica nanoparticles, virus-like particles, peptide- or protein-based particles, lipid-polymer particles, nanolipoprotein particles, and combinations thereof.

[0028] In some embodiments, the present invention provides HA-tagged huCA4 as a capture agent for resin-based screening of AAV / antibody-engineered systems. In some embodiments, the present invention provides HA-tagged rhCA4 as a capture agent for resin-based screening of AAV / antibody-engineered systems. In some embodiments, the rhCA4 sequence is provided in Table 2.

[0029] In some aspects, the present invention provides for the administration of compositions comprising the peptides provided herein to a subject. In some embodiments, the compositions of the present invention are administered via systemic bloodstream delivery. In some embodiments, the compositions of the present invention are administered via local direct injection. In some embodiments, the present invention provides compositions comprising the AAV and / or peptides of the present invention. In some embodiments, the compositions are injectable. In some embodiments, the compositions are ointments. In some embodiments, the compositions are oral compositions. In some embodiments, the compositions are oral compositions. Attached Figure Description

[0030] Figure 1 An overview of the selection process for the CA4-binding peptide of the present invention is provided.

[0031] Figure 2 The present invention provides a unique CA4-AAV combination composition.

[0032] Figure 3 An option for msCar4-based resin with low-dose library input (1.6e11 vg) is provided.

[0033] Figure 4An option for msCar4-based resin with high-dose library input (8e11 vg) is provided.

[0034] Figure 5 AAV variant performance is provided in resin-based selections at both low and high doses.

[0035] Figure 6 The performance of AAV variants in cell-based selection at different doses is provided.

[0036] Figure 7 The AAV variant performance was provided across a selection of LY6A resin-based formulations at low doses (Sample 1, 1.6e11 vg) and high doses (Sample 2, 8e11 vg).

[0037] Figure 8 A library pool generated using the method provided in this invention is provided.

[0038] Figure 9 Data on AAV association with huCA4 after round 2 selection at a low library input dose (5e10 vg) are provided.

[0039] Figure 10 Data on AAV binding with huCA4 after round 2 selection at a high library input dose (2.5e11 vg) are provided.

[0040] Figure 11 Data for independent round 1 selected replicas 2 of huCA4 combined with AAV at low (sample 1, 1.6e11 vg) and high (sample 2, 8e11) library input doses are provided.

[0041] Figure 12 Data are provided for independent first-round selected replicas 3 of huCA4 combined with AAV at low (sample 1, 1.6e11 vg) and high (sample 2, 8e11) library input doses.

[0042] Figure 13 The second round of dropdown selection results for rhCA4 combined with AAV is provided for low (sample 1, 5e10 vg) and high (sample 2, 2.5e11 vg) library input doses.

[0043] Figure 14 Independent first-round selection replicas 2 of rhCA4-AAV were provided at low (sample 1, 1.6e11 vg) and high (sample 2, 8e11) library input doses.

[0044] Figure 15Independent first-round selection replicas 2 of rhCA4-AAV were provided at low (sample 1, 1.6e11 vg) and high (sample 2, 8e11) library input doses.

[0045] Figure 16 Data on the second round of dropdown selection results for huCA4 combined with AAV at a low library input dose (5e10 vg) are provided.

[0046] Figure 17 Data on the second round of dropdown selection results for huCA4 combined with AAV at a high library input dose (2.5e11 vg) are provided.

[0047] Figure 18 Data from the second round of dropdown selections for rhCA4 combined with AAV are provided for low (sample 1, 5e10 vg) and high (sample 2, 2.5e11 vg) library input doses.

[0048] Figure 19 Data from pull-down assays evaluating the binding of individual AAV variants to huCA4 are provided.

[0049] Figure 20 Data are provided for evaluating huCA4-enhanced cell transduction in Alpha 43, 45, 48, and 49.

[0050] Figure 21 A pull-down assay was provided to evaluate the binding of individual AAV variants to rhCA4.

[0051] Figure 22 An overview of mice with humanized CA4 is provided.

[0052] Figure 23 Data on the binding performance of huCA4 to AAV in mice are provided.

[0053] Figure 24 Cell-based selection data for huCA4 binding to AAV are provided.

[0054] Figure 25 Data from the third round of dropdown selection results for huCA4 combined with AAV at low (Sample 1, 1.6e10 vg) and high (Sample 2, 7.8e10 vg) library input doses are provided.

[0055] Figure 26 Cell-based selection data for rhCA4-AAV combination in round 3 are provided.

[0056] Figure 27The third round of dropdown selection effects of rhCA4 combined with AAV were provided at low (Sample 1, 1.6e10 vg) and high (Sample 2, 7.8e10 vg) library input doses. Detailed Implementation

[0057] In the following detailed description, reference is made to the accompanying drawings, which form part of the detailed description. In the drawings, like reference numerals generally identify like components unless the context otherwise requires. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that, as generally described herein and as shown in the drawings, aspects of this disclosure can be arranged, substituted, combined, separated, and designed in a variety of different configurations, all of which are expressly covered herein and form part of this disclosure.

[0058] All patents, published patent applications, other publications and sequences from GenBank, and other databases mentioned herein are incorporated in their entirety by reference to the relevant technologies.

[0059] Blood-brain barrier and CNS Transporting therapeutic and diagnostic materials (drugs, genes, antibodies, oligonucleotides) across the blood-brain barrier (BBB) ​​to the central nervous system (CNS) and across the blood-retinal barrier to the eyes poses a significant obstacle to addressing brain and eye diseases.

[0060] The blood-brain barrier (BBB) ​​is a complex, dynamic, and adaptive interface that controls the exchange of substances between the central nervous system (CNS) and the blood, preventing uncontrolled leakage of substances from the blood into the brain. The cells constituting the BBB primarily include brain endothelial cells, which constantly communicate with other cells in the CNS (e.g., astrocytes, microglia, neurons, mast cells, pericytes, and circulating immune cells) to adjust their behavior to meet the needs of the CNS, respond to pathological symptoms, and, in some cases, participate in the onset, maintenance, or progression of disease. The complexity of BBB function explains much of the difficulty in developing drugs that can cross the BBB. Utilizing receptors at the BBB interface could provide a way to cross the BBB.

[0061] Carbonic anhydrase IV Carbonic anhydrase IV is an isoenzyme belonging to the carbonic anhydrase family, a zinc metalloenzyme family. This isoenzyme catalyzes the reversible reaction of CO2 hydration, enabling the enzyme to regulate intracellular and extracellular CO2 and H2O. + and HCO3 -Concentration. Carbonic anhydrases are involved in a variety of biological processes, including respiration, calcification, acid-base balance, bone resorption, and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid. Carbonic anhydrases exhibit extensive diversity in tissue distribution and their subcellular localization. Mammalian carbonic anhydrases have at least seven genetically distinct isoenzymes, designated I-VII, each of which catalyzes the reversible hydration of carbon dioxide via the zinc hydroxide mechanism. Physiological functions regulated by carbonic anhydrases include, for example, the removal of HCO3- from the lungs through respiration. - HCO3 in the kidneys - Carbonic anhydrases are involved in various metabolic processes, including urea production, gluconeogenesis, and lipogenesis. They are also used for the reuse of bodily fluids, the production of aqueous humor in the eyes, cerebrospinal fluid in the brain, gastric juice production in the stomach, pancreatic juice, and bone resorption by osteoclasts. Members of the carbonic anhydrase family also play important roles in metabolic processes such as urea production, gluconeogenesis, and lipogenesis.

[0062] Unlike other soluble carbonic anhydrases that attach to the plasma membrane via transmembrane domains, carbonic anhydrase IV (CA4) is a glycosylphosphatidyl-inositol-anchored membrane isoenzyme. Carbonic anhydrase IV is widely conserved in vertebrates and exhibits a similar CNS expression profile in humans, with recent single-cell analysis of the human brain vascular system confirming CA4 expression in the human BBB. Carbonic anhydrase IV has been shown to regulate pH, which is associated with neural firing, and can influence neuronal function through ion-gated channels.

[0063] In some embodiments, the carbonic anhydrase IV disclosed herein is human carbonic anhydrase IV (CA4). CA4 is known to be located on the luminal surface of cerebral endothelial cells throughout the cortex and cerebellum, where it enzymatically regulates the carbon dioxide-bicarbonate balance. Human CA4 has previously been characterized as a 35-kDa protein with “high activity” in CO2 hydration and in catalyzing HCO3-. -Human CA4 exhibits higher dehydration activity than other isoenzymes. Generally, human CA4 contains an 18-amino acid signal sequence at the N-terminus of the protein translocated in the endoplasmic reticulum (ER), and a 260-amino acid "CA domain" containing the active site amino acid residues, showing 30-36% homology to cytoplasmic CA. At the C-terminus, an additional 27 amino acid residues, containing a hydrophobic sequence of 21 amino acids sufficient to cross the membrane, are preceded by a 6-amino acid signal sequence for GPI anchoring. Amino acid residue Ser 266 has been identified as the GPI anchor attachment site. Removal of the C-terminal hydrophobic domain present in the CA4 precursor has a significant impact on GPI anchoring, cell surface expression, and enzyme activity. Based on the amino acid sequence deduced from the nucleotide sequence, human CA4 does not contain the classic common site for N-glycosylation (Asn-Xxx-Ser / Thr). Human CA4 also does not contain oligosaccharide chains, while other mammalian carbonic anhydrase IV (e.g., mouse carbonic anhydrase IV (Car4)) is a glycoprotein with one or more oligosaccharide side chains.

[0064] In some embodiments, the carbonic anhydrase IV (Car4) disclosed herein as a receptor for enhancing BBB crossing can be any carbonic anhydrase IV, such as mouse Car4, human CA4, or its homologs or variants. Carbonic anhydrase IV homologs and / or variants can be derived from vertebrate species, including but not limited to mice, rats, humans, cattle, rabbits, monkeys, pigs, horses, rainbow trout, chimpanzees, squirrels, chickens, goats, and sheep. Carbonic anhydrase IV homologs from various species can be found in publicly available databases identifiable by those skilled in the art, including, for example, UniProt, NCBI, and SwissProt.

[0065] In some embodiments, the sequences of human CA4 (huCA4) and rhesus monkey CA4 (rhCA4) are provided in Table 2A. In some embodiments, carbonic anhydrase IV homologs and variants may be about or at least 50% identical to the sequences huCA4 and / or rhCA4 provided in Table 2A (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or values ​​or ranges between any two of these values). In some embodiments, huCA4 is at least 75%, about 80%, about 85%, about 90%, about 95%, or about 98% identical to the sequence provided in SEQ ID NO: 1. In some embodiments, huCA4 is at least 75%, about 80%, about 85%, about 90%, about 95%, or about 98% identical to the sequence provided in SEQ ID NO: 2. In some embodiments, rhCA4 is at least 75%, about 80%, about 85%, about 90%, about 95%, or about 98% identical to the sequence provided in SEQ ID NO: 3. In some embodiments, rhCA4 is at least 75%, about 80%, about 85%, about 90%, about 95%, or about 98% identical to the sequence provided in SEQ ID NO: 4.

[0066] In some embodiments, the human CA4 receptor disclosed herein as a receptor for enhancing BBB crossing comprises the sequence provided in WO2023 / 168333, which is incorporated herein by reference in its entirety. In some embodiments, the human CA4 receptor is provided in NCBI Reference Sequence: NM_000717.5, which is incorporated herein by reference in its entirety.

[0067] CREATE system In some embodiments, the CA4-binding peptides of this disclosure are isolated using the CREATE system, as described in Deverman et al. (Nature Biotechnology 34(2):204-209 (2016)) and International Patent Application Publications WO2015038958 and WO 2017100671, the contents of each of which are incorporated herein by reference in their entirety. “CREATE” or “Cre-based AAV targeting evolution” refers to an AAV capsid selection strategy that selects the capsid of a transduction target tissue (e.g., CNS or PNS) after intravenous injection. This method has been demonstrated in mouse models.

[0068] A library of AAV capsids with one or more target peptide insertions is developed and administered intravenously to transgenic mice. Specific targeting of these Cre-expressing transgenic mice can be developed; for example, GFAP-Cre mice can be used to target astrocytes. In some embodiments, the Cre / LoxP-mediated system can be used to knock out or overexpress and / or ectopically express CA4 to identify target peptides that interact with CA4.

[0069] Compared to other AAV serotypes (including parental AAV particles and capsids), variations in the target sequence and transgenic animal models enable the selection of AAV variants with desired transduction profiles, such as tropism towards neurons or astrocytes.

[0070] The CREATE method involves generating a library of targeting peptides and then assembling it into a viral genome backbone containing parental AAV capsid sequences. An AAV capsid library (AAV particles) is then generated, purified, and administered to transgenic animals (e.g., mice). Target tissues are collected, and AAV sequences are selectively recovered from Cre-expressing cells. These sequences are evaluated and characterized to identify targeting peptides that enrich the target tissue (i.e., enhance transduction or tropism). Target peptides and associated AAV particles can then be generated for further testing and characterization. This process is considered a round of evolution or selection. In some embodiments, more than one round of evolution is performed. Up to 15 rounds of selection can be performed.

[0071] More specifically, the CREATE system uses the rAAV-Cap-in-cis-lox viral genome, which contains regulatory elements of the AAV cap and AAV rep genes, as well as a Cre reversible switch. Because this viral genome lacks the fully functional rep gene required for AAV particle production, the rep is provided in trans form. Modified AAV2 / 9 Rep-Cap plasmids can be provided, with stop codons provided in the frame to prevent the expression of VP1-VP3 proteins.

[0072] The capsid library is generated using the rAAV-Cap-in-cis-lox viral genome as a backbone. A targeting peptide is inserted at any position into the parental AAV capsid protein (e.g., AAV9), thereby generating a fully functional AAV capsid protein and AAV particles. The targeting peptide can be designed by any method known in the art. In some embodiments, the targeting peptide is generated using polymerase chain reaction (PCR). AAV particles containing the capsid protein with the targeting peptide insertion are generated, and the viral genome encoding a reporter gene (e.g., GFP) is encapsulated therein. These AAV particles (or AAV capsid libraries) are then administered to transgenic mice via intravenous delivery to the tail vein. Administration of these capsid libraries to Cre-expressing mice results in the expression of the reporter gene payload in the target tissue due to Cre expression.

[0073] AAV particles and / or viral genomes can be recovered from target tissues to identify enriched target peptides and associated AAV particles, indicating enhanced transduction in the target tissue. Standard methods in the art, such as, but not limited to, next-generation sequencing (NGS), viral genome quantification, biochemical assays, immunohistochemistry, and / or imaging of target tissue samples, can be used to determine enrichment.

[0074] Target tissue can be any cell, tissue, or organ of the subject. As a non-limiting example, samples may be collected from the following: brain, spinal cord, dorsal root ganglia and related roots, liver, heart, gastrocnemius muscle, soleus muscle, pancreas, kidney, spleen, lung, adrenal gland, stomach, sciatic nerve, saphenous nerve, thyroid gland, eye (with or without optic nerve), pituitary gland, skeletal muscle (rectus femoris), colon, duodenum, ileum, jejunum, leg skin, superior cervical ganglion, bladder, ovary, uterus, prostate, testis, and / or any site identified as having a lesion or of interest.

[0075] The target peptides and associated AAV capsid proteins and AAV particles identified using the CREATE system include AAVPHP.B (PHP.B), AAVPHP.A (PHP.A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1-35, AAVPHP.B2 (PHP.B2), AAVPHP.B3 (PHP.B3), AAVPHP.N / PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, and AAV. PHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S / G2A12, AAVG2A15 / G2A3 (G2A3), AAVG2B4 (G2B4), AAVG2B5 (G2B5), and AAVPHP.S. In some embodiments, CREATE in mice is used to identify AAV capsids and / or targeting peptides with enhanced target tissue (e.g., CNS or PNS) transduction.

[0076] The CREATE system has been shown to be effective in identifying targeting peptides that enhance CNS transduction in mice following intravenous administration. However, translating mouse findings into human findings is not always straightforward. CREATE systems modified with non-transgenic animals or model systems more closely resembling humans may be helpful in identifying targeting peptides and associated AAV capsids and particles that can be used to treat human diseases. In some embodiments, the AAV interactors identified herein can be used to address this unmet need and to evaluate or generate new models for the design of human AAV capsids.

[0077] To adapt the CREATE method to non-transgenic animals, an alternative mechanism is needed to alter target tissues and / or cells to express Cre. In some embodiments, AAV Cre vectors can be used to transduce cells and induce subsequent Cre expression. In some embodiments, these AAV Cre vectors can be AAV1-Cre vectors. AAV Cre vectors can contain a viral genome with cell-type-specific promoters. These cell-type-specific promoters can be, but are not limited to, CAG, UBC, EF1α, synaptic protein, GFAP, MBP, VGLUT, VGAT, Nav1.8, albumin, TH, Chat, and / or any promoter known in the art.

[0078] In some embodiments, these AAV-Cre vectors are delivered to the target tissue via intra-organ parenchymal administration. In some embodiments, intra-organ parenchymal administration is applied directly to the dura mater of the subject. In some embodiments, intra-organ parenchymal administration is applied directly to the thalamus of the subject. In some embodiments, intra-organ parenchymal administration is applied directly to the cortex of the subject. In some embodiments, intra-organ parenchymal administration is applied indirectly to the cortex of the subject. In some embodiments, intra-organ parenchymal administration is applied simultaneously to one or more of the dura mater, thalamus, and / or cortex of the subject, and may be bilateral. In some embodiments, the subject is a non-human primate.

[0079] Regarding the CREATE method developed in mice, the AAV capsid library can be administered intravenously. In another embodiment, the AAV capsid library can be administered via intra-organ parenchymal delivery. In some embodiments, the AAV capsid library is administered before delivery of the AAV-Cre vector. In another embodiment, the AAV capsid library is administered after delivery of the AAV-Cre vector. The time between the administration of the AAV-Cre vector and the AAV capsid library can be seconds, minutes, hours, days, weeks, or years.

[0080] AAV capsid libraries may contain AAV particles that encode a viral genome encoding a reporter gene (e.g., GFP). Only cells in target tissues (e.g., CNS or DRG) that also express Cre (co-transduced by a Cre vector administered intra-organ parenchyma) will express the reporter gene. Target tissues can be collected and analyzed to identify AAV particles and targeting peptides that lead to target tissue enrichment, i.e., enhanced transduction. Standard methods in the art can be used to evaluate, analyze, or characterize sample tissues and AAV sequences, including but not limited to next-generation sequencing, viral genome quantification, biochemical assays, immunohistochemistry, and / or imaging.

[0081] CA4-binding peptide: In some embodiments, the present invention provides a CA4-binding peptide that can bind to carbonic anhydrase IV (e.g., human CA4 or its homologs or variants) disclosed herein, thereby increasing the permeability of the BBB (e.g., via transcytosis). In some embodiments, the increase in BBB permeability is achieved by altering (e.g., increasing or decreasing) the activity of carbonic anhydrase IV, such as by reducing its activity.

[0082] In some embodiments, the alteration of carbonic anhydrase IV activity is achieved by a targeting peptide binding to one or more active sites of carbonic anhydrase IV, including a zinc-binding site and a hydrophobic substrate-binding pocket. For example, the targeting peptide may bind to the zinc-binding site, the hydrophobic substrate-binding pocket, or both.

[0083] Some exemplary CA4-binding peptides and their corresponding AAVs that bind to mouse CA4 are provided in WO 2023 / 168333, which is incorporated herein by reference in its entirety.

[0084] Mouse CA4 is typically used to screen and develop AAVs to assess the BBB permeability of capsid-containing AAVs. However, there are differences in the AAV binding pockets between mouse CA4 and primate CA4. Specifically, engineered msCar4-binding AAVs, such as 9P31 or 9P36, which do not bind to rhCA4 or huCA4, have been identified in mice via directed evolution without mechanistic insights. Therefore, there is a need to identify AAVs capable of binding to primate or human CA4. This invention recognizes that such AAVs would be able to deliver therapeutic cargo across the CNS of primates and / or humans.

[0085] While methods for delivering therapeutic cargo across the CNS and eye via the BBB are being investigated, the AAVs under development are rodent-specific PHP.eB, and while PHP.eB is widely used in brain disease research, its therapeutic use in non-human primates (NHPs) and clinical trials remains limited. This inadequate efficacy translation from rodent to primate has prompted investigation into the molecular mechanisms of BBB transduction and crossing via engineered AAVs.

[0086] Without being bound by theory, the present invention provides, without mechanistic insights, the ability, through directed evolution to identify previously engineered msCar4-binding AAVs, such as 9P31 or 9P36, in mice that do not bind to rhCA4 or huCA4, due to protein differences in the AAV binding pocket between mouse CA4 and primate CA4.

[0087] Therefore, the need to identify potential membrane receptors in primates that could facilitate AAV delivery to the brain remains unmet. In some aspects, the present invention provides the utilization of pathways involving the carbonic anhydrase IV (CA4) pathway to deliver therapeutic cargo to the CNS via the BBB. For example, extensive CNS transduction via systemic delivery in mice using mouse carbonic anhydrase IV (msCar4) via engineered AAV capsids 9P31 and 9P36. CA4 and its functional mechanisms are conserved across rodents and primates (including humans) compared to previously identified AAV receptors such as the murine-restricted Ly6a.

[0088] CA4-binding peptides and AAV in humans or primates: In some respects, the present invention provides engineered AAVs that require combination with specific primate CA4 variants (such as rhCA4 or huCA4) intended for use as vectors in their final application. These AAVs are designed to cross the BBB and efficiently transduce the CNS of primates using a variety of non-invasive administration routes, including systemic administration.

[0089] Therefore, in some aspects, the present invention provides a method for increasing the permeability of the blood-brain barrier. In some embodiments, the method comprises providing a CA4-binding peptide (also referred to as a “targeting peptide”) capable of binding to primate and / or human carbonic anhydrase 4 (CA4 or CA-IV), thereby increasing the permeability of the blood-brain barrier.

[0090] In some respects, CA4-binding peptides are listed in Tables 1, 3, 4, and 5. In some embodiments, the targeting peptides of the present invention increase the permeability of the blood-brain barrier by at least 25%, 50%, 75%, 100%, or more compared to the absence of a targeting peptide.

[0091] In some embodiments, the CA4-binding peptide enhances the binding affinity of the viral vector or the non-viral vector to human CA4 or primate CA4. In some embodiments, the viral vector comprises an AAV vector. In some embodiments, the targeting peptide is part of the capsid protein of the AAV vector. In some embodiments, the AAV vector is a vector selected from the group consisting of: AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-DJ, human isolate hu.31, human isolate hu.32, rhesus monkey isolate rh.8, rhesus monkey isolate rh.10, and variants thereof. In some embodiments, the non-viral vector comprises lipid-based nanoparticles, polymer nanoparticles, inorganic nanoparticles, surfactant-based emulsions, nanowires, silica nanoparticles, peptide- or protein-based particles, lipid-polymer particles, nanolipoprotein particles, and combinations thereof.

[0092] Therefore, in some aspects, the present invention provides CA4-binding peptides that bind to human CA4 and / or primate CA4. Unless otherwise stated, the CA4-binding peptides mentioned herein are peptides that bind to human CA4 and / or primate CA4. In some embodiments, the CA4-binding peptide is inserted into the capsid protein of an AAV. In some preferred embodiments, the CA4-binding peptide is inserted between two adjacent amino acids of AA587-594 in SEQ ID NO: 1 of the AAV9 vector, or between functional equivalents of AA587-594 in an amino acid sequence that is at least 80% identical to that of SEQ ID NO: 1. In some embodiments, the targeting peptide is inserted between AA588-589 in SEQ ID NO: 1 of the AAV9 vector, or between functional equivalents of AA588-589 in an amino acid sequence that is at least 80% identical to that of SEQ ID NO: 1. In some embodiments, the CA4-binding peptide is listed in Tables 1, 3, 4, and 5.

[0093] In some aspects, the present invention provides AAV capsid proteins that bind to primate CA4 and / or human CA4. In some embodiments, these AAV capsid proteins include an insertion of a CA4-binding peptide. In some embodiments, these AAV capsid proteins or portions thereof that bind to primate and / or human CA4 are listed in Tables 1, 3, 4, and 5.

[0094] In some aspects, the present invention provides AAVs comprising an AAV capsid protein that binds to primate CA4 and / or human CA4. In some embodiments, these AAV capsid proteins comprise an insertion of a CA4-binding peptide. In some embodiments, these AAV capsid proteins or portions thereof that bind to primate and / or human CA4 are listed in Tables 1, 3, 4, and 5.

[0095] In some aspects, the present invention provides a recombinant adeno-associated virus (rAAV). In some embodiments, the rAAV comprises any of the AAV capsid proteins disclosed herein. In some embodiments, the rAAV comprises an AAV capsid protein containing a CA4-binding peptide having binding specificity to human CA4 and / or primate CA4, wherein the amino acid sequence of the targeting peptide is inserted between two adjacent amino acids or their functional equivalents in AA587-594 of the AAV9 capsid protein. In some embodiments, the two adjacent amino acids are AA588 and AA589. In some embodiments, the rAAV exhibits enhanced tropism for this nervous system compared to rAAV without the targeting peptide. In some embodiments, the rAAV is capable of transducing this nervous system at a transduction efficiency at least twice that of rAAV without the targeting peptide.

[0096] In some embodiments, the AAV or rAAV of the present invention is used to cross the BBB and efficiently transduce the CNS of primates using a variety of non-invasive administration routes, including systemic administration.

[0097] In some embodiments, the methods of the present invention enable these AAVs to be more readily applicable in preclinical testing and clinical intervention for neurological diseases. Additionally, in some embodiments, peptide modifications of these AAVs are further recruited for non-viral delivery, such as for antibodies, antibody-drug conjugates, oligonucleotides, enzymes, proteins, larger synthetic molecules, exosomes, nanoparticles, and contrast agents.

[0098] In some aspects, the present invention provides carbonic anhydrase IV (CA4) binding peptides listed in Tables 1, 3, 4, and 5. In some aspects, the present invention provides AAV listed in Tables 1, 3, 4, and 5. These CA4 binding peptides and AAVs listed in Tables 1, 3, 4, and 5 provide genetic pathways for the central nervous system of primates after noninvasive systemic delivery. In some embodiments, the present invention provides 9-amino acid peptides listed in Tables 4 and 5.

[0099] In some embodiments, the present invention provides the AAV provided in Tables 4 and 5. In some embodiments, the AAV comprises a 7-amino acid peptide insertion at position 588 / 589 of AAV9 and an AQ or DG at positions 587 to 588.

[0100] In some aspects, the present invention provides huCA4 binding AAV Alpha 43, 45, 48, and 49. In some embodiments, huCA4 binding AAV-binding peptides are listed in Table 1. In some preferred embodiments, the present invention provides huCA40 binding AAV peptides: DGVVHETVR; DGVVGVNIR; DGEVGLTVR; DGIVGSTIR.

[0101] In some embodiments, the 9-amino acid peptide sequences are listed in Table 1 (hereinafter). Table 1 provides the 9-amino acid peptide sequences following position 586 of the AAV variants that can be individually tested for binding to huCA4 or rhCA4.

[0102] Table 1: 9-amino acid peptide sequences:

[0103] In some embodiments, Table 2A provides exemplary sequences of huCA4 and rhCA4 in this invention.

[0104] Table 2A: Human CA4 and Rhesus Monkey CA4 Sequences:

[0105] Table 2B provides exemplary AAV sequences for AAV9 capsid protein and CA4 binding to AAV, wherein the amino acid-binding peptide of the present invention is inserted.

[0106] Table 2B: AAV capsid protein sequence:

[0107] In some embodiments, Table 3 provides CA4-binding 9-amino acid peptides. Table 3 provides exemplary 9-amino acid CA4-binding peptides and related AAV variants.

[0108] Table 3: CA4-binding 9-amino acid peptide sequence and AAV:

[0109] In some embodiments, Table 4 provides 9-meric amino acid peptides that bind to human CA4 (huCA4). In some preferred embodiments, the 9-meric amino acid peptides in Table 4 are located after position 586 of the AAV9 capsid protein or its functional equivalent.

[0110] Table 4: Human CA-IV-bound 9-mer amino acid peptides

[0111] In some embodiments, Table 5 provides 9-meric amino acid peptides that bind to rhesus monkey CA4 (rhCA4). In some preferred embodiments, the 9-meric amino acid peptides in Table 5 are located after position 586 of the AAV9 capsid protein or its functional equivalent.

[0112] Table 5: Rhesus monkey CA-IV-binding 9-mer amino acid peptides

[0113] Screening for CA-IV binding peptides and / or AAV: In some aspects, the present invention provides methods for screening peptides that bind to human CA4 and / or primate CA4. In some embodiments, the methods provided in the present invention utilize an animal-free workflow to assess the BBB permeability of peptides and AAV in humans and / or primates. In some embodiments, the assessment is performed by resin-based screening. Figure 1An overview of the methods used in this invention is provided. The AAV9-based library was generated using the M-CREATE method with a random 7-amino acid peptide insertion between positions 588 / 589 and with either AQ (wild-type) or DG at positions 587 to 588 (NNK library pool). (Ravindra Kumar, S., Miles, TF, Chen, X., Brown, D., Dobreva, T., Huang, Q., Ding, X., Luo, Y., Einarsson, PH, Greenbaum, A. et al. (2020). Multiple Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types. *Nature Methods*) 17 , 541-550. 10.1038 / s41592-020-0799-7.

[0114] In some embodiments, the present invention provides HA-tagged huCA4 as a capture agent for resin-based screening of AAV / antibody-engineered systems. In some embodiments, the present invention provides HA-tagged rhCA4 as a capture agent for resin-based screening of AAV / antibody-engineered systems. In some embodiments, the rhCA4 sequence is provided in Table 2.

[0115] In some embodiments, the engineered receptor protein has its C-terminal GPI signal peptide replaced with an HA tag, thereby allowing attachment to an anti-HA magnetic resin. Then, with or without the HA-tagged receptor (negative control), an AAV library pool is incubated overnight with the anti-HA resin to enrich receptor-binding variants, followed by rigorous washing to remove non-specific bindings.

[0116] Figure 2 The present invention provides unique CA4-binding AAV compositions. Three NNK library pools with different AAV variant distributions were independently generated. Two rounds of selection were performed on one NNK pool, generating approximately 9,000 unique sequences in the first round and approximately 700 leading CA4-binding AAVs in the second round. Approximately 700 variants were also selected from the other two NNK pools. The final result consists of two pools containing 2005 unique AAVs with binding affinity for huCA4 and 1979 AAVs with binding affinity for rhCA4.

[0117] The effectiveness of this in vitro resin-based screening method for identifying promising AAV variants was evaluated. Small pools of approximately 18,000 unique AAV variants on the resin were tested, including positive controls 9P31 and 9P36 containing mouse Car4 (msCar4) protein. msCar4 pull-down assays resulted in high enrichment and enhancement of 9P31 and 9P36. Figure 3 and Figure 4 It exhibits consistent and robust performance at both low doses (Sample 1, 1.6e11 vg) and high dose inputs (Sample 2, 8e11 vg). Figure 5 In some embodiments, the formulas used to calculate enrichment and enhancement are defined as follows.

[0118]

[0119]

[0120] Ri: The relative percentage of AAV variant i in the sample.

[0121] Figure 3 A selection of msCar4-based resins with a low-dose library input (1.6e11 vg) is provided. Red dots (top right of the figure) indicate 9P31 and 9P36, while orange dots represent AAV variants with enrichment >10 and enhancement >10. Yellow dots show all other AAV variants recovered on the resin.

[0122] Figure 4 A selection of msCar4-based resins with high-dose library input (8e11 vg) is provided. Red dots (top right of the figure) indicate 9P31 and 9P36, while orange dots represent AAV variants with enrichment >10 and enhancement >10. Yellow dots show all other AAV variants recovered on the resin.

[0123] It is worth noting that, in some advantageous aspects of the present invention, resin-based selection provides a better signal-to-noise ratio by recovering AAV variants of HEK293 adherent cells successfully transduced to express msCar4, compared to cell-based selection methods. Figure 5 and Figure 6 ).

[0124] Figure 5 AAV variant performance is provided in resin-based selections at both low and high doses.

[0125] Figure 6The performance of AAV variants in cell-based selection at different doses was provided. HEK293 cells were transfected in 6-well plates with pUC18 (negative control) or CA4 plasmid. After two days, 1.25e10 vg (sample 1) or 5e10 vg (sample 2) of AAV was added to the wells and incubated for 24 hours. Cells were then collected, lysed, and RNA extracted. RNA samples were subjected to RT-PCR and PCR amplification for NGS analysis.

[0126] from Figure 5 and Figure 6 As is evident from the data provided, resin-based selection offers a better signal-to-noise ratio by recovering AAV variants of successfully transduced HEK293 adherent cells compared to cell-based selection methods.

[0127] The selection method was validated using LY6A on resin and the same library input pool, which included positive controls PHP.eB and PHP.B. Huang, Q., Chan, KY, Tobey, IG, Chan, YA, Poterba, T., Boutros, CL, Balazs, AB, Daneman, R., Bloom, JM, Seed, C., and Deverman, BE (2019). Delivering genes across the blood-brain barrier: LY6A, a novel cellular receptor for AAV-PHP.B capsids. PLoS One. 14, e0225206. 10.1371 / journal.pone.0225206; Chan, KY, Jang, MJ, Yoo, BB, Greenbaum, A., Ravi, N., Wu, WL, Sanchez-Guardado, L., Lois, C., Mazmanian, SK, Deverman, BE, and Gradinaru, V. (2017). Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. *Nature Neuroscience* 20 ,1172-1179. 10.1038 / nn.4593.

[0128] Figure 7 The performance of AAV variants in the LY6A-based selection process is presented across low doses (Sample 1, 1.6e11 vg) and high doses (Sample 2, 8e11 vg). The red dot (top right) indicates PHP.eB and PHP.B (including codon replicas). Other dot color codes are provided on the right. Notably, the selection process effectively enriched PHP.eB and PHP.B (…). Figure 7 ).

[0129] A randomized AAV library pool (with AAV9 as the parental capsid) containing a 7-amino acid peptide insertion at position 588 / 589 and either AQ (wild-type) or DG at positions 587 to 588 (NNK library pool). Figure 1 The NNK library was generated using the evolutionary approach provided in Kumar 2020. Considering that 2.56e9 variants (theoretical library size, including unstable AAVs) could generate only about 1e7 AAV variants, three independent pools of NNK libraries with uniformly distributed AAV variants were thus generated. Figure 8 Pools of libraries generated using these methods are provided. For one of the NNK pools, two rounds of selection were performed. The first round generated approximately 9,000 unique sequences as input for the second round, and approximately 700 of the top variants were selected as CA4 combined with AAV ( Figure 9 and Figure 10 ). Figure 9 and Figure 10This represents the huCA4-binding AAVs after the second round of selection at low library input doses (5e10 vg) and high library input doses (2.5e11 vg), respectively. The final pool of huCA4-binding AAVs consists of... Figure 9 and 10 The green dots represent the variants. Additionally, approximately 700 variants were selected from two other NNK pools. Figure 11 and Figure 12 ). Figure 11 Data for independent round 1 selected replicas 2 of huCA4 combined with AAV at low (sample 1, 1.6e11 vg) and high (sample 2, 8e11) library input doses are provided. Figure 12 Independent first-round selected replicas 3 of huCA4-binding AAV at low (sample 1, 1.6e11 vg) and high (sample 2, 8e11) library input doses are provided. The final pool of huCA4-binding AAV was obtained from... Figure 11 and 12 The green dots represent [the data].

[0130] Similarly, a pull-down function combining rhCA4 and AAV was also implemented. Figure 13 The second round of dropdown selection results for rhCA4 combined with AAV is provided for low (sample 1, 5e10 vg) and high (sample 2, 2.5e11 vg) library input doses. Figure 14 Independent first-round selection replicas 2 of rhCA4-AAV were provided at low (sample 1, 1.6e11 vg) and high (sample 2, 8e11) library input doses. Figure 15 Independent first-round selection replicas 2 of rhCA4-binding AAV were provided at low (sample 1, 1.6e11 vg) and high (sample 2, 8e11) library input doses. The final pool of rhCA4-binding AAV was generated by... Figure 13 , 14 The green dots in 15 represent this.

[0131] Ultimately, a pool of 2005 unique huCA4 combined with AAV and another pool of 1979 rhCA4 combined with AAV were obtained. Figure 2 (13-15). Tables 4 and 5 list the selected 9-amino acid sequences following position 586.

[0132] Among the selected AAV variants, a group of AAVs listed in Table 1 were chosen, and additional individual pull-down and cell infectivity assays were performed. These experiments confirmed that AAVs bind to either huCA4 or rhCA4. For the pull-down assay, each individual AAV variant was incubated overnight with anti-HA resin with or without HA-labeled CA4 protein (negative control), followed by multiple washings of the resin and elution of the enriched AAVs with SDS loading buffer. The amount of AAV in the samples and negative control was assessed using Western blotting with anti-AAV and anti-HA antibodies. The results were validated by the pull-down assay, with Alpha 33, 41, 42, 43, 44, 45, 48, 49, 50, and 52 showing significant binding to huCA4. Figure 16 Data from the second round of dropdown selections for huCA4 combined with AAVs at a low library input dose (5e10 vg) are provided. A set of AAVs (represented by green, purple, and blue dots; color scheme provided on the right) were selected for individual characterization. Green dots correspond to Alpha 43, Alpha 45, Alpha 48, and Alpha 49. Figure 17 Data from the second round of dropdown selections for huCA4 combined with AAVs at a high library input dose (2.5e11 vg) are provided. A set of AAVs (represented by green, purple, and blue dots; color scheme provided on the right) were selected for individual characterization. Green dots correspond to Alpha 43, Alpha 45, Alpha 48, and Alpha 49. Figure 18 Data from the second round of dropdown selections for rhCA4 combined with AAVs are provided at low (Sample 1, 5e10 vg) and high (Sample 2, 2.5e11 vg) library input doses. A set of AAVs (represented by green and blue dots; color scheme provided on the right) were selected for individual characterization. Green dots correspond to Omega 2, 5, 6, 8, and 11.

[0133] Figure 19 Data are provided for a pull-down assay evaluating the binding of individual AAV variants to huCA4. AAV was incubated overnight with anti-HA resin, with or without HA-labeled huCA4 protein (negative control). After multiple washes, the enriched AAV was eluted with SDS loading buffer. The amount of AAV in the samples and negative control was assessed by Western blotting using anti-AAV and anti-HA antibodies.

[0134] In cell infectivity assays, Alpha 43, 45, 48, and 49 cells exhibited enhanced huCA4 transduction. For these assays, HEK293 adherent cells were transfected with either pUC18 or CA4 plasmids, and subsequently transduced with AAV carrying the EGFP gene two days later. Intracellular fluorescence signals were monitored in 96-well plates using fluorescence microscopy after 24 hours of incubation with AAV and cells. Figure 20 Data are provided for evaluating huCA4-enhanced cell transduction in Alpha 43, 45, 48, and 49.

[0135] Similar experiments were conducted to verify the binding of Omega 2, 5, 6, 8, and 11 to rhCA4 in pull-down assays. Figure 21 A pull-down assay was provided to evaluate the binding of individual AAV variants to rhCA4. AAV was incubated overnight with anti-HA resin, with or without HA-labeled rhCA4 protein (negative control). After multiple washes, the enriched AAV was eluted with SDS loading buffer. The amount of AAV in the samples and negative control was assessed by Western blotting using anti-AAV and anti-HA antibodies.

[0136] Subsequently, the binding of huCA4 to AAV was evaluated in mice with humanized BBB. Figure 22 An overview of mice with humanized CA4 is provided. The huCA4 gene was transiently introduced into endothelial cells using AAV1.X1, a vector specific to these cells. Three weeks later, systemic injection of potential huCA4-binding AAV carrying EGFP cargo was performed to evaluate BBB transcytosis efficiency.

[0137] Figure 23 Data on the binding performance of huCA4 to AAV in mice are provided. Mice were systemically injected with either EGFP packaged with huCA4-only AAV binding or with both AAV1.X1:CAG-huCA4 and huCA4-AAV binding. Apha2, Apha45, Apha48, and Apha49 were tested. Figure 23 The expression shown is 3 weeks after injection.

[0138] For the aforementioned AAV variant pool, a third round of cell-based selection and dropdown selection was performed. Alpha 41, 59-64 showed promising huCA4-enhanced infectivity and huCA4 binding, as did the validated Alpha 43, 45, 48, and 49. Figure 24 Cell-based selection data on huCA4 binding to AAV were provided. HEK293 cells were transfected with pUC18 or huCA4 plasmids for 48 hours, and then transduced using a pool of 2005 AAV libraries containing huCA4-binding variants. Figure 2Green dots: Alpha 43, 45, 48, and 49 with enhanced huCA4 infectivity; Blue dots: Other test variants with huCA4-independent infectivity; Red dots: Alpha 41 and Alpha 59-64, selected from round 3 as individual test candidates.

[0139] Figure 25 Data from the third round of dropdown selections for huCA4 binding to AAV are provided at low (Sample 1, 1.6e10 vg) and high (Sample 2, 7.8e10 vg) library input doses. Green dots: Alphas 43, 45, 48, and 49 with huCA4 binding; orange and red dots show the top variants in the third round of cell-based selection. Red dots indicate Alphas 41 and Alphas 59-64, which were selected for individual testing.

[0140] Figure 26 Cell-based selection data for rhCA4-binding AAV in round 3 are provided. HEK293 cells were transfected with pUC18 or rhCA4 plasmids for 48 hours, and then transduced using a pool of 1979 AAV libraries containing rhCA4-binding variants. Figure 2 Orange dots: Leading variants with enhanced rhCA4 infectivity; Blue dots: Other omega variants with rhCA4-independent infectivity previously tested.

[0141] Figure 27 The effects of the third round of dropdown selection on rhCA4-AAV binding at low (Sample 1, 1.6e10 vg) and high (Sample 2, 7.8e10 vg) library input doses are presented. Orange dots: top variants from the third round of cell-based selection. Among them, Omega 33-52 was selected for individual testing.

[0142] Cross-BBB delivery of treatment goods The present invention has the advantageous recognition that systemic delivery of adeno-associated virus (AAV) provides a more widely distributed, non-invasive method compared to direct brain or eye injection, which is particularly beneficial for diffuse neurological disorders.

[0143] In some embodiments, the payload or therapeutic cargo to be delivered to the nervous system is a biomolecule, a non-biological molecule, or a combination thereof. In some embodiments, the biomolecule is selected from the group consisting of nucleic acid sequences, proteins, peptides, lipids, polysaccharides, and any combination thereof. In some embodiments, the payload is a therapeutic molecule. In some embodiments, the nucleic acid sequence to be delivered to the nervous system comprises one or more of the following: a) a sequence encoding a nutrient factor, growth factor, or other soluble factor that may be released from the transduced cell and affect the survival or function of the cell and / or surrounding cells; b) DNA for restoring protein function in a human or animal carrying a gene mutation; c) DNA encoding a protein capable of controlling or altering the activity or state of a cell; d) DNA encoding a protein or nucleic acid for assessing the state of a cell; e) DNA and / or associated guide RNA for genome engineering; f) a sequence for genome editing via homologous recombination; g) a DNA sequence encoding therapeutic RNA; h) shRNA or artificial miRNA delivery systems; or i) a DNA sequence affecting the splicing of endogenous genes.

[0144] Therefore, in some aspects, the present invention provides methods for increasing the permeability of the blood-brain barrier to deliver therapeutic cargo across the BBB to the central nervous system and / or the eyes. In some embodiments, the present invention provides AAV bound to rhesus monkey CA4 (rhCA4) or human CA4 (huCA4) for CNS therapeutic cargo delivery in both preclinical and clinical applications in these species. In other advantageous aspects of the invention, CA4 is also highly concentrated in the brain, eyes, and intestines of primates compared to other reported transport proteins with broader expression patterns, such as transferrin receptors. Therefore, the methods for delivering therapeutic cargo across the CNS provided by the present invention may reduce off-target organ delivery.

[0145] In some aspects, the present invention provides a delivery system for delivering peptides in the CNS. In some embodiments, the delivery system comprises (1) a CA4-binding peptide specific for primate CA4 and / or human CA4; and (2) a pharmaceutical agent. In some embodiments, the CA4-binding peptide (1) is displayed on the surface of the delivery system; or (2) is partially embedded in the delivery system. In some embodiments, the delivery system may be selected from the group consisting of nanoparticles, nanotubes, nanowires, dendritic macromolecules, liposomes, liposomes and aqueous liposomes, polymer vesicles and nonionic surfactant vesicles, foams, hydrogels, cubes, quantum dots, exosomes, macrophages, and combinations thereof. In some embodiments, the delivery system comprises a viral vector or a nonviral vector. In some embodiments, the delivery system comprises nanoparticles selected from the group consisting of lipid-based nanoparticles, polymer nanoparticles, inorganic nanoparticles, surfactant-based emulsions, nanowires, silica nanoparticles, virus-like particles, peptide- or protein-based particles, lipid-polymer particles, nanolipoprotein particles, and combinations thereof.

[0146] In some embodiments, the delivery system comprises a viral vector or a non-viral vector. For example, the viral vector may comprise an adenovirus vector, an adeno-associated virus (AAV) vector, a lentiviral vector, or a retroviral vector. In some embodiments, the viral vector is an AAV vector, and the target peptide may be part of the capsid protein of the AAV vector.

[0147] Adeno-associated virus (AAV) and recombinant AAV (rAAV) In some embodiments, the delivery system for delivering the payload across the BBB is an AAV vector. In some embodiments, AAV is a replication-defective parvovirus with a single-stranded DNA genome of approximately 4.7 kb in length, comprising 145 nucleotide inverted terminal repeats (ITRs). ITRs play a crucial role in the integration of AAV DNA into the host cell genome. When AAV infects a host cell, the viral genome integrates into the host chromosome, resulting in latent infection of the cell. In natural systems, helper viruses (e.g., adenovirus or herpesvirus) provide the genes that allow the production of AAV virus in infected cells. In the case of adenovirus, genes E1A, E1B, E2A, E4, and VA provide helper functions. Following infection with a helper virus, the AAV provirus is rescued and amplified, producing both AAV and adenovirus. In the absence of a recombinant AAV vector with Rep and / or Cap genes, AAV may be non-integrating.

[0148] In some embodiments, the AAV vector may contain coding regions for one or more proteins of interest. The AAV vector may include a 5' AAV ITR, a 3' AAV ITR, a promoter, and a restriction site downstream of the promoter to allow insertion of a polynucleotide encoding one or more proteins of interest, wherein the promoter and restriction site are located downstream of the 5' AAV ITR and upstream of the 3' AAV ITR. In some embodiments, the AAV vector includes post-transcriptional regulatory elements downstream of the restriction site and upstream of the 3' AAV ITR.

[0149] Viral vectors may include additional sequences that adapt the vector for replication and integration in eukaryotes. In other embodiments, the viral vectors disclosed herein may include shuttle elements that adapt the vector for replication and integration in both prokaryotes and eukaryotes. In some embodiments, viral vectors may include additional transcription and translation initiation sequences, such as promoters and enhancers; and additional transcription and translation terminators, such as polyadenylation signals. Various regulatory elements that may be included in AAV vectors have been described in US2012 / 0232133, which is hereby incorporated by reference in its entirety.

[0150] The AAV serotypes used to derive the AAV capsid protein can be different. The AAV capsid can be derived from AAV9 or its variants. The AAV capsid can be derived from AAVs selected from: AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, human isolate hu.31, human isolate hu.32, rhesus monkey isolate rh.8, and rhesus monkey isolate rh.10. In some embodiments, the AAV capsid protein may be derived from the following AAV serotypes: AAV9, AAV9 K449R (or K449RAAV9), AAV1, AAVrhl0, AAV-DJ, AAV-DJ8, AAV5, AAVPHP.B (PHP.B), AAVPHP.A (PHP.A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1-35, AAVPHP.B2 (PHP.B2), AAVPHP.B 3 (PHP.B3), AAVPHP.N / PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B- DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP (3), AAVPHP.B-SNP, AAVP HP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPH P.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S / G2A12, AAVG2 Al5 / G2A3 (G2A3), AAVG2B4 (G2B4), AAVG2B5 (G2B5), PHP.S, AAV2, AAV2G9, AAV3, AA V3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2 , AAV8, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.6 8. AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12,AAV42-1b、AAV42-2、AAV42-3a、AAV42-3b、AAV42-4、AAV42-5a、AAV42-5b、AAV42-6b、AAV42-8、AAV42-10、AAV42-1b AV42-11、AAV42-12、AAV42-13、AAV42-15、AAV42-aa、AAV43-1、AAV43-12、AAV43-20、AAV43-21、AAV43-23、A AV43-25、AAV43-5、AAV44.1、AAV44.2、AAV44.5、AAV223.1、AAV223.2、AAV223.4、AAV223.5、AAV223.6、AAV2 23.7、AAVl-7 / rh.48、AAVl-8 / rh.49、AAV2-l5 / rh.62、AAV2-3 / rh.6l、AAV2-4 / rh.50、AAV2-5 / rh.5l、AAV3. l / hu.6、AAV3.l / hu.9、AAV3-9 / rh.52、AAV3-1l / rh.53、AAV4- 8 / r11.64、AAV4-9 / rh.54、AAV4-l9 / rh.55、AAV5-3 / rh.57、AAV5-22 / rh.58、AAV7.3 / hu.7、AAVl6.8 / hu.10、AAVl6.l2 / hu.1 1、AAV29.3 / bb.1、AAV29.5 / bb.2、AAVl06. l / hu.37、AAV1 l4.3 / hu.40、AAVl27.2 / hu.4l、AAVl27.5 / hu.42、AAVl28.3 / hu.44、AAVl30.4 / hu.48、AAVl45. l / hu.53、AAVl45.5 / hu.54、AAVl45.6 / hu.55、AAVl6l. l0 / hu.60、AAVl6l.6 / hu.6l、AAV33. l2 / hu.l7、AAV33.4 / hu.l5、AAV33.8 / hu.l6、AAV52 / hu.l9、AAV52.l / hu.20、AAV58.2 / hu.25、AAVA3.3、AAVA3.4、AAVA3.5、AAVA3.7、AAVC1、AAVC2、AAVC5、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2、AAVrh.60、AAVrh.44、AAVrh.65、AAVrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu.12、AAVH6, AAVH-1 / hu.1, AAVH-5 / hu.3, AAVLG-l0 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN72l-8 / rh.43, AAVCh.5, AAVCh.5Rl, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu. 9、AAVhu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.l6、AAVhu.17、AAVhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23、2、AAVhu.24、AAVhu.25、AAVhu. u.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.40、AAVhu.41、AAVhu.42、AAVhu.43、AA Vhu.44、AAVhu.44Rl、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48Rl、AAVhu.48R2、AAVhu.48R3、AAVhu.49、AAVhu.5 1、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVhu.14 / 9、AAVhu. u.t19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、AAVrh.14、AAVrh.17、AAVrh.18、AAVrh.19、AAVrh.20、AAVrh. rh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37、AAVrh.38、AAVrh.38AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.6l, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8RR533 A mutant, AAAV, BAAV, goat AAV, bovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhErl.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhERl.23, AAVhEr3.1, AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV shuffle 100-1, AAV shuffle 100-3, AAV shuffle 100-7, AAV shuffle 10-2, AAV shuffle 10-6, AAV shuffle 10-8, AAV shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, Ideal AAV (ttAAV), EGRENN AAV 10, Japanese AAV 10 serotype, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAVCBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-El, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAVCHt-6.8, AAV CHt-P1, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAVCKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-AAV CLv-13, AAV Clvl-7, AAV Clvl-8, AAV Clvl-9, AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-D1、AAVCLv-D2、AAV CLv-D3、AAV CLv-D4、AAV CLv-D5、AAV CLv-D6、AAV CLv-D7、AAV CLv-D8、AAVCLv-El、AAV-CLv-KAV、AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAVCLv-M1, AAV CLv-M11, AAV CLv-M2, AAV CLv-M1, AAV CLv-M2, AAV CLv-M5, AAV-CLv-AAV CLv-M7, AAV CLv-M8, AAVCLv-M9, AAV CLv-R1, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, RAVCLv-AAV CLv-R8, AAV CLv-R9, AAV CSp-l, AAV CSp-l0, AAV CSp-11, AAV CSp-2, AAVCSp-3, AAV CSp-4, AAV CSp-6, AAV Csp-7, AAV Cp-8 CSp-8. l0、AAV CSp-8.2、AAVCSp-8.4、AAV CSp-8.5、AAV CSp-8.6、AAV CSp-8.7、AAV CSp-8.8、AAV CSp-8.9、AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC 11、AAVF12 / HSC12、AAVF13 / HSC13、AAVF14 / HSC14、AAVF15 / HSC15、AAVF16 / HSC1 6、AAVF17 / HSC17、AAVF2 / HSC2、AAVF3 / HSC3、AAVF4 / HSC4、AAVF5 / HSC5、AAVF6 / H SC6、AAVF7 / HSC7、AAVF8 / HSC8、AAVF9 / H SC9, the AAV system is not allowed to be used in the SC9 system.

[0151] The AAV vector may be AAV9 having the amino acid sequences provided in Table 2 or an amino acid sequence having at least 70% sequence identity with the amino acid sequences provided in Table 2 (e.g., at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher). In some embodiments, the AAV vector is a variant AAV vector having at least 70% sequence identity with the amino acid sequence of any known AAV9 variant (e.g., at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or higher).

[0152] In some embodiments, the AAV vectors disclosed herein can be used as AAV transfer vectors carrying a transgene encoding a protein of interest (e.g., a target peptide) for the generation of a recombinant AAV virus capable of expressing the protein of interest in a host cell. Therefore, recombinant AAV viruses (rAAVs) are also disclosed herein. rAAVs may contain the AAV capsid protein described herein.

[0153] rAAV can contain chimeric AAV capsids. A “chimeric” AAV capsid refers to a capsid having exogenous amino acids or amino acid sequences. rAAV can contain mosaic AAV capsids. A “mosaic” AAV capsid refers to a capsid composed of two or more capsid proteins or polypeptides, each derived from a different AAV serotype. rAAV can be the result of transcapsidation, which in some cases refers to packaging an inverted terminal repeat (ITR) sequence from a first serotype into the capsid of a second serotype, where the first and second serotypes are not identical. In some cases, the capsid gene of the parental AAV serotype can be pseudotyped, meaning that an ITR from a first AAV serotype (e.g., AAV1) is used in the capsid from a second AAV serotype (e.g., AAV9), where the first and second AAV serotypes are not identical. As a non-limiting example, a pseudotyped AAV serotype containing both an AAV1 ITR and an AAV9 capsid protein can be represented as AAV1 / 9. rAAV may additionally or alternatively include a capsid that has been engineered to express an exogenous ligand-binding moiety (e.g., a receptor) or a modified natural receptor.

[0154] In some embodiments, the rAAV capsid protein comprises substitutions or insertions of one or more amino acids in the amino acid sequence of the AAV capsid protein. In some cases, the rAAV capsid protein described herein has an insertion or substitution of an amino acid heterologous to that of the wild-type AAV capsid protein, at the position of the inserted or substituted amino acid. In some embodiments, at the position of the inserted or substituted amino acid, the amino acid is not endogenous to the wild-type AAV capsid protein. The amino acid may be a naturally occurring amino acid at the same or equivalent amino acid position as the insertion or substitution in different AAV capsid proteins. The AAV capsid protein from which the engineered AAV capsid protein of this disclosure is derived may be referred to as a “parental” or “wild-type” AAV capsid protein or “corresponding unmodified capsid protein.” In some cases, the parental AAV capsid protein has a serotype selected from the following: AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. The complete genome of AAV-1 is available in GenBank accession NC_002077; the complete genome of AAV-2 is available in GenBank accession NC_001401 and in Srivastava et al., *Journal of Virology*, 45: 555-564 (1983); the complete genome of AAV-3 is available in GenBank accession NC_1829; the complete genome of AAV-4 is available in GenBank accession NC_001829; the genome of AAV-5 is available in GenBank accession AF085716; the complete genome of AAV-6 is available in GenBank accession NC_001862; at least portions of the genomes of AAV-7 and AAV-8 are available in GenBank accessions AX753246 and AX753249, respectively; the genome of AAV-9 is available in Gao et al., *Journal of Virology*, 78: 6381-6388. Provided in (2004); AAV-10 genome in Molecular Therapy ( Mol.Ther. The AAV-11 genome is provided in Virology, 13(1): 67-76 (2006); Virology The AAV-12 genome is available in Genbank accession number DQ813647; the AAV-13 genome is available in Genbank accession number EU285562. At least a portion of the AAV-DJ genome is available in Grimm, D. et al. Journal of Virology Provided in 82, 5887-5911 (2008).

[0155] In some embodiments, the rAAV vectors disclosed herein may carry a transgene encoding a CA4-binding peptide as described herein that can bind to human CA4 and / or primate CA4. The targeting peptide may be part of the rAAV capsid. The AAV capsid protein is also disclosed herein. The AAV capsid protein may contain the targeting peptide disclosed herein.

[0156] The position of the targeting peptide within the capsid protein can vary. In some embodiments, the targeting peptide may be inserted between two adjacent amino acids in AA586-595 of the AAV9 capsid protein (e.g., between AA586 and AA587, AA587 and AA588, AA588 and AA589, AA589 and AA590, AA590 and AA591, AA591 and AA592, AA592 and AA593, AA593, AA594 and AA595) or between its functional equivalents in other AAV capsid proteins. The AAV vector may be selected from the following vectors: AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-DJ, human isolate hu.31, human isolate hu.32, rhesus monkey isolate rh.8, rhesus monkey isolate rh.10, or variants thereof. In some embodiments, the AAV vector is AAV9 or a variant or derivative thereof. For example, the AAV capsid protein comprises or is composed of the following amino acid sequences provided in Table 2 or at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or values ​​or ranges between any two of these values) identical to the sequences provided in Table 2.

[0157] The targeting peptide can be inserted between AA588-589 of the AAV9 capsid protein or between its functional equivalents in other AAV capsid proteins. The two adjacent amino acids can be AA588-589. In some embodiments, the targeting peptide is inserted between AA587-590 of the AAV9 capsid protein or between its functional equivalents in other AAV capsid proteins.

[0158] Uses of AAV carriers and rAAVs for payload delivery In some aspects, the present invention provides compositions for delivering a payload (e.g., a pharmaceutical agent) to a target environment (such as the nervous system of a subject). The composition may comprise an AAV comprising (1) the AAV capsid protein disclosed herein, and (2) the pharmaceutical agent to be delivered to the target environment (e.g., the nervous system) of the subject.

[0159] The target environment can be the CNS, the peripheral nervous system (PNS), or a combination thereof. The target environment can be brain endothelial cells, neurons, capillaries in the brain, small arteries in the brain, arteries in the brain, or a combination thereof. In some embodiments, the target environment can be in the eye of a primate or a human.

[0160] The agent to be delivered may comprise nucleic acids, peptides, small molecules, aptamers, or combinations thereof. The AAV vectors disclosed herein can be efficiently transduced into a target environment (e.g., the central nervous system), for example, for the delivery of nucleic acids. In some embodiments, a method for delivering a nucleic acid sequence to the nervous system is provided. A protein may be part of the capsid of the AAV. The AAV may contain the nucleic acid sequence to be delivered to the nervous system. The AAV can then be administered to a subject.

[0161] Nucleic acid sequences intended for delivery to the nervous system may include one or more of the following: a) sequences encoding nutrient factors, growth factors, or other soluble factors that may be released from the transduced cell and affect the survival or function of that cell and / or surrounding cells; b) DNA (e.g., genomic or cDNA sequences) for restoring protein function in humans or animals carrying gene mutations; c) DNA encoding proteins that can be used to control or alter the activity or state of cells; d) DNA encoding proteins or nucleic acids used to assess the state of cells; e) DNA and / or associated guide RNAs for genome engineering; f) sequences for genome editing via homologous recombination; g) DNA sequences encoding therapeutic RNAs; h) shRNA or artificial miRNA delivery systems; or i) DNA sequences that affect the splicing of endogenous genes.

[0162] In some embodiments, the vector may also contain regulatory control elements known to those skilled in the art to influence the expression of desired intracellular polynucleotide-encoded RNA and / or protein products by the subject.

[0163] Functionally, the expression of polynucleotides can be at least partially controlled by operatively linked regulatory elements that regulate the transcription of polynucleotides, the transport, processing, and stability of the RNA encoded by the polynucleotides, and, where appropriate, the translation of the transcript. Specific examples of expression control elements are promoters, typically located at the 5' end of the transcribed sequence. Another example is enhancers, which can be located at the 5' or 3' end of the transcribed sequence or within the transcribed sequence. Another example of regulatory elements is microRNA recognition sequences. Another example is introns and splice donor and acceptor sequences that regulate intron splicing. Another example of regulatory elements is transcription termination signals and / or polyadenylation sequences.

[0164] Expression control elements and promoters include elements and promoters that are active in specific tissue or cell types, referred to herein as “tissue-specific expression control elements / promoters.” Tissue-specific expression control elements are typically active in specific cells or tissues (e.g., in the liver, brain, central nervous system, spinal cord, eye, retina, or lung). Expression control elements are typically active in these cells, tissues, or organs because they are recognized by transcriptional activator proteins or other transcriptional regulators specific to that particular cell, tissue, or organ type.

[0165] Expression control elements also include ubiquitous or heterogeneous promoters / enhancers capable of driving the expression of polynucleotides in many different cell types. Such elements include, but are not limited to, cytomegalovirus (CMV) immediate early promoter / enhancer sequences, Rous sarcoma virus (RSV) promoter / enhancer sequences, CMV, chicken β-actin, rabbit β-globin (CAG) promoter / enhancer sequences, and other viral promoters / enhancers active in various mammalian cell types; promoter / enhancer sequences from ubiquitous or heterogeneously expressed mammalian genes, including but not limited to β-actin, ubiquitin, or EF1α; or synthetic elements not found in nature.

[0166] Expression control elements can also confer expression in a regulated manner, meaning that a signal or stimulus increases or decreases the expression of an operably linked polynucleotide. A regulated element that increases the expression of an operably linked polynucleotide in response to a signal or stimulus is also called an "inducible element" (i.e., it is induced by a signal). Specific examples include, but are not limited to, hormone (e.g., steroid) inducible promoters. A regulated element that decreases the expression of an operably linked polynucleotide in response to a signal or stimulus is called an "inhibitory element" (i.e., the signal decreases expression so that expression increases when the signal is removed or absent). Generally, the amount of increase or decrease conferred by such elements is proportional to the amount of signal or stimulus present: the greater the amount of signal or stimulus, the greater the increase or decrease in expression.

[0167] In some preferred aspects, the present invention provides a method for treating or diagnosing conditions related to the CNS and / or the eye by administering a composition comprising the peptides or AAVs provided herein. In some embodiments, the peptides and / or AAVs of the present invention allow for the delivery across the BBB of therapeutic goods for treating the CNS and / or the eye. In some embodiments, the present invention provides a method for treating brain conditions. In some embodiments, the brain conditions treated by the compositions of the present invention include brain cancer, neurodegeneration, and glioblastoma. In some embodiments, the eye-related condition is glaucoma.

[0168] In some embodiments, the present invention provides compositions for delivering a pharmaceutical agent to the nervous system of a subject in need. In some embodiments, the composition comprises an AAV comprising (1) the AAV capsid protein disclosed herein and (2) a pharmaceutical agent to be delivered to the nervous system of the subject; optionally wherein the nervous system is the central nervous system (CNS), the peripheral nervous system (PNS), or a combination thereof. In some embodiments, the nervous system is brain endothelial cells, neurons, capillaries in the brain, small arteries in the brain, arteries in the brain, or a combination thereof. In some embodiments, the composition is a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers. In some embodiments, the pharmaceutical agent to be delivered comprises nucleic acids, peptides, small molecules, aptamers, or a combination thereof.

[0169] Nucleic acids (e.g., heterologous nucleic acids) may contain 5' ITRs and 3' ITRs. Agents may contain DNA sequences encoding proteins (e.g., nutrient factors, growth factors, or soluble proteins). Nucleic acids may contain promoters operatively linked to polynucleotides encoding, for example, proteins or RNA agents. Promoters may be able to induce transcription of polynucleotides. Transcription of polynucleotides can produce transcripts. Nucleic acids may contain one or more of the following: 5' UTRs, 3' UTRs, small promoters, enhancers, splicing signals, polyadenylation signals, terminators, one or more silencer effector binding sequences, protein degradation signals, and internal ribosome entry elements (IRES). Silencer effectors may contain microRNAs (miRNAs), pre-miRNAs, small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), their precursors, their derivatives, or combinations thereof. Silencer effectors may be able to bind to one or more silencer effector binding sequences, thereby reducing transcript stability and / or decreasing transcript translation. In some embodiments, the silencing effector comprises one or more miRNA binding sites (e.g., miR-122 binding site). The miRNA binding site is an operatively linked regulatory element that is typically located in the 3'UTR of the transcribed sequence. Binding of the miRNA to the target transcript (complexed with the RNA-induced silencing complex RISC) can reduce the expression of the target transcript through translational repression and / or transcript degradation.

[0170] The polynucleotide may further comprise transcript stabilizing elements. Transcript stabilizing elements may comprise the marmot hepatitis post-translational regulatory element (WPRE), bovine growth hormone polyadenylation (bGH-polyA) signal sequence, human growth hormone polyadenylation (hGH-polyA) signal sequence, or any combination thereof. The nucleic acid may be or may encode an RNA agent. The RNA agent may comprise one or more of the following: dsRNA, siRNA, shRNA, pre-miRNA, pri-miRNA, miRNA, stRNA, lncRNA, piRNA, and snoRNA. The RNA agent inhibits or suppresses the expression of the gene of interest in the cell. In some embodiments, the gene of interest may be selected from SOD1, MAPT, APOE, HTT, C90RF72, TDP-43, APP, BACE, SNCA, ATXN1, ATXN2, ATXN3, ATXN7, SCN1A-SCN5A, and SCN8A-SCN11A. Nucleic acids may further contain polynucleotides encoding one or more secondary proteins, and the protein and the one or more secondary proteins may contain a protein synthesis circuit. Nucleic acids may contain single-stranded AAV (ssAAV) vectors or self-complementary AAV (scAAV) vectors.

[0171] Promoters can contain ubiquitous promoters. Commonly used promoters can be selected from: cytomegalovirus (CMV) immediate early promoter; CMV promoter; viral simian virus 40 (SV40) (e.g., early or late); Moloney murine leukemia virus (MoMLV) LTR promoter; Rous sarcoma virus (RSV) LTR; RSV promoter; herpes simplex virus (HSV) (thymidine kinase) promoter; H5, P7.5, and P11 promoters from vaccinia virus; elongation factor 1-α (EF1a) promoter; early growth response 1 (EGR1); ferritin H (FerH); ferritin L (FerL); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); eukaryotic translation initiation factor 4A1 (EIF4A1); heat shock 70 kDa protein 5 (HSPA5); heat shock protein 90 kDa β member 1 (HSP90B1); heat shock protein 70 kDa (HSP70); β-kinin (β-KIN); human ROSA. 26 loci; ubiquitin C promoter (UBC); phosphoglycerate kinase-1 (PGK) promoter; 3-phosphoglycerate kinase promoter; cytomegalovirus enhancer; human β-actin (HBA) promoter; chicken β-actin (CBA) promoter; CAG promoter; CBH promoter; or any combination thereof.

[0172] The promoter can be an inducible promoter, such as a tetracycline-responsive promoter, a TRE promoter, a Tre3G promoter, a ecdysone-responsive promoter, a cumate-responsive promoter, a glucocorticoid-responsive promoter, an estrogen-responsive promoter, a PPAR-γ promoter, a RU-486-responsive promoter, or a combination thereof.

[0173] Promoters can include tissue-specific promoters and / or lineage-specific promoters. Tissue-specific promoters can be liver-specific thyroxine-binding globulin (TBG) promoters, insulin promoters, glucagon promoters, somatostatin promoters, pancreatic polypeptide (PPY) promoters, synaptic protein-1 (Syn) promoters, creatine kinase (MCK) promoters, mammalian myofibril (DES) promoters, α-myosin heavy chain (a-MHC) promoters, or cardiac troponin T (cTnT) promoters. Tissue-specific promoters can be neuron-specific promoters, such as synaptic protein-1 (Syn) promoters, CaMKIIa promoters, calcium / calmodulin-dependent protein kinase IIa promoters, tubulin α I promoters, neuron-specific enolase promoters, platelet-derived growth factor β chain promoters, TRPV1 promoters, Nav1.7 promoters, Nav1.8 promoters, Nav1.9 promoters, or Advillin promoters. Tissue-specific promoters may be or include muscle-specific promoters, such as the MCK promoter.

[0174] The promoter may contain intron sequences. The promoter may contain bidirectional promoters and / or enhancers. In some embodiments, the enhancer may be a CMV enhancer. One or more cells of the subject may contain an endogenous version of a nucleic acid sequence (e.g., a gene), and the promoter may contain or may be derived from the endogenous version of the promoter. In some embodiments, one or more cells of the subject contain an endogenous version of a nucleic acid sequence, and the sequence is not truncated relative to the endogenous version.

[0175] The promoter length can vary, for example, less than 1 kb. In other embodiments, the promoter is greater than 1 kb. The length of the promoter can be 200 bp, 210 bp, 220 bp, 230 bp, 240 bp, 250 bp, 260 bp, 270 bp, 280 bp, 290 bp, 300 bp, 310 bp, 320 bp, 330 bp, 340 bp, 350 bp, 360 bp, 370 bp, 380 bp, 390 bp, 400 bp, 410 bp, 420 bp, 430 bp, 440 bp, 450 bp, 460 bp, 470 bp, 480 bp, 490 bp, 500 bp, 510 bp, 520 bp, 530 bp, 540 bp, 550 bp, 560 bp, 570 bp, 580 bp, 590 The bp values ​​are 600 bp, 610 bp, 620 bp, 630 bp, 640 bp, 650 bp, 660 bp, 670 bp, 680 bp, 690 bp, 700 bp, 710 bp, 720 bp, 730 bp, 740 bp, 750 bp, 760 bp, 770 bp, 780 bp, 790 bp, 800 bp, or any two of these values, or greater than 800 bp. The promoter can provide sustained expression of the therapeutic gene product in target tissues (such as, but not limited to, the CNS) for a certain period of time.The expression of therapeutic gene expression products can persist for the following time periods: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 1 hour, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months. 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 ​​years, 49 years, 50 years, 55 years, 60 years, 65 years, or a number or range between any two of these values, or greater than 65 years.

[0176] The rAAV disclosed herein may comprise one or more nucleic acids disclosed herein. The nucleic acid may comprise a polynucleotide encoding a protein. The nucleic acid may be or may encode an RNA agent. The nucleic acid may comprise a promoter operatively linked to the polynucleotide encoding a protein. As disclosed herein, in some embodiments, the gene is operatively linked to a suitable regulatory element. The one or more genes of the nucleic acid may comprise siRNA, shRNA, antisense RNA oligonucleotide, antisense miRNA, trans-splicing RNA, guide RNA, single guide RNA, crRNA, tracrRNA, trans-splicing RNA, pre-mRNA, mRNA, or any combination thereof. The one or more genes of the nucleic acid may comprise one or more synthetic protein circuit components. The one or more genes of the nucleic acid may comprise an entire synthetic protein circuit comprising one or more synthetic protein circuit components. The one or more genes of the nucleic acid may comprise two or more synthetic protein circuits.

[0177] Proteins can be any protein, including those that are naturally occurring and those that are not. Examples include, but are not limited to, luciferase; fluorescent proteins (e.g., GFP); growth hormone (GH) and its variants; insulin-like growth factor (IGF) and its variants; granulocyte colony-stimulating factor (G-CSF) and its variants; erythropoietin (EPO) and its variants; insulin, such as proinsulin, preinsulin, insulin, insulin analogs, etc.; antibodies and their variants, such as hybrid antibodies, chimeric antibodies, humanized antibodies, monoclonal antibodies; antigen-binding fragments (Fab fragments) of antibodies, single-chain variable fragments (scFV fragments) of antibodies; dystrophin and its variants; coagulation factors and their variants; cystic fibrosis transmembrane transduction regulator (CFTR) and its variants; and interferons and their variants.

[0178] Pharmaceutical Composition In some aspects, the present invention provides for the administration of compositions comprising the peptides provided herein to a subject. In some embodiments, the compositions of the present invention are administered via systemic bloodstream delivery. In some embodiments, the compositions of the present invention are administered via local direct injection. In some embodiments, the present invention provides compositions comprising the AAV and / or peptides of the present invention. In some embodiments, the compositions are injectable. In some embodiments, the compositions are ointments. In some embodiments, the compositions are oral compositions. In some embodiments, the compositions are oral compositions.

[0179] This document also discloses pharmaceutical compositions comprising one or more rAAV viruses (or other delivery systems) disclosed herein and one or more pharmaceutically acceptable carriers. The compositions may also contain additional components such as diluents, stabilizers, excipients, and adjuvants. As used herein, a “pharmaceutically acceptable” carrier, excipient, diluent, adjuvant, or stabilizer is non-toxic (preferably inert) to cells or subjects exposed thereto at the doses and concentrations used, or has an acceptable level of toxicity as determined by those skilled in the art. The carrier, diluent, and adjuvant may include buffer solutions such as phosphates, citrates, or other organic acids; antioxidants such as ascorbic acid; low molecular weight peptides (e.g., less than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween™, Pluronics™, or polyethylene glycol (PEG). In some embodiments, a physiologically acceptable carrier is an aqueous pH buffer solution.

[0180] The methods disclosed herein include delivering a drug agent to the nervous system of a subject. In some embodiments, the method includes providing an AAV carrier comprising the AAV capsid protein disclosed herein. In some embodiments, the AAV carrier comprises a drug agent to be delivered to the nervous system. In some embodiments, the method includes administering the AAV carrier to the subject. The composition may be used for intravenous administration. The composition may be used for systemic administration. In some embodiments, the invention provides for local administration of the pharmaceutical composition. The subject may be an adult animal.

[0181] The titer of rAAV to be administered will vary depending on, for example, the specific rAAV, administration method, treatment target, individual, and targeted cell type, and can be determined using standard methods in the art. As will readily apparent to those skilled in the art, the useful in vivo dose of the recombinant virus to be administered and the specific administration method will vary depending on age, weight, severity of distress, the animal species being treated, the specific recombinant virus expressing the protein of interest, and the specific purpose of the recombinant virus employed. The determination of the effective dose level, i.e., the dose level required to achieve the desired outcome, can be accomplished by those skilled in the art using routine pharmacological methods. Typically, human clinical application of the product begins at a low dose level, which is increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish the useful dose and route of administration of the composition identified by the methods of the present invention using established pharmacological methods.

[0182] The effective dose and dosage of a pharmaceutical composition for the prevention or treatment of the diseases or conditions disclosed herein are defined by an observed beneficial response in relation to the disease or condition or its symptoms. A beneficial response includes prevention, relief, prevention, or cure of the disease or condition or its symptoms. In some embodiments, a beneficial response is measured by detecting a measurable improvement in the presence, level, or activity of a subject's biomarkers, transcriptomic risk profile, or gut microbiome. As used herein, "improvement" means a change in presence, level, or activity to that observed in normal individuals (e.g., individuals without the disease or condition). Where the therapeutic rAAV composition is ineffective or does not adequately relieve the disease or condition or its symptoms, the dose and / or route of administration may be changed, or an additional agent may be administered to the subject together with the therapeutic rAAV composition. In some embodiments, when a patient begins a regimen of the therapeutic rAAV composition, the patient also gradually discontinues (e.g., by gradually reducing the dose) a second treatment regimen.

[0183] In some embodiments, the pharmaceutical compositions according to this disclosure are administered at dose levels sufficient to deliver the following doses daily, once a day, or multiple times to achieve the desired therapeutic, diagnostic, or preventative effect: about 0.0001 mg / kg to about 100 mg / kg, about 0.001 mg / kg to about 0.05 mg / kg, about 0.005 mg / kg to about 0.05 mg / kg, about 0.001 mg / kg to about 0.005 mg / kg, about 0.05 mg / kg to about 0.5 mg / kg, about 0.01 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 40 mg / kg, about 0.5 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 25 mg / kg of subject body weight. It should be understood that those skilled in the art can convert the above dose concentrations into vg / kg or viral genome or into total viral genome.

[0184] The recombinant virus disclosed herein can be administered to subjects in need (e.g., humans). There are no particular limitations on the route of administration. For example, a therapeutically effective amount of the recombinant virus can be administered to a subject via standard methods in the art. Administration can be systemic. Administration can be intravenous.

[0185] Non-limiting examples of routes include intramuscular, intravaginal, intravenous, intraperitoneal, subcutaneous, epidermal, intradermal, rectal, intraocular, lung, intracranial, intraosseous, oral, buccal, general, or nasal administration. In some embodiments, the recombinant virus is administered to the subject via systemic transduction. In some embodiments, the recombinant virus is administered to the subject via intramuscular injection. In some embodiments, rAAV is administered to the subject via parenteral routes (e.g., via intravenous, intramuscular, or subcutaneous injection), via surface scratching, or via inoculation into the subject's body cavity. The route of administration and serotype of the AAV component of the rAAV virus can be readily determined by those skilled in the art taking into account the infection and / or disease state being treated and the target cells / tissues expressing the protein of interest. In some embodiments, intravenous administration of rAAV can be advantageous. The variant AAVs provided herein can advantageously provide intravenous administration of a vector with enhanced tropism to the CNS.

[0186] In some embodiments, the subject is a primate, and the agent is delivered to the endothelial cells and / or neurons of the nervous system. The nervous system may be the central nervous system (CNS). The efficiency of the agent delivered to the endothelial cells of the subject's nervous system may be at least 1.5 times, 2 times, or 3 times the efficiency of the agent delivered to the neurons of the nervous system. In some embodiments, the efficiency of the agent delivered to the endothelial cells of the subject's nervous system is more than 3 times the efficiency of the agent delivered to the neurons of the nervous system (e.g., 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or any number or range between these values).

[0187] The methods disclosed herein include methods for delivering a drug agent (e.g., a therapeutic agent) to cells. In some embodiments, the method includes contacting an AAV carrier comprising the AAV capsid protein disclosed herein with a cell. In some embodiments, the AAV carrier comprises a drug agent to be delivered to the nervous system. In some embodiments, the cell is an endothelial cell or a neuron. In some embodiments, the AAV carrier is contacted with the cell in vitro, in vivo, or ex vivo. The cell may be present in a tissue, organ, or subject. The cell may be a brain endothelial cell, a neuron, a cell in a capillary in the brain, a cell in a small artery in the brain, a cell in an artery in the brain, a cell in the cerebral vascular system, or a combination thereof.

[0188] The AAV vector can be the AAV9 vector or a variant thereof. In some embodiments, the AAV vector is selected from the following vectors: AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, human isolate hu.31, human isolate hu.32, rhesus monkey isolate rh.8, rhesus monkey isolate rh.10, or variants thereof. The serotype of the AAV vector may be different from the serotype of the AAV capsid.

[0189] The variant AAV capsid may contain tropism for tissues or cells of the central nervous system (CNS). Target cells may be neurons, neural stem cells, astrocytes, or tumor cells. Target cells may be located in the brain or spinal cord. Target cells may include antigen-presenting cells, dendritic cells, macrophages, nerve cells, brain cells, astrocytes, microglia, and neurons. In some embodiments, the target cells are endothelial cells.

[0190] The actual administration of rAAV can be accomplished using any physical method that delivers rAAV into the nervous system of a subject. For example, the rAAV disclosed herein can be advantageously administered intravenously for delivery to the CNS. As disclosed herein, the capsid protein of rAAV can be modified to target specific target environments of interest, such as the central nervous system, and to enhance tropism toward the target environment of interest (e.g., CNS tropism). The pharmaceutical composition can be prepared, for example, as an injectable formulation.

[0191] A therapeutically effective dose of rAAV can be administered to the subject at different time points. For example, rAAV can be administered to the subject before, during, or after the onset of the disease or condition. rAAV can also be administered to the subject before, during, or after the onset of a disease or condition (e.g., Huntington's disease (HD), Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, spinal muscular ataxia (types I and II), Friedreich ataxia, spinocerebellar ataxia, and any lysosomal storage disease involving CNS cells, including but not limited to Krabby's disease, Sandhof's disease, Tay-Sachs disease, Gaucher disease (types I, II, or 111), Niemann-Pick disease (NPC1 or NPC2 deficiency), Heller syndrome, Pompe disease, Batten disease, or any combination thereof), chronic pain, or a combination thereof. In some embodiments, rAAV is administered to the subject during remission of the disease or condition. In some embodiments, rAAV is administered to the subject before the onset of the disease or condition. In some embodiments, rAAV is administered to subjects at risk of developing a disease or condition.

[0192] In some embodiments, this document discloses formulations of pharmaceutically acceptable excipient and carrier solutions suitable for delivering the compositions described herein, as well as suitable dosages and treatment regimens for using a particular composition described herein in a variety of treatment regimens. In some embodiments, the amount of therapeutic gene expression product in each therapeutically useful composition may be prepared in such a manner that a suitable dose will be obtained at any given unit dose of the compound. Those skilled in the art of preparing such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and thus, a variety of dosages and treatment regimens may be desired. In some cases, the compositions are suitable pharmaceutical compositions disclosed herein that can be delivered intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intraventricularly, intramuscularly, intrathecally, orally, intraperitoneally, orally or nasally by direct injection to one or more cells, tissues, or organs. In some embodiments, the rAAVs disclosed herein can be advantageously administered intravenously for delivery to the CNS.

[0193] In some embodiments, the pharmaceutical form of AAV-based viral compositions suitable for injection includes sterile aqueous solutions or dispersions and sterile powders, which are reconstituted into sterile injectable solutions or dispersions immediately before use. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. Suitable flowability can be maintained, for example, by using coatings such as lecithin, or, in the case of dispersions, by maintaining the desired particle size and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it will be preferred to include isotonic agents, such as sugars or sodium chloride. Prolonged absorption of the injectable composition can be achieved by using agents with delayed absorption in the composition, such as aluminum monostearate and gelatin.

[0194] In some embodiments, for administration of injectable aqueous solutions, the solution may be suitably buffered, for example, if necessary, and the liquid diluent is first isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Some variations in dosage will inevitably occur depending on the condition of the subject being treated. The person responsible for administration will determine the appropriate dosage for the individual subject in any circumstances. Furthermore, for human administration, the formulation should meet the sterility, pyrogenicity, and general safety and purity standards required by the FDA's Office of Biologics Standards.

[0195] This document discloses sterile injectable solutions comprising the compositions disclosed herein (e.g., rAAV compositions), which are prepared by incorporating the compositions disclosed herein, in the desired amount, along with several other components listed above, into a suitable solvent, followed by filtration sterilization. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile medium containing a basic dispersion medium and other desired components from those listed above. In cases where sterile powders are used to prepare sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any other desired components from a previously sterile filtered solution. Injectable solutions can be advantageous for systemic administration (e.g., intravenous administration).

[0196] In some embodiments, the present invention provides kits comprising the compositions disclosed herein. Kits for treating or preventing diseases or conditions of the CNS, PNS, or a target organ or environment (e.g., the CNS) are also disclosed herein. In some cases, the disease or condition is cancer, pathogen infection, neurological disorder, muscle disease, or immune disorder, as described herein. In one embodiment, the kit may comprise a therapeutic or preventative composition containing an effective amount of a composition of rAAV particles encapsulating the nucleic acid provided herein and the rAAV capsid protein disclosed herein. In another embodiment, the kit may comprise a therapeutic or preventative composition containing an effective amount of unit-dose rAAV-modified cells (“modified cells”) expressing a therapeutic nucleic acid as described herein. In some embodiments, the kit comprises a sterile container that may contain the therapeutic composition; such a container may be a box, ampoule, bottle, vial, tube, bag, pouch, blister pack, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing medicines.

[0197] In some embodiments, rAAV is provided together with instructions for administering rAAV to a subject who has or is at risk of developing a disease or condition. The instructions typically include information about the use of the composition for the treatment or prevention of a disease or condition.

[0198] The kit may include allogeneic cells. In some embodiments, the kit includes cells that may contain genomically modified cells. In some embodiments, the kit includes "off-the-shelf" cells. In some embodiments, the kit includes cells that can be expanded for clinical use. In some embodiments, the kit contains inclusions for research purposes.

[0199] In some embodiments, the instructions include at least one of the following: a description of the therapeutic rAAV composition; a dosage table and administration for the treatment or prevention of the diseases or symptoms disclosed herein; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. These instructions may be printed directly on the container (if present), or as a label applied to the container, or as separate paper pages, brochures, cards, or folders provided with or applied to the container. In some embodiments, the instructions provide a procedure for administering rAAV alone to a subject. In some embodiments, the instructions provide procedures for administering rAAV to a subject at least about 1 hour (hr), 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, or up to 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after or before administration of any other therapeutic agent disclosed herein. In some cases, these instructions provide that rAAV is prepared for intravenous injection. In some cases, these instructions provide that rAAV is prepared for intranasal administration.

[0200] The disease or condition may include neurological disorders or conditions. For example, neurological disorders or conditions can include epilepsy; Dravet syndrome; Lennox-Gastaut syndrome; myoclonic seizures; juvenile myoclonic epilepsy; refractory epilepsy; schizophrenia; juvenile spasms; West syndrome; infantile spasms; refractory infantile spasms; Alzheimer's disease; Creutzfeld-Jakob's syndrome / disease; bovine spongiform encephalopathy (BSE); prion-associated infections; diseases involving mitochondrial dysfunction; diseases involving P-amyloid and / or tauopathy; Down syndrome; hepatic encephalopathy; Huntington's disease; motor neuron disease; amyotrophic lateral sclerosis (ALS); olivopontocerebellar atrophy; postoperative cognitive deficit (POCD); systemic lupus erythematosus; systemic sclerosis; Sjögren's syndrome. Syndrome); neuronal ceroid lipofuscin deposition syndrome; neurodegenerative cerebellar ataxia; Parkinson's disease; Parkinson's dementia; mild cognitive impairment; cognitive deficits in various forms of mild cognitive impairment; cognitive deficits in various forms of dementia; boxing dementia; vascular and prefrontal dementia; cognitive impairment; learning disability; eye injury; eye disease; eye condition; glaucoma; retinopathy; macular degeneration; head, brain, or spinal cord injury; head, brain, or spinal cord trauma; convulsions; epileptic convulsions; epilepsy; temporal lobe epilepsy; myoclonic epilepsy; tinnitus; movement disorder; chorea; Huntington's disease Chorea; Athetosis; Dystonia; Mechanical repetition; Twitching; Tardive dyskinesia; Tic disorder; Spasmodic torticollis; Blepharospasm; Focal and generalized dystonia; Nystagmus; Hereditary cerebellar ataxia; Corticobasal degeneration; Tremor; Essential tremor; Addiction; Anxiety disorder; Panic disorder; Social anxiety disorder (SAD); Attention deficit hyperactivity disorder (ADHD); Attention deficit syndrome (ADS); Restless legs syndrome (RLS); Childhood hyperactivity disorder; Autism; Dementia; Alzheimer's disease Alzheimer's disease dementia; Korsakoff syndrome dementia; Korsakoff syndrome; vascular dementia; HIV-related dementia; HIV-1 encephalopathy; AIDS encephalopathy; AIDS dementia complex; AIDS-related dementia; major depressive disorder; major depressive disorder; depression; memory loss; stress; bipolar mania-depressive disorder; drug tolerance; opioid tolerance; movement disorders; fragile X syndrome; irritable bowel syndrome (IBS); migraine;Multiple sclerosis (MS); muscle spasms; pain; chronic pain; acute pain; inflammatory pain; neuropathic pain; post-traumatic stress disorder (PTSD); schizophrenia; spastic state; Tourette's syndrome; eating disorders; food addiction; bulimia; agoraphobia; generalized anxiety disorder; obsessive-compulsive disorder; panic disorder; social phobia; phobia; substance-induced anxiety disorder; paranoia; schizoaffective disorder; schizophrenia-like disorder; substance-induced psychotic disorder; hypertension; or any combination thereof.

[0201] In some preferred embodiments, the present invention provides a method for treating brain disorders. In some preferred embodiments, the brain disorder is brain cancer. In some other preferred embodiments, the present invention provides a method for treating eye-related disorders. In some preferred embodiments, the eye-related disorder is glaucoma. In some embodiments, the present invention provides treatment of brain and / or eye disorders by administering a composition comprising the CA4-binding peptide or CA4-binding AAV of the present invention.

[0202] By incorporating via reference Throughout this disclosure, references and citations have been made to other literature, such as patents, patent applications, patent publications, journals, books, papers, online content, and publicly accessible databases. All such documents are incorporated herein by reference in their entirety for all purposes.

[0203] equivalent Based on the entire contents of this document, including references to scientific and patent literature cited herein, various modifications to the invention and many other embodiments thereof will become apparent to those skilled in the art, except for those shown and described herein. The subject matter of this document contains important information, illustrations, and guidance that may be adapted to practice the invention in its various embodiments and equivalents.

Claims

1. A carbonic anhydrase IV-binding peptide, selected from the group consisting of the carbonic anhydrase IV-binding peptides listed in Tables 1, 3, 4 and 5.

2. The carbonic anhydrase IV-binding peptide according to claim 1, wherein the carbonic anhydrase IV-binding peptide is selected from the group consisting of the 9-mer carbonic anhydrase IV-binding peptides listed in Tables 4 and 5.

3. An AAV capsid protein comprising the carbonic anhydrase IV-binding peptide according to claim 1 or 2.

4. The AAV capsid protein according to claim 3, wherein the AAV capsid protein comprises AAV9 as a parental sequence.

5. An AAV capsid comprising the AAV capsid protein according to claim 3 or 4.

6. The AAV cape of claim 5, wherein the AAV cape further comprises therapeutic or diagnostic goods to be delivered to the central nervous system (CNS) of a primate or human.

7. The AAV capsid of claim 6, wherein the therapeutic or diagnostic cargo is delivered to the therapeutic target in the CNS after crossing the blood-brain barrier (BBB).

8. The AAV capsid according to claim 5 or 6, wherein the therapeutic cargo is selected from the group consisting of nucleic acids, antibodies, peptides and small molecules.

9. The AAV capsid protein of claim 3, wherein the AAV capsid protein is Alpha43, 45, 48 and 49 listed in Table 1.

10. The AAV capsid protein of claim 9, wherein the AAV capsid protein binds to human CA4.

11. The AAV capsid protein according to claim 9 or 10, wherein the AAV capsid protein comprises a peptide selected from the group consisting of: DGVVHETVR (SEQ ID NO: 7); DGVVGVNIR (SEQ ID NO: 8); DGEVGLTVR (SEQ ID NO: 9); DGIVGSTIR (SEQ ID NO: 10).

12. A composition comprising the carbonic anhydrase IV-binding peptide, AAV capsid protein, and / or AAV as described in claims 1 to 11.

13. The composition of claim 12, wherein the composition is an injectable formulation.

14. The composition of claim 13, wherein the composition further comprises a pharmaceutically acceptable excipient.

15. A method for treating or diagnosing a condition by administering a composition comprising the carbonic anhydrase IV-binding peptide, AAV capsid protein, and / or AAV as claimed in claims 1 to 11.

16. The method of claim 15, wherein the symptom is a brain disease or an eye disease.

17. The method of claim 16, wherein the brain disease is selected from the group consisting of brain cancer, neurodegeneration, and glioblastoma.

18. The method of claim 16, wherein the eye disease is glaucoma.

19. The method of claim 15, wherein the composition is administered via systemic bloodstream delivery.

20. The method of claim 15, wherein the composition is administered by local injection.

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