Target for effecting receptor-mediated control of therapeutic biodistribution and efficacy

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). In some embodiments, the BBB is LRP6 across the receptor. Also disclosed herein are recombinant adeno-associated viruses (rAAVs) with increased specificity and transduction efficiency across BBB, as well as related compositions and methods of treating various diseases and conditions.

Description

[0001] Related applications

[0002] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Serial No. 63 / 420,002, filed on October 27, 2022, and U.S. Provisional Patent Application Serial No. 63 / 465,814, filed on May 11, 2023. The contents of these related applications are incorporated herein by reference in their entirety for all purposes.

[0003] Statement Regarding Federally Funded Research and Development

[0004] This invention was made with government support under Grant No. NS111369 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0005] References to sequence listings

[0006] This application is filed in electronic format with a sequence listing. The sequence listing is provided as a 51,181-byte file named 30KJ-365863-US Sequence Listing, created on October 23, 2023. The electronic format of the sequence listing is incorporated herein by reference in its entirety. Technical Field

[0007] The present disclosure relates generally to the field of gene delivery. More specifically, methods and compositions for crossing the blood-brain barrier are disclosed. Background Art

[0008] Targeting therapeutic and research molecules to tissues and cell types of interest and away from tissues and cell types that mediate potentially dangerous side effects is a fundamental issue in drug development. This is particularly true for molecules targeting the brain. The blood-brain barrier (BBB) severely restricts the properties of therapeutic and research molecules that, after systemic administration into the peripheral bloodstream, may enter the central nervous system (CNS).

[0009] There is a need to use gain-of-function screening rather than loss-of-function screening and to screen both naturally evolved and laboratory evolved AAVs. There is a need for methods to reverse engineer novel targets that determine the therapeutic biodistribution of products of naturally evolved and directed evolution of adeno-associated viruses (AAVs). Summary of the Invention

[0010] Disclosed herein is a method for increasing the permeability of the blood-brain barrier. In some embodiments, the method comprises providing a targeting peptide capable of binding to low-density lipoprotein receptor-related protein 6 (LRP6), thereby increasing the permeability of the blood-brain barrier.

[0011] In some embodiments, the targeting peptide binds to YWTD Domain 1 and / or Domain 2 of LRP6. In some embodiments, the permeability of the blood-brain barrier is increased by at least 25%, 50%, 75%, 100% or more compared to the absence of the targeting peptide.

[0012] Disclosed herein are methods for delivering a payload to a subject's nervous system. In some embodiments, the method comprises: providing a targeting peptide or a derivative thereof capable of binding to low-density lipoprotein receptor-related protein 6 (LRP6), 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.

[0013] In some embodiments, the delivery system comprises nanoparticles, nanotubes, nanowires, dendrimers, liposomes, ethosomes and aquasomes, polymer vesicles and non-ionic 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. In some embodiments, the targeting peptide enhances the binding affinity of the viral vector or the non-viral vector to LRP6. 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, polymeric nanoparticles, inorganic nanoparticles, surfactant-based emulsions, nanowires, silica nanoparticles, peptide- or protein-based particles, lipid-polymer particles, nanolipoprotein particles, and combinations thereof.

[0014] In some embodiments, the payload to be delivered to the nervous system is a biological molecule, a non-biological molecule, or a combination thereof. In some embodiments, the biological molecule is selected from the group consisting of: a nucleic acid sequence, a protein, a peptide, a lipid, a polysaccharide, 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 trophic factor, a 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 that restores protein function to a human or animal carrying a genetic mutation in a gene; c) DNA that encodes a protein that can be used to control or alter the activity or state of a cell; d) DNA that encodes 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 by homologous recombination; g) a DNA sequence encoding a therapeutic RNA; h) an shRNA or artificial miRNA delivery system; or i) a DNA sequence that affects the splicing of an endogenous gene.

[0015] In some embodiments, the LRP6 is mouse LRP6. In some embodiments, the LRP6 has an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the LRP6 is macaque LRP6. In some embodiments, the LRP6 has an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the LRP6 is human LRP6. In some embodiments, the LRP6 has an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 31. In some embodiments, upon binding, the targeting peptide is capable of interacting with: (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having an amino acid sequence of SEQ ID NO: 31; or (2) one or more positions functionally equivalent to S96, S114, E115, R141, W157, W183, or W242 in LRP6 having an amino acid sequence of SEQ ID NO: 31.

[0016] In some embodiments, the targeting peptide is inserted between two adjacent amino acids in AA587-594 of SEQ ID NO: 11 of the AAV9 vector, or between functional equivalents of AA587-594 in an amino acid sequence at least 80% identical to SEQ ID NO: 11. In some embodiments, the targeting peptide is inserted between AA588-589 of SEQ ID NO: 11 of the AAV9 vector, or between functional equivalents of AA588-589 in an amino acid sequence at least 80% identical to SEQ ID NO: 11.

[0017] The AAV vector can be conjugated to, for example, a nanoparticle, a second molecule, or a combination thereof. The administration can be, for example, systemic administration. In some embodiments, the administration is intravenous administration or intrathecal administration.

[0018] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a subject suffering from or at risk for one or more of the following: chronic pain, Friedreich's ataxia, Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), spinal muscular atrophy type I and II (SMA I and II), Friedreich's ataxia (FA), spinocerebellar ataxia, multiple sclerosis (MS), chronic traumatic encephalopathy (CTE), HIV-1 associated dementia, or a lysosomal storage disease involving cells in the CNS. In some embodiments, the lysosomal storage disease involving cells within the CNS is Krabbe disease, Sandhoff disease, Tay-Sachs, Gaucher disease (type I, II, or III), Niemann-Pick disease (NPC1 or NPC2 deficiency), Hurler syndrome, Pompe disease, or Batten disease. In some embodiments, the subject is a subject who has, is at risk of, or has had a stroke, traumatic brain injury, epilepsy, or spinal cord injury.

[0019] Disclosed herein include adeno-associated virus (AAV) capsid proteins. In some embodiments, the AAV capsid protein comprises a targeting peptide with binding specificity to LRP6. In some embodiments, the targeting peptide is part of the capsid protein of the rAAV vector. In some embodiments, the targeting peptide is inserted between two adjacent amino acids in AA587-594 of SEQ ID NO: 11, or between functional equivalents of AA587-594 in an amino acid sequence that is at least 80% identical to SEQ ID NO: 11. In some embodiments, when bound to LRP6, the targeting peptide is capable of interacting with: (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 of LRP6 having an amino acid sequence of SEQ ID NO: 31; or (2) one or more positions functionally equivalent to S96, S114, E115, R141, W157, W183, or W242 of LRP6 having an amino acid sequence of SEQ ID NO: 31. In some embodiments, the AAV 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.

[0020] Disclosed herein include recombinant adeno-associated viruses (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 comprising a targeting peptide with binding specificity to low-density lipoprotein receptor-related protein 6 (LRP6), wherein the amino acid sequence of the targeting peptide is inserted between two adjacent amino acids in AA587-594 of the AAV capsid protein or their functional equivalents. In some embodiments, the two adjacent amino acids are AA588 and AA589. In some embodiments, upon binding to LRP6, the targeting peptide is capable of interacting with: (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having an amino acid sequence of SEQ ID NO: 31; or (2) one or more positions functionally equivalent to S96, S114, E115, R141, W157, W183, or W242 in LRP6 having an amino acid sequence of SEQ ID NO: 31. In some embodiments, the rAAV has enhanced tropism for the nervous system relative to an rAAV that does not contain the targeting peptide. In some embodiments, the rAAV is capable of transducing the nervous system at an efficiency that is at least twice that of an rAAV that does not contain the targeting peptide.

[0021] Disclosed herein are compositions for delivering an agent to the nervous system of a subject in need thereof. In some embodiments, the composition comprises an AAV comprising (1) an AAV capsid protein disclosed herein and (2) an 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, arterioles 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 agent to be delivered comprises a nucleic acid, a peptide, a small molecule, an aptamer, or a combination thereof.

[0022] Disclosed herein include antibodies or fragments thereof. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence with binding specificity to LRP6. In some embodiments, the antibody or fragment thereof is a bispecific antibody comprising at least one Fab with specificity for LRP6. Disclosed herein include antibody conjugates comprising any one of the antibodies or fragments thereof disclosed herein. In some embodiments, the antibody conjugate further comprises a therapeutic agent or a detectable label. Disclosed herein include peptides or derivatives or conjugates thereof that are specific for low-density lipoprotein receptor-related protein 6 (LRP6). Disclosed herein include nucleic acids. In some embodiments, the nucleic acid comprises the sequence of any one of the antibodies or fragments thereof disclosed herein or any one of the peptides or derivatives or conjugates disclosed herein.

[0023] Disclosed herein include delivery systems. In some embodiments, the delivery system comprises (1) a targeting peptide specific for low-density lipoprotein receptor-related protein 6 (LRP6); and (2) an 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 can be selected from the group consisting of: nanoparticles, nanotubes, nanowires, dendrimers, liposomes, ethosomes and aquasomes, polymer vesicles and non-ionic surfactant vesicles, foams, hydrogels, cubes, quantum dots, exosomes, macrophages, and combinations thereof. In some embodiments, the delivery system comprises a viral vector or a non-viral 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.

[0024] Disclosed herein are methods for designing targeting peptides specific for low-density lipoprotein receptor-related protein 6 (LRP6). In some embodiments, the methods comprise: generating by computer simulation one or more targeting peptides, each of which is capable of interacting with: (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having an amino acid sequence of SEQ ID NO: 31; or (2) one or more positions functionally equivalent to S96, S114, E115, R141, W157, W183, or W242 in LRP6 having an amino acid sequence of SEQ ID NO: 31.

[0025] In some embodiments, generating the one or more targeting peptides by computer simulation comprises: generating a plurality of candidate peptides by computer simulation; performing a computer-assisted docking simulation on each of the plurality of candidate peptides bound to LRP6; and analyzing the structure of LRP6 bound to one or more of the plurality of candidate peptides to identify one or more targeting peptides capable of interacting with: (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having an amino acid sequence of SEQ ID NO: 31; or (2) one or more positions functionally equivalent to S96, S114, E115, R141, W157, W183, or W242 in LRP6 having an amino acid sequence of SEQ ID NO: 31.

[0026] In some embodiments, the method comprises: obtaining a binding score for each of the plurality of candidate peptides that bind to LRP6; and selecting one or more candidate peptides from the plurality of candidate peptides whose binding scores are above a threshold as targeting peptides specific for LRP6. The method may comprise: comparing the binding scores of two or more of the plurality of candidate peptides to rank the candidate peptide sequences. In some embodiments, obtaining the binding score for each of the plurality of candidate peptide sequences comprises (1) counting the total number of atoms in the interface between the candidate peptide and LRP6; (2) counting the total number of atoms in the candidate peptide that clash with LRP6; (3) obtaining a binding angle for the candidate peptide; and (4) obtaining a binding depth for the candidate peptide.

[0027] Disclosed herein are agents that bind to a protein selected from the group consisting of interleukin 3 (IL3), family with sequence similarity 234 member A (FAM234A), glycoprotein 2 (GP2), dipeptidyl peptidase-4 (DPP4), Dickkopf WNT signaling pathway inhibitor 3 (DKK3), alanyl aminopeptidase (ANPEP), epiphycan (EPYC), and LRP6. In some embodiments, the agent is selected from the group consisting of an antibody or fragment thereof, an aptamer, a small molecule, a nucleic acid, and a peptide. In some embodiments, the antibody or fragment thereof comprises an Fc domain. In some embodiments, the antibody or fragment thereof is a single chain variable fragment (scFv), a single domain antibody, an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a disulfide-linked Fv, a scFv, a single domain antibody, a bifunctional antibody, a multispecific antibody, a bispecific antibody (dual specific antibody), an anti-idiotypic antibody, a bispecific antibody (bispecific antibody) or a functionally active epitope binding fragment thereof. In some embodiments, the nucleic acid is a miRNA, shRNA, and siRNA or an oligonucleotide.

[0028] In some embodiments, the agent is conjugated to a detectable label. In some embodiments, the detectable label is selected from the group consisting of: biotin, a fluorophore, a luminescent or bioluminescent marker, a radiolabel, an enzyme, an enzyme substrate, a quantum dot, a contrast agent, a metal particle, a magnetic particle, and any combination thereof. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is a targeting peptide. In some embodiments, the targeting peptide is part of a delivery system, wherein the delivery system comprises a payload to be delivered to the cell. In some embodiments, the delivery system comprises a viral vector or a non-viral vector. In some embodiments, the viral vector comprises an AAV vector. In some embodiments, the targeting peptide is part of the capsid protein of an 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.

[0029] In some embodiments, the payload to be delivered to the cell is a biological molecule, a non-biological molecule, or a combination thereof. In some embodiments, the biological molecule is selected from the group consisting of: a nucleic acid sequence, a protein, a peptide, a lipid, a polysaccharide, 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 trophic factor, a 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) a DNA that restores protein function to a human or animal carrying a genetic mutation in a gene; c) a DNA that encodes a protein that can be used to control or change the activity or state of a cell; d) a DNA that encodes a protein or nucleic acid for assessing the state of a cell; e) a DNA and / or associated guide RNA for genome engineering; f) a sequence for genome editing by homologous recombination; g) a DNA sequence encoding a therapeutic RNA; h) an shRNA or artificial miRNA delivery system; or i) a DNA sequence that affects the splicing of an endogenous gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Non-limiting exemplary data are shown, demonstrating that surface plasmon resonance (SPR) confirmed direct binding interactions between the LRP6 extracellular domain and both AAV-X1.1 and AAV.CAP-Mac. No binding to AAV9 was detected.

[0031] Figure 2 Shown are exemplary computational modeling of engineered AAV insert peptides with the extracellular domain of human LRP6 in AlphaFold2, illustrating the binding of X1.1 to domain 2 and CAP-Mac to either domain 1 or domain 2.

[0032] Figure 3 Exemplary data are shown for AAV-X1 and AAV.CAP-Mac, showing enhanced potency on HEK293 cells compared to standard AAV9-based AAV that does not interact with the human receptor.

[0033] Figure 4 Non-limiting exemplary data are presented showing that knockdown of endogenous LRP6 in HEK293 cells by siRNA selectively reduces the efficacy of AAV-X1.1 and AAV.CAP-Mac.

[0034] Figure 5 Non-limiting exemplary screening libraries and result confirmation are shown. Test AAVs and controls were added to fixed microarray slides.

[0035] Figure 6 Non-limiting exemplary screening libraries and result confirmation are shown. Test AAVs and controls were added to fixed microarray slides.

[0036] Figures 7A-7D Non-limiting exemplary data related to confirmation and specificity screening are shown. Test AAV was added to the fixed confirmation slide using the direct fixation method (AAV was not removed). Anti-AAV9 antibody was used, followed by detection with AF647 anti-mIgG H+L. Anti-AAV9, clone HL2372, was provided by Merck, catalog number MABF2309-100UL, 1:500 dilution. Detection antibody: AlexaFluor 647 anti-mIgG H+L.

[0037] Figure 8A-8B Non-limiting exemplary data related to confirmation and specificity screening are shown. Test and control AAVs were added to fixed confirmation slides using a direct fixation method (AAV was not removed). Anti-AAV9 antibodies were used, followed by detection with AF647 anti-mIgG H+L. Anti-AAV9, clone HL2372, was provided by Merck, catalog number MABF2309-100UL, 1:500 dilution. Detection antibody: AlexaFluor 647 anti-mIgG H+L.

[0038] Figure 9A-9B Non-limiting exemplary data related to confirmation and specificity screening are shown. Controls were added to the fixed confirmation slide using the direct fixation method (samples were not removed). Anti-adeno-associated virus 9, clone HL2372, provided by Merck, catalog number MABF2309-100UL, 1:500 dilution. Detection antibody: Alexa Fluor 647 anti-mIgG H+L.

[0039] Figure 10 Data related to the overall summary of specific hits (eg, weak intensity and above) are shown.

[0040] Figure 11 Data related to the overall summary of specific hits (eg, weak intensity and above) are shown.

[0041] Figures 12A-12E Non-limiting exemplary data showing that high throughput screening identified AAV-binding human proteins are shown. Figure 12AA schematic diagram of AAV cell microarray screening is shown. DNA oligonucleotides encoding individual membrane proteins are chemically coupled to glass slides in a known pattern, and cells grown on these slides are reverse-transfected, thereby forming spots of cells overexpressing a specific known protein. Each protein is expressed in duplicate at two different locations on the slide. When AAV is applied to the slide, enhanced binding is detected from duplicate cell spots overexpressing the cognate AAV receptor. Figure 12B Data related to known AAV capsid receptor interactions (such as AAVR and LY6A with AAV-PHP.eB) are shown, and these interactions were used to optimize streptavidin-based detection conditions for biotinylated capsids with two sets of replicate spots. Anti-TGFBR2 antibody was used as a non-AAV positive control. Figure 12C The interaction of AAVR and LY6A with AAV9.CAP-B22 is shown to optimize direct detection conditions of anti-AAV9 antibodies against unmodified capsids with two sets of replicate spots. Anti-TGFBR2 antibody was used as a non-AAV control. Figure 12D It is shown that in the case of two sets of replicate spots, pooled AAV capsid screening conditions were optimized by varying the concentration of individual capsids within the pool to maximize the signal-to-noise ratio after direct detection with anti-AAV9 antibodies. Figure 12E Data are presented showing that the pooled screens generated preliminary hits that were deconvoluted by the individual capsid screens. Novel potential capsid binding proteins were identified by direct detection with anti-AAV9 antibodies. Fluorescent proteins reverse-transfected with each receptor were detected under transfection control conditions. No condition was treated with anti-AAV9 antibodies alone. DPP4, IL3, and DKK3 were identified in all individual AAV screens and likely represent interactions outside of the engineered regions of AAV9. LY6A, GP2, LRP6, FAM234A, ANPEP, CSF2, and EPYC specifically bound to at least one engineered capsid.

[0042] Figures 13A-13D Non-limiting exemplary data related to species- and serotype-specific interactions between AAV9 and IL3 are shown. Figure 13A Shown is a schematic diagram of a surface plasmon resonance (SPR) experiment in which IL3-Fc was captured on a Protein A sensor chip and an AAV analyte flowed over the sensor. Figure 13B Shown are graphs of data demonstrating that SPR confirmed serotype-specific interactions of AAV9 with the human immunomodulatory cytokine IL3. Figure 13C Shown are graphs of data demonstrating SPR confirmation of AAV9 binding to macaque, but not marmoset or mouse IL3. Figure 13DData related to MS / MS analysis are shown. Bis(sulfosuccinimidyl) suberate (BS3) of cross-linked AAV9 and hsIL3 was extracted from a PAGE gel and subjected to MS / MS analysis, which identified two intermolecular cross-links with high-confidence XlinkX scores.

[0043] Figures 14A-14D Non-limiting exemplary data showing that primate brain-enhanced AAVs acquire interactions with LRP6 are shown. Figure 14A A non-limiting exemplary sketch showing that AAV capsid-specific hits ranked by expression levels in human brain endothelial cells revealed that the highly conserved LRP6 is a potential receptor for BBB crossing is shown. Figure 14B Shown are graphs showing SPR confirmation of direct binding interactions of engineered capsids AAV9-X1.1 and CAP-Mac with human LRP6. Figure 14C Shown are AlphaFold models of the X1 and CAP-Mac polypeptides, which predict selective interaction with human LRP6 YWTD domain 1 (E1). Figure 14D SPR data showing mouse LRP6-E1E2 and LRP6-E3E4 (the minimal stable extracellular domain fragment formed by cooperative folding) confirm that AAV9-X1.1 and CAP-Mac bind only to LRP6-E1E2.

[0044] Figures 15A-15F Non-limiting exemplary data showing that LRP6 modulates CNS function of engineered AAV in mice are shown. Figure 15A Schematic diagram of Lrp6 conditional knockout via serial AAV injections. Lrp6Cre conditional knockout mice were systemically injected with AAV1-X1 containing Cre or mCherry, creating cohorts of mice with varying LRP6 expression. After a defined period of expression, these cohorts were injected with either AAV9-PHP.eB or AAV9-X1.1 containing eGFP. By switching serotypes, neutralizing antibodies were evaded and the vector's dependency on Lrp6 could be assessed in vivo. Figure 15B Representative sagittal brain and liver images from a conditional Lrp6 knockout experiment are shown. Imaging parameters were independently optimized for AAV9-X1.1 and AAV9-PHP.eB secondary dose conditions. Figure 15C Quantification of AAV potency is shown, demonstrating that conditional knockout of Lrp6 in the mouse brain selectively and potentially reduces AAV9-X1.1 brain and liver potency. Data points are the average of two sections per tissue region per animal, with the physiological regions of interest consistent across four experimental cohorts. Bars represent the mean. Figure 15D-15E showed that AAV9-X1.1 can be used in macaques ( Figure 15D ) and human primary brain microvascular endothelial cell cultures ( Figure 15E ) with enhanced potency, but with Mesd inhibition of LRP6, its potency decreased to that of AAV9. The bars represent the mean. Immunofluorescence of mouse and infant macaque brain tissue revealed that LRP6 expression was consistently high across brain endothelial cells as well as neurons and astrocytes ( Figure 15F ).

[0045] Figure 16 Non-limiting exemplary data related to individual characterization of pooled AAVs prior to comprehensive screening are shown. Individual AAVs were tested at different doses to determine the optimal signal-to-noise ratio for each capsid, while confirming detection of known interactors KIAA0319L (AAVR) and LY6A (only for CAP-B22). AAV binding detected on duplicate spots of the same protein is indicated by arrows.

[0046] Figures 17A-17C Data related to cell culture potency assay validation of high-throughput screening hits are shown. Figure 17A Depicted are representative images and quantification results showing that transient overexpression of mouse and human GP2 in HEK293T cells enhances the efficacy of CAP-Mac and AAV9-X1.1, with stronger effects on the human protein. The scale indicates the degree of infection (minimum, 0.01; maximum, 0.17) and the total brightness of each signal area (minimum, 0.11; maximum, 0.45). Figure 17B Depicted are representative images and quantification of results showing that transient overexpression of mouse and human FAM234A in HEK293T cells enhances the efficacy of PHP.eB and CAP-B22, with a stronger effect on the mouse protein. The extent of infection (minimum, 0.04; maximum, 0.07) and the total brightness per signal area (minimum, 0.16; maximum, 0.29) are shown. Figure 17C Depicted are representative images and quantification results showing that transient overexpression of human ANPEP and DPP4 did not enhance the efficacy of AAV9-X1.1 and AAV9, respectively. Scale bar indicates 200 μm.

[0047] Figures 18A-18B Non-limiting exemplary data related to SPR confirmation of selected screening hits are shown. Figure 18A ) or human GP2-Fc ( Figure 18B ) immobilized on a Protein A chip to assess interactions between AAV analytes. In contrast to the cell microarray screening, no interaction between AAV9 and DKK3 was observed, whereas AAV9-X1.1 acquired the ability to directly bind to human GP2, consistent with the results of the cell microarray screening and cell culture potency assays.

[0048] Figures 19A-19B Shown is a non-limiting exemplary graph of annotated MS / MS spectra of AAV9 cross-linked to human IL3 by BS3.

[0049] Figures 20A-20D Non-limiting exemplary data are shown for LRP6 binding to X1 peptides and AAV-BI30 of various serotypes. Figure 20A SPR data showing the complete human LRP6 extracellular domain confirm that the X1 insertion peptide can modularly bind LRP6 to multiple serotypes. Figure 20B Shown are SPR data showing that AAV-BI30 confirms its binding interaction with mouse LRP6-E1E2 but not LRP6-E3E4. Figure 20C Shown are exemplary AlphaFold models that predict binding of XI and CAP-Mac variable region VIII peptides to WYTD domains 1 or 2 (e.g., E1E2), but in some examples cannot confidently assign a specific binding pose. Figure 20D Non-limiting exemplary data showing that LRP5 WYTD domain 1 does not bind AAV-X1.1 despite having high sequence similarity to LRP6 are shown.

[0050] Figure 21 Described are non-limiting exemplary data showing that X1.1 and CAP-Mac bind to LRP6 and AAVR but not LRP5. Pull-down assays performed with the extracellular domains of mouse LRP6, mouse LRP5, and the human AAVR PDK2 domain against AAV9, AAV9-X1.1, and CAP-Mac traps are shown. Asterisks indicate LRP6 binding interactions acquired by X1.1 and CAP-Mac during directed evolution of the parental AAV9 capsid.

[0051] Figures 22A-22B Non-limiting exemplary data related to cell culture potency assay validation of LRP6 interaction are shown. Figure 22A Schematic diagram showing Mesd chaperone function and LRP6 domain-dependent inhibition by recombinant Mesd and SOST proteins. Figure 22B Quantification of AAV potency showing the effects of transient overexpression of LRP receptors and LRP6 inhibition is shown: Degree of infection (minimum, 0.04; maximum, 0.23) and total brightness per signal area (minimum, 0.04; maximum, 0.51).

[0052] Figures 23A-23B Non-limiting exemplary data are shown regarding the efficacy of X1 AAV in mouse liver and human primary cell culture. Figure 23ARepresentative liver images from a conditional Lrp6 knockout experiment are shown. Identical imaging parameters were used in all conditions. Figure 23B Depicted are data showing that AAV1-X1 has enhanced potency in primary human brain microvascular endothelial cell cultures, with potency reduced to AAV1 levels upon Mesd inhibition of LRP6. Bars represent mean values. DETAILED DESCRIPTION

[0053] In the detailed description that follows, reference is made to the accompanying drawings that form a part of this detailed description. In the accompanying drawings, similar symbols generally identify similar components unless the context indicates otherwise. 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 illustrated in the accompanying drawings, aspects of the present disclosure may be arranged, substituted, combined, separated, and designed into a variety of different configurations, all of which are expressly encompassed herein and form a part of the disclosure.

[0054] All patents, published patent applications, other publications, and sequences from GenBank, and other databases mentioned herein are incorporated by reference in their entirety with respect to the relevant art.

[0055] Disclosed herein is a method for increasing the permeability of the blood-brain barrier. In some embodiments, the method comprises providing a targeting peptide capable of binding to low-density lipoprotein receptor-related protein 6 (LRP6), thereby increasing the permeability of the blood-brain barrier.

[0056] Disclosed herein are methods 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 LRP6, wherein the targeting peptide is part of a delivery system, and wherein the delivery system comprises the payload to be delivered to the nervous system; and administering the delivery system to the subject.

[0057] Disclosed herein include adeno-associated virus (AAV) capsid proteins. In some embodiments, the AAV capsid protein comprises a targeting peptide with binding specificity to LRP6. Disclosed herein include 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, the AAV capsid protein comprising a targeting peptide with binding specificity to LRP6, wherein the amino acid sequence of the targeting peptide is inserted between two adjacent amino acids in AA587-594 of the AAV capsid protein or a functional equivalent thereof.

[0058] Disclosed herein include compositions for delivering an agent to the nervous system of a subject in need thereof. In some embodiments, the composition comprises an AAV comprising (1) an AAV capsid protein disclosed herein and (2) an 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.

[0059] Disclosed herein include antibodies or fragments thereof. In some embodiments, the antibodies or fragments thereof comprise an amino acid sequence that has binding specificity for LRP6. Disclosed herein include peptides or derivatives or conjugates thereof that have specificity for LRP6. Disclosed herein include nucleic acids. In some embodiments, the nucleic acid comprises the sequence of any of the antibodies or fragments thereof disclosed herein or any of the peptides or derivatives or conjugates disclosed herein.

[0060] Disclosed herein include delivery systems. In some embodiments, the delivery system comprises (1) a targeting peptide specific for low-density lipoprotein receptor-related protein 6 (LRP6); and (2) an agent.

[0061] Disclosed herein are methods for designing targeting peptides specific for LRP6. In some embodiments, the methods comprise: generating by computer simulation one or more targeting peptides, each of which is capable of interacting with: (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having an amino acid sequence of SEQ ID NO: 31; or (2) one or more positions functionally equivalent to S96, S114, E115, R141, W157, W183, or W242 in LRP6 having an amino acid sequence of SEQ ID NO: 31.

[0062] Disclosed herein include agents that are capable of binding to a protein selected from the group consisting of interleukin 3 (IL3), family with sequence similarity 234 member A (FAM234A), glycoprotein 2 (GP2), dipeptidyl peptidase-4 (DPP4), Dickkopf WNT signaling pathway inhibitor 3 (DKK3), alanyl aminopeptidase (ANPEP), Epiphycan (EPYC), and LRP6.

[0063] definition

[0064] Unless otherwise defined, technical terms and scientific terms used herein all have the same meaning as that generally understood by those of ordinary skill in the art to which the present disclosure belongs.See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd edition, J. Wiley & Sons (New York, NY, 1994); Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY, 1989). For the purposes of this disclosure, the following terms are defined below.

[0065] As used herein, the terms "nucleic acid" and "polynucleotide" are interchangeable and may refer to any nucleic acid, whether composed of phosphodiester bonds or modified bonds, such as phosphotriester bonds, phosphoamidate bonds, siloxane bonds, carbonate bonds, carboxymethyl ester bonds, acetamidate bonds, carbamate bonds, thioether bonds, bridged phosphoramidate bonds, bridged methylenephosphonate bonds, bridged phosphoramidate bonds, bridged phosphoramidate bonds, bridged methylenephosphonate bonds, phosphorothioate bonds, methylphosphonate bonds, phosphorodithioate bonds, bridged phosphorothioate bonds, or sultone bonds, and combinations of such bonds. The terms "nucleic acid" and "polynucleotide" also specifically include nucleic acids composed of bases other than the five biological bases (adenine, guanine, thymine, cytosine, and uracil).

[0066] As used herein, the term "vector" may refer to a vehicle for carrying or transferring nucleic acid. Non-limiting examples of vectors include plasmids and viruses (eg, AAV viruses).

[0067] As used herein, the term "construct" may refer to a recombinant nucleic acid that has been generated for the purpose of expressing a specific nucleotide sequence, or a recombinant nucleic acid to be used to construct other recombinant nucleotide sequences.

[0068] As used herein, the term "plasmid" may refer to a nucleic acid that can be used to replicate a recombinant DNA sequence in a host organism. The sequence may be double-stranded DNA.

[0069] The term "viral genome" refers to a nucleic acid sequence flanked by cis-acting nucleic acid sequences that mediate the packaging of the nucleic acid into viral capsids. For example, for AAV and parvovirus, "inverted terminal repeats" (ITRs) located at the 5' and 3' ends of the viral genome are known to have this function, and ITRs can mediate the packaging of heterologous viral genomes (e.g., non-wild-type viral genomes) into viral capsids.

[0070] The term "element" refers to a separate or different portion of something, such as a nucleotide sequence having a separate function in a longer nucleic acid sequence. The terms "regulatory element" and "expression control element" are used interchangeably herein, and refer to nucleic acid molecules that can affect the expression of an operably connected encoding sequence in a specific host organism. These terms are widely used in and encompass all elements that promote or regulate transcription, including core elements, upstream elements, enhancers, and response elements required for the basic interaction of promoters, RNA polymerases, and transcription factors (see, for example, Lewin, "Gene V (Genes V)" (Oxford University Press, Oxford) 847-873 pages). Exemplary regulatory elements in prokaryotes include promoters, operator sequences, and ribosome bind sites. Regulatory elements for eukaryotic cells can include, but are not limited to, transcription and translation control sequences, such as promoters, enhancers, splicing signals, polyadenylation signals, terminators, protein degradation signals, internal ribosome entry elements (IRES), 2A sequences, etc., which provide and / or regulate the expression of the encoding sequence in the host cell and / or the generation of encoded polypeptides.

[0071] As used herein, the term "promoter" is a nucleotide sequence that allows RNA polymerase to bind and direct gene transcription. Typically, a promoter is located in the 5' non-coding region of a gene, close to the transcription start site of the gene. The sequence elements that initiate transcription in a promoter are typically characterized by a common nucleotide sequence. Examples of promoters include, but are not limited to, promoters from bacteria, yeast, plants, viruses, and mammals (including humans). Promoters can be inducible, repressible, and / or constitutive. Inducible promoters initiate transcriptional levels from the raising of DNA in response to some changes in culture conditions (such as temperature changes) under their control.

[0072] As used herein, the term "enhancer" refers to a regulatory element that increases the efficiency of transcription, regardless of the enhancer's distance or orientation relative to the transcription start site.

[0073] As used herein, the term "operably linked" is used to describe the connection between a regulatory element and a gene or its coding region. Typically, gene expression is controlled by one or more regulatory elements, such as, but not limited to, constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. A gene or coding region is referred to as being "operably linked" or "operably connected" or "operably associated" with a regulatory element, which means that the gene or coding region is controlled or influenced by the regulatory element. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.

[0074] As used herein, the term "polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "polypeptides", and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also referred to as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids and does not refer to the specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other terms used to refer to one or more chains of two or more amino acids are included within the definition of "polypeptide", and the term "polypeptide" can be used to replace any of these terms or to be used interchangeably therewith. The term "polypeptide" is also intended to refer to products of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization with known protection / blocking groups, proteolytic cleavage, or modification of non-naturally occurring amino acids. The polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. The polypeptide can be generated in any way, including by chemical synthesis.

[0075] As used herein, the term "variant" can refer to a polynucleotide or polypeptide having a sequence substantially similar to a reference polynucleotide or polypeptide. In the case of a polynucleotide, compared with a reference polynucleotide, a variant can have a deletion, substitution, and / or addition of one or more nucleotides at a 5' end, a 3' end, and / or one or more internal sites. The similarity and / or otherness of sequences between a variant and a reference polynucleotide can be detected using conventional techniques known in the art, such as polymerase chain reaction (PCR) and hybridization techniques. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated by using site-directed mutagenesis. Typically, variants of polynucleotides, including but not limited to DNA, can have at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to a reference polynucleotide, as determined by sequence alignment programs known to those skilled in the art. In the case of polypeptides, variants can have one or more amino acid deletions, substitutions, additions compared to a reference polypeptide. Sequence similarity and / or differences between variants and reference polypeptides can be detected using conventional techniques known in the art, such as Western blotting. Generally, variants of a polypeptide can have at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to a reference polypeptide as determined by sequence alignment programs known to those of skill in the art.

[0076] As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences refers to the nucleotide bases or amino acid residues that are identical in the two sequences when aligned for maximum correspondence over a specified comparison window. When referring to percentage sequence identity or similarity of proteins, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, in which an amino acid residue is substituted with a functionally equivalent residue having similar physiochemical properties and, therefore, does not alter the functional properties of the molecule.

[0077] As used herein, amino acid whose functional equivalent residues generally refer to other amino acid residues having substantially similar physiochemical and stereochemical properties to the original amino acid. Physiological chemical properties include water solubility (hydrophobicity or hydrophilicity), dielectric and electrochemical properties, physiological pH, partial charge (positive, negative or neutral) of the side chain, and other properties that can be identified by those skilled in the art. Stereochemical properties include amino acid whose spatial and conformational arrangement and its chirality. For example, in the sense of the present disclosure, glutamic acid is considered to be a residue that is functionally equivalent to aspartic acid. Tyrosine and tryptophan are considered to be residues that are functionally equivalent to phenylalanine. Arginine and lysine are considered to be residues that are functionally equivalent to histidine.

[0078] As used herein, the term "binding" refers to a non-covalent interaction between macromolecules (e.g., between two proteins and / or peptides). Macromolecules are said to be "associated" or "interacting" or "bound" when in a non-covalent interaction state (e.g., when molecule X is said to interact with molecule Y, this means that molecule X binds to molecule Y in a non-covalent manner). A binding interaction can be characterized by a dissociation constant (Kd), for example, a Kd of 10 or less. -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M, 10 -15 M or a value or range between any two of these values. Kd can depend on environmental conditions, such as pH and temperature. "Affinity" refers to the strength of binding, and increased binding affinity is associated with a lower Kd.

[0079] As used herein, the words "specific," "specifically," or "specificity" refer to recognition, contact, and formation of a stable complex between the first and second molecules, as well as substantially less recognition, contact, or formation of a stable complex to no recognition, contact, or formation of a stable complex between each of the first and second molecules and any other molecules that may be present. Exemplary specific binding is antibody-antigen interaction, cell receptor-ligand interaction, polynucleotide hybridization, enzyme-substrate interaction, etc. As used herein, the term "specificity" refers to the unique association of a component with a specific complex of which it is a part. As used herein, the term "specificity" refers to the unique binding of a sequence to a single polynucleotide that is a complementary sequence. A "stable complex" means a detectable complex, and does not require any arbitrary level of stability, although higher stability is generally preferred. As used herein, the terms "specific," "specifically," or "specificity" refer to the ability of a targeting peptide to form a stable complex with a target protein, such as LRP6, wherein there is significantly less to no binding to macromolecules other than the target protein that may be present. As used herein, the terms "specific," "specifically," or "specificity" when referring to the binding of a targeting peptide to a target protein, LRP6, also refer to the ability of the target protein to form a stable complex with the targeting peptide, wherein there is significantly less to no binding to candidate peptides other than the identified targeting peptide that may be present.

[0080] The term "AAV" or "adeno-associated virus" refers to a dependent parvovirus within the Parvovirus genus of viruses. For example, the AAV can be an AAV derived from a naturally occurring "wild-type" virus, an rAAV genome packaged into a capsid derived from a capsid protein encoded by a naturally occurring cap gene, and / or an rAAV genome packaged into a capsid derived from a capsid protein encoded by a non-natural capsid cap gene. Non-limiting examples of AAV include AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), AAV type 11 (AAV11), AAV type 12 (AAV12), AAV type DJ (AAV-DJ), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. In some cases, AAV is described as "primate AAV," which refers to an AAV that infects primates. Similarly, AAV can infect bovine animals (e.g., "bovine AAV," etc.). In some cases, AAV is wild-type or naturally occurring. In some cases, AAV is recombinant.

[0081] As used herein, the term "AAV capsid" refers to a capsid protein or peptide of an adeno-associated virus. In some cases, the AAV capsid protein is configured to encapsulate genetic information (e.g., heterologous nucleic acid, transgene, therapeutic nucleic acid, viral genome). In some cases, the AAV capsid of the present disclosure is a variant AAV capsid, meaning that in some cases it is a parent or wild-type AAV capsid that has been modified in the amino acid sequence of the parent AAV capsid protein.

[0082] As used herein, the term "AAV genome" may refer to a nucleic acid polynucleotide encoding genetic information associated with a virus. In some cases, the genome comprises a nucleic acid sequence flanked by AAV inverted terminal repeat (ITR) sequences. The AAV genome may be a rAAV genome generated using recombinant genetics methods, and the rAAV genome may comprise a heterologous nucleic acid (e.g., a transgene) that includes and / or is flanked by ITR sequences.

[0083] The term "rAAV" refers to "recombinant AAV". In some embodiments, the recombinant AAV has an AAV genome in which some or all of the rep and cap genes have been replaced with heterologous sequences. As used interchangeably herein, the terms "AAV particle", "AAV nanoparticle" or "AAV vector" refer to an AAV virus or virion comprising an AAV capsid within which is packaged a heterologous DNA polynucleotide or "genome" comprising a nucleic acid sequence flanked by AAV ITR sequences. In some cases, the AAV particle is modified relative to the parental AAV particle.

[0084] The term "cap gene" refers to a nucleic acid sequence encoding a capsid protein that forms or contributes to the formation of the viral capsid or protein coat. In the case of AAV, the capsid protein may be VP1, VP2, or VP3. For other parvoviruses, the name and number of the capsid proteins may vary.

[0085] The term "rep gene" refers to the nucleic acid sequence encoding the nonstructural proteins (rep78, rep68, rep52 and rep40) required for viral replication and production.

[0086] The terms "native" and "wild-type" are used interchangeably herein and may refer to a form of a polynucleotide, gene or polypeptide as found in nature, with its own regulatory sequences, if any.

[0087] As used herein, "endogenous" refers to the native form of a polynucleotide, gene, or polypeptide in its natural location in an organism or in the genome of an organism. An "endogenous polynucleotide" includes a naturally occurring polynucleotide in its natural location in the genome of an organism. An "endogenous gene" includes a naturally occurring gene in its natural location in the genome of an organism. An "endogenous polypeptide" includes a naturally occurring polypeptide in its natural location in an organism.

[0088] As used herein, "heterologous" refers to a polynucleotide, gene or polypeptide that is not normally present in a host organism but is introduced into a host organism. A "heterologous polynucleotide" includes a natural coding region or portion thereof that is reintroduced into a source organism in a form different from the corresponding natural polynucleotide. A "heterologous gene" includes a natural coding region or portion thereof that is reintroduced into a source organism in a form different from the corresponding natural gene. For example, a heterologous gene may include a natural coding region that is a part of a chimeric gene that contains a non-natural regulatory region that is reintroduced into a natural host. A "heterologous polypeptide" includes a natural polypeptide that is reintroduced into a source organism in a form different from the corresponding natural polypeptide. Subject genes and proteins can be fused with other genes and proteins to produce chimeric or fusion proteins. According to the embodiments disclosed in this theme, useful genes and proteins include not only the full-length sequences specifically exemplified, but also parts, segments and / or fragments (including continuous fragments and internal and / or terminal deletions compared to the full-length molecules), variants, mutants, chimeras and fusions thereof of these sequences.

[0089] As used herein, the term "exogenous" gene is meant to encompass all genes that do not naturally occur within the genome of an individual. For example, miRNAs can be introduced exogenously by a virus (e.g., AAV nanoparticles).

[0090] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen-binding fragment or single chain thereof. Thus, the term "antibody" includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule that has biological activity that binds to an antigen. Examples of this class include, but are not limited to, a heavy or light chain complementary determining region (CDR) or ligand binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region or any portion thereof, or at least a portion of a binding protein.

[0091] As used herein, the term "antibody fragment" or "antigen-binding fragment" is a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of the structure, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spirochetes, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts as an antibody by binding to a specific antigen to form a complex.

[0092] As used herein, "subject" refers to an animal that is the object of treatment, observation or experiment. "Animal" includes cold-blooded and warm-blooded vertebrates and invertebrates, such as fish, crustaceans, reptiles and particularly mammals. As used herein, "mammal" refers to an individual belonging to the mammal class, and includes but is not limited to humans, livestock animals and farm animals, zoo animals, sports animals and pet animals. Non-limiting examples of mammals include mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cattle, horses, primates, such as monkeys, chimpanzees and apes and particularly humans. In certain embodiments, mammals are humans. However, in certain embodiments, mammals are not humans. In certain embodiments, subjects are rodents (e.g., rats or mice). In certain embodiments, subjects are primates (e.g., humans or monkeys).

[0093] As used herein, the term "treatment" refers to an intervention made in response to a disease, illness or physiological condition that a patient exhibits. The goal of treatment may include, but is not limited to, one or more of the following: alleviating or preventing symptoms, slowing down or preventing the progression or worsening of a disease, illness or condition, and alleviating a disease, illness and condition. The terms "treatment" and "treat" include, for example, therapeutic treatments, preventive treatments, and applications in which one of the subjects is reduced to develop a disease or other risk factor. Treatment does not require a complete cure of the disease and encompasses embodiments in which one of the symptoms or potential risk factors is reduced. In certain embodiments, "treatment" refers to both therapeutic treatments and preventive or prophylactic measures. Subjects requiring treatment include subjects who have been affected by a disease or illness or an undesirable physiological condition, as well as subjects who are to prevent a disease, illness or an undesirable physiological condition. As used herein, the term "prevention" refers to any activity that alleviates the burden of those symptoms that an individual later exhibits. This can be done at the primary, secondary, and / or tertiary prevention levels, where: a) primary prevention avoids the development of symptoms / disorders / conditions; b) secondary prevention activities are aimed at the early stages of treatment of the condition / disorder / symptom, thereby increasing the chances of intervention to prevent the progression of the condition / disorder / symptom and the onset of symptoms; and c) tertiary prevention reduces the negative impact of an already established condition / disorder / symptom by, for example, restoring function and / or reducing any condition / disorder / symptom or associated complications. The term "prevention" does not require 100% elimination of the likelihood of an event. Rather, it means that the likelihood of an event occurring has been reduced in the presence of a compound or method.

[0094] As used herein, the term "effective amount" refers to an amount sufficient to affect beneficial or desired biological and / or clinical outcomes.

[0095] As used herein, the term "pharmaceutically acceptable" carrier is a carrier that is non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. A "pharmaceutically acceptable" carrier can be, but is not limited to, an organic or inorganic, solid or liquid excipient suitable for the selected mode of application (such as oral application or injection), and is administered in the form of conventional pharmaceutical preparations, such as solids such as tablets, granules, powders, capsules, and liquids such as solutions, emulsions, and suspensions. Typically, a physiologically acceptable carrier is a pH buffered aqueous solution, such as a phosphate buffer or a citrate buffer. A physiologically acceptable carrier may also contain one or more of the following: antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin; gelatin; immunoglobulins; hydrophilic polymers, such as polyvinyl pyrrolidone; amino acids; 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 nonionic surfactants, such as Tween TM , polyethylene glycol (PEG) and Pluronics TM Adjuvants, stabilizers, emulsifiers, lubricants, binders, pH adjusters, control agents, isotonicity agents and other conventional additives may also be added to the carrier.

[0096] As described herein, products of natural and directed evolution of adeno-associated viruses (AAVs) are used to reverse engineer novel targets for determining therapeutic biodistribution. The identified targets open new avenues for the rational engineering of potent and specific therapeutics for humans. Cell-based microarray screening technology was used to probe the interactions of AAV with human cell surface and secreted proteins. The screening revealed a set of novel protein interactions, divided into (1) protein interactions targeting AAV serotype 9 (AAV9) and (2) protein interactions that selectively target engineered AAVs evolved from AAV9. These new targets include proteins that can enhance therapeutic targeting to specific cell types and tissues, as well as targets that help proteins evade host immune responses. The disclosed target proteins allow for target-based engineering to introduce and / or modulate interactions with these proteins to alter therapeutic biodistribution, efficacy, and patient immune responses.

[0097] Provided herein are novel targets (e.g., targets of viral vectors) GP2, DPP4, IL-3, DKK3, FAM234A, LRP6, ANPEP, CSF2, and EPYC that can be used to modify the targeting of biological agents or chemicals in vitro or in vivo following intravenous, intrathecal, direct, or other routes of administration. In some embodiments, GP2, DPP4, IL-3, DKK3, FAM234A, LRP6, ANPEP, CSF2, and / or EPYC can be used to cross the BBB in mammalian systems by biological or chemical molecules.

[0098] Provided herein are AAVs whose capsids have been engineered directly against GP2, DPP4, IL-3, DKK3, FAM234A, LRP6, ANPEP, CSF2, and / or EPYC, or have been combined with molecules engineered directly against GP2, DPP4, IL-3, DKK3, FAM234A, LRP6, ANPEP, CSF2, and / or EPYC.

[0099] Provided herein are antibodies, scFabs, scFvs, and / or alternative protein scaffolds engineered directly against GP2, DPP4, IL-3, DKK3, FAM234A, LRP6, ANPEP, CSF2, and / or EPYC. Provided herein are peptides engineered directly against GP2, DPP4, IL-3, DKK3, FAM234A, LRP6, ANPEP, CSF2, and / or EPYC. Provided herein are small molecules engineered directly against GP2, DPP4, IL-3, DKK3, FAM234A, LRP6, ANPEP, CSF2, and / or EPYC.

[0100] Provided herein are small molecules and biopharmaceutical conjugates (e.g., antibody drug conjugates, ADCs) targeting GP2, DPP4, IL-3, DKK3, FAM234A, LRP6, ANPEP, CSF2, and / or EPYC. Provided herein are bispecific antibodies containing at least one Fab targeting GP2, DPP4, IL-3, DKK3, FAM234A, LRP6, ANPEP, CSF2, and / or EPYC.

[0101] As disclosed herein, AAV capsids engineered directly against GP2, DPP4, IL-3, DKK3, FAM234A, LRP6, ANPEP, CSF2, and / or EPYC can be used to deliver DNA or ASO molecules, therapeutic proteins, small therapeutic molecules, or other biologics in vitro and in vivo.

[0102] Disclosed herein are agents that can bind to a protein selected from the group consisting of interleukin 3 (IL3), family with sequence similarity 234 member A (FAM234A), glycoprotein 2 (GP2), dipeptidyl peptidase-4 (DPP4), Dickkopf WNT signaling pathway inhibitor 3 (DKK3), alanyl aminopeptidase (ANPEP), Epiphycan (EPYC), and LRP6. In some embodiments, the agent is selected from the group consisting of an antibody or fragment thereof, an aptamer, a small molecule, a nucleic acid, and a peptide.

[0103] The antibody or its fragment may comprise an Fc domain. The antibody or its fragment may be a single-chain variable fragment (scFv), a single domain antibody, an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a disulfide-linked Fv, a scFv, a single domain antibody, a bifunctional antibody, a multispecific antibody, a bispecific antibody, an anti-idiotypic antibody, a bispecific antibody, or a functionally active epitope binding fragment thereof. The nucleic acid may be a miRNA, shRNA, and siRNA or an oligonucleotide.

[0104] The agent can be conjugated to a detectable label. In some embodiments, the detectable label is selected from the group consisting of biotin, a fluorophore, a luminescent or bioluminescent marker, a radiolabel, an enzyme, an enzyme substrate, a quantum dot, a contrast agent, a metal particle, a magnetic particle, and any combination thereof. The agent can be a therapeutic agent.

[0105] The agent can be a targeting peptide. The targeting peptide can be part of a delivery system, wherein the delivery system comprises a payload to be delivered to the cell. The delivery system can comprise a viral vector or a non-viral vector. The viral vector can comprise an AAV vector. The targeting peptide can be part of the capsid protein of an 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. The non-viral vector can comprise 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.

[0106] The payload to be delivered to the cell can be a biological molecule, a non-biological molecule, or a combination thereof. In some embodiments, the biological molecule is selected from the group consisting of: a nucleic acid sequence, a protein, a peptide, a lipid, a polysaccharide, and any combination thereof. The payload can be a therapeutic molecule. The nucleic acid sequence to be delivered to the nervous system can include one or more of the following: a) a sequence encoding a trophic factor, a growth factor, or other soluble factor that may be released from the transduced cell and affect the survival rate or function of the cell and / or surrounding cells; b) a DNA (e.g., a genomic or cDNA sequence) that restores protein function to a human or animal carrying a genetic mutation in a gene; c) a DNA encoding a protein that can be used to control or change the activity or state of a cell; d) a DNA encoding a protein or nucleic acid for assessing the state of a cell; e) a DNA and / or associated guide RNA for genome engineering; f) a sequence for genome editing by homologous recombination; g) a DNA sequence encoding a therapeutic RNA; h) an shRNA or artificial miRNA delivery system; or i) a DNA sequence that affects the splicing of an endogenous gene.

[0107] Lipoprotein receptor-related protein 6 (LRP6)

[0108] Disclosed herein is a method for increasing the permeability of the blood-brain barrier. In some embodiments, the method comprises providing a targeting peptide capable of binding to low-density lipoprotein receptor-related protein 6 (LRP6), thereby increasing the permeability of the blood-brain barrier. In some embodiments, the targeting peptide binds to YWTD domain 1 and / or domain 2 (also referred to as E1 and E2 domains) of LRP6. The permeability of the blood-brain barrier can be increased by at least or about 25%, 50%, 75%, 100%, or more (e.g., at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 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%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125 %, 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 a number or range between any two of these values).

[0109] Disclosed herein are methods for delivering a payload to a subject's nervous system. In some embodiments, the method comprises: providing a targeting peptide or a derivative thereof capable of binding to LRP6, wherein the targeting peptide is part of a delivery system, and wherein the delivery system comprises the payload to be delivered to the nervous system; and administering the delivery system to the subject. The delivery system can comprise nanoparticles, nanotubes, nanowires, dendrimers, liposomes, ethosomes and aquasomes, polymersomes and nonionic surfactant vesicles, foams, hydrogels, cubes, quantum dots, exosomes, macrophages, and any combination thereof. The delivery system can comprise a viral vector or a non-viral vector.

[0110] In certain embodiments, the targeting peptide enhances the binding affinity of the viral vector or the non-viral vector to LRP6. For example, the binding affinity of the viral vector to LRP6 can be enhanced by at least about 2 times (e.g., 2 times, 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 a numerical value or range between any of these values). The viral vector can include an AAV vector. The targeting peptide can be a part of the capsid protein of an 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. The non-viral vector may comprise 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.

[0111] The payload to be delivered to the nervous system can be a biological molecule, a non-biological molecule, or a combination thereof. In some embodiments, the biological molecule is selected from the group consisting of: a nucleic acid sequence, a protein, a peptide, a lipid, a polysaccharide, and any combination thereof. The payload can be a therapeutic molecule. The nucleic acid sequence to be delivered to the nervous system can include one or more of the following: a) a sequence encoding a trophic factor, a growth factor, or other soluble factor that may be released from the transduced cell and affect the survival rate or function of the cell and / or surrounding cells; b) a DNA (e.g., a genomic DNA or cDNA sequence) that restores protein function to a human or animal carrying a genetic mutation in a gene; c) a DNA encoding a protein that can be used to control or change the activity or state of a cell; d) a DNA encoding a protein or nucleic acid for assessing the state of a cell; e) a DNA and / or associated guide RNA for genome engineering; f) a sequence for genome editing by homologous recombination; g) a DNA sequence encoding a therapeutic RNA; h) an shRNA or artificial miRNA delivery system; or i) a DNA sequence that affects the splicing of an endogenous gene.

[0112] The LRP6 gene encodes a member of the low-density lipoprotein (LDL) receptor gene family. The LDL receptor is a transmembrane cell surface protein that participates in receptor-mediated endocytosis of lipoproteins and protein ligands. The protein encoded by this gene acts as a receptor for Wnt or as a co-receptor with Frizzled protein, thereby transmitting the typical Wnt / β-catenin signaling cascade. Through its interaction with the Wnt / β-catenin signaling cascade, this gene plays a role in regulating cell differentiation, proliferation and migration, as well as the development of many cancer types. This protein undergoes γ-secretase-dependent RIP- (regulated intramembrane proteolysis) processing. The NCBI gene ID is 4040. The Ensembl ID is ENSG00000070018. The ID is 603507 and the UniProtKB / Swiss-Pro ID is 075581.

[0113] LRP6 can be mouse LRP6. In some embodiments, LRP6 has an amino acid sequence that has at least 80% (e.g., at least or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) sequence identity to the amino acid sequence of SEQ ID NO: 29. LRP6 can be macaque LRP6. In some embodiments, LRP6 has an amino acid sequence that has at least 80% (e.g., at least or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) sequence identity to the amino acid sequence of SEQ ID NO: 30. The LRP6 can be human LRP6. In some embodiments, LRP6 has an amino acid sequence that has at least 80% (e.g., at least or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) sequence identity to the amino acid sequence of SEQ ID NO:31.

[0114] The methods disclosed herein may comprise providing a targeting peptide capable of binding to LRP6 or a derivative thereof. Upon binding, the targeting peptide is capable of interacting with: (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having an amino acid sequence of SEQ ID NO: 31; or (2) one or more positions functionally equivalent to S96, S114, E115, R141, W157, W183, or W242 in LRP6 having an amino acid sequence of SEQ ID NO: 31. The targeting peptide can be inserted between two adjacent amino acids in AA587-594 of SEQ ID NO: 11 of the AAV9 vector, or between functional equivalents of AA587-594 in an amino acid sequence at least 80% identical to SEQ ID NO: 11. The targeting peptide can be inserted between AA588-589 of SEQ ID NO: 11 of the AAV9 vector, or between functional equivalents of AA588-589 in an amino acid sequence at least 80% identical to SEQ ID NO: 11. The targeting peptide can be inserted between or in place of AA452-460 of SEQ ID NO: 11 of the AAV9 vector, or between functional equivalents of AA588-589 in an amino acid sequence at least 80% identical to SEQ ID NO: 11.

[0115] In some embodiments, the targeting peptide can comprise or consist of an amino acid sequence having at least 80% identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or a numerical value or range between any two of these values) to any one of the sequences of SEQ ID NOs: 1-10. In some embodiments, the targeting peptide comprises or consists of an amino acid sequence to any one of the sequences of SEQ ID NOs: 1-10. In some embodiments, the targeting peptide comprises at least four consecutive amino acids to any one of the sequences of SEQ ID NOs: 1-10.

[0116] Targeting peptides

[0117] Disclosed herein are targeting peptides and related AAV particles comprising capsid proteins with one or more targeting peptide insertions for enhancing or improving the transduction of target tissues (e.g., cells of the CNS or PNS). In some embodiments, the targeting peptide can guide the AAV particles to cells or tissues of the CNS. The cells of the CNS can be, but are not limited to, neurons (e.g., excitatory, inhibitory, motor, sensory, autonomous, sympathetic, parasympathetic, Purkinje, Betz, etc.), glial cells (e.g., microglia, astrocytes, oligodendrocytes) and / or supporting cells of the brain, such as immune cells (e.g., T cells). The tissues of the CNS can be, but are not limited to, the cortex (e.g., frontal lobe, parietal lobe, occipital lobe, temporal lobe), thalamus, hypothalamus, striatum, putamen, caudate nucleus, hippocampus, entorhinal cortex, basal ganglia, or deep cerebellar nuclei. In some embodiments, the targeting peptide can guide the AAV particles to cells or tissues of the PNS. The cells or tissues of the PNS can be, but are not limited to, dorsal root ganglia (DRG). Targeting peptides can direct AAV particles to the CNS (eg, cortex) following intravenous administration. Targeting peptides can direct AAV particles to the PNS (eg, DRG) following intravenous administration.

[0118] The length of targeting peptide may be different. In certain embodiments, the length of targeting peptide is 3-20 amino acid. As limiting examples, the length of targeting peptide can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 3-5, 3-8, 3-10, 3-12, 3-15, 3-18, 3-20, 5-10, 5-15, 5-20, 10-12, 10-15, 10-20, 12-20 or 15-20 amino acid.

[0119] In some embodiments, a targeting peptide is provided that is capable of binding to low-density lipoprotein receptor-related protein 6 (LRP6) or a derivative thereof. Upon binding, the targeting peptide is capable of interacting with: (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having an amino acid sequence of SEQ ID NO: 31; or (2) one or more positions functionally equivalent to S96, S114, E115, R141, W157, W183, or W242 in LRP6 having an amino acid sequence of SEQ ID NO: 31.

[0120] The targeting peptide of the present disclosure can be identified and / or designed by any method known in the art. As non-limiting examples, such as Deverman et al. (" Nature Biotechnology (Nature Biotechnology) " 34 (2): 204-209 (2016)) and International Patent Application Publication No. WO 2015038958, No. WO2017100671 and No. WO2020028751, the CREATE system described can be used as the mode of identifying the targeting peptide in mice or other research animals (such as but not limited to non-human primates), wherein the content of each document of these documents is incorporated herein by reference in its entirety. Targeting peptide can be designed by computer simulation. As assessed by those skilled in the art, the peptide designed by computer simulation can be tested in any in vitro or in vivo model.

[0121] Targeting peptide and relevant AAV particle can be identified from the library of the AAV capsid that comprises targeting peptide variant.In certain embodiments, targeting peptide can be 7 amino acid sequences (7 aggressiveness).In another embodiment, targeting peptide can be 9 amino acid sequences (9 aggressiveness).The generation of targeting peptide or design method also may be different, and wherein limiting examples comprises random peptide selection, site saturation mutagenesis and / or the optimization of peptide specific region (for example, side joint area or central core).

[0122] In certain embodiments, the targeting peptide library comprises a targeting peptide of 7 amino acids (7 aggressiveness) randomly generated by PCR. In certain embodiments, the targeting peptide library comprises amino acid whose targeting peptide has 3 mutations. In certain embodiments, the amino acid whose mutations are continuous amino acids. In certain embodiments, the amino acid whose mutations are not continuous amino acids. In certain embodiments, the parent's targeting peptide is 7 aggressiveness. In certain embodiments, the parent's peptide is 9 aggressiveness.

[0123] In some embodiments, the targeting peptide library comprises 7-mer targeting peptides, wherein the amino acid and / or flanking sequence of the targeting peptide are evolved by site-saturation mutagenesis of 3 consecutive amino acids. In some embodiments, NNK (N=any base; K=G or T) codons are used to generate site-saturation mutant sequences.

[0124] In silico screening of targeting peptides

[0125] Disclosed herein are methods for designing targeting peptides specific for LRP6. In some embodiments, the methods comprise: generating by computer simulation one or more targeting peptides, each of which is capable of interacting with: (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having an amino acid sequence of SEQ ID NO: 31; or (2) one or more positions functionally equivalent to S96, S114, E115, R141, W157, W183, or W242 in LRP6 having an amino acid sequence of SEQ ID NO: 31.

[0126] Generating the one or more targeting peptides by computer simulation may comprise: generating a plurality of candidate peptides by computer simulation; performing a computer-assisted docking simulation on each of the plurality of candidate peptides bound to LRP6; and analyzing the structure of LRP6 bound to one or more of the plurality of candidate peptides to identify one or more targeting peptides capable of interacting with: (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having an amino acid sequence of SEQ ID NO: 31; or (2) one or more positions functionally equivalent to S96, S114, E115, R141, W157, W183, or W242 in LRP6 having an amino acid sequence of SEQ ID NO: 31.

[0127] The computer simulation method can provide a high-throughput method for screening a large number of candidate peptides and identifying targeted peptides with desired specificity. In some embodiments, the candidate peptide may comprise a portion of an AAV capsid protein. In some embodiments, the candidate peptide is a portion of an AAV capsid protein. The method may include constructing one or more peptide-receptor models (e.g., performing computer-assisted docking simulations) for each candidate peptide compounded with LRP6. A molecular model of a peptide-protein complex can be constructed using any reasonable computational peptide design and docking method, database, program, or algorithm described herein or known in the art. Exemplary computational modeling methods, public databases, and programs include, but are not limited to, AlphaFold (e.g., AlphaFold2 and AlphaFold-Multimer provided by DeepMind, available on github.com / deepmind / alphafold), RoseTTAFold (github.com / RosettaCommons / RoseTTAFold), AutoDock, DOCK, FlexX, GOLD, OSPREY, SCWRL, PyMol, SWISS-MODEL

[0128] (academic.oup.com / nar / article / 46 / W1 / W296 / 5000024), Protein Data Bank (PDB) (available through the websites of member organizations, e.g., PDBe - pdbe.org, PDBj - pdbj.org, RCSB - rcsb.org / pdb, and BMRB - bmrb.wisc.edu), Phyre2 (nature.com / articles / nprot.2015.053), and RaptorX (nature.com / articles / nprot.2012.085).

[0129] The method can further comprise using visualization software (such as PyMol, Qlucore Omics Explorer, WebMol, InsightII, Discovery Studio 2.1 and other software that those skilled in the art can identify) to evaluate the characteristics or parameters related to the interaction between the candidate peptide and LRP6. The characteristics or parameters evaluated can include interfacial energy and physical and geometric scoring. The characteristics or parameters related to the interaction between the candidate peptide and LRP6 can include measuring surface complementarity, solvent accessible surface area, solvation free energy, electrostatic interaction energy, van der Waals energy (van der Waals energy) and / or total molecular mechanics energy. The method can also include determining the total number of atoms in the interface, the total number of atoms in the peptide that conflict with LRP6, the binding angle of the peptide and / or the binding depth of the peptide in each presumed peptide-receptor complex model. The method can also include identifying the lowest energy conformation of the LRP6 complex. The energy score of each conformation can be determined by calculating the interaction energy between the peptide and LRP6, including electrostatic energy, desolvation energy, van der Waals energy, as those skilled in the art will understand.

[0130] In some embodiments, the targeting peptides can be assigned a binding score and sorted based on the binding score. A threshold value can be applied to identify the desired targeting peptide. The method can include obtaining a binding score for each of the multiple candidate peptides that bind to LRP6; and selecting one or more candidate peptides with a binding score higher than the threshold value as a targeting peptide with binding specificity (or high binding affinity) to LRP6.

[0131] A combination of physical and geometric scoring parameters, including interface energy, binding angle, and binding pocket depth calculations, can be used to generate a binding score. In some embodiments, a binding score for each candidate peptide sequence in the plurality of candidate peptide sequences can be obtained by: (1) counting the total number of atoms in the interface between the candidate peptide and LRP6; (2) counting the total number of atoms in the candidate peptide that clash with LRP6; (3) obtaining a binding angle for the candidate peptide; and / or (4) obtaining a binding depth for the candidate peptide.

[0132] In some embodiments, the number of atoms in the interface of candidate peptide and LRP6 can be the total number of atoms in the cut-off distance between the intervening atoms of candidate peptide and LRP6. In various embodiments, the cut-off distance can vary. In certain embodiments, the cut-off distance is approximately, at most, at most about 5 dusts (for example, 2 dusts, 3 dusts, 4 dusts or 5 dusts). In various embodiments, the number of intervening atoms between candidate peptide and LRP6 can be different. For example, the number of intervening atoms can be approximately or can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or numerical value or scope between any value in these values.

[0133] The total number of atoms in the peptide that clash with LRP6 can be the total number of atoms within the clash distance. The clash distance can be defined as the distance between the peptide and the receptor atoms when a geometric clash occurs. In some embodiments, the clash distance can be about 1 angstrom.

[0134] The binding angle of a peptide can be defined as the angle between the carrier of the LRP6 anchor from the LRP6 center of gravity and the carrier from the LRP6 center of gravity to the peptide center of gravity. Depth of binding can be defined as the difference normalized relative to the small radius between the point closest to the center of LRP6 on the peptide and the small radius of the ellipsoidal shell of LRP6. The binding score can be the sum of the contact score, binding angle, and depth of binding (calculated based on the total number of atoms and the total number of conflicting atoms in the interface of the candidate peptide and LRP6). In certain embodiments, the binding score is equal to or greater than 0. For example, if the sum of the contact score, binding angle, and depth of binding is negative, the binding score is defined as 0. , 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, or a value or range between any two of these values.

[0135] In some embodiments, the binding score (B 肽 ) can be defined as follows:

[0136]

[0137] The method can further comprise selecting one or more candidate peptides having a binding score higher than a threshold value among the multiple candidate peptides as a targeting peptide with binding specificity to LRP6. The threshold value can be an arbitrary value defined by the user. In certain embodiments, the threshold value can be a binding score of a targeting peptide known to have binding specificity to LRP6. In certain embodiments, the result from individual paired competition (relative score between two competitor peptides) can be assembled into a peptide competition metric, which ranks the set of candidate peptides according to their receptor-binding probability encoded in the AlphaFold2 neural network.

[0138] CREATE system

[0139] In some embodiments, the targeting peptides disclosed herein are isolated by the CREATE system, as described in Deverman et al. (Nature Biotechnology 34(2):204-209 (2016)) and International Patent Application Publication Nos. WO 2015038958 and WO 2017100671, the contents of each of which are incorporated herein by reference in their entirety. "CREATE" or "AAV targeted evolution based on Cre recombination" refers to an AAV capsid selection strategy that selects capsids that transduce target tissues (e.g., CNS or PNS) after intravenous injection. This approach has been demonstrated in mouse models.

[0140] A library of AAV capsids with one or more targeting peptides inserted 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 LRP6 to identify targeting peptides that interact with LRP6.

[0141] Variation in targeting sequences, as well as transgenic animal models, enables the selection of AAV variants with desired transduction profiles, such as tropism for neurons or astrocytes, as compared to other AAV serotypes (including parental AAV particles and capsids).

[0142] The CREATE method involves generating a library of targeting peptides, which are then assembled into a viral genome backbone comprising the parent AAV capsid sequence. 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 cells expressing Cre. These sequences are evaluated and characterized to identify targeting peptides that enrich the target tissue (i.e., enhance transduction or tropism). Targeting peptides and related AAV particles can then be generated for further testing and characterization. This process is considered to be 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.

[0143] More specifically, the CREATE system uses an rAAV-Cap-in-cis-lox viral genome that contains regulatory elements for the AAV cap and AAV rep genes, along with a Cre reversible switch. Because this viral genome lacks the fully functional rep gene required for AAV particle production, rep is provided in trans. Modified AAV2 / 9 Rep-Cap plasmids are available that incorporate an in-frame stop codon to prevent expression of the VP1-VP3 proteins.

[0144] Capsid library is generated using rAAV-Cap-in-cis-lox viral genome as a skeleton. The targeting peptide is inserted into the parent AAV capsid protein (e.g., AAV9) at any position to generate fully functional AAV capsid protein and AAV particles. The targeting peptide can be designed by any method known in the art. In certain embodiments, polymerase chain reaction (PCR) is used to generate targeting peptides. AAV particles comprising capsid proteins with targeting peptide insertions 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 by intravenous delivery to the tail vein. Due to the expression of Cre, the expression of reporter gene payloads in target tissues will be generated by administering these capsid libraries to mice expressing Cre.

[0145] AAV particles and / or viral genomes can be recovered from the target tissue to identify enriched targeting peptides and associated AAV particles, indicating enhanced transduction of 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.

[0146] The target tissue can be any cell, tissue or organ of the subject. As non-limiting examples, samples can be collected from the 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, testicles and / or any site identified as having a lesion or site of interest.

[0147] Targeting peptides and related 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, 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 transduction of target tissues (e.g., CNS or PNS).

[0148] The CREATE system has been shown to be effective in identifying targeting peptides that enhance transduction of the CNS in mice following intravenous administration. However, translating findings from mouse studies to human studies is not always straightforward. Modifying the CREATE system in non-transgenic animals or more human-like model systems may facilitate the identification of 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 human AAV capsid design.

[0149] In order to adapt the CREATE method to non-transgenic animals, another mechanism is needed to modify the 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. The AAV Cre vector can comprise a viral genome with a cell type-specific promoter. These cell type-specific promoters can be, but are not limited to, CAG, UBC, EF1α, synaptophysin, GFAP, MBP, VGLUT, VGAT, Nav1.8, parvalbumin, TH, ChaT, and / or any promoter known in the art.

[0150] In some embodiments, these AAV-Cre vectors are delivered to the target tissue by intraparenchymal administration. In some embodiments, intraparenchymal administration is directly administered to the dura mater of the subject. In some embodiments, intraparenchymal administration is directly administered to the thalamus of the subject. In some embodiments, intraparenchymal administration is directly administered to the cortex of the subject. In some embodiments, intraparenchymal administration is indirectly administered to the cortex of the subject. In some embodiments, intraparenchymal administration is simultaneously administered to one or more of the dura mater, thalamus, and / or cortex of the subject, and may be bilaterally administered. In some embodiments, the subject is a non-human primate.

[0151] As for the CREATE method developed in mice, the AAV capsid library can be administered intravenously. In another embodiment, the AAV capsid library can be administered by intraparenchymal delivery. In some embodiments, the AAV capsid library is administered before the delivery of the AAV-Cre vector. In another embodiment, the AAV capsid library is administered after the delivery of the AAV-Cre vector. The length of time between the administration of the AAV-Cre vector and the AAV capsid library can be seconds, minutes, hours, days, weeks, or years.

[0152] The AAV capsid library can comprise AAV particles comprising viral genomes encoding reporter genes (e.g., GFP). In target tissues (e.g., CNS or DRG), only cells that also express Cre (co-transduced by the Cre vector administered in the organ parenchyma) will express the reporter gene. Target tissues can be collected and analyzed to identify the AAV particles and targeting peptides that lead to enrichment of target tissues, i.e., enhanced transduction. Standard methods in the art can be used to assess, 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.

[0153] Antibody and peptide derivatives

[0154] Disclosed herein also include antibodies or fragments thereof comprising amino acid sequences that have binding specificity to the target proteins disclosed herein (e.g., LRP6). Disclosed herein also include peptide derivatives or conjugates thereof that have specificity to the target proteins disclosed herein (e.g., LRP6).

[0155] In some embodiments, the antibody or fragment thereof may further comprise an Fc domain. In some embodiments, the antibody or fragment thereof is a single-chain variable fragment (scFv), a single domain antibody, an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a disulfide-linked Fv, a scFv, a single domain antibody, a bifunctional antibody, a multispecific antibody, a bispecific antibody (dual specific antibody), an anti-idiotypic antibody, a bispecific antibody (bispecific antibody), or a functionally active epitope-binding fragment thereof.

[0156] In some embodiments, the antibody or fragment thereof is a bispecific antibody comprising at least one Fab specific for LRP6 or another protein identified herein (eg, GP2). The bispecific antibody may comprise another binding site for a different antigen.

[0157] In some embodiments, the antibody or its fragment cannot elicit a deleterious immune response in the subject (e.g., human) to be treated. In some embodiments, the antibodies, fragments, variants, or derivatives thereof of the present disclosure are modified using techniques recognized in the art to reduce their immunogenicity. For example, the antibody can be a humanized antibody, a primed antibody, a deimmunized antibody, or a chimeric antibody.

[0158] In some embodiments, the antibody, its fragment, variant or derivative may further comprise a chemical moiety that is not naturally associated with the antibody. For example, the antibody or its fragment may comprise a flexible linker, or may be modified to add a functional moiety, such as a detectable label. The antibody, its fragment, variant or derivative may be modified, for example, by covalent or non-covalent attachment of a chemical moiety to the antibody so that the connection does not interfere with or prevent the antibody from binding to the epitope. In some embodiments, the chemical moiety may be conjugated to the antibody using any technique known in the art.

[0159] The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding peptides, antibodies, fragments, variants or derivatives thereof of the present disclosure. For example, the polynucleotides of the present disclosure can encode the heavy chain and light chain variable regions of antibodies, fragments, variants or derivatives thereof on the same polynucleotide molecule or a separate polynucleotide molecule. In some embodiments, the polynucleotides of the present disclosure can encode portions of the heavy chain and light chain variable regions (e.g., CDR regions) of antibodies, fragments, variants or derivatives thereof on the same polynucleotide molecule or a separate polynucleotide molecule.

[0160] Payload delivery

[0161] Disclosed herein include methods and delivery systems for delivering a payload (e.g., a therapeutic agent) to a target tissue, such as the nervous system. In some embodiments, the method comprises providing a targeting peptide or derivative thereof that can bind to LRP6. The targeting peptide can be part of a delivery system, and the delivery system can include a payload to be delivered to the nervous system. The method can further comprise administering the delivery system to the subject.

[0162] In some embodiments, the delivery system comprises nanoparticles, nanotubes, nanowires, dendrimers, liposomes, ethosomes and aquasomes, polymer vesicles and non-ionic surfactant vesicles, foams, hydrogels, cubes, quantum dots, exosomes, macrophages, and combinations thereof. In some embodiments, the delivery system comprises a nanoparticle 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.

[0163] In some embodiments, the delivery system comprises a viral vector or a non-viral vector. For example, the viral vector can comprise an adenoviral 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 can be a part of the capsid protein of the AAV vector.

[0164] Adeno-associated virus (AAV) and recombinant AAV (rAAV)

[0165] In certain embodiments, the delivery system for delivering payload across the BBB is an AAV vector. In certain embodiments, AAV is a replication-deficient parvovirus with a single-stranded DNA genome of about 4.7 kb in length, including 145 nucleotide inverted terminal repeats (ITRs). ITR plays an important role in the integration of AAV DNA into the host cell genome. When AAV infects host cells, the viral genome is integrated into the host's chromosome, thereby causing latent infection of the cell. In natural systems, helper viruses (e.g., adenoviruses or herpes viruses) provide genes that allow the production of AAV viruses in infected cells. In the case of adenovirus, genes E1A, E1B, E2A, E4 and VA provide auxiliary functions. After infection with helper viruses, the AAV provirus is rescued and amplified, and both AAV and adenovirus are produced. In the case of a recombinant AAV vector without Rep and / or Cap genes, AAV can be non-integrated.

[0166] In some embodiments, the AAV vector may comprise coding regions for one or more proteins of interest. The AAV vector may comprise 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 the restriction site are located downstream of the 5'AAV ITR and upstream of the 3'AAV ITR. In some embodiments, the AAV vector comprises a post-transcriptional regulatory factor-element downstream of the restriction site and upstream of the 3'AAV ITR.

[0167] The viral vector may include additional sequences that adapt the vector to replication and integration in eukaryotes. In other embodiments, the viral vectors disclosed herein may include a shuttle element that adapts the vector to replication and integration in both prokaryotes and eukaryotes. In some embodiments, the viral vector 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.

[0168] The AAV serotype used to derive the AAV capsid protein can be different. The AAV capsid can be derived from AAV9 or a variant thereof. The AAV capsid can be derived from an AAV selected from the group consisting of: 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 can be derived from an AAV serotype selected from the group consisting of: AAV9, AAV9 K449R (or K449RAAV9), AAV1, AAVrh10, 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 A15 / 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、AAV42-11、AAV42-12、AAV42-13、AAV42-15、AAV42 2-aa、AAV43-1、AAV43-12、AAV43-20、AAV43-21、AAV43-23、AAV43-25、AAV43-5、AAV44.1、AAV44.2、AAV44.5、AAV223.1、AAV223.2、AAV223.4、AAV223.5 AV223.6、AAV223.7、AAV1-7 / rh.48、AAV1-8 / rh.49、AAV2-15 / rh.62、AAV2-3 / rh.61、AAV2-4 / rh.50、AAV2-5 / rh.51、AAV3.1 / rh.6、AAV3.1 / rh.6、AAV3-9 / rh.61 rh.52、AAV3-11 / rh.53、AAV4-8 / r11.64、AAV4-9 / rh.54、AAV4-19 / rh.55、AAV5-3 / rh.57、AAV5-22 / rh.58、AAV7.3 / hu.7、AAV16.8 / hu.10、AAV16.12 / hu.10 1、AAV29.3 / bb.1、AAV29.5 / bb.2、AAV106.1 / hu.37、AAV114.3 / hu.40、AAV127.2 / hu.41、AAV127.5 / hu.42、AAV128.3 / hu.44、AAV130.4 / hu.48、AAV145.1 / hu.53、AAV145.5 / hu.54、AAV145.6 / hu.55、AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、AAV52 / hu .19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAVA3.3、AAVA3.4、AAVA3.5、AAVA3.7、AAVC1、AAVC2、AAVC5、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVrh.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、AAVrh.12、AAVh6、AAVrh.AAVH-1 / hu.1、AAVH-5 / hu.3、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVN721-8 / rh.43、AAVCh.5、AAVCh.5R1、AAVcy.2、AAVcy.3、AAVcy4. 、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. hu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.24、AAVhu.25、AAVhu.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、AAVhu. .44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu.48R2、AAVhu.48R3、AAVhu.49、AAVhu.51、 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. .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.61, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV, BAAV, goat AAV, bovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, 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-PAEC 12, 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 SM 100-10, 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, AAV CBr-7.8, AAV CBr-B7.3, AAVCBr-B7.4, AAV CBr-E1, 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, AAV CHt-6.8, AAV CHt-P1, AAVCHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAVCKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B1, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-H1, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-F1, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1, AAV CLv1-1, AAV Clv1-10, AAV CLv1-2, AAV CLv-12, AAV CLv1-3AAV CLv-13, AAV CLv1-4, AAV Clv1-7, AAV Clv1-8, AAV Clv1-9, AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-1-AV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-E1, AAV CLv-K1, AAV-CLv-AAV 3 CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-M1, AAV CLv-M11, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-MAV7 CLv-M8, AAV CLv-M9, AAVCLv-R1, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R6, AAV CLv-R7, AAV CLv-R2 CLv-R9、AAV CSp-1、AAV CSp-10、AAV CSp-11、AAV CSp-2、AAV CSp-3、AAVCSp-4、AAV CSp-6、AAV CSp-7、AAV CSp-8CSp-18AAV、AAV. CSp-8.2、AAV CSp-8.4、AAVCSp-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 / HSC1 1、AAVF12 / HSC12、AAVF13 / HSC13、AAVF14 / HSC14、AAVF15 / HSC15、AAVF16 / HSC16、 AAVF17 / HSC17,AAVF2 / HSC2,AAVF3 / HSC3,AAVF4 / HSC4,AAVF5 / HSC5,AAVF6 / HSC6 、AAVF7 / HSC7、AAVF8 / HSC8、AAVF9 / HSC9、 The AAV system is not available in the United States.

[0169] The AAV vector can be an AAV9 having the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 70% sequence identity (e.g., at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the AAV vector is a variant AAV vector having at least 70% sequence identity (e.g., at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence of any one of SEQ ID NOs: 11-18.

[0170] 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 targeting peptide) to produce a recombinant AAV virus that can express the protein of interest in a host cell. Thus, disclosed herein also includes recombinant AAV viruses (rAAV). rAAV can comprise the AAV capsid proteins described herein.

[0171] rAAV can comprise a chimeric AAV capsid. A "chimeric" AAV capsid refers to a capsid having an exogenous amino acid or amino acid sequence. rAAV can comprise a mosaic AAV capsid. A "mosaic" AAV capsid refers to a capsid composed of two or more capsid proteins or polypeptides, each of which is derived from a different AAV serotype. rAAV can be the result of transcapsidation, which in some cases refers to packaging an inverted terminal repeat sequence (ITR) from a first serotype into a capsid of a second serotype, wherein the first serotype and the second serotype are not identical. In some cases, the capsid gene of the parent AAV serotype can be pseudotyped, which means that the ITR from the first AAV serotype (e.g., AAV1) is used in a capsid from a second AAV serotype (e.g., AAV9), wherein the first AAV serotype and the second AAV serotype are not identical. As a non-limiting example, a pseudotyped AAV serotype comprising AAV1 ITRs and AAV9 capsid proteins can be represented as AAV1 / 9. The rAAV may additionally or alternatively comprise a capsid that has been engineered to express an exogenous ligand-binding moiety (eg, a receptor) or a modified native receptor.

[0172] In some embodiments, the rAAV capsid protein comprises a replacement or insertion 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 replacement of an amino acid heterologous to the wild-type AAV capsid protein, and the amino acid is at the amino acid position of the insertion or replacement. In some embodiments, at the amino acid position of the insertion or replacement, the amino acid is not endogenous to the wild-type AAV capsid protein. The amino acid can be a naturally occurring amino acid in the same or equivalent amino acid position as the insertion replaced in different AAV capsid proteins. The AAV capsid protein from which the engineered AAV capsid protein of the present disclosure is produced can be referred to as a "parent" or "wild-type" AAV capsid protein or a "corresponding unmodified capsid protein". In some cases, the parent 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 provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and in Srivastava et al., J. Virol., 45:555-564 (1983); the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the complete genome of AAV-5 is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862; at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., J. Virol., 78:6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006); the AAV-11 genome is provided in Virology, 330(2):375-383 (2004); portions of the AAV-12 genome are provided in Genbank Accession No. DQ813647; and portions of the AAV-13 genome are provided in Genbank Accession No. EU285562. At least a portion of the AAV-DJ genome is provided in Grimm, D. et al., J. Virol. 82, 5887-5911 (2008).

[0173] In some embodiments, the rAAV vectors disclosed herein may carry a transgene encoding a targeting peptide described herein that is capable of binding specifically to LRP6. The targeting peptide may be part of the rAAV capsid. Disclosed herein also include AAV capsid proteins. The AAV capsid proteins may contain the targeting peptides disclosed herein.

[0174] The position of the targeting peptide in the capsid protein can vary. In some embodiments, the targeting peptide can be inserted between two adjacent amino acids in AA586-595 of the AAV9 capsid protein (e.g., between AA586 and AA587, between AA587 and AA588, between AA588 and AA589, between AA589 and AA590, between AA590 and AA591, between AA591 and AA592, between AA592 and AA593, between AA593, AA594 and AA595) or between functional equivalents thereof in other AAV capsid proteins. The AAV vector can be 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, 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 consists of SEQ ID NO: 11 or an amino acid sequence that is 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 a number or range between any two of these values) identical to SEQ ID NO: 11.

[0175] 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.

[0176] When bound to LRP6, the targeting peptide is capable of interacting with: (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having an amino acid sequence of SEQ ID NO: 31; or (2) one or more positions functionally equivalent to S96, S114, E115, R141, W157, W183, or W242 in LRP6 having an amino acid sequence of SEQ ID NO: 31.

[0177] The binding specificity between the targeting peptide carried by rAAV and LRP6 can be screened using the AAV targeted evolution (CREATE) method (M-CREATE) based on multiple Cre recombination. In M-CREATE, rAAV nucleic acid contains a marker sequence flanked by two lox sequences. rAAV is administered to animals (e.g., mice), wherein the gene encoding the Cre recombinase is expressed only in target cells (e.g., endothelial cells in the brain). The marker sequence of rAAV that can enter target cells is reversed by the Cre recombinase expressed in the target cells. Due to the lack of specific rAAV nucleic acid for target cells, Cre recombinase cannot be contacted, so its marker sequence will not be reversed. Therefore, after sequencing the nucleic acid of the rAAV recovered from the animal, the targeting peptide of the rAAV containing the reverse marker sequence is specific to the target cell. The M-CREATE method is described in detail in U.S. Patent Publication US20170166926A1, the contents of which are incorporated herein by reference as a whole for all purposes.

[0178] The generation of viral vectors can be achieved using any suitable genetic engineering techniques well known in the art, including but not limited to standard techniques for restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, such as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY (1989)). Viral vectors can incorporate sequences from the genome of any known organism. The sequences can be incorporated in their native form or modified in any way to obtain the desired activity. For example, the sequences can contain insertions, deletions, or substitutions.

[0179] Use of AAV vectors and rAAV for payload delivery

[0180] Disclosed herein are also compositions for delivering a payload (e.g., a pharmaceutical agent) to a target environment (e.g., the nervous system of a subject). The composition can include an AAV comprising (1) an AAV capsid protein disclosed herein, and (2) a pharmaceutical agent to be delivered to a target environment (e.g., the nervous system) of a subject.

[0181] 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, arterioles in the brain, arteries in the brain, or a combination thereof.

[0182] The agent to be delivered can include nucleic acids, peptides, small molecules, aptamers, or combinations thereof. The AAV vectors disclosed herein can be effectively transduced into a target environment (e.g., CNS), for example, for delivering nucleic acids. In some embodiments, a method for delivering a nucleic acid sequence to the nervous system is provided. A protein can be part of the capsid of an AAV. The AAV can include a nucleic acid sequence to be delivered to the nervous system. The AAV can then be administered to a subject.

[0183] The nucleic acid sequence to be delivered to the nervous system may include one or more of the following: a) a sequence encoding a trophic 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) a DNA (e.g., a genomic DNA or cDNA sequence) that restores protein function to humans or animals carrying a genetic mutation in a gene; c) a DNA encoding a protein that can be used to control or alter the activity or state of a cell; d) a DNA encoding a protein or nucleic acid for assessing the state of a cell; e) a DNA and / or associated guide RNA for genome engineering; f) a sequence for genome editing by homologous recombination; g) a DNA sequence encoding a therapeutic RNA; h) an shRNA or artificial miRNA delivery system; or i) a DNA sequence that affects the splicing of an endogenous gene.

[0184] In some embodiments, the vector may further comprise regulatory control elements known to those skilled in the art to affect the expression of RNA and / or protein products encoded by the polynucleotide in the desired cells of a subject.

[0185] Functionally, the expression of a polynucleotide can be controlled at least in part by operably connected regulatory elements such that these elements regulate the transcription of the polynucleotide, the transportation, processing and stability of the RNA encoded by the polynucleotide, and, where appropriate, the translation of the regulatory transcript. A specific example of an expression control element is a promoter typically located 5' of the transcribed sequence. Another example of an expression control element is an enhancer that can be located 5' or 3' of the transcribed sequence or within the transcribed sequence. Another example of a regulatory element is a recognition sequence for microRNA. Another example of a regulatory element is an intron and splice donor and splice acceptor sequences that regulate the splicing of the intron. Another example of a regulatory element is a transcription termination signal and / or a polyadenylation sequence.

[0186] Expression control elements and promoters include those that are active in specific tissues 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 regulatory factors that are unique to the specific cell, tissue, or organ type.

[0187] Expression control elements also include ubiquitous or promiscuous promoters / enhancers that can drive 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 promiscuous mammalian genes, including but not limited to β-actin, ubiquitin, or EF1α; or synthetic elements that do not occur in nature.

[0188] Expression control elements can also be given expression in a controllable manner, that is, signals or stimulations increase or reduce the expression of operably connected polynucleotides. The controllable elements that increase the expression of operably connected polynucleotides in response to signals or stimulations are also referred to as "inducible elements" (that is, they are induced by signals). Specific examples include but are not limited to hormone (for example, steroid) inducible promoters. The controllable elements that reduce the expression of operably connected polynucleotides in response to signals or stimulations are referred to as "inhibitory elements" (that is, signals reduce expression so that expression increases when signals are removed or do not exist). Generally, the amount of increase or decrease given by such elements is directly proportional to the amount of the signal or stimulation present: the larger the amount of signal or stimulation, the larger the increase or reduction degree of expression.

[0189] The nucleic acid (e.g., heterologous nucleic acid) may comprise a 5'ITR and a 3'ITR. The agent may comprise a DNA sequence encoding a protein (e.g., a nutrient, a growth factor, or a soluble protein). The nucleic acid may comprise a promoter operably linked to a polynucleotide encoding, for example, a protein or RNA agent. The promoter may be capable of inducing transcription of the polynucleotide. Transcription of the polynucleotide may generate transcripts. The nucleic acid may comprise one or more of the following: a 5'UTR, a 3'UTR, a small promoter, an enhancer, a splicing signal, a polyadenylation signal, a terminator, one or more silencer effector binding sequences, a protein degradation signal, and an internal ribosome entry element (IRES). The silencer effector may comprise a microRNA (miRNA), a precursor microRNA (pre-miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a precursor thereof, a derivative thereof, or a combination thereof. The silencer effector may be capable of binding to the one or more silencer effector binding sequences, thereby reducing the stability of the transcript and / or reducing the translation of the transcript. In some embodiments, the silencing effector comprises one or more miRNA binding sites (e.g., miR-122 binding sites). MiRNA binding sites are operably linked regulatory elements that are typically located in the 3'UTR of the transcribed sequence. The binding of miRNA to the target transcript (complexed with the RNA-induced silencing complex RISC) can reduce the expression of the target transcript by translational inhibition and / or transcript degradation.

[0190] The polynucleotide may further comprise a transcript stabilizing element. The transcript stabilizing element may comprise a woodchuck hepatitis post-translational regulatory element (WPRE), a bovine growth hormone polyadenylation (bGH-polyA) signal sequence, a 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: 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 certain embodiments, the gene of interest may be selected from SOD1, MAPT, APOE, HTT, C9ORF72, TDP-43, APP, BACE, SNCA, ATXN1, ATXN2, ATXN3, ATXN7, SCN1A-SCN5A, and SCN8A-SCN11A. The nucleic acid may further comprise a polynucleotide encoding one or more secondary proteins, and the protein and the one or more secondary proteins may comprise a synthetic protein circuit. The nucleic acid may comprise a single-stranded AAV (ssAAV) vector or a self-complementary AAV (scAAV) vector.

[0191] The promoter may comprise a ubiquitous promoter. The ubiquitous promoter may be selected from the group consisting of the cytomegalovirus (CMV) immediate early promoter; the CMV promoter; the viral simian virus 40 (SV40) (e.g., early or late); the Moloney murine leukemia virus (MoMLV) LTR promoter; the Rous sarcoma virus (RSV) LTR; the RSV promoter; the herpes simplex virus (HSV) (thymidine kinase) promoter; the H5, P7.5, and P11 promoters from vaccinia virus; the elongation factor 1-alpha (EF1-alpha) promoter. F1a) 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 beta member 1 (HSP90B1); heat shock protein 70 kDa (HSP70); β-kinesin (β-KIN); human ROSA 26 locus; 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.

[0192] The promoter can be an inducible promoter, such as a tetracycline-responsive promoter, a TRE promoter, a Tre3G promoter, an ecdysone-responsive promoter, a cumate-responsive promoter, a glucocorticoid-responsive promoter, and an estrogen-responsive promoter, a PPAR-γ promoter, a RU-486-responsive promoter, or a combination thereof.

[0193] The promoter may comprise a tissue-specific promoter and / or a lineage-specific promoter. The tissue-specific promoter may be a liver-specific thyroxine-binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, an alpha-myosin heavy chain (a-MHC) promoter, or a cardiac troponin T (cTnT) promoter. The tissue-specific promoter may be a neuron-specific promoter, such as a synapsin-1 (Syn) promoter, a CaMKIIa promoter, a calcium / calmodulin-dependent protein kinase IIa promoter, a microtubule protein αI promoter, a neuron-specific enolase promoter, a platelet-derived growth factor β chain promoter, a TRPV1 promoter, a Nav1.7 promoter, a Nav1.8 promoter, a Nav1.9 promoter, or an Advillin promoter. The tissue-specific promoter may be or comprise a muscle-specific promoter, such as the MCK promoter.

[0194] The promoter may comprise an intron sequence. The promoter may comprise a bidirectional promoter and / or an enhancer. In certain embodiments, the enhancer may be a CMV enhancer. One or more cells of an experimenter may comprise an endogenous version (e.g., gene) of a nucleotide sequence, and the promoter may comprise or may be derived from an endogenous version of the promoter. In certain embodiments, one or more cells of an experimenter comprise an endogenous version of a nucleotide sequence, and the sequence is not truncated relative to the endogenous version.

[0195] The length of the promoter 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 bp, 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, 810 bp, 820 bp, 830 bp, 840 bp, 850 bp, 860 bp, 87 In some embodiments, the promoter may be a promoter comprising at least one of the following: 1) a promoter comprising at least one of the following: 2) a promoter comprising at least one of the following: 1) a promoter comprising at least one of the following: 2) a promoter comprising at least one of the following: 3) a promoter comprising at least one of the following: 4) a promoter comprising at least one of the following: 5) a promoter comprising at least one of the following: 6) a promoter comprising at least one of the following: 7) a promoter comprising at least one of the following: 8) a promoter comprising at least one of the following: 9) a promoter comprising at least one of the following: 10) a promoter comprising at least one of the following: 11) a promoter comprising at least one of the following: 12) a promoter comprising at least one of the following: 13) a promoter comprising at least one of the following: 14) a promoter comprising at least one of the following: 15) a promoter comprising at least one of the following: 16) a promoter comprising at least one of the following: 17) a promoter comprising at least one of the following: 18) a promoter comprising at least one of the following: 19) a promoter comprising at least one of the following: 20) a promoter comprising at least one of the following: 20) a promoter comprising at least one of the following:Expression of the therapeutic gene expression product can continue 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 months, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65 years, or a value or range between any two of these values or greater than 65 years.

[0196] As used herein, "protein of interest" can be any protein, including naturally occurring and non-naturally occurring proteins. In some embodiments, the polynucleotides encoding one or more proteins of interest can be present in one of the AAV vectors disclosed herein, wherein the polynucleotides are operably linked to a promoter. In some cases, the promoter can drive expression of the protein of interest in a host cell (e.g., an endothelial cell). In some embodiments, the protein of interest is an anti-tau antibody, an anti-AB antibody, and / or an ApoE isoform.

[0197] The protein can comprise aromatic L-amino acid decarboxylase (AADC), survival motor neuron 1 (SMN1), frataxin (FXN), cystic fibrosis transmembrane conductance regulator (CFTR), factor X (FIX), RPE65, retinoid isomerohydrolase (RPE65), sarcoglycan alpha (SGCA) and sarcoplasmic / endoplasmic reticulum Ca2+-ATPase (SERCA2a), ApoE2, GBA1, GRN, ASP A, CLN2, GLB1, SGSH, NAGLU, IDS, NPC1, GAN, CFTR, GDE, OTOF, DYSF, MYO7A, ABCA4, F8, CEP290, CDH23, DMD, ALMS1, or any combination thereof.

[0198] The protein may comprise a disease-associated protein. In certain embodiments, the expression level of the disease-associated protein is associated with the occurrence and / or progression of the disease. The protein may comprise methyl CpG binding protein 2 (MeCP2), DRK1A, KAT6A, NIPBL, HDAC4, UBE3A, EHMT1, one or more genes NPHP1, LIMK1 encoded on chromosome 9q34.3, one or more genes P53, TPI1, FGFR1 and related genes encoded on chromosome 7q11.23, RA1, SHANK3, CLN3, NF-1, TP53, PFK, CD40L, CYP19A1, PGRN, CHRNA7, PMP22, CD40LG, derivatives thereof, or any combination thereof.

[0199] In certain embodiments, nucleic acid can comprise the DNA (for example, cDNA or genomic DNA sequence) of protein encoding, to control or monitor the activity or state of cell, and / or for evaluating the state of cell.Protein can comprise fluorescence activity, polymerase activity, protease activity, phosphatase activity, kinase activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity or its any combination.Protein can comprise nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidative activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosidase activity, acetyltransferase activity, deacetylase activity, adenylation activity, deadenylation activity or its any combination.Protein can comprise nuclear localization signal (NLS) or nuclear export signal (NES).

[0200] The protein can include CRE recombinase, GCaMP, cell therapy components, knockdown gene therapy components, cell surface exposed epitopes, or any combination thereof. The protein can include a chimeric antigen receptor. The protein can include a diagnostic agent (e.g., green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), TagRFP, Dronpa, Padron, mApple, mCherry, mruby3, rsCherry, rsCherryRev, derivatives thereof, or any combination thereof).

[0201] In some embodiments, the nucleic acid may comprise a DNA (e.g., a cDNA or genomic DNA sequence) or a guide RNA encoding a protein for gene editing; or a DNA sequence for genome editing by homologous recombination. The protein may comprise a programmable nuclease. In some embodiments, the programmable nuclease is selected from: SpCas9 or its derivatives; VRER, VQR, EQR SpCas9; xCas9-3.7; eSpCas9; Cas9-HF1; HypaCas9; evoCas9; HiFi Cas9; ScCas9; StCas9; NmCas9; SaCas9; CjCas9; CasX; Cas9 H940A nickase; Cas12 and its derivatives; dcas9-APOBEC1 fusion, BE3 and dcas9 deaminase fusion; dcas9-Krab, dCas9-VP64, dCas9-Tet1 and dcas9 transcriptional regulator fusions; Dcas9 fluorescent protein fusions; Cas13 fluorescent protein fusions; RCas9 fluorescent protein fusions; Cas13 adenosine deaminase fusions. Programmable nucleases can include zinc finger nucleases (ZFNs) and / or transcription activator-like effector nucleases (TALENs). Programmable nucleases can include Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), zinc finger nucleases, TAL effector nucleases, meganucleases, MegaTAL, Tev-m TALEN, MegaTev, homing endonucleases, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr 5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, C2c1, C2c3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, derivatives thereof, or any combination thereof. Nucleic acids and / or rAAVs may comprise polynucleotides encoding (i) targeting molecules and / or (ii) donor nucleic acids. Targeting molecules may be capable of being associated with programmable nucleases. Targeting molecules may comprise single-stranded DNA or single-stranded RNA.The targeting molecule may comprise a single guide RNA (sgRNA).

[0202] The rAAV disclosed herein may comprise one or more nucleic acids of the 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 operably linked to the polynucleotide encoding the protein. As disclosed herein, in some embodiments, the gene is operably 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.

[0203] The protein can be any protein, including naturally occurring and non-naturally occurring proteins. Examples include, but are not limited to, luciferase; fluorescent proteins (e.g., GFP); growth hormone (GH) and variants thereof; insulin-like growth factor (IGF) and variants thereof; granulocyte colony-stimulating factor (G-CSF) and variants thereof; erythropoietin (EPO) and variants thereof; insulin, such as proinsulin, preproinsulin, insulin, insulin analogs, etc.; antibodies and variants thereof, such as hybrid antibodies, chimeric antibodies, humanized antibodies, monoclonal antibodies; antigen-binding fragments of antibodies (Fab fragments), single-chain variable fragments of antibodies (scFV fragments); anti-dystrophin and variants thereof; coagulation factors and variants thereof; cystic fibrosis transmembrane conductance regulator (CFTR) and variants thereof; and interferons and variants thereof.

[0204] Examples of proteins of interest include, but are not limited to, luciferase; fluorescent proteins (e.g., GFP); growth hormone (GH) and variants thereof; insulin-like growth factor (IGF) and variants thereof; granulocyte colony-stimulating factor (G-CSF) and variants thereof; erythropoietin (EPO) and variants thereof; insulin, such as proinsulin, preproinsulin, insulin, insulin analogs, etc.; antibodies and variants thereof, such as hybrid antibodies, chimeric antibodies, humanized antibodies, monoclonal antibodies; antigen-binding fragments of antibodies (Fab fragments), single-chain variable fragments of antibodies (scFV fragments); dystrophin and variants thereof; coagulation factors and variants thereof; CFTR and variants thereof; and interferons and variants thereof.

[0205] In some embodiments, the protein of interest is a therapeutic protein or a variant thereof. Non-limiting examples of therapeutic proteins include blood factors such as β-globin, hemoglobin, tissue plasminogen activator and coagulation factors; colony stimulating factor (CSF); interleukins such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, etc.; growth factors such as keratinocyte growth factor (GF), stem cell factor (SCF), fibroblast growth factor (FGF, such as basic FGF and acidic FGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), bone morphogenetic protein (BMP), epidermal growth factor (EGF), growth differentiation factor-9 (GDF-9), hepatocellular carcinoma ... , myostatin (GDF-8), nerve growth factor (NGF), neurotrophic factors, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor α (TGF-α), transforming growth factor β (TGF-β), etc.; soluble receptors, such as soluble TNF-α receptor, soluble VEGF receptor, soluble interleukin receptor (e.g., soluble IL-1 receptor and soluble type II IL-1 receptor), soluble γ / δ T cell receptor, ligand binding fragments of soluble receptors, etc.; enzymes, such as β-glucosidase, imiglucerase, β-glucocerebrosidase and arabinocerebrosidase; enzyme activators, such as Tissue plasminogen activator; chemokines, such as IP-10, monokine induced by interferon-γ (Mig), Groa / IL-S, RANTES, MIP-1α, MIP-1β, MCP-1, PF-4, etc.; angiogenic agents, such as vascular endothelial growth factor (VEGF, such as VEGF121, VEGF165, VEGF-C, VEGF-2), transforming growth factor-β, basic fibroblast growth factor, glioma-derived growth factor, angiopoietin, angiopoietin-2, etc.; anti-angiogenic agents, such as soluble VEGF receptor; protein vaccines; neuroactive peptides, such as nerve growth factor (NGF), bradykinin, cholecystokinin, gastrin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dynorphin, warfarin, neurotensin, motilin, thyroid-stimulating hormone, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, sleep peptide, etc.; thrombolytics; atrial natriuretic peptide; relaxin; glial fibrillary acidic protein; follicle-stimulating hormone (FSH); human alpha-1 antitrypsin; leukemia inhibitory factor (LIF); transforming growth factor (TGF); tissue factor, luteinizing hormone; macrophage-activating factor;Tumor necrosis factor (TNF); neutrophil chemoattractant factor (NCF); nerve growth factor; tissue inhibitor of metalloproteinases; vasoactive intestinal peptide; angiopoietin; angiotropin; fibrinogen; hirudin; IL-1 receptor antagonist; etc. Some other non-limiting examples of proteins of interest include ciliary neurotrophic factor (CNTF); brain-derived neurotrophic factor (BDNF); neurotrophins 3 and 4 / 5 (NT-3 and 4 / 5); glial cell line-derived neurotrophic factor (GDNF); aromatic amino acid decarboxylase (AADC); hemophilia-related coagulation proteins, such as factor IX or factor X; dystrophin or mini-dystrophin; lysosomal acid lipase; phenylalanine hydroxylase (PAH); glycogen storage disease-related enzymes, such as glucose-6-phosphatase, acid maltase, glycogen debranching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase (e.g., PHKA2), glucose transporter (e.g., GLUT2), aldolase A, β-enolase, and glycogen synthase; lysosomal enzymes (e.g., β-N-acetylhexosaminidase A); and any variants thereof.

[0206] The protein of interest can be, for example, an active fragment of a protein, such as any of the aforementioned proteins, a fusion protein comprising some or all of two or more proteins, or a fusion protein including all or part of the aforementioned proteins.

[0207] The viral vector may comprise a polynucleotide comprising a coding region for two or more proteins of interest. The two or more proteins of interest may be identical or different from one another. In some embodiments, the two or more proteins of interest are related polypeptides, such as the light and heavy chains of the same antibody.

[0208] The protein of interest can be a multi-subunit protein. For example, the protein of interest can comprise two or more subunits or two or more independent polypeptide chains. In some embodiments, the protein of interest can be an antibody, including but not limited to antibodies of various isotypes (e.g., IgG1, IgG2, IgG3, IgG, IgA, IgD, IgE, and IgM); monoclonal antibodies produced by any means known to those skilled in the art, including antigen-binding fragments of monoclonal antibodies; humanized antibodies; chimeric antibodies; single-chain antibodies; antibody fragments, such as Fv, F(ab')2, Fab', Fab, Facb, scFv, etc.; provided that the antibody is able to bind to the antigen. In some embodiments, the antibody is a full-length antibody. In some embodiments, the protein of interest is not an immunoadhesin.

[0209] In some embodiments, the resulting targeting molecules can be used in methods and / or therapies related to in vivo gene transfer applications in long-lived cell populations. In some embodiments, these methods and / or therapies can be applied to any rAAV-based gene therapy, including, for example, spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), Parkinson's disease, Friedreich's ataxia, Pompe disease, Huntington's disease, Alzheimer's disease, Batten disease, lysosomal storage diseases, glioblastoma multiforme, Rett syndrome, Leber's congenital amaurosis, chronic pain, stroke, spinal cord injury, traumatic brain injury, and lysosomal storage diseases. In addition, rAAV can also be used for in vivo delivery of transgenes for non-therapeutic scientific research (such as optogenetics), gene overexpression, gene knockdown with shRNA or miRNA, endogenous miRNA modulation using miRNA sponges or decoys, recombinase delivery for conditional gene deletion, conditional (recombinase-dependent) expression, or gene editing with CRISPR, TALEN, and zinc finger nucleases.

[0210] In certain embodiments, gene encoding can stimulate the immunogenic material of immune response (for example, adaptive immune response), for example, from the antigenic peptide or protein of pathogen.The expression of antigen can stimulate the adaptive immune system of health to provide adaptive immune response.Therefore, it is contemplated that some embodiments of nucleic acid provided herein can be used as the vaccine (for example, as vaccine) of preventing and treating or treating infectious disease.

[0211] As described herein, the nucleotide sequence encoding the protein can be modified to improve the expression efficiency of the protein. The methods that can be used to improve the transcription and / or translation of the gene are not particularly limited herein. For example, the nucleotide sequence can be modified to better reflect the use of the host codon to increase gene expression (e.g., protein production) in the host (e.g., mammal).

[0212] The gene expression degree in target cell can change.The amount of protein expressed in subject (for example, subject's CNS) can change.For example, in some embodiments, protein can be expressed in following amount in subject: at least about 9 μ g / ml, at least about 10 μ g / ml, at least about 50 μ g / ml, at least about 100 μ g / ml, at least about 200 μ g / ml, at least about 300 μ g / ml, at least about 400 μ g / ml, at least about 500 μ g / ml, at least about 600 μ g / ml, at least about 700 μ g / ml, at least about 800 μ g / ml, at least about 900 μ g / ml or at least about 1000 μ g / ml. In some embodiments, the protein is expressed in the subject at about 9 μg / ml, about 10 μg / ml, about 50 μg / ml, about 100 μg / ml, about 200 μg / ml, about 300 μg / ml, about 400 μg / ml, about 500 μg / ml, about 600 μg / ml, about 700 μg / ml, about 800 μg / ml, about 900 μg / ml, about 1000 μg / ml, about 1500 μg / ml, about 2000 μg / ml, about 2500 μg / ml, or a range between any two of these values. One skilled in the art will appreciate that the method requires that the level of expression of the protein vary depending on non-limiting factors, such as the particular protein and the subject being treated, and one skilled in the art can readily determine an effective amount of protein using routine methods known in the art without undue experimentation.

[0213] Medicament (for example, therapeutic agent) can be an inducer of cell death. Medicament can induce cell death through non-endogenous cell death pathway (for example, bacterial pore-forming toxin). In certain embodiments, medicament (for example, protein encoded by nucleic acid) can be a pro-survival protein. In certain embodiments, medicament is a regulator of the immune system. Medicament can activate adaptive immune response and innate immune response or both. In certain embodiments, nucleic acid encoding can stimulate immunogenic materials of immune response (for example, adaptive immune response), such as antigenic peptides or proteins from pathogens. The expression of antigens can stimulate the adaptive immune system of the body to provide adaptive immune response. Therefore, it is conceivable that some embodiments of the compositions provided herein can be used as vaccines (for example, as vaccines) for preventing and treating infectious diseases. Protein can include CRE recombinase, GCaMP, cell therapy components, knock down gene therapy components, epitopes exposed on the cell surface, or any combination thereof. In certain embodiments, protein includes CFTR, GDE, OTOF, DYSF, MYO7A, ABCA4, F8, CEP290, CDH23, DMD, and ALMS1.

[0214] Medicament (for example, therapeutic agent) can comprise non-protein coding gene such as RNA medicament, for example, the sequence of coding antisense RNA, RNAi, shRNA and microRNA (miRNA), miRNA sponge or decoy, for the recombinase delivery of conditional gene deletion, conditional (recombinase-dependent) expression, including those required for gene editing components described herein. Non-protein coding gene can also encode tRNA, rRNA, tmRNA, piRNA, double-stranded RNA, snRNA, snoRNA and / or long non-coding RNA (IncRNA). In some embodiments, RNA medicament can comprise non-natural or modified nucleotides (for example, pseudouridine). In some embodiments, non-protein coding gene can regulate the expression or activity of target gene or gene expression product. For example, RNA described herein can be used to suppress gene expression in target cells (for example, cells in the central nervous system (CNS)). In some embodiments, the inhibition of gene expression refers to inhibition of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100%. In some embodiments, the protein product of the target gene is inhibited by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%. The gene can be a wild-type gene or a gene with at least one mutation. The target protein can be a wild-type protein or a protein with at least one mutation.

[0215] Examples of genes encoding therapeutic proteins include sequences associated with signal transduction biochemical pathways, such as genes or polynucleotides (e.g., signal transducers) associated with signal transduction biochemical pathways. In some embodiments, the methods and compositions disclosed herein comprise knockdown of endogenous signal transducers, accompanied by tuned expression of proteins comprising appropriate versions of signal transducers. Examples of DNA or RNA sequences contemplated herein include sequences of disease-associated genes or polynucleotides. "Disease-associated" genes or polynucleotides refer to any genes or polynucleotides that produce transcription or translation products at abnormal levels or in abnormal forms in cells derived from tissues affected by the disease, compared to tissues or cells of non-disease controls. It can be a gene that is expressed at an abnormally high level; it can be a gene that becomes expressed at an abnormally low level, wherein the altered expression is associated with the occurrence and / or progression of the disease. Disease-associated genes also refer to genes with mutations or genetic variations that directly result in or are in linkage disequilibrium with genes that cause the disease. The transcribed or translated products can be known or unknown and can be at normal or abnormal levels. Signal transducers can be associated with one or more diseases or conditions. In some embodiments, the disease or condition is characterized by abnormal signaling of one or more signal transducers disclosed herein. In some embodiments, the activation level of a signal transducer is associated with the onset and / or progression of the disease or condition. The activation level of a signal transducer can directly or indirectly contribute to the cause of the disease or condition.

[0216] Many proteins (e.g., enzymes) are secreted and can play a cross-correction role. For these proteins, the AAV disclosed herein can be used to deliver genetic material into brain endothelial cells, thereby converting these cells into biofactories to produce therapeutic agents and distributing these therapeutic agents to other cell types. For example, the production of Sparcl1 / Hevin protein secreted in brain endothelial cells can rescue the thalamocortical synaptic loss phenotype of Hevin KO mice. This proof of concept supports a brain endothelial cell biofactory model for producing enzymes, antibodies, or other biotherapeutics, thereby providing a new type of treatment for diseases such as lysosomal storage diseases.

[0217] In some embodiments, rAAVs having a capsid protein comprising one or more targeting peptides disclosed herein can be used to deliver genes to specific cell types in a subject's target environment. For example, rAAVs can be used to deliver genes to neurons and glia in the nervous system (including PNS, CNS, or both) of a subject (e.g., a mammal). The compositions and methods disclosed herein can be used, for example, to: (i) reduce expression of mutant huntingtin in patients with Huntington's disease by, for example, incorporating a huntingtin-specific microRNA expression cassette into the rAAV genome and packaging the rAAV genome into variant rAAV for delivery, for example, via the vasculature, (ii) deliver a functional copy of a tachin gene to patients with Friedreich's ataxia, (iii) restore expression of an enzyme that is critical for normal lysosomal function in patients whose enzyme expression is deficient due to a genetic mutation (e.g., patients with Neimann-Pick disease, mucopolysaccharidosis III, and / or Gaucher's disease), (iv) use rAAV to generate animal models of disease, or a combination thereof.

[0218] A subject in need thereof can be a subject having or at risk of developing one or more of the following: chronic pain, Friedreich's ataxia, Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), spinal muscular atrophy type I and II (SMA I and II), Friedreich's ataxia (FA), spinocerebellar ataxia, multiple sclerosis (MS), chronic traumatic encephalopathy (CTE), HIV-1 associated dementia, or a lysosomal storage disease involving cells within the CNS. The lysosomal storage disease can be Krabbe disease, Sandhoff disease, Tay-Sachs disease, Gaucher disease (type I, II, or III), Niemann-Pick disease (NPC1 or NPC2 deficiency), Hurler syndrome, Pompe disease, or Batten disease.

[0219] In some embodiments, the subject is suffering from an acute condition or injury. A subject in need thereof can be a subject suffering from, at risk of suffering from, or who has suffered from: a stroke, a traumatic brain injury, epilepsy, or a spinal cord injury.

[0220] Pharmaceutical compositions and methods of administration

[0221] Also disclosed herein are pharmaceutical compositions comprising one or more rAAV viruses (or other delivery systems) and one or more pharmaceutically acceptable carriers of the rAAV viruses disclosed herein. The compositions may also include additional ingredients, such as diluents, stabilizers, excipients, and adjuvants. As used herein, a "pharmaceutically acceptable" carrier, excipient, diluent, adjuvant, or stabilizer is nontoxic (preferably inert) to cells or subjects exposed thereto at the dosage and concentration employed, or has an acceptable toxicity level determined by one skilled in the art. Carriers, diluents, and adjuvants may include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides (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 dextrins; 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 TM 、Pluronics TM Or polyethylene glycol (PEG).In some embodiments, the physiologically acceptable carrier is an aqueous pH buffered solution.

[0222] Disclosed herein are methods for delivering an agent to the nervous system of a subject. In some embodiments, the method comprises providing an AAV vector comprising an AAV capsid protein disclosed herein. In some embodiments, the AAV vector comprises the agent to be delivered to the nervous system. In some embodiments, the method comprises administering the AAV vector to the subject. The composition can be administered intravenously. The composition can be administered systemically. The agent can be delivered to the intraventricular epithelial lining in the brain of the subject, the central canal of the spinal cord, capillaries in the brain, arterioles in the brain, arteries in the brain, or a combination thereof. The subject can be an adult animal.

[0223] The titer of rAAV to be administered will vary depending on, for example, the specific rAAV, the mode of administration, the therapeutic goal, the individual and the cell type being targeted, and can be determined by standard methods in the art. As will be readily apparent to those skilled in the art, the useful in vivo dose of the recombinant virus to be administered and the specific mode of administration will vary depending on the age, weight, severity of the affliction and species of animal being treated, the specific recombinant virus expressing the protein of interest used, and the specific use of the recombinant virus employed. The determination of the effective dose level, i.e., the dose level required to achieve the desired result, can be accomplished by those skilled in the art using routine pharmacological methods. Typically, human clinical use of the product begins with a lower dose level, with the dose level increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration of the compositions identified by the methods of the present invention using established pharmacological methods.

[0224] The exact dosage can be determined on a drug-by-drug basis, and in most cases, some generalizations can be made regarding dosage. In some embodiments, rAAV for delivering an agent to a subject's nervous system (e.g., CNS) can be administered to a subject, for example, by injection, at a dose of between 1×10 10 The viral genomes (vg) of recombinant viruses / subject (kg) were compared with 2×10 14 vg / kg, for example, between 5×10 11 vg / kg and 5×10 12 In some embodiments, the dose of rAAV administered to a subject does not exceed 2×10 14 In some embodiments, the dose of rAAV administered to a subject does not exceed 5×10 12 In some embodiments, the dose of rAAV administered to a subject does not exceed 5×10 11 vg / kg.

[0225] The effective dose and dosage of the pharmaceutical composition for preventing or treating the diseases or conditions disclosed herein are defined by the observed beneficial response associated with the disease or condition or the symptoms of the disease or condition. A beneficial response comprises preventing, alleviating, stopping or curing the disease or condition or the symptoms of the disease or condition. In some embodiments, a beneficial response is measured by detecting a measurable improvement in the presence, level or activity of a subject's biomarker, transcriptome risk profile or intestinal microbiome. As used herein, "improvement" refers to a shift in the presence, level or activity to that observed in a normal individual (e.g., an individual not suffering from the disease or condition). In the case where the therapeutic rAAV composition is ineffective or does not fully alleviate the disease or condition or the symptoms of the disease or condition, the dosage and / or route of administration can be changed, or another agent can be administered to the subject together with the therapeutic rAAV composition. In some embodiments, when the patient begins the regimen of the therapeutic rAAV composition, the patient also gradually stops (e.g., the dosage is gradually reduced) the second treatment regimen.

[0226] In some embodiments, the pharmaceutical compositions of the present disclosure are administered at a dosage level sufficient to deliver the following daily, once a day, or multiple times a day to obtain the desired therapeutic, diagnostic, or preventive 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 dosage concentrations into vg per kg or viral genome or into total viral genomes.

[0227] In some embodiments, the concentration of infectious particles in a dose of the pharmaceutical composition is at least or about 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 or 10 17 In some cases, the concentration of infectious particles was 2 × 10 7, 2×10 8 , 2×10 9 , 2×10 10 , 2×10 11 , 2×10 12 , 2×10 13 , 2×10 14 , 2×10 15 , 2×10 16 , 2×10 17 or a range between any two of these values. In some cases, the concentration of infectious particles is 3 × 10 7 , 3×10 8 , 3×10 9 , 3×10 10 , 3×10 11 , 3×10 12 , 3×10 13 , 3×10 14 , 3×10 15 , 3×10 16 , 3×10 17 or a range between any two of these values. In some cases, the concentration of infectious particles is 4 × 10 7 , 4×10 8 , 4×10 9 , 4×10 10 , 4×10 1 , 4×10 12 , 4×10 13 , 4×10 14 , 4×10 15 , 4×10 16 , 4×10 17 or a range between any two of these values. In some cases, the concentration of infectious particles is 5 × 10 7 , 5×10 8 , 5×10 9 , 5×10 10 , 5×10 11 , 5×10 12 , 5×10 13 , 5×10 14 , 5×10 15 , 5×10 16 , 5×10 17 or a range between any two of these values. In some cases, the concentration of infectious particles is 6 × 10 7 , 6×10 8 , 6×10 9 , 6×10 10 , 6×10 11, 6×10 12 , 6×10 13 , 6×10 14 , 6×10 15 , 6×10 16 , 6×10 17 or a range between any two of these values. In some cases, the concentration of infectious particles was 7 × 10 7 , 7×10 8 , 7×10 9 , 7×10 10 , 7×10 11 , 7×10 12 , 7×10 13 , 7×10 14 , 7×10 15 , 7×10 6 , 7×10 17 or a range between any two of these values. In some cases, the concentration of infectious particles was 8 × 10 7 , 8×10 8 , 8×10 9 , 8×10 10 , 8×10 11 , 8×10 12 , 8×10 13 , 8×10 14 , 8×10 15 , 8×10 16 , 8×10 17 or a range between any two of these values. In some cases, the concentration of infectious particles was 9 × 10 7 , 9×10 8 , 9×10 9 , 9×10 10 , 9×10 11 , 9×10 12 , 9×10 13 , 9×10 14 , 9×10 15 , 9×10 16 , 9×10 17 Or a range between any two of these values.

[0228] The recombinant viruses disclosed herein can be administered to a subject (e.g., a human) in need thereof. The route of administration is not particularly limited. For example, a therapeutically effective amount of the recombinant virus can be administered to a subject via standard routes in the art. Administration can be systemic. Administration can be intravenous.

[0229] The limiting examples of approach include intramuscular, intravaginal, intravenous, intraperitoneal, subcutaneous, epidermal, intradermal, rectal, intraocular, lung, intracranial, intraosseous, oral, cheek, whole body or nose. In certain embodiments, the recombinant virus is applied to the subject by systemic transduction. In certain embodiments, the recombinant virus is applied to the subject by intramuscular injection. In certain embodiments, rAAV is applied to the subject by parenteral route (for example, by intravenous, intramuscular or subcutaneous injection), by surface scratching or by being inoculated into the body cavity of the subject. The route of administration and serotype of the AAV components of rAAV virus can be easily determined by those skilled in the art in view of the infection treated and / or morbid state and the target cell / tissue of the protein being paid close attention to. In certain embodiments, it can be advantageous to administer rAAV by intravenous administration. The variant AAV provided herein can advantageously provide the intravenous administration of the carrier with enhanced tropism to CNS.

[0230] In certain embodiments, the subject is a primate, and medicament is delivered to endothelial cells and / or neurons of the nervous system. Nervous system can be central nervous system (CNS). The efficiency of the medicament delivered to the endothelial cells of the nervous system of the subject can be at least 1.5 times, 2 times or 3 times of the efficiency of the medicament delivered to the neurons of the nervous system. In certain embodiments, the efficiency of the medicament delivered to the endothelial cells of the nervous system of the subject is more than 3 times (for example, 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 the numeral or scope between any value in these values) of the medicament delivered to the neurons of the nervous system.

[0231] Disclosed herein are methods for delivering an agent (e.g., a therapeutic agent) to a cell. In some embodiments, the method comprises contacting an AAV vector comprising an AAV capsid protein disclosed herein with a cell. In some embodiments, the AAV vector comprises an 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 vector is contacted with the cell in vitro, in vivo, or ex vivo. The cell may be present in a tissue, an organ, or a 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 vasculature, or a combination thereof.

[0232] The AAV vector can be an AAV9 vector or a variant thereof. 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, human isolate hu.31, human isolate hu.32, rhesus monkey isolate rh.8, rhesus monkey isolate rh.10, or a variant thereof. The serotype of the AAV vector can be different from the serotype of the AAV capsid.

[0233] The variant AAV capsid may comprise a tropism for tissues or cells of the central nervous system (CNS). The target cell may be a neuronal cell, a neural stem cell, an astrocyte, or a tumor cell. The target cell may be located in the brain or spinal cord. The target cell may comprise an antigen presenting cell, a dendritic cell, a macrophage, a nerve cell, a brain cell, astrocyte, microglia, and a neuron. In some embodiments, the target cell is an endothelial cell.

[0234] The actual administration of rAAV can be accomplished by using any physical method for transporting 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 so that rAAV targets a specific target environment of interest, such as the central nervous system, and enhances tropism to the target environment of interest (e.g., CNS tropism). The pharmaceutical composition can be prepared, for example, as an injectable formulation.

[0235] The recombinant virus to be used can be used in liquid or freeze-dried form (in combination with one or more suitable preservatives and / or protective agents to protect the virus during the freeze-drying process). For gene therapy (e.g., gene therapy for neurological disorders that can be alleviated by specific gene products), a therapeutically effective dose of a recombinant virus expressing a therapeutic protein is administered to a host in need of such treatment. The use of the recombinant virus disclosed herein in the preparation of a medicament for inducing immunity in a host or providing gene therapy to the host is within the scope of this application.

[0236] In cases where human dosages of rAAV have been established for at least some condition, those same dosages or dosages between about 0.1% and 500%, more preferably between about 25% and 250%, of the established human dosages may be used. In cases where human dosages have not been established, as would be the case with a newly discovered pharmaceutical composition, the dosages may be determined based on the ED. 50 or ID 50 Suitable human dosages can be extrapolated from the values or other appropriate values derived from in vitro or in vivo studies, as qualified by toxicity studies and efficacy studies in animals.

[0237] The rAAV of therapeutically effective amount can be applied to the subject at different time points.For example, rAAV can be applied to the subject before, during or after the subject has suffered from disease or illness.rAAV can also be applied to the subject before, during or after disease or illness (for example, Huntington's disease (HD), Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, spinal muscular atrophy (type I and type II), Friedreich's ataxia, spinocerebellar ataxia and any lysosomal storage disease related to CNS cells, these lysosomal storage diseases include but are not limited to Krabbe disease, Sandhoff disease, Tay-Sachs disease, Gaucher disease (type I, type II or type I), Niemann-Pick disease (NPC1 or NPC2 deficiency), Hurler syndrome, Pompe disease, Batten disease or its any combination), chronic pain or its combination occurs.In certain embodiments, rAAV is applied to the subject during disease or illness remission.In certain embodiments, rAAV is applied before the disease or illness outbreak of the subject. In some embodiments, rAAV is administered to a subject at risk for developing a disease or condition.

[0238] The disease or disorder may comprise a disease or disorder of the nervous system. For example, the neurological disease or disorder 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-related infections; diseases involving mitochondrial dysfunction; diseases involving beta-amyloid and / or tauopathy; Down's 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 lipofuscinosis; 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; dementia pugilistica; vascular and prefrontal dementia; cognitive impairment; learning disabilities; eye injury; eye diseases; eye conditions; 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 disorders; chorea; Huntington's disease chorea); athetosis; dystonia; repetitive movements; tics; tardive dyskinesia; tic disorders; spasmodic torticollis; blepharospasm; focal and generalized dystonias; nystagmus; hereditary cerebellar ataxias; corticobasal degeneration; tremor; essential tremor; addiction; anxiety disorders; 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; dementia associated with HIV infection; HIV-1 encephalopathy; AIDS encephalopathy; AIDS dementia complex; AIDS-related dementia; major depressive disorder; major depressive disorder; depression; memory loss; stress; bipolar manic-depressive disorder; medication 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; spasticity; Tourette's syndrome; eating disorders; food addiction; binge eating disorder; agoraphobia; generalized anxiety disorder; obsessive-compulsive disorder; panic disorder; social phobia; phobia; substance-induced anxiety disorder; delusional disorder; schizoaffective disorder; schizophreniform disorder; substance-induced psychotic disorder; hypertension; or any combination thereof.

[0239] In some embodiments, disclosed herein are formulations of pharmaceutically acceptable excipients and carrier solutions suitable for delivering the compositions described herein, as well as suitable dosages and treatment regimens for using the specific compositions described herein in a variety of treatment regimens. In some embodiments, the amount of the therapeutic gene expression product in each therapeutically useful composition can be prepared in such a way that a suitable dosage is obtained in 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 in this way, a variety of dosages and treatment regimens may be desired. In some cases, the composition is a pharmaceutical composition of a suitable formulation disclosed herein, which is intraocular, intravitreal, parenteral, subcutaneous, intravenous, intracerebro-ventricularly, intramuscularly, intrathecally, orally, intraperitoneally, orally or nasally by inhalation or by direct injection and is delivered to one or more cells, tissues, or organs by direct injection. In some embodiments, the rAAV disclosed herein can be advantageously administered intravenously for delivery to the CNS.

[0240] In some embodiments, the pharmaceutical form of the AAV-based viral composition suitable for injectable use includes sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), a suitable mixture thereof, and / or a vegetable oil. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. 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 an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable composition can be achieved by using an agent that delays absorption in the composition, such as aluminum monostearate and gelatin.

[0241] In certain embodiments, for the use of injectable aqueous solution, for example, if necessary, this solution can be buffered compatibly, and the liquid diluent is first isotonic with enough saline or glucose.These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.Depend on the condition of disease of the experimenter being treated, some changes will inevitably occur in dosage.The personnel responsible for using will determine the appropriate dose for individual experimenter under any circumstances.In addition, for the mankind, use, composite should meet the sterility, pyrogenicity and general safety and purity standard required by FDA biological product standard office.

[0242] Disclosed herein are sterile injectable solutions comprising the compositions disclosed herein (e.g., rAAV compositions) prepared by incorporating the compositions disclosed herein in the desired amount into an appropriate solvent along with several of the other ingredients enumerated above, as needed, followed by filter sterilization. Typically, dispersions are prepared by incorporating the various sterile active ingredients into a sterile vehicle containing a base dispersion medium and the desired other ingredients from those enumerated above. In the case of sterile powders for preparing sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze drying techniques, which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile filtered solution thereof. Injectable solutions can be advantageous for systemic administration (e.g., by intravenous administration).

[0243] Also provided herein are formulations in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of proteins), and are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. After formulation, the solution will be administered in a therapeutically effective amount in a manner compatible with the dosage formulation. The formulation is easily administered in various dosage forms such as injectable solutions and drug release capsules.

[0244] Formulations for intranasal administration may comprise a coarse powder comprising the active ingredient and having an average particle size of about 0.2 μm to 500 μm. Such formulations are administered in the manner of snuff, for example by rapidly inhaling into the nasal cavity from a powder container held near the nose. Formulations suitable for nasal administration may, for example, comprise as little as about 0.1% (w / w) and as much as 100% (w / w) of the active ingredient, and may comprise one or more of the additional ingredients described herein. Pharmaceutical compositions may be prepared, packaged and / or sold as formulations suitable for buccal administration. Such formulations may, for example, be in the form of tablets and / or lozenges made using conventional methods, and may, for example, comprise 0.1% to 20% (w / w) of the active ingredient, the remainder comprising an orally soluble and / or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternatively, formulations suitable for buccal administration may comprise a powdered and / or aerosolized and / or atomized solution and / or suspension comprising the active ingredient. Such powdered, aerosolized and / or aerosolized formulations, when dispersed, may have an average particle and / or droplet size in the range of about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.

[0245] The appropriate dose and dosage for administration to a subject will be determined by factors including, but not limited to, the particular therapeutic rAAV composition, the disease condition and its severity, the identity of the subject in need of treatment (e.g., weight, sex, age), and may be determined based on the specific circumstances surrounding the case, including, for example, the particular agent being administered, the route of administration, the condition being treated, and the subject or host being treated.

[0246] The amount of AAV composition and the timing of administration of such compositions will be within the ability of those skilled in the art having the benefit of the present teachings. However, in some embodiments, administration of a therapeutically effective amount of the disclosed compositions can be achieved by a single administration, e.g., a single injection of a sufficient number of infectious particles to provide a therapeutic benefit to a patient receiving such treatment. This is made possible, at least in part, by the fact that certain target cells (e.g., neurons) do not divide, thereby eliminating the need for multiple or chronic administration.

[0247] In some embodiments, it may be advantageous to provide multiple or sequential administrations of an AAV vector composition over a relatively short or relatively extended period of time (as may be determined by the medical practitioner overseeing the administration of such compositions). For example, the number of infectious particles administered to a mammal may be about 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 1012 , 10 13 The amount of infectious particles per ml may be on the order of 100,000 or even higher, given in a single dose or divided into two or more administrations, as may be required to achieve therapy for the particular disease or condition being treated. Indeed, in certain embodiments, it may be desirable to administer two or more different AAV vector compositions, alone or in combination with one or more other therapeutic agents, to achieve the desired effect of a particular therapeutic regimen. In various embodiments, the daily dose and unit dose vary depending on a number of variables, including but not limited to the activity of the therapeutic rAAV composition used, the disease or condition being treated, the mode of administration, the needs of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0248] The targeting peptides described herein can be used to generate rAAVs with enhanced CNS tropism, wherein the capsid proteins are derived from different AAV serotypes (e.g., AAV9 and AAV1). In some embodiments, this can advantageously provide for the administration of two or more different AAV vector compositions without inducing an immune response in a subject.

[0249] The frequency of administration of the rAAV virus can vary. For example, the rAAV virus can be administered to the subject about once a week, about once every two weeks, about once a month, about once every six months, about once a year, about once every two years, about once every three years, about once every four years, about once every five years, about once every six years, about once every seven years, about once every eight years, about once every nine years, about once every ten years, or about once every fifteen years. In some embodiments, the rAAV virus is administered to the subject at most about once a week, at most about once every two weeks, at most about once a month, at most about once every six months, at most about once a year, at most about once every two years, at most about once every three years, at most about once every four years, at most about once every five years, at most about once every six years, at most about once every seven years, at most about once every eight years, at most about once every nine years, at most about once every ten years, or at most about once every fifteen years.

[0250] Disclosed herein are kits comprising compositions disclosed herein. Also disclosed herein are kits for treating or preventing diseases or conditions of the CNS, PNS, or target organs or environments (e.g., CNS). In some cases, the disease or condition is cancer, pathogen infection, nervous system disease, muscle disease, or immune disorder, such as those described herein. In one embodiment, the kit may include a therapeutic or preventive composition containing an effective amount of a composition of rAAV particles, the rAAV particles encapsulating nucleic acids provided herein and rAAV capsid proteins disclosed herein. In another embodiment, the kit may include a therapeutic or preventive composition containing an effective amount of cells ("modified cells") modified by rAAV as described herein in a unit dosage form expressing therapeutic nucleic acids. In some embodiments, the kit includes a sterile container that may contain a therapeutic composition; such containers may be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister packs, or other suitable container forms known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for accommodating drugs.

[0251] In some embodiments, the rAAV is provided with instructions for administering the rAAV to a subject having or at risk of developing a disease or condition. The instructions generally can include information regarding the use of the composition for treating or preventing a disease or condition.

[0252] The kit may include allogeneic cells. In some embodiments, the kit includes cells that may include genomic modifications. 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.

[0253] In some embodiments, the instructions include at least one of the following: a description of the therapeutic rAAV composition; dosage schedules and administration for treating or preventing a disease or condition disclosed herein; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. These instructions can be printed directly on the container (if present), or as a label applied to the container or as a separate sheet, brochure, card, or folder provided in or with 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 the 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 an additional therapeutic agent disclosed herein. In some cases, the instructions provide for the rAAV to be formulated for intravenous injection. In some cases, the instructions provide for the rAAV to be formulated for intranasal administration.

[0254] Examples

[0255] Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure in any way.

[0256] Example 1

[0257] Targets for achieving receptor-mediated control of therapeutic biodistribution and efficacy

[0258] This example describes a screening method for identifying binding partners that mediate the tropism of engineered AAVs.

[0259] A panel of six AAVs, including AAV9, AAV.CAP-Mac (also known as CAP-C1), AAV.CAP-B22, AAV-X1.1 (also known as X1B10), AAV.MaCPNS1 (also known as PNS1), and AAV.MaCPNS2 (also known as PNS2), was added to fixed HEK293 cells expressing 6019 human plasma membrane proteins, secreted proteins, and cell surface tether proteins in duplicate, as well as 397 human heterodimers. The AAVs were combined so that 3×10 4particles / cell of each of AAV9, AAV.CAP-Mac, AAV.CAP-B22, AAV.MaCPNS1, AAV.MaCPNS2, and 1×10 4 After adding AAV, the sample was fixed directly. Receptor-bound AAV was labeled first with an anti-AAV9 antibody and then with a secondary detection antibody.

[0260] Subsequently, 22 library hits were identified in a confirmation screen that also included various positive and negative controls and were probed with each AAV separately. The screening results are shown in Figure 5-Figure 11 middle.

[0261] Three novel targets were identified for AAV9: dipeptidyl peptidase 4 (DPP4), interleukin-3 (IL-3), and Dickkopf-related protein 3 (DKK3). Without being bound by any particular theory, the role of IL-3 in T cell activation and immune responses to pathogens provides a method for modulating the remarkable tolerogenic host immune response of AAV. Without being bound by any particular theory, the role of DKK3 as an inhibitor of WNT signaling provides a method for modulating the shuttling activity of DKK3 to WNT-expressing cells and tissues. The binding of endogenous secreted factors such as IL-3 and DKK3 may also function to hide viral particles in "self" to reduce the immune response.

[0262] AAV.CAP-B22's novel protein target is FAM234A. New targets specific for AAV-X1.1 include pancreatic secretory granule membrane major glycoprotein GP2 (GP2), low-density lipoprotein receptor-related protein 6 (LRP6), aminopeptidase N (ANPEP), granulocyte-macrophage colony-stimulating factor (CSF2), and epiphycan (EPYC). These proteins collectively contribute to the altered tropism of AAV-X1.1 relative to AAV9.

[0263] LRP6 is strongly expressed in the human BBB and is a target for AAV-X1.1 to enhance its efficacy in the CNS across species. AAV-X1.1 and CAP-Mac were found to bind strongly and directly to the human LRP6 extracellular domain by SPR, with CAP-Mac binding appearing to be more potent, whereas no such interaction was observed with AAV9 ( Figure 1 Computational modeling of the AAV-X1.1 inserted peptide to the extracellular domain of human LRP6 showed binding to domain 2, while the inserted peptide of CAP-Mac was shown to bind to either domain 1 or domain 2 ( Figure 2 Both engineered AAVs showed enhanced potency on human HEK293 cells (which endogenously express LRP6) compared to standard AAV9-based AAVs ( Figure 3 Knockout of endogenous LRP6 in HEK293 cells selectively reduced the efficacy of both AAV-X1.1 and CAP-Mac ( Figure 4 ).

[0264] These targets allow for target-based engineering of AAVs to modulate biodistribution, potency, and host immune interactions. These targets also allow for target-based engineering of therapeutic antibodies, peptides, Fabs, scFvs, nanobodies, and alternative protein scaffolds, as well as ASOs and small molecules, to obtain properties of native and engineered AAVs.

[0265] Table 1: Test and control samples

[0266]

[0267] method

[0268] Library screening

[0269] The direct fixation method (after adding the sample, without removing the AAV and fixing the slide) was used to screen the samples containing 3 × 10 4 particles / cell of CAP-C1, CAP-B22, PNS1, PNS2, and AAV9 (control AAV), as well as 1×10 4 Binding of a test AAV pool of X1B10 containing 100 particles / cell to fixed HEK293 cells / slide (18 slide sets, n=2 slides per slide set) expressing replicates of 6019 human plasma membrane proteins, secreted proteins, and cell surface-tethered human secreted proteins, as well as 397 human heterodimers. All transfection efficiencies exceeded the minimum threshold. Anti-AAV9 antibody (anti-adeno-associated virus 9, clone HL2372, provided by Merck, catalog number MABF2309-100UL, 1:500 dilution) was used, followed by AlexaFluor647 anti-mIgG H+L detection antibody. A total of 22 library hits (replicate spots) were identified by analyzing fluorescence (AF647 and ZsGreen1) on ImageQuant. Intensity (signal to background) ranged from very weak to moderate / strong.

[0270] Confirmation / Specificity Screening:

[0271] Vectors encoding all 22 hits, plus vectors encoding mouse SCA1 (LY6A) and KIAA0319L, and control vectors encoding CD86 and EGFR, were spotted in duplicate on new slides and used to reverse transfect human HEK293 cells. All transfection efficiencies exceeded the minimum threshold. Direct fixation was performed as described above (n = 2 slides per treatment, NB. Controls were also fixed after sample incubation). After fixation, 3 × 10 4 CAP-C1, CAP-B22, PNS1, PNS2, or AAV9 (control AAV) particles / cell, 1×10 4 Transfected HEK293 cells were treated with 0.5 μg / mL of X1B10 (0.5 μg / mL) or no test molecule (secondary only; negative control). The slides were analyzed as described above. Hits were classified as specific or nonspecific (e.g., positive or negative control also appeared).

[0272] Table 2: Dot pattern

[0273]

[0274]

[0275] In summary, the test AAVs CAP-C1, CAP-B22, PNS1, PNS2, AAV9 (control AAV), and X1B10 were screened for binding to human HEK293 cells expressing 6019 human plasma membrane proteins, secreted proteins, and cell surface-tethered secreted proteins, as well as 397 human heterodimers. CAP-C1 showed significant specific interactions with GP2, DPP4, IL3, and DKK3. CAP-B22 showed significant specific interactions with one of its primary targets, mouse SCA1 (LY6A), as well as with DPP4, FAM234A, IL3, and DKK3. X1B10 showed specific interactions with GP2, DPP4, LRP6, ANPEP, CSF2, IL3, DKK3, and EPYC. PNS1, PNS2, and AAV9 (control AAV) all showed significant specific interactions with DPP4, IL3, and DKK3.

[0276] Example 2

[0277] The human cell surface-AAV interactome identifies LRP6 as a blood-brain barrier transcytosis receptor and targets immune cells Identification of the cytokine IL3 as an AAV9 binder

[0278] Adeno-associated virus (AAV) is a fundamental gene delivery tool in basic science and clinical therapy. However, its application may be hindered by a lack of in-depth mechanistic understanding, particularly of engineered vectors generated by directed evolution. As described herein, an unbiased human cell microarray platform is suitable for determining the extracellular and cell surface interactomes of natural and engineered AAVs. Naturally evolved and serotype-specific interactions of AAV9 with human interleukin 3 (IL3) have been identified, which, without being bound by any particular theory, may play a role in the host immune response. In addition, specific interactions of laboratory-evolved low-density lipoprotein receptor-related protein 6 (LRP6) with engineered capsids have been identified, which can cross the blood-brain barrier after intravenous administration in non-human primates. The unbiased cell microarray screening method also allows the identification of off-target tissue binding interactions of engineered brain-enriched AAVs, thereby providing information on the peripheral organ tropism and side effects of the vector. These results enable the confident application of engineered AAVs in a variety of organisms and open up target information engineering for providing improved viral and non-viral vectors for non-invasive therapeutic delivery to the brain.

[0279] Adeno-associated virus (AAV) has become the gene delivery vector of choice both on the benchtop and in the clinic. Systemic administration of AAV allows non-invasive targeting, particularly of large or distributed biological structures, but access to the brain from the periphery is restricted by the blood-brain barrier (BBB), a complex biological structure that regulates the entry of molecules into the central nervous system (CNS). Systemic administration of AAV also exposes the vector to the host immune system and off-target tissues. Brain targeting efficiency after systemic administration of native serotypes is low, often requiring high doses that increase cost and may induce adverse side effects. Therefore, improved vectors are needed to fully realize the therapeutic potential of AAV gene therapy.

[0280] AAV capsid engineering, particularly through directed evolution approaches, has been shown to significantly improve its potency in desired cell types and tissues following systemic intravenous delivery. However, because AAV capsids are used across species, the enhanced tropism of many engineered vectors may vary. This is problematic for human clinical trials, as capsids developed in non-human species that perform poorly upon translation may not only fail to provide therapeutic benefit but may also render patients ineligible for future therapies by inducing neutralizing antibodies.

[0281] This translational challenge of AAV engineering through directed evolution also presents an opportunity to better understand the fundamental mechanisms of drug delivery to the brain. Directed evolution of engineered capsids with enhanced BBB-crossing capabilities provides a platform for investigating the most efficient pathways across this barrier. While recent advances have demonstrated that engineered AAVs can utilize multiple BBB-crossing receptors, the mechanisms of vector enhancement in the primate brain remain largely unexplored.

[0282] To address this challenge, as described herein, Retrogenix cellular microarrays of the human membrane proteome and secretome were adapted to screen for interactions between native and engineered AAV capsids and host cells. This allowed for rapid analysis of over 90% of the human membrane proteome and secretome, including key protein classes such as receptors, transporters, and cytokines. Using this broad, unbiased screen, several novel AAV interactions were identified that impacted host immune responses (binding of human interleukin-3 (IL3) to AAV9), enhanced cross-species BBB crossing (through binding of AAV9-X1.1 and CAP-Mac to low-density lipoprotein receptor-related protein 6 (LRP6)), and off-target tissue tropism (through binding of AAV9-X1.1 and CAP-Mac to glycoprotein 2 (GP2)). Understanding the mechanisms of action of systemic AAV, through methods such as those used here, will be crucial for successful vector translation and enable the design of improved vectors and other therapeutic protein forms for specific targets.

[0283] High-throughput screening of AAV binding partners

[0284] To screen for AAV-binding proteins, Retrogenix cell microarrays of the human membrane proteome and secretome were used, in which DNA oligonucleotides encoding human membrane and secretory proteins were immobilized at known locations on the slide ( Figure 12A HEK293 cells were then grown on slides and individually reverse transfected with the corresponding patterned oligonucleotides. AAVs that directly interact with a given protein will preferentially bind to cells expressing that protein; other slide locations define nonspecific background binding. To increase confidence in binding specificity, each protein was patterned at two different locations (four locations were presented during initial optimization) ( Figure 12B-12C Screening conditions were optimized for two different detection methods: biotin labeling and direct detection using previously identified AAV and interacting protein pairs (1) AAV9 with AAVR (KIAA0319L) and (2) PHP.eB with mouse LY6A ( Figure 12B-12C). Biotinylated capsids can be detected with fluorescent streptavidin, and unlabeled capsids can be detected with antibodies targeting epitopes distinct from those of the variable regions IV and VIII of the normally engineered capsids. In some embodiments, the level of capsid primary amine labeling must be adjusted so that surface modification does not interfere with key capsid binding interactions. The best signal-to-noise ratio (calculated as the mean intensity of the positive control spot compared to the mean intensity of the rest of the slide) for replicate spots has been found to be obtained by directly immobilizing cell-bound AAV without washing.

[0285] Next, direct capsid detection and validation conditions were investigated with a panel of AAV capsids, including AAV9 and five engineered AAV9 variants with enhanced potency in the CNS following systemic administration in nonhuman primates (NHPs) ( Tables 3 and Figure 12D Testing these capsids individually revealed that all, except MaCPNS1, exhibited detectable AAVR binding (the exception may be due to the geometry of the capsid variable region VIII insertion), whereas only CAP-B22 interacted with mouse LY6A (presumably through the PHP.eB loop in variable region VIII) ( Figure 12D and Figure 16 To enable higher throughput screening, the six capsids were tested as a pool. Pooled testing required additional dose optimization, first for the individual and then for the overall background binding levels of the included capsids (Table 5). The optimal dose was determined to minimize background binding while still distinguishing the unique interaction of CAP-B22 with mouse LY6A from the five non-LY6A interacting capsids ( Figure 12D ).

[0286] Following these controls, the six-capsid pool was tested in a comprehensive screen against approximately 6,400 proteins (including 6,000 human plasma membrane proteins, secreted and cell surface-tethered proteins), as well as approximately 400 heterodimers. Twenty-two library hits were identified in which each replicate showed a stronger signal than background. To assign these hits to specific capsids, a subsequent deconvolution screen was performed on each individual capsid in the pool ( Figure 12E ). DNA oligomers of the 22 identified hits, along with a positive control CD86, were affixed to new slides in duplicate positions. A negative control condition without AAV analyte and a positive control condition with CTLA4-Fc (CD86 binder) were also included. Hits that included both membrane-localized and secreted proteins were successfully assigned to capsids. Some of these interactions were unique to specific AAV9 variants, while others were common across all capsids tested (Table 4).

[0287] Validation of individual AAV binding interactions

[0288] To validate binders from the cell microarray screen, the hits were tested for their ability to enhance AAV potency in cell culture ( Figures 17A-17C ) and characterized the capsid binding interaction by surface plasmon resonance (SPR) ( Figure 13A and Figures 18A-18B This reduced the candidate receptors to a subset of validated interactors (Table 4). In analyzing these interactions, the identified interaction of AAV9 and all its laboratory-evolved derivatives with the human immunomodulatory protein interleukin-3 (IL3) was particularly striking because AAV is relatively well tolerated by the immune system. IL3 is produced by activated T cells as part of the inflammatory response to viral infection, thereby triggering the expansion of various immune cells and activating plasmacytoid dendritic cells that secrete type I interferons. Using SPR, AAV9 was found to bind to human IL3, but not to the closely related natural serotypes AAV8 and AAVrh10 ( Figure 13B ). IL3 from different species was next tested, and AAV9 was found to bind to human and macaque IL3 (83% sequence identity), but not to marmoset or mouse IL3 (69% and 27% sequence identity, respectively) ( Figure 13B ), which suggests that there are differences between New World monkeys and Old World monkeys.

[0289] To understand the species and serotype specificity of the IL3-AAV9 interaction, the structure of the binding complex was investigated. Since functional AAV ligands are likely to have weak and dynamic monomeric interactions, avidity was exploited by flowing 60-mer AAV9 capsids over dimeric IL3-Fc captured by Protein A to detect all biologically significant interactions. Although high avidity appeared in SPR experiments, the apparent affinity of this interaction was only consistent with a high nM interaction. Therefore, IL3-bound AAV9 was chemically cross-linked followed by mass spectrometry (XL-MS). Using bis(sulfosuccinimidyl) suberate (BS3) cross-linker, two cross-links between the proteins were detected ( Figure 13D and Figures 19A-19B These crosslinks placed IL3 at the base of the 3-fold spike and in the 2-fold valley, but no binding model could be generated.

[0290] We then assessed whether validated AAV interactors could potentially explain the enhanced brain tropism of the engineered capsids. Screening hits were ranked based on their expression levels in endothelial cells of the human BBB, noting the specific interaction of low-density lipoprotein receptor-related protein 6 (LRP6) with AAV9-X1.1. Figure 14A). This capsid protein showed enhanced brain endothelial-specific tropism in mice, which shifted to enhanced neuronal tropism in macaques (Table 3). Although AAV9-X1.1 contains modifications of AAV9 in both variable regions IV and VIII (Table 3), the tropism of AAV9-X1.1 can be shifted to other native serotypes, such as AAV1 and AAV-DJ, simply by transferring the variable region VIII insertion of AAV9-X1.1. By SPR, it was confirmed that the X1 peptide inserted in the variable region VIII confers the ability of AAV1-X1 and AAVDJ-X1, but not the unmodified parental serotype, to bind to LRP6 ( Figure 20A This suggests that the functional form of the X1 peptide in different AAV serotypes in vivo corresponds to the LRP6-binding form.

[0291] LRP6 is a core receptor in the canonical Wnt signaling pathway and plays a developmental and homeostatic role in many tissues. The high LRP6 sequence conservation across species (98% and 99.5% sequence identity between human LRP6 and mouse or macaque LRP6, respectively) was compared to AAV9, which has enhanced tropism compared to AAV9-X1.1 in rodents and primates. A similar enhancement in tropism across species was also observed for CAP-Mac (Table 3), which was engineered in marmosets and had enhanced neuronal tropism in macaques compared to the enhanced endothelial tropism of AAV9 in marmosets. Therefore, the interaction of CAP-Mac with the extracellular domain of human LRP6 was also tested by SPR ( Figure 14B As with IL3, avidity was used to ensure capture of weak but functionally important interactions. Unlike its parental capsid, AAV9, which has sub-nM apparent affinity, both AAV9-X1.1 and CAP-Mac strongly bound to human LRP6-Fc.

[0292] LRP6 has many endogenous WNT signaling partners, with binding sites spanning extracellular YWTD domains 1 and 2 (E1E2) or domains 3 and 4 (E3E4). AlphaFold-Multimer was applied to construct AAV interaction complex models, which predicted that X1 and CAP-Mac variable region VIII peptides bind to LRP6 YWTD domain 1 ( Figure 14C Although the cooperative folding of E1 and E2 complicates testing of the individual domains, SPR results with mouse LRP6 extracellular domain fragments were consistent with model predictions, where an LRP6-E1E2 but not an LRP6-E3E4 interaction was observed ( Figure 14DAAV-BI30, another engineered capsid specifically expressed in mouse brain endothelium, was also tested (Table 3) and found to also bind to LRP6-E1E2, but not LRP6-E3E4 ( Figure 20B Pull-down assays confirmed that both AAV9-X1.1 and CAP-Mac bound to the full-length extracellular domain of mouse LRP6, but not to the closely related full-length extracellular domain of LRP5 ( Figure 21 On the other hand, AAV9 only binds to PKD2 of AAVR, which has been reported previously.

[0293] AAV9-X1.1 and CAP-Mac potently infected HEK293 cells, with AAV9-X1.1 being more potent ( Figure 22B To determine whether this potency was mediated by endogenous LRP6 expression in HEK293 cells, the vector was tested with LRP6 inhibitors. Mesoderm development LRP partner (Mesd), a natural endoplasmic reticulum chaperone and recombinant extracellular inhibitor of LRP5 and LRP6, and sclerostin (SOST), which inhibits LRP6 by binding only to E1E2, significantly reduced the potency of AAV9-X1.1 ( Figures 22A-22B Importantly, neither Mesd nor SOST inhibited the potency of PHP.eB in overexpressing LY6A cells. Transient overexpression of human LRP6 enhanced the potency of both capsids, with CAP-Mac being the more potent ( Figure 22B This effect was largely maintained when the truncated LRP6-E1E2 was used. As expected from the pull-down assay, transient overexpression of LRP5 did not enhance the efficacy of CAP-Mac or AAV9-X1.1. Together, these results support a specific functional interaction between LRP6 and both CAP-Mac and AAV9-X1.1, although the two capsids may have different functional sensitivities to LRP6 expression levels. While AAV9-X1.1 effectively binds LRP6 at low endogenous expression levels of LRP6, CAP-Mac exhibits enhanced efficacy following transient overexpression of LRP6.

[0294] Notably, in addition to the expected CNS tropism acquired through these AAV capsid engineering efforts, the two LRP6-binding engineered capsids also acquired interactions with the GPI-linked protein glycoprotein 2 (GP2), which is specifically expressed in the pancreas and exerts antimicrobial effects in a secreted form in the intestine ( Figure 17A 、 Figures 18A-18B and Table 4). In cell culture, GP2 enhanced the potency of both capsids, with the effect being stronger for the human protein than for the mouse ( Figure 17A ).

[0295] FAM234A, which binds CAP-B22 in a cell microarray screen, was found to be expressed at low levels across many neuronal types in the brain. Although FAM234A has been identified in disease association studies, a specific molecular function has not yet been assigned to this protein. In cell culture, FAM234A enhanced the efficacy of both CAP-B22 and PHP.eB, with the mouse receptor showing a stronger effect than the human protein. Figure 17B This suggests that the interaction is driven by the capsid consensus variable region VIII insertion sequence (Table 3).

[0296] Engineered AAVs exploit LRP6 at the blood-brain barrier in mice and primates

[0297] Repeated administration of the same serotype is complicated by the production of host neutralizing antibodies in response to previous AAV exposure. Serotype-switched XI vectors, such as AAV1-X1, have been shown to be amenable to secondary systemic administration to mice previously exposed to AAV9-based vectors. This property was exploited to determine the in vivo effects of the interaction of AAV9-X1.1 with LRP6 ( Figure 15A ). AAV1-X1 packaged with mCherry targeting the brain endothelium was systemically administered to Lrp6 Cre conditional knockout mice as a control or Cre recombinase. Three weeks later, AAV9-based PHP.eB or AAV9-X1.1 packaged with eGFP were systemically delivered. Regardless of the AAV1-X1 cargo, PHP.eB showed characteristically strong brain transduction, while in AAV1-X1-transfected cells of Lrp6 knockout mice administered with AAV1-X1, the brain endothelial tropism of AAV9-X1.1 was significantly reduced ( Figure 15B-15C ), confirming the necessity of LRP6 for capsid BBB entry in vivo. In the liver, which also expresses LRP6, AAV9-X1.1 capsids showed enhanced potency compared to PHP.eB ( Figure 23A Under Lrp6 knockout conditions, decreased liver transduction by AAV9-X1.1 was also observed ( Figure 15B-15C ).

[0298] To confirm that LRP6 interaction mediates the brain efficacy of AAV9-X1.1 in primates, the vector was tested on cultured macaque and human primary brain microvascular endothelial cells (PBMECs) ( Figure 15D-15E ). In PBMECs from both species, AAV9-X1.1 was significantly more potent than its parental AAV9. On the other hand, the LRP6 inhibitor Mesd selectively reduced the potency of AAV9-X1.1 in PBMECs to the level of AAV9. A similar LRP6-dependent enhancement of the potency of AAV1-X1 compared to AAV1 was also observed in human PBMECs ( Figure 23B The similarity of the responses of AAV1-X1 and AAV9-X1.1 supports the SPR experiments ( Figure 20A ), indicating that the X1 peptide is necessary and sufficient for targeting the capsid to the BBB through its interaction with LRP6.

[0299] Recent advances in capsid engineering have enabled AAV vectors to more efficiently cross the blood-brain barrier (BBB) in rodents and non-human primates after systemic administration, but the limited understanding of the mechanisms of transcytosis (particularly in humans) has hindered the predictable translation and additional rational design of these and other non-viral BBB-crossing molecules. This translation challenge also provides an opportunity to better understand the biology of the BBB and AAV vectors. To date, only a few targets, such as the transferrin receptor, have been used for research or therapy. This article discloses a pipeline for finding homologous receptors for engineered AAV, focusing on the human membrane proteome and secretome. This disclosure validates the utility of cell microarray screening in identifying natural and engineered AAV receptors. This article identifies LRP6 as a novel and highly conserved target for blood-brain barrier transcytosis by AAV9-X1.1, a potent engineered capsid in primate neurons, and human IL3 as an interacting partner of AAV9. These findings enable the use of identified receptors for targeted drug delivery across multiple therapeutic modalities such as small molecules, antibodies, or oligonucleotides.

[0300] The safety and immune tolerance of delivery vectors are key considerations for AAV entry into the clinic, as adverse reactions may occur. The immunomodulatory potential of the IL3-AAV9 interaction may include providing host neutralization mechanisms, or stealth mechanisms in which AAV evades the immune system or uses decoy receptors to weaken its response. In addition to activated T cells, IL3 is also continuously secreted by astrocytes in the brain to reprogram microglia and combat Alzheimer's disease. Therefore, AAV9 interacts with IL3, which is shared by all AAV9-based engineered capsids with enhanced BBB crossing, and in some embodiments, may affect processes beyond the immune tolerance of the vector itself in the context of healthy and diseased brain. Importantly, AAV9 isolates from human clinical tissues bind to human and macaque (83% AA identity), but not to marmoset or mouse IL3 (69% and 27% AA identity, respectively). Without being bound by any particular theory, this species-dependent interaction may decouple the safety profiles of rodent and primate AAVs, particularly in the context of neurodegeneration.

[0301] Without being bound by any particular theory, the high sequence conservation of LRP6 (98% AA identity between mouse and human) may explain the enhanced tropism of AAV capsids targeting this receptor across species.

[0302] Both AAV9-X1.1 and CAP-Mac also showed binding to GP2 (not present in the CNS), suggesting that, without being bound by any particular theory, this interaction may have piggybacked on the functional enhancement provided by LRP6 binding during directed evolution selection. This is also supported by the finding that AAV interacts more strongly with human GP2 than with the mouse protein that emerged during the directed evolution of AAV9-X1.1. These findings highlight the importance of extensive unbiased interaction screening, as disclosed herein, for constructing a comprehensive safety profile of engineered capsids prior to clinical trials.

[0303] Studying the diversity of mechanisms by which natural and engineered AAVs cross the BBB can also prepare for defense against future pathogens. Just as antibiotic resistance is testing the modern world, a concern is that rapidly evolving pathogens will develop “BBB resistance”—the ability to enter the brain and cause severe disease (as some retroviruses, including HIV-1, have already done). A recent worrying example is the discovery of SARS-CoV-2 capsid protein in the brains of patients with long COVID and associated with neuropsychiatric symptoms. By screening existing pathogens and their possible molecular evolution against the growing catalog of human BBB transcytosis receptors, including transferrin receptor, insulin receptor, CD98hc, CA4, and LRP6 disclosed in this invention, we can anticipate outbreaks of pathogens that could cause neuropsychiatric sequelae.

[0304] This article provides methods for efficiently screening natural AAV serotypes and engineered variants against the human proteome. This disclosure expands the limited list of targets for enhanced BBB crossing in primates. These findings suggest new strategies for the successful clinical translation of engineered AAV, provide targets for the development of non-viral therapeutic modalities, and highlight potential vulnerabilities of future pathogens.

[0305] Table 3: Capsid engineering details and in vivo tropism of vectors used in this study

[0306]

[0307] Table 4: Summary of identified AAV interactions. AAV9 hits were observed in all engineered derivatives.

[0308]

[0309] Table 5: Doses of individual vectors within the pool to optimize signal-to-noise ratio

[0310]

[0311] Table 6 below shows the amino acid sequences of exemplary targets of AAV identified herein. Table 7 shows the binding site residues on human LRP6.

[0312] Table 6: Amino acid sequences of exemplary targets identified

[0313]

[0314]

[0315]

[0316]

[0317]

[0318] Table 7: Human LRP6 E1 domain binding site residues

[0319]

[0320] method

[0321] Viral vector production

[0322] Briefly, HEK293 cells were triple transfected with capsid, genome, and helper plasmids. The medium was exchanged the next day, then harvested and replaced two days later. Five days after transfection, the medium and cells were harvested and processed for AAV purification. The cells were lysed in a high salt solution and treated with salt-activated nuclease. The culture medium was PEG-precipitated and resuspended in a salt-activated nuclease solution. Both solutions were added to an iodixanol density column, ultracentrifuged, and extracted from the 40% / 60% interface. Finally, the AAV was buffer-exchanged, concentrated, titrated, and assayed for endotoxins (for vectors destined for non-human primates) using the Piece LAL Chromogenic Endotoxin Kit (Cat. No. A39552).

[0323] Retrogenix Cell Microarray

[0324] Retrogenix cell microarrays were performed for AAV analytes as described below. Prescreening optimization was performed on HEK293 cells and slides of cells overexpressing mouse LY6A and human AAVR (KIAA0319L), TGFBR2, and EGFR. Transfection efficiency was verified to exceed the minimum threshold before analyte application. 6 × 10 cells per HEK293 cell were used. 4 AAV was added to the fixed cells at a concentration of 100 AAV particles.

[0325] By adding 1 × 10 13 Biotinylated AAV was generated by incubating AAV with a 10,000-fold molar ratio of NHS-PEG4-biotin (Thermo A39259) at room temperature for 2 hours. The reaction was quenched with 1M Tris, pH 8, before buffer exchange, concentration, and AAV re-titration. Biotinylated AAV was detected on fixed HEK293 cells by AF647-labeled streptavidin. Unlabeled AAV on fixed HEK293 cells was detected by anti-AAV9 clone HL2372 (Merck, MABF2309-100UL) at a dilution of 1:500, followed by detection with AF647-labeled anti-mIgG H+L.

[0326] To achieve an appropriate signal-to-noise ratio (which is necessary to minimize false positives and false negatives), untagged AAV was screened individually and as a pool using anti-AAV9 detection at various concentrations. The final test pool was screened against fixed HEK293 cells / slide expressing approximately 6,000 human plasma membrane proteins, secreted proteins, and cell surface-tethered human secreted proteins, as well as approximately 400 human heterodimers (each in duplicate). Hits were identified using ImageQuant as spots observed in duplicate. After screening, 22 identified hits and a CD86 positive control protein were spotted on new slides for individual AAV testing in deconvolution screening. A negative control condition containing no analyte and a positive control condition with CTLA4-Fc (which interacts with CD86) were also included.

[0327] Protein preparation

[0328] Lyophilized mouse LRP6 (AA20-1366) was labeled with a 6xHis tag, and the N-terminal (E1E2) and C-terminal half (E3E4) fragments of mouse LRP6 (N-half: AA 20-628, C-half: AA 629-1244) were labeled with Fc (mouse IgG 2a), and full-length human LRP6 (AA 20-1368) was labeled with Fc (IgG1), and LRP5 (AA1-1383) was labeled with 6xHis tag, and SOST protein was purchased from Bio-Techne (catalog numbers 2960-LR-025, 9950-LR-050, 9954-LR-050, 1505-LR-025, 7344-LR-025 / CF, 1406-ST). Mesd protein was purchased from SinoBiological (catalog number 10949-H08H). All proteins were mixed in Dulbecco's phosphate buffered saline (DPBS, Gibco) at the desired concentration before use. TM ) in the refactoring.

[0329] Interleukin-3 proteins from human (AA 1-152), mouse (AA1-166), marmoset (AA1-143), and macaque (AA1-144) were triple-labeled with Fc-Myc-6xHis, human and mouse GP2 (hGP2: AA 1-518, mGP2: AA 1-515) were triple-labeled with Fc-Myc-6xHis, and human and mouse DKK3 (hDKK3: AA 1-350, mDKK3: AA 1-349) were triple-labeled with Fc-Myc-6xHis using ExpiFectamine according to the manufacturer's manual. TM (Thermo Fisher Scientific) was used at 3 × 10 6 The cells were transfected into Expi293F at a density of 10 viable cells / ml. TM (Thermo Fisher Scientific) cells, and after 120 hours, the secreted proteins in the culture medium were collected and cleared using a 0.45 μm PVDF vacuum filter (Sigma Millipore). Each His-tagged protein in the culture medium was captured using Ni-NTA resin (Qiagen) and eluted with DPBS containing 150 mM imidazole.

[0330] The human adeno-associated virus receptor (AAVR) PKD2 domain (AA 401-498) tagged with 6xHis was purified using methods known in the art. Briefly, PKD2 was expressed in BL21(DE3)-RIPL E. coli. The cells were lysed by ultrasound and the insoluble fraction was removed by centrifugation. The cleared lysate was applied to a Ni-NTA column (Qiagen) and eluted with DPBS containing 250 mM imidazole.

[0331] Surface plasmon resonance (SPR)

[0332] A Sierra SPR-32 (Bruker) loaded with a Protein A sensor chip was used. Fc fusion proteins in HBS-P+ buffer (GE Healthcare) were immobilized at the following capture levels: for 13B- Figure 13C 、 Figure 14D 、 Figures 18A-18B and Figures 20A-20D , 600-800 response units (RU), and for Figure 14B , 1200-1500 RU. AAV was injected at a flow rate of 10 μL per minute for 240 seconds, followed by a 600-second dissociation. AAV concentrations ranged from 2.4 × 10 per mL to 12 vg and were performed at 2-fold dilution intervals. A regeneration step with 10 mM glycine, pH 1.5 was performed between each cycle. All kinetic data were double referenced.

[0333] Pull-down assay

[0334] Pull-down assays were typically performed as described below. Briefly, the capture AAV was mixed with His-tagged bait protein and Ni-NTA resin in a binding buffer of DPBS containing 20 mM imidazole and stirred on an orbital mixer at 4 ° C for 1 hour. The resin was then collected with a spin column, washed twice with 10 column volumes of binding buffer, and eluted with 45 μL of DPBS containing 150 mM imidazole. Western blot analysis was performed on the eluate using anti-VP1 / VP2 / VP3 (ARP, catalog number 03-61058) and anti-6xHis (Abcam, catalog number abl8184) antibodies.

[0335] HEK293 cell culture potency assay

[0336] HEK293T cells in Dulbecco's Modified Eagle Medium (DMEM) containing 5% fetal bovine serum (FBS), 1% non-essential amino acids (NEAA) and 100U of penicillin-streptomycin per mL were cultured in 6-well plates at 37° C. under 5% CO . At 80% confluence, cells were transiently transfected with 2.53 μg of plasmid DNA encoding membrane protein hits from the Retrogenix cell microarray screen. Cells were transferred to 96-well plates at 20% confluence and incubated in FluoroBrite supplemented with 0.5% FBS, 1% NEAA, 100U of penicillin-streptomycin per mL, 1xGlutaMAX, and 15 μM HEPES. TMMaintained in DMEM. Plates were imaged 24 hours after AAV application on a Keyence BZ-X700 (4x objective). For experiments with protein inhibitors, Mesd (26 ug / ml) and SOST (0.2 μg / ml) were added 4 hours before AAV addition. NucBlue TM Live ReadyProbes TM Reagent (Hoechst 33342) was added to each well to aid autofocus. Image quantification was performed using a custom Python image processing pipeline.

[0337] Primary cell culture potency assay

[0338] Human brain microvascular endothelial cells (ScienCell Research Laboratories, catalog number 1000) and cynomolgus monkey primary brain microvascular endothelial cells (CellBiologics, catalog number MK-6023) were cultured according to the instructions provided by the supplier. 4 Cell cultures were treated with viral vectors packaged with the single-stranded AAV genome CAG-eGFP at a multiplicity of infection (MOI) of 1:1 (4 wells per vector). One day after the infection procedure, the fluorescence expression of the cultures was examined and quantified.

[0339] animal

[0340] Adult (6-8 weeks old) homozygous B6;129S-Lrp6tml.lVari / J mice (Jackson Labs, No. 026267) were retro-orbitally administered with 1×10 12 vg of AAV1-X1 containing Ef1a-mCherry or Ef1A-Cre (N=6 in each case). After 3 weeks, these mice were re-administered with 1×10 12 vg of PHP.eB or AAV-X1.1 packaged with CAG-eGFP (N = 3 for each condition). Mice were randomly assigned to specific AAV conditions. The experimenters were not blinded to any experiments in this study.

[0341] Lrp6 conditional knockout tissue preparation and imaging

[0342] Mice were anesthetized with Euthasol (pentobarbital sodium and phenytoin sodium solution, Virbac AH) and perfused transcardially with approximately 50 mL of 0.1 M PBS with a pH of 7.4, followed by an equal volume of 0.1 M PBS containing 4% paraformaldehyde (PFA). Collected organs were fixed overnight at 4°C in 4% PFA, cleaned, and stored in 0.1 M PBS with 0.05% sodium azide at 4°C. Leica VT1200 vibrating microtome was used to prepare 100 μm brain slices, which were imaged using a Plan-Apochromat 10x 0.45M27 (working distance, 2.0 mm) objective lens on a Zeiss LSM 880 confocal microscope. Images were analyzed using Zen Black 2.3SP1 (Zeiss) and ImageJ.

[0343] AlphaFold structural modeling

[0344] The structure of the complex of LRP6 ECD and AAV-X1 or AAV.CAP-Mac VR-VIII peptide was modeled using the cloud-based implementation of AlphaFold-Multimer-v3 provided in ColabFold v2.3.5. The input contained two sequences: the surface-exposed residues of VR-VIII of AAV-X1 (587-AQGNNTRSVAQAQTG-594, SEQ ID NO: 35) or AAV-CAP-Mac (587-AQLNTTKPIAQAQTG-594, SEQ ID NO: 36) and the extracellular domain of human LRP6 (UniProt entry 075581, residues 20-1370). Google Lab Notebook was run using an A100 SXM4 40GB GPU. Five structural models were generated using a protocol with up to 20 recycles, and MSAs were generated using MMseqs2 (UniRef+Environmental) and templates from PDB70. Structural models were ranked using a weighted combination of pTM and iPTM scores.

[0345] In at least some of the foregoing embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, unless such replacement is technically infeasible. It will be understood by those skilled in the art that various other omissions, additions, and modifications may be made to the above-described methods and structures without departing from the scope of the claimed subject matter. All such modifications and alterations are intended to fall within the scope of the subject matter defined by the appended claims.

[0346] With respect to the use of substantially any plural and / or singular terms herein, one skilled in the art can convert the plural to the singular and / or the singular to the plural where appropriate to the context and / or application. For clarity, various singular / plural permutations may be explicitly listed herein. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Any reference herein to "or" is intended to encompass "and / or" unless otherwise indicated.

[0347] Those of ordinary skill in the art will understand that, in general, the terms used herein, and particularly in the appended claims (e.g., the bodies of the appended claims), are generally intended to be “open-ended” terms (e.g., the term “including” should be read as “including but not limited to,” the term “having” should be read as “having at least,” the term “includes” should be read as “including but not limited to,” etc.). Those of ordinary skill in the art will further understand that if a specific number of an introduced claim recitation is intended, such intent will be expressly recited in the claim, and in the absence of such recitation, no such intent is present. For example, to aid understanding, the following appended claims may contain use of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed as implying that the introduction of a claim recitation by the indefinite article "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrase "one or more" or "at least one" and the indefinite article "a" or "an" (e.g., "a" and / or "an" should be interpreted as meaning "one or more" or "at least one"); the same is true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is expressly recited, one skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the direct recitation of "two recitations" without other modifiers means at least two recitations or two or more recitations). Furthermore, in those instances where a convention similar to “at least one of A, B, and C, etc.” is used, generally speaking, such convention is intended to be used in the sense that one skilled in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention similar to “at least one of A, B, or C, etc.” is used, generally speaking, such convention is intended to be used in the sense that one skilled in the art would understand the convention (e.g., “a system having at least one of A, B, or C” would include, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art will further understand that, in fact, any transitional words and / or phrases presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one, either, or both of the terms.

[0348] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0349] As will be understood by those skilled in the art, for any and all purposes, such as providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations thereof. Any listed range can be readily identified as fully describing and allowing the same range to be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily decomposed into a lower third, a middle third, and an upper third, etc. As will be understood by those skilled in the art, all language such as "at most," "at least," "greater than," "less than," etc. includes the referenced number and refers to a range that can be subsequently decomposed into the subranges discussed above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to a group having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to a group having 1, 2, 3, 4, or 5 articles, etc.

[0350] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. A method for increasing the permeability of the blood-brain barrier, the method comprising: Provided are targeting peptides capable of binding to low-density lipoprotein receptor-related protein 6 (LRP6), thereby increasing the permeability of the blood-brain barrier.

2. The method according to claim 1, wherein the targeting peptide binds to YWTD domain 1 and / or domain 2 of LRP6.

3. The method of any one of claims 1 to 2, wherein the permeability of the blood-brain barrier is increased by at least 25%, 50%, 75%, 100% or more as compared to the absence of the targeting peptide.

4. A method of delivering a payload to the nervous system of a subject, the method comprising: Providing a targeting peptide or a derivative thereof capable of binding to low-density lipoprotein receptor-related protein 6 (LRP6), 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 The delivery system is administered to the subject.

5. The method of claim 4, wherein the delivery system comprises nanoparticles, nanotubes, nanowires, dendrimers, liposomes, ethosomes and aquasomes, polymersomes and niosomes, foams, hydrogels, cubes, quantum dots, exosomes, macrophages, and any combination thereof.

6. The method of claim 4, wherein the delivery system comprises a viral vector or a non-viral vector. 7 . The method according to claim 6 , wherein the targeting peptide enhances the binding affinity of the viral vector or the non-viral vector to LRP6.

8. The method of any one of claims 6 to 7, wherein the viral vector comprises an AAV vector; and optionally wherein the targeting peptide is part of a capsid protein of the AAV vector.

9. The method of any one of claims 6 to 8, wherein 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.

10. The method of any one of claims 6 to 8, wherein the non-viral vector comprises lipid-based nanoparticles, polymeric nanoparticles, inorganic nanoparticles, surfactant-based emulsions, nanowires, silica nanoparticles, peptide- or protein-based particles, lipid-polymer particles, nanolipoprotein particles, and combinations thereof.

11. The method of any one of claims 4 to 10, wherein the payload to be delivered to the nervous system is a biomolecule, a non-biological molecule, or a combination thereof; and optionally wherein the biomolecule is selected from the group consisting of: a nucleic acid sequence, a protein, a peptide, a lipid, a polysaccharide, and any combination thereof.

12. The method of any one of claims 4 to 11, wherein the payload is a therapeutic molecule.

13. The method according to any one of claims 11 to 12, wherein the nucleic acid sequence to be delivered to the nervous system comprises one or more of: a) sequences encoding trophic factors, growth factors or other soluble factors that may be released from the transduced cells and affect the survival or function of the cells and / or surrounding cells; b) DNA that restores protein function to humans or animals carrying a genetic mutation in a gene; c) DNA encoding a protein that can be used to control or alter the activity or state of a cell; d) DNA encoding a protein or nucleic acid for evaluating the status of a cell; e) DNA and / or related guide RNA for genome engineering; f) sequences for genome editing by homologous recombination; g) a DNA sequence encoding a therapeutic RNA; h) shRNA or artificial miRNA delivery systems; or i) DNA sequences that affect the splicing of endogenous genes.

14. The method of any one of claims 1 to 13, wherein the LRP6 is mouse LRP6; and optionally wherein the LRP6 has an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

29.

15. The method of any one of claims 1 to 13, wherein the LRP6 is cynomolgus monkey LRP6; and optionally wherein the LRP6 has an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

30.

16. The method of any one of claims 1 to 13, wherein the LRP6 is human LRP6; and optionally wherein the LRP6 has an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

31.

17. The method of any one of claims 1 to 16, wherein upon binding, the targeting peptide is capable of interacting with: (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having an amino acid sequence of SEQ ID NO: 31; or (2) one or more positions functionally equivalent to S96, S114, E115, R141, W157, W183, or W242 in LRP6 having an amino acid sequence of SEQ ID NO:

31.

18. The method according to any one of claims 7 to 17, wherein the targeting peptide is inserted between two adjacent amino acids in AA587-594 of SEQ ID NO: 11 of the AAV9 vector, or between functional equivalents of AA587-594 in an amino acid sequence that is at least 80% identical to SEQ ID NO:

11.

19. The method according to any one of claims 7 to 18, wherein the targeting peptide is inserted between AA588-589 of SEQ ID NO: 11 of the AAV9 vector, or between functional equivalents of AA588-589 in an amino acid sequence that is at least 80% identical to SEQ ID NO:

11.

20. The method of any one of claims 7 to 19, wherein the AAV vector is conjugated to a nanoparticle, a second molecule, or a combination thereof.

21. The method of any one of claims 4 to 20, wherein the administration is systemic administration; and optionally wherein the administration is intravenous administration or intrathecal administration.

22. The method of any one of claims 4 to 21, wherein the subject is a mammal; and optionally wherein the subject is a human.

23. The method of any one of claims 4 to 22, wherein the subject is a subject having or at risk for one or more of: chronic pain, Friedreich's ataxia, Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), spinal muscular atrophy type I and II (SMAI and II), Friedreich's ataxia (FA), spinocerebellar ataxia, multiple sclerosis (MS), chronic traumatic encephalopathy (CTE), HIV-1 associated dementia, or a lysosomal storage disease involving cells within the CNS; and optionally wherein the lysosomal storage disease involving cells within the CNS is Krabbe disease, Sandhoff disease, Tay-Sachs disease, Gaucher disease, or disease (type I, II, or III), Niemann-Pick disease (NPC1 or NPC2 deficiency), Hurler syndrome, Pompe disease, or Batten disease.

24. The method of any one of claims 4 to 23, wherein the subject is a subject suffering from, at risk of suffering from, or has suffered from a stroke, traumatic brain injury, epilepsy, or spinal cord injury.

25. An adeno-associated virus (AAV) capsid protein comprising a targeting peptide having binding specificity for LRP6.

26. The AAV capsid protein of claim 25, wherein the targeting peptide is part of the capsid protein of the rAAV vector.

27. The AAV capsid protein according to any one of claims 25 to 26, wherein the targeting peptide is inserted between two adjacent amino acids in AA587-594 of SEQ ID NO: 11, or between functional equivalents of AA587-594 in an amino acid sequence that is at least 80% identical to SEQ ID NO:

11.

28. The AAV capsid protein of any one of claims 25 to 27, wherein when bound to LRP6, the targeting peptide is capable of interacting with: (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having an amino acid sequence of SEQ ID NO: 31; or (2) one or more positions functionally equivalent to S96, S114, E115, R141, W157, W183, or W242 in LRP6 having an amino acid sequence of SEQ ID NO:

31.

29. The AAV capsid protein of any one of claims 25 to 28, wherein AAV 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.

30. A recombinant adeno-associated virus (rAAV) comprising the AAV capsid protein according to any one of claims 25 to 29.

31. A recombinant adeno-associated virus (rAAV), comprising an AAV capsid protein, wherein the AAV capsid protein comprises a targeting peptide with binding specificity to low-density lipoprotein receptor-related protein 6 (LRP6), wherein the amino acid sequence of the targeting peptide is inserted between two adjacent amino acids in AA587-594 of the AAV capsid protein or their functional equivalents.

32. The rAAV of any one of claims 30 to 31 , wherein the two adjacent amino acids are AA588 and AA589.

33. The rAAV of any one of claims 31 to 32, wherein when bound to LRP6, the targeting peptide is capable of interacting with: (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having an amino acid sequence of SEQ ID NO: 31; or (2) one or more positions functionally equivalent to S96, S114, E115, R141, W157, W183, or W242 in LRP6 having an amino acid sequence of SEQ ID NO:

31.

34. The rAAV of any one of claims 30 to 33, wherein the rAAV has enhanced tropism for the nervous system relative to an rAAV not comprising the targeting peptide.

35. The rAAV of claim 34, wherein the rAAV is capable of transducing the nervous system with an efficiency at least 2-fold greater than the transduction efficiency of a rAAV not comprising the targeting peptide.

36. A composition for delivering an agent to the nervous system of a subject in need thereof, the composition comprising an AAV comprising (1) an AAV capsid protein according to any one of claims 25 to 29 and (2) an 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.

37. The composition for use of claim 36, wherein the nervous system is brain endothelial cells, neurons, capillaries in the brain, arterioles in the brain, arteries in the brain, or combinations thereof.

38. A composition for use according to any one of claims 36 to 37, wherein the composition is a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers.

39. The composition for use of any one of claims 36 to 38, wherein the agent to be delivered comprises a nucleic acid, a peptide, a small molecule, an aptamer, or a combination thereof.

40. An antibody or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence that has binding specificity for LRP6.

41. The antibody or fragment thereof of claim 40, wherein the antibody or fragment thereof is a bispecific antibody comprising at least one Fab specific for LRP6.

42. An antibody conjugate comprising the antibody or fragment thereof according to any one of claims 40 to 41, wherein the antibody conjugate further comprises a therapeutic agent or a detectable label.

43. A peptide or derivative or conjugate thereof which is specific for low-density lipoprotein receptor-related protein 6 (LRP6).

44. A nucleic acid comprising a sequence encoding the antibody or fragment thereof according to any one of claims 40 to 42 or the peptide or derivative or conjugate thereof according to claim 43.

45. A delivery system comprising (1) a targeting peptide specific for low-density lipoprotein receptor-related protein 6 (LRP6); and (2) a pharmaceutical agent.

46. The delivery system of claim 45, wherein the targeting peptide is (1) displayed on the surface of the delivery system; or (2) partially embedded in the delivery system.

47. The delivery system of any one of claims 45 to 46, wherein the delivery system is selected from the group consisting of nanoparticles, nanotubes, nanowires, dendrimers, liposomes, ethosomes and aquasomes, polymersomes and non-ionic surfactant vesicles, foams, hydrogels, cubes, quantum dots, exosomes, macrophages, and combinations thereof.

48. The delivery system of any one of claims 45 to 47, wherein the delivery system comprises a viral vector or a non-viral vector.

49. according to the delivery system described in any one in claim 45 to 47, wherein said delivery system comprises the nanoparticle 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.

50. A method for designing a targeting peptide specific for low-density lipoprotein receptor-related protein 6 (LRP6), the method comprising: One or more targeting peptides are generated by computer simulation, each of which is capable of interacting with: (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202 or H226 in LRP6 having an amino acid sequence of SEQ ID NO: 31; or (2) one or more positions functionally equivalent to S96, S114, E115, R141, W157, W183 or W242 in LRP6 having an amino acid sequence of SEQ ID NO:

31.

51. according to the method for claim 50, wherein generating described one or more targeting peptides by computer simulation comprises: Generate multiple candidate peptides through computer simulation; performing a computer-assisted docking simulation on each of the plurality of candidate peptides that bind to LRP6; and The structure of LRP6 bound to one or more candidate peptides from the plurality of candidate peptides is analyzed to identify one or more targeting peptides capable of interacting with: (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having an amino acid sequence of SEQ ID NO: 31; or (2) one or more positions functionally equivalent to S96, S114, E115, R141, W157, W183, or W242 in LRP6 having an amino acid sequence of SEQ ID NO:

31.

52. The method according to any one of claims 50 to 51, comprising: obtaining a binding score for each of the plurality of candidate peptides that binds to LRP6; and One or more candidate peptides having a binding score higher than a threshold value among the plurality of candidate peptides are selected as targeting peptides specific for LRP6.

53. The method of claim 52, comprising comparing the binding scores of two or more candidate peptides in the plurality of candidate peptides to rank candidate peptide sequences.

54. The method of any one of claims 52 to 53, wherein obtaining the binding score for each candidate peptide sequence in a plurality of candidate peptide sequences comprises (1) counting the total number of atoms in the interface between the candidate peptide and LRP6; (2) counting the total number of atoms in the candidate peptide where the atoms conflict with LRP6; (3) obtaining the binding angle of the candidate peptide; and (4) obtaining the binding depth of the candidate peptide.

55. An agent that is capable of binding to a protein selected from the group consisting of interleukin 3 (IL3), family with sequence similarity 234 member A (FAM234A), glycoprotein 2 (GP2), dipeptidyl peptidase-4 (DPP4), Dickkopf WNT signaling pathway inhibitor 3 (DKK3), alanyl aminopeptidase (ANPEP), Epiphycan (EPYC) and LRP6.

56. The agent of claim 55, wherein the agent is selected from the group consisting of an antibody or fragment thereof, an aptamer, a small molecule, a nucleic acid, and a peptide.

57. The agent of claim 56, wherein the antibody or fragment thereof comprises an Fc domain, and / or wherein the antibody or fragment thereof is a single-chain variable fragment (scFv), a single domain antibody, an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a disulfide-linked Fv, a scFv, a single domain antibody, a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, or a functionally active epitope-binding fragment thereof.

58. The agent of claim 56, wherein the nucleic acid is miRNA, shRNA, and siRNA or an oligonucleotide.

59. The agent of any one of claims 55 to 57, wherein the agent is conjugated to a detectable label; optionally wherein the detectable label is selected from the group consisting of: biotin, a fluorophore, a luminescent or bioluminescent marker, a radiolabel, an enzyme, an enzyme substrate, quantum dots, a contrast agent, a metal particle, a magnetic particle, and any combination thereof.

60. The agent of any one of claims 55 to 58, wherein the agent is a therapeutic agent.

61. The medicament of any one of claims 55 to 57, wherein the medicament is a targeting peptide; optionally, the targeting peptide is part of a delivery system, wherein the delivery system comprises a payload to be delivered to a cell.

62. The agent of claim 61, wherein the delivery system comprises a viral vector or a non-viral vector.

63. The agent of claim 62, wherein the viral vector comprises an AAV vector; and optionally wherein the targeting peptide is part of a capsid protein of the AAV vector.

64. The agent of claim 63, wherein 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.

65. The agent of claim 62, wherein the non-viral vector comprises lipid-based nanoparticles, polymeric nanoparticles, inorganic nanoparticles, surfactant-based emulsions, nanowires, silica nanoparticles, peptide- or protein-based particles, lipid-polymer particles, nanolipoprotein particles, and combinations thereof.

66. The agent of any one of claims 61 to 65, wherein the payload to be delivered to the cell is a biomolecule, a non-biological molecule, or a combination thereof; and optionally wherein the biomolecule is selected from the group consisting of: a nucleic acid sequence, a protein, a peptide, a lipid, a polysaccharide, and any combination thereof.

67. The agent of any one of claims 61 to 66, wherein the payload is a therapeutic molecule.

68. The agent according to any one of claims 66 to 67, wherein the nucleic acid sequence to be delivered to the nervous system comprises one or more of: a) sequences encoding trophic factors, growth factors or other soluble factors that may be released from the transduced cells and affect the survival or function of the cells and / or surrounding cells; b) DNA sequences that restore protein function to humans or animals carrying a genetic mutation in a gene; c) DNA sequences encoding proteins that can be used to control or alter the activity or state of cells; d) a DNA sequence encoding a protein or nucleic acid for assessing the status of a cell; e) DNA and / or related guide RNA for genome engineering; f) sequences for genome editing by homologous recombination; g) a DNA sequence encoding a therapeutic RNA; h) shRNA or artificial miRNA delivery systems; or i) DNA sequences that affect the splicing of endogenous genes.

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