Methods and Compositions for Trans-Blood Brain Barrier Delivery of Agents

Abstract

The present invention is based on the development of artificial targeting sequences that enhance the penetration of reagents into cells and across the blood-brain barrier, compositions comprising such sequences, and methods of using the same. Provided herein are AAVs comprising a capsid protein containing a targeting sequence and a transgene, preferably a therapeutic or diagnostic transgene. Also provided herein are methods of delivering a transgene to a cell, the methods comprising contacting the cell with an AAV or fusion protein as described herein.

Description

[0001] Priority Claim

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 696,422, filed Jul. 11, 2018. The foregoing is hereby incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on Jul. 11, 2019, is named 29618-0200WO1_SL.txt and is 58,834 bytes in size. Technical Field

[0005] Described herein are sequences that enhance the penetration of reagents across the blood-brain barrier, compositions comprising such sequences, and methods of using the same. Background Art

[0006] The delivery of therapeutic agents, including gene therapy reagents, has hampered the development of treatments for many conditions. The blood-brain barrier (BBB) is a major obstacle to the delivery of drugs to the mammalian central nervous system (CNS), particularly to the human brain, for the treatment of conditions including neurodegenerative diseases such as Parkinson's disease; Alzheimer's disease; Huntington's disease; amyotrophic lateral sclerosis; and multiple sclerosis. Summary of the Invention

[0007] The present invention is based on the development of artificial targeting sequences that enhance the penetration of reagents into cells and across the blood-brain barrier.

[0008] Accordingly, the present disclosure provides an AAV capsid protein, such as an engineered AAV capsid protein, comprising a targeting sequence comprising at least four contiguous amino acids from the sequences TVSALFK (SEQ ID NO:8); TVSALK (SEQ ID NO:4); KLASVT (SEQ ID NO:83); or KFLASVT (SEQ ID NO:84). In some embodiments, the AAV capsid protein comprises a targeting sequence comprising at least five contiguous amino acids from the sequences TVSALK (SEQ ID NO:4); TVSALFK (SEQ ID NO:8); KLASVT (SEQ ID NO:83); or KFLASVT (SEQ ID NO:84). In some embodiments, the AAV capsid protein comprises a targeting sequence comprising at least six contiguous amino acids from the sequences TVSALK (SEQ ID NO:4); TVSALFK (SEQ ID NO:8); KLASVT (SEQ ID NO:83); or KFLASVT (SEQ ID NO:84).

[0009] In some embodiments, the AAV is AAV9; other AAVs known in the art (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8 and variants thereof and other AAVs known in the art or described herein) can also be used.

[0010] In some embodiments, the AAV capsid protein comprises AAV9 VP1 (e.g., SEQ ID NO:85).

[0011] In some embodiments, the targeting sequence is inserted at a position corresponding to between amino acids 588 and 589 of SEQ ID NO:85 in the capsid protein.

[0012] The present disclosure also provides a nucleic acid encoding an AAV capsid protein comprising a targeting sequence as described herein.

[0013] Furthermore, the present disclosure provides an AAV comprising a capsid protein comprising a targeting sequence as described herein. In some embodiments, the AAV further comprises a transgene, preferably a therapeutic or diagnostic transgene. Therapeutic transgenes can include, for example, cDNA that restores protein function, guide RNAs for gene editing, RNAs, or miRNAs.

[0014] The present disclosure also provides targeting sequences comprising V[S / p][A / m / t / ]L (SEQ ID NO:79), TV[S / p][A / m / t / ]L (SEQ ID NO:80), TV[S / p][A / m / t / ]LK (SEQ ID NO:81), or TV[S / p][A / m / t / ]LFK (SEQ ID NO:82). In some embodiments, the targeting sequences comprise VPALR (SEQ ID NO:1); VSALK (SEQ ID NO:2); TVPALR (SEQ ID NO:3); TVSALK (SEQ ID NO:4); TVPMLK (SEQ ID NO:12); TVPTLK (SEQ ID NO:13); FTVSALK (SEQ ID NO:5); LTVSALK (SEQ ID NO:6); TVSALFK (SEQ ID NO:8); TVPALFR (SEQ ID NO:9); TVPMLFK (SEQ ID NO:10) or TVPTLFK (SEQ ID NO:11). Also provided are fusion proteins comprising a targeting sequence linked to a heterologous (e.g., non-AAV VP1) sequence, and AAV capsid proteins (e.g., AAV9 VP1) comprising a targeting sequence. In some embodiments, the targeting sequence is inserted at positions corresponding to amino acids 588 and 589 of SEQ ID NO:85.

[0015] Further provided herein are nucleic acids encoding the targeting sequences, fusion proteins, or AAV capsid proteins described herein, and AAVs comprising a capsid protein comprising a targeting sequence. In some embodiments, the AAV further comprises a transgene, preferably a therapeutic or diagnostic transgene. Therapeutic transgenes can include, for example, cDNA that restores protein function, guide RNAs for gene editing, RNAs, or miRNAs.

[0016] Also provided herein are methods of delivering a transgene to a cell, the methods comprising contacting the cell with an AAV or fusion protein described herein. In some embodiments, the cell is in a living subject, e.g., a mammalian subject. In some embodiments, the cell is in a tissue selected from the brain, spinal cord, dorsal root ganglion, heart, or muscle, and combinations thereof. In some embodiments, the cell is a neuron (optionally a dorsal root ganglion neuron), astrocyte, cardiomyocyte, or myocyte.

[0017] In some embodiments, the subject has a neurodegenerative disease, epilepsy; stroke; spinocerebellar ataxia; Canavan's disease; Metachromatic Leukodystrophy; spinal muscular atrophy; Friedreich’s ataxia; X-linked centronuclear myopathy; lysosomal storage disease; Barth Syndrome; Duchenne muscular dystrophy; Wilson’s disease; or type 1 Crigler-Najjar syndrome. In some embodiments, the neurodegenerative disease is Parkinson's disease; Alzheimer's disease; Huntington's disease; amyotrophic lateral sclerosis; and multiple sclerosis.

[0018] In some embodiments, the subject has a brain cancer, and the method comprises administering an AAV encoding an anti-cancer agent. In some embodiments, the anti-cancer agent is HSV.TK1, and the method further comprises administering ganciclovir.

[0019] In some embodiments, the cells are located in the brain of the subject, and the AAV is administered by parenteral delivery (e.g., by intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular delivery); intracerebral; or intrathecal delivery (e.g., by lumbar injection, cisterna magna injection, or intracerebral parenchymal injection).

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0021] Other features and advantages of the invention will become apparent from the following detailed description, the drawings, and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1A-1B An exemplary strategy for engineering AAV9 by inserting a cell-penetrating peptide (CPP) into the capsid of AAV9 is described. Figure 1AIt is a 3D model of AAV9 virus. The individual CPP inserted between amino acids 588 and 589 (VP1 numbering) in the capsid is shown on the 3-fold axis where receptor binding may occur. Figure 1B Describe the method for generating individual AAV. Three plasmids including pRC (engineered or non-engineered), pHelper, and pAAV were co-transfected into HEK 293T cells, and AAV was harvested and purified using an iodixanol gradient.

[0023] Figure 2A-2B Show representative images of mouse brain sections and their quantitative analysis after intravenous administration of low-dose candidate AAV. Mice with a mixed genetic background were used. The candidate AAVs differed in the CPP they inserted (see Table 3), but all expressed nuclear red fluorescent protein (RFP) as a reporter. For further screening, candidate AAVs with low yields were excluded. The dose of AAV was 1×10 10 vg (viral genome). Figure 2A Each white dot in [[]] represents an RFP-labeled cell. In Figure 2B , *P<0.05, relative to AAV9, ANOVA.

[0024] Figure 2C-2D Describe the representative images of mouse brain sections and their quantitative analysis after intravenous administration of AAV.CPP.11 and AAV.CPP.12 in repeated experiments. AAV.CPP.11 and AAV.CPP.12 contain CPP BIP1 and CPP BIP2 respectively (see Table 3). The dose of AAV was increased to 1×10 11 vg. The candidate AAVs expressed nuclear red fluorescent protein (RFP) as a reporter. Figure 2C Each white dot in [[]] represents an RFP-labeled cell. In Figure 2D , *P<0.05, **P<0.01, relative to AAV9, ANOVA.

[0025] Figure 3AOptimization of the BIP targeting sequence for further engineering of AAV9 towards better brain transduction. BIP1 (VPALR, SEQ ID NO:1), which enables AAV9 to transduce the brain more efficiently (as in AAV.CPP.11), is derived from the rat protein Ku70. The human, mouse, and rat Ku70 proteins differ in their exact amino acid sequences. BIP2 (VSALK, SEQ ID NO:2) is a "synthetic" peptide related to BIP1 as in AAV.CPP.12. Further engineering focused on the VSALK sequence, with the hope of minimizing the species specificity of the ultimately engineered AAV. To generate new targeting sequences, target amino acids were added to the VSALK sequence and, in other cases, the positions of individual amino acids were switched (switched). All new BIP2-derived sequences were again inserted into the AAV9 capsid to generate new candidate AAVs for screening. The sequences that emerged in turn were SEQ ID NO:69, 70, 71, 1 - 6, 72, 7, and 8.

[0026] Figure 3B-3C Describes representative images of mouse brain sections and their quantitative analysis after intravenous administration of more candidate AAVs. All candidate AAVs express nuclear red fluorescent protein (RFP) as a reporter protein. The dose of AAV was 1×10 11 vg per animal. Figure 3B Each white dot in represents an RFP-labeled cell. AAV.CPP.16 and AAV.CPP.21 were identified as top hits for their strong and widespread brain transduction. In Figure 3C , *P<0.05, **P<0.01, ***P<0.001, relative to AAV9, ANOVA.

[0027] Figure 3D Describes the quantitative analysis of transduction efficiency in the liver after intravenous administration of candidate AAVs. The percentage of transduced liver cells is shown. The dose of AAV was 1×10 11 vg per animal. ***P<0.001, relative to AAV9, ANOVA.

[0028] Figure 4A-4E Describes the screening of selected candidate AAVs in an in vitro spheroid model of the human blood-brain barrier. Figure 4A Illustrates spheroids composed of human microvascular endothelial cells that form a barrier on the surface, as well as human pericytes and astrocytes inside the spheroids. Evaluates the ability of candidate AAVs to penetrate from the surrounding medium into the interior of the spheroids and transduce the internal cells. Figure 4B-4D Shows graphs of spheroids treated with AAV9, AAV.CPP.16, and AAV.CPP.21. Figure 4EShow the relative RFP intensity of spheroids treated with different AAVs. ***P < 0.001, relative to AAV9, ANOVA.

[0029] Figure 5A-5B Describe the representative images and their quantitative analysis of brain sections after intravenous administration of AAV9, AAV.CPP.16, and AAV.CPP.21 in C57BL / 6J inbred mice. All candidate AAVs express nuclear red fluorescent protein (RFP) as a reporter protein. The dose of AAV was 1 × 10 12 vg per animal. Figure 5A Each white dot in Figure 5B represents an RFP-labeled cell. In

[0030] Figure 6A-6B Describe the representative images and their quantitative analysis of brain sections after intravenous administration of AAV9, AAV.CPP.16, and AAV.CPP.21 in BALB / cJ inbred mice. All candidate AAVs express nuclear red fluorescent protein (RFP) as a reporter protein. The dose of AAV was 1 × 10 12 vg per animal. Figure 6A Each white dot in Figure 6B represents an RFP-labeled cell. In

[0031] Figure 7A-7B Describe the representative images and their quantitative analysis of brain sections after intravenous administration of high doses of AAV.CPP.16 and AAV.CPP.21 in C57BL / 6J inbred mice. The two candidate AAVs express nuclear red fluorescent protein (RFP) as a reporter protein. The dose of AAV was 4 × 10 12 vg per animal. Figure 7A Each white dot in Figure 7B represents an RFP-labeled cell. In

[0032] Figure 8A Show that AAV.CPP.16 and AAV.CPP.21 transduce adult neurons (labeled by NeuN antibody) across multiple brain regions including the cortex, midbrain, and hippocampus in mice. Transduced neurons are co-labeled by NeuN antibody and RFP. 4 × 10 12 vg of AAV was intravenously administered to adult C57BL / 6J mice (6 weeks old).

[0033] Figure 8B Describe that AAV.CPP.16 and AAV.CPP.21 show enhanced ability to target the spinal cord and motor neurons of mice compared to AAV9. 4 × 1010 AAV of vg was intravenously administered to neonatal mice (1 day after birth). Motor neurons in the ventral horn of the spinal cord were observed using CHAT antibody staining. Co-localization of RFP and CHAT signals showed specific transduction of motor neurons.

[0034] Figure 9A Describe that AAV.CPP.16 shows enhanced ability to target the heart of adult mice compared to AAV9. 1×10 11 vg of AAV was intravenously administered to adult C57BL / 6J mice (6 weeks old). The percentage of RFP-labeled cells relative to all DAPI-stained cells was shown. *P<0.05, Student's test.

[0035] Figure 9B Describe that AAV.CPP.16 shows enhanced ability to target the skeletal muscle of adult mice compared to AAV9. 1×10 11 vg of AAV was intravenously administered to adult C57BL / 6J mice (6 weeks old). The percentage of RFP-labeled cells relative to all DAPI-stained cells was shown. *P<0.05, Student's test.

[0036] Figure 9C Describe that AAV.CPP.16 shows enhanced ability to target the dorsal root ganglion (DRG) of adult mice compared to AAV9. 1×10 11 vg of AAV was intravenously administered to adult C57BL / 6J mice (6 weeks old). The percentage of RFP-labeled cells relative to all DAPI-stained cells was shown. *P<0.05, Student's test.

[0037] Figure 10A Describe that after intravenous administration to non-human primates, AAV.CPP.16 and AAV.CPP.21 show enhanced ability to transduce brain cells in the primary visual cortex compared to AAV9. 2×10 13 vg / kg of AAV-CAG-AADC (as a reporter gene) was intravenously injected into 3-month-old cynomolgus monkeys with low pre-existing neutralizing antibodies. AAV-transduced cells (shown in black) were observed using antibody staining against AADC. The square area in the left figure was magnified as shown in the right figure. AAV.CPP.16 transduced significantly more cells compared to AAV9. AAV.CPP.21 also transduced more cells compared to AAV9, although its effect was less obvious compared to AAV.CPP.16.

[0038] Figure 10BDescribe the enhanced ability of AAV.CPP.16 and AAV.CPP.21 to transduce brain cells in the parietal cortex relative to AAV9 after intravenous administration to non-human primates. 2×10 13 vg / kg AAV-CAG-AADC (as a reporter gene) was intravenously injected into 3-month-old cynomolgus monkeys with low pre-existing neutralizing antibodies. AAV-transduced cells (shown in black) were observed using antibody staining against AADC. The square area in the left panel was magnified as shown in the right panel. AAV.CPP.16 transduced significantly more cells relative to AAV9. AAV.CPP.21 also transduced more cells relative to AAV9, although the effect was less obvious compared to AAV.CPP.16.

[0039] Figure 10C Describe the enhanced ability of AAV.CPP.16 and AAV.CPP.21 to transduce brain cells in the thalamus relative to AAV9 after intravenous administration to non-human primates. 2×10 13 vg / kg AAV-CAG-AADC (as a reporter gene) was intravenously injected into 3-month-old cynomolgus monkeys with low pre-existing neutralizing antibodies. AAV-transduced cells (shown in black) were observed using antibody staining against AADC. The square area in the left panel was magnified as shown in the right panel. AAV.CPP.16 transduced significantly more cells relative to AAV9. AAV.CPP.21 also transduced more cells relative to AAV9, although the effect was less obvious compared to AAV.CPP.16.

[0040] Figure 10D Describe the enhanced ability of AAV.CPP.16 and AAV.CPP.21 to transduce brain cells in the cerebellum relative to AAV9 after intravenous administration to non-human primates. 2×10 13 vg / kg AAV-CAG-AADC (as a reporter gene) was intravenously injected into 3-month-old cynomolgus monkeys with low pre-existing neutralizing antibodies. AAV-transduced cells (shown in black) were observed using antibody staining against AADC. The square area in the left panel was magnified as shown in the right panel. Both AAV.CPP.16 and AAV.CPP.21 transduced significantly more cells relative to AAV9.

[0041] Figure 11A-11BIt is described that AAV.CPP.16 and AAV.CPP.21 do not bind to LY6A. LY6A serves as a receptor for AAV.PHP.B, and its variants include AAV.PHP.eB (as described in US9102949, US20170166926), and mediates the potent crossing of the BBB by AAV.PHP.eB in certain mouse strains (Hordeaux et al. Mol Ther 2019 27(5):912 - 921; Huang et al. 2019, dx.doi.org / 10.1101 / 538421). Overexpression of mouse LY6A in cultured 293 cells significantly increases the binding of AAV.PHP.eB to the cell surface( Figure 11A ). Conversely, overexpression of LY6A does not increase viral binding to AAV9, AAV.CPP.16 or AAV.CPP.21( Figure 11B ). This indicates that AAV.CPP.16 or AAV.CPP.21 does not share LY6A with AAV.PHP.eB as a receptor.

[0042] Figure 12A-12C It is described that AAV.CPP.21 can be used for systemic delivery of therapeutic genes to brain tumors in a mouse model of glioblastoma (GBM). As Figure 11A , intravenously administered AAV.CPP.21 - H2BmCherry is shown to target tumor masses, especially the tumor expanding frontier. In Figure 11B -11C, when combined with the prodrug ganciclovir, AAV.CPP.21 is used to systemically deliver the "suicide gene" HSV.TK1, causing shrinkage of brain tumor masses. HSV.TK1 converts the originally "dormant" ganciclovir into a tumor - killing drug. *P < 0.05, Student's test.

[0043] Figure 13 It is described that compared with AAV9, when locally injected into the adult mouse brain, AAV.CPP.21 causes more extensive and potent transduction of brain tissue. Intracerebral injection of AAV (1×10 11 vg) was performed in adult mice (>6 weeks old), and brain tissue was harvested and examined 3 weeks after AAV injection. **P < 0.01, Student's test. Detailed Description

[0044] Difficulties associated with trans-BBB delivery have hampered the development of therapeutic agents for treating brain disorders including cancer and neurodegenerative diseases. Adeno-associated virus (AAV) has emerged as an important research and clinical tool for delivering therapeutic genes to the brain, spinal cord, and eye; see, e.g., US9102949; US 9585971; and US20170166926. However, existing AAVs, including AAV9, have limited efficiency in crossing the BBB or only work in certain non-primate species.

[0045] Through rational design and targeted screening based on known cell-penetrating peptides (CPPs) (see, e.g., Gomez et al., Bax-inhibiting peptides derived from Ku70 and cell-penetrating pentapeptides. Biochem. Soc. Trans. 2007; 35(Pt 4):797–801), it has been found that targeting sequences, when engineered into the capsid of AAV, can increase the efficiency of gene delivery to the brain by up to three orders of magnitude. These methods are used to engineer an AAV vector that significantly reduces tumor size in animal models of glioblastoma.

[0046] Targeting sequence

[0047] The methods of the present invention identify a number of potential targeting peptides that enhance penetration through the BBB, for example, when inserted into the capsid of an AAV such as AAV1, AAV2, AAV8, or AAV9, or when conjugated chemically or by expression as a fusion protein with a biological agent such as an antibody or other large biomolecule.

[0048] In some embodiments, the targeting peptide comprises a sequence of at least 5 amino acids. In some embodiments, the amino acid sequence comprises at least 4, such as 5, consecutive amino acids of the sequences VPALR (SEQ ID NO:1) and VSALK (SEQ ID NO:2).

[0049] In some embodiments, the targeting peptide comprises X 1 X 2 X 3 X 4 X 5 of the sequence, wherein:

[0050] (i) X 1 、X 2 、X 3 、X 4 are any four different amino acids of V, A, L, I, G, P, S, T, or M; and

[0051] (ii) X 5 is K, R, H, D, or E (SEQ ID NO:73).

[0052] In some embodiments, the targeting peptide comprises a sequence of at least 6 amino acids. In some embodiments, the amino acid sequence comprises at least 4, such as 5 or 6, consecutive amino acids of the sequences TVPALR (SEQ ID NO:3), TVSALK (SEQ ID NO:4), TVPMLK (SEQ ID NO:12), and TVPTLK (SEQ ID NO:13).

[0053] In some embodiments, the targeting peptide comprises X 1 X 2 X 3 X 4 X 5 X 6 of the sequence, wherein:

[0054] (i) X 1 is T;

[0055] (ii) X 2 、X 3 、X 4 、X 5 are any four different amino acids of V, A, L, I, G, P, S, T, or M; and

[0056] (iii) X 6 is K, R, H, D, or E (SEQ ID NO:74). In some embodiments, the targeting peptide comprises X 1 X 2 X 3 X 4 X 5 X 6 of the sequence, wherein:

[0057] (i) X 1 、X 2 、X 3 、X 4 are any four different amino acids from V, A, L, I, G, P, S, T, or M;

[0058] (ii) X 5 is K, R, H, D, or E; and

[0059] (iii) X 6 is E or D (SEQ ID NO:75).

[0060] In some embodiments, the targeting peptide comprises a sequence of at least 7 amino acids. In some embodiments, the amino acid sequence comprises at least 4, such as 5, 6, or 7 consecutive amino acids of the sequences FTVSALK (SEQ ID NO:5), LTVSALK (SEQ ID NO:6), TVSALFK (SEQ ID NO:8), TVPALFR (SEQ ID NO:9), TVPMLFK (SEQ ID NO:10), and TVPTLFK (SEQ ID NO:11). In some other embodiments, the targeting peptide comprises X 1 X 2 X 3 X 4 X 5 X 6 X 7 of the sequence, wherein:

[0061] (i) X 1 is F, L, W, or Y;

[0062] (ii) X 2 is T;

[0063] (iii) X 3 X 4 X 5 X 6 are any four different amino acids of V, A, L, I, G, P, S, T, or M; and

[0064] (iv) X 7 is K, R, H, D, or E (SEQ ID NO:76). In some embodiments, the targeting peptide comprises X 1 X 2 X 3 X 4 X 5 X 6 X 7 of the sequence, wherein:

[0065] (i) X 1 is T;

[0066] (ii) X 2 X 3 X 4 X 5 are any four different amino acids of V, A, L, I, G, P, S, T, or M;

[0067] (iii) X 6 is K, R, H, D, or E; and

[0068] (iv) X7 is E or D (SEQ ID NO:77).

[0069] In some embodiments, the targeting peptide comprises X 1 X 2 X 3 X 4 X 5 X 6 X 7 of the sequence, wherein:

[0070] (i) X 1 , X 2 , X 3 , X 4 are any four different amino acids of V, A, L, I, G, P, S, T, or M;

[0071] (ii) X 5 is K, R, H, D, or E;

[0072] (iii) X 6 is E or D; and

[0073] (iv) X 7 is A or I (SEQ ID NO:78).

[0074] In some embodiments, the targeting peptide comprises the sequence of V[S / p][A / m / t / ]L (SEQ ID NO:79), where the capital letters are preferred at that position. In some embodiments, the targeting peptide comprises the sequence of TV[S / p][A / m / t / ]L (SEQ ID NO:80). In some embodiments, the targeting peptide comprises the sequence of TV[S / p][A / m / t / ]LK (SEQ ID NO:81). In some embodiments, the targeting peptide comprises the sequence of TV[S / p][A / m / t / ]LFK. (SEQ ID NO:82).

[0075] In some embodiments, the targeting peptide is not composed of VPALR (SEQ ID NO:1) or VSALK (SEQ ID NO:2).

[0076] Table 1 lists specific exemplary amino acid sequences comprising the above 5-, 6- or 7-amino acid sequences.

[0077] Table 1 - Targeting Sequences

[0078]

[0079]

[0080] Targeting peptides including reverse sequences can also be used, for example, KLASVT (SEQ ID NO:83) and KFLASVT (SEQ ID NO:84).

[0081] The targeting peptides disclosed herein can be modified according to methods known in the art for generating peptidomimetics. See, for example, Qvit et al., Drug Discov Today. February 2017; 22(2):454–462; Farhadi and Hashemian, Drug Des Devel Ther. 2018; 12:1239–1254; Avan et al., Chem. Soc. Rev., 2014, 43, 3575-3594; Pathak, et al., Indo American Journal of Pharmaceutical Research, 2015.8; Kazmierski, W.M., editor, Peptidomimetics Protocols, Human Press (Totowa NJ 1998); Goodman et al., editors, Houben-Weyl Methods of Organic Chemistry: Synthesis of Peptides and Peptidomimetics, Thiele Verlag (New York 2003); and Mayo et al., J. Biol. Chem., 278:45746 (2003). In some cases, these modified peptidomimetic forms of the peptides and fragments disclosed herein exhibit enhanced in vivo stability relative to non-peptidomimetic peptides.

[0082] Methods for generating peptidomimetics include substituting one or more, such as all, of the amino acids in the peptide sequence with D-amino acid enantiomers. Such sequences are referred to herein as "retro" sequences. In another method, the order of the amino acid residues from the N-terminus to the C-terminus is reversed such that the order of the amino acid residues from the N-terminus to the C-terminus of the original peptide becomes the order of the amino acid residues from the C-terminus to the N-terminus in the modified peptidomimetic. Such sequences can be referred to as "inverso" sequences.

[0083] Peptidomimetics can be in retro and inverso forms, i.e., the "retro-inverso" form of the peptides disclosed herein. The new peptidomimetics can be composed of D-amino acids arranged such that the order of the amino acid residues from the N-terminus to the C-terminus in the peptidomimetic corresponds to the order of the amino acid residues from the C-terminus to the N-terminus in the original peptide.

[0084] Other methods for preparing peptidomimetics include replacing one or more amino acid residues in a peptide with chemically distinct but recognized amino acid functional analogs, i.e., artificial amino acid analogs. Artificial amino acid analogs include β-amino acids, β-substituted β-amino acids (“β 3 -amino acids”), phosphorus-containing analogs of amino acids such as -aminophosphonic acid and -aminophosphinic acid, and amino acids with non-peptide bonds. Artificial amino acids can be used to generate peptidomimetics such as peptoid oligomers (e.g., peptoid amide or ester analogs), β-peptides, cyclic peptides, oligoureas or oligourethane peptides; or heterocyclic molecules. Exemplary reverse-inverse targeting peptidomimetics include KLASVT and KFLASVT, where the sequences include all D-amino acids. These sequences can be modified, for example, by biotinylation at the amino terminus and amidation at the carboxyl terminus.

[0085] AAV

[0086] Viral vectors for use in the methods and compositions of the present invention include recombinant retroviruses, adenoviruses, adeno-associated viruses, alphaviruses, and lentiviruses, which include the targeting peptides described herein and optionally a transgene for expression in a target tissue.

[0087] The preferred viral vector system for delivering nucleic acids in the present method is adeno-associated virus (AAV). AAV is a small non-enveloped virus with a 25 nm capsid. No disease is known or has been shown to be associated with the wild-type virus. AAV has a single-stranded DNA (ssDNA) genome. AAV has been shown to exhibit long-term episomal transgene expression, and AAV has demonstrated excellent transgene expression in the brain, particularly in neurons. Vectors of AAV containing as few as 300 base pairs can be packaged and integrated. The space limit for foreign DNA is approximately 4.7 kb. AAV vectors such as those described in Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985) can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see, for example, Hermonat et al., Proc. Natl. Acad. Sci. USA 81:6466-6470 (1984); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford et al., Mol. Endocrinol. 2:32-39 (1988); Tratschin et al., J. Virol. 51:611-619 (1984); and Flotte et al., J. Biol. Chem. 268:3781-3790 (1993). There are many alternative AAV variants (over 100 have been cloned), and AAV variants have been identified based on desired properties. In some embodiments, the AAV is AAV1, AAV2, AAV4, AAV5, AAV6, AV6.2, AAV7, AAV8, AAV9, rh.10, rh.39, rh.43, or CSp3; for CNS use, in some embodiments, the AAV is AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, or AAV9. As an example, AAV9 has been shown to cross the blood-brain barrier relatively effectively. Using the method of the present invention, the AAV capsid can be genetically engineered to increase penetration across the BBB, or increase penetration into a specific tissue, by inserting a targeting sequence as described herein into the capsid protein, for example, between amino acids 588 and 589 of the AAV9 capsid protein VP1.

[0088] The exemplary wild-type AAV9 capsid protein VP1 (Q6JC40-1) sequence is shown below:

[0089]

[0090] Accordingly, the present disclosure provides an AAV comprising one or more of the targeting peptide sequences described herein, such as an AAV comprising a capsid protein comprising the targeting sequence described herein, the capsid protein being, for example, a capsid protein of SEQ ID NO:1 in which the targeting peptide sequence has been inserted between, for example, amino acids 588 and 589 of the sequence.

[0091] In some embodiments, the AAV further comprises a transgene sequence (i.e., a heterologous sequence), such as a transgene encoding a therapeutic agent as described herein or known in the art, or a reporter protein such as a fluorescent protein (an enzyme that catalyzes a reaction to produce a detectable product), or a cell surface antigen. The transgene is preferably linked to a sequence that promotes / drives the expression of the transgene in the target tissue.

[0092] Exemplary transgenes for use as therapeutic agents include neuronal apoptosis inhibitory protein (NAIP), nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), tyrosine hydroxylase (TH), GTP-cyclohydrolase (GTPCH), amino acid decarboxylase (AADC), aspartoacylase (ASPA), blood factors such as β-globin, hemoglobin, tissue plasminogen activator, and blood coagulation factors; colony stimulating factor (CSF); interleukins such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, etc.; growth factors such as keratinocyte growth factor (KGF), stem cell factor (SCF), fibroblast growth factor (FGF, such as basic FGF and acidic FGF), hepatocyte growth factor (HGF), insulin-like growth factors (IGFs), bone morphogenetic protein (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 (PDGF), thrombopoietin (TPO), transforming growth factor α (TGF-α), transforming growth factor β (TGF-β), etc.; soluble receptors such as soluble TNF-α receptor, soluble VEGF receptor, soluble interleukin receptors (e.g., soluble IL-1 receptor and soluble type II IL-1 receptor), soluble γ / δ T cell receptor, ligand-binding fragments of soluble receptors, etc.; enzymes such as α-glucosidase, imiglucerase, β-glucocerebrosidase, and alglucerase; enzyme activators such as tissue plasminogen activator; chemokines such as IP-10, monokine induced by interferon γ (Mig), Groα / IL-8, RANTES, MIP-1α, MIP-1β, MCP-1, PF-4, etc.; angiogenic agents such as vascular endothelial growth factors (VEGFs, e.g., VEGF121, VEGF165, VEGF-C, VEGF-2), transforming growth factor-β, basic fibroblast growth factor, glioma-derived growth factor, angiopoietin, angiopoietin-2; etc.; anti-angiogenic agents such as soluble VEGF receptor; protein vaccines;Neuroactive peptides, such as nerve growth factor (NGF), bradykinin, cholecystokinin, gastrin, secretin, oxytocin, gonadotropin-releasing hormone, β-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dynorphin, warfarin, neurotensin, motilin, thyroid-stimulating hormone, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, sleep peptide, etc.; thrombolytics; atrial natriuretic peptide; relaxin; glial fibrillary acidic protein; follicle-stimulating hormone neuropeptide (FSH); human α-1 antitrypsin; leukemia inhibitory factor (LIF); transforming growth factors (TGFs); tissue factor, luteinizing hormone; macrophage activating factor; tumor necrosis factor (TNF); neutrophil chemotactic factor (NCF); nerve growth factor; tissue inhibitor of metalloproteinase; vasoactive intestinal peptide; angiopoietin; angiotrophin; fibrin; hirudin; IL-1 receptor antagonist; etc. Some other examples of target proteins include ciliary neurotrophic factor (CNTF); neurotrophin 3 and 4 / 5 (NT-3 and 4 / 5); glial cell line-derived neurotrophic factor (GDNF); aromatic amino acid decarboxylase (AADC); hemophilia-related clotting proteins, such as factor VIII, factor IX, factor X; dystrophin or minidystrophin; lysosomal acid lipase; phenylalanine hydroxylase (PAH); enzymes related to glycogen storage diseases, such as glucose-6-phosphatase, acid maltase, glycogen debranching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase (e.g., PHKA2), glucose transporter (e.g., GLUT2), aldolase A, β-enolase, and glycogen synthase; lysosomal enzymes (e.g., β-N-acetylhexosaminidase A); and any variants thereof.

[0093] The transgenes can also encode antibodies, e.g., immune checkpoint inhibitory antibodies against PD-L1, PD-1, CTLA-4 (cytotoxic T-lymphocyte-associated protein-4; CD152); LAG-3 (lymphocyte activation gene 3; CD223); TIM-3 (T-cell immunoglobulin and mucin domain 3; HAVCR2); TIGIT (T-cell immunoreceptor with Ig and ITIM domains); B7-H3 (CD276); VSIR (V-set immunoregulatory receptor, also known as VISTA, B7H5, C10orf54); BTLA30 (B- and T-lymphocyte attenuator, CD272); GARP (glycoprotein A repetitions predominant); PVRIG (PVR-related immunoglobulin domain-containing); or VTCN1 (V-set domain containing T cell activation inhibitor 1, also known as B7-H4).

[0094] Other transgenes can include small or inhibitory nucleic acids that alter / reduce target gene expression, e.g., siRNA, shRNA, miRNA, antisense oligonucleotides, or long non-coding RNAs that alter gene expression (see, e.g., WO2012087983 and US20140142160), or CRISPR Cas9 / cas12a and guide RNAs.

[0095] The virus can also include one or more sequences that promote transgene expression, e.g., one or more promoter sequences; enhancer sequences, e.g., 5' untranslated region (UTR) or 3' UTR; polyadenylation sites; and / or insulator sequences. In some embodiments, the promoter is a brain tissue-specific promoter, e.g., a neuron-specific or glial-specific promoter. In certain embodiments, the promoter is the promoter of a gene selected from: neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), MeCP2, adenomatous polyposis coli (APC), ionized calcium-binding adapter molecule 1 (Iba-1), synapsin I (SYN), calcium / calmodulin-dependent protein kinase II, tubulin αI, neuron-specific enolase, and platelet-derived growth factor β chain. In some embodiments, the promoter is a pan-cellular promoter, e.g., cytomegalovirus (CMV), β-glucuronidase (GUSB), ubiquitin C (UBC), or Rous sarcoma virus (RSV) promoter. The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) can also be used.

[0096] In some embodiments, the AAV also has one or more additional mutations that increase delivery to a target tissue such as, for example, the CNS, or reduce off-tissue targeting, such as mutations that reduce liver delivery when delivery to the CNS, heart, or muscle is desired (e.g., as described in Pulicherla et al. (2011) Mol Ther 19:1070-1078); or additional targeting peptides are added, e.g., as described in Chen et al. (2008) Nat Med 15:1215-1218 or Xu et al., (2005) Virology 341:203-214 or US9102949; US9585971; and US20170166926. See also Gray and Samulski (2011) “Vector design and considerations for CNS applications,” in Gene Vector Design and Application to Treat Nervous System Disorders ed. Glorioso J., ed. (Washington, DC: Society for Neuroscience;), 1–9, available at sfn.org / ~ / media / SfN / Documents / Short%20Courses / 2011%20Short%20Course%20I / 2011_SC1_Gray.ashx.

[0097] Targeting peptides as tags / fusions

[0098] The targeting peptides described herein can also be used to increase the penetration of other (heterologous) molecules across the BBB, for example, by conjugation to a molecule or by expression as part of a fusion protein with, for example, an antibody or other large biomolecule. These can include genome editing proteins or complexes (e.g., TALEs, ZFNs, base editors, and CRISPR RNPs and guide RNAs comprising gene editing proteins such as Cas9 or Cas12a fused (e.g., at the N-terminus, C-terminus, or internally) to the peptides described herein) in addition to the therapeutic agents or reporter proteins described herein and those listed in Table 2. The fusion / complex does not include any additional sequences from Ku70, e.g., comprises heterologous non-Ku70 sequences and is not naturally occurring.

[0099] In some embodiments, the targeting sequence that is part of the non-AAV fusion protein does not include VPALR (SEQ ID NO:1) or VSALK (SEQ ID NO:2), or is not composed of VPALR (SEQ ID NO:1) or VSALK (SEQ ID NO:2).

[0100] Method of Use

[0101] The methods and compositions described herein can be used to deliver any composition, such as a target sequence, to tissues such as the central nervous system (brain), heart, muscle, or dorsal root ganglia or spinal cord (peripheral nervous system). In some embodiments, the method includes delivery to a specific brain region, such as the cortex, cerebellum, hippocampus, substantia nigra, amygdala. In some embodiments, the method includes delivery to neurons, astrocytes, glial cells, or cardiomyocytes.

[0102] In some embodiments, the methods and compositions such as AAV are used to deliver nucleic acid sequences to a subject having a disease such as a CNS disease; see, e.g., US9102949; US 9585971; and US20170166926. In some embodiments, the subject has a condition listed in Table 2; in some embodiments, the vector is used to deliver a therapeutic agent listed in Table 2 for treating the corresponding disease listed in Table 2. The therapeutic agent can be delivered, for example, as nucleic acid by a viral vector, where the nucleic acid encodes a therapeutic protein or other nucleic acids, such as antisense oligonucleotides, siRNA, shRNA, etc.; or as a fusion protein / complex with a targeting peptide as described herein.

[0103] Table 2 - Diseases

[0104]

[0105] In some embodiments, the compositions and methods are used for treating brain cancer. Brain cancers include gliomas (e.g., glioblastoma multiforme (GBM)), metastases (e.g., from lung cancer, breast cancer, melanoma, or colon cancer), meningiomas, pituitary adenomas, and acoustic neuromas. The compositions include a targeting peptide linked to an anti-cancer agent, such as a "suicide gene" that induces apoptosis in target cells (e.g., HSV.TK1, cytosine deaminase (CD) from herpes simplex virus or Escherichia coli, or Escherichia coli purine nucleoside phosphorylase (PNP) / fludarabine; see Krohne et al., Hepatology. September 2001; 34(3):511-8; Dey and Evans, "Suicide Gene Therapy by Herpes Simplex Virus-1 Thymidine Kinase (HSV-TK)" (2011) DOI:10.5772 / 18544), and the targeting peptide is an immune checkpoint inhibitory antibody known in the art or described herein. For example, an AAV vector comprising a targeting peptide as described herein can be used to deliver the "suicide gene" HSV.TK1 to a brain tumor. HSV.TK1 converts the otherwise "dormant" ganciclovir into an anti-tumor drug. Thus, the method can include systemic administration, such as intravenously, of an AAV (e.g., AAV9) comprising a targeting peptide as described herein and encoding HSV.TK1 together with the prodrug ganciclovir to a subject diagnosed with brain cancer.

[0106] Drug Compositions and Methods of Administration

[0107] The methods described herein include a pharmaceutical composition using a targeting peptide as an active ingredient.

[0108] The pharmaceutical composition generally includes a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc., which are compatible with drug administration.

[0109] The pharmaceutical composition is usually formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular, or injection or infusion administration. Thus, the delivery can be systemic or local.

[0110] Methods for formulating suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st Edition, 2005; and the Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY) series of books. For example, solutions or suspensions for parenteral application may include the following components: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate, or phosphate, and reagents for adjusting muscle elasticity such as sodium chloride or glucose. The pH can be adjusted with an acid or a base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0111] Pharmaceutical compositions suitable for injection use may include sterile aqueous solutions (water-soluble) or dispersions, as well as sterile powders for the extemporaneous preparation of sterile injection solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ) phosphate buffered saline (PBS). In all cases, the composition must be sterile and should flow to an extent that is easy to inject. It should be stable under the production and storage conditions and must prevent the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or a dispersion medium, which contains, for example, water, ethanol, polyols (such as glycerin, propylene glycol, and liquid polyethylene glycols, etc.), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by using coatings such as lecithin, in the case of dispersions by maintaining the required particle size and by using surfactants. The action of preventing microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents in the composition, such as sugars, polyols, such as mannitol, sorbitol, sodium chloride. Prolonged absorption of the injection composition can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0112] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound with one or a combination of the above-listed ingredients into a suitable solvent and then filtering sterilizing. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those listed 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 which yield a powder of the active ingredient and any other required ingredients from its previously sterile filtered solution.

[0113] In one embodiment, the therapeutic compound is prepared with a carrier that will protect the therapeutic compound from rapid elimination from the body, such as as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using standard techniques or, for example, are commercially available from Alza Corporation and NovaPharmaceuticals, Inc. Liposome suspensions, including liposomes targeted to selected cells with monoclonal antibodies against cell antigens, can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those of skill in the art, for example, as described in U.S. Patent No. 4,522,811.

[0114] The pharmaceutical composition can be included in a kit, container, package, or dispenser together with instructions for administration. For example, a composition comprising an AAV comprising a targeting peptide as described herein and a nucleic acid encoding HSV.TK1 can be provided in a kit together with ganciclovir.

[0115] Examples

[0116] The invention is further described in the following examples which do not limit the scope of the invention as set forth in the claims.

[0117] Materials and Methods

[0118] The following materials and methods were used in the following examples.

[0119] 1. Generation of capsid variants

[0120] To generate the capsid mutant plasmids, DNA fragments encoding cell-penetrating peptides (Table 3) were synthesized using CloneEZ seamless cloning technology (GenScript) and inserted into the backbone of the AAV9 Rep-cap plasmid (pRC9) between amino acid positions 588 and 589 (VP1 amino acid numbering). CPP BIP1 (VPALR, SEQ ID NO:1) and BIP2 (VSALK, SEQ ID NO:2) and their derivatives such as TVSALK (SEQ ID NO:4) in AAV.CPP.16 and TVSALFK (SEQ ID NO:8) in AAV.CPP.21 are derived from the Ku70 protein, and the sequence of the Ku protein is as follows:

[0121]

[0122] In addition, the VP1 protein sequences of AAV9, AAV.CPP.16, and AAV.CPP.21 are provided as follows:

[0123]

[0124] 2. Generation of recombinant AAV

[0125] Recombinant AAV was packaged using a standard triple plasmid co-transfection protocol (pRC plasmid, pHelper plasmid, and pAAV plasmid). pRC9 (or its variant), pHelper, and pAAV carrying the transgene (such as nuclear-directed RFP H2B-mCherry driven by the ubiquitous EF1a promoter) were co-transfected into HEK 293T cells using polyethyleneimine (PEI, Polysciences). The rAAV vectors were collected from the serum-free medium at 72 h and 120 h post-transfection, and the rAAV vectors were collected from the cells at 120 h post-transfection. The AAV particles in the medium were concentrated using a PEG precipitation method with 8% PEG-8000 (wt / vol). The cell pellet containing the virus particles was resuspended and lysed by sonication. The combined virus vectors from the PEG precipitation and cell lysate were treated with DNase and RNase at 37 °C for 30 minutes and then purified by iodixanol gradient (15%, 25%, 40%, and 60%) using ultracentrifugation (VTi 50 rotor, 40,000 r.p.m, 18 °C, 1 h). The rAAV was then concentrated using a Millipore Amicon filter unit (UFC910008, 100K MWCO) and formulated in Dulbecco's phosphate-buffered saline (PBS) containing 0.001% Pluronic F68 (Gibco).

[0126] 3. AAV titration method

[0127] Virus titer was determined by measuring the genomic copies of anti-DNAse using quantitative PCR. pAAV-CAG-GFP was digested with PVUII (NEB) to generate free ends of the plasmid ITR and used to generate a standard curve. Virus samples were incubated with DNase I to eliminate contaminating DNA, then treated with sodium hydroxide to lyse the viral capsid and release the viral genome. Quantitative PCR was performed using the ITR forward primer 5'-GGAACCCCTAGTGATGGAGTT (SEQ ID NO:91) and the ITR reverse primer 5'-CGGCCTCAGTGAGCGA (SEQ ID NO:92). Vector titers were normalized relative to rAAV-2 reference standard materials (RSMs, ATCC, catalog number: VR-1616, Manassas, VA).

[0128] 4. Administration of AAV in mice

[0129] For intravenous administration, AAV diluted in sterile saline (0.2 ml) was administered by tail vein injection in adult mice (older than 6 weeks). The animals were allowed to survive for three weeks and then euthanized for tissue harvest. For intracerebral injection, AAV diluted in PBS (10 μl) was injected using a Hamilton syringe, coordinates from bregma: right 1.0 mm, posterior 0.3 mm, depth 2.6 mm. All animal studies were conducted in an AAALAC-accredited facility approved by IACUC.

[0130] 5. Mouse tissue processing

[0131] Anesthetized animals were perfused through the heart with cold phosphate-buffered saline (PBS), followed by 4% paraformaldehyde (PFA). Tissues were fixed in 4% PFA overnight, then soaked in 30% sucrose solution for two days, and then embedded in OCT and snap-frozen. Typically, 80-μm-thick brain sections were cut for natural fluorescence imaging, and 40-μm-thick brain sections were cut for IHC.

[0132] 6. In vitro human BBB spheroid model

[0133] Hot 1% agarose (w / v, 50 μl) was added to a 96-well plate to cool / cure. Then, primary human astrocytes (Lonza Bioscience), human brain microvascular pericytes (HBVP, ScienCell Research Laboratories), and human cerebral microvascular endothelial cells (hCMEC / D3; Cedarlane) were seeded into the agarose gel at a ratio of 1:1:1 (1500 cells of each type). The cells were incubated at 37 °C in 5% CO 2Incubate in an incubator for 48 - 72 hours for the spontaneous assembly of multicellular BBB spheroids. It has been reported that a multicellular barrier is formed at the periphery of the spheroids, mimicking the blood - brain barrier. Add AAV - H2B - mCherry to the culture medium, and after 4 days, fix all the spheroids with 4% PFA. Transfer the spheroids to Nunc Lab - Tek II thin - glass 8 - well chamber slides (Thermo Scientific), and image them using a Zeiss LSM710 confocal microscope. Examine the RFP signal intensity inside the spheroids and use it as a "readout".

[0134] 7. AAV Administration in Non - Human Primates (NHP)

[0135] All NHP studies were conducted by a CRO in an AAALAC - accredited facility approved by the IACUC. Cynomolgus monkeys were pre - screened for the presence of little or no pre - existing neutralizing antibodies against AAV9 (<1:5 titer). Inject AAV diluted in PBS / 0.001% F68 intravenously (through the cephalic vein or femoral vein) using a peristaltic pump. Three weeks later, perfuse the animals with PBS followed by 4% PFA. Then collect the tissues and process them for paraffin embedding and sectioning.

[0136] 8. Immunohistochemistry

[0137] Float - stain mouse tissue sections with primary antibodies diluted in PBS containing 10% donkey serum and 2% Triton X - 100. The primary antibodies used include: chicken anti - GFP (1:1000); rabbit anti - RFP (1:1000); mouse anti - NeuN (1:500); rat anti - GFAP (1:500); goat anti - GFAP (1:500); mouse anti - CD31 (1:500). Apply secondary antibodies conjugated to fluorophores of Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 647 at a dilution of 1:200 to the host species of the primary antibody.

[0138] For paraffin sections of NHP tissues, perform DAB staining to visualize the cells transduced by AAV - AADC. Rabbit anti - AADC antibody (1:500, Millipore) is used as the primary antibody.

[0139] 9. AAV Binding Assay

[0140] Culture HEK293T cells at 37°C in 5% CO 2Incubate in an incubator. One day after inoculating HEK293T cells into a 24-well plate at a density of 250,000 cells per well, transfect the cDNA plasmid of LY6A into the cells using a transfection mixture of 200 ul DMEM (31053028; Gibco), 1 ug DNA plasmid, and 3 ug PEI. Forty-eight hours after transfection, place the cells on ice and cool for 10 minutes. Then replace the medium with 500 ul of ice-cold serum-free DMEM medium containing rAAV-mCherry with an MOI of 10,000. After incubating on ice for 1 hour, wash the cells presumably bound with AAV on the surface 3 times with cold PBS, and then perform genomic DNA isolation. Quantify the virus particles bound to the cells by qPCR using primers specific for mCherry, and normalize it to the HEK293T genome using human GCG as a reference.

[0141] 10. Mouse model of glioblastoma

[0142] All experiments were conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Brigham and Women’s Hospital and Harvard Medical School. Syngeneic immunocompetent C57BL / 6 female mice weighing 20 + / - 1 g (Envigo) were used. Intracranially inject GL261-Luc (100,000 mouse glioblastoma cells) resuspended in 2 μL phosphate-buffered saline (PBS) using a 10 μL syringe (80075; Hamilton) with a 26-gauge needle. Use a stereotactic frame to locate the implantation site (coordinates of bregma in mm: right 2, anterior 0.5, cortical depth 3.5). Seven days later, administer 200 ul of AAV-HSV-TK1 (1E+12 viral genomes, IV) once, and administer ganciclovir (50 mg / kg) daily for 10 days.

[0143] Example 1. Modification of AAV9 capsid

[0144] To identify peptide sequences that enhance the penetration of biomolecules or viruses across the blood-brain barrier, use the AAV peptide display technology. Insert the individual cell-penetrating peptides listed in Table 3 between amino acids 588 and 589 (VP1 numbering) into the AAV9 capsid, as Figure 1A shown. The insertion is carried out by modifying the RC plasmid, which is one of the three plasmids co-transfected for AAV packaging. Figure 1B An exemplary schematic diagram showing the experiment. Generate individual AAV variants and screen them separately. For more details, see Materials and Methods #1-3.

[0145] Table 3

[0146]

[0147] #, SEQ ID NO:

[0148] Syn, synthesis

[0149] Example 2. First-round in vivo screening

[0150] AAV expressing nuclear RFP (H2B-RFP) was intravenously injected into adult mice with a mixed C57BL / 6 and BALB / c genetic background. Three weeks later, the brain tissues were harvested and sectioned to show RFP-labeled cells ( Figure 2A and 2C the white dots in Figure 2B and 2D were quantified respectively). CPP BIP1 and BIP2 were inserted into the capsids of AAV.CPP.11 and AAV.CPP.12 respectively. For more details, see Materials and Methods #4-5.

[0151] Example 3. Optimization of modification of AAV9 capsid

[0152] AAV.CPP.11 and AAV.CPP.12 were further engineered by optimizing the BIP targeting sequence. The BIP insert was derived from the protein Ku70 (for the complete sequence, see Figure 3A and Materials / Methods #1). The BIP sequence VSALK selected as the "synthesis" source was focused on to minimize the potential species specificity of the engineered AAV vectors. AAVs were generated and the brain transduction efficiencies of the AAVs were tested respectively compared with AAV9 (see Figure 3B -C). Three weeks after IV injection of some AAV variants delivering the reporter gene RFP, the percentages of cell transduction in the mouse liver are shown in Figure 3D . For more details, see Materials and Methods #1-5.

[0153] Example 4. In vitro model - BBB penetration screening

[0154] An in vitro spheroid BBB model was used to screen the ability of some AAV variants to cross the human BBB. The spheroids contain human microvascular endothelial cells that form a barrier on the surface, as well as human pericytes and astrocytes. For AAVs carrying nuclear RFP as a reporter protein, their ability to penetrate from the surrounding medium into the interior of the spheroids and transduce the internal cells was evaluated. Figure 4A The experimental schematic diagram is shown. Figure 4B -D show the results of wt AAV9, AAV.CPP.16, and AAV.CPP.21 respectively, and those and other peptides in Figure 4EQuantification in the model. In this model, peptides 11, 15, 16, and 21 produced the greatest penetration into the spheroids. For more details, see Materials and Methods #6.

[0155] Example 5. In Vivo BBB Penetration Screening

[0156] In the experiments conducted as described above for Example 2, AAV.CPP.16 and AAV.CPP.21 were selected for further evaluation in an in vivo model. All AAVs carried nuclear RFP as a reporter protein. After intravenous administration to adult C57BL / 6J mice ( Figure 5A white dots in the midbrain sections, quantified in Figure 5B and adult BALB / c mice ( Figure 6A white dots in the midbrain sections, quantified in Figure 6B ), both showed enhanced ability to transduce brain cells relative to AAV9.

[0157] High doses of AAV.CPP.16 and AAV.CPP.21 (4 × 10 12 vg per mouse, IV administration) led to widespread brain transduction in mice. Both AAVs carried nuclear RFP as a reporter protein ( Figure 7A white dots in the midbrain sections, quantified in Figure 7B ).

[0158] Example 6. In Vivo Distribution of Modified AAVs

[0159] As Figure 8A shown, AAV.CPP.16 and AAV.CPP.21 preferentially targeted neurons (labeled with NeuN antibody) in multiple brain regions of mice including the cortex, midbrain, and hippocampus. Both AAVs carried nuclear RFP as a reporter protein.

[0160] AAV.CPP.16 and AAV.CPP.21 also showed enhanced ability to target spinal cord and motor neurons in mice relative to AAV9. All AAVs carried nuclear RFP as a reporter protein and were administered intravenously to neonatal mice (4 × 10 10 vg). Motor neurons were observed using CHAT antibody staining. Figure 8B In, co - localization of RFP and CHAT signals showed specific transduction of motor neurons.

[0161] The relative ability of AAV - CAG - H2B - RFP and AAV.CPP.16 - CAG - H2B - RFP to transduce various tissues in mice was also evaluated. Intravenous injection of 1 × 10 11vg. The number of transduced cells was normalized to the total number of cells labeled by DAPI nuclear staining. The results showed that AAV.CPP.16 was more effective than AAV9 in targeting tissues of the heart ( Figure 9A );skeletal muscle ( Figure 9B ), and dorsal root ganglia ( Figure 9C ) in mice.

[0162] Example 7. BBB Penetration in a Non-Human Primate Model

[0163] Three-month-old cynomolgus monkeys were intravenously injected with 2×10 13 vg / kg AAV-CAG-AADC (as a reporter gene). AAV-transduced cells (shown in black) were observed by staining with an antibody against AADC. As Figure 10A -D shows, after intravenous administration to non-human primates, AAV.CPP.16 and AAV.CPP.21 showed enhanced ability to transduce brain cells compared to AAV9. AAV.CPP.16 transduced significantly more cells in the primary visual cortex ( Figure 10A ), parietal cortex ( Figure 10B ), thalamus ( Figure 10C ), and cerebellum ( Figure 10D ) than wt AAV9. For more details, see Materials and Methods #7-8.

[0164] Example 8. AAV.CPP.16 and AAV.CPP.21 Do Not Bind to LY6A

[0165] LY6A serves as a receptor for AAV.PHP.eB and mediates the potent action of AAV.PHP.eB across the BBB in certain mouse strains. Overexpression of mouse LY6A in cultured 293 cells significantly increased the binding of AAV.PHP.eB to the cell surface (see Figure 11A ). In contrast, overexpression of LY6A did not increase viral binding to AAV9, AAV.CPP.16, or AAV.CPP.21 (see Figure 11B ). This indicates that AAV.CPP.16 or AAV.CPP.21 does not share LY6A as a receptor with AAV.PHP.eB. For more details, see Materials and Methods #9.

[0166] Example 9. Delivery of a Therapeutic Protein to the Brain Using AAV.CPP.21

[0167] AAV.CPP.21 was used to systemically deliver the "suicide gene" HSV.TK1 in a mouse model of brain tumor. HSV.TK1 converts the originally "dormant" ganciclovir into a tumor-killing drug. Intravenous administration of AAV.CPP.21-H2BmCherry (Figure 12A , the lower left and middle right figures) show the targeted tumor mass, especially the tumor expansion boundary. As Figure 12B -C shows, when combined with the prodrug ganciclovir, systemic delivery of the "suicide gene" HSV.TK1 using AAV.CPP.21 causes shrinkage of the brain tumor mass. These results indicate that AAV.CPP.21 can be used for systemic delivery of therapeutic genes to brain tumors. For more details, see Materials and Methods #10.

[0168] Example 10. Intracerebral administration of AAV.CPP.21

[0169] In addition to systemic administration (e.g., in Example 2), AAV as described herein was locally administered to the brains of mice. Intracerebral injection of AAV9-H2B-RFP and AAV.CPP.21-H2B-RFP ( Figure 13 ) resulted in a more extensive and higher-intensity RFP signal in the brain slices treated with AAV.CPP.21 relative to the brain slices treated with AAV9. For more details, see Materials and Methods #4.

[0170] Other embodiments

[0171] It should be understood that although the present invention has been described in connection with the detailed description of the invention, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are also within the scope of the appended claims. Sequence Listing <110> THE BRIGHAM AND WOMEN'S HOSPITAL, INC. <120> Methods and Compositions for Delivery of Agents across the Blood-Brain Barrier <130> 29618-0200WO1 <140> <141> <150> 62 / 696,422 <151> 2018-07-11 <160> 104 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <400> 1 Val Pro Ala Leu Arg 1 5 <210> 2 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 2 Val Ser Ala Leu Lys 1 5 <210> 3 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 3 Thr Val Pro Ala Leu Arg 1 5 <210> 4 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 4 Thr Val Ser Ala Leu Lys 1 5 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 5 Phe Thr Val Ser Ala Leu Lys 1 5 <210> 6 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 6 Leu Thr Val Ser Ala Leu Lys 1 5 <210> 7 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 7 Thr Phe Val Ser Ala Leu Lys 1 5 <210> 8 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 8 Thr Val Ser Ala Leu Phe Lys 1 5 <210> 9 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 9 Thr Val Pro Ala Leu Phe Arg 1 5 <210> 10 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 10 Thr Val Pro Met Leu Phe Lys 1 5 <210> 11 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 11 Thr Val Pro Thr Leu Phe Lys 1 5 <210> 12 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 12 Thr Val Pro Met Leu Lys 1 5 <210> 13 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 13 Thr Val Pro Thr Leu Lys 1 5 <210> 14 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 14 Val Pro Met Leu Lys 1 5 <210> 15 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 15 Val Pro Thr Leu Lys 1 5 <210> 16 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 16 Val Pro Met Leu Lys Glu 1 5 <210> 17 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 17 Val Pro Thr Leu Lys Asp 1 5 <210> 18 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 18 Val Pro Ala Leu Arg Asp 1 5 <210> 19 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 19 Val Ser Ala Leu Lys Glu 1 5 <210> 20 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 20 Val Ser Ala Leu Lys Asp 1 5 <210> 21 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 21 Thr Ala Val Ser Leu Lys 1 5 <210> 22 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 22 Thr Ala Leu Val Ser Lys 1 5 <210> 23 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 23 Thr Val Leu Ser Ala Lys 1 5 <210> 24 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 24 Thr Leu Val Ser Ala Lys 1 5 <210> 25 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 25 Thr Met Val Pro Leu Lys 1 5 <210> 26 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 26 Thr Met Leu Val Pro Lys 1 5 <210> 27 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 27 Thr Val Leu Pro Met Lys 1 5 <210> 28 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 28 Thr Leu Val Pro Met Lys 1 5 <210> 29 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 29 Thr Thr Val Pro Leu Lys 1 5 <210> 30 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 30 Thr Thr Leu Val Pro Lys 1 5 <210> 31 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 31 Thr Val Leu Pro Thr Lys 1 5 <210> 32 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 32 Thr Leu Val Pro Thr Lys 1 5 <210> 33 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 33 Thr Ala Val Pro Leu Arg 1 5 <210> 34 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 34 Thr Ala Leu Val Pro Arg 1 5 <210> 35 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 35 Thr Val Leu Pro Ala Arg 1 5 <210> 36 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 36 Thr Leu Val Pro Ala Arg 1 5 <210> 37 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 37 Thr Ala Val Ser Leu Lys Glu 1 5 <210> 38 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 38 Thr Ala Leu Val Ser Lys Glu 1 5 <210> 39 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 39 Thr Val Leu Ser Ala Lys Glu 1 5 <210> 40 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 40 Thr Leu Val Ser Ala Lys Glu 1 5 <210> 41 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 41 Thr Met Val Pro Leu Lys Glu 1 5 <210> 42 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 42 Thr Met Leu Val Pro Lys Glu 1 5 <210> 43 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 43 Thr Val Leu Pro Met Lys Glu 1 5 <210> 44 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 44 Thr Leu Val Pro Met Lys Glu 1 5 <210> 45 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 45 Thr Thr Val Pro Leu Lys Asp 1 5 <210> 46 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 46 Thr Thr Leu Val Pro Lys Asp 1 5 <210> 47 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 47 Thr Val Leu Pro Thr Lys Asp 1 5 <210> 48 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 48 Thr Leu Val Pro Thr Lys Asp 1 5 <210> 49 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 49 Thr Ala Val Pro Leu Arg Asp 1 5 <210> 50 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 50 Thr Ala Leu Val Pro Arg Asp 1 5 <210> 51 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 51 Thr Val Leu Pro Ala Arg Asp 1 5 <210> 52 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 52 Thr Leu Val Pro Ala Arg Asp 1 5 <210> 53 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 53 Thr Ala Val Ser Leu Phe Lys 1 5 <210> 54 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 54 Thr Ala Leu Val Ser Phe Lys 1 5 <210> 55 <211> 7 <212> PRT <213> artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 55 Thr Val Leu Ser Ala Phe Lys 1 5 <210> 56 <211> 7 <212> PRT <213> artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 56 Thr Leu Val Ser Ala Phe Lys 1 5 <210> 57 <211> 7 <212> PRT <213> artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 57 Thr Met Val Pro Leu Phe Lys 1 5 <210> 58 <211> 7 <212> PRT <213> artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 58 Thr Met Leu Val Pro Phe Lys 1 5 <210> 59 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 59 Thr Val Leu Pro Met Phe Lys 1 5 <210> 60 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 60 Thr Leu Val Pro Met Phe Lys 1 5 <210> 61 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 61 Thr Thr Val Pro Leu Phe Lys 1 5 <210> 62 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 62 Thr Thr Leu Val Pro Phe Lys 1 5 <210> 63 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 63 Thr Val Leu Pro Thr Phe Lys 1 5 <210> 64 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 64 Thr Leu Val Pro Thr Phe Lys 1 5 <210> 65 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 65 Thr Ala Val Pro Leu Phe Arg 1 5 <210> 66 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 66 Thr Ala Leu Val Pro Phe Arg 1 5 <210> 67 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 67 Thr Val Leu Pro Ala Phe Arg 1 5 <210> 68 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 68 Thr Leu Val Pro Ala Phe Arg 1 5 <210> 69 <211> 8 <212> PRT <213> Homo sapiens <400> 69 Lys Phe Thr Val Pro Met Leu Lys 1 5 <210> 70 <211> 8 <212> PRT <213> Mus sp. <400> 70 Lys Leu Thr Val Pro Thr Leu Lys 1 5 <210> 71 <211> 8 <212> PRT <213> Rattus sp. <400> 71 Lys Phe Thr Val Pro Ala Leu Arg 1 5 <210> 72 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 72 Lys Phe Thr Val Ser Ala Leu Lys 1 5 <210> 73 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic peptide <220> <221> MOD_RES <222> (1)..(4) <223> V, A, L, I, G, P, S, T, or M <220> <221> MOD_RES <222> (5)..(5) <223> K, R, H, D, or E <220> <223> For a detailed description of substitutions and preferred embodiments, see the submitted specification <400> 73 Xaa Xaa Xaa Xaa Xaa 1 5 <210> 74 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: synthetic peptide <220> <221> MOD_RES <222> (2)..(5) <223> V, A, L, I, G, P, S, T, or M <220> <221> MOD_RES <222> (6)..(6) <223> K, R, H, D, or E <220> <223> For a detailed description of substitutions and preferred embodiments, see the submitted specification <400> 74 Thr Xaa Xaa Xaa Xaa Xaa 1 5 <210> 75 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: synthetic peptide <220> <221> MOD_RES <222> (1)..(4) <223> V, A, L, I, G, P, S, T, or M <220> <221> MOD_RES <222> (5)..(5) <223> K, R, H, D, or E <220> <221> MOD_RES <222> (6)..(6) <223> E or D <220> <223> For a detailed description of substitutions and preferred embodiments, see the submitted specification <400> 75 Xaa Xaa Xaa Xaa Xaa Xaa 1 5 <210> 76 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <220> <221> MOD_RES <222> (1)..(1) <223> F, L, W, or Y <220> <221> MOD_RES <222> (3)..(6) <223> V, A, L, I, G, P, S, T, or M <220> <221> MOD_RES <222> (7)..(7) <223> K, R, H, D, or E <220> <223> For a detailed description of substitutions and preferred embodiments, see the submitted specification <400> 76 Xaa Thr Xaa Xaa Xaa Xaa Xaa 1 5 <210> 77 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <220> <221> MOD_RES <222> (2)..(5) <223> V, A, L, I, G, P, S, T, or M <220> <221> MOD_RES <222> (6)..(6) <223> K, R, H, D, or E <220> <221> MOD_RES <222> (7)..(7) <223> E or D <220> <223> For a detailed description of substitutions and preferred embodiments, see the submitted specification <400> 77 Thr Xaa Xaa Xaa Xaa Xaa Xaa 1 5 <210> 78 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Peptide <220> <221> MOD_RES <222> (1)..(4) <223> V, A, L, I, G, P, S, T, or M <220> <221> MOD_RES <222> (5)..(5) <223> K, R, H, D, or E <220> <221> MOD_RES <222> (6)..(6) <223> E or D <220> <221> MOD_RES <222> (7)..(7) <223> A or I <220> <223> For a detailed description of substitutions and preferred embodiments, see the submitted specification <400> 78 Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 <210> 79 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <220> <221> MOD_RES <222> (2)..(2) <223> S or P <220> <221> MOD_RES <222> (3)..(3) <223> A, M, or T <220> <223> For a detailed description of substitutions and preferred embodiments, see the submitted specification <400> 79 Val Xaa Xaa Leu 1 <210> 80 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <220> <221> MOD_RES <222> (3)..(3) <223> S or P <220> <221> MOD_RES <222> (4)..(4) <223> A, M, or T <220> <223> For a detailed description of substitutions and preferred embodiments, see the submitted specification <400> 80 Thr Val Xaa Xaa Leu 1 5 <210> 81 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: synthetic peptide <220> <221> MOD_RES <222> (3)..(3) <223> S or P <220> <221> MOD_RES <222> (4)..(4) <223> A, M, or T <220> <223> For a detailed description of substitutions and preferred embodiments, see the submitted specification <400> 81 Thr Val Xaa Xaa Leu Lys 1 5 <210> 82 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: synthetic peptide <220> <221> MOD_RES <222> (3)..(3) <223> S or P <220> <221> MOD_RES <222> (4)..(4) <223> A, M, or T <220> <223> For a detailed description of substitutions and preferred embodiments, see the submitted specification <400> 82 Thr Val Xaa Xaa Leu Phe Lys 1 5 <210> 83 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 83 Lys Leu Ala Ser Val Thr 1 5 <210> 84 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 84 Lys Phe Leu Ala Ser Val Thr 1 5 <210> 85 <211> 736 <212> PRT <213> Adeno-associated virus 9 <400> 85 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro 20 25 30 Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly 145 150 155 160 Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro 180 185 190 Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly 195 200 205 Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn 260 265 270 Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285 Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295 300 Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile 305 310 315 320 Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn 325 330 335 Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu 340 345 350 Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro 355 360 365 Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp 370 375 380 Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe 385 390 395 400 Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu 405 410 415 Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430 Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser 435 440 445 Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser 450 455 460 Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro 465 470 475 480 Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn 485 490 495 Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn 500 505 510 Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys 515 520 525 Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly 530 535 540 Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile 545 550 555 560 Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser 565 570 575 Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln 580 585 590 Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln 595 600 605 Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620 Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met 625 630 635 640 Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650 655 Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr 660 665 670 Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685 Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn 690 695 700 Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val 705 710 715 720 Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730 735 <210> 86 <211> 609 <212> PRT <213> Human <400> 86 Met Ser Gly Trp Glu Ser Tyr Tyr Lys Thr Glu Gly Asp Glu Glu Ala 1 5 10 15 Glu Glu Glu Gln Glu Glu Asn Leu Glu Ala Ser Gly Asp Tyr Lys Tyr 20 25 30 Ser Gly Arg Asp Ser Leu Ile Phe Leu Val Asp Ala Ser Lys Ala Met 35 40 45 Phe Glu Ser Gln Ser Glu Asp Glu Leu Thr Pro Phe Asp Met Ser Ile 50 55 60 Gln Cys Ile Gln Ser Val Tyr Ile Ser Lys Ile Ile Ser Ser Asp Arg 65 70 75 80 Asp Leu Leu Ala Val Val Phe Tyr Gly Thr Glu Lys Asp Lys Asn Ser 85 90 95 Val Asn Phe Lys Asn Ile Tyr Val Leu Gln Glu Leu Asp Asn Pro Gly 100 105 110 Ala Lys Arg Ile Leu Glu Leu Asp Gln Phe Lys Gly Gln Gln Gly Gln 115 120 125 Lys Arg Phe Gln Asp Met Met Gly His Gly Ser Asp Tyr Ser Leu Ser 130 135 140 Glu Val Leu Trp Val Cys Ala Asn Leu Phe Ser Asp Val Gln Phe Lys 145 150 155 160 Met Ser His Lys Arg Ile Met Leu Phe Thr Asn Glu Asp Asn Pro His 165 170 175 Gly Asn Asp Ser Ala Lys Ala Ser Arg Ala Arg Thr Lys Ala Gly Asp 180 185 190 Leu Arg Asp Thr Gly Ile Phe Leu Asp Leu Met His Leu Lys Lys Pro 195 200 205 Gly Gly Phe Asp Ile Ser Leu Phe Tyr Arg Asp Ile Ile Ser Ile Ala 210 215 220 Glu Asp Glu Asp Leu Arg Val His Phe Glu Glu Ser Ser Lys Leu Glu 225 230 235 240 Asp Leu Leu Arg Lys Val Arg Ala Lys Glu Thr Arg Lys Arg Ala Leu 245 250 255 Ser Arg Leu Lys Leu Lys Leu Asn Lys Asp Ile Val Ile Ser Val Gly 260 265 270 Ile Tyr Asn Leu Val Gln Lys Ala Leu Lys Pro Pro Pro Ile Lys Leu 275 280 285 Tyr Arg Glu Thr Asn Glu Pro Val Lys Thr Lys Thr Arg Thr Phe Asn 290 295 300 Thr Ser Thr Gly Gly Leu Leu Leu Pro Ser Asp Thr Lys Arg Ser Gln 305 310 315 320 Ile Tyr Gly Ser Arg Gln Ile Ile Leu Glu Lys Glu Glu Thr Glu Glu 325 330 335 Leu Lys Arg Phe Asp Asp Pro Gly Leu Met Leu Met Gly Phe Lys Pro 340 345 350 Leu Val Leu Leu Lys Lys His His Tyr Leu Arg Pro Ser Leu Phe Val 355 360 365 Tyr Pro Glu Glu Ser Leu Val Ile Gly Ser Ser Thr Leu Phe Ser Ala 370 375 380 Leu Leu Ile Lys Cys Leu Glu Lys Glu Val Ala Ala Leu Cys Arg Tyr 385 390 395 400 Thr Pro Arg Arg Asn Ile Pro Pro Tyr Phe Val Ala Leu Val Pro Gln 405 410 415 Glu Glu Glu Leu Asp Asp Gln Lys Ile Gln Val Thr Pro Pro Gly Phe 420 425 430 Gln Leu Val Phe Leu Pro Phe Ala Asp Asp Lys Arg Lys Met Pro Phe 435 440 445 Thr Glu Lys Ile Met Ala Thr Pro Glu Gln Val Gly Lys Met Lys Ala 450 455 460 Ile Val Glu Lys Leu Arg Phe Thr Tyr Arg Ser Asp Ser Phe Glu Asn 465 470 475 480 Pro Val Leu Gln Gln His Phe Arg Asn Leu Glu Ala Leu Ala Leu Asp 485 490 495 Leu Met Glu Pro Glu Gln Ala Val Asp Leu Thr Leu Pro Lys Val Glu 500 505 510 Ala Met Asn Lys Arg Leu Gly Ser Leu Val Asp Glu Phe Lys Glu Leu 515 520 525 Val Tyr Pro Pro Asp Tyr Asn Pro Glu Gly Lys Val Thr Lys Arg Lys 530 535 540 His Asp Asn Glu Gly Ser Gly Ser Lys Arg Pro Lys Val Glu Tyr Ser 545 550 555 560 Glu Glu Glu Leu Lys Thr His Ile Ser Lys Gly Thr Leu Gly Lys Phe 565 570 575 Thr Val Pro Met Leu Lys Glu Ala Cys Arg Ala Tyr Gly Leu Lys Ser 580 585 590 Gly Leu Lys Lys Gln Glu Leu Leu Glu Ala Leu Thr Lys His Phe Gln 595 600 605 Asp <210> 87 <211> 608 <212> PRT <213> Mouse <400> 87 Met Ser Glu Trp Glu Ser Tyr Tyr Lys Thr Glu Gly Glu Glu Glu Glu 1 5 10 15 Glu Glu Glu Glu Ser Pro Asp Thr Gly Gly Glu Tyr Lys Tyr Ser Gly 20 25 30 Arg Asp Ser Leu Ile Phe Leu Val Asp Ala Ser Arg Ala Met Phe Glu 35 40 45 Ser Gln Gly Glu Asp Glu Leu Thr Pro Phe Asp Met Ser Ile Gln Cys 50 55 60 Ile Gln Ser Val Tyr Thr Ser Lys Ile Ile Ser Ser Asp Arg Asp Leu 65 70 75 80 Leu Ala Val Val Phe Tyr Gly Thr Glu Lys Asp Lys Asn Ser Val Asn 85 90 95 Phe Lys Asn Ile Tyr Val Leu Gln Asp Leu Asp Asn Pro Gly Ala Lys 100 105 110 Arg Val Leu Glu Leu Asp Gln Phe Lys Gly Gln Gln Gly Lys Lys His 115 120 125 Phe Arg Asp Thr Val Gly His Gly Ser Asp Tyr Ser Leu Ser Glu Val 130 135 140 Leu Trp Val Cys Ala Asn Leu Phe Ser Asp Val Gln Leu Lys Met Ser 145 150 155 160 His Lys Arg Ile Met Leu Phe Thr Asn Glu Asp Asp Pro His Gly Arg 165 170 175 Asp Ser Ala Lys Ala Ser Arg Ala Arg Thr Lys Ala Ser Asp Leu Arg 180 185 190 Asp Thr Gly Ile Phe Leu Asp Leu Met His Leu Lys Lys Pro Gly Gly 195 200 205 Phe Asp Val Ser Val Phe Tyr Arg Asp Ile Ile Thr Thr Ala Glu Asp 210 215 220 Glu Asp Leu Gly Val His Phe Glu Glu Ser Ser Lys Leu Glu Asp Leu 225 230 235 240 Leu Arg Lys Val Arg Ala Lys Glu Thr Lys Lys Arg Val Leu Ser Arg 245 250 255 Leu Lys Phe Lys Leu Gly Glu Asp Val Val Leu Met Val Gly Ile Tyr 260 265 270 Asn Leu Val Gln Lys Ala Asn Lys Pro Phe Pro Val Arg Leu Tyr Arg 275 280 285 Glu Thr Asn Glu Pro Val Lys Thr Lys Thr Arg Thr Phe Asn Val Asn 290 295 300 Thr Gly Ser Leu Leu Leu Pro Ser Asp Thr Lys Arg Ser Leu Thr Tyr 305 310 315 320 Gly Thr Arg Gln Ile Val Leu Glu Lys Glu Glu Thr Glu Glu Leu Lys 325 330 335 Arg Phe Asp Glu Pro Gly Leu Ile Leu Met Gly Phe Lys Pro Thr Val 340 345 350 Met Leu Lys Lys Gln His Tyr Leu Arg Pro Ser Leu Phe Val Tyr Pro 355 360 365 Glu Glu Ser Leu Val Ser Gly Ser Ser Thr Leu Phe Ser Ala Leu Leu 370 375 380 Thr Lys Cys Val Glu Lys Glu Val Ile Ala Val Cys Arg Tyr Thr Pro 385 390 395 400 Arg Lys Asn Val Ser Pro Tyr Phe Val Ala Leu Val Pro Gln Glu Glu 405 410 415 Glu Leu Asp Asp Gln Asn Ile Gln Val Thr Pro Gly Gly Phe Gln Leu 420 425 430 Val Phe Leu Pro Tyr Ala Asp Asp Lys Arg Lys Val Pro Phe Thr Glu 435 440 445 Lys Val Thr Ala Asn Gln Glu Gln Ile Asp Lys Met Lys Ala Ile Val 450 455 460 Gln Lys Leu Arg Phe Thr Tyr Arg Ser Asp Ser Phe Glu Asn Pro Val 465 470 475 480 Leu Gln Gln His Phe Arg Asn Leu Glu Ala Leu Ala Leu Asp Met Met 485 490 495 Glu Ser Glu Gln Val Val Asp Leu Thr Leu Pro Lys Val Glu Ala Ile 500 505 510 Lys Lys Arg Leu Gly Ser Leu Ala Asp Glu Phe Lys Glu Leu Val Tyr 515 520 525 Pro Pro Gly Tyr Asn Pro Glu Gly Lys Val Ala Lys Arg Lys Gln Asp 530 535 540 Asp Glu Gly Ser Thr Ser Lys Lys Pro Lys Val Glu Leu Ser Glu Glu 545 550 555 560 Glu Leu Lys Ala His Phe Arg Lys Gly Thr Leu Gly Lys Leu Thr Val 565 570 575 Pro Thr Leu Lys Asp Ile Cys Lys Ala His Gly Leu Lys Ser Gly Pro 580 585 590 Lys Lys Gln Glu Leu Leu Asp Ala Leu Ile Arg His Leu Glu Lys Asn 595 600 605 <210> 88 <211> 608 <212> PRT <213> Rat <400> 88 Met Ser Glu Trp Glu Ser Tyr Tyr Lys Thr Glu Gly Glu Glu Glu Glu 1 5 10 15 Glu Glu Glu Gln Ser Pro Asp Thr Asn Gly Glu Tyr Lys Tyr Ser Gly 20 25 30 Arg Asp Ser Leu Ile Phe Leu Val Asp Ala Ser Arg Ala Met Phe Glu 35 40 45 Ser Gln Gly Glu Asp Glu Leu Thr Pro Phe Asp Met Ser Ile Gln Cys 50 55 60 Ile Gln Ser Val Tyr Thr Ser Lys Ile Ile Ser Ser Asp Arg Asp Leu 65 70 75 80 Leu Ala Val Val Phe Tyr Gly Thr Glu Lys Asp Lys Asn Ser Val Asn 85 90 95 Phe Lys Ser Ile Tyr Val Leu Gln Asp Leu Asp Asn Pro Gly Ala Lys 100 105 110 Arg Val Leu Glu Leu Asp Arg Phe Lys Gly Gln Gln Gly Lys Lys His 115 120 125 Phe Arg Asp Thr Ile Gly His Gly Ser Asp Tyr Ser Leu Ser Glu Val 130 135 140 Leu Trp Val Cys Ala Asn Leu Phe Ser Asp Val Gln Phe Lys Met Ser 145 150 155 160 His Lys Arg Ile Met Leu Phe Thr Asn Glu Asp Asp Pro His Gly Asn 165 170 175 Asp Ser Ala Lys Ala Ser Arg Ala Arg Thr Lys Ala Ser Asp Leu Arg 180 185 190 Asp Thr Gly Ile Phe Leu Asp Leu Met His Leu Lys Lys Arg Gly Gly 195 200 205 Phe Asp Val Ser Leu Phe Tyr Arg Asp Ile Ile Ser Ile Ala Glu Asp 210 215 220 Glu Asp Leu Gly Val His Phe Glu Glu Ser Ser Lys Leu Glu Asp Leu 225 230 235 240 Leu Arg Lys Val Arg Ala Lys Glu Thr Lys Lys Arg Val Leu Ser Arg 245 250 255 Leu Lys Phe Lys Leu Gly Lys Asp Val Ala Leu Met Val Gly Val Tyr 260 265 270 Asn Leu Val Gln Lys Ala Asn Lys Pro Phe Pro Val Arg Leu Tyr Arg 275 280 285 Glu Thr Asn Glu Pro Val Lys Thr Lys Thr Arg Thr Phe Asn Val Asn 290 295 300 Thr Gly Ser Leu Leu Leu Pro Ser Asp Thr Lys Arg Ser Leu Thr Phe 305 310 315 320 Gly Thr Arg Gln Ile Val Leu Glu Lys Glu Glu Thr Glu Glu Leu Lys 325 330 335 Arg Phe Asp Glu Pro Gly Leu Ile Leu Met Gly Phe Lys Pro Met Val 340 345 350 Met Leu Lys Asn His His Tyr Leu Arg Pro Ser Leu Phe Leu Tyr Pro 355 360 365 Glu Glu Ser Leu Val Asn Gly Ser Ser Thr Leu Phe Ser Ala Leu Leu 370 375 380 Thr Lys Cys Val Glu Lys Glu Val Ile Ala Val Cys Arg Tyr Thr Ala 385 390 395 400 Arg Lys Asn Val Ser Pro Tyr Phe Val Ala Leu Val Pro Gln Glu Glu 405 410 415 Glu Leu Asp Asp Gln Asn Ile Gln Val Thr Pro Ala Gly Phe Gln Leu 420 425 430 Val Phe Leu Pro Tyr Ala Asp Asp Lys Arg Lys Val Pro Phe Thr Glu 435 440 445 Lys Val Met Ala Asn Pro Glu Gln Ile Asp Lys Met Lys Ala Ile Val 450 455 460 Gln Lys Leu Arg Phe Thr Tyr Arg Ser Asp Ser Phe Glu Asn Pro Val 465 470 475 480 Leu Gln Gln His Phe Arg Asn Leu Glu Ala Leu Ala Leu Asp Met Met 485 490 495 Glu Ser Glu Gln Val Val Asp Leu Thr Leu Pro Lys Val Glu Ala Ile 500 505 510 Lys Lys Arg Leu Gly Ser Leu Ala Asp Glu Phe Lys Glu Leu Val Tyr 515 520 525 Pro Pro Gly Tyr Asn Pro Glu Gly Lys Ile Ala Lys Arg Lys Ala Asp 530 535 540 Asn Glu Gly Ser Ala Ser Lys Lys Pro Lys Val Glu Leu Ser Glu Glu 545 550 555 560 Glu Leu Lys Asp Leu Phe Ala Lys Gly Thr Leu Gly Lys Leu Thr Val 565 570 575 Pro Ala Leu Arg Asp Ile Cys Lys Ala Tyr Gly Leu Lys Ser Gly Pro 580 585 590 Lys Lys Gln Glu Leu Leu Glu Ala Leu Ser Arg His Leu Glu Lys Asn 595 600 605 <210> 89 <211> 742 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic polypeptide <400> 89 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro 20 25 30 Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly 145 150 155 160 Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro 180 185 190 Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly 195 200 205 Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn 260 265 270 Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285 Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295 300 Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile 305 310 315 320 Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn 325 330 335 Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu 340 345 350 Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro 355 360 365 Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp 370 375 380 Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe 385 390 395 400 Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu 405 410 415 Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430 Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser 435 440 445 Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser 450 455 460 Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro 465 470 475 480 Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn 485 490 495 Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn 500 505 510 Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys 515 520 525 Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly 530 535 540 Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile 545 550 555 560 Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser 565 570 575 Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Thr Val Ser Ala 580 585 590 Leu Lys Ala Gln Ala Gln Thr Gly Trp Val Gln Asn Gln Gly Ile Leu 595 600 605 Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile 610 615 620 Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu 625 630 635 640 Met Gly Gly Phe Gly Met Lys His Pro Pro Pro Gln Ile Leu Ile Lys 645 650 655 Asn Thr Pro Val Pro Ala Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys 660 665 670 Leu Asn Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu 675 680 685 Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu 690 695 700 Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala 705 710 715 720 Val Asn Thr Glu Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg 725 730 735 Tyr Leu Thr Arg Asn Leu 740 <210> 90 <211> 743 <212> PRT <213> Synthetic sequence <220> <223> Description of synthetic sequence: Synthetic polypeptide <400> 90 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro 20 25 30 Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly 145 150 155 160 Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro 180 185 190 Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly 195 200 205 Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn 260 265 270 Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285 Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295 300 Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile 305 310 315 320 Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn 325 330 335 Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu 340 345 350 Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro 355 360 365 Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp 370 375 380 Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe 385 390 395 400 Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu 405 410 415 Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430 Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser 435 440 445 Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser 450 455 460 Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro 465 470 475 480 Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn 485 490 495 Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn 500 505 510 Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys 515 520 525 Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly 530 535 540 Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile 545 550 555 560 Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser 565 570 575 Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Thr Val Ser Ala 580 585 590 Leu Phe Lys Ala Gln Ala Gln Thr Gly Trp Val Gln Asn Gln Gly Ile 595 600 605 Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro 610 615 620 Ile Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His Pro Ser Pro 625 630 635 640 Leu Met Gly Gly Phe Gly Met Lys His Pro Pro Pro Gln Ile Leu Ile 645 650 655 Lys Asn Thr Pro Val Pro Ala Asp Pro Pro Thr Ala Phe Asn Lys Asp 660 665 670 Lys Leu Asn Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val 675 680 685 Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro 690 695 700 Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser Asn Asn Val Glu Phe 705 710 715 720 Ala Val Asn Thr Glu Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr 725 730 735 Arg Tyr Leu Thr Arg Asn Leu 740 <210> 91 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic primer <400> 91 ggaaccccta gtgatggagt t 21 <210> 92 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic primer <400> 92 cggcctcagt gagcga 16 <210> 93 <211> 18 <212> PRT <213> Unknown <220> <223> Description of unknown SynB1 sequence <400> 93 Arg Gly Gly Arg Leu Ser Tyr Ser Arg Arg Arg Phe Ser Thr Ser Thr 1 5 10 15 Gly Arg <210> 94 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 94 His Ala Arg Ile Lys Pro Thr Phe Arg Arg Leu Lys Trp Lys Tyr Lys 1 5 10 15 Gly Lys Phe Trp 20 <210> 95 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 95 Pro Leu Ser Ser Ile Phe Ser Arg Ile Gly Asp Pro 1 5 10 <210> 96 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 96 Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu Lys Ala Leu Ala Ala Leu 1 5 10 15 Ala Lys Lys Ile Leu 20 <210> 97 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 97 Val Arg Leu Pro Pro Pro Val Arg Leu Pro Pro Pro Val Arg Leu Pro 1 5 10 15 Pro Pro <210> 98 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 98 Val Glu Leu Pro Pro Pro Val Glu Leu Pro Pro Pro Val Glu Leu Pro 1 5 10 15 Pro Pro <210> 99 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 99 Lys Gly Thr Tyr Lys Lys Lys Leu Met Arg Ile Pro Leu Lys Gly Thr 1 5 10 15 <210> 100 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 100 Pro Pro Arg Pro Pro Arg Pro Pro Arg 1 5 <210> 101 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic peptide <400> 101 Pro Pro Arg Pro Pro Arg Pro Pro Arg Pro Pro Arg Pro Pro Arg 1 5 10 15 <210> 102 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 102 Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <210> 103 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: synthetic peptide <400> 103 Leu Arg Arg Glu Arg Gln Ser Arg Leu Arg Arg Glu Arg Gln Ser Arg 1 5 10 15 <210> 104 <211> 9 <212> PRT <213> Human immunodeficiency virus 1 <400> 104 Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5

Claims

1. An adeno-associated virus (AAV) vector, which comprises an AAV9 VP1 capsid protein, wherein the AAV9 VP1 capsid protein comprises a targeting peptide inserted at positions corresponding to amino acids 588 and 589 of SEQ ID NO:85, and wherein the targeting peptide consists of TVSALFK (SEQ ID NO:8) or TVSALK (SEQ ID NO:4).

2. The AAV vector according to claim 1, wherein the AAV is AAV9.

3. The AAV vector according to claim 1, which further comprises a transgene.

4. The AAV vector according to claim 3, wherein the transgene encodes a therapeutic agent.

5. The AAV vector according to claim 1, which comprises a non-coding RNA.

6. The AAV vector according to claim 5, wherein the non-coding RNA is shRNA, siRNA or miRNA.

7. The AAV vector according to claim 3, wherein the delivery of the transgene to an organ or tissue is enhanced as compared to an AAV vector comprising the transgene and an AAV9 VP1 capsid protein without the targeting peptide.

8. The AAV vector according to claim 7, wherein the organ or tissue is the brain or the central nervous system.

9. The AAV vector according to claim 5, wherein the delivery of the non-coding RNA to an organ or tissue is enhanced as compared to an AAV vector comprising the non-coding RNA and an AAV9 VP1 capsid protein without the targeting peptide.

10. The AAV vector according to claim 9, wherein the organ or tissue is the brain or the central nervous system.

11. A composition, which comprises the AAV vector according to claim 1 and a pharmaceutically acceptable carrier.

12. An AAV9 VP1 capsid protein, which comprises a targeting peptide inserted at positions corresponding to amino acids 588 and 589 of SEQ ID NO:85, wherein the targeting peptide consists of TVSALFK (SEQ ID NO:8) or TVSALK (SEQ ID NO:4).

13. A nucleic acid, which encodes the AAV9 VP1 capsid protein according to claim 12.

14. Use of the AAV vector according to any one of claims 1 to 10 in the preparation of a reagent for delivering a transgene to a cell by a method comprising contacting the cell with the AAV vector.

15. The use according to claim 14, wherein the cell is a neuron, an astrocyte, a cardiomyocyte, or a myocyte.

16. The use according to claim 15, wherein the neuron is a dorsal root ganglion neuron.

17. The use according to claim 14, wherein the cell is located in a living subject.

18. The use according to claim 17, wherein the subject is a mammalian subject.

19. The use according to claim 17, wherein the cell is located in a tissue selected from the group consisting of the brain, the spinal cord, the dorsal root ganglion, the heart, the muscle, and combinations thereof.

20. The use according to claim 17, wherein the subject has a neurodegenerative disease, epilepsy; stroke; spinocerebellar ataxia; Canavan disease; metachromatic leukodystrophy; spinal muscular atrophy; Friedreich's ataxia; X-linked centronuclear myopathy; lysosomal storage disease; Barth syndrome; Duchenne muscular dystrophy; Wilson's disease; or type 1 Crigler-Najjar syndrome.

21. The use according to claim 20, wherein the neurodegenerative disease is Parkinson's disease; Alzheimer's disease; Huntington's disease; amyotrophic lateral sclerosis; or multiple sclerosis.

22. The use according to claim 17, wherein the subject has a brain cancer, and the method comprises administering an AAV vector encoding an anti-cancer agent.

23. The use according to claim 22, wherein the anti-cancer agent is HSV.TK1, and the method further comprises administering ganciclovir.

24. The use according to claim 17, wherein the cells are located in the brain of the subject, and the AAV vector is administered by parenteral delivery; intracerebral delivery; or intrathecal delivery.

25. The use according to claim 24, wherein the parenteral delivery is via intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular delivery.

26. The use according to claim 24, wherein the intrathecal delivery is via lumbar injection, cisterna magna injection, or intracerebral parenchymal injection.

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