Methods and compositions for drug delivery across the blood-brain barrier

Abstract

To provide artificial targeting sequences that enhance permeation of agents into cells and across the blood-brain barrier, and compositions comprising the sequences.SOLUTION: The present invention provides an adeno-associated virus (AAV) vector comprising an AAV capsid, wherein the AAV capsid comprises a peptide insert, and the peptide insert comprises 5 to 7 consecutive amino acids of TVSALFK (sequence number 8) or 5 to 6 consecutive amino acids of TVSALK (sequence number 4), as well as a composition for use in a method of delivering a transgene to a cell, the method comprising the AAV vector.SELECTED DRAWING: Figure 3C-D

Description

Technical Field

[0001] Priority Claim This application claims the benefit of U.S. Provisional Patent Application No. 62 / 696,422, filed Jul. 11, 2018. The entire content of the foregoing is hereby incorporated by reference into this specification.

[0002] Sequence Listing This application is electronically filed in ASCII format and includes a sequence listing which is hereby incorporated by reference into this specification in its entirety. The ASCII copy, created on Jul. 11, 2019, has the name 29618-0200WO1_SL.txt and a size of 5 8,834 bytes.

[0003] Sequences that enhance the penetration of drugs across the blood-brain barrier, compositions containing such sequences, and methods of using these are described herein.

Background Art

[0004] The delivery of therapeutic agents such as gene therapies is an obstacle to the development of treatments for many pathologies. The blood-brain barrier (BBB) is an important obstacle to drug delivery to the central nervous system (CNS) of mammals (particularly delivery to the human brain) for treating pathologies (e.g., neurodegenerative diseases such as Parkinson's disease; Alzheimer's disease; Huntington's disease; amyotrophic lateral sclerosis; and multiple sclerosis).

Summary of the Invention

[0005] The present invention is based on the development of artificial targeting sequences that enhance the penetration of drugs into cells and across the blood-brain barrier.

[0006] Thus, the sequences TVSALFK (SEQ ID NO: 8); TVSALK (SEQ ID NO: 4); KL​​​​​​​​ At least four from ASVT (sequence number 83) or KFLASVT (sequence number 84) AAV capsid protein containing a targeting sequence containing a sequence of 1 consecutive amino acids (for example) For example, a modified AAV capsid protein is provided herein. Several implementations In this state, this AAV capsid protein has the sequence TVSALK (sequence number 4); TVSA LFK(sequence number 8); KLASVT(sequence number 83); or KFLASVT(sequence number 83) It contains a targeting sequence comprising at least five consecutive amino acids from (number 84). In some embodiments, this AAV capsid protein has the sequence TVSALK(SEQ ID NO: 4); TVSALFK (sequence number 8); KLASVT (sequence number 83); or KFLA Targeting containing at least six consecutive amino acids from SVT (SEQ ID NO: 84) Includes arrays.

[0007] In some embodiments, this AAV is AAV9, but other AAVs known in the art (For example, AAV1, 2, 3, 4, 5, 6, 7, 8 and their variants, as well as Other materials known in the art or described herein may also be used.

[0008] In some embodiments, this AAV capsid protein is AAV9 VP1 (for example) (Includes sequence number 85).

[0009] In some embodiments, this targeting sequence is amino acid 588 of SEQ ID NO: 85 It is inserted into the capsid protein at a corresponding position between and 589.

[0010] AAV capsid proteins containing the targeting sequences described herein Nucleic acids that do so are also provided herein.

[0011] In addition, AAVs comprising a capsid protein comprising a targeting sequence described herein are provided. In some embodiments, the AAV further comprises a transgene, preferably either a therapeutic transgene or a diagnostic transgene. Examples of therapeutic transgenes include, for example, cDNA that restores protein function, guide RNA for gene editing, RNA , or miRNA. Targeting sequences comprising V[S / p][A / m / t / ]L (SEQ ID NO: 79), TV[S / p][A / m / t / ]

[0012] L (SEQ ID NO: 80), TV[S / p][A / m / t / ]LK (SEQ ID NO: 81), or T V[S / p][A / m / t / ]LFK (SEQ ID NO: 82) are also provided herein. In some embodiments, this targeting sequence is VPA LR (SEQ ID NO: 1); VSALK (SEQ ID NO: 2); TVPALR (SEQ ID NO: 3); TVS ALK (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). Fusion proteins comprising a targeting sequence linked to a heterologous (e.g., non-AAV PV1) sequence, and A AV capsid proteins (e.g., AAV9 VP1) comprising a targeting sequence are also provided. In some embodiments, this targeting sequence is inserted at positions corresponding to amino acids 588 and 589 of SEQ ID NO: 85.

[0013] ​​​​​​ In addition, the targeting arrays, fusion proteins, or AAs described herein nucleic acids encoding the V capsid protein and an AAV comprising a capsid protein comprising a targeting array are provided herein. In some embodiments, this AAV further comprises a transgene, preferably a therapeutic transgene or a diagnostic transgene. Examples of therapeutic transgenes can include, for example, a cDNA that restores protein function, a guide RNA, RNA, or miRNA for gene editing.

[0014] Furthermore, a method for delivering a transgene to a cell, the method comprising contacting the cell with an AAV or a fusion protein described herein is provided herein. In some embodiments, the cell is present in a living subject (e.g., a mammalian subject). In some embodiments, the cell is present in a tissue selected from the brain, spinal cord, dorsal root ganglia, heart, or muscle, and combinations thereof. In some embodiments, the cell is a nerve cell (optionally a dorsal root ganglia nerve cell), astrocyte, cardiomyocyte, or muscle cell.

[0015] In some embodiments, 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 disease; or Klippel-Trenaunay syndrome type 1. In some embodiments, the neurodegenerative disease is Parkinson's ​​​​​​​​​​​​​​N'Son's disease; Alzheimer's disease; Huntington's disease; Amyotrophic lateral sclerosis; and multiple sclerosis It is a disease.

[0016] In some embodiments, the subject has brain cancer, and this method codes for an anticancer agent. This includes administering AAV. In some embodiments, this anticancer agent is HSV. This is TK1, and this method further includes administering ganciclovir.

[0017] In some embodiments, these cells are present in the brain of the subject, and this AAV is delivered parenterally. (For example, by intravenous delivery, intra-arterial delivery, subcutaneous delivery, intraperitoneal delivery, or intramuscular delivery) ); intracerebral; or intrathecal delivery (e.g., by lumbar injection, cisterna magna injection, or intraparenchymal injection) ) is administered by [method].

[0018] Unless otherwise defined, all technical and scientific terms used herein are the same as those used in this document. It has the same meaning as generally understood by those skilled in the art to which the term "Ming" belongs. In this specification, Methods and materials for use in this invention are described, but other suitable methods and materials known in the art are also described. Methods and materials may also be used. The materials, methods, and examples are merely examples and are not intended to limit you. Not illustrated. All publications, patent applications, patents, arrangements, and data mentioned herein. Database entries and other references are incorporated as a whole by reference. In the event of a shield, this specification, including its definitions, shall prevail.

[0019] Other features and advantages of the present invention are described in the detailed description and drawings below, as well as in the claims. This will become clear. [Brief explanation of the drawing]

[0020] [Figure 1] This document illustrates an exemplary strategy for manipulating AAV9 by inserting cell-permeable peptides (CPPs) into its capsid. Figure 1A is a 3D model of the AAV9 virus. Individual CPPs inserted into the capsid between amino acids 588 and 589 (VP1 numbering) will be shown on the triple axis where receptor binding likely occurs. Figure 1B shows the method for producing individual AAVs. Three plasmids, containing pRC (manipulated or unmanipulated), p helper, and pAAV, are co-transfected into HEK 293T cells, AAVs are recovered, and purified using an iodixanol gradient. [Figure 2A] Representative images and quantitative analyses of mouse brain sections after intravenous administration of low-dose candidate AAVs are shown. Mice with mixed genetic backgrounds were used. Candidate AAVs all express nuclear red fluorescent protein (RFP) as a reporter, although they differ in inserted CPP (see Table 3). Candidate AAVs with low production yields were excluded for further screening. The AAV dose was 1 × 10¹⁰ vg (viral genome) per animal. Each white dot in Figure 2A represents an RFP-labeled cell. [Figure 2B] Representative images and quantitative analyses of mouse brain sections after intravenous administration of low-dose candidate AAVs are shown. Mice with mixed genetic backgrounds were used. Candidate AAVs all express nuclear red fluorescent protein (RFP) as a reporter, although they differ in inserted CPPs (see Table 3). Candidate AAVs with low production yields were excluded for further screening. The AAV dose was 1 × 10¹⁰ vg (viral genome) per animal. In Figure 2B, *P<0.05, vs. AAV9, ANOVA. [Figure 2C]Representative images and quantitative analyses of mouse brain sections after intravenous administration of AAV.CPP.11 and AAV.CPP.12 in repeated experiments are shown. AAV.CPP.11 and AAV.CPP.12 contain CPP BIP1 and CPP BIP2, respectively (see Table 3). The AAV dose was increased up to 1 × 10¹¹ vg per animal. Candidate AAVs express nuclear red fluorescent protein (RFP) as a reporter. Each white dot in Figure 2C represents an RFP-labeled cell. [Figure 2D] Representative images and quantitative analyses of mouse brain sections after intravenous administration of AAV.CPP.11 and AAV.CPP.12 in repeated experiments are shown. AAV.CPP.11 and AAV.CPP.12 contain CPP BIP1 and CPP BIP2, respectively (see Table 3). The AAV dose was increased up to 1 × 10¹¹ vg per animal. Candidate AAVs express nuclear red fluorescent protein (RFP) as a reporter. In Figure 2D, *P<0.05, **P<0.01, vs. AAV9, ANOVA. [Figure 3A] This paper describes the optimization of BIP targeting sequences for further manipulating AAV9 towards better brain transduction. BIP1 (VPALR, SEQ ID NO: 1), which enables more efficient transduction of AAV9 into the brain (as in AAV.CPP.11), is derived from the rat protein Ku70. Human, mouse, and rat Ku70 proteins have different exact amino acid sequences. BIP2 (VSALK, SEQ ID NO: 2), as in AAV.CPP.12, is a “synthetic” peptide associated with BIP1. Further manipulation focuses on the VSALK sequence, with the expectation of minimizing species specificity of the ultimately manipulated AAV. To generate novel targeting sequences, the desired amino acids are added to the VSALK sequence, and in other cases, the positions of individual amino acids are swapped. To generate novel candidate AAVs for screening, all novel BIP2-derived sequences are reinserted into the AAV9 capsid. The sequences listed in order are SEQ ID NOs: 69, 70, 71, 1-6, 72, 7, and 8. [Figure 3B]Representative images and quantitative analyses of mouse brain sections after intravenous administration of a wider range of candidate AAVs are shown. All candidate AAVs express nuclear red fluorescent protein (RFP) as a reporter. The AAV dose was 1 × 10¹¹ vg per animal. Each white dot in Figure 3B represents an RFP-labeled cell. AAV.CPP.16 and AAV.CPP.21 were identified as top hits, exhibiting robust and widespread brain transduction. [Figure 3C-D] Figure 3C shows representative images of mouse brain sections after intravenous administration of more candidate AAVs and their quantitative analysis. All candidate AAVs express nuclear red fluorescent protein (RFP) as a reporter. The dose of AAV is 1 × 10¹¹ vg per animal. AAV.CPP.16 and AAV.CPP.21 were identified as top hits with robust and widespread brain transduction. In Figure 3C, *P<0.05, **P<0.01, ***P<0.001, vs. AAV9, ANOVA. Figure 3D shows a quantitative analysis of transduction efficiency in the liver after intravenous administration of candidate AAVs. Represents the percentage of transduced liver cells. The dose of AAV is 1 × 10¹¹ vg per animal. ***P<0.001, vs. AAV9, ANOVA. [Figure 4A-B] This shows the screening of selected candidate AAVs in an in vitro spheroid model of the human blood-brain barrier. Figure 4A shows a spheroid containing human microvascular endothelial cells that form the barrier on its surface, and human pericytes and astrocytes inside the spheroid. Candidate AAVs were evaluated for their ability to penetrate from the surrounding culture medium into the spheroid and transduce cells inside. Figure 4B shows images of spheroids treated with AAV9, AAV.CPP.16, and AAV.CPP.21. [Figure 4C-D] This report describes the screening of selected candidate AAVs in an in vitro spheroid model of the human blood-brain barrier. Candidate AAVs were evaluated for their ability to penetrate from the surrounding culture medium into the spheroid and transduce cells within it. Figures 4C–4D show images of spheroids treated with AAV9, AAV.CPP.16, and AAV.CPP.21. [Figure 4E]This shows the screening of selected candidate AAVs in an in vitro spheroid model of the human blood-brain barrier. Figure 4E shows the relative RFP intensity of spheroids treated with various AAVs. ***P<0.001, vs. AAV9, ANOVA. [Figure 5A] Representative images and quantitative analyses of brain sections after intravenous administration of AAV9, AAV.CPP.16, and AAV.CPP.21 in C57BL / 6J inbred mice are shown. All candidate AAVs express nuclear red fluorescent protein (RFP) as a reporter. The AAV dose is 1 × 10¹² vg per animal. Each white dot in Figure 5A represents an RFP-labeled cell. [Figure 5B] Representative images and quantitative analyses of brain sections after intravenous administration of AAV9, AAV.CPP.16, and AAV.CPP.21 in C57BL / 6J inbred mice are shown. All candidate AAVs express nuclear red fluorescent protein (RFP) as a reporter. The AAV dose is 1 × 10¹² vg per animal. In Figure 5B, *P<0.05, ***P<0.001, ANOVA. [Figure 6A] Representative images and quantitative analyses of brain sections after intravenous administration of AAV9, AAV.CPP.16, and AAV.CPP.21 in BALB / cJ inbred mice are shown. All candidate AAVs express nuclear red fluorescent protein (RFP) as a reporter. The AAV dose is 1 × 10¹² vg per animal. Each white dot in Figure 6A represents an RFP-labeled cell. [Figure 6B] Representative images and quantitative analyses of brain sections after intravenous administration of AAV9, AAV.CPP.16, and AAV.CPP.21 in BALB / cJ inbred mice are shown. All candidate AAVs express nuclear red fluorescent protein (RFP) as a reporter. The AAV dose is 1 × 10¹² vg per animal. In Figure 6B, ***P<0.001, ANOVA. [Figure 7A]Representative images and quantitative analyses of brain sections after intravenous administration of high doses of AAV.CPP.16 and AAV.CPP.21 in C57BL / 6J inbred mice are shown. Both candidate AAVs express nuclear red fluorescent protein (RFP) as a reporter. The AAV dose is 4 × 10¹² vg per animal. Each white dot in Figure 7A represents an RFP-labeled cell. [Figure 7B] Representative images and quantitative analyses of brain sections after intravenous administration of high doses of AAV.CPP.16 and AAV.CPP.21 in C57BL / 6J inbred mice are shown. Both candidate AAVs express nuclear red fluorescent protein (RFP) as a reporter. The AAV dose is 4 × 10¹² vg per animal. In Figure 7B, *P<0.05, Student's test. [Figure 8A-1] This study demonstrates that AAV.CPP.16 and AAV.CPP.21 transduce mature neurons (labeled with NeuN antibody) across multiple brain regions in mice (e.g., cerebral cortex, midbrain, and hippocampus). Transduced neurons are co-labeled with NeuN antibody and RFP. Adult C57BL / 6J mice (6 weeks old) were administered 4 × 10¹² vg of AAV intravenously. [Figure 8A-2] This is a continuation of Figure 8A-1. [Figure 8B] This study demonstrates that AAV.CPP.16 and AAV.CPP.21 exhibit enhanced ability compared to AAV9 in targeting mouse spinal cord and motor neurons. Neonatal mice (1 day postnativity) were intravenously administered 4 × 10¹⁰ vg of AAV. Motor neurons in the anterior horn of the spinal cord were visualized using CHAT antibody staining. Co-localization of RFP and CHAT signaling suggests specific transduction of motor neurons. [Figure 9A] This study demonstrates that AAV.CPP.16 exhibits enhanced ability compared to AAV9 in targeting the heart of adult mice. Adult C57BL / 6J mice (6 weeks old) were intravenously administered 1 × 10¹¹ vg of AAV. The percentage represents the ratio of RFP-labeled cells to total DAPI-stained cells. *P<0.05, Student's test. [Figure 9B]This study demonstrates that AAV.CPP.16 exhibits enhanced ability compared to AAV9 in targeting skeletal muscle in adult mice. Adult C57BL / 6J mice (6 weeks old) were intravenously administered 1 × 10¹¹ vg of AAV. The percentage of RFP-labeled cells relative to all DAPI-stained cells is shown. *P<0.05, Student's test. [Figure 9C] This study demonstrates that AAV.CPP.16 exhibits enhanced ability compared to AAV9 in targeting the dorsal root ganglia (DRG) of adult mice. Adult C57BL / 6J mice (6 weeks old) were intravenously administered 1 × 10¹¹ vg of AAV. The percentage of RFP-labeled cells relative to all DAPI-stained cells is shown. *P<0.05, Student's test. [Figure 10A] This study demonstrates that AAV.CPP.16 and AAV.CPP.21 exhibit enhanced ability to transduce brain cells in the primary visual cortex after intravenous administration in non-human primates compared to AAV9. AAVs-CAG-AADC (as a reporter gene) was administered intravenously at a dose of 2 × 10¹³ vg / kg to 3-month-old cynomolgus monkeys with limited existing neutralizing antibodies. AAV-transduced cells (shown in black) were visualized using antibody staining against AADC. The rectangular areas in the left panel are magnified in the right panel. AAV.CPP.16 transduced significantly more cells than AAV9. AAV.CPP.21 also transduced more cells than AAV9, but its effect was less pronounced compared to AAV.CPP.16. [Figure 10B]This study demonstrates that AAV.CPP.16 and AAV.CPP.21 exhibit enhanced ability to transduce brain cells in the parietal lobe cortex after intravenous administration in non-human primates compared to AAV9. AAVs-CAG-AADC (as a reporter gene) was administered intravenously at a dose of 2 × 10¹³ vg / kg to 3-month-old cynomolgus monkeys with limited existing neutralizing antibodies. AAV-transduced cells (shown in black) were visualized using antibody staining against AADC. The rectangular areas in the left panel are magnified in the right panel. AAV.CPP.16 transduced significantly more cells than AAV9. AAV.CPP.21 also transduced more cells than AAV9, but its effect was less pronounced compared to AAV.CPP.16. [Figure 10C] This study demonstrates that AAV.CPP.16 and AAV.CPP.21 exhibit enhanced ability to transduce thalamic brain cells after intravenous administration in non-human primates compared to AAV9. AAVs-CAG-AADC (as a reporter gene) was administered intravenously at a dose of 2 × 10¹³ vg / kg to 3-month-old cynomolgus monkeys with limited existing neutralizing antibodies. AAV-transduced cells (shown in black) were visualized using antibody staining against AADC. The rectangular areas in the left panel are magnified in the right panel. AAV.CPP.16 transduced significantly more cells than AAV9. AAV.CPP.21 also transduced more cells than AAV9, but its effect was less pronounced compared to AAV.CPP.16. [Figure 10D] This study demonstrates that AAV.CPP.16 and AAV.CPP.21 exhibit enhanced ability to transduce cerebellar brain cells after intravenous administration in non-human primates compared to AAV9. AAVs-CAG-AADC (as a reporter gene) was intravenously injected at a dose of 2 × 10¹³ vg / kg into 3-month-old cynomolgus monkeys with limited existing neutralizing antibodies. AAV-transduced cells (shown in black) were visualized using antibody staining against AADC. The rectangular areas in the left panel are enlarged in the right panel. Both AAV.CPP.16 and AAV.CPP.21 transduced significantly more cells compared to AAV9. [Figure 11]We demonstrate that AAV.CPP.16 and AAV.CPP.21 do not bind to LY6A. LY6A functions as a receptor for AAV.PHP.B and its variants (e.g., AAV.PHP.eB (U.S. Patent No. 9102949, U.S. Patent Application Publication No. 20170166926)) and mediates the robust effect of AAV.PHP.eB across the blood-brain barrier 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 293 cultured 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 suggests that AAV.CPP.16 or AAV.CPP.21 do not share LY6A as a receptor with AAV.PHP.eB. [Figure 12A] We demonstrate that therapeutic genes can be systemically delivered to glioblastoma (GBM) brain tumors in a mouse model using AAV.CPP.21. Figure 11A shows that intravenously administered AAV.CPP.21-H2BmCherry targeted the tumor (particularly the tumor's spreading frontier). [Figure 12B] This study demonstrates that therapeutic genes can be systemically delivered to glioblastoma (GBM) brain tumors in a mouse model using AAV.CPP.21. Figure 11B shows that the use of AAV.CPP.21 for systemic delivery of the “suicide gene” HSV.TK1, when combined with the prodrug ganciclovir, results in a reduction in brain tumor size. HSV.TK1 otherwise transforms “dormant” ganciclovir into a tumor killer. *P<0.05, Student's test. [Figure 12C]This study demonstrates that therapeutic genes can be systemically delivered to glioblastoma (GBM) brain tumors in a mouse model using AAV.CPP.21. Figure 11C shows that the use of AAV.CPP.21 for systemic delivery of the “suicide gene” HSV.TK1, when combined with the prodrug ganciclovir, results in a reduction in brain tumor size. HSV.TK1 otherwise converts “dormant” ganciclovir into a tumor killer. *P<0.05, Student's test. [Figure 13-1] This study demonstrates that when administered locally to the brains of adult mice, AAV.CPP.21 induces more extensive and robust transduction of brain tissue compared to AAV9. Adult mice (>6 weeks old) were injected intracerebrally with AAV (1 × 10¹¹vg), and brain tissue was collected and examined 3 weeks after AAV injection. **P<0.01, Student's test.** [Figure 13-2] This is a continuation of Figure 13-1. [Modes for carrying out the invention]

[0021] The difficulty of delivering blood beyond the blood-brain barrier is a challenge for treating brain disorders such as cancer and neurodegenerative disorders. This is hindering the development of therapeutic drugs for the delivery of therapeutic genes to the brain, spinal cord, and eyes. Adeno-associated virus (AAV) is emerging as an important research and clinical tool. For example, U.S. Patent No. 9102949; U.S. Patent No. 9585971; and U.S. (See Specification No. 20170166926). However, existing AAV9s and others AAVs have limited cross-border efficiency across the blood-branch barrier (BBB) ​​or only function in some non-primate species. It is one of the following.

[0022] Rational design and targeted screening based on known cell-permeable peptides (CPPs) (For example, Gomez et al., Bax-inhibiting peptides derived from Ku70 and cell-pen See etrating pentapeptides. Biochem. Soc. Trans. 2007;35(Pt 4):797-801. When manipulated into the AAV capsid via this process, the efficiency of gene delivery to the brain can be increased by up to three orders of magnitude. Targeting sequences that improve the condition have been discovered. Using this method, animals with glioblastoma One such AAV vector that dramatically reduces tumor size in the model is used in the manipulation. It was created from [the source].

[0023] Targeting array This method allows for, for example, AAV (e.g., AAV1, AAV2, AAV8, or A When inserted into the capsid of AV9, or chemically or as a fusion protein Expression conjugates it to biological agents (e.g., antibodies or other large biomolecules). If this occurs, many potential targeting peptides that enhance penetration through the BBB will be present. It was decided.

[0024] In some embodiments, this targeting peptide is at least five amino acids The sequence includes the sequence VPALR(sequence number). In some embodiments, this amino acid sequence is the sequence VPALR(sequence number). No. 1) and VSALK (Sequence No. 2) at least 4 (e.g., 5) consecutive meshes Contains anoacids.

[0025] In some embodiments, this targeting peptide is arranged in X1X2X3X4X5 Including columns, here (i) X1, X2, X3, X4 are from among V, A, L, I, G, P, S, T, or M Any four different amino acids; (ii) X5 is K, R, H, D, or E (Sequence ID 73).

[0026] In some embodiments, this targeting peptide consists of at least six amino acids. The sequence includes the sequence TVPALR(sequence). In some embodiments, this amino acid sequence is the sequence TVPALR(sequence). Number 3), TVSALK (SEQ ID NO: 4), TVPMLK (SEQ ID NO: 12), and TVP TLK (SEQ ID NO: 13) consists of at least four (e.g., five or six) consecutive amino acids. Includes.

[0027] In some embodiments, this targeting peptide is X1X2X3X4X5X6 Includes an array of (i) X1 is T; (ii) X2, X3, X4, X5 are among V, A, L, I, G, P, S, T, or M Any four different amino acids; (iii) X6 is K, R, H, D, or E (Sequence ID 74).

[0028] In some embodiments, this targeting peptide is X1X2X3X4X5X6 Includes an array of (i) X1, X2, X3, X4 are from V, A, L, I, G, P, S, T, or M Any four different amino acids; (ii) X5 is K, R, H, D, or E; (iii) X6 is either E or D (Sequence ID 75).

[0029] In some embodiments, this targeting peptide consists of at least seven amino acids. The sequence includes the sequence FTVSALK( Column number 5), LTVSALFK (sequence number 6), TVSALFK (sequence number 8), TVPA LFR (SEQ ID NO: 9), TVPMLFK (SEQ ID NO: 10), and TVPTLFK (SEQ ID NO: 9), Number 11) at least four (e.g., five, six, or seven) consecutive amino acids Includes. In some other embodiments, this targeting peptide is X1X2X3X4 Includes an X5X6X7 array, (i) X1 is F, L, W, or Y; (ii) X² is T; (iii) X3, X4, X5, X6 are V, A, L, I, G, P, S, T, or M Any four different amino acids within it; (iv) X7 is K, R, H, D, or E (Sequence ID 76).

[0030] In some embodiments, this targeting peptide is X1X2X3X4X5X6 Includes the X7 array, (i) X1 is T; (ii) X2, X3, X4, X5 are among V, A, L, I, G, P, S, T, or M Any four different amino acids; (iii) X6 is K, R, H, D, or E; (iv) X7 is either E or D (Sequence ID 77).

[0031] In some embodiments, this targeting peptide is X1X2X3X4X5X6 Includes the X7 array, (i) X1, X2, X3, X4 are from among V, A, L, I, G, P, S, T, or M Any four different amino acids; (ii) X5 is K, R, H, D, or E; (iii) X6 is either E or D; (iv) X7 is either A or I (Sequence ID 78).

[0032] In some embodiments, this targeting peptide is V[S / p][A / m / t Includes the sequence / ]L (sequence number 79) (uppercase letters are preferred where uppercase letters are present). In some embodiments, this targeting peptide is TV[S / p][A / m / t / Includes the sequence ]L (sequence number 80). In some embodiments, this targeting peptide Chido contains the sequence TV[S / p][A / m / t / ]LK (sequence number 81). In this embodiment, the targeting peptide is TV[S / p][A / m / t / ]LF Contains the sequence K. (sequence number 82).

[0033] In some embodiments, this targeting peptide is VPALR (SEQ ID NO: 1) Or it is not VSALK (sequence number 2).

[0034] The amino acid sequences of specific examples containing the aforementioned sequences of 5, 6, or 7 amino acids are shown in the table. List them in 1.

[0035] [Table 1-1]

[0036] [Table 1-2]

[0037] Targeting peptides containing inverted sequences (e.g., KLASVT (SEQ ID NO: 83)) KFLASVT (sequence number 84) can also be used.

[0038] The targeting peptides disclosed herein are used to produce peptide mimetics. This can be modified according to methods known in this field. For example, Qvit et al., Drug Discov Today. 2017 Feb; 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., Ind o American Journal of Pharmaceutical Research, 2015. 8;Kazmierski, WM, ed., P eptidomimetics Protocols, Human Press (Totowa NJ 1998); Goodman et al., eds., Ho uben-Weyl Methods of Organic Chemistry: Synthesis of Peptides and Peptidomimetic s, Thiele Verlag (New York 2003); and Mayo et al., J. Biol. Chem., 278:45746 ( See 2003). In some cases, the peptides disclosed herein and This modified peptide-mimicking version of the fragment is, compared to non-peptide-mimicking peptides, invisibly This demonstrates enhanced stability in the volt.

[0039] Methods for creating peptide mimes involve selecting one or more amino acids from the peptide sequence. This includes substituting (for example, all) with D-amino acid enantiomers. Such sequences are... In the specification, it is referred to as a "retro" sequence. Alternatively, it refers to the N-terminus of an amino acid residue. The order from the N-terminus to the C-terminus is reversed, and as a result, the order from the N-terminus to the C-terminus of the original peptide is reversed. In the modified peptide mime, the order of amino acid residues is from the C-terminus to the N-terminus. This results in a sequence of residues. Such a sequence can be called an "inverso" sequence.

[0040] Peptide mimes can be both retroversions and inversoversions, immediately This could be a "retro-inverso" version of the peptide disclosed herein. This novel peptide mimeology has an amino acid sequence from the N-terminus to the C-terminus. The order of the residues matches the order of amino acid residues from the C-terminus to the N-terminus in the original peptide. It can be composed of D-amino acids arranged in a specific way.

[0041] Other methods for producing peptide mimes involve one or more amino acid residues in the peptide. These are chemically distinct but recognized functional analogues of this amino acid (i.e., artificial). This includes substitution with an amino acid analog. As an artificial amino acid analog, β-amino acids, β-substituted β-amino acids ("β 3 -amino acids), amino acid phosphite analogs (for example, ∀ -aminophosphonic acid and ∀-aminophosphinic acid), and having non-peptide bonds Examples of amino acids include those used to create peptide mimes (e.g., peptides). Oligomers (e.g., peptoidamides or ester analogs), β-peptides, cyclic compounds To create peptides, oligoureas, or oligocarbamate peptides; or heterocyclic molecules) It is possible. As an exemplary retro-inverse targeting peptide mimetic, KLASV Examples include T and KFLASVT (these sequences contain all D-amino acids). These sequences can be modified, for example, by biotinylation of the amino terminus and amidation of the carboxyl terminus. It can be modified.

[0042] AAV The viral vectors used in this method and composition are as described herein. The targeting peptide and the optionally selected transgene for expression in the target tissue. Recombinant retroviruses, adenoviruses, adeno-associated viruses, and alphaviruses are included. Examples include S and lentiviruses.

[0043] A preferred viral vector system useful for nucleic acid delivery in this method is adeno-associated viral It is a rus (AAV). AAV is a small non-enveloped utensil with a 25nm capsid. It is a virus. It is known or has been shown to be associated with the wild-type virus. There is no disease. AAV has a single-stranded DNA (ssDNA) genome. AAV has long It has been shown that AAV exhibits episomal transgene expression throughout the brain (especially in nerve cells). It shows excellent transgene expression in cells. It contains only 300 base pairs of AAV. The vectors can be packaged and integrated. The space for exogenous DNA is approximately 4.7k It is restricted to b. AAV vector (e.g., Tratschin et al., Mol. Cell. Biol. 5) DNA can be introduced into cells using the method described in 3251-3260 (1985). AV vectors have been used to introduce various nucleic acids into various cell types (for example, Hermon at 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 (1 988);Tratschin et al., J. Virol. 51:611-619 (1984); and Flotte et al., J. Bio See l. Chem. 268:3781-3790 (1993). Numerous alternative AAV variants (10 (More than 0 variants have been cloned) and AAV variants are based on desired characteristics. They have been identified. In some embodiments, the AAVs are AAV1, AAV2, AAV4, AAV5, AAV6, AV6.2, AAV7, AAV8, AAV9, rh.10, rh. 39, rh.43, or CSp3; in the case of use in CNS, several practices In terms of morphology, AAVs are AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, Alternatively, it could be AAV9. For example, AAV9 can cross the blood-brain barrier to a certain extent efficiently. It has been shown that this method can be used to achieve the targeting distribution described herein. Insertion of a column into a capsid protein (e.g., AAV between amino acids 588 and 589) 9. Insertion into the capsid protein VP1) allows the AAV capsid to cross the blood-brain barrier. Alternatively, genetic engineering may be used to increase penetration into specific tissues.

[0044] An example of the wild-type AAV9 capsid protein VP1 (Q6JC40-1) sequence is shown below. That is correct.

[0045] [ka]

[0046] Therefore, one of the targeting peptide sequences described herein or AAV containing multiple elements (for example, a capsule containing the targeting sequences described herein) AAV containing a doprotein (e.g., a capsid protein including SEQ ID NO: 1) The targeting peptide sequence is, for example, between amino acids 588 and 589, this sequence An AAV, which is inserted into the above, is provided herein.

[0047] In some embodiments, this AAV also uses a transgene sequence (i.e., a heterologous sequence), e.g. For example, a coding code for a therapeutic agent described herein or known in the art. Entering a gene, or a reporter protein, such as a fluorescent protein, to detect a product This also includes an enzyme or cell surface antigen that catalyzes the resulting reaction. This transgene is preferably It is linked to a sequence that promotes / drives the expression of this transgene in the target tissue.

[0048] Examples of transgenes for therapeutic use include: nerve cells Apoptosis inhibitory protein (NAIP), nerve growth factor (NGF), glial cell-derived growth factor Child (GDNF), brain-derived growth factor (BDNF), ciliary neurotrophic factor (CNTF), tyrofoam Synhydroxylase (TH), GTP-cyclohydrolase (GTPCH), amino acids Decarboxylase (AADC), aspartoacylase (ASPA), blood factors, for example For example, β-globin, hemoglobin, tissue plasminogen activator, and coagulation factors. Colony-stimulating factor (CSF); interleukins, e.g., IL-1, IL-2, IL -3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9 etc; growth factors, e.g. For example, keratinocyte growth factor (KGF), stem cell factor (SCF), fibroblast growth factor (FGF, e.g., basic FGF and acidic FGF), hepatocyte growth factor (HGF), Surin-like growth factor (IGF), bone morphogenetic protein (BMP), epidermal growth factor (EGF), Growth and differentiation factor-9 (GDF-9), hepatocellular carcinoma-derived growth factor (HDGF), myostatin ( GDF-8), nerve growth factor (NGF), neurotrophin, platelet-derived growth factor (P DGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-α), shape Transforming growth factor beta (TGF-β) etc.; soluble receptors, e.g. soluble TNF-α receptor Body, soluble VEGF receptors, soluble interleukin receptors (e.g., soluble IL-1 receptors) (Condition and soluble type II IL-1 receptor), soluble gamma / delta T cell receptor, soluble Receptor ligand-binding fragments, etc.; enzymes, e.g., α-glucosidase, imiglucerase, β-glucocerebrosidase and alglucerase; enzyme activators, e.g., combination Plasminogen activator; chemokines, e.g., IP-10, interferon - Gamma-induced monokines (Mig), Groa / IL-8, RANTES, MIP-1α, MIP-1β, MCP-1, PF-4, etc.; angioplasting agents, e.g., vascular endothelium. Growth factors (VEGF, e.g., VEGF121, VEGF165, VEGF-C, VEG) F-2), transforming growth factor-beta, basic fibroblast growth factor, glioma-derived growth factor Angiogenin, angiogenin-2, etc.; anti-angiogenic agents, for example, soluble VEGF receptors Content; protein vaccine; neuroactive peptides, e.g., nerve growth factor (NGF), Dikinin, cholecystokinin, gastrin, secretin, oxytocin, gonadotropin Endorphins, beta-endorphin, enkephalin, substance P, somatos Tatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dynorph Warfarin, neurotensin, motilin, thyroid-stimulating hormone, neuropeptides Y, progesterone, calcitonin, insulin, glucagon, vasopressin, angiotherapy Symptoms II, thyroid-stimulating hormone-releasing hormone, vasoactive intestinal peptide, sleep peptide Etc.; thrombolytic agents; atrial natriuretic peptide; relaxin; glial fiber acidic protein Quality; follicle-stimulating hormone (FSH); human alpha-1 antitrypsin; leukemia suppressor (LIF); Transforming Growth Factor (TGF); Tissue Factor; Progesterone; Macrophage Activity Sexualization factors; Tumor necrosis factor (NTF); Neutrophil chemotactic factor (NCF); Nerve growth factor; Tissue Metalloproteinase inhibitors; vasoactive intestinal peptides; angiogenin; angiotropin Pins; fibrin; hirudin; IL-1 receptor antagonists, etc. Some other examples include: ciliary neurotrophic factor (CNTF); neuro Trophin 3 and 4 / 5 (NT-3 and 4 / 5); glial cell-derived neurotrophic factor (G DNF; Aromatic amino acid decarboxylase (AADC); Hemophilia-related coagulation protein Factors, e.g., factor VIII, factor IX, factor X; dystrophin or mini-dystrophin Trophins; lysosomal acid lipase; phenylalanine hydroxylase (PAH); sugars Enzymes related to the underlying disease, such as glucose-6-phosphatase, acid maltase, glyco Glycogen debranching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, Muscle phosphofructokinase, phosphorylase kinase (e.g., PHKA2), glucose Transporters (e.g., GLUT2), aldolase A, β-enolase, and glycans Cogen synthase; lysosomal enzyme (e.g., beta-N-acetylhexosaminidase) Ze A); and any variant thereof.

[0049] This transgene can also encode antibodies, such as PD-L1, PD-1, and C. TLA-4 (Cytotoxic T-lymphocyte-associated protein-4; CD152); LAG-3 ( Lymphocyte activation gene 3; CD223); TIM-3 (T-cell immunoglobulin domain) and mucin domain 3; HAVCR2); TIGIT (Ig domain and ITIM domain) Main T-cell immune receptor); B7-H3 (CD276); VSIR (V-set Also known as immunomodulatory receptor, VISTA, B7H5, C10orf54); B TLA 30 (B- and T- lymphocyte attenuators, CD272); GARP (glycopropyl alcohol) Protein A repeat; dominant; PVRIG (contains PVR-related immunoglobulin domain); also VTCN1 (V-set domain-containing T cell activation inhibitor 1, also known as B7-H4) It can also encode immune checkpoint inhibitor antibodies against (ru).

[0050] Other transgenes include those that alter / reduce the expression of target genes, either small or repressive. Sex nucleic acids can be cited, for example, siRNA, shRNA, miRNA, antisense nucleic acids. Rigo, or long non-coding RNAs that alter gene expression (e.g., International Publication No. 2012) Brochure No. 087983 and U.S. Patent Application Publication No. 20140142160 (See also), or CRISPR Cas9 / cas12a, and guide RNA These could be cited.

[0051] The virus may also contain one or more sequences that promote the expression of the transgene, for example. , one or more promoter sequences; enhancer sequences, e.g., 5' untranslated region ( UTR) or 3'UTR; polyadenylation site; and / or insulator It may also include columns. In some embodiments, this promoter is a brain tissue-specific promoter. - For example, a neuron-specific promoter or a glial cell-specific promoter. In a particular embodiment, this promoter promotes a gene selected from the following: The culprits are: nerve nuclei (NeuN), glial fibrillation acidic protein (GFAP), and MeCP2. Adenomatous polyposis of the large intestine (APC), ionized calcium-binding adapter molecule 1 (Ib a-1) Synapsin I (SYN), calcium / calmodulin-dependent protein quinapsin -ase II, tubulin alpha I, nerve cell-specific enolase, and platelet-derived growth Factor beta chain. In some embodiments, this promoter is a pan-cell type promoter. - For example, cytomegalovirus (CMV), beta-glucuronidase, (GU promoters of SB, ubiquitin C (UBC), or Roussarcoma virus (RSV) The woodchuck hepatitis virus post-transcription reaction element (WPRE) can also be used.

[0052] In some embodiments, AAV also increases delivery to target tissue (e.g., CNS). It may also have one or more additional mutations, or (for example, Pulicherla et al. (2 011) Reduce extra-tissue targeting (as described in Mol Ther 19:1070-1078) One or more additional mutations (for example, intended for delivery to the CNS, heart, or muscle) They also have mutations that reduce delivery to the liver if present, or for example, Chen et al. . (2008) Nat Med 15:1215-1218 or Xu et al., (2005) Virology 341:203-214 if U.S. Patent No. 9,102,949; U.S. Patent No. 9,585,971; and U.S. Patent Other targeting as described in Patent Application Publication No. 20170166926 It also involves peptide addition. sfn.org / ~ / media / SfN / Documents / Short%20Courses / 2011%20Sho Gray and Samulski (2011) “Vector” is available at rt%20Course%20I / 2011_SC1_Gray.ashx design and considerations for CNS applications,” in Gene Vector Design and Appl ication to Treat Nervous System Disorders ed. Glorioso J., editor. (Washington, See also DC: Society for Neuroscience; 1-9.

[0053] Targeting peptides as tags / fusions Using the targeting peptides described herein, other ( Could the penetration of (different) molecules also be increased, for example, by conjugation of this molecule? Or, for example, expression as part of a fusion protein with an antibody or other large biomolecule. These can be increased further. These include the therapeutic agents or reporters described herein. In addition to those listed in Table 2, genome editing proteins or genome editing complexes. (For example, the peptides described herein (for example, at the N-terminus, C-terminus, or internally) TA containing a gene editing protein (e.g., Cas9 or Cas12a) fused to the tether LE, ZFN, Base editor, and CRISPR RNP, and Possible examples include guide RNA. The fusion / complex is any other sequence derived from Ku70. It does not contain (for example, heterogeneous non-Ku70 sequences) and does not exist in nature.

[0054] In some embodiments, the target used as part of a non-AAV fusion protein The sequence does not contain VPALR (sequence number 1) or VSARK (sequence number 2), or not.

[0055] How to use Using the methods and compositions described herein, any composition (e.g., objective) (arrangement of) tissues (e.g., central nervous system (brain), heart, muscles, or dorsal root ganglia or spine) It can be delivered to the spinal cord (peripheral nervous system). In some embodiments, this method can be used to deliver to a specific area of ​​the brain. This includes delivery to various regions (e.g., the cerebral cortex, cerebellum, hippocampus, substantia nigra, amygdala). Several implementations In this context, the method involves delivery to nerve cells, astrocytes, glial cells, or cardiomyocytes.

[0056] In some embodiments, this method and composition (e.g., AAV) are used to distribute nucleic acids. The column is a disease (for example, CNS diseases; for example, U.S. Patent No. 9102949; ibid. Specification No. 9585971; and U.S. Patent Application Publication No. 20170166926 To be delivered to an object having (see reference). In some embodiments, this object is in Table 2. The states listed are enumerated, and several embodiments, using vectors, are enumerated in Table 2. To treat the corresponding disease, the therapeutic drugs listed in Table 2 will be delivered. Therapeutic agents can be delivered, for example, as nucleic acids via viral vectors (this nucleic acid is for therapeutic use Proteins or other nucleic acids (e.g., antisense oligonucleotides, siRNA, shRNA, etc.) (encoding) or fusion with targeting peptides described herein (Can be delivered as an protein / complex).

[0057] [Table 2]

[0058] In some embodiments, this composition and method are used to treat brain cancer. For example, glioblastoma (e.g., glioblastoma multiforme (GBM)), metastasis (e.g., lung cancer, breast cancer, Examples include melanoma (or metastasis from colon cancer), meningioma, pituitary adenoma, and acoustic neuroma. This composition includes a targeting peptide linked to the following anticancer agents: for example. "Suicide genes" that induce apoptosis in target cells (e.g., HSV.TK1, simple Cytosine deaminase (C) derived from herpesvirus or Escherichia coli. D) or Escherichia coli purine nucleoside phosphorylase (PNP) / fludarabine; Krohne et al., Hepatology. 2001 Sep;34(3):511-8; Dey and Evans, “Suicide Gene Therapy by Herpes Simplex Virus-1 Thymidine Kinase (HSV-TK)” (20 11) See DOI: 10.5772 / 18544), known in the art or described herein. Immunotherapy checkpoint inhibitor antibodies. For example, the targeted antibodies described herein. Using an AAV vector containing a ing peptide, the "suicide gene" HSV.TK was used to target brain tumors. It can deliver 1. HSV.TK1 delivers ganciclovir, which would otherwise be "dormant". Convert it into a tumor killer. Therefore, this method is used for targeting the tumors described herein. AAV (e.g., AAV9) containing a compound peptide and encoding HSV.TK1, and The drug ganciclovir is administered systemically (e.g., intravenously) to patients diagnosed with brain cancer. This may include doing so.

[0059] Pharmaceutical composition and administration method The method described herein involves a medical device containing the targeting peptide as the active ingredient. This includes the use of pharmaceutical compositions.

[0060] Pharmaceutical compositions typically contain a pharmaceutically acceptable carrier. In addition, the phrase "pharmaceutically acceptable carrier" refers to physiological saline, solvent, dispersion suitable for pharmaceutical administration. This includes media, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc.

[0061] Pharmaceutical compositions are typically formulated to suit their intended route of administration. Examples of administration routes include parenteral administration, such as intravenous administration, intra-arterial administration, and dermatological administration. This includes administration via the throat, intraperitoneal cavity, intramuscular, injection, or infusion. Therefore, delivery may be systemic or local.

[0062] Methods for formulating appropriate pharmaceutical compositions are known in this field, for example, Remington: The S Science and Practice of Pharmacy, 21st ed., 2005; and Drugs and the Pharmaceutical I'm looking for books from the series *cal Sciences: A Series of Textbooks and Monographs* (Dekker, NY). See reference. For example, the following ingredients are used as a solution or suspension for parenteral administration: Possible examples: Sterile diluents, e.g., water for injection, physiological saline, fixative oil, polyethylene Recall, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents, for example. Benzyl alcohol or methylparaben; antioxidants, e.g., ascorbic acid or Sodium bisulfite; chelating agent, e.g., ethylenediaminetetraacetic acid; buffering agent, e.g., Acetates, citrates, or phosphates, and osmotic pressure modifiers, such as sodium chloride. Glucose or sodium hydroxide. Adjust the pH with an acid or base such as hydrochloric acid or sodium hydroxide. Obtain this parenteral formulation in ampoules, disposable syringes, or glass or plastic containers. It can be enclosed in multiple-dose vials made of stick material.

[0063] A sterile aqueous solution (water-soluble) or dispersion is a pharmaceutical composition suitable for injectable use. This could include sterile powders for the immediate preparation of sterile, injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, and Cremophor EL. TM (BASF, Parsippany, NJ) or phosphate-buffered saline (PBS) These include: In all cases, the composition must be sterilized and easy to use. It should be a fluid insofar as it can be injected. This composition is manufactured and stored It should be stable under certain conditions and protected from the action of microorganisms such as bacteria and fungi. It must be. The carrier is, for example, water, ethanol, polyol (for example, glycerol) Polyethylene glycol, propylene glycol, and liquid polyethylene glycol, etc., and their appropriate properties. It may be a solvent or dispersion medium containing a fine mixture. For example, a coating such as lecithin. Through use, in the case of dispersion, the necessary particle size is maintained, and in the case of use of surfactants This allows for the maintenance of more appropriate fluidity. It also helps prevent microbial action, using various antimicrobial and antimicrobial agents. Antimicrobial agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosulfame) This can be achieved by using an isotonic agent (e.g., sugar, polyhydric acid) in the composition. In many cases, this composition contains an isotonic agent (e.g., sugar, polyhydric acid). It is preferable to include alcohol (e.g., mannitol, sorbitol, sodium chloride). It seems that the sustained absorption of this injectable composition is due to the absorption delaying agent in this composition. For example, this can be achieved by including aluminum monostearate and gelatin. ru.

[0064] Along with one or a combination of the components listed above, in a suitable solvent, the necessary A certain amount of the active compound is incorporated, and if necessary, subsequently sterilized by filtration. Injectable solutions can be prepared. Generally, dispersions contain active compounds on a basic dispersion medium. By incorporating it into a sterile vehicle that includes other necessary components from those listed below, It is prepared. In the case of sterile powders for the preparation of sterile injectable solutions, preferred The preparation methods are vacuum drying and freeze-drying, which are used to obtain the solution from a pre-sterilized and filtered solution. This yields powders of the active ingredient and any additional desired ingredients.

[0065] In one embodiment, the therapeutic compound is protected from rapid elimination from the body. Protective carriers (e.g., controlled release such as implants and microencapsulated delivery systems) Prepared together with the formulation. A biodegradable and biocompatible polymer (e.g., ethylene vinyl acetate) is used. , polyacid anhydride, polyglycolic acid, collagen, polyorthoester, and polylactic acid ) may be used. Such formulations can be prepared using standard techniques, or for example Alza Corporation and Nova Pharmaceuticals It can be commercially obtained from , Inc. Liposome suspension (monoc for cellular antigens) (including liposomes that are targeted to selected cells by ronal antibodies) are also pharmaceutically acceptable. These can be used as carriers. For example, U.S. Patent No. 4,522,811. As described in the book, it can be prepared according to methods known to those skilled in the art.

[0066] This pharmaceutical composition should be packaged together with the administration instructions in a kit, container, pack, or dispenser. This may include, for example, the targeting peptides described herein and HSV.T A kit containing a composition including AAV with nucleic acid encoding K1, along with ganciclovir. It can be provided. [Examples]

[0067] The present invention is described in the following embodiments, which do not limit the scope of the present invention as described in the claims. Further details will be provided.

[0068] material and method In the following embodiments, the following materials and methods were used.

[0069] 1. Generation of capsid variants To generate capsid variant plasmids, cell-permeable peptides (Table 3) are used. Synthesize the DNA fragments to be cloned (GenScript), and CloneEZ seamless clone Using the GenScript technique, amino acid positions 588 and 589 (VP1 The skeleton of the AAV9 Rep-cap plasmid (pRC9) between amino acid numbering Inserted into CPP BIP1(VPALR, Array No. 1) and BIP2(VSALK , SEQ ID NO: 2), and their derivatives (e.g., TVSA in AAV.CPP.16) LK (SEQ ID NO: 4), and TVSALFK (SEQ ID NO: 8) in AAV.CPP.21) These are derived from the Ku70 protein, and their sequences are listed below.

[0070] [ka]

[0071] In addition, the VP1 tanks of the AAV9, AAV.CPP.16, and AAV.CPP.21 The protein sequence is listed below.

[0072] [ka]

[0073] [ka]

[0074] 2. Manufacturing of recombinant AAV Standard 3-plasmid cotransfection protocol (pRC plasmid, pHel Recombinant AAV is packaged using perplasmids and pAAV plasmids. It worked. Transgenes (for example, nuclear-oriented, driven by the ubiquitous EF1a promoter) p RC9 (or its variants), p helper, and pAAV are used in polyethyleneimine Cotransferring HEK 293T cells using (PEI, Polysciences) The rAAV vector was transfected at 72 hours and 120 hours. Later, the cells were recovered from serum-free medium and 120 hours after transfection. Then they were collected. The AAV particles in this culture medium were treated with 8% PEG-8000 (weight / volume). The cells were concentrated using PEG precipitation. The cell pellet containing the virus particles was resuspended and subjected to ultrasonic testing. The cells were dissolved by wave treatment. A combined viral vector was obtained from the PEG precipitate and cell lysates. The mixture was treated with DNase and RNase at 37°C for 30 minutes, and then ultracentrifuged. Iojiki (VTi 50 rotor, 40,000 rpm, 18℃, 1 hour) Purified by a Sanol gradient (15%, 25%, 40%, and 60%). Then, r AAV, Millipore Amicon filter unit (UFC910008, Concentrated using 100K MWCO, with 0.001% Pluronic F68(G The formulation was prepared with Dulbecco's phosphate-buffered saline (PBS) containing ibco.

[0075] 3.AAV titration We will measure the viral titer by measuring the DNase-resistant genome copy using quantitative PCR. Further determination was made. pAAV-CAG-GFP was digested with PVIII(NEB) and plasmid Free ends for ITR were generated and used to generate the standard curve. Virus samples were DNA Incubate with -se I to remove contaminating DNA, then treat with sodium hydroxide. The viral capsid was lysed to release the viral genome. Quantitative PCR was performed using ITR film. Word primer 5'-GGAACCCCTAGTGATGGAGTT (Sequence ID 91) and ITR reverse primer 5'-CGGCCTCAGTGAGCGA (SEQ ID NO: 9 2) was performed using the following method: Vector titer was measured using the rAAV-2 reference standard (RSM, ATC). Normalized to C, catalog number: VR-1616, Manassas, VA).

[0076] 4. Administration of AAV in mice For intravenous administration, AAV diluted with sterile saline (0.2 ml) was administered to adult mice. The drug was administered by tail vein injection to animals (over 6 weeks of age). The animals were then kept alive for 3 weeks. The animal was euthanized and tissue was collected. For intracerebral injection, AA was diluted with PBS (10 ul). Point V at a coordinate 1.0 mm to the right, 0.3 mm posterior, and 2.6 mm deep from the cross suture, Hamilt Injection was administered using a syringe. All animal studies were conducted using AA approved by IACUC. The procedure was carried out at an ALAC-certified facility.

[0077] 5. Processing of mouse tissue Anesthetized animals were perfused intracardiacly with cold phosphate-buffered saline (PBS), followed by 4% Paraformaldehyde (PFA) was perfused intracardiacly. The tissue was then post-cured overnight with 4% PFA. After fixing, it was immersed in a 30% sucrose solution for two days, then embedded in an OCT and snagged. The tissue was frozen. Typically, 80 μm thick brain sections were cut for spontaneous fluorescence imaging. Brain sections 40 μm thick were cut for intravascular coagulation (IHC).

[0078] 6. In vitro human BBB spheroid model Place hot 1% agarose (weight / volume, 50 µl) into a 96-well plate and let it cool. Then, the primary human astrocytes (Lonza) were placed on this agarose gel. Bioscience), Human brain microvascular pericytes (HBVP, ScienCell R esearch Laboratories), and human brain microvascular endothelial cells (hCM) EC / D3 (Cedarlane) in a 1:1:1 ratio (1500 cells of each type) The cells were seeded and incubated in a 5% CO2 incubator at 37°C for 48–72 hours. This allowed for the spontaneous construction of multicellular BBB spheroids. In the surrounding area, it has been reported that a multicellular barrier mimicking the blood-brain barrier (BBB) ​​is formed. AAVs-H2B-mCherry was added to the culture medium, and after 4 days, all spheroids were removed. Fixation was performed using 4% PFA and Nunc Lab-Tek II thin glass 8-welt. Transfer to a Thermo Scientific cover glass and use Zeiss LS Imaged using an M710 confocal microscope. The strength of the RFP signal within this spheroid. I looked up the degree and used it as a "readout".

[0079] 7. AAV administration in non-human primates (NHPs) All NHP trials are conducted by CROs at AAALAC-accredited facilities approved by IACUC. The experiment was conducted in cynomolgus monkeys, which have almost no neutralizing antibodies against AAV9 or have We pre-screened for whether or not it would occur (<1:5 titer). PBS / 0.001%F AAV diluted with 68 is administered using a peristaltic pump (via the cephalic vein or femoral vein). It was administered intravenously. Three weeks later, the animals were perfused transcardially with PBS, followed by transcardial perfusion of 4% PFA. The tissue was then perfused. Subsequently, the tissue was collected and processed for paraffin embedding and sectioning. did.

[0080] 8. Immunohistochemistry Primary antibody diluted in PBS containing 10% donkey serum and 2% Triton X-100. Floating staining of mouse tissue sections was performed using the following primary antibodies: Examples 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). Alexa Fluor 488, A LEXA Fluor 555, or Alexa Fluor 647 fluorocarbon The secondary antibody conjugated to A was diluted 1:200 and tested for the host species of the primary antibody. I used it.

[0081] For paraffin sections of NHP tissue, perform DAB staining and then AAV-AADC staining. More transduced cells were visualized. Rabbit anti-AADC antibody (1:500, Milli (pore) was used as the primary antibody.

[0082] 9. AAV binding assay HEK293T cells were cultured in a 5% CO2 incubator at 37°C. HEK293 to a 24-well plate at a cell density of 250,000 cells per well One day after T cell seeding, 200 µl of DMEM (31053028; Gibco), 1 Using a transfection mixture of ug of DNA plasmid and 3ug of PEI Then, these cells were transiently transfected with the LY6A cDNA plasmid. 48 hours after infection, the cells were placed on ice and cooled for 10 minutes. Then, Prepare the culture medium with 500 µl of ice-cold serum containing rAAVs-mCherry at an MOI of 10,000. The medium was changed to free DMEM medium. After incubation on ice for 1 hour, presumably Cells with AAV bound to their surface were washed three times with cold PBS, and then the genomic DNA was isolated. The virus particles bound to the cells were removed using a primer specific to mCherry. Quantification was performed using qPCR, and human GCG was used as a reference for HEK293T. It was normalized to the genome.

[0083] 10. Mouse model of glioblastoma All experiments were conducted at Brigham and Women's Hospital and At Harvard Medical School, I studied Animal Care and In accordance with protocols approved by the Use Committees (IACUC) The procedure was performed. Syngeneic immune-responsive C57BL / 6 female mammograms weighing 20+ / -1g (Envigo) were administered. A mortar was used. GL261- was resuspended in 2 μl of phosphate-buffered saline (PBS). Luc (100,000 mouse glioblastoma cells) was injected using a 26-gauge needle (80075; Ham It was injected intracranially using a 10 μl syringe equipped with an ilton. Stereotactic fixation frame Using the map, the position of the transplant site was determined (coordinates from the cross suture (mm): 2 to the right, 0.5 Anterior, 3.5 to the cerebral cortex). 7 days later, 200 ul of AAV-HSV-TK1 (1 E+12 viral genomes, IV) administered once, followed by ganciclovir (50 mg / kg). It was administered daily for 10 days.

[0084] [Example 1] AAV9 Capsid Modification Peptide sequences that may enhance the penetration of biomolecules or viruses across the blood-brain barrier. AAV peptide display technology was used to identify the following: (Listed in Table 3) Individual cell-permeable peptides, as shown in Figure 1A, have amino acids 588 and 58 It was inserted into the AAV9 capsid between 9 (VP1 numbering). This insertion was performed on the AAV pack. RC plasmid is one of the three plasmids that are co-transfected for caging. This was carried out by modifying Rasmid, and Figure 1B shows an exemplary schematic diagram of this experiment. Each AAV variant was manufactured and screened separately. For further details... See Materials and Methods #1-3.

[0085] [Table 3]

[0086] [Example 2] First round of in vivo screening AAV expressing nuclear RFP (H2B-RFP) was used on a C57BL / 6 gene background and B Adult mice with a mixed ALB / c gene background were intravenously injected. Three weeks later, brain tissue was examined. The cells were collected, sectioned, and identified as RFP-labeled (white dots in Figures 2A and 2C). (These are quantified in Figures 2B and 2D, respectively). CPP BIP1 and BIP2 These were inserted into the capsids of AAV.CPP.11 and AAV.CPP.12, respectively. For more details, see Materials and Methods ##4-5.

[0087] [Example 3] Optimization of the modified AAV9 capsid By optimizing the BIP targeting sequence, AAV.CPP.11 and AA V.CPP.12 were further engineered. The BIP insert is derived from the protein Ku70 (see Figure 3A and Materials / Methods #1 for the complete sequence). The "synthetic" BIP sequence VSALK, a source of variability, was selected as a research focus to minimize the potential species specificity of the engineered AAV vectors. AAVs were produced and tested separately for brain transduction efficiency compared to AAV9 (see Figures 3B-C). The percentage of cell transduction in the mouse liver at 3 weeks after IV injection of several AAV variants delivering the reporter gene RFP is shown in Figure 3D. For more details, see Materials and Methods ##1-5.

[0088] [Example 4] In vitro model - BBB penetration screening A subset of AAV variants was screened for their ability to cross the human BBB using an in vitro spheroid BBB model. This spheroid contains human microvascular endothelial cells that form tight junctions on the surface, along with human pericytes and astrocytes. AAVs with nuclear RFP as a reporter were evaluated for their ability to penetrate from the surrounding medium into the interior of this spheroid and transduce cells inside. Figure 4A shows the outline of the experiment. Figures 4B-D show the results for wt AAV9, AAV.CPP.16, and AAV.CPP.21, respectively, and these and other peptides are quantified in Figure 4E. In this model, the peptides D11, 15, 16, and 21 caused the greatest penetration into the spheroid. For further details, see Materials and Methods #6.

[0089] [Example 5] In Vivo BBB Penetration Screening In the experiment conducted as described above for Example 2, for further evaluation in the in vivo model, AAV.CPP.16 and AAV.CPP.21 were selected. All AAVs had nuclear RFP as a reporter. Both showed enhanced ability to transduce brain cells after intravenous administration compared to AAV9 in C57BL / 6J adult mice (white dots in the brain sections in Figure 5A, quantified in Figure 5B) and BALB / c adult mice (white dots in the brain sections in Figure 6A, quantified in Figure 6B).

[0090] High doses of AAV.CPP.16 and AAV.CPP.21 (4 × [[ID= 26]] 10 12 vg, IV administration) resulted in extensive brain transduction in mice. Both AAVs had nuclear RFP as a reporter (white dots in the brain sections in Figure 7A, quantified in Figure 7B).

[0091] [Example 6] In Vivo Distribution of Modified AAVs As shown in Figure 8A, AAV.CPP.16 and AAV.CPP.21 preferentially targeted neurons (labeled [[ID=4 3]]by NeuN antibody) across multiple brain regions (e.g., cerebral cortex, midbrain, and hippocampus) in mice. Both AAVs had nuclear RFP as a reporter.

[0092] AAV.CPP.16 and AAV.CPP.21 also study the spinal cord and motor nerves of mice. In transcellular targeting, it also showed enhanced capability compared to AAV9. All AAVs are reported It contains nuclear RFP as a component and was administered intravenously to neonatal mice (4 times × 10 10 v g) Motor neurons were visualized using CHAT antibody staining. RFP signature in Figure 8B The co-localization of NAL and CHAT signaling suggested specific transduction in motor neurons. .

[0093] AAV-CAG-H2B-RFP and AAV.C are transducers that induce various tissues in mice. The relative capabilities of PP.16-CAG-H2B-RFP were also evaluated. 1 / 10 11 VG intravenously The injection was administered. The number of transduced cells was compared to the total number of cells labeled by DAPI nuclear staining. The data was then normalized. This result shows that AAV.CPP.16 is found in mouse cardiac tissue (Figure 9A); In targeting skeletal muscle tissue (Figure 9B) and dorsal root ganglion tissue (Figure 9C), AAV9 and It was shown to be more efficient by comparison.

[0094] [Example 7] BBB penetration in non-human primate models 2 x 10 13 vg / kg of AAVs-CAG-AADC (as a reporter gene) It was intravenously injected into 3-month-old cynomolgus monkeys. AAV transducer cells (shown in black) were administered to A Visualization was performed using antibody staining against ADC, as shown in Figures 10A-D, AAV.C PP.16 and AAV.CPP.21, compared to AAV9, are venous in non-human primates. It showed enhanced ability to induce plasma transfer into brain tissue after internal administration. AAV.CPP.16 is a primary visual In the occipital lobe (Figure 10A), parietal cortex (Figure 10B), thalamus (Figure 10C), and cerebellum (Figure 10D ), significantly more cells were transduced compared to wt AAV9. For further details, please refer to Materials and Methods ##7-8.

[0095] [Example 8] AAV.CPP.16 and AAV.CPP.21 do not bind to LY6A LY6A functions as a receptor for AAV.PHP.eB and mediates the robust effect of AAV.PHP.eB in the crossing of 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). Conversely, overexpression of LY6A did not increase the viral binding for AAV9, AAV.CPP.16, or AAV.CPP.21 (see Figure 11B). This suggests that AAV.CPP.16 or AAV .CPP.21 does not share LY6A as a receptor with AAV.PHP.eB . For further details, please refer to Materials and Methods #9.

[0096] [Example 9] Delivery of therapeutic proteins to the brain using AAV.CPP.21 Using AAV.CPP.21, the "suicide gene" HSV.TK1 was systemically delivered to a mouse model of brain tumors. HSV.TK1 converts ganciclovir, which would otherwise be "dormant," into a tumor killing agent. Intravenous administration of AAV.CPP.21-H2BmCherry (Figure 12A, lower left and right central panels) was shown to target tumors (especially the frontiers where tumors spread). As shown in Figures 12B-C, the "suicide gene" H The use of AAV.CPP.21 for whole-body delivery of SV.TK1 allows for prodrug delivery. When used in combination with a certain ganciclovir, it resulted in a reduction in brain tumor size. These results indicate that This demonstrates that therapeutic genes can be delivered systemically to brain tumors using AAV.CPP.21. For further details, please refer to Materials and Methods #10.

[0097] [Example 10] Intracerebral administration of AAV.CPP.21 In addition to systemic administration (for example, in Example 2), the AAV described herein may be administered to the mouth It was administered locally to the brain. AAV9-H2B-RFP and AAV.CPP.21-H2 Intracerebral injection of B-RFP (Figure 13) compared brain sections treated with AAV9 showed that AA Extensive and high-intensity RFP signals were observed in brain sections treated with V.CPP.21. For further details, please refer to Materials and Methods #4.

[0098] Other Embodiments The present invention is described in conjunction with its detailed description, but the above description is in accordance with the attached claims. The scope of this invention, as defined by the range, is intended to be illustrative and not limited. It must be understood that this has not been done. Other aspects, advantages, and modifications are described below. It is within the scope of the request. Various embodiments of the present invention are shown below. 1. An AAV capsid protein containing an amino acid sequence that includes at least four consecutive amino acids from the sequence TVSALFK (SEQ ID NO: 8); TVSALK (SEQ ID NO: 4); KLASVT (SEQ ID NO: 83); or KFLASVT (SEQ ID NO: 84). 2. The AAV capsid protein described in 1 above, comprising an amino acid sequence containing at least five consecutive amino acids from the sequence TVSALK (SEQ ID NO: 4); TVSALFK (SEQ ID NO: 8); KLASVT (SEQ ID NO: 83); or KFLASVT (SEQ ID NO: 84). 3. The AAV capsid protein described in 1 above, comprising an amino acid sequence containing at least six consecutive amino acids from the sequence TVSALK (SEQ ID NO: 4); TVSALFK (SEQ ID NO: 8); KLASVT (SEQ ID NO: 83); or KFLASVT (SEQ ID NO: 84). 4. The AAV is AAV9, which is the AAV capsid protein described in any of items 1 to 3 above. 5. An AAV capsid protein as described in any of items 1-4 above, including AAV9 VP1. 6. The AAV capsid protein described in item 5 above, wherein a targeting sequence is inserted at the positions corresponding to amino acids 588 and 589 of sequence number 85. 7. A nucleic acid encoding the AAV capsid protein described in any of items 1 to 6 above. 8. AAV containing the capsid protein described in any of items 1-6 above. 9. The AAV described in 8 above, further comprising a transgene, preferably a therapeutic transgene. 10. Targeting sequences containing 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). 11. The targeting sequence is the targeting sequence described in 10 above, which includes VPALR (sequence number 1); VSALK (sequence number 2); TVPALR (sequence number 3); TVSALK (sequence number 4); TVPMLK (sequence number 12); TVPTLK (sequence number 13); FTVSALK (sequence number 5); LTVSALFK (sequence number 6); TVSALFK (sequence number 8); TVPALFR (sequence number 9); TVPMLFK (sequence number 10); or TVPTLFK (sequence number 11). 12. A fusion protein comprising the targeting sequence described in item 10 or 11 above and a heterologous sequence. 13. An AAV capsid protein containing the targeting sequence described in item 10 or 11 above. 14. The AAV capsid protein described in item 13 above, including AAV9 VP1. 15. The AAV capsid protein described in 14 above, wherein the targeting sequence is inserted at the positions corresponding to amino acids 588 and 589 of the sequence number. 16. A nucleic acid encoding a targeting sequence, fusion protein, or AAV capsid protein as described in any of items 10 to 16 above. 17. AAV containing the capsid protein described in any of items 13-15 above. 18. The AAV according to 17 above, further comprising a transgene, preferably a therapeutic transgene. 19. A method for delivering a transgene to a cell, comprising bringing the cell into contact with the AAV described in 1-9, 17, or 18 above. 20. The method according to 19 above, wherein the cells are nerve cells (optionally selected dorsal root ganglion cells), astrocytes, cardiomyocytes, or myocytes. 21. The cell is present in a living subject, according to the method described in 19 above. 22. The method described in 19 above, wherein the subject is a mammal. 23. The method according to any one of 20 to 22 above, wherein the cells are present in tissue selected from the brain, spinal cord, dorsal root ganglia, heart, or muscle, and combinations thereof. 24. The method according to 23 above, wherein the subject has a neurodegenerative disease, epilepsy; stroke; spinocerebellar ataxia; Canavan disease; metachromatic leukodystrophy; spinal muscular atrophy; Friedreich's ataxia; X-linked central nucleus myopathy; lysosomal storage disorder; Barth syndrome; Duchenne muscular dystrophy; Wilson's disease; or Crigler-Nadjar syndrome type 1. 25. The method according to 24 above, wherein the neurodegenerative disease is Parkinson's disease; Alzheimer's disease; Huntington's disease; amyotrophic lateral sclerosis; and multiple sclerosis. 26. The method according to 23, wherein the subject has brain cancer, and the method comprises administering an AAV encoding an anticancer agent. 27. The method according to 26, wherein the anticancer agent is HSV.TK1, and the method further comprises administering ganciclovir. 28. The method according to any one of 22 to 27 above, wherein the cells are present in the brain of the subject, and the AAV is administered by parenteral delivery; intracerebral delivery; or intrathecal delivery. 29. The parenteral delivery is by intravenous delivery, intra-arterial delivery, subcutaneous delivery, intraperitoneal delivery, or intramuscular delivery, as described in 28 above. 30. The intrathecal delivery is by lumbar injection, cisterna magna injection, or intraparenchymal injection, as described in 28 above.

Claims

1. An adeno-associated virus (AAV) vector comprising an AAV capsid, wherein the AAV capsid comprises a peptide insert, the peptide insert consists of TVSALFK (SEQ ID NO: 8) or TVSALK (SEQ ID NO: 4), the AAV capsid is an AAV9 capsid, and the peptide insert is located between amino acids 588 and 589 of SEQ ID NO:

85.

2. The AAV vector according to claim 1, further comprising an introduced gene sequence.

3. The AAV vector according to claim 2, wherein the introduced gene sequence encodes a therapeutic protein or nucleic acid.

4. The AAV vector according to claim 1, further comprising a non-coding RNA that alters the expression of a target gene.

5. The AAV vector according to claim 4, wherein the non-coding RNA is shRNA, siRNA, or miRNA.

6. A composition for use in a method for delivering a transgene to a cell, comprising an AAV vector according to any one of claims 1 to 5, wherein the method comprises contacting the cell with the AAV vector.

7. The composition according to claim 6, wherein the cells are nerve cells, astrocytes, cardiomyocytes, or muscle cells.

8. The composition according to claim 6, wherein the cells are dorsal root ganglion neurons.

9. The composition according to claim 6, wherein the cells are present in a living subject.

10. The composition according to claim 9, wherein the subject is a mammal.

11. The composition according to claim 6, wherein the cells are present in tissue selected from the brain, spinal cord, dorsal root ganglia, heart, or muscle, and combinations thereof.

12. The composition according to claim 9, wherein the subject is a neurodegenerative disease, epilepsy; stroke; spinocerebellar ataxia; Canavan disease; metachromatic leukodystrophy; spinal muscular atrophy; Friedreich's ataxia; X-linked central nucleus myopathy; lysosomal storage disorder; Barth syndrome; Duchenne muscular dystrophy; Wilson's disease; or Crigler-Nadjar syndrome type 1.

13. The composition according to claim 12, wherein the neurodegenerative disease is Parkinson's disease; Alzheimer's disease; Huntington's disease; amyotrophic lateral sclerosis; or multiple sclerosis.

14. The composition according to claim 9, wherein the subject has brain cancer, the AAV vector comprises a transgene sequence encoding an anticancer drug, and the anticancer drug is a protein.

15. The composition according to claim 14, wherein the anticancer drug is HSV. TK1, and the method further comprises administering ganciclovir.

16. The composition according to claim 9, wherein the cells are present in the brain of the subject, and the composition is administered by parenteral delivery; intracerebral delivery; or intrathecal delivery.

17. The composition according to claim 16, wherein the parenteral delivery is by intravenous delivery, intra-arterial delivery, subcutaneous delivery, intraperitoneal delivery, or intramuscular delivery.

18. The intrathecal delivery is by lumbar injection, cisterna magna injection, or intraparenchymal injection, as described in claim 16. The composition of.

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