Targets for receptor-mediated control of therapeutic drug biodistribution and efficacy

Abstract

Disclosed herein are novel blood-brain barrier (BBB) ​​crossing receptors at the interface of the BBB, targeting peptides and derivatives thereof capable of binding to the novel receptors, and related methods for using the receptors to increase BBB permeability and deliver drugs to the nervous system (e.g., CNS). In some embodiments, the BBB crossing receptor is LRP6. Disclosed herein also include recombinant adeno-associated viruses (rAAVs) with increased specificity and transduction efficiency across the BBB, as well as related compositions and methods for treating various diseases and conditions.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 420,002, filed October 27, 2022; and U.S. Provisional Patent Application No. 63 / 465,814, filed May 11, 2023. The contents of these related applications are incorporated herein by reference in their entirety for all purposes.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED R&D 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.

[0003] Reference to sequence listing This application is filed together with an electronic Sequence Listing, which is provided as a file entitled 30KJ-365863-US_SequenceListing, created on October 23, 2023, and is 51,181 bytes in size. The information in the electronic Sequence Listing is incorporated herein by reference in its entirety.

[0004] background Field 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 technology]

[0005] Description of Related Technology Targeting therapeutic and research molecules to target tissues and cell types of interest and avoiding tissues and cell types that mediate potentially harmful side effects is a fundamental challenge for drug development. This is particularly true for molecules that target the brain. The blood-brain barrier (BBB) ​​severely restricts the nature of therapeutic and research molecules that can enter the central nervous system (CNS) upon systemic administration into the peripheral bloodstream.

[0006] There is a need to use gain-of-function rather than loss-of-function screens, and to screen both naturally and laboratory-evolved AAVs. There is a need for methods to reverse engineer novel targets that determine the biodistribution of therapeutic agents that are the product of natural and directed evolution of adeno-associated viruses (AAVs). Summary of the Invention [Means for solving the problem]

[0007] Abstract Disclosed herein is a method for increasing the permeability of the blood-brain barrier. In some embodiments, the method includes 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.

[0008] 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.

[0009] Disclosed herein is a method for delivering a payload to the nervous system of a subject. In some embodiments, the method includes providing a targeting peptide or a derivative thereof that can bind to low-density lipoprotein receptor-related protein 6 (LRP6), wherein the targeting peptide is part of a delivery system, and the delivery system comprises a payload to be delivered to the nervous system; and administering the delivery system to the subject.

[0010] In some embodiments, the delivery system comprises nanoparticles, nanotubes, nanowires, dendrimers, liposomes, ethosomes and aquasomes, polymersomes and niosomes, foams, hydrogels, cubosomes, 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 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 isolate rh.8, rhesus isolate rh.10, and variants thereof. In some embodiments, non-viral vectors include 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.

[0011] In some embodiments, the payload delivered to the nervous system is a biomolecule, a non-biomolecule, or a combination thereof. In some embodiments, 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. In some embodiments, the payload is a therapeutic molecule. In some embodiments, the nucleic acid sequence delivered to the nervous system includes one or more of: a) a sequence encoding a trophic factor, a growth factor, or other soluble factor that can be released from a 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 that has a genetic mutation in that 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 used to evaluate the state of a cell; e) DNA and / or related guide RNA for genome manipulation; f) a sequence for genome editing via 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.

[0012] In some embodiments, LRP6 is mouse LRP6. In some embodiments, LRP6 has an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29. In some embodiments, LRP6 is macaque LRP6. In some embodiments, LRP6 has an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 30. In some embodiments, LRP6 is human LRP6. In some embodiments, LRP6 has an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 31. In some embodiments, upon binding, the targeting peptide can interact with (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having the 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 the amino acid sequence of SEQ ID NO: 31.

[0013] In some embodiments, the targeting peptide is inserted between two adjacent amino acids at AA587-594 of SEQ ID NO: 11 of the AAV9 vector, or a functional equivalent 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 a functional equivalent of AA588-589 in an amino acid sequence at least 80% identical to SEQ ID NO: 11.

[0014] The AAV vector can be, for example, conjugated to nanoparticles, second molecules, or combinations thereof.Administered can be, for example, systemic administration.In some embodiments, administered is intravenous or intrathecal administration.

[0015] In some embodiments, the subject is a mammal. In some embodiments, the subject is human. In some embodiments, the subject is a subject suffering from 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 types 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 in the CNS is Krabbe disease, Sandhoff disease, Tay-Sachs disease, Gaucher disease (types I, II, or III), Niemann-Pick disease (NPC1 or NPC2 deficiency), Hurler syndrome, Pompe disease, or Batten disease. In some embodiments, the subject has suffered from, is at risk of developing, or has suffered from a stroke, traumatic brain injury, epilepsy, or spinal cord injury.

[0016] Disclosed herein are adeno-associated virus (AAV) capsid proteins. In some embodiments, the AAV capsid protein comprises a targeting peptide having binding specificity for LRP6. In some embodiments, the targeting peptide is part of the capsid protein of an rAAV vector. In some embodiments, the targeting peptide is inserted between two adjacent amino acids at AA587-594 of SEQ ID NO: 11, or a functional equivalent of AA587-594 in an amino acid sequence at least 80% identical to SEQ ID NO: 11. In some embodiments, upon binding to LRP6, the targeting peptide can interact with (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having the 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 the 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 isolate rh.8, rhesus isolate rh.10, and variants thereof.

[0017] Disclosed herein are recombinant adeno-associated viruses (rAAVs). In some embodiments, the rAAVs comprise any of the AAV capsid proteins disclosed herein. In some embodiments, the rAAVs comprise an AAV capsid protein comprising a targeting peptide having binding specificity for low-density lipoprotein receptor-related protein 6 (LRP6), wherein the amino acid sequence of the targeting peptide is inserted between two adjacent amino acids at AA587-594 of the AAV capsid protein, or a functional equivalent thereof. In some embodiments, the two adjacent amino acids are AA588 and AA589. In some embodiments, upon binding to LRP6, the targeting peptide can interact with (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having the 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 the amino acid sequence of SEQ ID NO: 31. In some embodiments, the rAAV has enhanced tropism for the nervous system compared to rAAV that does not contain a targeting peptide. In some embodiments, the rAAV can transduce the nervous system at least two-fold more efficiently than rAAV that does not contain a targeting peptide.

[0018] Disclosed herein is a composition for use in delivering a drug to the nervous system of a subject in need thereof. In some embodiments, the composition comprises (1) an AAV capsid protein disclosed herein, and (2) an AAV comprising a drug to be delivered to the subject's nervous system; optionally, 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, brain capillaries, brain arterioles, brain arteries, or a combination thereof. In some embodiments, the composition is a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers. In some embodiments, the drug to be delivered comprises a nucleic acid, a peptide, a small molecule, an aptamer, or a combination thereof.

[0019] Disclosed herein includes antibodies or fragments thereof. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence having binding specificity for LRP6. In some embodiments, the antibody or fragment thereof is a bispecific antibody comprising at least one Fab having specificity for LRP6. Disclosed herein includes antibody conjugates comprising any 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 includes peptides or derivatives or conjugates thereof having specificity for low density lipoprotein receptor-related protein 6 (LRP6). Disclosed herein includes nucleic acids. In some embodiments, the nucleic acid comprises a sequence encoding any of the antibodies or fragments thereof, or any of the peptides or derivatives or conjugates thereof disclosed herein.

[0020] Disclosed herein is a delivery system. In some embodiments, the delivery system comprises (1) a targeting peptide having specificity for low-density lipoprotein receptor-related protein 6 (LRP6); and (2) a drug. In some embodiments, the targeting peptide is (1) displayed on the surface of the delivery system; or (2) partially embedded in the delivery system. In some embodiments, the delivery system is selected from the group consisting of nanoparticles, nanotubes, nanowires, dendrimers, liposomes, ethosomes and aquasomes, polymersomes and niosomes, foams, hydrogels, cubosomes, 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, polymeric 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.

[0021] Disclosed herein is a method for designing a targeting peptide having specificity for low-density lipoprotein receptor-related protein 6 (LRP6). In some embodiments, the method includes in silico generating one or more targeting peptides that can interact with (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having the 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 the amino acid sequence of SEQ ID NO: 31.

[0022] In some embodiments, the step of generating one or more targeting peptides in silico includes the steps of generating a large number of candidate peptides in silico; performing computer-assisted docking simulations for each of the large number of candidate peptides that bind to LRP6; and analyzing the structure of LRP6 bound to one or more of the large number of candidate peptides to identify one or more targeting peptides that can interact with (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having the 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 the amino acid sequence of SEQ ID NO: 31.

[0023] The method may include obtaining a binding score for each of a number of candidate peptides that bind to LRP6; and selecting one or more of the number of candidate peptides that have a binding score above a threshold as a targeting peptide with specificity for LRP6. The method may include comparing the binding scores of two or more of the number of candidate peptides to rank the candidate peptide sequences. In some embodiments, obtaining a binding score for each of the number of candidate peptide sequences includes: (1) counting the total number of atoms at the interface between the candidate peptide and LRP6; (2) counting the total number of atoms in the candidate peptide, where the atoms are in conflict with LRP6; (3) obtaining the bond angle of the candidate peptide; and (4) obtaining the binding depth of the candidate peptide.

[0024] Disclosed herein are agents capable of binding to a protein selected from the group consisting of interleukin 3 (IL3), family 234 member A with sequence similarity (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, an Fv fragment, a disulfide-linked Fv, an scFv, a single-domain antibody, a diabody, a multispecific antibody, a bispecific antibody, an anti-idiotypic antibody, a diabody, or a functionally active epitope-binding fragment thereof. In some embodiments, the nucleic acid is an miRNA, shRNA, and siRNA, or an oligonucleotide.

[0025] 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, an imaging 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, and the delivery system comprises a payload to be delivered to a 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 a 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 isolate rh.8, rhesus isolate rh.10, and variants thereof. In some embodiments, the non-viral vector comprises a lipid-based nanoparticle, a polymeric nanoparticle, an inorganic nanoparticle, a surfactant-based emulsion, a nanowire, a silica nanoparticle, a peptide- or protein-based particle, a lipid-polymer particle, a nanolipoprotein particle, and combinations thereof.

[0026] In some embodiments, the payload delivered to the cell is a biomolecule, a non-biomolecule, or a combination thereof. In some embodiments, 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. In some embodiments, the payload is a therapeutic molecule. In some embodiments, the nucleic acid sequence delivered to the nervous system includes one or more of: a) a sequence encoding a trophic factor, a growth factor, or other soluble factor that can 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 humans or animals that have a genetic mutation in that gene; c) DNA that encodes a protein that can be used to control or change the activity or state of a cell; d) DNA that encodes a protein or nucleic acid used to evaluate the state of a cell; e) DNA and / or related guide RNA for genome manipulation; f) a sequence for genome editing via 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 endogenous genes. [Brief explanation of the drawings]

[0027] [Figure 1] Figure 1 shows non-limiting exemplary data showing that surface plasmon resonance (SPR) confirms direct binding interactions between the LRP6 extracellular domain and both AAV-X1.1 and AAV.CAP-Mac. Binding is not detected with AAV9.

[0028] [Figure 2] Figure 2 shows exemplary computational modeling in AlphaFold2 of engineered AAV-inserted peptides with the human LRP6 extracellular domain, showing binding to domain 2 for X1.1 and to either domain 1 or domain 2 for CAP-Mac.

[0029] [Figure 3]Figure 3 shows exemplary data for AAV-X1 and AAV.CAP-Mac, demonstrating enhanced potency in HEK293 cells compared to standard AAV9-based AAVs that have not acquired interactions with human receptors.

[0030] [Figure 4] FIG. 4 presents non-limiting exemplary data demonstrating that siRNA-mediated knockdown of endogenous LRP6 in HEK293 cells selectively reduces the potency of AAV-X1.1 and AAV.CAP-Mac.

[0031] [Figure 5] Figure 5 shows a non-limiting exemplary screening library and validation of the results. Test AAVs and controls were added to fixed microarray slides.

[0032] [Figure 6] Figure 6 shows a non-limiting exemplary screening library and validation of the results. Test AAVs and controls were added to fixed microarray slides.

[0033] [Figure 7A] Figures 7A-7D show non-limiting exemplary data for confirmation and specificity screening. Test AAV was added to fixed confirmation slides using the direct fixation method (AAV is not removed). Detection was performed using an anti-AAV9 antibody followed by AF647 anti-mIgG H+L. Anti-adeno-associated virus 9, clone HL2372, supplied by Merck, catalog number MABF2309-100UL, 1:500 dilution. Detection antibody: AlexaFluor647 anti-mIgG H+L. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 7D] Same as above.

[0034] [Figure 8A]Figures 8A-8B show non-limiting exemplary data for validation and specificity screening. Test and control AAVs were added to fixed validation slides using the direct fixation method (AAV is not removed). Detection was performed using an anti-AAV9 antibody followed by AF647 anti-mIgG H+L. Anti-adeno-associated virus 9, clone HL2372, supplied by Merck, catalog number MABF2309-100UL, 1:500 dilution. Detection antibody: AlexaFluor647 anti-mIgG H+L. [Figure 8B] Same as above.

[0035] [Figure 9A] Figures 9A-9B show non-limiting exemplary data for confirmation and specificity screening. Controls were added to fixed confirmation slides using the direct fixation method (sample not removed). Anti-adeno-associated virus 9, clone HL2372, supplied by Merck, catalog number MABF2309-100UL, 1:500 dilution. Detection antibody: AlexaFluor 647 anti-mIgG H+L. [Figure 9B] Same as above. [Figure 9C] Same as above.

[0036] [Figure 10-1] FIG. 10 shows data for a global overview of specific hits (eg, weak intensity and above). [Figure 10-2] Same as above.

[0037] [Figure 11-1] FIG. 11 shows data for a global overview of specific hits (eg, weak intensity and above). [Figure 11-2] Same as above.

[0038] [Figure 12A]Figures 12A-12E show non-limiting, exemplary data demonstrating high-throughput screening to identify AAV-binding human proteins. Figure 12A shows a schematic of the AAV cell microarray screen. DNA oligos encoding individual membrane proteins were chemically coupled to slides in known patterns, and cells grown on the slides were reverse transfected, thereby creating spots of cells overexpressing specific known proteins. Each protein was expressed in duplicate at two different slide locations. When AAV was applied to the slide, enhanced binding was detected from duplicate cell spots overexpressing the cognate AAV receptor. Figure 12B shows data on known AAV capsid-receptor interactions, e.g., AAVR and LY6A with AAV-PHP.eB, which were used to optimize conditions for streptavidin-based detection of biotinylated capsids using two sets of replicate spots. An anti-TGFBR2 antibody was used as a non-AAV positive control. Figure 12C shows the use of AAVR and LY6A interactions with AAV9.CAP-B22 to optimize conditions for direct anti-AAV9 antibody detection of unmodified capsids using two sets of replicate spots. An anti-TGFBR2 antibody was used as a non-AAV control. Figure 12D shows that pooled AAV capsid screening conditions were optimized by varying the concentration of individual capsids within the pool to maximize signal to noise after direct detection with anti-AAV9 antibodies using two sets of replicate spots. Figure 12E shows data demonstrating that pooled screening yielded preliminary hits that were deconvoluted by individual capsid screening. Potential novel capsid-associated proteins were identified by direct detection with anti-AAV9 antibodies. Transfection control conditions detected fluorescent proteins reverse-transfected with their respective receptors. The no condition was treated with anti-AAV9 antibodies only. DPP4, IL3, and DKK3 were identified in all individual AAV screens and may represent interactions outside the engineered region of AAV9.LY6A, GP2, LRP6, FAM234A, ANPEP, CSF2, and EPYC specifically bind to at least one engineered capsid. [Figure 12B] Same as above. [Figure 12C] Same as above. [Figure 12D] Same as above. [Figure 12E] Same as above.

[0039] [Figure 13A] Figures 13A-13D show non-limiting, exemplary data regarding species- and serotype-specific interactions between AAV9 and IL3. Figure 13A shows a schematic of a surface plasmon resonance (SPR) experiment in which IL3-Fc is captured on a protein A sensor chip and AAV analyte is flowed over the sensor. Figure 13B shows a graph of data demonstrating SPR confirming the serotype-specific interaction of AAV9 with the human immunomodulatory cytokine IL3. Figure 13C shows a graph of data demonstrating SPR confirming AAV9 binding to macaque IL3, but not marmoset or mouse IL3. Figure 13D shows data regarding MS / MS analysis. Bis(sulfosuccinimidyl) suberate (BS3)-crosslinked AAV9 and hsIL3 were extracted from a PAGE gel and analyzed by MS / MS, identifying two intramolecular crosslinks with high-confidence XlinkX scores. [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 13D] Same as above.

[0040] [Figure 14A]Figures 14A-14D show non-limiting, exemplary data demonstrating that primate brain-enhanced AAVs acquire interactions with LRP6. Figure 14A shows a non-limiting, exemplary animation demonstrating that aligning AAV capsid-specific hits with human brain endothelial cell expression levels reveals highly conserved LRP6 as a potential receptor for BBB crossing. Figure 14B shows a graph demonstrating that SPR confirms that the engineered capsids AAV9-X1.1 and CAP-Mac acquire direct binding interactions with human LRP6. Figure 14C shows an AlphaFold model of the X1 and CAP-Mac peptides predicting selective interactions with human LRP6 YWTD domain 1 (E1). Figure 14D shows SPR data for mouse LRP6-E1E2 and LRP6-E3E4 (minimal stable extracellular domain fragments resulting from cooperative folding), confirming that AAV9-X1.1 and CAP-Mac bind exclusively to LRP6-E1E2. [Figure 14B] Same as above. [Figure 14C] Same as above. [Figure 14D] Same as above.

[0041] [Figure 15A]Figures 15A-15F show non-limiting, exemplary data demonstrating that LRP6 modulates the CNS function of engineered AAVs in mice. Figure 15A shows a schematic of Lrp6 conditional knockout by sequential AAV injections. Lrp6 Cre conditional knockout mice were systemically injected with AAV1-X1, which packaged either Cre or mCherry, to generate cohorts of mice differing in their LRP6 expression. After allowing time for expression, these cohorts were injected with AAV9-PHP.eB or AAV9-X1.1, which packaged eGFP. Serotype switching allows for evasion of neutralizing antibodies and assessment of vector dependency on Lrp6 in vivo. Figure 15B shows representative sagittal brain and liver images from a conditional Lrp6 knockout experiment. Imaging parameters were independently optimized for the second dose of AAV9-X1.1 and AAV9-PHP.eB. Figure 15C shows a graph of AAV potency quantification demonstrating that conditional knockout of Lrp6 in mouse brain selectively and potently reduces the brain and liver potency of AAV9-X1.1. Data points are the average of two sections per tissue region per animal, corresponding to the physiological region of interest across four experimental cohorts. Bars represent mean values. Figures 15D-E show data demonstrating that AAV9-X1.1 enhanced potency in macaque (Figure 15D) and human primary brain microvascular endothelial cell cultures (Figure 15E), which was reduced to AAV9 levels by Mesd inhibition of LRP6. Bars represent mean values. Immunofluorescence of mouse and infant macaque brain tissue reveals consistently high LRP6 expression throughout the brain endothelium as well as neurons and astrocytes (Figure 15F). [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 15D] Same as above. [Figure 15E] Same as above. [Figure 15F] Same as above.

[0042] [Figure 16]Figure 16 shows non-limiting exemplary data on the individual characteristics of pooled AAVs before full screening. Individual AAVs were tested at various doses to determine the optimal signal-to-noise ratio for different capsids, confirming the detection of LY6A only for the known interactors KIAA0319L (AAVR) and CAP-B22. AAV binding detected in duplicate spots of the same protein is indicated by an arrow.

[0043] [Figure 17A] Figures 17A-C show data on cell culture potency assay validation of high-throughput screening hits. Figure 17A shows representative images and quantification demonstrating transient overexpression of mouse and human GP2 in HEK293T cells, which resulted in enhanced potency for CAP-Mac and AAV9-X1.1, with stronger effects for the human proteins. Scales indicate the degree of infection (min 0.01; max 0.17) and total light intensity per signal area (min 0.11; max 0.45). Figure 17B shows representative images and quantification demonstrating transient overexpression of mouse and human FAM234A in HEK293T cells, which resulted in enhanced potency for PHP.eB and CAP-B22, with stronger effects for the mouse proteins. Scales indicate the degree of infection (min 0.04; max 0.07) and total light intensity per signal area (min 0.16; max 0.29). Figure 17C shows representative images and quantification demonstrating that transient overexpression of human ANPEP and DPP4 did not result in enhanced potency for AAV9-X1.1 and AAV9, respectively. Scale bar indicates 200 μm. [Figure 17B] Same as above. [Figure 17C] Same as above.

[0044] [Figure 18A]Figures 18A-18B show non-limiting exemplary data regarding SPR validation of selected screening hits. Immobilization of human DKK3-Fc (Figure 18A) or human GP2-Fc (Figure 18B) on a Protein A chip allowed for evaluation of AAV-analyte interactions. In contrast to the cell microarray screening, no interaction was observed for AAV9 with DKK3, while AAV9-X1.1 acquired direct binding ability to human GP2, consistent with the cell microarray screening and cell culture potency assay. [Figure 18B] Same as above.

[0045] [Figure 19A] 19A-19B show non-limiting exemplary graphs of annotated MS / MS spectra of AAV9 crosslinking to human IL3 via BS3. [Figure 19B] Same as above.

[0046] [Figure 20A] Figures 20A-20D show non-limiting, exemplary data regarding LRP6 binding to the X1 peptide and AAV-BI30 in multiple serotypes. Figure 20A shows data showing SPR of the complete human LRP6 extracellular domain, confirming that the X1 inserted peptide module enables LRP6 binding across multiple serotypes. Figure 20B shows SPR data confirming the binding interaction of AAV-BI30 with mouse LRP6-E1E2, but not LRP6-E3E4. Figure 20C shows an exemplary AlphaFold model predicting that the X1 and CAP-Mac variable region VIII peptides bind to WYTD domain 1 or 2 (e.g., E1E2), although in some embodiments, specific binding poses cannot be confidently assigned. Figure 20D shows non-limiting, exemplary data demonstrating that, despite their high degree of sequence similarity, LRP5 WYTD domain 1, unlike that of LRP6, does not bind to AAV-X1.1. [Figure 20B] Same as above. [Figure 20C] Same as above. [Figure 20D] Same as above.

[0047] [Figure 21] Figure 21 presents non-limiting exemplary data showing that X1.1 and CAP-Mac bind to LRP6 and AAVR but not LRP5. Pull-down assays of AAV9, AAV9-X1.1, and CAP-Mac preys with the extracellular domains of mouse LRP6, mouse LRP5, and human AAVR PDK2 domains are shown. Asterisks indicate LRP6-binding interactions acquired by X1.1 and CAP-Mac during directed evolution from the parent capsid AAV9.

[0048] [Figure 22A] Figures 22A-B show non-limiting exemplary data for validation of cell culture potency assays of LRP6 interaction. Figure 22A shows a schematic of Mesd chaperone function and LRP6 domain-dependent inhibition by recombinant Mesd and SOST proteins. Figure 22B shows quantification of AAV potency demonstrating the effects of transient overexpression of the LRP receptor and LRP6 inhibition. The extent of infection (min 0.04; max 0.23) and total luminosity per signal area (min 0.04; max 0.51) were measured. [Figure 22B] Same as above.

[0049] [Figure 23A] Figures 23A-B show non-limiting, exemplary data regarding the efficacy of X1 AAV in mouse liver and human primary cell cultures. Figure 23A shows representative liver images from a conditional Lrp6 knockout experiment. The same imaging parameters were applied to all conditions. Figure 23B presents data showing that AAV1-X1 enhanced efficacy in human primary brain microvascular endothelial cell cultures, where Mesd inhibition of LRP6 reduced efficacy to AAV1 levels. Bars represent mean values. [Figure 23B] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0050] Detailed Description In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols typically identify like elements, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant 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 the aspects of the present disclosure, as generally described herein and illustrated in the drawings, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated and made a part of this disclosure herein.

[0051] 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.

[0052] Disclosed herein is a method for increasing the permeability of the blood-brain barrier. In some embodiments, the method includes 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.

[0053] Disclosed herein is a method for delivering a payload to the nervous system of a subject. In some embodiments, the method includes: providing a targeting peptide or a derivative thereof that can bind to LRP6, wherein the targeting peptide is part of a delivery system, and the delivery system comprises a payload that is delivered to the nervous system; and administering the delivery system to a subject.

[0054] Disclosed herein is an adeno-associated virus (AAV) capsid protein. In some embodiments, the AAV capsid protein comprises a targeting peptide having binding specificity for LRP6. Disclosed herein is a recombinant adeno-associated virus (rAAV). In some embodiments, the rAAV comprises any of the AAV capsid proteins disclosed herein. In some embodiments, the rAAV comprises an AAV capsid protein comprising a targeting peptide having binding specificity for LRP6, wherein the amino acid sequence of the targeting peptide is inserted between two adjacent amino acids at AA587-594 of the AAV capsid protein, or a functional equivalent thereof.

[0055] Disclosed herein are compositions for use in delivering agents to the nervous system of a subject in need thereof. In some embodiments, the compositions comprise (1) an AAV capsid protein disclosed herein, and (2) an AAV comprising an agent to be delivered to the nervous system of the subject; optionally, the nervous system is the central nervous system (CNS), the peripheral nervous system (PNS), or a combination thereof.

[0056] The present disclosure includes antibodies or fragments thereof. In some embodiments, the antibody or fragment thereof comprises an amino acid sequence having binding specificity for LRP6. The present disclosure includes peptides or derivatives or conjugates thereof having specificity for LRP6. The present disclosure includes nucleic acids. In some embodiments, the nucleic acid comprises a sequence encoding any of the antibodies or fragments thereof, or any of the peptides or derivatives or conjugates thereof disclosed herein.

[0057] Disclosed herein is a delivery system. In some embodiments, the delivery system includes: (1) a targeting peptide having specificity for low-density lipoprotein receptor-related protein 6 (LRP6); and (2) a drug.

[0058] Disclosed herein is a method for designing a targeting peptide with specificity for LRP6. In some embodiments, the method comprises in silico generation of one or more targeting peptides that can interact with (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having the 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 the amino acid sequence of SEQ ID NO: 31.

[0059] Disclosed herein are agents 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. definition

[0060] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this disclosure belongs.See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., 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 purpose of this disclosure, the following terms are defined below.

[0061] As used herein, the terms "nucleic acid" and "polynucleotide" are interchangeable and can refer to any nucleic acid, whether it is composed of phosphodiester linkages or modified linkages, such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sultone linkages, and combinations of such linkages.The terms "nucleic acid" and "polynucleotide" also specifically include nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).

[0062] The term "vector," as used herein, can refer to a vehicle for carrying or transferring a nucleic acid. Non-limiting examples of vectors include plasmids and viruses (e.g., AAV viruses).

[0063] The term "construct," as used herein, may refer to a recombinant nucleic acid created for the purpose of expressing a specific nucleotide sequence or used in the construction of other recombinant nucleotide sequences.

[0064] 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.

[0065] The term "viral genome" refers to a nucleic acid sequence flanking a cis-acting nucleic acid sequence that mediates the packaging of nucleic acids into viral capsids. For example, for AAV and parvovirus, it is known that the "inverted terminal repeats" (ITRs) located at the 5' and 3' ends of the viral genome have this function, and that the ITRs can mediate the packaging of heterologous, e.g., non-wild-type, viral genomes into viral capsids.

[0066] The term "element" can refer to a separate or distinct part of something, for example, a nucleic acid sequence with a separate function within 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 linked coding sequence in a specific host organism. These terms are used broadly to encompass all elements that promote or regulate transcription, including promoters, core elements required for the basic interaction of RNA polymerase and transcription factors, upstream elements, enhancers, and response elements (see, for example, Lewin, "Genes V" (Oxford University Press, Oxford) pages 847-873). Exemplary regulatory elements in prokaryotes include promoters, operator sequences, and ribosome binding sites. Regulatory elements used in eukaryotic cells may include, but are not limited to, transcriptional and translational control sequences, such as promoters, enhancers, splicing signals, polyadenylation signals, terminators, proteolytic signals, internal ribosome entry elements (IRES), 2A sequences, and the like, that provide and / or regulate the expression of a coding sequence and / or the production of the encoded polypeptide in a host cell.

[0067] As used herein, the term "promoter" refers to a nucleotide sequence that allows RNA polymerase to bind and direct the transcription of a gene. Typically, promoters are located in the 5' non-coding region of a gene, proximal to the transcription start site of the gene. Sequence elements within a promoter that function in initiating transcription are often characterized by a consensus 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 increased levels of transcription from DNA under their control in response to some change in culture conditions, such as a change in temperature.

[0068] As used herein, the term "enhancer" refers to a type of regulatory element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription.

[0069] 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 placed under the control of one or more regulatory elements, such as, but not limited to, a constitutive or inducible promoter, a tissue-specific regulatory element, and an enhancer. A gene or coding region is said to be "operably linked" or "operably linked" or "operably associated" with a regulatory element, meaning 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 causes the transcription or expression of the coding sequence.

[0070] As used herein, the term "polypeptide" is intended to encompass the singular "polypeptide" and the plural "polypeptides" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids is included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modifications with non-naturally occurring amino acids. Polypeptides may be derived from natural biological sources or may be produced by recombinant technology, although not necessarily translated from a designed nucleic acid sequence. It may be made in any manner, including by chemical synthesis.

[0071] As used herein, the term "variant" can refer to a polynucleotide or polypeptide having a sequence substantially similar to that of a reference polynucleotide or polypeptide. In the case of a polynucleotide, a variant can have one or more nucleotide deletions, substitutions, and / or additions at the 5'-end, 3'-end, and / or one or more internal sites compared to the reference polynucleotide. The sequence similarity and / or difference 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 created by using site-directed mutagenesis. Generally, polynucleotide variants, 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 with reference polynucleotides, 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, or additions compared with reference polypeptides.The sequence similarity and / or difference between variants and reference polypeptides can be detected using conventional techniques known in the art, such as Western blot. Generally, a variant 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.

[0072] 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 in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentages of sequence identity or similarity are used in reference to proteins, it is recognized that non-identical residue positions often differ by conservative amino acid substitutions, in which amino acid residues are replaced with functionally equivalent residues that have similar physicochemical properties and therefore do not change the functional properties of the molecule.

[0073] As used herein, a functionally equivalent residue of an amino acid may typically refer to another amino acid residue having substantially similar physiochemical and stereochemical characteristics to the original amino acid. Physiological properties include water solubility (hydrophobic or hydrophilic), dielectric and electrochemical properties, physiological pH, side chain partial charge (positive, negative, or neutral), and other properties identifiable to those skilled in the art. Stereochemical characteristics include the spatial and conformational arrangement of amino acids and their chirality. For example, glutamic acid is considered to be a functionally equivalent residue to aspartic acid within the meaning of the present disclosure. Tyrosine and tryptophan are considered to be functionally equivalent residues to phenylalanine. Arginine and lysine are considered to be functionally equivalent residues to histidine.

[0074] As used herein, the term "binding" refers to a non-covalent interaction between macromolecules (e.g., between two proteins and / or peptides). In a non-covalent interaction, the macromolecules are said to be "associated" or "interact" or "bound" (e.g., when molecule X is said to interact with molecule Y, it means that molecule X binds to molecule Y in a non-covalent manner). A binding interaction is characterized by a dissociation constant (Kd), e.g., 10 -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 The binding affinity may be characterized by a Kd of less than or equal to M, or a number or range between any two of these values. Kd may depend on environmental conditions, such as pH and temperature. "Affinity" refers to the strength of binding, with increased binding affinity correlating with a decreased Kd.

[0075] The terms "specific," "specifically," or "specificity," when used herein with respect to the binding of a first molecule to a second molecule, refer to the recognition, contact, and formation of a stable complex between the first molecule and the second molecule, with substantially low to no recognition, contact, and formation of a stable complex between each of the first and second molecules and other molecules that may be present. Exemplary specific bindings include antibody-antigen interactions, cell receptor-ligand interactions, polynucleotide hybridization, enzyme-substrate interactions, and the like. The term "specific," when used herein with respect to molecular components of a complex, refers to the unique association of that component into the specific complex of which it is a part. The term "specific," when used herein with respect to the sequence of a polynucleotide, refers to the unique association of that sequence with a single polynucleotide that is complementary to the sequence. A "stable complex" refers to a complex that is detectable and does not require any particular level of stability, although greater stability is generally preferred. The terms "specific," "specifically," or "specificity," as used herein with respect to the binding of a targeting peptide to a target protein, e.g., LRP6, refer to the ability of the targeting peptide to form a stable complex with the target protein with substantially low to no binding to macromolecules other than the target protein that may be present. The terms "specific," "specifically," or "specificity," as used herein with respect to the binding of a targeting peptide to a target protein, e.g., LRP6, also refer to the ability of the protein to form a stable complex with the targeting peptide with substantially low to no binding to candidate peptides other than the identified targeting peptide that may be present.

[0076] The term "AAV" or "adeno-associated virus" refers to a dependent parvovirus within the Parvoviridae genus of viruses. For example, the AAV may be derived from a naturally occurring "wild-type" virus, a rAAV genome packaged in a capsid derived from a capsid protein encoded by a naturally occurring cap gene, and / or a rAAV genome packaged in a capsid derived from a capsid protein encoded by a non-native capsid 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 DJ type (AAV-DJ), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV and ovine AAV.In some examples, AAV is described as " primate AAV ", which refers to the AAV that infects primates. Similarly, AAV can infect bovine animals (e.g., "bovine AAV"). In some cases, the AAV is wild-type or naturally occurring. In some cases, the AAV is recombinant.

[0077] The term "AAV capsid" as used herein refers to the capsid protein or peptide of adeno-associated virus. In some cases, the AAV capsid protein is configured to encapsidate 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, which means that in some cases, the parent or wild-type AAV capsid is modified in the amino acid sequence of the parent AAV capsid protein.

[0078] The term "AAV genome" as used herein may refer to a nucleic acid polynucleotide encoding the genetic information of a virus. In some cases, the genome comprises a nucleic acid sequence flanking the AAV inverted terminal repeat (ITR) sequence. The AAV genome may be a rAAV genome created using recombinant genetic methods, which may comprise a heterologous nucleic acid (e.g., a transgene) comprising and / or flanking the ITR sequence.

[0079] The term "rAAV" refers to "recombinant AAV." In some embodiments, recombinant AAV has an AAV genome in which some or all of the rep and cap genes are replaced with heterologous sequences. The terms "AAV particle," "AAV nanoparticle," or "AAV vector," which are used interchangeably herein, refer to an AAV virus or virion that includes an AAV capsid that packages a heterologous DNA polynucleotide, or a "genome" that includes a nucleic acid sequence flanking the AAV ITR sequence. In some cases, AAV particles are modified relative to parent AAV particles.

[0080] The term "cap gene" refers to a nucleic acid sequence that encodes a capsid protein that forms or contributes to the formation of the viral capsid or protein shell. In the case of AAV, the capsid protein may be VP1, VP2, or VP3. For other parvoviruses, the names and number of capsid proteins may vary.

[0081] The term "rep genes" refers to nucleic acid sequences that encode nonstructural proteins (rep78, rep68, rep52 and rep40) required for viral replication and production.

[0082] The terms "native" and "wild-type" are used interchangeably herein and may refer to a form of a polynucleotide, gene, or polypeptide that is found in nature along with its own regulatory sequences, if present.

[0083] 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 native polynucleotide in its natural location in the genome of an organism. An "endogenous gene" includes a native gene in its natural location in the genome of an organism. An "endogenous polypeptide" includes a native polypeptide in its natural location in an organism.

[0084] As used herein, "heterologous" refers to a polynucleotide, gene, or polypeptide that has been introduced into a host organism but is not normally found in the host organism. A "heterologous polynucleotide" includes a native coding region, or a portion thereof, that has been reintroduced into the source organism in a form that differs from the corresponding native polynucleotide. A "heterologous gene" includes a native coding region, or a portion thereof, that has been reintroduced into the source organism in a form that differs from the corresponding native gene. For example, a heterologous gene may include a native coding region that is part of a chimeric gene that includes a non-native regulatory region that is reintroduced into the native host. A "heterologous polypeptide" includes a native polypeptide that has been reintroduced into the source organism in a form that differs from the corresponding native polypeptide. Genes and proteins of interest can be fused to other genes and proteins to generate chimeric or fusion proteins. Genes and proteins useful according to embodiments of the present disclosure include not only the specifically exemplified full-length sequences, but also portions, segments, and / or fragments of these sequences (including contiguous fragments, and internal and / or terminal deletions compared to the full-length molecule), variants, mutants, chimeras, and fusions thereof.

[0085] The term "exogenous" gene, as used herein, is meant to encompass all genes that do not naturally occur in the genome of an individual. For example, miRNAs can be exogenously introduced by viruses, such as AAV nanoparticles.

[0086] 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 thereof, or a single chain. Thus, the term "antibody" includes any protein or peptide containing molecule comprising at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples include, but are not limited to, a heavy or light chain complementarity-determining region (CDR) or a 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.

[0087] The term "antibody fragment" or "antigen-binding fragment," as used herein, refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds with the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0088] 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, and reptiles, particularly mammals. "Mammal," as used herein, refers to an individual belonging to the class Mammalia, including, but not limited to, humans, domestic and farm animals, zoo animals, sport animals, and pet animals. Non-limiting examples of mammals include mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; and primates, such as monkeys, chimpanzees, and apes, particularly humans. In some embodiments, the mammal is a human. However, in some embodiments, the mammal is not a human. In some embodiments, the subject is a rodent (e.g., a rat or a mouse). In some embodiments, the subject is a primate (e.g., a human or a monkey).

[0089] As used herein, the term "treatment" refers to an intervention made in response to a disease, disorder, or physiological condition exhibited by a patient. The goals of treatment may include, but are not limited to, one or more of: alleviating or preventing symptoms; slowing or halting the progression or worsening of a disease, disorder, or condition; and ameliorating a disease, disorder, or condition. The terms "treat" and "treatment" include, for example, therapeutic treatment, prophylactic treatment, and applications that reduce a subject's risk of developing a disorder or other risk factors. Treatment does not require a complete cure of the disorder, but encompasses embodiments that reduce symptoms or underlying risk factors. In some embodiments, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already affected by a disease, disorder, or undesirable physiological condition, and those in whom the disease, disorder, or undesirable physiological condition is to be prevented. As used herein, the term "prevention" refers to any activity that reduces the burden on an individual after they exhibit these symptoms. This can be done at the primary, secondary, and / or tertiary prevention levels, where a) primary prevention avoids the occurrence of symptoms / disorders / conditions; b) secondary prevention activities are directed at the early stages of treating a condition / disorder / symptom, thereby increasing the opportunity for intervention to prevent the progression of the condition / disorder / symptom and the appearance of symptoms; and c) tertiary prevention reduces the negative impact of an already established condition / disorder / symptom, for example, by restoring function and / or reducing any condition / disorder / symptom or associated complications. The term "prevent" does not require 100% elimination of the possibility of an event. Rather, it refers to the possibility of the occurrence of an event being reduced in the presence of a compound or method.

[0090] As used herein, the term "effective amount" refers to an amount sufficient to effect beneficial or desired biological and / or clinical results.

[0091] As used herein, the term "pharmaceutically acceptable" carrier refers to a carrier that is non-toxic to cells or mammals exposed to it at the dosages and concentrations used. A "pharmaceutically acceptable" carrier can be, but is not limited to, an organic or inorganic, solid or liquid excipient that is suitable for the selected mode of application, for example, oral application or injection, and is administered in the form of a conventional pharmaceutical preparation, for example, a solid such as a tablet, granule, powder, capsule, or liquid such as a solution, emulsion, suspension, or the like. In many cases, the physiologically acceptable carrier is an aqueous pH buffer solution, for example, a phosphate buffer or a citrate buffer. Physiologically acceptable carriers 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 polyvinylpyrrolidone, 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 non-ionic surfactants such as Tween®, polyethylene glycol (PEG), and Pluronic®. Auxiliaries, stabilizers, emulsifiers, lubricants, binders, pH adjusting agents, isotonicity agents, and other conventional additives may also be added to the carrier.

[0092] As described herein, the products of natural and directed evolution in adeno-associated viruses (AAVs) were used to reverse engineer novel targets that determine the biodistribution of therapeutics. The identified targets open new avenues for the rational engineering of potent and specific therapeutics in humans. Cell-based microarray screening techniques were used to explore AAV interactions with human cell surface and secreted proteins. The screening revealed a panel of novel protein interactions grouped as (1) those targeting AAV serotype 9 (AAV9) and (2) those selectively targeting engineered AAVs evolved from AAV9. These new targets include proteins that can enhance the targeting of therapeutics to specific cell types and tissues, as well as targets that help proteins evade the host immune response. The disclosed target proteins enable novel target-based engineering to introduce and / or modulate interactions with these proteins to alter the biodistribution, efficacy, and immune response of therapeutics.

[0093] Provided herein are novel targets (e.g., of viral vectors) GP2, DPP4, IL-3, DKK3, FAM234A, LRP6, ANPEP, CSF2, and EPYC that can be used to modify the targeting of biologics or chemicals in vitro or in vivo after intravenous, intrathecal, direct, or other 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 with biologics or chemical molecules.

[0094] Provided herein are AAVs whose capsids are directly engineered against GP2, DPP4, IL-3, DKK3, FAM234A, LRP6, ANPEP, CSF2, and / or EPYC, or are combined with molecules directly engineered against GP2, DPP4, IL-3, DKK3, FAM234A, LRP6, ANPEP, CSF2, and / or EPYC.

[0095] Provided herein are antibodies, scFabs, scFvs, and / or alternative protein scaffolds directly engineered against GP2, DPP4, IL-3, DKK3, FAM234A, LRP6, ANPEP, CSF2, and / or EPYC. Provided herein are peptides directly engineered against GP2, DPP4, IL-3, DKK3, FAM234A, LRP6, ANPEP, CSF2, and / or EPYC. Provided herein are small molecules directly engineered against GP2, DPP4, IL-3, DKK3, FAM234A, LRP6, ANPEP, CSF2, and / or EPYC.

[0096] Provided herein are small molecule and biologic-drug conjugates (e.g., antibody-drug conjugates, ADCs) that target GP2, DPP4, IL-3, DKK3, FAM234A, LRP6, ANPEP, CSF2, and / or EPYC. Provided herein are bispecific antibodies containing at least one Fab that target GP2, DPP4, IL-3, DKK3, FAM234A, LRP6, ANPEP, CSF2, and / or EPYC.

[0097] As disclosed herein, AAV capsids directly engineered against GP2, DPP4, IL-3, DKK3, FAM234A, LRP6, ANPEP, CSF2, and / or EPYC can be used for delivery of DNA or ASO molecules, therapeutic proteins, therapeutic small molecules, or other biologics in vitro and in vivo.

[0098] Disclosed herein are agents capable of binding to a protein selected from the group consisting of interleukin 3 (IL3), family 234 member A with sequence similarity (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.

[0099] The antibody or fragment thereof may comprise an Fc domain. The antibody or fragment thereof 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, an scFv, a single-domain antibody, a diabody, 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 an miRNA, shRNA, siRNA, or an oligonucleotide.

[0100] The agent may 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 radioactive label, an enzyme, an enzyme substrate, a quantum dot, an imaging agent, a metal particle, a magnetic particle, and any combination thereof. The agent may be a therapeutic agent.

[0101] The agent may be a targeting peptide. The targeting peptide may be part of a delivery system, which contains a payload to be delivered to cells. The delivery system may include a viral vector or a non-viral vector. The viral vector may include an AAV vector. The targeting peptide may be part of a 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 isolate rh.8, rhesus isolate rh.10, and variants thereof. The non-viral vector may include 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.

[0102] The payload delivered to the cell can be a biomolecule, a non-biomolecule, or a combination thereof. In some embodiments, 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. The payload can be a therapeutic molecule. The nucleic acid sequence 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 can be released from the transduced cell and affect the survival or function of the cell and / or surrounding cells; b) DNA (e.g., a genomic or cDNA sequence) that restores protein function to a human or animal carrying a genetic mutation in that 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 used to evaluate the state of a cell; e) DNA and / or associated guide RNA for genome manipulation; f) a sequence for genome editing via 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. Lipoprotein receptor-related protein 6 (LRP6)

[0103] The present disclosure includes a method for increasing the permeability of the blood-brain barrier. In some embodiments, the method includes providing a targeting peptide that can bind 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 domain) of LRP6. The blood-brain barrier permeability is at least 25%, 50%, 75%, 100%, or more, or at least 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%, 126%, 127%, 128%, 12 %, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values).

[0104] Disclosed herein is a method for delivering a payload to a subject's nervous system. In some embodiments, the method includes providing a targeting peptide or a derivative thereof capable of binding to LRP6, wherein the targeting peptide is part of a delivery system, and the delivery system comprises a payload to be delivered to the nervous system; and administering the delivery system to the subject. The delivery system may include nanoparticles, nanotubes, nanowires, dendrimers, liposomes, ethosomes and aquasomes, polymersomes and niosomes, foams, hydrogels, cubosomes, quantum dots, exosomes, macrophages, and any combination thereof. The delivery system may include a viral vector or a non-viral vector.

[0105] In some embodiments, targeting peptide enhances the binding affinity of viral vectors or non-viral vectors to LRP6.For example, the binding affinity of viral vectors to LRP6 can be enhanced by at least about 2 times (for example, 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 any number or range between these values).The viral vector can include 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 isolate rh.8, rhesus isolate rh.10, and variants thereof. Non-viral vectors may include 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.

[0106] The payload delivered to the nervous system can be a biomolecule, a non-biomolecule, or a combination thereof. In some embodiments, 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. The payload can be a therapeutic molecule. The nucleic acid sequence 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 can be released from the transduced cell and affect the survival or function of the cell and / or surrounding cells; b) DNA (e.g., genomic DNA or cDNA sequence) that restores protein function to humans or animals that have a genetic mutation in that 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 used to evaluate the state of a cell; e) DNA and / or related guide RNA for genome manipulation; f) a sequence for genome editing via 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 endogenous genes.

[0107] The LRP6 gene encodes a member of the low-density lipoprotein (LDL) receptor gene family. The LDL receptor is a transmembrane cell surface protein involved in receptor-mediated endocytosis of lipoproteins and protein ligands. The protein encoded by this gene functions as a receptor or together with Frizzled, a co-receptor for Wnt, thereby transducing the classical Wnt / beta-catenin signaling cascade. Through its interaction with the Wnt / beta-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 gamma-secretase-dependent RIP (regulated intramembrane proteolysis) processing. The NCBI gene ID is 4040. The Ensembl ID is ENSG00000070018. The OMIM® ID is 603507. The UniProtKB / Swiss-Pro ID is O75581.

[0108] The LRP6 can be mouse LRP6. In some embodiments, the LRP6 has an amino acid sequence that has at least 80% sequence identity (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 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) to the amino acid sequence of SEQ ID NO: 29. The LRP6 can be macaque LRP6. In some embodiments, LRP6 has an amino acid sequence that has at least 80% sequence identity (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 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) to the amino acid sequence of SEQ ID NO: 30. LRP6 may be human LRP6. In some embodiments, LRP6 has an amino acid sequence having at least 80% sequence identity (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 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) to the amino acid sequence of SEQ ID NO: 31.

[0109] The methods disclosed herein may include providing a targeting peptide capable of binding to LRP6 or a derivative thereof. Upon binding, the targeting peptide may be capable of interacting with (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having the 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 the amino acid sequence of SEQ ID NO: 31. The targeting peptide may be inserted between two adjacent amino acids in AA587-594 of SEQ ID NO: 11 of the AAV9 vector, or a functional equivalent 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 a functional equivalent 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 replace AA452-460 of SEQ ID NO: 11 of the AAV9 vector, or a functional equivalent of AA588-589 in an amino acid sequence at least 80% identical to SEQ ID NO: 11.

[0110] In some embodiments, the targeting peptide may 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 number 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 the amino acid sequence of any of the sequences of SEQ ID NOs: 1-10. In some embodiments, the targeting peptide comprises at least four contiguous amino acids from any of the sequences of SEQ ID NOs: 1-10. Targeting Peptides

[0111] Disclosed herein are targeting peptides and related AAV particles comprising capsid proteins with one or more targeting peptide inserts for enhanced or improved delivery of target tissues (e.g., cells of the CNS or PNS). In some embodiments, the targeting peptide may direct the AAV particle to a cell or tissue of the CNS. Cells of the CNS may include, but are not limited to, neurons (e.g., excitatory, inhibitory, motor, sensory, autonomic, sympathetic, parasympathetic, Purkinje, Betz, etc.), glial cells (e.g., microglia, astrocytes, oligodendrocytes), and / or brain support cells, such as immune cells (e.g., T cells). Tissues of the CNS may include, but are not limited to, the cortex (e.g., frontal, parietal, occipital, temporal), thalamus, hypothalamus, striatum, putamen, caudate nucleus, hippocampus, entorhinal cortex, basal ganglia, or deep cerebellar nuclei. In some embodiments, the targeting peptide may direct AAV particles to cells or tissues of the PNS. The cells or tissues of the PNS may be, but are not limited to, dorsal root ganglia (DRG). The targeting peptide may direct AAV particles to the CNS (e.g., cortex) after intravenous administration. The targeting peptide may direct AAV particles to the PNS (e.g., DRG) after intravenous administration.

[0112] The targeting peptide may vary in length. In some embodiments, the targeting peptide is 3 to 20 amino acids in length. By way of non-limiting example, the targeting peptide may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 3 to 5, 3 to 8, 3 to 10, 3 to 12, 3 to 15, 3 to 18, 3 to 20, 5 to 10, 5 to 15, 5 to 20, 10 to 12, 10 to 15, 10 to 20, 12 to 20, or 15 to 20 amino acids in length.

[0113] In some embodiments, a targeting peptide or its derivative is provided that can bind to low density lipoprotein receptor-related protein 6 (LRP6).When binding, the targeting peptide can be capable of interacting with (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having the 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 the amino acid sequence of SEQ ID NO:31.

[0114] The targeting peptides of the present disclosure may be identified and / or designed by any method known in the art. As a non-limiting example, the CREATE system described in Deverman et al., (Nature Biotechnology 34(2):204-209 (2016)), and International Patent Application Publication Nos. WO2015038958, WO2017100671, and WO2020028751, the contents of each of which are incorporated herein by reference in their entirety, may be used to identify targeting peptides in mice or other research animals, such as, but not limited to, non-human primates. Targeting peptides may also be designed in silico. Peptides designed in silico may be tested in any in vitro or in vivo model that is evaluated by those skilled in the art.

[0115] Targeting peptides and related AAV particles may be identified from a library of AAV capsids composed of targeting peptide variants. In some embodiments, the targeting peptide may be a seven amino acid sequence (7-mer). In other embodiments, the targeting peptide may be a nine amino acid sequence (9-mer). Targeting peptides may also vary in the way they are created or designed, non-limiting examples of which include random peptide selection, site-saturation mutagenesis, and / or optimization of specific regions of the peptide (e.g., flanking regions or central core).

[0116] In some embodiments, the targeting peptide library comprises targeting peptides seven amino acids (7-mer) in length randomly generated by PCR. In some embodiments, the targeting peptide library comprises targeting peptides with three mutated amino acids. In some embodiments, these three mutated amino acids are consecutive amino acids. In some embodiments, these three mutated amino acids are not consecutive amino acids. In some embodiments, the parent targeting peptide is a 7-mer. In some embodiments, the parent peptide is a 9-mer.

[0117] In some embodiments, the targeting peptide library comprises 7-mer targeting peptides, and amino acids in the targeting peptides and / or flanking sequences are evolved by site-saturation mutagenesis of three consecutive amino acids. In some embodiments, NNK (N=any base; K=G or T) codons are used to create site-saturation mutant sequences. In silico screening of targeting peptides

[0118] Disclosed herein is a method for designing a targeting peptide with specificity for LRP6. In some embodiments, the method comprises in silico generation of one or more targeting peptides that can interact with (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having the 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 the amino acid sequence of SEQ ID NO: 31.

[0119] The step of generating one or more targeting peptides in silico may include the steps of generating a large number of candidate peptides in silico; performing computer-assisted docking simulations for each of the large number of candidate peptides that bind to LRP6; and analyzing the structure of LRP6 bound to one or more of the large number of candidate peptides to identify one or more targeting peptides that can interact with (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having the 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 the amino acid sequence of SEQ ID NO: 31.

[0120] In silico methods can provide a high-throughput approach for screening a large number of candidate peptides and identifying targeting peptides with desired specificity. In some embodiments, the candidate peptides may comprise a portion of an AAV capsid protein. In some embodiments, the candidate peptides are portions 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 in complex with LRP6. Molecular models may be constructed for peptide-protein complexes using any rational computer 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 provided by DeepMind, and AlphaFold-Multimer, available at github.com / deepmind / alphafold), RoseTTAFold (github.com / RosettaCommons / RoseTTAFold), AutoDock, DOCK, FlexX, GOLD, OSPREY, SCWRL, PyMol, SWISS-MODEL (academic.oup.com / nar / article / 46 / Wl / W296 / 5000024), Protein Data Bank (PDB) (available via member organization websites, 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).

[0121] The method can further include evaluating properties or parameters associated with the interaction between the candidate peptide and LRP6 using visualization software such as PyMol, Qlucore Omics Explorer, WebMol, Insight II, Discovery Studio 2.1, and others identifiable to those skilled in the art. The evaluated properties or parameters can include interfacial energy and physical and geometric scoring. Evaluating properties or parameters associated with 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, and / or total molecular mechanics energy. The method can also include determining the total number of interfacial atoms, the total number of atoms in the peptide clashing with LRP6, the bond angle of the peptide, and / or the binding depth of the peptide for each putative 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, and van der Waals energy, as will be understood by those skilled in the art.

[0122] In some embodiments, targeting peptides can be assigned a binding score and ranked based on the binding score. A threshold value can be set to identify desired targeting peptides. The method can include obtaining a binding score for each of a number of candidate peptides that bind to LRP6, and selecting one or more of the candidate peptides that have a binding score above the threshold as a targeting peptide with binding specificity (or high binding specificity) to LRP6.

[0123] A combination of physical and geometric scoring parameters, including interfacial energy, bond angle, and binding pocket depth calculations, can be used to generate a binding score. In some embodiments, the binding score for each of a number of candidate peptide sequences can be obtained by: (1) counting the total number of atoms at the interface between the candidate peptide and LRP6; (2) counting the total number of atoms in the candidate peptide that are in conflict with LRP6; (3) obtaining the bond angles of the candidate peptide; and / or (4) obtaining the binding depth of the candidate peptide.

[0124] The total number of atoms at the interface between the candidate peptide and LRP6 may be the total number of atoms within a cutoff distance between the atoms at the interface of the candidate peptide and the atoms at the interface of LRP6. The cutoff distance may vary in different embodiments. In some embodiments, the cutoff distance is about 5 angstroms, at most 5 angstroms, at most about 5 angstroms (e.g., 2 angstroms, 3 angstroms, 4 angstroms, or 5 angstroms). The number of atoms at the interface between the candidate peptide and LRP6 may vary in different embodiments. For example, the number of atoms at the interface can be about 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 a number or range between any of these values, 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 a number or range between any of these values.

[0125] The total number of atoms in the peptide that collide with LRP6 can be the total number of atoms within the collision distance. The collision distance can be defined as the distance at which a geometric collision occurs between the peptide and the receptor atom. In some embodiments, the collision distance can be about 1 angstrom.

[0126] The bond angle of a peptide can be defined as the angle between the vector from the LRP6 centroid to the LRP6 anchor and the vector from the LRP6 centroid to the peptide centroid. The bond depth can be defined as the difference in distance between the closest point on the peptide to the LRP6 center and the minor radius of the ellipsoidal shell of LRP6 normalized by the minor radius. The bond score can be the sum of contact scores calculated based on the total number of atoms at the interface between the candidate peptide and LRP6, the total number of clashing atoms, the bond angle, and the bond depth. In some embodiments, the bond score is equal to or greater than 0. For example, if the sum of the contact score, bond angle, and bond depth is negative, the bond score is defined as 0. The binding score of a targeting peptide that binds to LRP6 can be about 0, 1, 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, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, or a number or range between any two of these values.

[0127] In some embodiments, the binding score (B ペプチド ) can be defined as follows:

number

[0128] The method may further comprise selecting one or more of the multiple candidate peptides having a binding score above a threshold as a targeting peptide having binding specificity for LRP6. The threshold may be any value defined by the user. In some embodiments, the threshold may be the binding score of a targeting peptide known to have binding specificity for LRP6. In some embodiments, the results from each pairwise comparison (the relative scores between two competing peptides) may be aggregated into a peptide competition metric that ranks the set of candidate peptides according to their receptor-binding probability, as coded in the AlphaFold2 neural network. CREATE System

[0129] In some embodiments, the targeting peptides of the present disclosure are isolated via the CREATE system described in Deverman et al., (Nature Biotechnology 34(2):204-209 (2016)), and International Patent Application Publication Nos. WO2015038958 and WO2017100671, the contents of each of which are incorporated herein by reference in their entirety. "CREATE" or "Cre recombination-based AAV targeted evolution" refers to an AAV capsid selection strategy that selects for capsids that transduce target tissues (e.g., CNS or PNS) after intravenous injection. This method has been demonstrated in a mouse model.

[0130] A library of AAV capsids with one or more targeting peptide inserts has been developed and intravenously administered to transgenic mice. These transgenic Cre-expressing mice can be developed for specific targeting, for example, GFAP-Cre mice can be used to target astrocytes. In some embodiments, a 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.

[0131] Variation of the targeting sequence and transgenic animal model allows for the selection of AAV variants with desired transduction profiles, e.g., tropism for neurons or astrocytes, compared to other AAV serotypes, including parental AAV particles and capsids.

[0132] The CREATE method involves creating a library of targeting peptides that are then assembled into a viral genome backbone containing the parent AAV capsid sequence. The AAV capsid library (AAV particles) is then created, purified, and administered to a transgenic animal (e.g., a mouse). Target tissues are collected, and AAV sequences are selectively recovered from Cre-expressing cells. These sequences are evaluated and characterized to identify targeting peptides that result in enrichment (i.e., enhanced transduction or targeting) in the target tissue. Targeting peptides and related AAV particles can then be created for further testing and characterization. This process is considered a round of evolution or selection. In some embodiments, two or more rounds of evolution are performed. As many as 15 rounds of selection may be performed.

[0133] More specifically, the CREATE system uses a rAAV-Cap-in-cis-lox viral genome containing regulatory elements for the AAV cap and AAV rep genes and a Cre reversible switch. Because this viral genome lacks a fully functional rep gene, required for AAV particle production, rep is provided in trans. A modified AAV2 / 9 Rep-Cap plasmid may be provided in which a stop codon is provided in frame to prevent expression of the VP1-VP3 proteins.

[0134] A capsid library is created using the rAAV-Cap-in-cis-lox viral genome as a backbone. A targeting peptide is inserted into the parent AAV capsid protein (e.g., AAV9) at any position that results in the creation of a fully functional AAV capsid protein and AAV particles. Targeting peptides may be designed by any method known in the art. In some embodiments, targeting peptides are created using polymerase chain reaction (PCR). AAV particles containing capsid proteins with targeting peptide inserts are created, and a viral genome encoding a reporter (e.g., GFP) is packaged therein. These AAV particles (or AAV capsid libraries) are then administered to transgenic mice via intravenous delivery to the tail vein. Administration of these capsid libraries to Cre-expressing mice results in the expression of the reporter payload in target tissues due to Cre expression.

[0135] AAV particles and / or viral genomes may be recovered from the target tissue to identify enriched targeting peptides and associated AAV particles that exhibit enhanced transduction of the target tissue. Enrichment may be determined using standard methods in the art, including, but not limited to, next-generation sequencing (NGS), viral genome quantification, biochemical assays, immunohistochemistry, and / or imaging of the target tissue sample.

[0136] The target tissue may be any cell, tissue, or organ of interest. By way of non-limiting example, samples may be collected from the brain, spinal cord, dorsal root ganglion and associated 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, ovaries, uterus, prostate, testes, and / or any site identified as having a lesion or being of interest.

[0137] Targeting peptides and associated AAV capsid proteins and AAV particles identified using the CREATE system include AAVPHP.B (PHP.B), AAVPHP.A (PHP.A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1-35, AAVPHP.B2 (PHP.B2), AAVPHP.B3 (PHP.B3), AAVPHP.N / PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, and AAVPHP.B- AQP, AAVPHP.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-ST P, 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).

[0138] The CREATE system has proven effective in identifying targeting peptides for enhanced transduction into the CNS of mice after intravenous administration.However, the transfer of knowledge from mice to humans is not always straightforward.The modification of the CREATE system for non-transgenic animals or model systems similar to humans can help identify targeting peptides and related AAV capsids and particles useful for the treatment of human diseases.In some embodiments, the AAV interactors identified herein can be used to address this unmet need and evaluate or create new models for the design of AAV capsids in humans.

[0139] To adapt the CREATE method to non-transgenic animals, a different mechanism is required to modify the target tissue and / or induce Cre expression in cells. In some embodiments, AAV Cre vectors may be used to transduce cells and then induce Cre expression. In some embodiments, these AAV Cre vectors may be AAV1-Cre vectors. AAV Cre vectors may contain a viral genome with a cell-type-specific promoter. These cell-type-specific promoters may be, but are not limited to, CAG, UBC, EF1α, synapsin, GFAP, MBP, VGLUT, VGAT, Nav1.8, parvalbumin, TH, ChaT, and / or any promoter known in the art.

[0140] In some embodiments, these AAV-Cre vectors are delivered to the target tissue by intraparenchymal administration. In some embodiments, the intraparenchymal administration is directly to the subject's putamen. In some embodiments, the intraparenchymal administration is directly to the subject's thalamus. In some embodiments, the intraparenchymal administration is directly to the subject's cortex. In some embodiments, the intraparenchymal administration is indirectly to the subject's cortex. In some embodiments, the intraparenchymal administration is simultaneously to one or more of the subject's putamen, thalamus, and / or cortex, and may be bilaterally administered. In some embodiments, the subject is a non-human primate.

[0141] Regarding the CREATE method developed in mice, the AAV capsid library can be administered intravenously.In another embodiment, the AAV capsid library can be administered 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 time between the administration of the AAV-Cre vector and the AAV capsid library can be several seconds, several minutes, several hours, several days, several weeks, or several years.

[0142] The AAV capsid library may contain AAV particles containing a viral genome encoding a reporter (e.g., GFP). Only those cells in the target tissue (e.g., CNS or DRG) that also express Cre (co-transduced by the intraparenchymal Cre vector) will express the reporter. The target tissue may be collected and analyzed to identify AAV particles and targeting peptides that result in enrichment, i.e., enhanced transduction, in the target tissue. Sample tissues and AAV sequences may be evaluated, analyzed, or characterized using standard methods in the art, including, but not limited to, next-generation sequencing, viral genome quantification, biochemical assays, immunohistochemistry, and / or imaging. Antibody and Peptide Derivatives

[0143] The present disclosure also includes antibodies or fragments thereof comprising an amino acid sequence having binding specificity for a target protein (e.g., LRP6) disclosed herein. The present disclosure also includes peptide derivatives or conjugates thereof having specificity for a protein (e.g., LRP6) disclosed herein.

[0144] In some embodiments, the antibody or fragment thereof can 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, an scFv, a single-domain antibody, a diabody, a multispecific antibody, a bispecific antibody, an anti-idiotypic antibody, a diabody, or a functionally active epitope-binding fragment thereof.

[0145] In some embodiments, the antibody or fragment thereof is a bispecific antibody comprising at least one Fab with specificity for LRP6 or another protein identified herein (e.g., GP2). Bispecific antibodies may comprise separate binding sites directed against different antigens.

[0146] In some embodiments, the antibody or fragment thereof is unable to induce a harmful immune response in the treated subject, e.g., in a human. In some embodiments, the antibodies, fragments, variants, or derivatives of the present disclosure are modified to reduce their immunogenicity using art-recognized techniques. For example, the antibody may be a humanized antibody, a primatized antibody, a deimmunized antibody, or a chimeric antibody.

[0147] In some embodiments, an antibody, fragment, variant, or derivative thereof can further comprise a chemical moiety not naturally associated with the antibody. For example, the antibody or fragment thereof can comprise a flexible linker or be modified to add a functional moiety, such as a detectable label. The antibody, fragment, variant, or derivative thereof can be modified, for example, by covalent or non-covalent attachment of a chemical moiety to the antibody so that the attachment does not interfere with or prevent the antibody from binding to the epitope. In some embodiments, the chemical moiety can be conjugated to the antibody using any technique known in the art.

[0148] The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the peptides, antibodies, fragments, variants, or derivatives thereof of the present disclosure. For example, the polynucleotides of the present disclosure can encode the heavy and light chain variable regions of an antibody, fragments, variants, or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules. In some embodiments, the polynucleotides of the present disclosure can encode portions of the heavy and light chain variable regions of an antibody (e.g., CDR regions), fragments, variants, or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules. Payload Delivery

[0149] Disclosed herein are 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 includes providing a targeting peptide capable of binding to LRP6 or a derivative thereof. The targeting peptide can be part of a delivery system, and the delivery system can include a payload delivered to the nervous system. The method can further include administering the delivery system to a subject.

[0150] In some embodiments, the delivery system comprises nanoparticles, nanotubes, nanowires, dendrimers, liposomes, ethosomes and aquasomes, polymersomes and niosomes, foams, hydrogels, cubosomes, quantum dots, exosomes, macrophages, and combinations thereof. In some embodiments, the delivery system comprises nanoparticles selected from lipid-based nanoparticles, polymeric 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.

[0151] 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 part of the capsid protein of the AAV vector. Adeno-associated virus (AAV) and recombinant AAV (rAAV)

[0152] In some embodiments, the delivery system for delivering a payload across the BBB is an AAV vector. In some embodiments, AAV is a replication-deficient parvovirus, and its single-stranded DNA genome is approximately 4.7 kb long, including a 145-nucleotide inverted terminal repeat (ITR). The ITR plays a role in the integration of AAV DNA into the host cell genome. When AAV infects a host cell, the viral genome is integrated into the host cell chromosome, resulting in latent infection of the cell. In natural systems, helper viruses (e.g., adenoviruses or herpesviruses) provide genes that enable the production of AAV virus in infected cells. In the case of adenovirus, the genes E1A, E1B, E2A, E4, and VA provide helper functions. Upon infection with the helper virus, the AAV provirus is rescued and amplified, and both AAV and adenovirus are produced. In the example of a recombinant AAV vector that does not have the Rep and / or Cap genes, AAV can be non-integrating.

[0153] In some embodiments, the AAV vector can comprise the coding region of one or more proteins of interest.The AAV vector can comprise a 5'AAV ITR, a 3'AAV ITR, a promoter, and a restriction site downstream of the promoter that allows the insertion of a polynucleotide that encodes one or more proteins of interest, and 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 element downstream of the restriction site and upstream of the 3'AAV ITR.

[0154] Viral vectors can comprise additional sequences that make the vector suitable for replication and integration in eukaryotes.In other embodiments, the viral vectors disclosed herein can comprise shuttle elements that make the vector suitable for replication and integration in both prokaryotes and prokaryotes.In some embodiments, viral vectors can comprise 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 can be included in AAV vectors are described in US2012 / 0232133, the entire contents of which are incorporated herein by reference.

[0155] The AAV serotype used to derive the AAV capsid protein can vary. The AAV capsid can be derived from AAV9 or a variant thereof. The AAV capsid can be derived from an AAV selected from AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, human isolate hu.31, human isolate hu.32, rhesus isolate rh.8, and rhesus isolate rh.10. In some embodiments, the AAV capsid protein is derived from AAV9, AAV9 K449R (or K449R AAV9), AAV1, AAVrhl0, AAV-DJ, AAV-DJ8, AAV5, AAVPHP.B(PHP.B), AAVPHP.A(PHP.A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1-35, AAVPHP.B2(PH P.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, A AVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.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, AAVG2 Al 5 / G2A3(G2A3), AAVG2B4(G2B4), AAVG2B5(G2B5), PHP.S, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV6, AAV6.1, A AV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, 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、AAV42-6b AV42-8、AAV42-10、AAV42-11、AAV42-12、AAV42-13、AAV42-15、AAV42-aa、AAV43-1、AAV43-12、AAV43-20、AAV43-21、AAV43-23、AAV43-25、AAV43-5、AAV 44.1、AAV44.2、AAV44.5、AAV223.1、AAV223.2、AAV223.4、AAV223.5、AAV223.6、AAV223.7、AAVl-7 / rh.48、AAVl-8 / rh.49、AAV2-l5 / rh.62、AAV2-3 / rh.6l、AAV2-4 / rh.50、AAV2-5 / rh.5l、AAV3.l / hu.6、AAV3.l / hu.9、AAV3-9 / rh.52、AAV3-1l / rh.53、AAV4-8 / r11.64、AAV4-9 / rh.54、AAV4-l9 / rh.55、AAV5 -3 / rh.57、AAV5-22 / rh.58、AAV7.3 / hu.7、AAVl6.8 / hu.10、AAVl6.l2 / hu.11、AAV29.3 / bb.1、AAV29.5 / bb.2、AAVl06.l / hu.37、AAV1l4.3 / hu.40、AAVl27.2 / hu.4l、AAVl27.5 / hu.42、AAVl28.3 / hu.44、AAVl30.4 / hu.48、AAVl45.l / hu.53、AAVl45.5 / hu.54、AAVl45.6 / hu.55、AAVl6l.l0 / hu.60、AAVl6l.6 / hu.6l、AAV33.l2 / hu.l7、AAV33.4 / hu.l5、AAV33.8 / hu.l6、AAV52 / hu.l9、AAV52.l / hu.20、AAV58.2 / hu.25、AAVA3.3、AAVA3.4、AAVA3.5、AAVA3.7、AAVC1、AAVC2、AAVC5、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2、AAVrh.60、AAVrh.44、AAVrh.65、AAVrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu.12、AAVH6、AAVH-1 / hu.1、AAVH-5 / hu.3、AAVLG-l0 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVN72l-8 / rh.43、AAVCh.5、AAVCh.5Rl、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、AAVhu.1、AAVhu. 2、AAVhu.3、AAVhu.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu.9、AAVhu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.l6、AAVhu.17、AAVhu.18、AAVhu.20、AAVhu. Vhu.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.3 7、AAVhu.39、AAVhu.40、AAVhu.41、AAVhu.42、AAVhu.43、AAVhu.44、AAVhu.44Rl、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48 、AAVhu.48Rl、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. AVhu.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.13 R、AAVrh.14、AAVrh.17、AAVrh.18、AAVrh.19、AAVrh.20、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.6l, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533 A mutant, AAAV, BAAV, goat AAV, bovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhErl.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhERl.23, AAVhEr3.1, AAV2.5T、AAV-PAEC、AAV-LK01、AAV-LK02、AAV-LK03、AAV-LK04、AAV-LK05、AAV-LK06、AAV-LK07、A AV-LK08、AAV-LK09、AAV-LK10、AAV-LK11、AAV-LK12、AAV-LK13、AAV-LK14、AAV-LK15、AAV-L K16、AAV-LK17、AAV-LK18、AAV-LK19、AAV-PAEC2、AAV-PAEC4、AAV-PAEC6、AAV-PAEC7、AAV-P AEC8、AAV-PAEC11、AAV-PAEC12、AAV-2-pre-miRNA-101、AAV-8h、AAV-8b、AAV-h、AAV-b、AAV BC 10-2、AAVシャッフル100-1、AAVシャッフル100-3、AAVシャッフル100-7、AAVシ10-2 AAV 10-6 AAV 10-8 AAV 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.53、AAV4-8 / 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 / h u.28、AAV46.6 / hu.29、AAV128.1 / hu.43、トゥルータイプAAV(ttAAV)、EGRENN AAV 10, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-74, AAV CBr-7.5. CBr-7.7、AAV CBr-7.8、AAV CBr-B7.3、AAV CBr-B7.4、AAV CBr-El、AAV CBr-E2、AAV CBr-E3、AAV CBr-E4、AAV CBr-E5、AAV CBr-e5、AAV CBr-E6、AAV CBr-E7、AAV CBr-E8、AAV CHt-1、AAV CHt-2、AAV CHt-3、AAV CHt-6.1、AAV CHt-6.1、AAV CHt-6.5、AAV CHt-6.6、AAV CHt-6.7、AAV CHt-6.8、AAV CHt-P1、AAV CHt-P2、AAV CHt-P5、AAV CHt-P6、AAV CHt-P8、AAV CHt-P9、AAV CKd-1、AAV CKd-10、AAV CKd-2、AAV CKd-3、AAV CKd-4、AAV CKd-6、AAV CKd-7、AAV CKd-8、AAV CKd-Bl、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 CLvl-l、AAV CLvl-lO、AAV CLvl-2、AAV CLvl-l2、AAV CLvl-3、AAV CLv-13、AAV CLvl-4、AAV Clvl-7、AAV Clvl-8、AAV Clvl-9、AAV CLv-2、AAV CLv-3. , AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-D1, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-El, AAV CLv-Kl, AAV CLv-K3, AAV 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-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-R1, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-l, AAV CSp-l0, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.l0, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, 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 / HSC, AAVF9 / HSC9, its variants, hybrids or chimeras of any of the aforementioned AAV serotypes, or any combination thereof, and may be derived from an AAV serotype selected therefrom. <00'00761><00'00762><00'00763> It should be noted that in the original text, "AAVF8 / HSC8" seems to be incomplete as "HSC" is not fully defined. Also, the Chinese part is translated as accurately as possible based on the context, but the overall text structure might need further clarification depending on the specific technical background. And the 7 - digit tags ,

[0156] , are preserved as they are.The AAV vector can be 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.

[0157] In some embodiments, the AAV vector disclosed herein can be used as an AAV transfer vector to carry 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 host cells.Therefore, what is disclosed herein also includes recombinant AAV virus (rAAV).rAAV can include the AAV capsid protein described herein.

[0158] rAAV can contain chimeric AAV capsids. A "chimeric" AAV capsid refers to a capsid having an exogenous amino acid or amino acid sequence. rAAV can also contain mosaic AAV capsids. A "mosaic" AAV capsid refers to a capsid composed of two or more capsid proteins or polypeptides, each derived from a different AAV serotype. rAAV can be the result of transcapsidation, which in some cases refers to the packaging of an inverted terminal repeat (ITR) from a first serotype into a capsid of a second serotype, where the first and second serotypes are not the same. In some cases, the capsid gene of a parent AAV serotype can be pseudotyped, meaning that the ITRs from a first AAV serotype (e.g., AAV1) are used in a capsid from a second AAV serotype (e.g., AAV9), where the first and second AAV serotypes are not the same. As a non-limiting example, a pseudotyped AAV serotype containing AAV1 ITRs and AAV9 capsid proteins can be designated AAV1 / 9. rAAV may additionally or alternatively contain a capsid that has been engineered to express an exogenous ligand-binding moiety (e.g., a receptor), or a modified native receptor.

[0159] In some embodiments, the rAAV capsid protein contains one or more amino acid substitutions or insertions in the amino acid sequence of the AAV capsid protein. The rAAV capsid proteins described herein have, in some cases, an amino acid insertion or substitution that is heterologous to the wild-type AAV capsid protein at the amino acid position of the insertion or substitution. In some embodiments, the amino acid at the amino acid position of the insertion or substitution is not endogenous to the wild-type AAV capsid protein. The amino acid may be a naturally occurring amino acid at the same or equivalent amino acid position as an insertion or substitution in a different AAV capsid protein. The AAV capsid protein from which the engineered AAV capsid protein of the present disclosure is produced may be referred to as the "parent" or "wild-type" AAV capsid protein, or the "corresponding unmodified capsid protein." In some cases, the parent AAV capsid protein has a serotype selected from AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.The complete genome of AAV-1 is provided in GenBank accession number NC_002077; the complete genome of AAV-2 is provided in GenBank accession number NC_001401 and Srivastava et al., J. Virol., 45: 555-564 (1983); the complete genome of AAV-3 is provided in GenBank accession number NC_1829; the complete genome of AAV-4 is provided in GenBank accession number NC_001829; the AAV-5 genome is provided in GenBank accession number AF085716; the complete genome of AAV-6 is provided in GenBank accession number NC_001862; at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank accession numbers AX753246 and AX753249, respectively; and 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); a portion of the AAV-12 genome is provided in Genbank accession number DQ813647; and a portion of the AAV-13 genome is provided in Genbank accession number EU285562. At least a portion of the AAV-DJ genome is provided in Grimm, D. et al. J. Virol. 82, 5887-5911 (2008).

[0160] In some embodiments, the rAAV vector disclosed herein can carry a transgene encoding the targeting peptide described herein that can bind to LRP6.The targeting peptide can be part of the capsid of rAAV.The present disclosure also includes AAV capsid proteins.The AAV capsid proteins can include the targeting peptide disclosed herein.

[0161] The location of the targeting peptide within the capsid protein can vary. In some embodiments, the targeting peptide can be inserted between two adjacent amino acids AA586-595 of the AAV9 capsid protein or its functional equivalent in other AAV capsid proteins (e.g., between AA586 and AA587, AA587 and AA588, AA588 and AA589, AA589 and AA590, AA590 and AA591, AA591 and AA592, AA592 and AA593, AA593, AA594 and AA595). The AAV vector can be selected from AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-DJ, human isolate hu.31, human isolate hu.32, rhesus isolate rh.8, rhesus 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 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.

[0162] The targeting peptide can be inserted between AA588 and AA589 of the AAV9 capsid protein or its functional equivalent in other AAV capsid proteins. The two adjacent amino acids can be AA588 and AA589. In some embodiments, the targeting peptide is inserted between AA587 and AA590 of the AAV9 capsid protein or its functional equivalent in other AAV capsid proteins.

[0163] Upon binding to LRP6, the targeting peptide may be capable of interacting with (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having the 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 the amino acid sequence of SEQ ID NO: 31.

[0164] The binding specificity between the targeting peptides carried by rAAV and LRP6 can be screened using the multiplexed Cre recombination-based AAV targeted evolution (CREATE) method (M-CREATE). In M-CREATE, the rAAV nucleic acid contains a target sequence flanked by two lox sequences. The rAAV is administered to an animal (e.g., a mouse) along with a gene encoding Cre recombinase, which is expressed only in target cells (e.g., endothelial cells in the brain). The target sequence of the rAAV, which can enter the target cells, is inverted by the Cre recombinase expressed in the target cells. The nucleic acid of the rAAV lacking specificity for the target cell does not have access to the Cre recombinase, and therefore the target sequence is not inverted. Therefore, after sequencing the nucleic acid of the rAAV recovered from the animal, the targeting peptide of the rAAV containing the inverted target sequence has specificity for the target cell. The M-CREATE method is described in detail in U.S. Patent Publication No. US20170166926A1, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0165] The creation of viral vectors can be achieved using any suitable genetic engineering technique known in the art, including, but not limited to, standard techniques of restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, as described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY (1989)). Viral vectors can incorporate sequences derived from the genome of any known organism. The sequences can be incorporated in their native form or can be modified in any way to obtain the desired activity. For example, the sequences can include insertions, deletions, or substitutions. Use of AAV vectors and rAAV for payload delivery

[0166] Disclosed herein also includes compositions for use in delivering a payload (e.g., a pharmaceutical agent) to a target environment, such as the nervous system of a subject. The composition can include (1) an AAV capsid protein disclosed herein, and (2) an AAV containing the agent to be delivered to the target environment (e.g., the nervous system) of the subject.

[0167] 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.

[0168] The pharmaceutical agent to be delivered may comprise nucleic acid, peptide, small molecule, aptamer, or a combination thereof.The AAV vector disclosed herein can be efficiently transduced into the target environment (e.g., CNS) to deliver, for example, nucleic acid.In some embodiments, a method for delivering nucleic acid sequence to the nervous system is provided.The protein can be part of the capsid of AAV.The AAV can comprise the nucleic acid sequence to be delivered to the nervous system.The AAV can then be administered to a subject.

[0169] The nucleic acid sequences delivered to the nervous system can include one or more of: a) sequences encoding trophic factors, growth factors, or other soluble factors that are released from the transduced cell and that may affect the survival or function of that cell and / or surrounding cells; b) DNA (e.g., genomic or cDNA sequences) that restore protein function to a human or animal carrying a genetic mutation in that 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 used to assess the state of a cell; e) DNA and / or associated guide RNA for performing genome manipulation; f) sequences for genome editing via homologous recombination; g) DNA sequences encoding therapeutic RNAs; h) shRNA or artificial miRNA delivery systems; or i) DNA sequences that affect the splicing of endogenous genes.

[0170] In some embodiments, the vector may also include regulatory control elements known to those of skill in the art that affect expression of the RNA and / or protein products encoded by the polynucleotide in the desired cells of a subject.

[0171] Functionally, the expression of a polynucleotide can be controlled, at least in part, by an operably linked regulatory element, such that the element modulates the transcription of the polynucleotide, the transport, processing, and stability of the RNA encoded by the polynucleotide, and, if appropriate, the translation of the transcript. A specific example of an expression control element is a promoter, which is usually located 5' of the transcribed sequence. Another example of an expression control element is an enhancer, which 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 intron splicing. Another example of a regulatory element is a transcription termination signal and / or a polyadenylation sequence.

[0172] 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 specific cells, tissues, or organs because they are recognized by transcriptional activator proteins or other transcriptional regulators that are specific to these cells, tissues, or organ types.

[0173] Expression control elements also include ubiquitous or promiscuous promoters / enhancers that can drive expression of a polynucleotide in many different cell types. Such elements include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter / enhancer sequence, the Rous sarcoma virus (RSV) promoter / enhancer sequence, the CMV, chicken β-actin, rabbit β-globin (CAG) promoter / enhancer sequence, and other viral promoters / enhancers active in a variety of mammalian cell types; promoter / enhancer sequences derived from ubiquitously or promiscuous mammalian genes, including, but not limited to, beta-actin, ubiquitin, or EF1 alpha; or synthetic elements that do not occur in nature.

[0174] Expression control elements can also confer expression in a regulatable manner, i.e., a signal or stimulus increases or decreases the expression of an operably linked polynucleotide. Regulatable elements that increase the expression of an operably linked polynucleotide in response to a signal or stimulus are also referred to as "inducible elements" (i.e., they are induced by the signal). Specific examples include, but are not limited to, hormone (e.g., steroid)-inducible promoters. Regulatable elements that decrease the expression of an operably linked polynucleotide in response to a signal or stimulus are referred to as "repressive elements" (i.e., the signal decreases expression, such that expression increases when the signal is removed or absent). Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal or stimulus present, with greater amounts of signal or stimulus resulting in a greater increase or decrease in expression.

[0175] The nucleic acid (e.g., a heterologous nucleic acid) can include a 5'ITR and a 3'ITR. The agent can include a DNA sequence encoding a protein (e.g., a trophic factor, a growth factor, or a soluble protein). The nucleic acid can include, for example, a promoter operably linked to a polynucleotide encoding a protein or RNA agent. The promoter can be capable of inducing transcription of the polynucleotide. Transcription of the polynucleotide can result in a transcript. The nucleic acid can include one or more of a 5'UTR, a 3'UTR, a mini-promoter, an enhancer, a splicing signal, a polyadenylation signal, a terminator, one or more silencer effector binding sequences, a proteolytic signal, and an internal ribosome entry element (IRES). The silencer effector can include 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. Silencer effector can bind to one or more silencer effector binding sequences, thereby reducing the stability of transcripts and / or reducing the translation of transcripts.In some embodiments, silencing effector comprises one or more miRNA binding sites (for example, miR-122 binding sites).MiRNA binding sites are typically operably linked to regulatory elements located in the 3'UTR of the transcribed sequence.The binding of miRNA to target transcripts (in complex with RNA-induced silencing complex, RISC) can reduce the expression of target transcripts through translation inhibition and / or transcript degradation.

[0176] The polynucleotide can further comprise a transcript stabilization element. The transcript stabilization element can 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 can be an RNA agent or can encode an RNA agent. The RNA agent can 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 a gene of interest in a cell. In some embodiments, the gene of interest can be selected from SOD1, MAPT, APOE, HTT, C90RF72, TDP-43, APP, BACE, SNCA, ATXN1, ATXN2, ATXN3, ATXN7, SCN1A-SCN5A, and SCN8A-SCN11A. The nucleic acid can further comprise a polynucleotide encoding one or more secondary proteins, wherein the protein and the one or more secondary proteins can comprise a synthetic protein circuit. The nucleic acid can comprise a single-stranded AAV (ssAAV) vector or a self-complementary AAV (scAAV) vector.

[0177] The promoter may include a ubiquitous promoter, such as a cytomegalovirus (CMV) immediate early promoter, a CMV promoter, a simian virus 40 (SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, a RSV promoter, a herpes simplex virus (HSV) (thymidine kinase) promoter, a H5, P7.5, and P11 promoter from vaccinia virus, and an elongation factor 1-associated promoter. The promoter may be selected from the group consisting of: EF1a promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa 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.

[0178] The promoter may 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, an RU-486-responsive promoter, or a combination thereof.

[0179] The promoter may include 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 α-myosin heavy chain (α-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 II promoter, a tubulin alpha I promoter, a neuron-specific enolase promoter, a platelet-derived growth factor beta 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 include a muscle-specific promoter, such as an MCK promoter.

[0180] The promoter may comprise an intron sequence. The promoter may comprise a bidirectional promoter and / or enhancer. In some embodiments, the enhancer may be a CMV enhancer. One or more cells of the subject may comprise an endogenous version of a nucleic acid sequence (e.g., a gene), and the promoter may comprise or be derived from the endogenous version of the promoter. In some embodiments, one or more cells of the subject comprise an endogenous version of a nucleic acid sequence, and the sequence is not shortened compared to the endogenous version.

[0181] Promoters can vary in length, for example, less than 1 kb. In other embodiments, promoters are greater than 1 kb. Promoters can be 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1 The promoter may have a length of 50, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 bp, or a number or range between any two of these values, or greater than 800 bp. The promoter may provide for expression of the therapeutic gene expression product for a period of time in a targeted tissue, for example, but not limited to, the CNS.Therapeutic gene expression products were measured at the following times: 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, 9th, 10th, 11th, 12th, 13th, 2 weeks, 15th, 16th, 17th, 18th, 19th, 20th, 3 weeks, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st, 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, 1 4 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 years, It can be 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 ​​years, 49 years, 50 years, 55 years, 60 years, 65 years, or a number or range between any two of these values, or a period of more than 65 years.

[0182] As used herein, "protein of interest" can be any protein, including naturally occurring and non-naturally occurring proteins. In some embodiments, the polynucleotide encoding one or more proteins of interest can be present in one of the AAV vectors disclosed herein, wherein the polynucleotide is operably linked to a promoter. In some examples, the promoter can drive the expression of the protein of interest in host cells (e.g., endothelial cells). In some embodiments, the protein of interest is an anti-tau antibody, an anti-AB antibody, and / or an ApoE isoform.

[0183] The proteins can include 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 sarco / 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.

[0184] The protein may include a disease-related protein. In some embodiments, the expression level of the disease-related protein correlates with the appearance and / or progression of the disease. The protein may include methyl-CpG binding protein 2 (MeCP2), DRK1A, KAT6A, NIPBL, HDAC4, UBE3A, EHMT1, one or more genes encoded on chromosome 9q34.3, NPHP1, LIMK1, one or more genes encoded on chromosome 7q11.23, P53, TPI1, FGFR1 and related genes, RA1, SHANK3, CLN3, NF-1, TP53, PFK, CD40L, CYP19A1, PGRN, CHRNA7, PMP22, CD40LG, derivatives thereof, or any combination thereof.

[0185] In some embodiments, the nucleic acid may comprise DNA (e.g., a cDNA or genomic DNA sequence) encoding a protein for controlling or monitoring a cellular activity or state and / or assessing a cellular state. The protein may comprise a fluorescent activity, a polymerase activity, a protease activity, a phosphatase activity, a kinase activity, a sumoylating activity, a desumoylating activity, a ribosylation activity, a deribosylation activity, a myristoylating activity, a demyristoylating activity, or any combination thereof. The protein may comprise a nuclease activity, a methyltransferase activity, a demethylase activity, a DNA repair activity, a DNA damage activity, a deamination activity, a dismutase activity, an alkylation activity, a depurination activity, an oxidation activity, a pyrimidine dimer formation activity, an integrase activity, a transposase activity, a recombinase activity, a polymerase activity, a ligase activity, a helicase activity, a photolyase activity, a glycosylase activity, an acetyltransferase activity, a deacetylase activity, an adenylation activity, a deadenylation activity, or any combination thereof. The protein may comprise a nuclear localization signal (NLS) or a nuclear export signal (NES).

[0186] The protein may include a CRE recombinase, a GCaMP, a cell therapy component, a knockdown gene therapy component, a cell surface-exposed epitope, or any combination thereof. The protein may include a chimeric antigen receptor. The protein may 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).

[0187] In some embodiments, the nucleic acid may comprise a DNA (e.g., a cDNA or genomic DNA sequence) encoding a protein for gene editing, or a guide RNA; or a DNA sequence for genome editing via homologous recombination. The protein may comprise a programmable nuclease. In some embodiments, the programmable nuclease is selected from SpCas9 or a derivative thereof; 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 fusions, BE3, and dcas9-deaminase fusions; 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 include Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), zinc finger nucleases, TAL effector nucleases, meganucleases, MegaTALs, Tev-m TALENs, 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, Cmr5, C The nucleic acid and / or rAAV may include mr6, 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. The nucleic acid and / or rAAV may include a polynucleotide encoding (i) a targeting molecule and / or (ii) a donor nucleic acid. The targeting molecule may be capable of associating with a programmable nuclease. The targeting molecule may include single-stranded DNA or single-stranded RNA. The targeting molecule may include a single guide RNA (sgRNA).

[0188] The rAAV disclosed herein can include one or more of the nucleic acids disclosed herein. The nucleic acid can include a polynucleotide encoding a protein. The nucleic acid can be an RNA agent or can encode an RNA agent. The nucleic acid can include a promoter operably linked to a polynucleotide encoding a protein. As disclosed herein, the gene, in some embodiments, is operably linked to appropriate regulatory elements. The one or more genes of the nucleic acid can include 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 can include components of one or more synthetic protein circuits. The one or more genes of the nucleic acid can include an entire synthetic protein circuit including one or more synthetic protein circuit components. The one or more genes of the nucleic acid can include two or more synthetic protein circuits.

[0189] Protein can be any protein, including naturally occurring protein and non-naturally occurring protein.Examples include but are not limited to luciferase; fluorescent protein (for example, GFP); growth hormone (GH) and its variant; insulin-like growth factor (IGF) and its variant; granulocyte colony-stimulating factor (G-CSF) and its variant; erythropoietin (EPO) and its variant; insulin, for example, proinsulin, preproinsulin, insulin, insulin analogue etc.; antibody and its variant, for example, hybrid antibody, chimeric antibody, humanized antibody, monoclonal antibody; antibody antigen-binding fragment (Fab fragment), antibody single-chain variable fragment (scFV fragment); dystrophin and its variant; coagulation factor and its variant; cystic fibrosis transmembrane conductance regulator (CFTR) and its variant; and interferon and its variant.

[0190] 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, e.g., proinsulin, preproinsulin, insulin, insulin analogs, etc.; antibodies and variants thereof, e.g., 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.

[0191] 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 clotting factors; colony-stimulating factors (CSF); interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, and the like; growth factors, such as keratinocyte growth factors (GF), stem cell factors (SCF), fibroblast growth factors (FGF, e.g., basic FGF and acidic FGF), hepatocyte growth factor (HGF), insulin-like growth factors (IGF), bone morphogenetic proteins (BMP), epidermal growth factor (EGF), growth differentiation factor-9 (GDF-9), hepatoma-derived growth factor (HDGF), myostatin (GDF-8), nerve growth factor (NGF), neurotrophins, platelet-derived growth factor (PGF), and the like. 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 gamma / delta T cell receptor, ligand-binding fragments of soluble receptors, enzymes, such as glucosidase, imiglucarase, beta-glucocerebrosidase, and alglucerase; enzyme activators, such as tissue plasminogen activator; chemokines, such as IP-10, interferon-gamma-induced monokine (Mig), Groa / IL-S, RANTES, MIP-1, a, MIPI-β, MCP-1, PF-4, etc.; angiogenic agents, such as vascular endothelial growth factors (VEGF, e.g., VEGF121, VEGF165, VEGF-C, VEGF-2), transforming growth factor-beta, basic fibroblast growth factor, glioma-derived growth factor, angiogenin, angiogenin-2, etc.; anti-angiogenic agents, such as soluble VEGF receptors; protein vaccines;Neuroactive peptides, such as nerve growth factor (NGF), bradykinin, cholecystokinin, gastritis, 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 These include antihistamines; 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, progesterone; macrophage-activating factor; tumor necrosis factor (TNF); neutrophil chemotactic factor (NCF); nerve growth factor; tissue inhibitor of metalloproteinases; vasoactive intestinal peptide; angiogenin; angiotropin; fibrin; hirudin; IL-1 receptor antagonists; and others. 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, e.g., factor IX or factor X; dystrophin or nini-dystrophin; lysosomal acid lipase; phenylalanine hydroxylase (PAH); glycogen storage disease-related enzymes, e.g., glucose-6-phosphate, acid maltase, glycogen debranching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase (e.g., PHKA2), glucose transporters (e.g., GLUT2), aldolase A, beta-enolase, and glycogen synthase; lysosomal enzymes (e.g., beta-N-acetylhexosaminidase A);and any variants thereof;

[0192] 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 part or all of two or more proteins, or a fusion protein comprising all or a portion of any of the aforementioned proteins.

[0193] The viral vector can comprise a polynucleotide comprising coding regions for two or more proteins of interest, and the two or more proteins of interest can be the same or different from each other. In some embodiments, the two or more proteins of interest are associated with polypeptides, such as the light and heavy chains of the same antibody.

[0194] The protein of interest may be a multi-subunit protein. For example, the protein of interest may comprise two or more subunits or two or more independent polypeptide chains. In some embodiments, the protein of interest may be an antibody, including, but not limited to, various antibody 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 capable of binding to an antigen. In some embodiments, the antibody is a full-length antibody. In some embodiments, the protein of interest is not an immunoadhesin.

[0195] In some embodiments, the resulting targeting molecules can be used in methods and / or therapies for in vivo gene transfer applications to long-lived cell populations, which 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, rAAVs can also be used for non-therapeutic scientific research, such as in vivo delivery of transgenes for optogenetics, gene overexpression, gene knockdown with shRNA or miRNA, modulation of endogenous miRNAs 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.

[0196] In some embodiments, the gene encodes immunogenic material that can stimulate an immune response (e.g., an adaptive immune response), such as, for example, an antigenic peptide or protein from a pathogen. Expression of the antigen can stimulate the body's adaptive immune system to provide an adaptive immune response. As such, it is contemplated that some embodiments of the nucleic acids provided herein can be used as (e.g., as) vaccines for the prevention or treatment of infectious diseases.

[0197] As described herein, the nucleotide sequence encoding a protein can be modified to improve the expression efficiency of the protein.The method that can be used to improve the transcription and / or translation of the gene herein is not particularly limited.For example, the nucleotide sequence can be modified to better reflect the codon usage frequency of the host, which increases gene expression (e.g., protein production) in the host (e.g., mammal).

[0198] The degree of gene expression in target cell can vary.The amount of protein expressed in subject (for example, CNS of subject) can vary.For example, in some embodiments, protein can be expressed in subject at the amount of at least about 9 μg / ml, at least about 10 μg / ml, at least about 50 μg / ml, at least about 100 μg / ml, at least about 200 μg / ml, at least about 300 μg / ml, at least about 400 μg / ml, at least about 500 μg / ml, at least about 600 μg / ml, at least about 700 μg / ml, at least about 800 μg / ml, at least about 900 μg / ml or at least about 1000 μg / ml. In some embodiments, the protein is expressed in a subject at an amount of 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. Those skilled in the art will understand that the expression level required for a protein for a method to be effective may vary depending on non-limiting factors, such as the particular protein and the subject being treated, and that an effective amount of protein can be easily determined by one of ordinary skill in the art using conventional methods known in the art without undue experimentation.

[0199] The agent (e.g., a therapeutic agent) can be an inducer of cell death. The agent can induce cell death through a non-intrinsic cell death pathway (e.g., a bacterial pore-forming toxin). In some embodiments, the agent (e.g., a protein encoded by the nucleic acid) can be a pro-survival protein. In some embodiments, the agent is a modulator of the immune system. The agent can activate an adaptive immune response, an innate immune response, or both. In some embodiments, the nucleic acid encodes immunogenic material that can stimulate an immune response (e.g., an adaptive immune response), such as, for example, an antigenic peptide or protein from a pathogen. Expression of the antigen can stimulate the body's adaptive immune system to provide an adaptive immune response. As such, it is contemplated that some embodiments of the compositions provided herein can be used as (e.g., as) a vaccine for the prevention or treatment of infectious diseases. The protein can include a CRE recombinase, a GCaMP, a cell therapy component, a knockdown gene therapy component, a cell surface-exposed epitope, or any combination thereof. In some embodiments, the proteins include CFTR, GDE, OTOF, DYSF, MYO7A, ABCA4, F8, CEP290, CDH23, DMD, and ALMS1.

[0200] Agents (e.g., therapeutic agents) may include non-protein-coding genes, such as RNA agents, e.g., sequences encoding antisense RNA, RNAi, shRNA, and microRNA (miRNA), miRNA sponges or decoys, recombinase delivery for conditional gene deletion, conditional (recombinase-dependent) expression, including those required for the gene editing components described herein. Non-protein-coding genes may also encode tRNA, rRNA, tmRNA, piRNA, double-stranded RNA, snRNA, snoRNA, and / or long non-coding RNA (lncRNA). In some embodiments, RNA agents may include unnatural or modified nucleotides (e.g., pseudouridine). In some embodiments, non-protein-coding genes may modulate the expression or activity of target genes or gene expression products. For example, the RNAs described herein may be used to inhibit gene expression in target cells, e.g., cells in the central nervous system (CNS). In some embodiments, inhibition of gene expression refers to at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% inhibition. In some cases, the protein product of the targeted gene is inhibited by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99%. The gene can be either a wild-type gene or a gene with at least one mutation. The targeted protein can be a wild-type protein or a protein with at least one mutation.

[0201] Examples of genes encoding therapeutic proteins include sequences associated with signal transduction biochemical pathways, such as signal transduction biochemical pathway-associated genes or polynucleotides (e.g., signal transducers). In some embodiments, the methods and compositions disclosed herein include knockdown of endogenous signal transducers, achieved by regulated expression of proteins, including appropriate versions of the signal transducers. Examples of DNA or RNA sequences contemplated herein include sequences for disease-associated genes or polynucleotides. A "disease-associated" gene or polynucleotide refers to any gene or polynucleotide that results in abnormal levels or abnormal forms of transcription or translation products in cells derived from disease-affected tissue compared to non-diseased control tissues or cells. This may be a gene that becomes expressed at abnormally high levels; or a gene that becomes expressed at abnormally low levels, where altered expression correlates with the onset and / or progression of the disease. A disease-associated gene also refers to a gene with a mutation or genetic variation that is directly responsible for the pathogenesis of the disease or is in linkage disequilibrium with a gene responsible for the pathogenesis of the disease. The transcription or translation product may be known or unknown, and may be at normal or abnormal levels. Signal transduction substances can be associated with one or more diseases or disorders. In some embodiments, diseases or disorders are characterized by the abnormal signal transduction of one or more signal transduction substances disclosed herein. In some embodiments, the activation level of signal transduction substances correlates with the appearance and / or progression of diseases or disorders. The activation level of signal transduction substances can be a direct or indirect cause of the pathogenesis of diseases or disorders.

[0202] Many proteins (e.g., enzymes) can be secreted and exhibit cross-correlation effects. For these, genetic material can be delivered to brain endothelial cells using the AAVs disclosed herein, transforming these cells into biofactories to produce and distribute therapeutic agents to other cell types. For example, production of secreted Sparcl1 / Hevin protein in brain endothelial cells can rescue the thalamocortical synapse loss phenotype of Hevin KO mice. This proof-of-concept supports the brain endothelial cell biofactory model for the production of enzymes, antibodies, or other biotherapeutics, providing a novel therapeutic approach for diseases such as lysosomal storage diseases.

[0203] In some embodiments, rAAVs having capsid proteins comprising one or more targeting peptides disclosed herein can be used to deliver genes to specific cell types in a target environment of a subject. For example, rAAVs can be used to deliver genes to neurons and glia in the nervous system (including the 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 the expression of mutant huntingtin in patients with Huntington's disease, e.g., by incorporating a huntingtin-specific microRNA expression cassette into the rAAV genome and packaging the rAAV genome into a variant rAAV for delivery, e.g., through the vasculature; (ii) deliver a functional copy of the frataxin gene to patients with Friedreich's ataxia; (iii) restore expression of an enzyme important for normal lysosomal function in patients lacking expression of the enzyme due to a genetic mutation (e.g., patients with Niemann-Pick disease, mucopolysaccharidosis III, and / or Gaucher disease); (iv) use rAAV to generate animal models of disease; or combinations thereof.

[0204] The subject in need may be suffering from or at risk of developing 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 types 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 may be Krabbe disease, Sandhoff disease, Tay-Sachs disease, Gaucher disease (types I, II, or III), Niemann-Pick disease (NPC1 or NPC2 deficiency), Hurler syndrome, Pompe disease, or Batten disease.

[0205] In some embodiments, the subject is suffering from an acute condition or injury. A subject in need may be suffering from, at risk of developing, or has suffered from a stroke, traumatic brain injury, epilepsy, or spinal cord injury. Pharmaceutical Compositions and Methods of Administration

[0206] Also disclosed herein are pharmaceutical compositions comprising one or more of the rAAV viruses (or other delivery systems) disclosed herein and one or more pharmaceutically acceptable carriers. The compositions can also include additional components, such as diluents, stabilizers, excipients, and adjuvants. As used herein, a "pharmaceutically acceptable" carrier, excipient, diluent, adjuvant, or stabilizer is non-toxic (preferably inert) to cells or subjects exposed to it at the dosage and concentration used, or has an acceptable level of toxicity as determined by one skilled in the art. Carriers, diluents and adjuvants can 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 dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween®, Pluronic®, or polyethylene glycol (PEG).In some embodiments, the physiologically acceptable carrier is an aqueous pH buffered solution.

[0207] Disclosed herein includes a method for delivering a drug to the nervous system of a subject. In some embodiments, the method includes providing an AAV vector comprising an AAV capsid protein disclosed herein. In some embodiments, the AAV vector comprises a drug to be delivered to the nervous system. In some embodiments, the method includes administering the AAV vector to a subject. The composition may be for intravenous administration. The composition may be for systemic administration. The drug may be delivered to the endothelial lining of the ventricles in the brain, the central canal of the spinal cord, capillaries in the brain, arterioles in the brain, arteries in the brain, or a combination thereof of the subject. The subject may be an adult animal.

[0208] The titer of the administered rAAV varies depending on, for example, the specific rAAV, the mode of administration, the goal of treatment, the individual, and the targeted cell type, and can be determined by standard methods in the art. As is readily apparent to those skilled in the art, the useful in vivo dosage and specific mode of administration of the administered recombinant virus 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 for which the recombinant virus is employed. Determining the effective dosage level, which is the dosage level required to achieve the desired result, can be achieved by those skilled in the art using routine pharmacological methods. Typically, human clinical application of the product begins at a lower dosage level, and the dosage level is increased until the desired effect is achieved. Alternatively, accepted in vitro studies can be used to establish the useful dose and route of administration of the composition identified by this method using established pharmacological methods.

[0209] The exact dosage can in most cases be determined on a drug-by-drug basis, and some generalizations regarding dosage can be made. In some embodiments, rAAV for delivery of an agent to a subject's nervous system (e.g., CNS) is administered at a dose of 1 x 10 per kg of subject, e.g., via injection. 10 Recombinant virus viral genome (vg) ~ 2 x 10 per kg l4 vg, e.g., 5 x 10 11 vg / kg~5×l0 12 In some embodiments, the dose of rAAV administered to a subject is 2 x 10 vg / kg. 14 In some embodiments, the dose of rAAV administered to a subject is 5×10 per kg or less. 12 In some embodiments, the dose of rAAV administered to a subject is 5×10 vg or less. 11 vg or less.

[0210] Effective doses and dosages of pharmaceutical compositions for preventing or treating a disease or condition disclosed herein are defined by an observed beneficial response related to the disease or condition, or symptoms of the disease or condition. A beneficial response includes preventing, alleviating, halting, or curing the disease or condition, or 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 biomarker, transcriptomic risk profile, or gut microbiome in a subject. "Improvement," as used herein, refers to a shift in the presence, level, or activity toward that observed in a normal individual (e.g., an individual not suffering from the disease or condition). In instances where a therapeutic rAAV composition is not therapeutically effective or does not provide sufficient relief of the disease or condition, or symptoms of the disease or condition, the dosage and / or route of administration may be altered, or additional agents may be administered to the subject along with the therapeutic rAAV composition. In some embodiments, when a patient is initiated on a regimen of a therapeutic rAAV composition, the patient also stops the second treatment regimen (e.g., dose tapering).

[0211] In some embodiments, pharmaceutical compositions according to the present disclosure are administered one or more times daily at a dosage level sufficient to deliver 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 per day to achieve the desired therapeutic, diagnostic, or prophylactic effect. It will be appreciated that the above dosage concentrations can be converted to vg or viral genomes per kg or to total viral genomes administered by one skilled in the art.

[0212] In some embodiments, the dose of the pharmaceutical composition is at least 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , or 10 17 , 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 is 2 x 10 7 , 2 × 10 8 , 2 × 10 9 , 2 × 10 10 , 2 × 10 11 , 2 × 10 12 , 2 × 10 13 , 2 × 1014 , 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 x 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 x 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 x 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 x 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 , 6×1016 , 6×10 17 , or a range between any two of these values. In some cases, the concentration of infectious particles is 7 x 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 is 8 x 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 is 9 x 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.

[0213] The recombinant virus 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 a route standard in the art. Administration can be systemic administration. Administration can be intravenous administration.

[0214] Non-limiting examples of routes include intramuscular, intravaginal, intravenous, intraperitoneal, subcutaneous, epicutaneous, intradermal, rectal, intraocular, pulmonary, intracranial, intraosseous, oral, buccal, systemic, or nasal. In some embodiments, the recombinant virus is administered to the subject by systemic transduction. In some embodiments, the recombinant virus is administered to the subject by intramuscular injection. In some embodiments, the rAAV is administered to the subject by a parenteral route (e.g., by intravenous, intramuscular, or subcutaneous injection), by surface scarification, or by inoculation into a body cavity of the subject. The route of administration and the serotype of the AAV component of the rAAV virus can be easily determined by one skilled in the art, taking into account the infection and / or disease state to be treated and the target cell / tissue for expressing the protein of interest. In some embodiments, it may be advantageous to administer the rAAV via intravenous administration. The variant AAVs provided herein can advantageously provide intravenous administration of the vector with enhanced tropism to the CNS.

[0215] In some embodiments, the subject is a primate, and the agent is delivered to endothelial cells and / or neurons of the nervous system. The nervous system can be the central nervous system (CNS). The agent can be delivered to endothelial cells of the subject's nervous system at least 1.5-fold, 2-fold, or 3-fold more efficiently than the agent is delivered to neurons of the nervous system. In some embodiments, the agent is delivered to endothelial cells of the subject's nervous system more than 3-fold more efficiently (e.g., 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or a number or range between any of these values) than the agent is delivered to neurons of the nervous system.

[0216] Disclosed herein includes methods for delivering an agent (e.g., a therapeutic agent) to a cell. In some embodiments, the method includes contacting a cell with an AAV vector comprising an AAV capsid protein disclosed herein. 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, contacting the AAV vector with the cell occurs 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 an arteriole in the brain, a cell in an artery in the brain, a cell in a cerebral blood vessel, or a combination thereof.

[0217] AAV vector can be AAV9 vector or its variant.In some embodiments, AAV vector is selected from AAV1, AAV2, AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, human isolate hu.31, human isolate hu.32, rhesus isolate rh.8, rhesus isolate rh.10 or its variant.The serotype of AAV vector can be different from the serotype of AAV capsid.

[0218] The variant AAV capsid may contain tropism for tissues or cells of the central nervous system (CNS). The target cells may be neurons, neural stem cells, astrocytes, or tumor cells. The target cells may be located in the brain or spinal cord. The target cells may include antigen-presenting cells, dendritic cells, macrophages, nervous system cells, brain cells, astrocytes, microglial cells, and neurons. In some embodiments, the target cells are endothelial cells.

[0219] The actual administration of rAAV can be achieved by using any physical method that transports rAAV to 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, such as the central nervous system, and enhances the tropism (e.g., CNS tropism) of the target environment.The pharmaceutical composition can be prepared, for example, as an injectable formulation.

[0220] The recombinant virus used can be available in liquid or lyophilized form (in combination with one or more suitable preservatives and / or protectants to protect the virus during the lyophilization process). For gene therapy (e.g., of neurological disorders that can be alleviated by a specific gene product), a recombinant virus expressing a therapeutically effective dose of a therapeutic protein is administered to a host in need of such treatment. Use of the recombinant viruses disclosed herein in the manufacture of a medicament for inducing immunity in a host or providing gene therapy to a host is within the scope of this application.

[0221] In instances where a human dosage for rAAV has been established for at least one condition, that same dosage or a dosage that is about 0.1% to 500%, more preferably about 25% to 250%, of the established human dosage can be used. When a human dosage has not been established, as is the case for newly discovered pharmaceutical compositions, a suitable human dosage can be determined based on the ED, as qualified by toxicity and efficacy studies in animals. 50 Or ID 50 values, or other appropriate values ​​derived from in vitro or in vivo studies.

[0222] A therapeutically effective amount of rAAV can be administered to a subject at various times. For example, rAAV can be administered to a subject before, during, or after the subject develops a disease or disorder. rAAV can also be administered to a subject before, during, or after the onset of a disease or disorder (e.g., Huntington's disease (HD), Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, spinal muscular atrophy type I and II, Friedreich's ataxia, spinocerebellar ataxia, and any lysosomal storage disease involving CNS cells, including but not limited to Krabbe disease, Sandhoff disease, Tay-Sachs disease, Gaucher disease (type I, II, or 111), Niemann-Pick disease (NPC1 or NPC2 deficiency), Hurler syndrome, Pompe disease, Batten disease, or any combination thereof), chronic pain, or a combination thereof. In some embodiments, rAAV is administered to a subject during the remission of a disease or disorder. In some embodiments, the rAAV is administered prior to the onset of a disease or disorder in a subject. In some embodiments, the rAAV is administered to a subject at risk of developing a disease or disorder.

[0223] The disease or disorder may include a neurological disease or disorder, such as epilepsy, Dravet syndrome, Lennox-Gastaut syndrome, myoclonic seizures, juvenile myoclonic epilepsy, intractable epilepsy, schizophrenia, juvenile spasms, West syndrome, infantile spasms, intractable infantile spasms, Alzheimer's disease, Creutzfeldt-Jakob 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, and postoperative cognitive impairment (POCD). ), systemic lupus erythematosus, systemic sclerosis, Sjögren's syndrome, neuronal ceroid lipofuscinosis, neurodegenerative cerebellar ataxia, Parkinson's disease, Parkinson's disease dementia, mild cognitive impairment, various forms of cognitive impairment in mild cognitive impairment, various forms of cognitive impairment in dementia, dementia pugilistica, vascular and frontal lobe dementia, cognitive impairment, learning disabilities, eye injury, eye disease, eye disorders, glaucoma, retinopathy, macular degeneration, head or brain or spinal cord injury, head or brain or spinal cord trauma, spasticity Spasms, epileptic convulsions, epilepsy, temporal lobe epilepsy, myoclonic epilepsy, tinnitus, dyskinesia, chorea, Huntington's chorea, athetosis, dystonia, stereotypies, ballismus, tardive dyskinesia, tic disorders, spasmodic torticollis, blepharospasm, focal and generalized dystonia, nystagmus, hereditary cerebellar ataxia, corticobasal degeneration, tremor, essential tremor, addiction, anxiety disorders, panic disorder, social anxiety disorder (SAD), attention deficit hyperactivity disorder (ADHD), attention deficit syndrome (ADS), Restless limbs syndrome (RLS), hyperkinesia in children, autism, dementia, dementia in Alzheimer's disease, dementia in Korsakoff's syndrome, Korsakoff's syndrome, vascular dementia, dementia related to HIV infection, HIV-1 encephalopathy, AIDS encephalopathy, AIDS dementia complex, AIDS-associated dementia, major depressive disorder, major depression, depression, memory loss, stress, bipolar manic-depressive disorder, drug tolerance, drug tolerance to opioids, movement disorders, fragile X syndrome, irritable bowel syndrome (IBS), migraine,The conditions may include 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.

[0224] In some embodiments, the present specification discloses the formulation 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 various treatment regimens. In some embodiments, the amount of 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. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations are considered by those skilled in the art of preparing such pharmaceutical formulations, and therefore various dosages and treatment regimens may be desirable. In some examples, the composition is suitably formulated into a pharmaceutical composition disclosed herein that is delivered to one or more cells, tissues, or organs via intraocular, intravitreal, parenteral, subcutaneous, intravenous, intracerebroventricular, intramuscular, intrathecal, oral, intraperitoneal, oral or nasal inhalation, or direct injection. In some embodiments, the rAAV disclosed herein can be advantageously administered intravenously for delivery to the CNS.

[0225] In some embodiments, pharmaceutical forms of AAV-based virus compositions suitable for injectable use include 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, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0226] In some embodiments, for administration of an injectable aqueous solution, for example, the solution is preferably buffered, and if necessary, the liquid diluent can first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Some variation in dosage may not necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for each individual subject. In addition, for human administration, preparations must meet the sterility, pyrogenicity, and general safety and purity standards required by the FDA Office of Biologics Standards.

[0227] Disclosed herein are sterile injectable solutions containing the compositions disclosed herein (e.g., rAAV compositions), which are prepared by incorporating the required amount of the compositions disclosed herein in an appropriate solvent, along with certain other ingredients as enumerated above, as needed, followed by sterile filtration. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof. Injectable solutions may be advantageous for systemic administration, for example, by intravenous administration.

[0228] Neutral or salt forms of the formulations are also provided herein. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of protein), and those formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. When formulated, solutions are administered in a manner compatible with the dosage formulation and in an amount that is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as injectable solutions, drug-release capsules, etc.

[0229] Formulations for intranasal administration may comprise a coarse powder containing the active ingredient and having an average particle size of about 0.2 μm to 500 μm. Such formulations are administered in the same manner as snuff is taken, for example, by rapid inhalation through the nasal passage from a container of powder held close to the nose. Formulations suitable for nasal administration may contain, for example, from about 0.1% (w / w) to about 100% (w / w) of the active ingredient, and may include one or more of the additional ingredients described herein. Pharmaceutical compositions may also be prepared, packaged, and / or sold in a formulation suitable for buccal administration. Such formulations may be in the form of, for example, tablets and / or lozenges made using conventional methods, and may contain, for example, 0.1% to 20% (w / w) of the active ingredient, the remainder comprising an orally dissolvable and / or disintegrable composition, and, optionally, one or more of the additional ingredients described herein. Alternatively, formulations suitable for buccal administration may comprise a powder and / or a sprayed and / or atomized solution and / or suspension comprising the active ingredient. Such powdered, sprayed, and / or atomized formulations, when dispersed, may comprise 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 of the additional ingredients described herein.

[0230] Suitable doses and dosages to be administered to a subject will be determined by factors including, but not limited to, the particular therapeutic rAAV composition, the disease state and its severity, the identity of the subject requiring treatment (e.g., weight, sex, age), and can be determined according to the particular circumstances surrounding the case, including, for example, the specific agent being administered, the route of administration, the condition being treated, and the subject or host being treated.

[0231] The amount of AAV composition and the number of times of administration of such composition are within the scope of a person skilled in the art who has the benefit of the present teachings.However, in some embodiments, the administration of a therapeutically effective amount of the disclosed composition can be achieved by a single administration, such as a single injection of a sufficient number of infectious particles, to provide a therapeutic benefit to patients undergoing such treatment.This is made possible, at least in part, by the fact that certain target cells (e.g., neurons) do not divide, eliminating the need for multiple or chronic administration.

[0232] In some embodiments, it is advantageous to provide multiple or sequential administrations of AAV vector compositions, either over a relatively short or relatively long period of time, as can be determined by the physician supervising the administration of such compositions. For example, the number of infectious particles administered to a mammal may be about 10, given in a single dose, or divided into two or more administrations, as may be necessary to achieve treatment of the particular disease or disorder being treated. 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or even higher infectious particles / ml. Indeed, in some 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, daily and unit dosages will vary depending on several 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 requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0233] The targeting peptides described herein can be used to create rAAVs with enhanced CNS tropism that have capsid proteins 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.

[0234] The dosing frequency of the rAAV virus can vary. For example, the rAAV virus can be administered to a subject about once per week, about once per two weeks, about once per month, about once per six months, about once per year, about once per two years, about once per three years, about once per four years, about once per five years, about once per six years, about once per seven years, about once per eight years, about once per nine years, about once per ten years, or about once per fifteen years. In some embodiments, the rAAV virus is administered to a subject at most about once per week, at most about once per two weeks, at most about once per month, at most about once per six months, at most about once per year, at most about once per two years, at most about once per three years, at most about once per four years, at most about once per five years, at most about once per six years, at most about once per seven years, at most about once per eight years, at most about once per nine years, at most about once per ten years, or at most about once per 15 years.

[0235] Disclosed herein are kits comprising the compositions disclosed herein. Also disclosed herein are kits for the treatment or prevention of a disease or condition of the CNS, PNS, or a target organ or environment (e.g., the CNS). In some examples, the disease or condition is cancer, a pathogen infection, a neurological disease, a muscular disease, or an immune disorder, such as those described herein. In one embodiment, the kit can include a therapeutic or prophylactic composition containing a composition of an effective amount of rAAV particles encapsulating a nucleic acid provided herein and an rAAV capsid protein of the present disclosure. In another embodiment, the kit can include a therapeutic or prophylactic composition containing an effective amount of rAAV-modified cells ("modified cells") described herein in a unit dosage form that expresses a therapeutic nucleic acid. In some embodiments, the kit includes a sterile container that can contain the therapeutic composition; such a container can be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or any other suitable container form known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0236] 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 can generally include information about using the composition to treat or prevent the disease or condition.

[0237] The kits can include allogeneic cells. In some embodiments, the kits include cells that may include genetic modifications. In some embodiments, the kits include "off-the-shelf" cells. In some embodiments, the kits include cells that can be expanded for clinical use. In some embodiments, the kits contain contents for research purposes.

[0238] In some embodiments, the instructions include at least one of the following: a description of the therapeutic rAAV composition; dosing schedules and administration for treating or preventing a disease or condition disclosed herein; cautions; warnings; indications; counter-indications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (if present), as a label attached to the container, or as a separate sheet, pamphlet, card, or foldout supplied in or with the container. In some embodiments, the instructions alone provide steps for administering rAAV to a subject. In some embodiments, the instructions provide for 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 up to 12 ... The instructions provide for administering rAAV to a subject 7 days later, or 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 before the administration. In some examples, the instructions provide that the rAAV is formulated for intravenous injection. In some examples, the instructions provide that the rAAV is formulated for intranasal administration. [Example]

[0239] Certain aspects of the above-discussed embodiments are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure in any way. Example 1 Targets for receptor-mediated control of therapeutic drug biodistribution and efficacy

[0240] A screen to identify binding partners that mediate the tropism of engineered AAV is described in this example.

[0241] 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, secreted, and cell surface-tethered proteins, and 397 human heterodimers in duplicate at 3 × 10 4 1 x 10 particles / cell each of AAV9, AAV.CAP-Mac, AAV.CAP-B22, AAV.MaCPNS1, and AAV.MaCPNS2 4 AAV was pooled to provide AAV-X1.1 particles / cells. After AAV addition, samples were directly fixed. Receptor-bound AAV was labeled with anti-AAV9 antibody followed by a secondary detection antibody.

[0242] Twenty-two library hits were then identified and probed individually with each AAV in a confirmatory screen that also included various positive and negative controls. The results of this screen are shown in Figures 5-11.

[0243] Three new 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 means for modulating AAV-specific permissive host immune responses. Without being bound by any particular theory, the role of DKK3 as a WNT signaling inhibitor provides a method for modulating the shuttle activity of DKK3 to WNT-expressing cells and tissues. Binding of endogenous and secreted factors such as IL-3 and DKK3 may also function to mask viral particles as "self" to minimize immune responses.

[0244] The new protein target for AAV.CAP-B22 is FAM234A. New targets specific to 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 alternative tropism of AAV-X1.1 relative to AAV9.

[0245] LRP6 is strongly expressed in the human BBB and is a target for the enhanced efficacy of AAV-X1.1 across CNS 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 stronger, whereas no such interaction was found for AAV9 (Figure 1). Computer modeling of the AAV-X1.1 inserted peptide with the human LRP6 extracellular domain indicates binding to domain 2, whereas the CAP-Mac inserted peptide shows binding to either domain 1 or domain 2 (Figure 2). Both of these engineered AAVs show enhanced efficacy against human HEK293 cells (which endogenously express LRP6) compared to standard AAV9-based AAVs (Figure 3). Knockdown of endogenous LRP6 in HEK293 cells selectively reduces the efficacy of both AAV-X1.1 and CAP-Mac (Figure 4).

[0246] These targets allow for target-based engineering of AAV to modulate biodistribution, efficacy, and host immune interactions. They 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 capture the properties of native and engineered AAV. [Table 1-1] [Table 1-2] methodology Library screening

[0247] 3×10 4 CAP-C1, CAP-B22, PNS1, PNS2 and AAV9 (control AAV), and 1 × 10 4 Test AAV pools containing X1B10 particles / cells were screened for binding to fixed HEK293 cells / slides expressing 6019 human plasma membrane proteins, secreted proteins, and cell surface-tethered human secreted proteins, as well as 397 human heterodimers in duplicate (18 slide sets, n = 2 slides per slide set) using the direct fixation method (slides were fixed without removing AAV after sample addition). All transfection efficiencies exceeded a minimum threshold. Anti-AAV9 antibody (anti-adeno-associated virus 9, clone HL2372, supplied by Merck, catalog number MABF2309-100UL, 1:500 dilution) was used, followed by AlexaFluor647 anti-mIgG H+L detection antibody. In total, 22 library hits (duplicate spots) were identified by analyzing fluorescence (AF647 and ZsGreen1) in ImageQuant. Signal intensity ranged from very weak to moderate / strong (signal over background). Confirmatory / specificity screening

[0248] Vectors encoding all 22 hits, plus vectors encoding mouse SCA1 (LY6A) and KIAA0319L, and control vectors encoding CD86 and EGFR were spotted in duplicate onto new slides and used to reverse transfect human HEK293 cells. All transfection efficiencies exceeded the minimum threshold. Transfected HEK293 cells were transfected at 3 × 10 cells per well using the direct fixation method described above. 41 × 10 particles / cell of CAP-C1, CAP-B22, PNS1, PNS2, or AAV9 (control AAV) 4 After fixation, particles / cells were treated with X1B10, 0.2 μg / mL CTLA4-mFc (positive control), or no test molecule (secondary only; negative control) (n = 2 slides per treatment; N.B., controls were also fixed after sample incubation). Slides were analyzed as described above. Hits were classified as specific or nonspecific (e.g., either positive or negative controls were also prepared). [Table 2-1] [Table 2-2]

[0249] 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 6,019 human plasma membrane proteins, secreted proteins, and cell surface-anchored secreted proteins and 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. Example 2 Human cell surface AAV interactome identifies LRP6 as a blood-brain barrier transcytosis receptor and the immune cytokine IL3 as an AAV9 binder

[0250] Adeno-associated viruses (AAVs) are fundamental gene delivery tools for basic science and clinical therapy. However, the lack of mechanistic insight, particularly for engineered vectors created by directed evolution, can hinder their application. As described herein, an unbiased human cell microarray platform was adapted to determine the extracellular and cell surface interactomes of native and engineered AAVs. We identified a naturally evolved serotype-specific interaction of AAV9 with human interleukin 3 (IL3), which, without being bound by any particular theory, may play a role in the host immune response. We also identified a laboratory-evolved low-density lipoprotein receptor-related protein 6 (LRP6) interaction specific to engineered capsids that cross the blood-brain barrier in non-human primates upon intravenous administration. The unbiased cell microarray screening approach also enabled the identification of off-target tissue-binding interactions of engineered brain-enriched AAVs, which informs the vector's peripheral organ tropism and side effects. These results enable the confident application of engineered AAVs in diverse organisms and allow for the targeted engineering of improved viral and non-viral vectors for non-invasive delivery of therapeutic agents to the brain.

[0251] Adeno-associated viruses (AAVs) have become the gene delivery vector of choice in bench and clinical settings. Systemic administration of AAVs allows for noninvasive targeting, particularly of large or dispersed biological structures. However, access from the periphery to the brain is limited by the blood-brain barrier (BBB), a complex biological structure that regulates molecular access to the central nervous system (CNS). Systemic administration of AAVs also exposes the vector to the host immune system and off-target tissues. The low efficiency of brain targeting after systemic administration with native serotypes often necessitates high doses that increase costs and can induce adverse side effects. Therefore, improved vectors are needed if AAV gene therapy is to realize its full therapeutic potential.

[0252] AAV capsid engineering, particularly by directed evolution, has been demonstrated to significantly improve efficacy in desired cell types and tissues after systemic intravenous delivery. However, when AAV capsids are applied across species, the enhanced tropism of many engineered vectors can vary. This is important for human clinical trials, where capsids developed in non-human species that function poorly when translated may not only fail to provide therapeutic benefit, but may also induce neutralizing antibodies, thereby excluding patients from future therapies.

[0253] This translational challenge of engineering AAVs 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 provides a platform with which researchers can explore the most effective pathways through this barrier. Recent developments suggest that engineered AAVs can utilize diverse BBB-crossing receptors, but the mechanisms of primate brain-enhanced vector delivery remain unexplored.

[0254] To address this challenge, as described herein, we adapted the Retrogenix human membrane proteome and secretome cell microarray to screen for native and engineered AAV capsid interactions with host cells. This enabled rapid assay of over 90% of the human membrane proteome and secretome, including important protein classes such as receptors, transporters, and cytokines. Using this broad, unbiased screen, we identified several novel AAV interactions with implications for host immune responses (human interleukin-3 (IL3) binding to AAV9), enhanced BBB crossing across species (through low-density lipoprotein receptor-related protein 6 (LRP6) binding by AAV9-X1.1 and CAP-Mac), and off-target tissue tropism (through glycoprotein 2 (GP2) binding by AAV9-X1.1 and CAP-Mac). Understanding the mechanism of action of systemic AAV by methods such as those used here is critical for successful vector translation and will enable the design of improved vectors and other therapeutic protein modalities for specific targets. High-throughput screening for AAV binding partners

[0255] To screen for AAV-binding proteins, we used Retrogenix human membrane proteome and secretome cell microarrays, in which DNA oligos encoding human membrane and secreted proteins were immobilized at known slide locations (Figure 12A). HEK293 cells were then grown on the slides and individually reverse-transfected with the corresponding pattern of oligos. AAVs that directly interact with a given protein preferentially bind to cells expressing that protein, while 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 for initial condition optimization) (Figures 12B-C). Screening conditions were optimized using previously identified AAVs and interacting protein pairs (1) AAV9 and AAVR (KIAA0319L) and (2) PHP.eB and mouse LY6A for two different detection methods: biotin tagging and direct detection (Figures 12B-C). Biotinylated capsids can be detected with fluorescent streptavidin, while unlabeled capsids are detected with antibodies whose epitopes are generally distinct from the engineered capsid variable regions IV and VIII. In some embodiments, the primary amine labeling level of the capsid must be adjusted so that the surface modification does not interfere with the capsid's critical binding interactions. We found that the best signal-to-noise ratio for duplicate spots (calculated as the average intensity across the positive control spot compared to the average intensity across the rest of the slide) was achieved by directly immobilizing cell-bound AAV without washing.

[0256] Next, we investigated direct capsid detection and validated conditions with a panel of AAV capsids, including AAV9 and five engineered AAV9 variants with enhanced potency in the CNS of non-human primates (NHPs) after systemic administration (Table 3 and Figure 12D). Testing of these capsids revealed that, individually, all AAVs except MaCPNS1 exhibited detectable AAVR binding (an exception that may be due to the placement of the capsid's variable region VIII insert), but only CAP-B22 interacted with mouse LY6A (potentially due to the PHP.eB loop in variable region VIII) (Figure 12D and Figure 16). To enable high-throughput screening, the six capsids were tested as a pool. Pooled testing required additional dosage optimization, first for individual capsids and then for the overall background binding levels of the included capsids (Table 5). An optimal dose was determined that minimized background binding while still allowing the specific interaction of CAP-B22 with mouse LY6A, distinct from the five non-LY6A-interacting capsids (Fig. 12D).

[0257] After these controls, the six capsid pools were tested in a full screen of approximately 6,400 proteins, including 6,000 human plasma membrane proteins, as well as secreted and cell surface-tethered proteins, and approximately 400 heterodimers. Twenty-two library hits were identified, each with duplicate spots that showed enhanced signal above background. To assign these hits to specific capsids, a follow-up deconvolution screen was performed with each individual capsid from the pool (Figure 12E). DNA oligos for the 22 identified hits, as well as a positive control, CD86, were immobilized in duplicate locations on new slides. Negative control conditions without AAV analyte and positive control conditions with CTLA4-Fc (a CD86 binder) were also included. Hits, including both membrane-localized and secreted proteins, were successfully assigned to capsids. Some of these interactions were unique to specific AAV9 variants, while others were conserved across all capsids tested (Table 4). Validation of individual AAV binding interactions

[0258] To validate binders from the cell microarray screen, hits were tested for their ability to enhance AAV potency in cell culture (Figures 17A-C), and capsid-binding interactions were characterized by surface plasmon resonance (SPR) (Figure 13A and Figures 18A-B). 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) stood out 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, triggering the expansion and proliferation of various immune cells and activating type I interferon-secreting plasmacytoid dendritic cells. Using SPR, we found that AAV9 binds to human IL3 but is not closely related to the native serotypes AAV8 and AAVrh10 (Figure 13B). IL3 from different species was then 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), suggesting differences between New and Old World monkeys.

[0259] To understand the species and serotype specificity of the IL3 interaction with AAV9, we examined the structure of the bound complex. Because functional AAV ligands can have weak, dynamic monomeric interactions, we exploited avidity by flowing 60-mer AAV9 capsids over protein A-captured dimeric IL3-Fc to detect all biologically relevant interactions. Despite this high avidity in SPR experiments, the apparent affinity of the interaction was consistent with only high-nM interactions. Therefore, we performed chemical cross-linking of IL3-bound AAV9 followed by mass spectrometry (XL-MS). Using bis(sulfosuccinimidyl) suberate (BS3) cross-linker, we detected two cross-links between the proteins (Figure 13D and Figures 19A-B). These cross-links position IL3 at the base of the three-fold symmetric spike and in the two-fold symmetric valley, but the generation of a binding model is not possible.

[0260] Validated AAV interactors were then evaluated for their potential to explain the enhanced brain tropism of the engineered capsid. Sorting the screening hits by their expression levels in human BBB endothelial cells highlighted the specific interaction of AAV9-X1.1 with low-density lipoprotein receptor-related protein 6 (LRP6) (Figure 14A). This capsid exhibits enhanced brain endothelium-specific tropism in mice, which shifts to enhanced neuronal tropism in macaques (Table 3). While AAV9-X1.1 contains modifications from AAV9 in both variable regions IV and VIII (Table 3), the tropism of AAV9-X1.1 can be transferred to other native serotypes, such as AAV1 and AAV-DJ, by transferring only the variable region VIII insert of AAV9-X1.1. SPR confirmed that the X1 peptide insertion in variable region VIII conferred LRP6 binding to AAV1-X1 and AAVDJ-X1, but not to their unmodified parental serotypes (Figure 20A), demonstrating that the functional modularity of the X1 peptide in different AAV serotypes in vivo corresponds to LRP6-binding modularity.

[0261] LRP6 is a coreceptor of the canonical Wnt signaling pathway, playing a role in development and homeostasis in many tissues. The high level of LRP6 sequence conservation across species (98% and 99.5% sequence identity conserved between human LRP6 and mouse or macaque LRP6, respectively) is consistent with the enhanced tropism of AAV9-X1.1 compared to AAV9 in rodents and primates. Similar enhancement of tropism across species was also observed for CAP-Mac, engineered in marmosets, with enhanced endothelial tropism in marmosets and enhanced neuronal tropism in macaques compared to AAV9 (Table 3). Therefore, CAP-Mac was also tested for interaction with the human LRP6 extracellular domain by SPR (Figure 14B). Like IL3, avidity was utilized to ensure weak but functionally important interactions were captured. Both AAV9-X1.1 and CAP-Mac bind strongly to human LRP6-Fc, unlike their parent capsid, AAV9, with sub-nM apparent affinities.

[0262] LRP6 has many endogenous WNT signaling partners with binding sites spanning either the extracellular YWTD domains 1 and 2 (E1E2) or domains 3 and 4 (E3E4). Using AlphaFold-Multimer, we constructed a model of the AAV interaction complex, which predicted that the X1 and CAP-Mac variable region VIII peptides bind to LRP6 YWTD domain 1 (Figure 14C). Although cofolding of E1 and E2 complicates testing of individual domains, SPR results from mouse LRP6 extracellular domain fragments were consistent with the model predictions, with interactions observed for LRP6-E1E2 but not LRP6-E3E4 (Figure 14D). We also tested AAV-BI30, another engineered capsid with specific expression in mouse brain endothelium (Table 3), and found that it also bound 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 that of the closely related LRP5 (Fig. 21). On the other hand, AAV9 bound only to PKD2 in AAVR, as previously reported.

[0263] AAV9-X1.1 and CAP-Mac potently infected HEK293 cells, with AAV9-X1.1 having a stronger effect (Figure 22B). To determine whether this potency was mediated by endogenous LRP6 expression in HEK293 cells, the vectors were tested with LRP6 inhibitors. The potency of AAV9-X1.1 was significantly reduced by mesoderm development LRP chaperone (Mesd), a native endoplasmic reticulum chaperone and recombinant extracellular inhibitor of LRP5 and LRP6, and sclerostin (SOST), which inhibits LRP6 by binding only to E1E2 (Figures 22A-B). Importantly, neither Mesd nor SOST inhibited the potency of PHP.eB in LY6A-overexpressing cells. Transient overexpression of human LRP6 boosted the potency of both capsids, with a stronger effect on CAP-Mac (Figure 22B). This effect was largely preserved when the truncated LRP6-E1E2 was used. As expected from our pull-down assay, transient overexpression of LRP5 did not enhance the potency 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. AAV9-X1.1 tethers LRP6 more productively at lower endogenous LRP6 expression levels, whereas CAP-Mac shows enhanced potency after transient overexpression of LRP6.

[0264] Notably, in addition to the intended CNS-targeting receptors achieved by these AAV capsid engineering efforts, both LRP6-binding engineered capsids also acquired interactions with the GPI-linked protein glycoprotein 2 (GP2), which has specific pancreatic expression and, in secreted form, plays an antibacterial role in the intestine (Figure 17A, Figures 18A-B, and Table 4). GP2 boosted the potency of both capsids in cell culture, with a stronger effect on human than mouse proteins (Figure 17A).

[0265] FAM234A, which bound to CAP-B22 in cell microarray screening, is found in the brain at low expression across many neuronal types. Although FAM234A was identified in disease association studies, a specific molecular function has not been assigned to this protein. FAM234A enhances the potency of both CAP-B22 and PHP.eB in cell culture, with the mouse receptor exhibiting a stronger effect than the human protein (Figure 17B). This suggests that the interaction is driven by the shared variable region VIII insert sequence of the capsid (Table 3). Engineered AAVs utilize LRP6 at the blood-brain barrier in mice and primates

[0266] Host neutralizing antibodies generated in response to previous exposure to AAV complicate repeated administration with the same serotype. We demonstrated that serotype-switched X1 vectors, such as AAV1-X1, allow for a second systemic administration in mice previously exposed to an AAV9-based vector. Taking advantage of this property, we determined the in vivo effect of AAV9-X1.1 LRP6 interaction (Figure 15A). Brain endothelial-targeted AAV1-X1, packaging either mCherry or Cre recombinase as a control, was systemically administered to Lrp6 Cre-conditional knockout mice. Three weeks later, either AAV9-based PHP.eB or eGFP-packaging AAV9-X1.1 was systemically delivered. Although PHP.eB demonstrated characteristic robust brain transduction regardless of the AAV1-X1 cargo, AAV9-X1.1 brain endothelial tropism was significantly reduced in AAV1-X1-transfected mice with Lrp6 knockout (Figures 15B-C), confirming the requirement of LRP6 for capsid BBB entry in vivo. AAV9-X1.1 capsids showed enhanced potency compared to PHP.eB in the liver, where LRP6 is also expressed (Figure 23A). Decreased AAV9-X1.1 liver transduction was also observed in the Lrp6 knockout condition (Figures 15B-C).

[0267] To confirm that LRP6 interaction mediates the brain efficacy of AAV9-X1.1 in primates, the vector was tested in macaque and human primary brain microvascular endothelial cells (PBMECs) in culture (Figures 15D-15E). AAV9-X1.1 was significantly more potent than its parental AAV9 in PBMECs from both species. On the other hand, the LRP6 inhibitor Mesd selectively reduced the potency of AAV9-X1.1 in PBMECs, returning it to AAV9 levels. A similar LRP6-dependent boost in potency by AAV1-X1 compared with AAV1 was also observed in human PBMECs (Figure 23B). The similarity in the responses of AAV1-X1 and AAV9-X1.1 supports SPR experiments showing that the X1 peptide is necessary and sufficient to target the capsid to the BBB via its interaction with LRP6 (Figure 20A).

[0268] Recent advances in capsid engineering have resulted in AAV vectors that can more efficiently cross the blood-brain barrier (BBB) ​​in rodents and non-human primates after systemic administration. However, predictable translation and further rational design of these and other non-viral BBB-crossing molecules, particularly in humans, is hindered by a limited understanding of transcytosis mechanisms. This translational challenge also represents an opportunity for better understanding 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. Disclosed herein is a pipeline for finding cognate receptors for engineered AAVs that focuses on the human membrane proteome and secretome. This disclosure validates the utility of cell microarray screening to identify receptors for native and engineered AAVs. We identify LRP6 as a novel, highly conserved target for blood-brain barrier transcytosis by AAV9-X1.1, a potent engineered capsid for primate neurons, and human IL3 as an interacting partner for AAV9. These findings allow for the exploitation of identified receptors for targeted drug delivery across diverse therapeutic modalities, e.g., small molecules, antibodies, or oligonucleotides.

[0269] Safety and immune tolerance of delivery vectors are important considerations as AAVs move into the clinic, as adverse reactions may occur. The immunomodulatory potential of IL3-AAV9 interactions may include providing a host neutralization mechanism or a cloaking mechanism for AAV to evade the immune system or dampen its response using a decoy receptor. In addition to activated T cells, IL3 is also constitutively secreted by astrocytes in the brain to reprogram microglia and combat Alzheimer's disease. Therefore, AAV9 interaction with IL3, shared by all AAV9-based engineered capsids for enhanced BBB crossing, may, in some embodiments, influence processes beyond the immune tolerance of the vector itself in healthy and diseased brain contexts. Importantly, AAV9 isolates from human clinical tissues bind to human and macaque IL3 (83% AA identity) but not marmoset or mouse IL3 (69% and 27% AA identity, respectively). Without being bound by any particular theory, this species-dependent interaction may contribute to the discrepancy between rodent and primate AAV safety profiles, particularly in the context of neurodegeneration.

[0270] The high degree of sequence conservation in LRP6 (98% AA identity between mouse and human) may explain, without being bound by any particular theory, the broad conservation across species of enhanced tropism by AAV capsids targeting this receptor.

[0271] Both AAV9-X1.1 and CAP-Mac also show binding to GP2, which is not present in the CNS, suggesting that the interaction may piggyback on the functional enhancement provided by LRP6 binding during directed evolutionary selection, although not bound by any particular theory. This is supported by the finding that AAV interacts more strongly with human GP2 than with the mouse protein present during the directed evolution of AAV9-X1.1. These findings highlight the importance of extensive, unbiased interaction screening as disclosed herein to establish a complete safety profile for engineered capsids prior to clinical trials.

[0272] Investigating the diverse mechanisms by which natural and engineered AAVs cross the BBB may also allow for preparedness in defense against additional pathogens. Just as antibiotic resistance challenges modern society, there is concern that rapidly evolving pathogens will develop "BBB resistance," the ability to access the brain and cause severe disease (as some retroviruses, including HIV-1, have already done). In a recent and troubling example, SARS-CoV-2 capsid proteins have been found in the brains of patients with long-term COVID and associated with neuropsychiatric symptoms. By screening existing pathogens and their potential molecular evolution against the growing catalog of human BBB transcytosis receptors (including transferrin receptor, insulin receptor, CD98hc, CA4, and, as disclosed here, LRP6), we can anticipate a proliferation of pathogens with neuropsychiatric sequelae.

[0273] Provided herein is a method for efficiently screening native 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 AAVs, provide targets for the development of non-viral therapeutic modalities, and highlight potential vulnerabilities to future pathogens. [Table 3] [Table 4] [Table 5-1] [Table 5-2]

[0274] Table 6 below shows the amino acid sequences of exemplary targets of AAV identified herein. Table 7 shows the residues of the binding site in human LRP6. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 7] method Viral vector production

[0275] Briefly, HEK293 cells were triple-transfected with capsid, genome, and helper plasmids. The medium was changed the next day, then collected and replaced two days later. Five days after transfection, the medium and cells were collected and processed for AAV purification. The cells were lysed in a high-salt solution and treated with salt-activated nuclease. The medium was PEG precipitated and resuspended in salt-activated nuclease solution. Both solutions were loaded onto an iodixanol density column, ultracentrifuged, and extracted from the 40% / 60% interface. Finally, the AAV was buffer-exchanged, concentrated, titered, and assayed for endotoxin using the Piece LAL chromogenic endotoxin kit (catalog number A39552) (for vectors designated for non-human primates). Retrogenix Cell Microarray

[0276] Retrogenix cell microarrays were performed as described below for AAV analytes. Pre-screening optimization was performed on slides of HEK293 cells and cells overexpressing mouse LY6A and human AAVR (KIAA0319L), TGFBR2, and EGFR. Transfection efficiency was verified to exceed a minimum threshold prior to analyte loading. AAV was administered at 6 x 10 per HEK293 cell. 4 AAV particles were added to the fixed cells at a concentration of 0.05%.

[0277] Biotinylated AAV was diluted in PBS with NHS-PEG4-biotin (Thermo A39259) at a 10,000-fold molar ratio of 1 × 10 per mL. 13 The viral genome (vg) was generated by incubation with AAV at room temperature for 2 hours. The reaction was quenched with 1 M Tris, pH 8, followed by buffer exchange, concentration, and AAV re-titering. Biotinylated AAV was detected in HEK293 cells after fixation with AF647-labeled streptavidin. Unlabeled AAV was detected in HEK293 cells after fixation with anti-AAV9 clone HL2372 (Merck, MABF2309-100UL) at a 1:500 dilution, followed by AF647-labeled anti-mIgG H+L.

[0278] To achieve a suitable signal-to-noise ratio necessary to minimize false positives and false negatives, unlabeled AAVs were screened individually and as pools at various concentrations using anti-AAV9 detection. The final test pools were screened in duplicate against fixed HEK293 cells / slides expressing approximately 6,000 human plasma membrane proteins, secreted proteins, and cell surface-tethered human secreted proteins, as well as approximately 400 human heterodimers. Hits were identified using ImageQuant as spots observed in duplicate. After screening, the 22 identified hits and a CD86 positive control protein were spotted onto new slides for testing of individual AAVs in deconvolution screening. Negative control conditions without analyte and positive control conditions with CTLA4-Fc (which interacts with CD86) were also included. Protein preparation

[0279] Lyophilized mouse LRP6 (AA20-1366) tagged with 6xHis tag, Fc (mouse IgG 2a N-terminal (E1E2) and C-terminal half (E3E4) fragments of mouse LRP6 tagged with AA20-628 (N half: AA20-628, C half: AA629-1244), full-length human LRP6 tagged with Fc (human IgG1) (AA20-1368), LRP5 tagged with 6xHis (AA1-1383), and SOST protein were purchased from Bio-Techne (catalog numbers 2960-LR-025, 9950-LR-050, 9954-LR-050, 1505-LR-025, 7344-LR-025 / CF, and 1406-ST, respectively). Mesd protein was purchased from SinoBiological (catalog number 10949-H08H). All proteins were reconstituted in Dulbecco's Phosphate Buffered Saline (DPBS, Gibco™) at the desired concentrations before use.

[0280] Interleukin-3 proteins from human (AA1-152), mouse (AA1-166), marmoset (AA1-143), and macaque (AA1-144) were triple-tagged with Fc-Myc-6×His, and Fc-Myc-6×His-tagged human and mouse GP2 (hGP2: AA1-518, mGP2: AA1-515), and Fc-Myc-6×His-tagged human and mouse DKK3 (hDKK3: AA1-350, mDKK3: AA1-349) were purified at 3 × 10 ribosomal concentrations using ExpiFectamine™ (Thermo Fisher Scientific) according to the manufacturer's instructions. 6 Expi293F™ (Thermo Fisher Scientific) cells were transfected at a density of 10 ...

[0281] The 6xHis-tagged human adeno-associated virus receptor (AAVR) PKD2 domain (AA401-498) was purified using methods known in the art. Briefly, PKD2 was expressed in BL21(DE3)-RIPL E. coli. Cells were lysed by sonication, and the insoluble fraction was clarified by centrifugation. The clarified lysate was applied to a Ni-NTA column (Qiagen) and eluted with DPBS containing 250 mM imidazole. Surface Plasmon Resonance (SPR)

[0282] 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 capture levels of 600-800 response units (RU) for Figures 13B-13C, 14D, 18A-18B, and 20A-20D, and 1200-1500 RU for Figure 14B. AAV was injected at a flow rate of 10 μL per minute for 240 seconds, followed by a 600-second dissociation. The AAV concentration was 2.4 × 10 per mL. 12 Starting at vg, the dilution proceeded in 2-fold intervals. A regeneration step with 10 mM glycine pH 1.5 was performed between each cycle. All kinetic data were double-reference subtracted. Pull-down assay

[0283] The pull-down assay was generally performed as described below. Briefly, the prey AAV was mixed with the His-tagged bait protein and Ni-NTA resin in a binding buffer of DPBS containing 20 mM imidazole on an orbital shaker at 4°C for 1 hour. The resin was then collected in a spin column, washed twice with 10 column volumes of binding buffer, and eluted in 45 μL of DPBS containing 150 mM imidazole. The eluate was analyzed by Western blot using anti-VP1 / VP2 / VP3 (ARP, catalog number 03-61058) and anti-6xHis (Abcam, catalog number ab18184) antibodies. HEK293 cell culture potency assay

[0284] HEK293T cells were cultured in 6-well plates in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum (FBS), 1% non-essential amino acids (NEAA), and 100 U penicillin-streptomycin per mL at 37°C in 5% CO2. At 80% confluency, 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% confluency and maintained in FluoroBrite™ DMEM supplemented with 0.5% FBS, 1% NEAA, 100 U penicillin-streptomycin per mL, 1× GlutaMAX, and 15 μM HEPES. Plates were imaged 24 hours after AAV challenge using a Keyence BZ-X700 (4x objective). For experiments using protein inhibitors, Mesd (26 μg / ml) and SOST (0.2 μg / ml) were added 4 hours before AAV addition. NucBlue™ Live ReadyProbes™ reagent (Hoechst 33342) was added to each well to aid in autofocusing. Image quantification was performed using a dedicated Python image processing pipeline. Primary cell culture potency assay

[0285] Human brain microvascular endothelial cells (ScienCell Research Laboratories, Catalog No. 1000) and primary cynomolgus monkey brain microvascular endothelial cells (CellBiologics, Catalog No. MK-6023) were cultured according to the instructions provided by the supplier. Cell cultures were then plated at 5 x 10 cells per well. 4 The cells were treated with a viral vector containing the single-stranded AAV genome CAG-eGFP at a multiplicity of infection (MOI) of 4 wells per vector. Fluorescence expression in the cultures was examined and quantified 1 day after the infection procedure. animal

[0286] Adult (6–8 week old) homozygous B6;129S-Lrp6tm1.1Vari / J mice (Jackson Labs #026267) were injected with 1 × 10 HIV-1 markers per animal. 12 Three weeks later, these mice were injected retroorbitally with 1 x 106 AAV1-X1 packaging either Ef1a-mCherry or Ef1A-Cre (N = 6 per condition). 12 Mice were re-administered with PHP.eB or AAV-X1.1 packaging 1000 mg of CAG-eGFP (N=3 per condition). Mice were randomly assigned to specific AAV conditions. The experiment was not blinded to any of the experiments performed in this study. Lrp6 conditional knockout tissue preparation and imaging

[0287] Mice were anesthetized with Euthasol (pentobarbital sodium and phenytoin sodium solution, Virbac AH) and transcardially perfused with approximately 50 mL of 0.1 M PBS, pH 7.4, followed by an equal volume of 4% paraformaldehyde (PFA) in 0.1 M PBS. Collected organs were post-fixed overnight at 4°C in 4% PFA, washed, and stored at 4°C in 0.1 M PBS with 0.05% sodium azide. 100 μm brain sections were prepared using a Leica VT1200 vibratome and imaged on a Zeiss LSM 880 confocal microscope using a Plan-Apochromat 10x0.45 M27 (working distance, 2.0 mm) objective. Images were analyzed using Zen Black 2.3 SP1 (Zeiss) and ImageJ. AlphaFold structural modeling

[0288] The complex structure of the LRP6 ECD and the 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 consisted of two sequences: the surface-exposed residues in 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 O75581, residues 20–1370). The Google Colaboratory notebook was run using an A100 SXM4 40GB GPU. Five structural models were generated using a protocol with a maximum of 20 recycles and MSA generated using templates from MMseqs2 (UniRef+Environmental) and PDB 70. Structural models were ranked using a weighted combination of pTM and iPTM scores.

[0289] In at least some of the previously described embodiments, one or more elements used in an embodiment may be used interchangeably in another embodiment unless such substitution is technically feasible. Those skilled in the art will recognize that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to be within the scope of the subject matter, as defined by the appended claims.

[0290] With respect to the use of virtually any plural and / or singular term herein, one of ordinary skill in the art can convert from plural to singular and / or from singular to plural as appropriate to the context and / or application. Various singular / plural permutations may be expressly set forth herein for clarity. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Any reference to "or" herein is intended to encompass "and / or" unless stated otherwise.

[0291] Those skilled in the art will understand that the terms used in this specification, in general, and in the appended claims in particular (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including, but not limited to," etc.). Where a specific number of introduced claim recitations are intended, such intention will be explicitly recited in the claim, and it will be further understood by those skilled in the art that, in the absence of such recitation, no such intention exists. For example, as an aid to understanding, the following appended claims may contain the 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 meaning that introducing a claim recitation with the indefinite article "a" or "an" means limiting any particular claim containing such an 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 an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be construed to mean "at least one" or "one or more"); the same applies to the use of definite articles used to introduce claim recitations. Additionally, even when a specific number of introduced claim recitations is explicitly recited, those skilled in the art will recognize that such recitation should be construed to mean at least the recited number (e.g., the bare recitation of "two recitations" without other modifiers means at least two recitations, or two or more recitations).Furthermore, in instances where a convention similar to "at least one of A, B, and C, etc." is used, such configuration is generally intended in the sense that one of ordinary skill 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 instances where a convention similar to "at least one of A, B, or C, etc." is used, such configuration is generally intended in the sense that one of ordinary skill 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.). It will be further understood by those skilled in the art that virtually any disjunction and / or phrase presenting two or more alternative conditions, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the conditions, either of the conditions, or both conditions.

[0292] Additionally, when features or aspects of the disclosure are described in terms of a Markush group, one of skill 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.

[0293] As will be understood by those skilled in the art, for any and all purposes, e.g., with respect to the provision of a specification, all ranges disclosed herein encompass any and all possible subranges and combinations of subranges. Any listed range can be readily recognized as fully descriptive and permitting division of that same range into at least one half, one third, one quarter, one fifth, one tenth, etc. As a non-limiting example, each range discussed herein can be readily divided into a lower third, middle third, upper third, etc. As will also be understood by those skilled in the art, all terms such as "up to," "at least," "greater than," "less than," etc., refer to ranges that are inclusive of the recited numbers and that can then be divided into subranges as discussed above. Finally, as will be understood by those skilled in the art, ranges include each individual member. Thus, for example, a group having 1 to 3 items refers to groups having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to groups having 1, 2, 3, 4, or 5 items, etc.

[0294] 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 not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. 1. A method for increasing the permeability of the blood-brain barrier, comprising: 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. A method comprising:

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

3. 3. The method of any one of claims 1-2, wherein 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.

4. 1. A method for 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 the delivery system comprises the payload to be delivered to the nervous system; and administering the delivery system to the subject. A method comprising:

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

6. 5. The method of claim 4, wherein the delivery system comprises a viral vector or a non-viral vector.

7. The method of claim 6, wherein the targeting peptide enhances the binding affinity of the viral vector or the non-viral vector to LRP6.

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

9. 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. 9. The method of any one of claims 6 to 8, wherein the non-viral vector comprises a lipid-based nanoparticle, a polymeric nanoparticle, an inorganic nanoparticle, a surfactant-based emulsion, a nanowire, a silica nanoparticle, a peptide- or protein-based particle, a lipid-polymer particle, a nanolipoprotein particle, and combinations thereof.

11. 11. The method of any one of claims 4 to 10, wherein the payload delivered to the nervous system is a biomolecule, a non-biomolecule, or a combination thereof; optionally, 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 nucleic acid sequence delivered to the nervous system is a) sequences encoding trophic factors, growth factors, or other soluble factors that may be released from the transduced cell and affect the survival or function of that cell and / or surrounding cells; b) DNA that restores protein function to humans or animals carrying a genetic mutation in that gene; c) DNA encoding a protein that can be used to regulate or alter the activity or state of a cell; d) DNA encoding a protein or nucleic acid used to assess the state of a cell; e) DNA and / or associated guide RNA for genome engineering; f) sequences for genome editing via homologous recombination; g) a DNA sequence encoding a therapeutic RNA; h) an shRNA or artificial miRNA delivery system; or i) DNA sequences that affect the splicing of endogenous genes The method of any one of claims 11 to 12, comprising one or more of:

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

31.

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

31. The method of any one of claims 1 to 16.

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

11.

19. 19. The method of 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 a functional equivalent of AA588-589 in an amino acid sequence at least 80% identical to SEQ ID NO:

11.

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

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

23. 23. The method of any one of claims 4 to 22, wherein the subject is suffering from or at risk of developing 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 types 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; and optionally, the lysosomal storage disease involving cells in the CNS is Krabbe disease, Sandhoff disease, Tay-Sachs, Gaucher disease (types I, II, or III), Niemann-Pick disease (NPC1 or NPC2 deficiency), Hurler syndrome, Pompe disease, or Batten disease.

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

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

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

27. 27. The AAV capsid protein of any one of claims 25 to 26, wherein the targeting peptide is inserted between two adjacent amino acids at AA 587-594 of SEQ ID NO: 11, or a functional equivalent of AA 587-594 in an amino acid sequence at least 80% identical to SEQ ID NO:

11.

28. Upon binding to LRP6, the targeting peptide is (1) R28, G158, E159, W183, A201, K202, or H226 in LRP6 having the amino acid sequence of SEQ ID NO:

31. One or more positions functionally equivalent to; or (2) S96, S114, E115, R141, W157, W183, or W242 in LRP6 having the amino acid sequence of SEQ ID NO:

31. The AAV capsid protein of any one of claims 25 to 27, wherein the targeting peptide can interact with one or more positions functionally equivalent to;

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

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

31. A recombinant adeno-associated virus (rAAV) comprising an AAV capsid protein comprising a targeting peptide having binding specificity for low density lipoprotein receptor-related protein 6 (LRP6), wherein the amino acid sequence of the targeting peptide is inserted between two adjacent amino acids at AA 587-594 of the AAV capsid protein, or a functional equivalent thereof.

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

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

31. The rAAV of any one of claims 31 to 32.

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

35. 35. The rAAV of claim 34, wherein the rAAV is capable of transducing the nervous system at least two times more efficiently than an rAAV that does not contain the targeting peptide.

36. 30. A composition for use in delivering a drug to the nervous system of a subject in need thereof, comprising: (1) an AAV capsid protein of any one of claims 25-29; and (2) an AAV comprising an drug to be delivered to the nervous system of the subject, wherein optionally the nervous system is the central nervous system (CNS), the peripheral nervous system (PNS), or a combination thereof.

37. 37. The composition for use of claim 36, wherein the nervous system is a brain endothelial cell, a neuron, a capillary in the brain, an arteriole in the brain, an artery in the brain, or a combination thereof.

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

39. 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 comprising an amino acid sequence having binding specificity for LRP6.

41. The antibody or fragment thereof according to claim 40, which is a bispecific antibody comprising at least one Fab having specificity 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 a derivative or conjugate thereof having specificity for low density lipoprotein receptor-related protein 6 (LRP6).

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

45. A delivery system comprising: (1) a targeting peptide having specificity for low density lipoprotein receptor-related protein 6 (LRP6); and (2) a drug.

46. 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. 47. The delivery system of any one of claims 45 to 46, selected from the group consisting of nanoparticles, nanotubes, nanowires, dendrimers, liposomes, ethosomes and aquasomes, polymersomes and niosomes, foams, hydrogels, cubosomes, quantum dots, exosomes, macrophages, and combinations thereof.

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

49. 48. The delivery system of any one of claims 45 to 47, comprising nanoparticles selected from the group consisting of lipid-based nanoparticles, polymeric 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 having specificity for low density lipoprotein receptor-related protein 6 (LRP6), comprising: (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having the 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 the amino acid sequence of SEQ ID NO:

31. Creating one or more targeting peptides in silico that can interact with each other. A method comprising:

51. generating said one or more targeting peptides in silico; generating a large number of candidate peptides in silico; Performing a computer-assisted docking simulation for each of the multiple candidate peptides that bind to LRP6; and Analyzing the structure of LRP6 bound to one or more of the multiple candidate peptides to identify one or more targeting peptides that can interact with (1) one or more positions functionally equivalent to R28, G158, E159, W183, A201, K202, or H226 in LRP6 having the 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 the amino acid sequence of SEQ ID NO:

31.

51. The method of claim 50, comprising:

52. Obtaining a binding score for each of the plurality of candidate peptides that bind to LRP6; and Selecting one or more of the plurality of candidate peptides having a binding score above a threshold as a targeting peptide having specificity for LRP6.

52. The method of any one of claims 50 to 51, comprising:

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

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

55. An agent 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. 56. The agent of claim 55, which 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 antibody or fragment thereof comprises an Fc domain and / or 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 57. The agent of claim 56, which is a fragment, Fv fragment, disulfide-linked Fv, scFv, single domain antibody, diabody, multispecific antibody, bispecific antibody, anti-idiotypic antibody, diabody, or functionally active epitope-binding fragment thereof.

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

59. 58. The agent of any one of claims 55 to 57, wherein the agent is conjugated to a detectable label; optionally, 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, an imaging agent, a metal particle, a magnetic particle, and any combination thereof.

60. The agent according to any one of claims 55 to 58, which is a therapeutic agent.

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

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

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

64. The method 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. 63. The method of claim 62, wherein the non-viral vector comprises a lipid-based nanoparticle, a polymeric nanoparticle, an inorganic nanoparticle, a surfactant-based emulsion, a nanowire, a silica nanoparticle, a peptide- or protein-based particle, a lipid-polymer particle, a nanolipoprotein particle, and combinations thereof.

66. 66. The agent of any one of claims 61 to 65, wherein the payload delivered to the cell is a biomolecule, a non-biomolecule, or a combination thereof; optionally, 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 nucleic acid sequence delivered to the nervous system is a) sequences encoding trophic factors, growth factors, or other soluble factors that may be released from the transduced cell and affect the survival or function of that cell and / or surrounding cells; b) a DNA sequence that restores protein function to a human or animal carrying a genetic mutation in that gene; c) DNA sequences that encode proteins that can be used to regulate or alter cellular activities or conditions; d) a DNA sequence encoding a protein or nucleic acid used to assess the state of a cell; e) DNA and / or associated guide RNA for genome engineering; f) sequences for genome editing via homologous recombination; g) a DNA sequence encoding a therapeutic RNA; h) an shRNA or artificial miRNA delivery system; or i) DNA sequences that affect the splicing of endogenous genes 68. The method of any one of claims 66 to 67, comprising one or more of:

Images