Methods and compositions for delivery of agents across the blood-brain barrier

Abstract

The present invention relates to methods and compositions for delivering agents across the blood brain barrier. The present invention is based on the development of artificial targeting sequences that enhance the penetration of agents into cells and across the blood brain barrier, compositions comprising the sequences, and methods of use thereof. Provided herein are AAVs comprising a capsid protein comprising a targeting sequence and a transgene, preferably a therapeutic or diagnostic transgene. Further, provided herein are methods of delivering a transgene to a cell comprising contacting the cell with the AAV or fusion protein described herein.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202510648887.3 (22) Application Date 2019.07.11 (30) Priority Data 62 / 696,422 2018.07.11 US (62) Divisional Application Data 201980059342.1 2019.07.11 (71) Applicant Brigham and Women's Hospital Address USA (72) Inventor Bei Fengfeng (74) Patent Agency Beijing Linda Liu Intellectual Property Agency (General Partnership) 11277 Patent Attorney Li Maojia Yan Junping (51) Int.Cl. C12N 15 / 864 (2006.01) C07K 14 / 015 (2006.01) C07K 19 / 00(2006.01) A61K 35 / 761(2015.01) A61K 47 / 46(2006.01) A61K 31 / 7088(2006.01) A61K 31 / 713(2006.01) A61K 48 / 00(2006.01) A61K 31 / 522(2006.01) A61K 38 / 45(2006.01) A61K 38 / 16(2006.01) A61P 35 / 00(2006.01) A61P 25 / 28(2006.01) A61P 25 / 16(2006.01) A61P 25 / 14 (2006.01) A61P 25 / 08 (2006.01) A61P 9 / 10 (2006.01) A61P 25 / 00 (2006.01) (54) Title of Invention: Methods and Compositions for Delivering Reagents Across the Blood-Brain Barrier (57) Abstract: This invention relates to methods and compositions for delivering reagents across the blood-brain barrier. The 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 said sequences, and methods of using them. This document provides AAV comprising a capsid protein containing a targeting sequence and a transgene, preferably a therapeutic or diagnostic transgene. Furthermore, this document provides a method for delivering the transgene to cells, the method comprising contacting said cells with the AAV or fusion protein described herein. Claims 2 pages, Description 28 pages, Sequence Listing (electronic publication), Drawings 33 pages, CN 120843603 A 2025.10.28 CN 1 20 84 36 03 A 1. An adeno-associated virus (AAV) vector comprising an AAV capsid, wherein the AAV capsid contains a peptide insert of up to 21 amino acids, and wherein the peptide insert comprises 5-7 amino acids of TVSALFK (SEQ ID NO:8).2. The AAV vector of claim 1, comprising a transgenic sequence. 3. The AAV vector of claim 2, wherein the transgenic sequence encodes a therapeutic agent. 4. The AAV vector of claim 1, comprising non-coding RNA. 5. The AAV vector of claim 4, wherein the non-coding RNA is shRNA, siRNA, or miRNA. 6. The AAV vector of claim 2, wherein delivery of the transgenic sequence to an organ or tissue is enhanced compared to an AAV vector comprising an AAV capsid without the peptide insert and the transgenic sequence. 7. The AAV vector of claim 6, wherein the organ or tissue: (i) comprises a permeability barrier, (ii) comprises epithelium containing tight junctions, or (iii) is the brain or central nervous system. 8. The AAV vector of claim 4, wherein delivery of the non-coding RNA to an organ or tissue is enhanced compared to an AAV vector comprising an AAV capsid without the peptide insert and the non-coding RNA. 9. The AAV vector of claim 8, wherein the organ or tissue: (i) comprises a permeability barrier, (ii) comprises epithelium containing tight junctions, or (iii) is the brain or central nervous system. 10. A composition comprising the AAV vector of any one of claims 1 to 9 and a pharmaceutically acceptable carrier. 11. Use of the AAV vector of any one of claims 1 to 9 in the preparation of an agent for delivering a transgene to cells by contacting the cells with the AAV vector. 12. An adeno-associated virus (AAV) vector comprising an AAV capsid, wherein the AAV capsid comprises a peptide insert of up to 21 amino acids, and wherein the peptide insert comprises 5 or 6 consecutive amino acids of TVSALK (SEQ ID NO: 4). 13. The AAV vector of claim 12, comprising a transgene sequence. 14. The AAV vector of claim 13, wherein the transgene sequence encodes a therapeutic agent. 15. The AAV vector of claim 12, comprising non-coding RNA. 16. The AAV vector of claim 15, wherein the non-coding RNA is shRNA, siRNA, or miRNA. 17. The AAV vector of claim 13, wherein the delivery of the transgenic sequence to an organ or tissue is enhanced relative to an AAV vector comprising an AAV capsid without the peptide insert and the transgenic sequence. 18. The AAV vector of claim 17, wherein the organ or tissue: (i) comprises a permeability barrier, (ii) comprises epithelium containing tight junctions, or (iii) is the brain or central nervous system.19. The AAV vector of claim 15, wherein delivery of the non-coding RNA to an organ or tissue is enhanced relative to an AAV vector comprising an AAV capsid without the peptide insert and the non-coding RNA. 20. The AAV vector of claim 19, wherein the organ or tissue: (i) comprises a permeability barrier; (ii) comprises epithelium containing tight junctions; or (iii) is the brain or central nervous system. 21. A composition comprising the AAV vector of any one of claims 12 to 20 and a pharmaceutically acceptable carrier. 22. Use of the AAV vector of any one of claims 12 to 20 in the preparation of a reagent for delivering transgenes to cells by means of contacting the cells with the AAV vector. 23. Use of an adeno-associated virus (AAV) vector in the preparation of a reagent for delivering a transgene to a subject, said AAV vector comprising (i) a transgene, and (ii) an AAV capsid containing a peptide insert of up to 21 amino acids, said peptide insert comprising 5-7 consecutive amino acids of TVSALFK (SEQ ID NO: 8) or 5 or 6 consecutive amino acids of TVSALK (SEQ ID NO: 4). 24. The use according to claim 23, wherein said transgene comprises a sequence encoding a therapeutic agent. 25. The use according to claim 23, wherein delivery of said transgene to an organ or tissue is enhanced compared to an AAV vector comprising an AAV capsid without said peptide insert and said transgene. 26. The use according to claim 25, wherein the transgene is delivered to (i) an organ or tissue comprising a barrier, (ii) an organ or tissue comprising epithelium containing tight junctions, (iii) the brain or central nervous system, (iv) the cortex, cerebellum, hippocampus, substantia nigra, thalamus, or amygdala, or (v) neurons, astrocytes, glial cells, or cardiomyocytes. 27. The use according to any one of claims 23 to 26, wherein the subject has a neurodegenerative disease or cancer. 28. The use according to claim 27, wherein the neurodegenerative disease is Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, epilepsy, stroke, spinocerebellar ataxia, or Canavan disease. 29. The use according to claim 27, wherein the cancer is brain cancer. 30. The use according to any one of claims 23 to 26, wherein the transgene comprises non-coding RNA. 31. An AAV capsid protein comprising a targeting sequence, said targeting sequence comprising TVSALFK (SEQ ID NO:8); TVSALK (SEQ ID NO:8)32. The AAV capsid protein of claim 31, comprising an amino acid sequence comprising TVSALK (SEQ ID NO:4); KLASVT (SEQ ID NO:83); or KFLASVT (SEQ ID NO:84). 33. The AAV capsid protein of claim 31, comprising an amino acid sequence comprising TVSALFK (SEQ ID NO:8). 34. The AAV capsid protein of claim 31, comprising an amino acid sequence of SEQ ID NO:89 or SEQ ID NO:90. 35. The AAV capsid protein of claim 31, comprising AAV9 VP1. 36. An AAV capsid protein comprising a targeting sequence comprising TV[S / p][A / m / t / ]L (SEQ ID NO:80), wherein the targeting sequence is inserted at positions corresponding to amino acids 588 and 589 of SEQ ID NO:85. 37. The AAV capsid protein of any one of claims 31 to 36, wherein the AAV is AAV9. 38. A nucleic acid encoding the AAV capsid protein according to any one of claims 31 to 36. 39. An AAV comprising the AAV capsid protein according to any one of claims 31 to 36. Claims 2 / 2 pages 3 CN 120843603 A Method and composition for delivering reagents across the blood-brain barrier

[0001] This application is a divisional application of patent application No. 201980059342.1 (International Application No. PCT / US2019 / 041386), filed July 11, 2019, entitled "Method and composition for delivering reagents across the blood-brain barrier".

[0002] Priority Claim

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 696,422, filed July 11, 2018. The entire foregoing is incorporated herein by reference.

[0004] Sequence Listing

[0005] This application includes a sequence listing, which is electronically submitted in ASCII format, the entirety of which is incorporated herein by reference. The ASCII copy created on July 11, 2019, is named 29618-0200WO1_SL.txt and is 58,834 bytes in size. Technical Field

[0006] This document describes sequences that enhance the penetration of agents across the blood-brain barrier, compositions including said sequences, and methods of using them. Background Art

[0007] The delivery of therapeutic agents, including gene therapy agents, has hindered the development of treatments for many conditions. The blood-brain barrier (BBB) ​​is the barrier between the delivery of drugs to the mammalian central nervous system (CNS), and particularly to the human brain, to treat conditions including Parkinson's disease.Major obstacles to the condition of neurodegenerative diseases such as Sjögren's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, and multiple sclerosis. Summary of the Invention

[0008] 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.

[0009] Therefore, this invention provides an AAV capsid protein, such as an engineered AAV capsid protein, comprising a targeting sequence containing at least four consecutive 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 containing at least five consecutive 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 includes a targeting sequence comprising at least six consecutive 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).

[0010] In some embodiments, the AAV is AAV9; other AAVs known in the art may also be used (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8 and variants thereof, and other AAVs known in the art or described herein).

[0011] In some embodiments, the AAV capsid protein includes AAV9VP1 (e.g., SEQ ID NO:85).

[0012] In some embodiments, the targeting sequence is inserted in the capsid protein at a position between amino acids 588 and 589 corresponding to SEQ ID NO:85.

[0013] This document also provides nucleic acids encoding AAV capsid proteins comprising the targeting sequences described herein.

[0014] Furthermore, this document provides an AAV comprising a capsid protein containing the target sequence as described herein. In some embodiments, the AAV further includes a transgene, preferably a therapeutic or diagnostic transgene. Therapeutic transgenes may include, for example, cDNA that restores protein function, guide RNA for gene editing, RNA, or miRNA.

[0015] This document also provides V[S / p][A / m / t / ]L (SEQ ID NO:79), TV[S / p][A / m / t / ]L (SEQ ID NO:80), and TV[S / p][A / m / t / ]LK (SEQ ID NO:79).Target sequences of VPALR (SEQ ID NO:1); VSALR (SEQ ID NO:2); TVPALR (SEQ ID NO:3); TVSALK (SEQ ID NO:4); TVPMLK (SEQ ID NO:12); TVPTLK (SEQ ID NO:13); FTVSLK (SEQ ID NO:5); LTVSLK (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) are also provided. Fusion proteins comprising a target sequence linked to a heterologous (e.g., non-AAV VP1) sequence are also provided, as well as AAV capsid proteins comprising the target sequence (e.g., AAV9 VP1). In some embodiments, the target sequence is inserted at positions 588 and 589 corresponding to SEQ ID NO:85.

[0016] Furthermore, this document provides nucleic acids encoding the target sequence, fusion protein, or AAV capsid protein as described herein, and AAVs comprising capsid proteins containing the target sequence. In some embodiments, the AAV further includes a transgene, preferably a therapeutic or diagnostic transgene. Therapeutic transgenes may include, for example, cDNA that restores protein function, guide RNA for gene editing, RNA, or miRNA.

[0017] Furthermore, this document provides a method of delivering a transgene to cells, the method comprising contacting the cells with the AAV or fusion protein described herein. In some embodiments, the cells are located in a living subject, for example, a mammalian subject. In some embodiments, the cells are located in a tissue selected from the brain, spinal cord, dorsal root ganglion, heart, or muscle, and combinations thereof. In some embodiments, the cells are neurons (optionally dorsal root ganglion neurons), astrocytes, cardiomyocytes, or myocytes.

[0018] In some embodiments, the subject has neurodegenerative diseases, epilepsy, stroke, spinocerebellar ataxia, Canavan's disease, metachromatic leukodystrophy, spinal muscular atrophy, Friedreich's ataxia, X-linked centronuclear myopathy, lysosomal storage disease, or Bartholin's disease.Barth Syndrome; Duchenne muscular dystrophy; Wilson's disease; or Crigler-Najjar syndrome type 1. In some embodiments, the neurodegenerative disease is Parkinson's disease; Alzheimer's disease; Huntington's disease; amyotrophic lateral sclerosis; and multiple sclerosis.

[0019] In some embodiments, the subject has brain cancer, and the method includes administering an AAV encoding an anticancer agent. In some embodiments, the anticancer agent is HSV.TK1, and the method further includes administering ganciclovir.

[0020] In some embodiments, the cells are located in the subject's brain, and the AAV is administered via parenteral delivery (e.g., via intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular delivery); intracerebral delivery; or intrathecal delivery (e.g., via lumbar injection, cerebellomedullary cistern injection, or intraparenchymal injection).

[0021] 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 pertains. Methods and materials used in this invention are described herein; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the definitions included in this specification shall prevail.

[0022] Other features and advantages of the invention will become apparent from the following detailed description and drawings, as well as the claims. Specification 2 / 28 pages 5 CN 120843603 A Brief Description of the Drawings

[0023] Figures 1A-1B illustrate an exemplary strategy for engineering AAV9 by inserting a cell-penetrating peptide (CPP) into the capsid of AAV9. Figure 1A is a 3D model of the AAV9 virus. Individual CPPs inserted between amino acids 588 and 589 (VP1 number) in the capsid are shown on a 3x axis, on which receptor binding may occur. Figure 1B illustrates the method for individual AAV production. Three plasmids, including pRC (engineered or unengineered), pHelper, and pAAV, were co-transfected into HEK 293T cells, and AAV was harvested and purified using an iodixanol gradient.

[0024] Figures 2A-2B show representative images and quantitative analysis of mouse brain slices after intravenous administration of low doses of candidate AAV. Mice with mixed genetic backgrounds were used. The candidate AAVs differed from the CPPs they were inserted into (see Table 3), but all expressed nuclear red fluorescent protein (RFP) as a reporter protein. For further screening, those with low yields were excluded.Candidate AAVs were selected. The AAV dose was 1 × 10¹⁰ vg (viral genome) per animal. Each white dot in Figure 2A represents an RFP-labeled cell. In Figure 2B, *P < 0.05, ANOVA relative to AAV9.

[0025] Figures 2C-2D depict representative images and quantitative analyses of mouse brain slices 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 AAV dose was increased to 1 × 10¹¹ vg per animal. The candidate AAVs express nuclear red fluorescent protein (RFP) as a reporter protein. Each white dot in Figure 2C represents an RFP-labeled cell. In Figure 2D, *P < 0.05, **P < 0.01, ANOVA relative to AAV9.

[0026] Figure 3A shows the optimization of the BIP targeting sequence for further engineering of AAV9 toward better brain transduction. BIP1 (VPALR, SEQ ID NO:1), which enables AAV9 to transduce the brain more effectively (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), like AAV.CPP.12, is a “synthetic” peptide associated with BIP1. Further engineering focuses on the VSALD sequence, aiming to minimize the species specificity of the ultimately engineered AAV. To generate new targeting sequences, target amino acids are added to the VSALD sequence, and in other cases, the positions of individual amino acids are switched. All the new sequences derived from BIP2 are then inserted back into the AAV9 capsid to generate new candidate AAVs for screening. The sequences appearing in sequence are SEQ ID NO:69, 70, 71, 1-6, 72, 7, and 8.

[0027] Figures 3B-3C depict representative images and quantitative analysis of mouse brain slices after intravenous administration of more candidate AAVs. All candidate AAVs expressed nuclear red fluorescent protein (RFP) as a reporter protein. The dose of AAV 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 for their strong and extensive brain transduction. In Figure 3C, *P<0.05, **P<0.01, ***P<0.001, ANOVA relative to AAV9.

[0028] Figure 3D depicts quantitative analysis of transduction efficiency in the liver after intravenous administration of candidate AAVs. Liver slices showing transduction are shown.Percentage of vitreous cells. The dose of AAV was 1 × 10¹¹ vg per animal. ***P < 0.001, ANOVA relative to AAV9.

[0029] Figures 4A-4E illustrate the screening of selected candidate AAVs in an in vitro spheroid model of the human blood-brain barrier. Figure 4A illustrates spheroids comprising human microvascular endothelial cells forming a barrier on the surface, and human pericytes and astrocytes inside the spheroids. The ability of candidate AAVs to penetrate from the surrounding media into the interior of the spheroids and transduce the internal cells was evaluated. Figures 4B-4D show graphs of spheroids treated with AAV9, AAV.CPP.16, and AAV.CPP.21. Figure 4E shows the relative RFP intensity of spheroids treated with different AAVs. ***P < 0.001, ANOVA relative to AAV9.

[0030] Figures 5A-5B depict representative images and quantitative analysis of brain slices from C57BL / 6J inbred mice after intravenous administration of AAV9, AAV.CPP.16, and AAV.CPP.21. All candidate AAVs expressed nuclear red fluorescent protein (RFP) as reporter proteins. The dose of AAV was 1 × 10¹² vg per animal. Each white dot in Figure 5A represents a cell labeled with RFP. In Figure 5B, *P < 0.05, ***P < 0.001, ANOVA.

[0031] Figures 6A-6B depict representative images and quantitative analysis of brain slices from BALB / cJ inbred mice after intravenous administration of AAV9, AAV.CPP.16, and AAV.CPP.21. All candidate AAVs expressed nuclear red fluorescent protein (RFP) as reporter proteins. The dose of AAV was 1 × 10¹² vg per animal. Each white dot in Figure 6A represents an RFP-labeled cell. In Figure 6B, ***P < 0.001, ANOVA.

[0032] Figures 7A-7B depict representative images and quantitative analysis of brain slices after intravenous administration of high doses of AAV.CPP.16 and AAV.CPP.21 in C57BL / 6J inbred mice. Both candidate AAVs expressed nuclear red fluorescent protein (RFP) as reporter proteins. The dose of AAV was 4 × 10¹² vg per animal. Each white dot in Figure 7A represents an RFP-labeled cell. In Figure 7B, *P < 0.05, Student's test.

[0033] Figure 8A shows the transduction of AAV.CPP.16 and AAV.CPP.21 into adult neurons (labeled by NeuN antibody) across multiple brain regions including the cortex, midbrain, and hippocampus in mice. Transducing neurons were co-labeled with NeuN antibody and RFP. Adult C57BL / 6J mice (6 weeks old) were intravenously administered 4 × 10¹² vg of AAV.

[0034] Figure 8B illustrates the enhanced ability of AAV.CPP.16 and AAV.CPP.21 to target spinal cord and motor neurons in mice relative to AAV9. 4 × 10¹⁰ vg of AAV was administered intravenously to newborn mice (day 1 after birth). Motor neurons in the ventral horn of the spinal cord were observed using CHAT antibody staining. Colocalization of RFP and CHAT signals indicates specific transduction of motor neurons.

[0035] Figure 9A illustrates the enhanced ability of AAV.CPP.16 to target the heart in adult mice relative to AAV9. 1 × 10¹¹ vg of AAV was administered intravenously to adult C57BL / 6J mice (6 weeks old). The percentage of RFP-labeled cells relative to all DAPI-stained cells is shown. *P < 0.05, Student's test.

[0036] Figure 9B illustrates the enhanced ability of AAV.CPP.16 to target skeletal muscle in adult mice relative to AAV9. 1 × 10¹¹ vg of AAV was administered intravenously to adult C57BL / 6J mice (6 weeks old). The percentage of RFP-labeled cells relative to all DAPI-stained cells is shown. *P < 0.05, Student's test.

[0037] Figure 9C illustrates the enhanced ability of AAV.CPP.16 to target the dorsal root ganglion (DRG) of adult mice relative to AAV9. 1 × 10¹¹ vg of AAV was administered intravenously to adult C57BL / 6J mice (6 weeks old). The percentage of RFP-labeled cells relative to all DAPI-stained cells is shown. *P < 0.05, Student's test.

[0038] Figure 10A illustrates the enhanced ability of AAV.CPP.16 and AAV.CPP.21 to transduce brain cells in the primary visual cortex relative to AAV9 after intravenous administration to non-human primates. 2 × 10¹³ 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. Cells transduced by AAV were observed using antibody staining against AADC (shown in black). A magnified square area in the left image is shown in the right image. 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 pronounced compared to AAV.CPP.16.

[0039] Figure 10B illustrates the enhanced transduction of brain cells in the parietal cortex by AAV.CPP.16 and AAV.CPP.21 compared to AAV9 after intravenous administration to non-human primates. 2 × 10¹³ 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. Cells transduced by AAV were observed using antibody staining against AADC (shown in black).Cells transduced by AAV are observed using staining (shown in black). A magnified square area in the left image is shown in the right image. AAV.CPP.16 transduces significantly more cells compared to AAV9. AAV.CPP.21 also transduces more cells compared to AAV9, although its effect is less pronounced compared to AAV.CPP.16.

[0040] Figure 10C illustrates the enhanced transduction of brain cells in the thalamus by AAV.CPP.16 and AAV.CPP.21 compared to AAV9 after intravenous administration to non-human primates. 2 × 10¹³ vg / kg AAV-CAG-AADC (as a reporter gene) was intravenously injected into 3-month-old cynomolgus monkeys with low levels of pre-existing neutralizing antibodies. Cells transduced by AAV were observed using staining with an antibody against AADC (shown in black). A magnified square area in the left image is shown in the right image. 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 pronounced compared to AAV.CPP.16.

[0041] Figure 10D illustrates the enhanced transduction of brain cells in the cerebellum by AAV.CPP.16 and AAV.CPP.21 compared to AAV9 after intravenous administration to non-human primates. 2 × 10¹³ vg / kg AAV-CAG-AADC (as a reporter gene) was intravenously injected into 3-month-old cynomolgus monkeys with low pre-existing neutralizing antibodies. Cells transduced by AAV were observed using antibody staining against AADC (shown in black). A magnified square area in the left image is shown in the right image. Both AAV.CPP.16 and AAV.CPP.21 transduced significantly more cells compared to AAV9.

[0042] Figures 11A-11B illustrate that AAV.CPP.16 and AAV.CPP.21 do not bind to LY6A. LY6A acts as a receptor for AAV.PHP.B, and its variants include AAV.PHP.eB (as described in US9102949, US20170166926), and mediate a strong transBBB effect of 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 increased the binding of AAV.PHP.eB to the cell surface (Figure 11A). Conversely, overexpression of LY6A did not increase the binding of AAV.PHP.eB to the cell surface.Viral binding of AAV9, AAV.CPP.16, or AAV.CPP.21 (Fig. 11B). This indicates that AAV.CPP.16 or AAV.CPP.21 does not share LY6A with AAV.PHP.eB as a receptor.

[0043] Figs. 12A-12C illustrate the use of AAV.CPP.21 for systemic delivery of therapeutic genes to brain tumors in a mouse model of glioblastoma (GBM). As shown in Fig. 12A, intravenously administered AAV.CPP.21-H2BmCherry is shown targeting the tumor mass, particularly the tumor expanding frontier. In Figs. 12B-12C, when combined with the prodrug ganciclovir, systemic delivery of the "suicide gene" HSV.TK1 using AAV.CPP.21 caused shrinkage of the brain tumor mass. HSV.TK1 converted the previously "dormant" ganciclovir into a tumor-killing drug. *P<0.05, Student's test.

[0044] Figure 13 illustrates that, compared to AAV9, AAV.CPP.21 elicits more extensive and potent brain tissue transduction when injected locally into the brain of adult mice. Intracerebral injection of AAV (1 × 10¹¹ 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

[0045] Difficulties associated with cross-BBB delivery have hindered the development of therapeutics for brain diseases, including cancer and neurodegenerative diseases. Adeno-associated viruses (AAVs) have become an important research and clinical tool for delivering therapeutic genes to the brain, spinal cord, and eyes; see, for example, US9102949; US 9585971; and US20170166926. However, existing AAVs, including AAV9, have limited cross-BBB efficiency or work only in certain non-primate species.

[0046] Through rational design and targeted screening based on known cell-penetrating peptides (CPPs) (see, for example, 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 AAVs, can increase gene delivery efficiency 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.

[0047] Targeting Sequences

[0048] The methods of the present invention identify a number of potential targeting peptides, such as those inserted into, for example, AAV1, AAV2,When AAV capsids such as AAV8 or AAV9 are used, or when conjugated chemically or by expression as a fusion protein with a biological agent such as an antibody or other large biomolecule, penetration through the BBB is enhanced. Specification 5 / 28 pages 8 CN 120843603 A

[0049] 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, for example 5, consecutive amino acids of the sequences VPALR (SEQ ID NO:1) and VSALRK (SEQ ID NO:2).

[0050] In some embodiments, the targeting peptide comprises the sequence X1X2X3X4X5, wherein:

[0051] (i) X1, X2, X3, X4 are any four different

[0052] amino acids of V, A, L, I, G, P, S, T, or M; and

[0053] (ii) X5 is K, R, H, D, or E (SEQ ID NO:73).

[0054] 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, for example 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).

[0055] In some embodiments, the targeting peptide comprises the sequence X1 X2 X3 X4 X5 X6, wherein:

[0056] (i) X1 is T;

[0057] (ii) X2, X3, X4, X5 are any four different amino acids of V, A, L, I, G, P, S, T, or M; and

[0058] (iii) X6 is K, R, H, D, or E (SEQ ID NO:74).

[0059] In some embodiments, the targeting peptide comprises the sequence X1X2X3X4X5X6, wherein:

[0060] (i) X1, X2, X3, X4 are any four different amino acids from V, A, L, I, G, P, S, T, or M;

[0061] (ii) X5 is K, R, H, D, or E; and

[0062] (iii) X6 is E or D (SEQ ID NO:75).

[0063] In some embodiments, the targeting peptide comprises a sequence of at least 7 amino acids. In some embodiments, the amino acid sequence comprises 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:75).At least four, for example five, six, or seven consecutive amino acids of (SEQ ID NO: 11). In some other embodiments, the targeting peptide comprises the sequence X1 X2 X3 X4 X5 X6 X7, wherein:

[0064] (i) X1 is F, L, W, or Y;

[0065] (ii) X2 is T;

[0066] (iii) X3, X4, X5, X6 are any four different amino acids of V, A, L, I, G, P, S, T, or M; and

[0067] (iv) X7 is K, R, H, D, or E (SEQ ID NO: 76).

[0068] In some embodiments, the targeting peptide comprises the sequence X1 X2 X3 X4 X5 X6 X7, wherein:

[0069] (i) X1 is T;

[0070] (ii) X2, X3, X4, X5 are any four different amino acids of V, A, L, I, G, P, S, T, or M;

[0071] (iii) X6 is K, R, H, D, or E; and

[0072] (iv) X7 is E or D (SEQ ID NO:77).

[0073] In some embodiments, the targeting peptide comprises the sequence X1X2X3X4X5X6X7, wherein:

[0074] (i) X1, X2, X3, X4 are any four different amino acids of V, A, L, I, G, P, S, T, or M;

[0075] (ii) X5 is K, R, H, D, or E;

[0076] (iii) X6 is E or D; and

[0077] (iv) X7 is A or I (SEQ ID NO: 78).

[0078] In some embodiments, the targeting peptide comprises the sequence V[S / p][A / m / t / ]L (SEQ ID NO: 79), wherein uppercase letters are preferred at that position. In some embodiments, the targeting peptide comprises the sequence TV[S / p][A / m / t / ]L (SEQ ID NO: 9 CN 120843603 A 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).

[0079] In some embodiments, the targeting peptide is not composed of VPALR (SEQ ID NO:1) or VSALRK (SEQ ID NO:2).

[0080] Table 1 lists specific exemplary amino acid sequences comprising the above-described 5, 6, or 7-amino acid sequences.

[0081] Table 1 - Targeting Sequences

[0082] Specification 7 / 28 Page 10 CN 120843603 A

[0083] Specification 8 / 28Page 11 CN 120843603 A

[0084]

[0085] Targeting peptides including reverse sequences may also be used, for example, KLASVT (SEQ ID NO:83) and KFLASVT (SEQ ID NO:84).

[0086] The targeting peptides disclosed herein may be modified according to methods known in the art for generating peptides. See, for example, Qvit et al., Drug Discov Today. Feb. 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, WM, ed., Peptidomimetics Protocols, Human Press (Totowa NJ 1998); Goodman et al., ed., 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 peptide-like forms of the peptides and fragments disclosed herein exhibit enhanced in vivo stability relative to non-peptide-like peptides.

[0087] Methods for generating peptide-like peptides include replacing one or more, for example, all amino acids in a peptide sequence with D-amino acid enantiomers. Such sequences are referred to herein as “retro” sequences. In another method, the N-terminal to C-terminal sequence of amino acid residues is reversed, such that the N-terminal to C-terminal sequence of the original peptide becomes the C-terminal to N-terminal sequence of the modified peptide-like peptide. Such sequences may be referred to as “inverso” sequences.

[0088] Peptides-like peptides can be in both retro and inverso forms, i.e., the “retro-inverso” form of the peptides disclosed herein. New peptide-like peptides may consist of D-amino acids arranged such that the N-terminal to C-terminal sequence of the peptide-like peptide corresponds to the C-terminal to N-terminal sequence of the original peptide.

[0089] Other methods for preparing peptides include using chemically different but recognized amino acid functional analogs, namely artificial amino acids.An acid analog replaces one or more amino acid residues in the peptide. Artificial amino acid analogs include β-amino acids, β-substituted β-amino acids (“β3-amino acids”), phosphorus-containing analogs of amino acids such as α-aminophosphonic acid and α-aminophosphinoic acid, and amino acids having non-peptide bonds. Peptides can be generated using artificial amino acids, such as peptide oligomers (e.g., peptide amides or ester analogs), β-peptides, cyclic peptides, oligoureas or oligocarbamate peptides; or heterocyclic molecules. Exemplary reverse-targeting peptides include KLASVT and KFLASVT, wherein the sequence comprises all D-amino acids. These sequences can be modified, for example, by biotinylation of the amino terminus and amidation of the carboxyl terminus.

[0090] AAV

[0091] Viral vectors used in the methods and compositions of the present invention include recombinant retroviruses, adenoviruses, adeno-associated viruses, alpha viruses, and lentiviruses, which include the targeting peptides described herein and optionally transgenes for expression in target tissues.

[0092] The preferred viral vector system for delivering nucleic acids in this method is adeno-associated virus (AAV). AAV is a tiny, non-enveloped virus with a 25 nm capsid. No disease is known or shown to be associated with wild-type viruses. AAV has a single-stranded DNA (ssDNA) genome. AAV has been shown to exhibit long-term free transgene expression, and AAV has demonstrated excellent transgene expression in the brain, particularly in neurons. Vectors containing as few as 300 base pairs of AAV can be packaged and integrated. The spatial restriction of the exogenous DNA is about 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. AAV vectors have been used to introduce various nucleic acids into different cell types (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. [Instructions 9 / 28 pages 12 CN 120843603 A] et al., J. Biol. Chem. 268:3781-3790 (1993). Many alternative AAV variants exist (over 100 have been cloned), and AAV variants have been identified based on desired characteristics. In some implementations, the AAV is AAV1, AAV2, AAV4, AAV5, AAV6, etc.AV6.2, AAV7, AAV8, AAV9, rh.10, rh.39, rh.43, or CSp3; for CNS use, in some embodiments, AAV is AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, or AAV9. As an example, AAV9 has been shown to be relatively efficient across the blood-brain barrier. Using the methods of the present invention, AAV capsids can be genetically engineered to increase penetration across the BBB or to increase penetration into specific tissues by inserting a targeting sequence as described herein into a capsid protein, for example, between amino acids 588 and 589 of the AAV9 capsid protein VP1.

[0093] An exemplary wild-type AAV9 capsid protein VP1 (Q6JC40-1) sequence is shown below:

[0094]

[0095] Therefore, this document provides AAVs comprising one or more of the target peptide sequences described herein, for example, AAVs comprising a capsid protein containing the target sequence described herein, said capsid protein being, for example, a capsid protein comprising, for example, SEQ ID NO:1 in which the target peptide sequence has been inserted into a sequence such as amino acids 588 and 589.

[0096] In some embodiments, the AAV further comprises a transgenic sequence (i.e., a heterologous sequence), for example, 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 a target tissue.

[0097] Exemplary transgenes used as therapeutic agents include neuronal apoptosis inhibitor protein (NAIP), nerve growth factor (NGF), glial cell-derived growth factor (GDNF), brain-derived growth factor (BDNF), ciliary neurotrophic factor (CNTF), tyrosine hydroxylase (TH), GTP-cyclohydrolase (GTPCH), amino acid decarboxylase (AADC), and aspartate acylase (ASPA). Blood factors, such as β-globin, hemoglobin, tissue plasminogen activator, and coagulation factors; colony-stimulating factor (CSF); interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, and IL-9; 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 factor (IGFs), bone morphogenetic protein (BMP), epidermal growth factor (EGF), growth differentiation factor-9 (GDF-9), hepatocellular carcinoma-derived growth factor (HDGF), and myostatin (GDF-8).Nerve growth factor (NGF), neurotrophic factors, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor α (TGF-α), transforming growth factor β (TGF-β), etc.; soluble receptors, such as soluble TNF-α receptors, soluble VEGF receptors, soluble interleukin receptors (e.g., soluble IL-1 receptors and soluble type II IL-1 receptors), soluble γ / δ T cell receptors, soluble receptor ligand-binding fragments, etc.; enzymes, such as α-glucosidase, imiglucosidase, β-glucocerebrosidase, and alglucerase; enzyme activators, such as tissue plasminogen activator; chemokines, such as IP-10, interferon-γ-induced mononuclear factor (Mig), Groa / IL-8, RANTES, MIP-1α, MIP-1β, MCP-1, etc. 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 receptors; protein vaccines; neuroactive peptides, such as nerve growth factor (NGF), bradykinin, cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasing hormone, β-endorphin, enkephalin, substance P, somatostatin, prolactin, glycopyrrolidone, growth hormone-releasing hormone, and serotonin. 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.; thrombolytic agents; atrial natriuretic peptide; relaxin; glial fibrillary acidic protein; follicle-stimulating hormone neuropeptide (FSH); human α-1 antitrypsin; leukemia inhibitory factor (LIF); transforming growth factor (TGFs); tissue factor; luteinizing hormone; macrophage activating factor; tumor necrosis factor (TNF); neutrophil chemotactic factor (NCF); nerve growth factor; tissue inhibitors of metalloproteinases; vasoactive intestinal peptide; angiopoietin; vascular nutrients; fibrin; hirudin; IL-1 receptor antagonists; etc. Other examples of target proteins include ciliary neurotrophic factor (CNTF); neurotrophin 3 and 4 / 5 (NT-3 and 4 / 5); glial cell-derived neurotrophic factor (GDNF); aromatic amino acid decarboxylases (AADCs); hemophilia-related clotting proteins, such as factor VIII, factor IX, and factor X; myoglobin or mini-myoglobin; lysosomal acid lipase; and phenylalanine.Aminohydroxylases (PAHs); enzymes associated with 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 transporters (e.g., GLUT2), aldolase A, β-enolase, and glycogen synthase; lysosomal enzymes (e.g., β-N-acetylhexosaminease A); and any variants thereof.

[0098] Transgenic genes can also encode antibodies, for example, those targeting 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 domain and mucin domain 3; HAVCR2); TIGIT (T-cell immune receptor with Ig and ITIM domains); B7-H3 (CD276); VSIR (V-set immunomodulatory receptor, also known as VISTA, B7H5, C10orf54); BTLA30 (B- and T-lymphocyte attenuation factor, CD272); GARP (glycoprotein A repetitions). Predominant); PVRIG (containing the PVR-associated immunoglobulin domain); or VTCN1 (containing the Vset domain of T-cell activation inhibitor 1, also known as B7-H4) immune checkpoint inhibitory antibody.

[0099] Other transgenes may include small or repressive nucleic acids that alter / depress the expression of the target gene, such as siRNA, shRNA, miRNA, antisense oligonucleotides, or long non-coding RNAs (see, for example, WO2012087983 and US20140142160), or CRISPR Cas9 / Cas12a and guide RNA.

[0100] The virus may also include one or more sequences that promote transgene expression, such as one or more promoter sequences; enhancer sequences, such as 5' untranslated region (UTR) or 3' UTR; polyadenylation sites; and / or insulator sequences. In some embodiments, the promoter is a brain tissue-specific promoter, such as a neuron-specific or glial-specific promoter. In some embodiments, the promoter is a promoter selected from the following genes: neuronal nucleus (NeuN), glial fibrillary acidic protein (GFAP), MeCP2, adenomatous polyposis (APC), ionized calcium-binding aptamer 1 (Iba-1), synaptic protein I (SYN), calcium / calmodulin-dependent protein kinase II, tubulin αI, neuron-specific enolase, and platelets.The promoter is a β-chain of cytomegalovirus. In some implementations, the promoter is a pancellular promoter, such as a cytomegalovirus (CMV), β-glucuronidase (GUSB), ubiquitin C (UBC), or Rous sarcoma virus (RSV) promoter. A post-transcriptional response element (WPRE) of marmot hepatitis virus may also be used.

[0101] In some embodiments, the AAV also has one or more additional mutations that increase delivery to target tissues such as the CNS, or decrease extra-tissue targeting, such as mutations that decrease liver delivery when delivery to the CNS, heart, or muscle is intended (e.g., as described in Pulicherla et al. (2011) Mol Ther 19:1070-1078); or add other targeting peptides, such as those 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

[0102] sfn.org / ~ / media / SfN / Documents / Short%20Courses / 2011%20Short%20Course%20I / 2011_SC1_Gray.ashx.

[0103] Targeting peptides as tags / fusions

[0104] The targeting peptides described herein can also be used, for example, by conjugating with molecules or by expressing, for example, antibodies or other large biomolecules as part of a fusion protein to increase the penetration of other (heterologous) molecules across the BBB. In addition to the therapeutic agents or reporter proteins described herein and those listed in Table 2, these may include genome editing proteins or complexes (e.g., TALE, ZFN, base editors, and CRISPR RNPs and guide RNAs comprising gene editing proteins such as Cas9 or Cas12a fused to peptides described herein (e.g., at the N-terminus, C-terminus, or internally). Fusions / complexes do not include any other sequences derived from Ku70, such as heterologous non-Ku70 sequences, and are not naturally occurring.

[0105] In some embodiments, the target sequence used as part of the non-AAV fusion protein does not include VPALR (SEQ ID NO:1) or VSALR (SEQ ID NO:2), or is not composed of VPALR (SEQ ID NO:1) or VSALR (SEQ ID NO:2).

[0106] Method of Use

[0107] 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 specific brain regions, such as the cortex, cerebellum, hippocampus, substantia nigra, amygdala. In some embodiments, the method includes delivery to neurons, astrocytes, glial cells, or cardiomyocytes.

[0108] In some embodiments, the method and compositions such as AAV are used to deliver nucleic acid sequences to subjects suffering from diseases such as CNS; see, for example, US9102949; US 9585971; and US20170166926. In some embodiments, the subject has the conditions listed in Table 2; in some embodiments, the vector is used to deliver the therapeutic agents listed in Table 2 to treat the corresponding diseases listed in Table 2. The therapeutic agents may be delivered, for example, via a viral vector as nucleic acid, the nucleic acid encoding a therapeutic protein or other nucleic acid, such as antisense oligonucleotides, siRNA, shRNA, etc., as described in CN 120843603 A on page 12 / 28 of its specification; or as a fusion protein / complex with a targeting peptide as described herein.

[0109] Table 2 - Diseases

[0110]

[0111] In some embodiments, the compositions and methods are used to treat brain cancer. Brain cancer includes gliomas (e.g., glioblastoma multiforme (GBM)), metastatic tumors (e.g., from lung cancer, breast cancer, melanoma, or colon cancer), meningiomas, pituitary adenomas, and acoustic neuromas. The composition comprises a targeting peptide linked to an anticancer 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. Sep 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), the targeting peptide being as described in this article.Immune checkpoint inhibitory antibodies known in the domain 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 turns the otherwise “dormant” ganciclovir into a tumor-killing drug. Thus, the method may include administering an AAV (e.g., AAV9) comprising a targeting peptide as described herein and encoding HSV.TK1 together with the prodrug ganciclovir, for example, systemically to a subject already diagnosed with brain cancer, such as intravenously.

[0112] Pharmaceutical Compositions and Methods of Administration

[0113] The methods described herein include pharmaceutical compositions using a targeting peptide as an active ingredient.

[0114] Pharmaceutical compositions generally comprise pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable carrier” includes saline, solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents and absorption delay agents, etc., that are compatible with drug administration.

[0115] Pharmaceutical compositions are typically formulated to be compatible with their 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, delivery can be systemic or local.

[0116] 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 Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions for parenteral administration may include the following components: sterile diluents, such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antimicrobial 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 agents for modulating muscle elasticity, such as sodium chloride or glucose. The pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0117] Pharmaceutical compositions suitable for injection may include sterile aqueous solutions (water-soluble) or dispersions, and sterile powders for the provisional preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL™ (BASF, Parsippany, NJ) phosphate-buffered saline (PBS). In all cases, this combinationThe material must be sterile and should flow easily for injection. It should be stable under production and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using coatings such as lecithin, in the case of dispersions by maintaining the desired particle size, and by using surfactants. Antimicrobial activity can be achieved using various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyols such as mannitol, sorbitol, and sodium chloride. Extended absorption of the injectable composition can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0118] A sterile injectable solution can be prepared by incorporating the desired amount of the active compound with one or a combination of the desired ingredients listed above into a suitable solvent, followed by filtration and sterilization. Typically, a dispersion is prepared by incorporating the active compound into a sterile medium comprising a basic dispersion medium and the desired other ingredients from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying, which yields powders of the active ingredient and any other desired ingredients from its previously sterile filtered solution.

[0119] In one embodiment, the therapeutic compound is prepared together with a carrier that protects the therapeutic compound from rapid elimination from the body, for example as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using standard techniques or are commercially available, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes targeting selected cells with monoclonal antibodies against cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0120] Pharmaceutical compositions can be included in a kit, container, package, or dispenser along with instructions for use. For example, a composition comprising an AAV containing a targeting peptide as described herein and a nucleic acid encoding HSV.TK1 can be provided in a kit along with ganciclovir. Instructions for Use 14 / 28 pages 17 CN 120843603 A

[0121] Examples

[0122] The invention is further described in the following embodiments, which do not limit the scope of the invention as set forth in the claims.

[0123] Materials and Methods

[0124] The following materials and methods are used in the following embodiments.

[0125] 1. Generation of Capsid Variants

[0126] In order to generate capsid variant plasmids, a DNA fragment (GenScript) encoding a cell-penetrating peptide (Table 3) was synthesized using the 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 number). 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, derived from the Ku70 protein, the sequence of which is as follows: Specification 15 / 28 pages 18 CN 120843603 A

[0127]

[0128] In addition, the VP1 protein sequences of AAV9, AAV.CPP.16 and AAV.CPP.21 are provided as follows: Specification 16 / 28 pages 19 CN 120843603 A

[0129]

[0130] 2. Generation of recombinant AAV

[0131] Recombinant AAV was packaged using a standard three-plasmid co-transfection protocol (pRC plasmid, pHelper plasmid and pAAV plasmid). pRC9 (or a variant thereof), pHelper, and transgenic pAAV (e.g., nuclear-directed RFP H2B-mCherry driven by the ubiquitous EF1a promoter, as per instructions, page 17 / 28, CN 120843603 A) were co-transfected into HEK 293T cells using polyethyleneimine (PEI, Polysciences). The rAAV vector was collected from serum-free medium at 72 h and 120 h post-transfection, and from cells at 120 h post-transfection. AAV particles in the medium were concentrated using PEG precipitation with 8% PEG-8000 (wt / vol). The cell pellet containing viral particles was resuspended and lysed by sonication. The viral vector derived from the combination of PEG precipitate and cell lysate was treated with DNase and RNase at 37°C for 30 min, followed by purification using an ultracentrifugation (VTi 50 rotor, 40,000 rpm, 18°C, 1 h) via an iodixanol gradient (15%, 25%, 40%, and 60%). The purified vector was then purified using a Millipore Amicon filter unit (UFC910008, 100K).rAAV was concentrated with MWCO and prepared in Duchenne phosphate-buffered saline (PBS) containing 0.001% Pluronic F68 (Gibco).

[0132] 3. AAV titer

[0133] Viral titer was determined by measuring the genomic copy number against DNase using quantitative PCR. pAAV-CAG-GFP was digested with PVUII (NEB) to generate the free ends of the plasmid ITR, which were used to generate a standard curve. Viral samples were incubated with DNase I to remove contaminating DNA, and then treated with sodium hydroxide to dissolve 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). The vector titer was normalized relative to the rAAV-2 reference standard material (RSMs, ATCC, catalog number: VR-1616, Manassas, VA).

[0134] 4. Administration of AAV in mice

[0135] For intravenous administration, AAV diluted in sterile saline (0.2 ml) was administered via tail vein injection in adult mice (over 6 weeks old). Animals were kept alive for three weeks and then euthanized for tissue harvesting. For intracerebral injection, AAV diluted in PBS (10 μl) was injected using a Hamilton syringe at coordinates from the anterior fontanelle: right 1.0 mm, posterior 0.3 mm, depth 2.6 mm. All animal studies were conducted in IALAC-approved AAALAC-accredited facilities.

[0136] 5. Mouse tissue processing

[0137] Anesthetized animals were perfused with cold phosphate-buffered saline (PBS), followed by 4% paraformaldehyde (PFA). Tissue was fixed overnight in 4% PFA, then soaked in 30% sucrose solution for two days, then embedded in OCT and flash-frozen. Typically, 80µm thick brain sections are cut for natural fluorescence imaging, and 40µm thick brain sections are used for IHC.

[0138] 6. In vitro human BBB spheroid model

[0139] Hot 1% agarose (w / v, 50µl) was added to 96-well plates for cooling / curing. Primary human astrocytes (Lonza Bioscience), human brain microvascular perivascular cells (HBVP, ScienCell Research Laboratories), and human brain microvascular endothelial cells (hCMEC / D3; Cedarlane) were then seeded into agarose gel at a 1:1:1 ratio (1500 cells of each type). The cells were incubated at 37°C in a 5% CO2 incubator for 48–72 hours to spontaneously assemble multicellular BBB spheroids. It has been reported that a multicellular barrier, mimicking the blood-brain barrier, was formed around the spheroids.AAV-H2B-mCherry was added to the culture medium, and after 4 days all spheroids were fixed with 4% PFA. The spheroids were then transferred to Nunc Lab-Tek II thin glass 8-well coverslips (Thermo Scientific) and imaged using a Zeiss LSM710 confocal microscope. The RFP signal intensity inside the spheroids was examined and used as a “reading”.

[0140] 7. AAV administration in non-human primates (NHP)

[0141] All NHP studies were performed by the CRO at an AAALAC-accredited facility approved by IACUC. Cynomolgus monkeys were pre-screened for little or no pre-existing neutralizing antibodies against AAV9 (titers <1:5). AAV diluted in PBS / 0.001% F68 was injected intravenously (via cephalic or femoral vein) using a peristaltic pump. After 3 weeks, the animals were perfused with PBS, followed by 4% PFA. Tissues were then collected and processed for paraffin embedding and sectioning.

[0142] 8. Immunohistochemistry manual, 18 / 28 pages, 21 CN 120843603 A

[0143] Mouse tissue sections were float-stained with primary antibodies diluted in PBS containing 10% donkey serum and 2% Triton X-100. The primary antibodies used included: 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); and mouse anti-CD31 (1:500). Secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 647 were applied to the host species of the primary antibody at a dilution of 1:200.

[0144] For paraffin sections of NHP tissue, DAB staining was performed to visualize cells transduced by AAV-AADC. Rabbit anti-AADC antibody (1:500, Millipore) was used as the primary antibody.

[0145] 9. AAV binding assay

[0146] HEK293T cells were incubated at 37°C in a 5% CO2 incubator. One day after seeding HEK293T cells into 24-well plates at a density of 250,000 cells per well, the cDNA plasmid of LY6A was transfected into the cells using a transfection mixture of 200 μL DMEM (31053028; Gibco), 1 μg DNA plasmid, and 3 μg PEI. Forty-eight hours after transfection, the cells were cooled on ice for 10 minutes. The medium was then replaced with 500 μL of ice-cold serum-free DMEM medium containing rAAV-mCherry with an MOI of 10,000. After incubation on ice for 1 hour, the cells suspected to be bound to AAV were washed three times with cold PBS, and then the AAV binding assay was performed.DNA was isolated from the genome. Viral particles binding to cells were quantified by qPCR using primers specific to mCherry and normalized to the HEK293T genome using human GCG as a reference.

[0147] 10. Mouse model of glioblastoma

[0148] All experiments were performed according to protocols approved by the Animal Conservation and Use Committee (IACUC) of Brigham and Women's Hospital and Harvard Medical School. Allogeneic immunized C57BL / 6 female mice weighing 20+ / -1g (Envigo) were used. GL261-Luc (100,000 mouse glioblastoma cells) resuspended in 2μL phosphate-buffered saline (PBS) was injected intracranially using a 10μl syringe (80075; Hamilton) with a 26-gauge needle. The implantation site (anterior fontanelle coordinates, in mm: right 2, anterior 0.5, cortical depth 3.5) was located using a stereotactic frame. After 7 days, 200 μL of AAV-HSV-TK1 (1E+12 viral genome, IV) was administered once, and ganciclovir (50 mg / kg) was administered daily for 10 days.

[0149] Example 1. Modification of AAV9 capsid

[0150] To identify peptide sequences that enhance the penetration of biomolecules or viruses across the blood-brain barrier, AAV peptide display technology was used. Single cell-penetrating peptides listed in Table 3 were inserted into the AAV9 capsid between amino acids 588 and 589 (VP1 number), as shown in Figure 1A. Insertion was performed by modifying the RC plasmid, one of three plasmids used for co-transfection for AAV packaging. Figure 1B shows an exemplary schematic diagram of the experiment. Individual AAV variants were generated and screened separately. For more details, see Materials and Methods #1-3.

[0151] Table 3 Specification 19 / 28 pages 22 CN 120843603 A

[0152]

[0153] #,SEQ ID NO:

[0154] Syn, Synthesis

[0155] Example 2. First round of in vivo screening

[0156] AAV expressing nuclear RFP (H2B-RFP) was intravenously injected into adult mice with mixed C57BL / 6 and BALB / c genetic backgrounds. After 3 weeks, brain tissue was harvested and sectioned to show RFP-labeled cells (white dots in Figures 2A and 2C, quantified in Figures 2B and 2D, respectively). CPP BIP1 and BIP2 were inserted into the capsids of AAV.CPP.11 and AAV.CPP.12, respectively. See Materials and Methods #4-5 for more details.

[0157] Example 3. Optimization of AAV9 capsid modification

[0158] AAV.CPP.11 and AAV.CPP.12 were further engineered by optimizing the BIP targeting sequence. The BIP insertion was derived from protein Ku70 (see Figure 3A and Materials / Methods #1 for the complete sequence). The BIP sequence VSALTK, chosen as the source of “synthesis,” was selected as the focus of the study to minimize the potential species specificity of the engineered AAV vectors. AAVs were generated and their brain transduction efficiency was tested, compared to AAV9 (see Figures 3B-3C). The percentage of cell transduction in mouse livers 3 weeks after IV injection of some AAV variants delivering the reporter gene RFP is shown in Figure 3D. For more details, see Materials and Methods #1-5.

[0159] Example 4. In vitro model - BBB penetration screening Instructions 20 / 28 pages 23 CN 120843603 A

[0160] The ability of some AAV variants to cross the human BBB was screened using an in vitro spheroid BBB model. The spheroids consist of 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 globular body and transduce internal cells was evaluated. Figure 4A shows a schematic diagram of the experiment. Figures 4B-D show the results for wt AAV9, AAV.CPP.16, and AAV.CPP.21, respectively, which, along with other peptides, are quantified in Figure 4E. In this model, peptides 11, 15, 16, and 21 produced the greatest penetration into the globular body. For more details, see Materials and Methods #6.

[0161] Example 5. In vivo BBB penetration screening

[0162] In the experiments performed 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. Following intravenous administration to adult C57BL / 6J mice (white dots in brain slices in Fig. 5A, quantified in Fig. 5B) and adult BALB / c mice (white dots in brain slices in Fig. 6A, quantified in Fig. 6B), both mice exhibited enhanced brain cell transduction capabilities relative to AAV9.

[0163] High doses of AAV.CPP.16 and AAV.CPP.21 (4 × 10¹² vg per mouse, IV administration) resulted in extensive brain transduction in mice. Both AAVs carry nuclear RFP as a reporter protein (white dots in brain slices in Fig. 7A, quantified in Fig. 7B).

[0164] Example 6. In vivo distribution of modified AAVs

[0165] As shown in Fig. 8A, AAV.CPP.16 and AAV.CPP.21 preferentially targeted neurons (labeled with NeuN antibody) across multiple brain regions in mice, including the cortex, midbrain, and hippocampus. Both AAVs carry nuclear RFP as a reporter protein.

[0166] AAV.CPP.16 and AAV.CPP.21 also showed enhanced targeting of spinal cord and motor neurons in mice relative to AAV9. All AAVs carried nuclear RFP as a reporter protein and were administered intravenously to newborn mice (4 × 10¹⁰ vg). Motor neurons were observed using CHAT antibody staining. In Figure 8B, colocalization of RFP and CHAT signals shows specific transduction of motor neurons.

[0167] The relative ability of AAV-CAG-H2B-RFP and AAV.CPP.16-CAG-H2B-RFP to transduce various tissues in mice was also evaluated. 1 × 10¹¹ vg was administered intravenously. The number of transduced cells was normalized to the total number of DAPI-labeled cells. The results showed that AAV.CPP.16 was more effective than AAV9 in targeting the heart (Figure 9A), skeletal muscle (Figure 9B), and dorsal root ganglia (Figure 9C) tissues in mice.

[0168] Example 7. BBB Penetration in a Non-Human Primate Model

[0169] Three-month-old cynomolgus monkeys were intravenously injected with 2 × 10¹³ vg / kg AAV-CAG-AADC (as a reporter gene). Cells transduced by AAV were observed using antibody staining against AADC (shown in black). As shown in Figures 10A-D, AAV.CPP.16 and AAV.CPP.21 showed enhanced transduction of brain cells relative to AAV9 after intravenous administration to non-human primates. AAV.CPP.16 transduced significantly more cells than wt AAV9 in the primary visual cortex (Figure 10A), parietal cortex (Figure 10B), thalamus (Figure 10C), and cerebellum (Figure 10D). For more details, see Materials and Methods #7-8.

[0170] Example 8. AAV.CPP.16 and AAV.CPP.21 do not bind to LY6A

[0171] LY6A acts as a receptor for AAV.PHP.eB and mediates a strong trans-BBB effect of AAV.PHP.eB 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 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 with AAV.PHP.eB as a receptor. For more details, see Materials and Methods #9.

[0172] Example 9. Delivery of therapeutic protein to the brain using AAV.CPP.21

[0173] AAV.CPP.21 was used to deliver the "suicide gene" HSV.TK1 systemically in a mouse model of brain tumors. HSV.TK1 delivers the originalThis "dormant" ganciclovir is transformed into a tumor-killing drug. Intravenous administration of AAV.CPP.21-H2BmCherry (Figure 12A, lower left and middle right images, 24 CN 120843603 A, 21 / 28 pages) showed targeting of the tumor mass, especially the tumor extension boundary. As shown in Figures 12B-12C, when combined with the prodrug ganciclovir, systemic delivery of the "suicide gene" HSV.TK1 using AAV.CPP.21 caused shrinkage of the brain tumor mass. These results suggest that AAV.CPP.21 can be used for systemic delivery of therapeutic genes to brain tumors. For more details, see Materials and Methods #10.

[0174] Example 10. Intracerebral administration of AAV.CPP.21

[0175] In addition to systemic administration (e.g., in Example 2), AAV was locally administered to the brain of mice as described herein. 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 brain slices treated with AAV.CPP.21 compared to brain slices treated with AAV9. See Materials and Methods #4 for more details.

[0176] Other Embodiments

[0177] It should be understood that although the invention has been described in conjunction with a 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.

[0178] Other aspects, advantages, and modifications are also within the scope of the appended claims.

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[0185] Specification page 28 / 28, 31 CN 120843603 A Figure 1A Figure 1B Specification Figure 1 / 33, 32 CN 120843603 A Figure 2A Specification Figure 2 / 33, 33 CN 120843603 A Figure 2B Instruction Manual Appendix 3 / 33 Page 34 CN 120843603 A Figure 2C Instruction Manual Appendix 4 / 33 Page 35 CN 120843603 A Figure 2D Instruction Manual Appendix 5 / 33 Page 36 CN120843603 A Figure 3A Instruction Manual Appendix 6 / 33 Page 37 CN 120843603 A Figure 3B Instruction Manual Appendix 7 / 33 Page 38 CN 120843603 A Figure 3C Figure 3D Instruction Manual Appendix 8 / 33 Page 39 CN 120843603 A Figure 4A Figure 4B Instruction Manual Appendix 9 / 33 Page 40 CN 120843603 A Figure 4C Figure 4D Instruction Manual Appendix 10 / 33 Page 41 CN 120843603 A Figure 4E Instruction Manual Appendix 11 / 33 Page 42 CN 120843603 A Figure 5A Instruction Manual Appendix 12 / 33 Page 43 CN 120843603 A Figure 5B Instruction Manual Appendix 13 / 33 Page 44 CN 120843603 A Figure 6A Instruction Manual Appendix 14 / 33 Page 45 CN 120843603 A Figure 6B Instruction Manual Drawing, Page 15 / 33 46 CN 120843603 A Figure 7A Instruction Manual Drawing, Page 16 / 33 47 CN 120843603 A Figure 7B Instruction Manual Drawing, Page 17 / 33 48 CN 120843603 A Figure 8A Instruction Manual Drawing, Page 18 / 33 49 CN 120843603 A Figure 8A Continued Instruction Manual Drawing, Page 19 / 33 50 CN 120843603 A Figure 8B Instruction Manual Drawing, Page 20 / 33 51 CN 120843603 A Figure 9A Instruction Manual Drawing, Page 21 / 33 52 CN 120843603 A Figure 9B Instruction Manual Drawing, Page 22 / 33 53 CN 120843603 A Figure 9C Instruction Manual Drawing, Page 23 / 33 54 CN 120843603 A Figure 10A Instruction Manual Appendix, Page 24 / 33, 55 CN 120843603 A Figure 10B Instruction Manual Appendix, Page 25 / 33, 56 CN 120843603 A Figure 10C Instruction Manual Appendix, Page 26 / 33, 57 CN 120843603 A Figure 10D Instruction Manual Appendix, Page 27 / 33, 58 CN 120843603 A Figure 11A Figure 11B Instruction Manual Appendix, Page 28 / 33, 59 CN 120843603 A Figure 12A Instruction Manual Appendix, Page 29 / 33, 60 CN 120843603 A Figure 12B DescriptionBook attached drawings, pages 30 / 33, 61 of CN 120843603 A, Figure 12C; Book attached drawings, pages 31 / 33, 62 of CN 120843603 A, Figure 13; Book attached drawings, pages 32 / 33, 63 of CN 120843603 A, Continued Figure 13; Book attached drawings, pages 33 / 33, 64 of CN 120843603 A, Abstract. Abdominal ultrasound examination method, system and device. Abstract: The present invention relates to methods and compositions for delivery of agents across the blood - brain barrier. The present invention is based on the development of artificial targeting sequences that enhance permeation of agents into cells and across the blood brain barrier, compositions comprising the sequences, and methods of use thereof. Provided herein is an AAV comprising a capsid protein comprising a targeting sequence and a transgene, preferably a therapeutic or diagnostic transgene. Further, provided herein are methods of delivering a transgene to a cell, the method comprising contacting the cell with an AAV or fusion protein described herein.

Claims

1. An adeno-associated virus (AAV) vector comprising an AAV capsid, wherein the AAV capsid contains a peptide intercalation of up to 21 amino acids, and wherein the peptide intercalation comprises 5-7 amino acids of TVSALFK (SEQ ID NO:8).

2. The AAV vector according to claim 1, wherein it comprises a transgenic sequence.

3. The AAV vector according to claim 2, wherein the transgenic sequence encodes a therapeutic agent.

4. The AAV vector according to claim 1, comprising non-coding RNA.

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

6. The AAV vector of claim 2, wherein the delivery of the transgenic sequence to an organ or tissue is enhanced compared to an AAV vector comprising an AAV capsid without the peptide insert and the transgenic sequence.

7. The AAV carrier according to claim 6, wherein the organ or tissue: (i) Includes permeability barriers, (ii) Including epithelium containing tight junctions, or (iii) is the brain or central nervous system.

8. The AAV vector of claim 4, wherein the delivery of the non-coding RNA to an organ or tissue is enhanced compared to an AAV vector comprising an AAV capsid without the peptide insert and the non-coding RNA.

9. The AAV carrier according to claim 8, wherein the organ or tissue: (i) Includes permeability barriers, (ii) Including epithelium containing tight junctions, or (iii) is the brain or central nervous system.

10. A composition comprising an AAV carrier according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

11. Use of the AAV vector according to any one of claims 1 to 9 in the preparation of a reagent for delivering transgenes to cells by means of contacting the cells with the AAV vector.

12. An adeno-associated virus (AAV) vector comprising an AAV capsid, wherein the AAV capsid contains a peptide insert of up to 21 amino acids, and wherein the peptide insert comprises 5 or 6 consecutive amino acids of TVSALK (SEQ ID NO:4).

13. The AAV vector according to claim 12, comprising a transgenic sequence.

14. The AAV vector of claim 13, wherein the transgenic sequence encodes a therapeutic agent.

15. The AAV vector of claim 12, comprising non-coding RNA.

16. The AAV vector according to claim 15, wherein the non-coding RNA is shRNA, siRNA, or miRNA.

17. The AAV vector of claim 13, wherein the delivery of the transgenic sequence to an organ or tissue is enhanced compared to an AAV vector comprising an AAV capsid without the peptide insert and the transgenic sequence.

18. The AAV carrier according to claim 17, wherein the organ or tissue: (i) Includes permeability barriers, (ii) Including epithelium containing tight junctions, or (iii) is the brain or central nervous system.

19. The AAV vector of claim 15, wherein the delivery of the non-coding RNA to an organ or tissue is enhanced compared to an AAV vector comprising an AAV capsid without the peptide insert and the non-coding RNA.

20. The AAV carrier of claim 19, wherein the organ or tissue: (i) Includes permeability barriers, (ii) Including epithelium containing tight junctions, or (iii) is the brain or central nervous system.

21. A composition comprising an AAV carrier according to any one of claims 12 to 20 and a pharmaceutically acceptable carrier.

22. Use of the AAV vector according to any one of claims 12 to 20 in the preparation of a reagent for delivering transgenes to cells by means of contacting the cells with the AAV vector.

23. Use of an adeno-associated virus (AAV) vector in the preparation of a reagent for delivering a transgene to a subject, the AAV vector comprising (i) a transgene, and (ii) an AAV capsid containing a peptide insert of up to 21 amino acids, wherein the peptide insert comprises 5-7 consecutive amino acids of TVSALFK (SEQ ID NO:8) or 5 or 6 consecutive amino acids of TVSALK (SEQ ID NO:4).

24. The use according to claim 23, wherein the transgene comprises a sequence encoding a therapeutic agent.

25. The use according to claim 23, wherein the delivery of the transgene to an organ or tissue is enhanced relative to an AAV vector comprising an AAV capsid without the peptide insert and the transgene.

26. The use according to claim 25, wherein the transgene is delivered to (i) Organs or tissues that include barriers, (ii) Including organs or tissues containing tightly connected epithelium, (iii) Brain or central nervous system (iv) Cortex, cerebellum, hippocampus, substantia nigra, thalamus, or amygdala, or (v) Neurons, astrocytes, glial cells, or cardiomyocytes.

27. The use according to any one of claims 23 to 26, wherein the subject has a neurodegenerative disease or cancer.

28. The use according to claim 27, wherein the neurodegenerative disease is Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, epilepsy, stroke, spinocerebellar ataxia, or Canavan disease.

29. The use according to claim 27, wherein the cancer is brain cancer.

30. The use according to any one of claims 23 to 26, wherein the transgene comprises non-coding RNA.

31. An AAV capsid protein comprising a targeting sequence, said targeting sequence comprising TVSALFK (SEQ ID NO:8); TVSALK (SEQ ID NO:4); KLASVT (SEQ ID NO:83); or KFLASVT (SEQ ID NO:84).

32. The AAV capsid protein according to claim 31, comprising an amino acid sequence including TVSALK (SEQ ID NO:4).

33. The AAV capsid protein according to claim 31, comprising an amino acid sequence including TVSALFK (SEQ ID NO:8).

34. The AAV capsid protein according to claim 31, comprising the amino acid sequence of SEQ ID NO:89 or SEQ ID NO:

90.

35. The AAV capsid protein of claim 31, comprising AAV9 VP1.

36. An AAV capsid protein comprising a targeting sequence comprising TV[S / p][A / m / t / ]L (SEQ ID NO: 80), wherein the targeting sequence is inserted at positions 588 and 589 corresponding to amino acids SEQ ID NO:

85.

37. The AAV capsid protein according to any one of claims 31 to 36, wherein the AAV is AAV9.

38. A nucleic acid encoding the AAV capsid protein according to any one of claims 31 to 36.

39. An AAV comprising the AAV capsid protein according to any one of claims 31 to 36.

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