Recombinant adeno-associated viruses and uses thereof

Abstract

To provide recombinant adeno-associated viruses (rAAVs) having capsid proteins engineered to include amino acid sequences that confer and / or enhance desired properties.SOLUTION: Provided are engineered capsid proteins comprising peptide insertions from heterologous proteins inserted within or near variable region IV (VR-IV) of virus capsid, such that the insertion is surface exposed on the AAV particle. The invention also provides capsid proteins that direct rAAVs to target tissues, in particular, capsid proteins comprising peptides derived from erythropoietin or dynein that are inserted into surface-exposed variable regions and target rAAVs to retinal tissue and / or neural tissue, including a central nervous system, and deliver therapeutics for treating neurological and / or eye disorders.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] 0. Sequence List This application contains a Sequence Listing which has been submitted electronically in ASCII format, the entirety of which is set forth below. The ASCII text created on March 29, 2020 is incorporated herein by reference. The file is named 38013_0002P1_SL.txt and is 637,86 It is 6 bytes.

[0002] The present invention relates to a method for the preparation of nucleic acid molecules that are modified to contain amino acid sequences that confer and / or enhance desired properties. In particular, the present invention relates to a recombinant adeno-associated virus (rAAV) having a modified capsid protein. The present invention relates to a method for the preparation of a nucleic acid sequence for a nucleic acid sequence comprising the steps of: (a) inserting a variable region of a viral capsid into an AAV particle such that the variable region is surface-exposed on the AAV particle; or, alternatively, in or near variable region IV (VR-IV) or, alternatively, in variable region VIII (VR-V III) a modified peptide insert from a heterologous protein inserted in or near The present invention also provides a capsid protein that induces rAAV to target tissues. Targeting rheumatoid proteins, particularly rAAV, to neural tissues including the retina and central nervous system; and or inserted into a surface-exposed variable region to improve transduction thereof, e.g., E. coli Capsid proteins containing peptides derived from lopoietin or dynein, and neurological disorders - Patents.com The present invention provides a delivery therapeutic agent for treating [Background technology]

[0003] The use of adeno-associated viruses (AAV) as gene delivery vectors has unmet medical needs. Dozens of naturally occurring AAV viruses are promising tools for treating many patient needs. AAV sequences have been reported in primate tissues, and surveys of the natural diversity of AAV sequences in primate tissues have revealed multiple We identified over 100 variants distributed across the clade of AAV. It is a single-stranded DNA virus with a relatively small genome and simple genetic components. AAV establishes latent infection without the aid of a helper virus. Inverted terminal repeats usually function as replication and packaging signals for vector generation. It contains the Rep gene and the Cap gene flanked by the ITRs. The capsid protein is The method comprises the steps of: carrying genomic DNA and determining tissue tropism for delivering DNA to target cells; It forms a capsid.

[0004] Due to their low pathogenicity and the promise of long-term targeted gene expression, recombinant AAV (rAAV) has In this study, therapeutic sequences were packaged into various capsids and used as gene transfer vectors. Such vectors have been used in preclinical gene therapy studies and include over 20 More than 100 gene therapy products are currently in clinical development. Recombinant AAVs, e.g., AAV9 have demonstrated desirable neurotropic properties and used recombinant AAV9 for the treatment of CNS diseases. Clinical trials are underway. However, there remains a need to improve the neurotropic properties of rAAV in human subjects. Attempts to raise the bar have had limited success.

[0005] For example, to deliver therapeutic agents in the treatment of disorders related to the central nervous system and the eye, particularly the retina. Vectors with Enhanced Neurotropic Properties for Use in Crossing the Blood-Brain Barrier for Immunotherapy - Patent application There remains a need for improved tissue-specific targeting for the delivery of therapeutic agents. There is also a need for rAAV vectors that have improved tissue-specific transduction and / or expression. Summary of the Invention

[0006] The desired properties, e.g., tissue targeting, transduction and / or integration of the rAAV genome, may be achieved by and / or improving the function of the capsid protein. In particular, the present invention provides a recombinant adeno-associated virus (rAAV) having an insert Variable region IV (VR-IV) of the viral capsid is surface-exposed on the AAV particle. or in or near variable region VIII (VR-VIII) Modified capsid proteins containing one or more peptide inserts from a heterologous protein. In certain embodiments, the insert comprises the AAV9 capsid protein (SEQ ID NO:1). 18, numbered in FIG. 8) This occurs immediately after the amino acid residue in the AVV9 capsid after amino acid 454 (i.e., between amino acids 454 and 455) or, in the capsid protein of different AAV types, between V9, including those following the residue corresponding to amino acid 454 (e.g., SEQ ID NOs: 110-117). 8 or 119-121), "corresponding" refers to the sequence alignment in FIG. For AAV types not included in the above list, the sequence of the AAV9 capsid protein (SEQ ID NO: 118) and and similar amino acids of AAV capsid proteins that will be well known in the art. It means that the sequences are aligned by sequence alignment. Therefore, the present invention is As numbered, the amino acid sequence corresponding to the amino acid residue at position 454 of AAV9 is Modified capsids containing a peptide insert from a heterologous protein inserted behind or near the capsid. In further specific embodiments, the insert comprises an AAV9 capsid protein. Amino acid sequence corresponding to amino acid 588 of protein (SEQ ID NO:118, as numbered in FIG. 8) immediately following residues (i.e., between amino acids 588 and 589), or in different AAV types in the capsid protein after the residue corresponding to amino acid 588 of AAV9 (e.g. For example, SEQ ID NOs: 110 to 117 or 119 to 121. The capsid protein is The capsid protein may be any AAV capsid protein, e.g., AAV plasminogen activator. AAV serotype 1 (SEQ ID NO: 110); AAV serotype 2 (SEQ ID NO: 111); AAV serotype 3 (SEQ ID NO: No. 112); AAV serotype 4 (SEQ ID NO: 113); AAV serotype 5 (SEQ ID NO: 114) 451 to 452 of AAV serotype 6 (SEQ ID NO: 115); AAV7 capsid (SEQ ID NO: 116) 461; 451 to 461 of AAV8 capsid (sequence number 117); AAV serotype 9 (sequence number No. 118; AAV serotype 9e (SEQ ID NO: 119); AAV serotype rh10 (SEQ ID NO: 120); AAV serotype rh20 (SEQ ID NO: 121); and AAV serotype hu.37 (SEQ ID NO: row number 122), AAV serotype rh39 (SEQ ID NO: 124), and AAV serotype rh74 (SEQ ID NO: 123 or SEQ ID NO: 154) (see FIG. 8).

[0007] Capsid proteins that direct rAAV to target tissues, particularly erythropoietin or dasminoids, are of peptides derived from dyneins (including axonemal or cytoplasmic dyneins) or the rAAV genome The present invention provides a method for the preparation of a cellular medicament comprising administering to a patient a medicament comprising administering to the patient a peptide that enhances tissue targeting and / or cellular uptake and / or integration. Also provided is a peptide that can be inserted into a surface-exposed variable region of an rAAV to nervous tissue, including the central nervous system, and retinal tissue to treat neurological and ophthalmic disorders. These peptides advantageously deliver therapeutic agents for the capsid protein to When incorporated into V particles, the capsid tyrosine kinase inhibitor was designed to make the inserted peptide surface-exposed. These peptides are inserted into the amino acid sequence of the AAV9 capsid (SEQ ID NO: No. 118 (see FIG. 8 for alignment) amino acids 262-273; 1; or immediately following or corresponding to one of the amino acids 585 to 593 or after one of the amino acids that correspond to the first codon encoding VP2. Acid residues, i.e., amino acid 138 of the AAV9 capsid and position 13 of the AAV9 capsid Inserted immediately after the amino acid corresponding to 8 (SEQ ID NO: 118, see FIG. 8 for alignment). In certain embodiments, the peptide inserted is peptide K of the axonemal dynein heavy chain. MQVPFQ (SEQ ID NO: 1); TLAAPFK (SEQ ID NO: 2); QQAAPSF (SEQ ID NO: No. 3); RYNAPFK (SEQ ID NO: 4); LKLPPIV (SEQ ID NO: 5); PFIKPF E (SEQ ID NO: 6); or at least four of one of TLSLPWK (SEQ ID NO: 7). or 5, 6, 7 or 8 consecutive amino acids thereof, or alternative Specifically, a non-linear epitope of erythropoietin, QEQLER, designated ARA290, was identified. 5, 6, 7, 8, 9, 10 or 11 consecutive amino acids of ALNSS (SEQ ID NO: 8). do.

[0008] and facilitating or increasing cellular uptake and / or integration of the rAAV genome. A modified capsid protein containing a peptide that targets a specific tissue, such as a peptide In one embodiment, the peptide is inserted into a surface-exposed variable region of the capsid protein. In this embodiment, the peptide is derived from bone (e.g., at least one of the sequences DDDDDDDD (SEQ ID NO: 9) at least 4 consecutive amino acids or at least 7 or 8 consecutive amino acids), brain (LSSRL At least 4 amino acids or at least 7 consecutive amino acids of DA (SEQ ID NO: 10) or 7 consecutive amino acids or 7, 8 or 9 consecutive amino acids), kidney (peptide CLPVASC (SEQ ID NO: 12) or LPVA At least 4 or 5 consecutive amino acids or peptides CLPVA of S (SEQ ID NO: 13) SC (SEQ ID NO: 12 or LPVAS (SEQ ID NO: 13)), muscle (ASSLNIA ( At least 4, 5, 6, or 7 consecutive amino acids of ASSLNIA (peptide of SEQ ID NO: 14), retinal cells (LGETTRP (SEQ ID NO: 15) or LA At least 4 consecutive amino acids or 5, 6, or 7 consecutive amino acids) in a cell for transduction or genomic integration, and / or or promotes, or transferrin receptor (HAIYPRH (SEQ ID NO: 17), TH RPPMWSPVWP (SEQ ID NO: 18), RTIGPSV (SEQ ID NO: 19), or CR At least 4 consecutive amino acids of TIGPSVC (SEQ ID NO: 20) or at least 7 or 7 consecutive amino acids). In certain embodiments, the peptide is CLPVASC (SEQ ID NO: 12) or is ASSLNIA (SEQ ID NO: 14) For example, the capsid of AAV9 contains this peptide inserted after position 454 The capsid preferentially targets capsid-bearing rAAV to the kidney compared to the liver. In other embodiments, the inserted peptide is at least 4 contiguous amino acids or Each has 7 or 8 consecutive amino acids, or the peptide SITLVKSTQTV (sequence 21) or TILSRSTQTG (sequence number 22) or QAVRTSL (sequence number SEQ ID NO: 23) or QAVRTSH (SEQ ID NO: 24). In another embodiment, the peptide is a sequence of 12 or fewer contiguous amino acids. Modified IgG antibodies having one or more amino acid substitutions that may improve or reduce immune neutralizing activity. Capsids are provided. Such amino acid modifications include A269S in AAV8, and other The corresponding substitutions in AAV-type capsids are S263F / S269T / A273 in AAV9 T, and the corresponding substitutions in other AAV capsids, W530R or Q4 in AAV9 74A, and corresponding substitutions in other AAV capsids. Capsids with substitutions are identified as NNN (asparagine) sequences at positions 498–500 of the AAV8 capsid. A substitution of AAA (alanine) at 496-498 of the AAV9 capsid Substitution of NNN (asparagine) with AAA (alanine) or other AAV capsids It may further have the corresponding substitution in:

[0009] Upon systemic, intravenous, intrathecal, intranasal, intraperitoneal, or intravitreal administration, one or more cells Modified capsids that enhance rAAV transduction in one or more tissues, including types A capsid protein is a surface exposed variable region (VR) of the capsid, e.g. For example, in VR-I, VR-IV or VR-VIII, or in the first amino acid of VP2 for example, immediately after residue 138 of the AAV9 capsid (amino acid sequence of SEQ ID NO: 118). or inserted immediately after the corresponding residues of another AAV capsid, or alternatively The peptides are modified with one or more of the amino acid substitutions described herein, and include at least one Transduction of AAV with modified capsids in various tissues was significantly higher than that of the corresponding unmodified capsids. The capsid protein is also increased upon administration compared to transduction of AAV with In certain embodiments, transduction is detected by detection of the transgene, such as GFP fluorescence. It is measured as follows.

[0010] In certain embodiments, the rAAV comprises a genome comprising a transgene of therapeutic interest. Provided herein is an rAAV incorporating a modified capsid as described herein. Packaging cells for producing the described rAAV are provided. Methods of treatment by delivery of modified rAAV and methods of treatment comprising the modified rAAV described herein Pharmaceutical compositions containing the rAAV having the modified capsid described herein are also provided. Methods of manufacture are also provided.

[0011] The present invention relates to human axonemal dynein heavy chain tails, ARA290, and other tissue-targeted or host-targeted polypeptides. The rAAV9 capsid and rAAV9 fusion proteins were modified with peptide inserts designed based on the fusion peptide. The construction of capsids modified with amino acid substitutions is illustrated by the examples set forth below.

[0012] 3.1. Implementation 1. At least 4 and at most 20 from a heterologous protein that is not a capsid protein Recombinant adeno-associated virus (rAAV) capsids containing peptide inserts of 10 consecutive amino acids The peptide insert is a capsid protein having the AAV9 capsid protein (sequence number 111111 of FIG. 8). Immediately following the amino acid residue corresponding to one of amino acids 451 to 461 in column 118) and when the capsid protein is packaged as an AAV particle, the peptide The recombinant adeno-associated virus (rAAV) capsid protein is a surface-exposed tide insert. Stuffy.

[0013] 2. The capsid protein is AAV serotype 1 (AAV1), serotype 2 (AAV2) , serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (A AV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8) ), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rh10 (AAVrh1 0), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype At least one of the serotypes hu.37 (AAVhu.37) or rh74 (AAVrh74) 2. The rAAV capsid protein of embodiment 1, which is from at least one AAV serotype. Stuffy.

[0014] 3. The peptide insert is in the sequence shown in FIG. 450 to 459 of the AAV1 capsid amino acid sequence (SEQ ID NO:110); 449 to 458 of the AAV2 capsid amino acid sequence (SEQ ID NO:111); 449 to 459 of the AAV3 capsid amino acid sequence (SEQ ID NO:112); 443 to 453 of the AAV4 capsid amino acid sequence (SEQ ID NO: 113); 442 to 445 of the AAV5 capsid amino acid sequence (SEQ ID NO:114); 450 to 459 of the AAV6 capsid amino acid sequence (SEQ ID NO: 115); 451 to 461 of the AAV7 capsid amino acid sequence (SEQ ID NO: 116); 451 to 461 of the AAV8 capsid amino acid sequence (SEQ ID NO: 117); 451 to 461 of the AAV9 capsid amino acid sequence (SEQ ID NO:118); 452 to 461 of the AAV9e capsid amino acid sequence (SEQ ID NO:119); 452 to 461 of the AAVrh10 capsid amino acid sequence (SEQ ID NO: 120); 452 to 461 of the AAVrh20 capsid amino acid sequence (SEQ ID NO:121); 452 to 461 of the AAVhu.37 capsid amino acid sequence (SEQ ID NO:122); 45 of the AAVrh74 capsid amino acid sequence (SEQ ID NO: 123 or SEQ ID NO: 154) 2~461; or 452 to 461 of the AAVrh39 capsid amino acid sequence (SEQ ID NO: 124) The rAAV capsid of embodiment 2 is provided immediately following one of the amino acid residues in High protein.

[0015] 4. The peptide inserts are aligned in FIG. 8 and are numbered according to their amino acid numbering. The amino acid residues I451, N452, G453, S454, and G45 of the AAV9 capsid 5, Q456, N457, Q458, Q459, T460, or L461 or immediately following amino acids I451, N452, G of the AAV9 capsid (SEQ ID NO: 118). 453, S454, G455, Q456, N457, Q458, Q459, T460, or L461, 3. The rAAV capsid protein of embodiment 3.

[0016] 5. The heterologous protein is a homing domain, a neutralizing antibody epitope, or a purified tag. The rAAV capsid protein of any preceding embodiment,

[0017] 6. The homing domain comprises: Neural tissue homing domain; axonemal or cytoplasmic dynein homing domains; bone homing domain; kidney-homing domain; muscle homing domain; endothelial cell homing domain; integrin receptor binding domain; transferrin receptor binding domain; a tumor cell targeting domain; or Retinal cell homing domain 6. The rAAV capsid protein of embodiment 5,

[0018] 7. The peptide insert comprises the amino acid sequence SITLVKSTQTV (SEQ ID NO:21), TILSRSTQTG (SEQ ID NO: 22), VVMVGEKPITITQHSVETEG ( SEQ ID NO: 25), RSSEEDKSTQTT (SEQ ID NO: 26), KMQVPFQ (SEQ ID NO: No. 1), LKLPPIV (SEQ ID NO: 5), PFIKPFE (SEQ ID NO: 6), TLSLPW K (SEQ ID NO: 7), QQAAPSF (SEQ ID NO: 3), RYNAPFK (SEQ ID NO: 4), T LAVPFK (SEQ ID NO: 27), TLAAPFK (SEQ ID NO: 2), LGETTRP (SEQ ID NO: No. 15), or at least four or less of LALGETTRP (SEQ ID NO: 16) each of which contains a dynein peptide or a dynein homing peptide of seven consecutive amino acids; 7. The rAAV capsid protein of embodiment 6, comprising:

[0019] 8. The peptide insert from the transferrin receptor binding domain comprises the amino acid At least one of the sequences RTIGPSV (SEQ ID NO: 19) or CRTIGPSVC (SEQ ID NO: 20) 7. The rAAV gene of embodiment 6, comprising at least 4 or at least 7 consecutive amino acids. Psid protein.

[0020] 9. The peptide insert from the retinal cell homing domain comprises the amino acid sequence LG Embodiments including ETTRP (SEQ ID NO: 15) or LALGETTRP (SEQ ID NO: 16) 7. The rAAV capsid protein according to embodiment 6.

[0021] 10. The LGETTRP (SEQ ID NO: 15) or LALGETTRP (SEQ ID NO: 16) ) The peptide insert is inserted into the AAV8 capsid protein or the AAV9 capsid protein. 10. The rAAV capsid protein of embodiment 9, which is present in the protein.

[0022] 11. The peptide insert is aligned in FIG. 8 and is numbered according to amino acid numbering. , wherein the AAV9 capsid protein (SEQ ID NO: 11 8) after amino acid S454 of the AAV9 capsid protein or

[0023] In any of the preceding embodiments, rAAV capsid proteins as described.

[0023] 12. The above method, wherein the capsid protein is not the AAV2 capsid protein. The rAAV capsid protein of any of the embodiments.

[0024] 13. Encoding the rAAV capsid protein according to any of the above embodiments, or a nucleotide sequence encoding an amino acid sequence sharing at least 80% identity therewith. A nucleic acid comprising a nucleotide sequence.

[0025] 14. The AA comprising the capsid protein encoded by the nucleotide sequence A packaged vector capable of expressing the nucleic acid of embodiment 13 to produce a V vector. Zing cells.

[0026] 15. An rAAV vector comprising a capsid protein according to any one of embodiments 1 to 12. -

[0027] 16. The rAAV vector of embodiment 15, further comprising a transgene.

[0028] 17. The rAAV vector of embodiment 15 or 16 and a pharma- ceutically acceptable carrier 23. A pharmaceutical composition comprising:

[0029] 18. A method for delivering a transgene to a cell, the method comprising: 6. The method of claim 5, wherein the transgene is delivered to the cell. The method is achieved.

[0030] 19. The transgene is delivered to a target tissue or target cell or cells thereof in a subject in need thereof. 17. A method of delivering a cell matrix to a subject, the method comprising the steps of: wherein the transgene is administered to the target tissue of the subject. The method of claim 1, wherein the

[0031] 20. A pharmaceutical composition for use in delivering a transgene to a cell, comprising: The composition comprises the rAAV vector of embodiment 16, and the cells are transfected with the vector. The pharmaceutical composition.

[0032] 21. For use in delivering a transgene to a target tissue in a subject in need thereof. A pharmaceutical composition comprising the rAAV vector of embodiment 16. wherein the peptide insert is a homing peptide, and the vector is The pharmaceutical composition is administered.

[0033] 22. The rAAV vector is administered systemically, intravenously, intrathecally, intranasally, or intraperitoneally. or for use according to any one of claims 18 to 21, wherein the composition is administered intravitreally or intraperitoneally. Pharmaceutical compositions.

[0034] 23. The target tissue, or the target cell or its cell matrix, and the vector is a peptide derived from the neural tissue homing domain. Includes inserts, and the vector comprises the peptide insert from the bone-homing domain. fruit, the kidney, and the vector comprises the peptide insert from the kidney-homing domain. Including, muscle, and the vector comprises the peptide insert from the muscle-homing domain. Including, the vector is an endothelial cell, the peptide from the endothelial cell homing domain Includes inserts, a cell expressing an integrin receptor, the vector comprising said peptide insert from the binding domain, a tumor cell expressing a transferrin receptor, comprising said peptide insert from a cytochrome P450 receptor binding domain, a tumor cell, the vector comprising a peptide insert from the tumor cell targeting domain. Contains, or a retinal cell, and the vector comprises a peptide from the retinal cell homing domain. Insert The method or pharmaceutical composition for use according to any one of embodiments 18 to 21, comprising:

[0035] 24. A recombinant adeno-associated virus (rAAV) capsid protein, comprising: The cytoprotein is A neural tissue homing protein or domain (provided that the peptide insert has the sequence TLA does not contain VPFK (SEQ ID NO:27); axonemal or cytoplasmic dynein homing domains; bone homing domain; kidney-homing domain; muscle homing domain; endothelial cell homing domain; integrin receptor binding domain; Transferrin receptor binding domain (wherein the peptide insert has the sequence RTIGPSV (SEQ ID NO:19) or CRTIGPSVC (SEQ ID NO:20); A tumor cell targeting domain; and Retinal cell homing domain (wherein the peptide insert is the sequence LGETTRP (SEQ ID NO: No. 15) nor LALGETTRP (SEQ ID NO: 16) At least four and up to four heterologous proteins or domains selected from the group consisting of containing a peptide insert of 20 consecutive amino acids at When the capsid protein is packaged into an AAV particle, the peptide The recombinant adeno-associated virus (rAAV) capsid protein, wherein the insert is surface exposed. quality.

[0036] 25. The neural tissue homing protein or retinal cell homing domain is human 25. The rAAV capsid protein of embodiment 24, which is an axonemal dynein (HAD) heavy chain tail. Quality.

[0037] 26. The peptide insert comprises at least one amino acid sequence from the dimerization domain of the HAD heavy chain tail. 26. The rAAV capsid of embodiment 25, comprising 4 and up to 12 consecutive amino acids. Compact quality.

[0038] 27. The peptide insert is (as shown in Figures 7A-7M): (DYH1_HUMAN UniProtKB-Q9P2D7 aa1-1542)( SEQ ID NO:97); (DYH2_HUMAN UniProtKB-Q9P225 aa1-1764)( SEQ ID NO:98); (aa1-1390 of DYH3_HUMAN UniProtKB-Q8TD57)( SEQ ID NO:99); (DYH5_HUMAN UniProtKB-Q8TE73 aa1-1941)( sequence number 100); (aa1 to 1433 of DYH6_HUMAN UniProtKB-Q9C0G6)( SEQ ID NO:101); (DYH7_HUMAN UniProtKB-Q8WXX0 aa1-1289)( SEQ ID NO:102); (DYH8_HUMAN UniProtKB-Q96JB1 aa1-1807)( SEQ ID NO:103); (DYH9_HUMAN UniProtKB-Q9NYC9 aa1-1831)( SEQ ID NO:104); (aa1 to 1793 of DYH10_HUMAN UniProtKB-Q8IVF4) (SEQ ID NO:105); (aa1 to 1854 of DYH11_HUMAN UniProtKB-Q96DT5) (SEQ ID NO:106); (aa1 to 1214 of DYH12_HUMAN UniProtKB-Q6ZR08) (SEQ ID NO:107); (DYH14_HUMAN UniProtKB-Q0VDD8 aa1-200)( SEQ ID NO:108); or (aa1-1794 of DYH17_HUMAN UniProtKB-Q9UFH2) (SEQ ID NO:109)

[0036] Embodiment 26, which comprises at least 4 and at most 12 consecutive amino acids from the group consisting of: The rAAV capsid protein described in.

[0039] 28. The peptide insert comprises any one of the axonemal dynein heavy chain sequences (Figures 7A-7M). In one embodiment, the amino acid sequence includes at least 4 and at most 12 consecutive amino acids from residues 1 to 200 of 28. The rAAV capsid protein of embodiment 27.

[0040] 29. The peptide insert is any one of the axonemal dynein heavy chain sequences of Figures 7A-7M. 28. The rAAV capsid protein of embodiment 27, comprising one to seven consecutive amino acids. .

[0041] 30. The peptide insert comprises any one of the dynein heavy chain sequences (Figures 7A-7M). 29. The rAAV encapsulation method of embodiment 28, comprising 7 consecutive amino acids from residues 1 to 200 of Side protein.

[0042] 31. The peptide insert is KMQVPFQ(SEQ ID NO:1); TLAAPFK (SEQ ID NO:2); QQAAPSF (SEQ ID NO:3); RYNAPFK (SEQ ID NO:4); LKLPPIV (SEQ ID NO:5); PFIKPFE (SEQ ID NO:6); or TLSLPWK (SEQ ID NO: 7) 26. The rAAV fragment of embodiment 25, comprising at least four consecutive amino acids of one of Psid protein.

[0043] 32. The peptide insert is KMQVPFQ(SEQ ID NO:1); TLAAPFK (SEQ ID NO:2); QQAAPSF (SEQ ID NO:3); RYNAPFK (SEQ ID NO:4); LKLPPIV (SEQ ID NO:5); PFIKPFE (SEQ ID NO:6); or TLSLPWK (SEQ ID NO: 7) 26. The rAAV capsid protein of embodiment 25, comprising a peptide from one of quality.

[0044] 33. The peptide insert comprises the amino acid sequence TLAAPFK (SEQ ID NO: 2). 33. The rAAV capsid protein of embodiment 32.

[0045] 34. The neural tissue homing protein is a mouse axonemal dynein (MAD) heavy chain tail. 25. The rAAV capsid protein of embodiment 24, wherein

[0046] 35. The neural tissue homing domain binds to a natural repair receptor and inhibits erythropoiesis. A non-specific EPO (erythropoietin) domain or a conformation of said domain 25. The rAAV capsid protein of embodiment 24, which is an analog of

[0047] 36. The peptide insert comprises at least one sequence selected from the group consisting of QEQLERALNSS (SEQ ID NO: 8). 36. The rAAV capsid of embodiment 35, comprising at least 4 and at most 11 consecutive amino acids. protein.

[0048] 37. The peptide insert comprises the ARA290 sequence QEQLERALNSS (SEQ ID NO:8). 37. The rAAV capsid protein of embodiment 36, wherein the rAAV capsid protein is

[0049] 38. The neural tissue homing protein is SRL (serine-arginine-lysine). 25. The rAAV capsid of embodiment 24, which is a brain-homing domain having a motif. protein.

[0050] 39. The peptide insert from the brain-homing domain has the amino acid sequence LSSR At least seven of LDA (SEQ ID NO: 10) or CLSSRLDAC (SEQ ID NO: 11) The rAAV capsid protein of embodiment 38, comprising consecutive amino acids.

[0051] 40. The axonemal or cytoplasmic dynein homing domain is a dynein light chain homing domain. 25. The rAAV capsid protein of embodiment 24, which is a domain.

[0052] 41. The peptide insert from the dynein light chain homing domain is SITLV KSTQTV (SEQ ID NO: 21), TILSRSTQTG (SEQ ID NO: 22), VVMVGE KPITITQHSVETEG (SEQ ID NO: 25), or RSSEEDKSTQTT (SEQ ID NO: The rAAV capsid protein of embodiment 40, which is one of the following:

[0053] 42. The bone-homing protein has a hydroxyapatite (HA) binding domain. 25. An rAAV capsid protein as described in embodiment 24.

[0054] 43. The peptide insert from the hydroxyapatite (HA) binding domain is at least six amino acid residues of the sequence DDDDDDDD (SEQ ID NO:9), 42. The rAAV capsid protein described in

[0055] 44. The kidney-homing domain has the amino acid sequence CLPVASC (SEQ ID NO: 12). 25. The rAAV capsid protein of embodiment 24, wherein

[0056] 45. The peptide insert from the kidney homing domain comprises the amino acid sequence LPV AS (SEQ ID NO: 13) or CLPVASC (SEQ ID NO: 12), The rAAV capsid protein is shown.

[0057] 46. ​​The peptide insert from the muscle-homing domain comprises the amino acid sequence ASS. The rAAV capsid protein of embodiment 24, which is LNIA (sequence number 14).

[0058] 47. The peptide insert has the amino acid sequence QAVRTSL (SEQ ID NO: 23) or Q 25. The rAAV capsid protein of embodiment 24, which is AVRTSH (SEQ ID NO: 24). quality.

[0059] 48. The peptide insert from the endothelial cell homing domain comprises the amino acid sequence S 25. The rAAV capsid protein of embodiment 24, which is IGYPLP (SEQ ID NO: 28). quality.

[0060] 49. The peptide insert from the integrin binding domain has the amino acid sequence CD The rAAV capsid protein according to embodiment 24, having CRGDCFC (SEQ ID NO: 29). Stuffy.

[0061] 50. The transferrin receptor binding domain is a transferrin domain, if or a conformational analog thereof, or an iron mimetic thereof. AAV capsid protein.

[0062] 51. The peptide insert from the transferrin domain has the sequence HAIYPR H (SEQ ID NO: 17) or THRPPMWSPVWP (SEQ ID NO: 18) rA according to embodiment 50, comprising 4 consecutive amino acids and up to 12 consecutive amino acids. AV capsid protein.

[0063] 52. The peptide insert comprises the amino acid sequence HAIYPRH (SEQ ID NO: 17) or T The rAAV capsid of embodiment 51, which is HRPPMWSPVWP (SEQ ID NO: 18). High protein.

[0064] 53. The peptide insert from the tumor cell targeting domain comprises the amino acid sequence NGR The rAAV capsid protein of embodiment 24, which is AHA (sequence number 30).

[0065] 54. The peptide insert comprises the amino acid residues (shown in FIG. 8): AAV1 capsid amino acid sequence (SEQ ID NO: 110) 138;262-272;450 ~459; or 585-593; 138;262-272;449 of the AAV2 capsid amino acid sequence (SEQ ID NO:111) ~458; or 584-592; 138;262-272;449 of the AAV3 capsid amino acid sequence (SEQ ID NO:112) ~459; or 585-593; AAV4 capsid amino acid sequence (SEQ ID NO: 113) 137;256-262;443 ~453; or 583~591; AAV5 capsid amino acid sequence (SEQ ID NO: 114) 137;252-262;442 ~445; or 574-582; AAV6 capsid amino acid sequence (SEQ ID NO: 115) 138;262-272;450 ~459;585~593; 138;263-273;451 of the AAV7 capsid amino acid sequence (SEQ ID NO:116) ~461;586~594; AAV8 capsid amino acid sequence (SEQ ID NO: 117) 138;263-274;452 ~461;587~595; AAV9 capsid amino acid sequence (SEQ ID NO: 118) 138;262-273;452 ~461;585~593; 138;262-273;45 of the AAV9e capsid amino acid sequence (SEQ ID NO:119) 2-461;585-593; 138; 263-274 of the AAVrh10 capsid amino acid sequence (SEQ ID NO: 120); 452-461;587-595; 138; 263-274 of the AAVrh20 capsid amino acid sequence (SEQ ID NO: 121); 452-461;587-595; 138; 263-274 of the AAVhu37 capsid amino acid sequence (SEQ ID NO: 122); 452-461;587-595; 13 of the AAVrh74 capsid amino acid sequence (SEQ ID NO: 123 or SEQ ID NO: 154) 8;263-274;452-461;587-595;or 138; 263-274 of the AAVrh39 capsid amino acid sequence (SEQ ID NO:124); 452~461;587~595 The rAAV capsid of any one of embodiments 24 to 53, which is immediately followed by one of High protein.

[0066] 55. Between amino acid residues 588 to 589 of the AAV9 capsid (SEQ ID NO: 118). or the amino acid residues of the AAV9 capsid as aligned in FIG. The amino acid sequence TLAAPFK (SEQ ID NO: 2) inserted correspondingly between 588 and 589 A recombinant AAV capsid protein comprising:

[0067] 56. Amino acids I451 to L461 of the AAV9 capsid (SEQ ID NO: 118) or Immediately after one of S268 or as aligned in FIG. Inserted corresponding to one of the amino acids I451 to L461 or S268 of the AV9 capsid. A recombinant AAV capsid protein containing the inserted amino acid sequence TLAAPFK (SEQ ID NO: 2) Quality.

[0068] 57. Between amino acid residues 588 to 589 of the AAV9 capsid (SEQ ID NO: 118). or the amino acid residues of the AAV9 capsid as aligned in FIG. The amino acid sequence QEQLERALNSS (SEQ ID NO: 588-589) was inserted correspondingly between Recombinant AAV capsid protein containing the ribozyme (No. 8).

[0069] 58. Amino acids I451 to L461 of the AAV9 capsid (SEQ ID NO: 118) or Immediately after one of S268 or as aligned in FIG. Inserted corresponding to one of the amino acids I451 to L461 or S268 of the AV9 capsid. A recombinant AAV capsid containing the inserted amino acid sequence QEQLERALNSS (SEQ ID NO: 8) High protein.

[0070] 59. The peptide insert is AAV9, as aligned in FIG. Amino acids 451 to 461, S268 or Q5 of the capsid protein (SEQ ID NO: 118) 88 immediately following an amino acid residue corresponding to any one of embodiments 24 to 54. The rAAV capsid protein described herein.

[0071] 60. The peptide insert is the AAV9 capsid protein (SEQ ID NO: 118) 59. The rAAV of embodiment 59, wherein the rAAV is located immediately after one of amino acids 451 to 461. Capsid protein.

[0072] 61. The peptide insertion is present in the eighth variable region (VR-VIII). An rAAV capsid protein according to any one of embodiments 24 to 53.

[0073] 62. The capsid protein is not the AAV2 capsid protein, 62. The rAAV capsid protein according to any one of embodiments 24 to 61.

[0074] 63. A vector encoding an rAAV capsid protein according to any one of embodiments 24 to 62. or a nucleic acid encoding an amino acid sequence that shares at least 80% identity therewith. A nucleic acid comprising an octide sequence.

[0075] 64. The AA comprising the capsid protein encoded by the nucleotide sequence A packaged vector capable of expressing the nucleic acid of embodiment 63 to produce a V vector. Zing cells.

[0076] 65. A rAAV vector comprising a capsid protein according to any one of embodiments 24 to 62. Tha.

[0077] 66. The rAAV vector of embodiment 65, further comprising a transgene.

[0078] 67. The rAAV vector according to embodiment 65 or 66 and a pharma- ceutically acceptable carrier 23. A pharmaceutical composition comprising:

[0079] 68. A method for delivering a transgene to a cell, the method comprising: 6. The method of claim 5, wherein the transgene is delivered to the cell. The method is achieved.

[0080] 69. A method for delivering a transgene to a target tissue in a subject in need thereof, comprising: The method comprises administering to the subject the rAAV vector of embodiment 66, The method, wherein a transgene is delivered to the subject.

[0081] 70. A pharmaceutical composition for use in delivering a transgene to a cell, comprising: The biological composition comprises the rAAV vector of embodiment 66, wherein the transgene is The pharmaceutical composition is delivered to a cell.

[0082] 71. For use in delivering a transgene to a target tissue in a subject in need thereof. A pharmaceutical composition comprising the rAAV vector of embodiment 66. wherein a transgene is delivered to the target tissue.

[0083] 72. The rAAV vector is administered systemically, intravenously, intrathecally, intranasally, or intraperitoneally. or for use according to any one of embodiments 68 to 71, wherein the composition is administered intravitreally or intraperitoneally. Pharmaceutical compositions.

[0084] 73. Embodiment 6, in which the vector is administered via lumbar puncture or via the cisterna magna. 8 to 71, or a pharmaceutical composition for use according to the method.

[0085] 74. A recombinant adeno-associated virus (rAAV) capsid protein, comprising: The cid proteins are TLAVPFK (SEQ ID NO: 27), RTIGPSV (SEQ ID NO: 19). , CRTIGPSVC (SEQ ID NO: 20), LGETTRP (SEQ ID NO: 15), or LA A pair of at least four consecutive amino acids from one of the sequences LGETTRP (SEQ ID NO: 16). including a peptide insert,

[0086] The peptide insert is aligned in the AAV9 capsid as shown in FIG. The amino acid residues immediately following those corresponding to amino acids 268, 454, or 588 of the endoprotein The recombinant adeno-associated virus (rAAV) capsid protein,

[0087] 75. The capsid protein is not the AAV2 capsid protein. 75. The rAAV capsid protein of embodiment 74.

[0088] 76. The peptide insert is the AAV9 capsid protein (SEQ ID NO: 118) Contains the amino acid sequence TLAVPFK (SEQ ID NO:27) between amino acid residues 454 and 455. 75. The rAAV capsid protein of embodiment 74.

[0089] 77. The peptide insert is the AAV9 capsid protein (SEQ ID NO: 118) Immediately following one of the amino acid residues 262 to 273 is the amino acid sequence TLAVPFK (SEQ ID NO: 75. The rAAV capsid protein of embodiment 74, comprising:

[0090] 78. The peptide insert is an amino acid sequence of the AAV8 capsid protein (SEQ ID NO: 117). The present invention relates to a method for producing a recombinant human ovarian cancer comprising the steps of: The rAAV capsid protein of embodiment 74.

[0091] 79. The peptide insert comprises the amino acid sequence LALGETTRP (SEQ ID NO: 16). 79. The rAAV capsid protein of embodiment 78.

[0092] 80. The peptide insert is the AAV8 capsid protein (SEQ ID NO: 117) The amino acid sequence LGETTRP (SEQ ID NO: 1) was inserted between amino acid residues 590 and 591. 5). The rAAV capsid protein of embodiment 74, comprising:

[0093] 81. The peptide insert comprises the amino acid sequence LALGETTRP (SEQ ID NO: 16). 81. The rAAV capsid protein of embodiment 80.

[0094] 82. The peptide insert is the AAV8 capsid protein (SEQ ID NO: 117) Immediately following amino acid residues 263 to 274, the amino acid sequence LGETTRP (SEQ ID NO: 15) is included. 75. The rAAV capsid protein of embodiment 74.

[0095] 83. The peptide insert comprises the amino acid sequence LALGETTRP (SEQ ID NO: 16). 83. The rAAV capsid protein of embodiment 82.

[0096] 84. A vector encoding an rAAV capsid protein according to any one of embodiments 74 to 83. or a nucleic acid encoding an amino acid sequence that shares at least 80% identity therewith. A nucleic acid comprising an octide sequence.

[0097] 85. The AA comprising the capsid protein encoded by the nucleotide sequence A packaged vector capable of expressing the nucleic acid of embodiment 84 to produce a V vector. Zing cells.

[0098] 86. A rAAV vector comprising a capsid protein according to any one of embodiments 74 to 83. Tha.

[0099] 87. The rAAV vector of embodiment 86, further comprising a transgene.

[0100] 88. The rAAV vector according to embodiment 86 or 87 and a pharma- ceutically acceptable carrier 23. A pharmaceutical composition comprising:

[0101] 89. A method for delivering a transgene to a cell, the method comprising the steps of: 6 or 87, wherein the transgene is The method further comprises delivering the antibody to the cell.

[0102] 90. A method for delivering a transgene to a target tissue in a subject in need thereof, comprising: The method comprises administering to the subject an rAAV vector of embodiment 86 or 87. wherein the transgene is delivered to the target tissue.

[0103] 91. A pharmaceutical composition for use in delivering a transgene to a cell, comprising: The pharmaceutical composition comprises the rAAV vector of embodiment 86 or 87, The pharmaceutical composition wherein the drug is delivered to the cell.

[0104] 92. For use in delivering a transgene to a target tissue in a subject in need thereof. A pharmaceutical composition comprising an rAAV according to any one of embodiments 86 to 87. The pharmaceutical composition comprising a vector, wherein the transgene is delivered to the target tissue.

[0105] 93. The target tissue is a retinal cell, and the peptide insert comprises the amino acid sequence LGE Embodiments including TTRP (SEQ ID NO: 15) or LALGETTRP (SEQ ID NO: 16) 89 to 92, or a pharmaceutical composition for use according to the method.

[0106] 94. The rAAV vector is administered systemically, intravenously, intrathecally, intranasally, or intraperitoneally. or for use according to any one of embodiments 89 to 93, wherein the composition is administered intravitreally or intraperitoneally. Pharmaceutical compositions.

[0107] 95. Embodiment 8, in which the vector is administered via lumbar puncture or via the cisterna magna. 9. The method according to claim 9 to 93, or a pharmaceutical composition for use.

[0108] 96. Recombinant AAV capsid protein, comprising a wild-type or unmodified capsid protein. The rAAV capsid protein comprises one or more amino acid substitutions to the AAV protein. AAV8 capsid protein with a 269S amino acid substitution (SEQ ID NO: 117), or S263G / S269R / A273T substitutions, or W503R or Q474A The one or more aAVs are AAV9 capsid proteins having a substitution (SEQ ID NO: 118). amino acid substitutions, or corresponding substitutions in the capsid protein of another AAV type capsid The recombinant AAV capsid protein.

[0109] 97. 498-NNN / AAA-500 for AAV8 capsid protein or is 496-NNN / AAA- for the AAV9 capsid protein (SEQ ID NO: 118) 498, or the corresponding substitution in the capsid protein of another AAV type capsid. 97. The recombinant AAV capsid protein of embodiment 96, comprising:

[0110] 98. A vector encoding an rAAV capsid protein according to embodiment 96 or 97, or a nucleotide sequence encoding an amino acid sequence sharing at least 80% identity therewith. A nucleic acid comprising a sequence.

[0111] 99. The AA comprising the capsid protein encoded by the nucleotide sequence A packaged vector capable of expressing the nucleic acid of embodiment 98 to produce a V vector. Zing cells.

[0112] 100. A rAA comprising any of the capsid proteins according to embodiment 96 or 97. V Vector.

[0113] 101. The rAAV vector of embodiment 100, further comprising a transgene.

[0114] 102. The rAAV vector according to embodiment 100 or 101 and a pharma- ceutical acceptable A pharmaceutical composition comprising a carrier.

[0115] 103. A method for delivering a transgene to a cell, the method comprising: 101, wherein the transgene is contacted with the rAAV vector of claim 102. The method of claim 1, wherein the

[0116] 104. A method for delivering a transgene to a target tissue in a subject in need thereof, comprising: The method includes administering to the subject an rAAV vector of embodiment 101, The method, wherein the transgene is delivered to the target tissue.

[0117] 105. A pharmaceutical composition for use in delivering a transgene to a cell, comprising: The pharmaceutical composition comprises the rAAV vector of embodiment 101, wherein the transgene is The pharmaceutical composition delivered to the cell.

[0118] 106. For use in delivering a transgene to a target tissue in a subject in need thereof. 102. A pharmaceutical composition comprising the rAAV vector of embodiment 101. wherein the transgene is delivered to the target tissue.

[0119] 107. The rAAV vector is administered systemically, intravenously, intrathecally, intranasally, or intraperitoneally. 107. The method or use according to any one of embodiments 102 to 106, wherein the pharmaceutical composition is administered intravitreally or intraperitoneally. A pharmaceutical composition for

[0120] 108. In embodiments, the vector is administered via lumbar puncture or via the cisterna magna. 102 to 106. A pharmaceutical composition for use according to the method or method of claim 102. [Brief description of the drawings]

[0121] [Figure 1] FIG. 1 shows a comparison of the capsid amino acid sequence including the adeno-associated virus type 9 VR-IV loop (AAV9 VR-IV) from residues L447 to R476 (residues 451-459 are shown in brackets) with corresponding regions in other AAVs. The figure discloses SEQ ID NOs: 87-92, 88, and 93-96, respectively, in order of appearance. [Diagram 2] FIG. 1 shows a protein model of the AAV capsid structure, showing the capsid variable regions VR-IV, VR-V, and VR-VIII. The box highlights the loop region of VR-IV that provides the surface-exposed amino acids as represented in the model. [Diagram 3]High packaging efficiency (titer) in terms of genome copies per mL (GC / mL) is shown for wild-type AAV9 and eight candidate modified rAAV9 vectors (1090, 1091, 1092, 1093, 1094, 1095, 1096, and 1097), each of which contains a FLAG insert immediately following a different site in the AAV9 VR-IV from residues I451 to Q458. All vectors were packaged with the luciferase transgene in 10 mL cultures; error bars represent standard error of the mean. [Figure 4] Figure 1 demonstrates surface exposure of the lVR-IV loop FLAG insert in each of eight candidate modified rAAV9 vectors (1090, 1091, 1092, 1093, 1094, 1095, 1096, and 1097), as confirmed by immunoprecipitation of packaged vectors by binding to anti-FLAG resin. [Diagram 5] Figure 1 shows transduction efficiency in Lec2 cells transduced with a capsid vector carrying the luciferase gene (as a transgene) packaged into either wild-type AAV9 (9-luc) or each of eight candidate modified (FLAG peptide inserted) rAAV9 vectors (1090, 1091, 1092, 1093, 1094, 1095, 1096, and 1097); transduction activity is expressed as percent luciferase activity (A), taking the activity of 9-luc as 100%, or as relative light units (RLU) per microgram of protein (B). [Figure 6A]1 shows a bar graph illustrating that inserts of various peptide lengths and compositions immediately after S454 of AAV9 can affect the efficiency of AAV particle production in packaging cells. Ten peptides of various compositions and lengths were inserted after S454 in AAV9 VR-IV. qPCR was performed on harvested supernatants of transfected suspension HEK293 cells 5 days after transfection. The results shown in the bar graph demonstrate that insert quality affects the ability of AAV particles to be produced and secreted by HEK293 cells, as indicated by the overall yield (titer). (Error bars represent standard error of the average peptide length marked in brackets on the Y-axis). [Figure 6B] Fluorescence images of transduced cell cultures of the following cell lines are shown: (6B) Lec2 cell line, (6C) HT-22 cell line, (6D) hCMEC / D3 cell line, and (6E) C2C12 cell line. AAV9 wild type and S454 inserted homing peptide capsids containing a GFP transgene were used to transduce the aforementioned cell lines. The P1 vector was not included in the images due to extremely low transduction efficiency, and the P8 vector was not included due to low titer. AAV9.S454.FLAG showed low transduction levels in all cell types tested. [Figure 6C] Fluorescence images of transduced cell cultures of the following cell lines are shown: (6B) Lec2 cell line, (6C) HT-22 cell line, (6D) hCMEC / D3 cell line, and (6E) C2C12 cell line. AAV9 wild type and S454 inserted homing peptide capsids containing a GFP transgene were used to transduce the aforementioned cell lines. The P1 vector was not included in the images due to extremely low transduction efficiency, and the P8 vector was not included due to low titer. AAV9.S454.FLAG showed low transduction levels in all cell types tested. [Figure 6D]Fluorescence images of transduced cell cultures of the following cell lines are shown: (6B) Lec2 cell line, (6C) HT-22 cell line, (6D) hCMEC / D3 cell line, and (6E) C2C12 cell line. AAV9 wild type and S454 inserted homing peptide capsids containing a GFP transgene were used to transduce the aforementioned cell lines. The P1 vector was not included in the images due to extremely low transduction efficiency, and the P8 vector was not included due to low titer. AAV9.S454.FLAG showed low transduction levels in all cell types tested. [Figure 6E] Fluorescence images of transduced cell cultures of the following cell lines are shown: (6B) Lec2 cell line, (6C) HT-22 cell line, (6D) hCMEC / D3 cell line, and (6E) C2C12 cell line. AAV9 wild type and S454 inserted homing peptide capsids containing a GFP transgene were used to transduce the aforementioned cell lines. The P1 vector was not included in the images due to extremely low transduction efficiency, and the P8 vector was not included due to low titer. AAV9.S454.FLAG showed low transduction levels in all cell types tested. [Figure 7A] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7B] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7C] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7D] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7E] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7F] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7G]1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7H] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7I] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7J] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7K] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7L] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7M] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 8-1] Alignment of AAV1-9e, rh10, rh20, rh39, and rh74 version 1 and version 2 with insertion sites for heterologous peptides after the VP2 start codon and in or near variable region 1 (VR-I), variable region 4 (VR-IV), and variable region 8 (VR-VIII) (all highlighted in gray), with the specific insertion site in variable region 8 (VR-VIII) of each capsid protein indicated by the symbol "#" (after amino acid residue 588 according to the AAV9 amino acid numbering). [Figure 8-2] This is a continuation of Figure 8-1. [Figure 8-3] This is a continuation of Figure 8-1. [Figure 8-4] This is a continuation of Figure 8-1. [Figure 8-5] This is a continuation of Figure 8-1. [Figure 9]1 shows the amino acid sequence for the recombinant AAV9 vector containing a peptide insert of ARA290 between Q588 and A589 (SEQ ID NO: 153); the ARA290 insert is shown in bold. [Figure 10] GFP (green fluorescent protein) transgene copies in mouse brain cells following administration of the AAV vectors: AAV9; AAV.PHP.eB, also referred to herein as AAV9e (AAV9 with the peptide TLAVPFK (sequence number 27) inserted between positions 588 and 589 and the modifications A587D / A588G); AAV.hDyn (AAV9 with TLAAPFK (sequence number 2) between positions 588 and 589); AAV.PHP.S (AAV9 with the peptide QAVRTSL (sequence number 23) between positions 588 and 589); and AAV.PHP.SH (AAV9 with the peptide QAVRTSH (sequence number 24) inserted between positions 588 and 589). [Figure 11] 1 shows the amino acid sequences for recombinant AAV9 vectors containing TLAAPFK (SEQ ID NO:2) peptide inserts between Q588 and A589 (A), between S268 and S269 in VR-III (B), and between S454 and G455 in VR-IV (C), with the TLAAPFK (SEQ ID NO:2) inserts shown in bold. [Figure 12] 1 shows the amino acid sequences for recombinant AAV9 vectors containing the KMQVPFQ (SEQ ID NO: 1) peptide inserts between Q588 and A589 (A), between S268 and S269 in VR-III (B), and between S454 and G455 in VR-IV (C), with the KMQVPFQ (SEQ ID NO: 1) inserts shown in bold. [Figure 13] 1 shows the amino acid sequences for recombinant AAV9 vectors containing peptide inserts of QQAAPSF (SEQ ID NO: 3) between Q588 and A589 (A), between S268 and S269 in VR-III (B), and between S454 and G455 in VR-IV (C), with the QQAAPSF (SEQ ID NO: 3) inserts shown in bold. [Figure 14]1 shows the amino acid sequences for recombinant AAV9 vectors containing RYNAPFK (SEQ ID NO: 4) peptide inserts between Q588 and A589 (A), between S268 and S269 in VR-III (B), and between S454 and G455 in VR-IV (C), with the RYNAPFK (SEQ ID NO: 4) inserts shown in bold. [Figure 15] 1 shows the amino acid sequences for recombinant AAV9 vectors containing LKLPPIV (SEQ ID NO:5) peptide inserts between Q588 and A589 (A), between S268 and S269 in VR-III (B), and between S454 and G455 in VR-IV (C), with the LKLPPIV (SEQ ID NO:5) inserts shown in bold. [Figure 16] 1 shows the amino acid sequences for recombinant AAV9 vectors containing PFIKPFE (SEQ ID NO: 6) peptide inserts between Q588 and A589 (A), between S268 and S269 in VR-III (B), and between S454 and G455 in VR-IV (C), with the PFIKPFE (SEQ ID NO: 6) inserts shown in bold. [Figure 17] 1 shows the amino acid sequences for recombinant AAV9 vectors containing the TLSLPWK (SEQ ID NO: 7) peptide inserts between Q588 and A589 (A), between S268 and S269 in VR-III (B), and between S454 and G455 in VR-IV (C), with the TLSLPWK (SEQ ID NO: 7) inserts shown in bold. [Figure 18] 1 shows the amino acid sequences for recombinant AAV8 vectors containing the LGETTRP (SEQ ID NO: 15) peptide inserts between N590 and T591 (A), between A269 and T270 in VR-III (B), and between T453 and T454 in VR-IV (C), with the LGETTRP (SEQ ID NO: 15) inserts shown in bold. [Figure 19]1 shows the amino acid sequences for recombinant AAV8 vectors containing the LALGETTRP (SEQ ID NO: 16) peptide inserts between N590 and T591 (A), between A269 and T270 in VR-III (B), and between T453 and T454 in VR-IV (C), with the LALGETTRP (SEQ ID NO: 16) inserts shown in bold. [Figure 20] Shown is an in vitro Transwell assay for AAV vectors crossing the blood-brain barrier (BBB) ​​cell layer (A) and results showing that AAV.hDyn (indicated by inverted triangles) crosses the BBB cell layer of the assay faster than AAV9 (squares) and faster and to a greater extent than AAV2 (circles) (B). [Figure 21] Figure 8 shows the results of next generation sequencing (NGS) analysis of brain gDNA from mice administered pools of modified and native capsids intravenously, revealing the relative abundance in mouse tissues of the different capsids in the pools. Three different pools were injected into mice. The dotted lines indicate which vectors were pooled together. Parental AAV9 was included in each pool as a control (pool 1: BC01, pool 2: BC31, pool 3: BC01). Barcodes for each capsid in the pools are listed in Tables 8a-8c. [Figure 22] Figure 1 shows the in vivo transduction profile of AAV.hDyn in female C57Bl / 6 mice, showing copy number / microgram gDNA in brain (A), liver (B), heart (C), lung (D), kidney (E), skeletal muscle (F), sciatic nerve (G), and ovary (H) in naive mice or mice injected with either AAV9 or AAV.hDyn, with AAV.hDyn demonstrating increased brain biodistribution compared to AAV9. [Figure 23] Images of immunohistochemical staining of brain sections from the striatum (A), hippocampus (B), and cortex (C) showing the distribution of GFP from AAV.hDyn throughout the brain reveal global transduction of the brain with the engineered vector. [Figure 24]1 shows the in vivo kidney to liver transduction efficiency ratios of genetically modified AAV9 vectors containing a homing peptide insert immediately following amino acid 454. Details of the homing peptides used in this study are outlined in Table 8. [Diagram 25] 1 shows the amino acid sequence for a recombinant AAV9 vector containing a TLAVPFK (SEQ ID NO:27) peptide insert between S454 and G455 of VR-IV, with the TLAVPFK (SEQ ID NO:27) insert shown in bold. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0122] Conferring desirable properties, such as tissue targeting, transduction, and integration of the rAAV genome; and / or a combination having a capsid protein modified to include an amino acid sequence that enhances Recombinant adeno-associated viruses (rAAVs) are provided. In particular, the capsid protein is The virus is designed to have a peptide insert that is surface-exposed when packaged as a particle. In or near variable region IV (VR-IV) of the capsid, 4 to 20, or 7 consecutive amino acids, and in embodiments, 12 or fewer consecutive amino acids Modified capsid proteins are provided that contain a peptide insert of, for example, human axoneme The dimerization domain of the heavy chain tail region of dynein and others described herein (Table 1A and 1B) to target specific tissues and / or enhance rAAV cellular uptake, transformation, and Recombinant capsid proteins with peptides inserted to facilitate transfer and / or genome integration Proteins, and rAAVs containing them, are also provided.

[0123] One or more amino acid substitutions that facilitate transduction and / or tissue tropism as described herein Also provided are modified capsids having the following structure: Also provided are pharmaceutical compositions thereof, nucleic acids encoding the capsid proteins, and targeted delivery, Modified Capsules for Improved Transduction and / or Treatment of Target Tissue-Associated Disorders Methods of Making and Using Capsid Proteins and rAAV Vectors Having Capsids - Patent application In particular, compositions comprising rAAV and erythropoietin or dapagliflozin are provided. Capsid proteins containing peptides derived from or related to dynein, To target rAAV to neural tissues, including the retina and / or central nervous system, and to treat neurological disorders and / or to facilitate delivery of therapeutic agents to treat disorders of the eye, particularly the retina. Methods for using the same are provided. a composition comprising an rAAV that includes a peptide insert that targets or homes to the Methods for using the same are also provided.

[0124] 5.1.Definition The term "AAV" or "adeno-associated virus" refers to a virus in the genus Parvoviridae. AAV refers to the naturally occurring "wild-type" virus in the AAV derived from the virus, the capsid protein encoded by the naturally occurring cap gene The rAAV genome packaged in a capsid containing a protein and / or a non-naturally occurring The capsid is packed into a capsid containing the capsid protein encoded by the cap gene. Examples of the latter include AAV derived from a caged rAAV genome. having a capsid protein that contains a peptide insertion within the amino acid sequence of the capsid present This includes AAVs.

[0125] The term "rAAV" refers to "recombinant AAV." In some embodiments, recombinant AAV V is an A variant in which part or all of the rep and cap genes have been replaced with heterologous sequences. Contains the AV genome.

[0126] The term "rep-cap helper plasmid" refers to a plasmid that contains the rep and cap genes of a virus. and a rAAV genome that provides the This refers to a plasmid that aids in the production of AAV from the genome.

[0127] The term "cap gene" refers to a gene that forms or helps form the capsid coat of a virus. In the case of AAV, the capsid protein is The polypeptide can be VP1, VP2, or VP3.

[0128] The term "rep gene" refers to a gene encoding a nonstructural protein required for viral replication and merging. It refers to a nucleic acid sequence that

[0129] As used herein, the terms "nucleic acid" and "nucleotide sequence" include those derived from DNA molecules. (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA and Combinations of DNA and RNA molecules or hybrid DNA / RNA molecules, as well as DNA or includes analogs of RNA molecules. Such analogs include inosine or tritiated Nucleotide analogs, including but not limited to bases, can be used to generate the nucleotides. Such analogs may also provide, for example, nuclease resistance or increased ability to cross cell membranes. These may include DNA or RNA molecules that contain modified backbones that confer beneficial attributes to the molecule, such as A nucleic acid or nucleotide sequence may be single-stranded or double-stranded, and may contain single-stranded and double-stranded portions. The nucleic acid may contain both double-stranded and triple-stranded portions, but is preferably double-stranded DNA.

[0130] As used herein, the terms "subject," "host," and "patient" are used interchangeably. As used herein, a subject is a mammal, e.g., a non-primate (e.g., a rat). animals (e.g., rats, pigs, horses, cats, dogs, rats, etc.) or primates (e.g., monkeys and humans), Or, in certain embodiments, a human.

[0131] As used herein, the term "therapeutic agent" refers to an agent that treats symptoms associated with a disease or disorder. Any agent that may be used in the treatment, management, or amelioration of a disease or disorder that is As used herein, a therapeutic gene refers to a gene that is involved in a function provided by a gene. A "therapeutically effective amount" is an amount effective to treat or manage a target disease or disorder. The amount of an agent that provides at least one therapeutic benefit when administered to a subject (e.g., Furthermore, the therapeutic efficacy of the agent of the present invention is The amount is determined by the amount of agent, alone or in combination with other therapies, that is effective in treating a disease or disorder. By "therapeutic benefit," it is meant providing at least one therapeutic benefit in the placement or management of a therapeutic agent.

[0132] As used herein, the term "prophylactic agent" refers to an agent that prevents, delays, or reduces the progression of a disease or disorder. or any agent that may be used in the inhibition or deactivation of a disease or disorder caused by a transgene. As used herein, "prophylactic efficacy" refers to a function provided by a The "amount" refers to the amount administered to a subject predisposed thereto in preventing or delaying a target disease or disorder. The amount of prophylactic agent that provides at least one prophylactic benefit when administered to a subject (e.g., a subject that is not otherwise specified by a transgene) is A prophylactically effective amount also refers to an amount of a product expressed by a target agent in preventing the onset of a target disease or disorder. or an amount of drug sufficient to slow the progression of a targeted disease or disorder. in an amount sufficient to delay or minimize the onset of the target disease or disorder; or to prevent recurrence or A prophylactically effective amount can refer to an amount sufficient to prevent or slow the spread of a target disease. It may refer to an amount of an agent sufficient to prevent or delay the progression of symptoms of a disease or disorder. In addition, a prophylactically effective amount for a prophylactic agent of the present invention, alone or in combination with other agents, In some embodiments, the amount of prophylactic agent has at least one prophylactic effect in preventing or delaying a disease or disorder. It means providing a benefit.

[0133] The prophylactic agents of the invention can be administered to a subject who is "predisposed" to the target disease or disorder. A subject who is "predisposed" to a disease or disorder is one who exhibits symptoms associated with the development of the disease or disorder, or or genetic makeup, environmental exposures, or other risks for such diseases or disorders The cause is present, but the symptoms are not yet at a level that can be diagnosed as a disease or disorder. For example, a person with a family history of a disease associated with the missing gene (provided by the transgene) In addition, patients who have undergone removal of the primary tumor may be predisposed to this condition. Patients with persistent occult tumors may qualify as predisposed to tumor recurrence .

[0134] "Central nervous system" ("CNS"), as used herein, refers to a system in which a circulating agent crosses the blood-brain barrier. refers to the nervous tissue that is reached after passing through the brain, optic nerves, cranial nerves, and spinal cord. The CNS also includes cerebrospinal fluid, which fills the central canal of the spinal cord and the ventricles of the brain.

[0135] 5.2. Recombinant AAV Capsids and Vectors One embodiment includes a peptide insert from a heterologous protein that is not an AAV protein. The capsid protein of recombinant adeno-associated virus (rAAV) is modified to AAV particles that contain peptide inserts when packaged into the AAV particle. In some embodiments, the peptide insert is an AAV 9. Capsid variable region IV (VR-IV) or other types of AAV capsids Within the corresponding region (i.e., without deleting any capsid amino acids), In some embodiments, the amino acid residues are present between the amino acids (see alignment in FIG. 8). The peptide insert can be inserted into the variable region VIII (VR-VIII) of the AAV9 capsid, or into another Within the corresponding region of the capsid for any AAV type (i.e., any capsid amino acid (Alignment in Figure 8) In some embodiments, the peptide insert directs the rAAV particle to a target tissue. and / or a heterologous vector that promotes rAAV uptake, transduction and / or genomic integration. From a protein or domain (not an AAV capsid protein or domain) The nucleic acid encoding the modified capsid protein and its variants, rA Packaging cells for expressing nucleic acids for generating AV vectors, transgenes Further, rAAV vectors and pharmaceutical compositions of the rAAV vectors, as well as transgenes The rAAV is used to deliver the antigen to a target cell type or tissue in a subject in need thereof. Methods of using the vectors are also provided.

[0136] In various embodiments, the target tissue is neural tissue, bone, kidney, muscle, eye / retina, or endometrium. The peptide insert may be in the tissue or in a specific receptor or tumor, e.g. For example, one or more specific cell types, e.g., those within a target tissue or its cellular matrix, may be targeted. The present invention provides a method for the preparation of a nucleic acid sequence that is derived from a heterologous protein or domain that specifically recognizes and / or binds to a nucleic acid sequence. In particular, peptides derived from erythropoietin or dynein, especially axonemal dynein or The cytoplasmic dynein heavy chain dimerization domain or any surface-exposed variable region of the cytoplasmic dynein Binding to or association with cytoplasmic dynein may target rAAV to neural tissue, The drug has the potential to cross the blood-brain barrier to reach the CNS and deliver therapeutic agents to treat neurological disorders. This includes reaching

[0137] 5.2.1 rAAV Vectors with Peptide Inserts The present inventors have surprisingly found that the VR-IV loop of the AAV9 capsid (see FIG. 2) Peptides were attached to the corresponding regions on the VR-IV loop of the capsids of other AAV types, as well as to nearby We found a suitable location for the insertion. Previous studies have investigated potential locations in various AAVs. However, none of them identified AAV9 VR-IV as suitable for this purpose (e.g. For example, Wu et al., 2000, “Mutational Analysis of the Adeno-Associated Virus Type 2(AAV2) Capsid Gene and Construction of AAV2 Vec tors with Altered Tropism,” J of Virolog y 74(18):8635-8647 ;Lochrie et al,2006,“ Adeno-associated virus(AAV)capsid genes isolated from rat and mouse liver genomi c DNA define two new AAV species distant ly related to AAV-5,” Virology 353:68-82 ;Shi and Bartlett,2003,“RGD Inclusion in VP3 Provides Adeno-Associated Virus Typ e 2(AAV2)-Based Vectors with a Heparan S ulfate-Independent Cell Entry Mechanism, ” Molecular Therapy 7(4):515525-;Nicklin et al.,2001,“Efficient and Selective AA V2-Mediated Gene Transfer Directed to Hu man Vascular Endothelial Cells” Molecula r Therapy 4(2):174-181;Grifman et al.,20 01,“Incorporation of Tumor-Targeting Pep tides into Recombinant Adeno-associated Virus Capsids,” Molecular Therapy 3(6):9 64-975;Girod et al.1999,“Genetic capsid modifications allow efficient re-targeti ng of adeno-associated virus type 2,”Na ture Medicine 3(9):1052-1056;Douar et al .,2003,“Deleterious effect of peptide in sertions in a permissive site of the AAV 2 capsid, “Virology 309:203-208; and Ponnazh agan, et al. 2001, J. of Virology 75(19):949 (See also 3-9501).

[0138] Thus, at the novel insertion points, particularly within the surface-exposed variable regions in the capsid coat, rAA carrying a peptide insert in or near the variable region IV of the capsid protein In some embodiments, the rAAV capsid protein is AV9 capsid protein (amino acid sequence SEQ ID NO: 118, AAV9 capsid amino acid sequence For alignment of the capsid protein amino acid sequences of other AAV serotypes with the Immediately after the amino acid residue corresponding to one of amino acids 451 to 461 of (i.e., connected by a peptide bond to the C-terminus) of the peptide insert, When the sesquioxane protein is packaged as an AAV particle, the peptide insert The peptide insert does not delete any residues of the AAV capsid protein. Generally, the peptide insert should be inserted to differentiate the capsid protein from that of other serotypes. These are present in variable (less conserved) regions of the protein and in surface-exposed loops.

[0139] Peptide insertions described as being inserted "at" a given site are those that occur in the wild-type virus. immediately following the residue normally found at that site in For example, an insertion at Q588 in AAV9 results in a peptide bond. The tide insertion is Q5 in the AAV9 wild-type capsid protein sequence (SEQ ID NO: 118). 88 and consecutive amino acids (A589). at or near (within 5, 10, 15 residues or within a structural loop that is the site of an insertion ) there is no deletion of amino acid residues.

[0140] In certain embodiments, the capsid protein is an AAV9 capsid protein, The insertion is located immediately following at least one of amino acid residues 451 to 461. In embodiments, the peptide insert comprises the AAV9 capsid (amino acid sequence SEQ ID NO: 118) Amino acids I451, N452, G453, S454, G455, Q456, N457, Q In certain embodiments, the amino acid sequence is located immediately after 458, Q459, T460, or L461. The peptide is located between residues S454 and G455 of the AAV9 capsid protein or the AAV AAV capsid proteins other than the 9 capsid protein (amino acid sequence SEQ ID NO: 118) It is inserted between residues corresponding to S454 and G455 of

[0141] In another embodiment, the heterologous peptide is surface-exposed when incorporated into an AAV vector. To achieve this, a heterologous target protein was inserted into the AAV capsid protein. Modified capsid proteins are provided that contain a targeting peptide. Such peptides include , preferably from the human axonemal dynein (HAD) heavy chain tail, or 1A and 1B or other targeting peptides for specific tissue types. do.

[0142] In other embodiments, the capsid protein is selected from AAV serotype 1 (AAV1), serotype 2 ( AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), blood serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (A AVrh8), serotype 9e (AAV9e), serotype rh10 (AAVrh10), serotype rh20(AAVrh20), serotype rh39(AAVrh39), serotype hu.37( AAVhu.37), and serotype rh74 (AAVrh74, versions 1 and 2) (Fig. 8), and the insert is from at least one AAV type selected from the following: immediately following an amino acid residue corresponding to at least one of the amino acid residues 451 to 461 The alignment of these different AAV serotypes is shown in Figure 8. The amino acid residues that are "matched" are aligned in the same position in the alignment as the residues in the reference sequence. The "corresponding" amino acid residues in the different capsid amino acid sequences are indicated as shown. In some specific embodiments, the peptide insert is an AAV 450 to 459 of AAV1 capsid (SEQ ID NO: 110); AAV2 capsid (SEQ ID NO: 111) 449-458 of AAV3 capsid (SEQ ID NO: 112); 449-459 of AAV4 capsid 443 to 453 of the AAV5 capsid (SEQ ID NO: 113); 42-445; 450-459 of AAV6 capsid (SEQ ID NO: 115); AAV7 capsid 451 to 461 of the AAV8 capsid (SEQ ID NO: 117); ~461; 451 to 461 of AAV9 capsid (SEQ ID NO: 118); AAV9e capsid 452 to 461 of (SEQ ID NO: 119); 4 of AAVrh10 capsid (SEQ ID NO: 120) 52-461; 452-461 of AAVrh20 capsid (SEQ ID NO: 121); AAVh 452 to 461 of u.37 (SEQ ID NO: 122); AAVrh74 (SEQ ID NO: 123 or 452 to 461 of SEQ ID NO: 154; or 452 of AAVrh39 (SEQ ID NO: 124) In some embodiments, rA is located immediately following one of the amino acid residues in The AV capsid protein is the AAV9 capsid protein (amino acid sequence of SEQ ID NO: 118). 8) (see FIG. 8 ) immediately following amino acid 588 (i.e., at its C-terminus). When the insert is contained and the capsid protein is packaged into an AAV particle, The peptide insert is surface exposed. In other embodiments, the rAAV capsid protein is Not immediately following amino acid 588 of AAV9 or the sequence corresponding to amino acid 588 of AAV9 It has a small peptide insert.

[0143] In another embodiment, the peptide comprises at least 4 consecutive amino acids of Tables 1A and 1B. or at least 7 consecutive amino acids, or exactly 7 consecutive amino acids, but in In the form of a targeting peptide, the targeting peptide comprises 12 or fewer consecutive amino acids, or a functional fragment thereof. In these cases, the capsid protein is classified as AAV serotype 1 (AAV1), serotype 2 (AAV2), Serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AA) V6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8) , serotype 9e (AAV9e), serotype rh10 (AAVrh10), serotype rh20 (A AVrh20), serotype rh39 (AAVrh39), serotype hu.37 (AAVhu. 37), and from serotype rh74 (AAVrh74, versions 1 and 2) (see Fig. 8 ). from at least one selected AAV type, and the peptide is incorporated into an AAV vector. The peptide is attached to the capsid protein at any point so that it is surface-exposed when the peptide is embedded in the capsid. In certain embodiments, the peptide is inserted into the AAV1 capsid (SEQ ID NO: 110). 138; 262-272; 450-459; or 585-593; AAV2 capsid 138; 262-272; 449-458; or 584-59 of (SEQ ID NO: 111) 2; 138 of the AAV3 capsid (SEQ ID NO: 112); 262-272; 449-459; or 585-593; 137; 256-26 of the AAV4 capsid (SEQ ID NO: 113) 2; 443-453; or 583-591; AAV5 capsid (SEQ ID NO: 114) 137;252-262;442-445;or 574-582;AAV6 capsid (SEQ ID NO:115) 138; 262-272; 450-459; 585-593; AA 138;263-273;451-461;586- of V7 capsid (SEQ ID NO: 116) 594; 138; 263-274; 452-46 of AAV8 capsid (SEQ ID NO: 117) 1;587-595;138;262-273 of AAV9 capsid (SEQ ID NO:118); 452-461; 585-593; 138 of AAV9e capsid (SEQ ID NO: 119); 2 62-273; 452-461; 585-593; AAVrh10 capsid (SEQ ID NO:1 20) 138; 263-274; 452-461; 587-595; AAVrh20 Psid (SEQ ID NO: 121) 138; 263-274; 452-461; 587-595 138 of AAVrh74 capsid (SEQ ID NO: 123 or SEQ ID NO: 154); 263 to 274; 452-461; 587-595, AAVhu37 capsid (SEQ ID NO: 122) 138; 263-274; 452-461; 587-595; or AAVrh39 Psid (SEQ ID NO: 124) 138; 263-274; 452-461; 587-595 (numbered in Figure 8) are inserted after

[0144] In some embodiments, the capsid protein is from an AAV other than serotype AAV2. In some embodiments, the peptide insertion is an AAV2 capsid protein. There is no amino acid residue immediately following the amino acid residue corresponding to amino acid 570 or 611 of In embodiments, the peptide insert comprises amino acids 587-58 of the AAV2 capsid protein. 8 (Schaffer et al., US 2014 / 02 94771). In some embodiments, the amino acid sequence DDDDDDDD (SEQ ID NO: 9) insert of the bone 1 peptide at amino acid 138 of the AAV2 capsid protein (Almeciga-Diaz et al., 2018, Pedia trRes.84:545).

[0145] Also provided are AAV vectors comprising the modified capsids. AV vectors are non-replicative and contain nucleoside encoding the rep or cap proteins. (These are used in packaging cells for the production of rAAV vectors.) In some embodiments, the AAV-based vector comprises one or more AAV In some embodiments, the AA V-based vectors are AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6. , AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.r h20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu3 7, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP .B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.L K03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV. HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.H SC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other rAAV particles, or a combination of two or more thereof. In some embodiments, the AAV-based vectors provided herein comprise a p53 gene. The targets are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, A AV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV .rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.A nc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV. PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV .HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC 5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AA V.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AA V.HSC14, AAV.HSC15, or AAV.HSC16 or other rAA V particles, or a combination of two or more of them. In some embodiments, the rAAV particles can be any of the AAV1, AAV2, AAV3, AAV 4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, A AV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV .rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM 4-1, AAV.hu37, AAV.Anc80, rAAV.Anc80L65, AAV .7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF , AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HS C3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, A AV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV .HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or is selected from AAV.HSC16, or a derivative, modification, or pseudotype thereof. At least the VP1, VP2 and / or VP3 sequences of the AAV capsid serotype being studied 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99. 5%, i.e., up to 100% identical capsid proteins. The AAV vector may contain a genome that includes a transgene encoding a therapeutic protein. .

[0146] In certain embodiments, recombinant human ovarian sarcoma vectors for use in the compositions and methods herein are The AAV is Anc80 or Anc80L65 (e.g., Zinn et al. , 2015, Cell Rep. 12(6):1056-1068 (incorporated by reference in its entirety). In certain embodiments, the compositions and methods herein include The recombinant AAV for use in the present invention is AAV.7m8 (including variants thereof) (e.g., For example, US9,193,956; US9,458,517; US9,587,282; U S2016 / 0376323 and WO2018 / 075798 (each of which is incorporated herein by reference in its entirety). (see, incorporated herein by reference). In certain embodiments, AAV for use in the compositions and methods are disclosed in US 9,585,971. In certain embodiments, the AAV vector is any AAV vector that is capable of expressing the AAV gene, such as AAV-PHP.B. AAV for use in the compositions and methods include AAV8 and serotype cy5, AAV2 / Rec with hybrid capsid sequences derived from rh20 or rh39 2 vector or AAV2 / Rec3 vector (see, e.g., Issa et al. , 2013, PLoS One 8(4):e60361 (for more information on these vectors, see In certain embodiments, the compositions described herein may be The AAV for use in the products and methods is any of the following, each of which in its entirety The AAV disclosed in US Pat. No. 2,282,133, which is incorporated herein by reference. 99;US7,906,111;US8,524,446;US8,999,678;U S8,628,966;US8,927,514;US8,734,809;US9,2 84,357;US9,409,953;US9,169,299;US9,193,9 56;US9,458,517;US9,587,282;US2015 / 037480 3;US2015 / 0126588;US2017 / 0067908;US2013 / 0 224836;US2016 / 0215024;US2017 / 0051257;PCT / US2015 / 034799; and PCT / EP2015 / 053335. In embodiments, the rAAV particles are disclosed in the following patents and patent applications, each of which is incorporated herein by reference in its entirety: (incorporated herein by reference thereto): U.S. Patent Nos. 7,282,199; 7,906,11 1;8,524,446;8,999,678;8,628,966;8,927,51 4;8,734,809;US9,284,357;9,409,953;9,169, 299; 9,193,956; 9,458,517; and 9,587,282; U.S. Patents. Publication No. 2015 / 0374803;2015 / 0126588;2017 / 006 7908;2013 / 0224836;2016 / 0215024;2017 / 0051 257; and International Patent Application Nos. PCT / US2015 / 034799; PCT / EP20 VP1, VP2 and / or VP3 of the AAV capsid disclosed in any one of US Pat. No. 15 / 053335. or at least 80% or more identical to the VP3 sequence, e.g., 85%, 85%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, 99.5%, etc., i.e., up to 100% identical capsid proteins Has quality.

[0147] In some embodiments, the rAAV particles are described in U.S. Pat. No. 9,840,719 and WO 2014 / 023363. Any AAV capsid disclosed in US Pat. No. 2015 / 013313, such as AAV.Rh 74 and RHM4-1, each of which is incorporated herein by reference in its entirety. In some embodiments, the rAAV particles are derived from WO201 4 / 172669 (incorporated herein by reference in its entirety). In some embodiments, the rAAV particles are dis et al., 2016, Gene Therapy 23:857-862 and and Georgiadis et al., 2018, Gene Therapy 25: 450, each of which is incorporated by reference in its entirety, In some embodiments, the rAAV particles comprise an AAV2 / 5 capsid. Any AAV capsid disclosed in WO2017 / 070491, such as F. (All of which is incorporated by reference herein in its entirety). In some embodiments, the rAAV particles Puzzo et al.,2017,Sci.Transl.Med.29(9) 418, which is incorporated by reference in its entirety. In some embodiments, the rAAV particles are capsids of AAV3 or AAV3B. Patent Nos. 8,628,966; US8,927,514; US9,923,120 and W Any of the AAV capsids disclosed in US Pat. No. 6,399,413, or US Pat. No. 6,399,413, for example, HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or H SC16, each of which is incorporated by reference in its entirety.

[0148] In some embodiments, the rAAV particles are prepared as described in International Application Publication No. WO2003 / 0520. 51 (see, for example, SEQ ID NO:2 of the '051 publication), WO2005 / 033321 (see, for example, , '321 publication), WO03 / 042397 (see, for example, ' 397 (see SEQ ID NOs: 2, 81, 85, and 97 of the same publication), WO2006 / 068888 ( See, for example, SEQ ID NOs: 1 and 3-6 of the '888 publication), WO2006 / 110689 (e.g. For example, see SEQ ID NOs: 5 to 38 of the '689 publication), WO2009 / 104964 (for example, See SEQ ID NOs: 1 to 5, 7, 9, 20, 22, 24 and 31 of the '964 publication), 0 / 127097 (see, for example, SEQ ID NOs: 5-38 of the '097 publication), and WO2015 / 191508 (see, for example, SEQ ID NOs: 80-294 of the '508 publication), and U.S. Patent Application Publication No. Publication No. 20150023924 (see, for example, SEQ ID NOs: 1, 5-10 of the '924 publication) No. 6,393,636, the contents of each of which are incorporated herein by reference in their entireties. In some embodiments, the rAAV particles have a sesquioxane protein. No. WO2003 / 052051 (see, for example, SEQ ID NO:2 of the '051 publication), WO200 5 / 033321 (see, for example, SEQ ID NOs: 123 and 88 of the '321 publication), WO03 / 042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of the '397 publication), WO 2006 / 068888 (see, for example, SEQ ID NOs: 1 and 3-6 of the '888 publication), WO2 006 / 110689 (see, for example, SEQ ID NOs: 5-38 of the '689 publication), WO2009 / 104964 (for example, SEQ ID NOs: 1 to 5, 7, 9, 20, 22, 24 and 31), WO2010 / 127097 (see, for example, SEQ ID NOs: 5 to 38 of the '097 publication) (see, for example, SEQ ID NOs: 80 to 294 of WO2015 / 191508) See, e.g., sequence numbers 1 and 2 of the '924 publication, and U.S. Patent Publication No. 20150023924. VP1, VP2 and / or V of the AAV capsid disclosed in At least 80% or more identical to the P3 sequence, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical capsid proteins has.

[0149] In additional embodiments, the rAAV particles comprise pseudotyped AAV capsids. In some embodiments, the pseudotyped AAV capsid is rAAV2 / 8 or rAAAV. V2 / 9 pseudotyped AAV capsid. Generating pseudotyped rAAV particles and methods for use are known in the art (e.g., Duan et al. l., J.Virol.,75:7662-7671(2001);Halbert e t al.,J.Virol.,74:1524-1532(2000);Zolotu khin et al., Methods 28:158-167(2002); and A Uricchio et al.,Hum.Molec.Genet.10:3075- 3081, (2001).

[0150] In certain embodiments, single-stranded AAV (ssAAV) may be used. Alternatively, a self-complementary vector, such as scAAV, may be used (see, e.g., Wu, 2007). ,Human Gene Therapy,18(2):171-82;McCarty et al,2001,Gene Therapy,8(16):1248-1254 ;US6,596,535;US7,125,717;and US7,456,683 (and each of which is incorporated herein by reference in its entirety).

[0151] Typically, a peptide insert comprises a sequence of consecutive amino acids from a heterologous protein or a domain thereof. The inserted peptide is typically a sequence similar to that of the protein or domain from which it is derived. Long enough to retain a particular biological sample function, property, or characteristic of the inserted The peptides typically resemble the native capsid protein without the insert. or substantially similarly, to allow the capsid protein to form a coat. In a preferred embodiment, the peptide insert is between about 4 and about 30 amino acids in length. The amino acid sequence may be about 4 to about 20, about 4 to about 15, about 5 to about 10, or about 7 amino acids in length. The peptide sequence for insertion is at least 4 amino acids in length and can be 5, 6, 7, It may be 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In embodiments, the peptide sequence is 16, 17, 18, 19, or 20 amino acids in length. In embodiments, the peptide is 7 amino acids, 10 amino acids, or 12 amino acids or less in length. Below.

[0152] A "peptide insert from a heterologous protein" in an AAV capsid protein is The capsid protein is introduced into the capsid of any AAV serotype. Non-limiting examples include the amino acid sequence of the humanized ribozyme in the AAV capsid protein. It includes peptides of the protein.

[0153] In some embodiments, the peptide insert is a homing protein or a homing protein thereof. from a targeting domain or a targeting protein or a targeting domain thereof. A "homing domain" or "homing protein" is a molecule that binds to a cytoplasmic target of a specific cell type. Specific cell types, tissue types, organs, tumor types, etc., including domains or proteins that preferentially or selectively target a target site, organ, or tumor relative to the target site. In the context of the present invention, a peptide from a homing protein or domain is , providing a peptide for insertion into the capsid protein to form the capsid coat, or AA V forms part of the vector and then induces the capsid, coat, or vector to produce a specific Target cell types, tissue types, organs, tumor types, etc., or target the AAV genome Homing proteins or domains may be used without limitation to promote uptake and / or integration. Typical examples include neural tissue homing domains, axonemal or cytoplasmic dynein homing domains, and , bone-homing domain, kidney-homing domain, muscle-homing domain, endothelial cell Cell-homing domain, retinal cell-homing domain, domain targeting specific cell receptors Domains such as integrin receptor binding domains and transferrin receptor binding domains. These include targeting peptides from other viruses, tumor cell targeting domains, and other targeting peptides. As used herein, the terms "homing" and "targeting" are used interchangeably. These peptides may also, or alternatively, mediate cellular uptake of rAAV in cells of a target tissue. The invention may facilitate transduction, transduction and / or genomic integration.

[0154] Use as a peptide insert in any of the AAV capsid sites described herein Examples of peptides for the purposes of the present invention are provided in Tables 1A-1B below, which include peptide functions. At least a four amino acid sequence thereof, or a seven amino acid sequence thereof, having a potential attribute In some embodiments, the contiguous portion includes up to 12 contiguous amino acids. For example, La akkonen and Vuorinen, 2010, “Homing peptid es as targeted delivery vehicles,” Integ rative Biology,2:326-337(review article) In certain embodiments, the recombinant AAV capsids and AAV vectors are The peptide inserts were inserted into the AAV capsid sequence in the manner shown in Table 1A below. and 1B, or at least 4, 5, 6, or 7 peptides thereof. In another embodiment, the peptide is modified to include an amino acid sequence. Positions 138, 262-273, and 451-4 of the amino acid sequence of the capsid (SEQ ID NO:118) 61, or amino acid residues 585-593, or in any other AAV serotype (See Figure 8 for capsid sequence alignment.) ). [Table 1A] [Table 1B] TIFF2025069170000004.tif52168 [Table 2] [Table 3A] TIFF2025069170000007.tif251169TIFF2025069170000008.tif188167 [Table 3B] TIFF2025069170000010.tif254168TIFF2025069170000011.tif195168

[0155] For example, a rAAV vector containing a transgene encoding glial derived growth factor (GDGF) The vector is used to treat / prevent / manage Parkinson's disease. For example, rAAV vectors can be administered intravenously, intrathecally, intranasally, and The antibody may be provided by intravenous and / or intraperitoneal administration.

[0156] In certain embodiments, the rAAV of the present invention is associated with a target tissue or target cell type. The target cell type (associated with the disorder or disease to be treated / prevented) including the cellular matrix Diseases or disorders associated with specific tissues or cell types are utilized in the delivery of have a greater effect on a particular tissue or cell type compared to other tissues of that cell type or the effects or symptoms of the disorder are manifested in a particular tissue or cell type. The method of delivering a transgene to a target tissue in a subject in need thereof comprises administering a peptide insert to the host. In the case of Parkinson's disease, the administration of rAAV, a targeting peptide, to the subject is For example, rAAV vectors containing peptide inserts that target rAAV to neural tissues have been used. In particular, the peptide insert can facilitate the rAAV crossing the blood-brain barrier and reaching the CNS. Such peptide inserts include neural tissue homing domains, e.g., These include those derived from the "EPO peptides" or "HAD peptides" described in the specification.

[0157] For example, capsid proteins containing the EPO peptide retarget AAV to the CNS, Capsid proteins containing EPO peptides can be used to cross the blood-brain barrier. It may further have a protective effect on CNS tissue, for example, EPO inserts may enhance the natural repair pathway. Binding to the body, activating the IRR biological switch, suppressing inflammation, and / or CNS modification In some embodiments, the rAAV comprising the EPO peptide of the present invention It is used in one or more of the following disorders: organ ischemic injury, stroke, myocardial infarction, renal injury. damage, kidney disease, brain injury, renal ischemia, limb ischemia, autoimmune encephalomyelitis, autoimmune neuritis, Multiple sclerosis, Guillain-Barre syndrome, neuropathic pain, diabetic complications, e.g. diabetic retinopathy and diabetic autonomic neuropathy, as well as sarcoidosis.

[0158] Neuropathic diseases or disorders, such as those described herein, may be used to treat neurological disorders. rAAV vectors containing peptide inserts from the tissue homing domain can be used Diseases / disorders related to nervous tissue include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), ), amyotrophic lateral sclerosis (ALS), Batten disease, Batten juvenile NCL, Canavan disease , chronic pain, Friedreich's ataxia, glioblastoma multiforme, Huntington's disease, late childhood Neuronal ceroid lipofuscinosis (LINCL), lysosomal storage disorder, Leber congenital Amaurosis, multiple sclerosis, Parkinson's disease, Pompe disease, Rett syndrome, spinal cord injury, spinal muscle The vectors are used to treat diseases or disorders including cerebrovascular disease (SMA), stroke, and traumatic brain injury. Transgenes for therapeutic / prophylactic benefit to subjects suffering from or at risk of developing It may further comprise a derivative (see Tables 3A-3B).

[0159] For bone-related diseases or disorders, bone homing therapy, such as those described herein, rAAV vectors containing peptide inserts from the binding domain can be used.

[0160] For kidney-related diseases or disorders, renal hospice, such as those described herein, rAAV vectors containing peptide inserts from the gaming domain can be used.

[0161] For muscle-related diseases or disorders, muscle homogenates, such as those described herein, are used. rAAV vectors containing peptide inserts from the gaming domain can be used.

[0162] For diseases or disorders associated with endothelial cells, endothelial cell therapy, such as those described herein, rAAV vectors containing peptide inserts from the epithelial cell homing domain can be used .

[0163] Diseases associated with integrin receptors or cells expressing specific integrin receptors or disorders, such as those described herein, rAAV vectors containing peptide inserts from the vectors can be used.

[0164] Diseases Associated with Transferrin Receptor or Cells Expressing Transferrin Receptor or for disorders, such as those described herein, transferrin receptor binding rAAV vectors containing peptide inserts from the domains can be used.

[0165] For tumor-related diseases or disorders, peptide insertion from the tumor cell targeting domain is A rAAV vector containing the input may be used.

[0166] The retinal cells comprising HAD peptides for a disease or disorder related to the retina or eye. rAAV vectors containing peptide inserts from the homing domain can be used. The tide insert enhances retinal tropism and targets rAAV to the subject's eye or retina. The term "retinal cells" refers to amacrine cells, bipolar cells, aqueous Squamous cells, Müller glial cells, photoreceptor cells (e.g., rods and cones), retinal ganglion cells ( For example, midget cells, parasol cells, bistratified cells retinal ganglion cells, giant retinal ganglion cells, and photosensitive ganglion cells, retinal pigment epithelium, and endothelial cells of the internal limiting membrane. Refers to one or more of the cell types found in or near the retina, including:

[0167] Typically, when the rAAV vector contains a peptide insert for retinal cell homing, the vector The vector is administered by in vivo injection, for example, by direct injection into the eye. rAAV containing peptide inserts to improve retinal tropism were administered intravitreally. In some embodiments, the rAAV for improved retinal tropism can be administered intraocularly. Thus, for example, administration via the pars plana into the vitreous or aqueous humor of the eye. In this study, rAAV was administered via peribulbar or subconjunctival injection to improve retinal tropism. One of the rAAV vectors with a peptide insert for retinal cell homing is The advantage of this is that the subject may avoid surgery, e.g., treatment delivered instead by injection. In certain embodiments, the therapeutic agent is administered intravitreally. In some embodiments, the rAAV vectors described herein are delivered by intravenous injection to treat eye-related diseases. Therapeutic efficacy for treating a disease or disorder, particularly a disease or disorder related to the retina of a subject. In more embodiments, the treatment comprises a single intravitreal injection, no more than two intravitreal injections. intravitreal injections, 3 or fewer intravitreal injections, 4 or fewer intravitreal injections, 5 or fewer intravitreal injections, or achieved after six or fewer intravitreal injections.

[0168] Diseases / disorders related to the eye or retina are referred to as "ophthalmic diseases." Non-limiting examples of ophthalmic diseases Anterior ischemic optic neuropathy; acute macular neuroretinopathy; Bardet-Biedl syndrome; Jett's disease; branch retinal vein occlusion; central retinal vein occlusion; congenital choroideremia; choroidal neovascularization Chorioretinal degeneration; Cone-rod dystrophy; Color vision disorders (e.g., achromatopsia, protanopia) congenital night blindness; diabetic uveitis; retina epiretinal disorders; hereditary macular degeneration; histoplasmosis; macular degeneration (e.g. acute macular degeneration , nonexudative age-related macular degeneration, exudative age-related macular degeneration); diabetic retinopathy; edema (e.g., Macular edema, cystoid macular edema, diabetic macular edema); glaucoma; Leber's congenital amaurosis; Leber's Barr hereditary optic neuropathy; macular telangiectasia; multifocal choroiditis; non-retinopathy diabetic retinal syndrome eye failure;ocular trauma;ocular tumors;proliferative vitreoretinopathy (PVR);retinopathy of prematurity;retinoschisis; Retinitis pigmentosa; retinal artery occlusive disease; retinal detachment; Stargardt's disease (fundus yellow); sympathetic ophthalmia; uveal spread; uveitic retinal disease; Usher syndrome; Vogt-Koyanagi-Hara or posterior ocular conditions associated with ocular laser or photodynamic therapy. can be.

[0169] The rAAV vectors of the invention can also be used to, inter alia, deliver oligonucleotides, drugs, imaging agents, inorganic nanoparticles, and the like. The present invention can facilitate the delivery, particularly targeted delivery, of nanoparticles, liposomes, and antibodies to target cells or tissues. rAAV vectors can also be used to specifically express non-coding DNA, RNA, or oligonucleotides. This can facilitate delivery, particularly targeted delivery, of nucleotides to target tissues.

[0170] The agent may be any pharmacologic agent known in the art and / or described herein. The rAAV molecules of the invention can be provided in any suitable form, either alone or in combination with other suitable compositions. The present invention may be administered in combination with other prophylactic and / or therapeutic agents.

[0171] The dosages and frequencies of administration provided herein are those that are therapeutically and prophylactically effective. The dosage and frequency of administration typically depend on the particular therapeutic or prophylactic agent being administered. The severity and type of disease, the route of administration, and the age, weight, response, and medical history of the patient. The procedure may vary according to the specific factors of each patient and is at the discretion of the practitioner and the circumstances of each patient. The appropriate regimen should be determined by considering such factors. For example, as reported in the literature and in the Physician's Desk Reference (56 th The dosages are selected by those skilled in the art according to the dosages recommended in the American Journal of Clinical Chemistry, Vol. 13, No. 1, 2002. The prophylactic and / or therapeutic agents may be administered repeatedly. The timing regimen in which such agents are administered and whether such agents are administered separately or as a mixture Several aspects of the procedure may be varied.

[0172] The amount of the agent of the invention that will be effective can be determined by standard clinical techniques. can be extrapolated from dose-response curves derived from in vitro or animal model test systems. For any agent used in the methods of the present invention, a therapeutically effective dose may be determined based on the cell culture The dose can be estimated initially from culture assays. The dose is determined in cell culture using the IC 50 (vinegar The concentration of the test compound that achieves a half-maximal inhibition of symptoms is determined by measuring the concentration of the test compound in the circulating plasma. Such information may be used in animal models to develop useful The plasma levels can be used, for example, by high performance liquid chromatography to determine the dosage more accurately. This can be measured by chromatography.

[0173] Prophylactic and / or therapeutic agents, and combinations thereof, may be suitably administered prior to use in humans. The compounds may be tested in any suitable animal model system. Such animal model systems include rats, mice, and , chickens, cows, monkeys, pigs, dogs, rabbits, etc. Any animal system well known in the art may be used. Such model systems include: It is widely used and well known to those skilled in the art. Animal model systems for CNS pathologies based on mice or other small mammals other than primates are available. It is used.

[0174] When the prophylactic and / or therapeutic agents of the invention are tested in animal models, they These compounds may be tested in clinical trials to establish their efficacy. The establishment of clinical trials is within the skill of the art. The method is carried out according to the general methodology known to those skilled in the art, and the optimal dosage and administration route of the agent of the present invention are determined. For example, clinical trials can be conducted to assess efficacy and toxicity in human patients. It may be designed to test the rAAV molecules of the invention for efficacy and toxicity.

[0175] The toxicity and efficacy of the prophylactic and / or therapeutic agents of the present invention can be evaluated, for example, by LD 50 (50 of the group % lethal dose) and ED 50 To determine the dose that is therapeutically effective in 50% of the population, This may be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between the dose and the effect is the therapeutic index, which is the LD 50 / ED 50 can be expressed as Prophylactic and / or therapeutic agents that exhibit large therapeutic indices are preferred. Alternatively, a therapeutic agent may be used, but minimizing potential damage to non-infected cells, thereby A delivery system that targets such drugs to the site of affected tissue to reduce side effects. Care should be taken to design

[0176] The rAAV molecules of the invention are generally useful in providing a desired therapeutic and / or prophylactic benefit. Data from cell culture assays and animal studies are consistent with those obtained from human studies. Ranges and / or schedules for dosages of prophylactic and / or therapeutic agents for use in The dosage of such drugs can be used to design a schedule that minimizes toxicity. No ED at all 50 The dosage will vary depending on the dosage form used and the circulating concentration range that includes may vary within this range depending upon the dosage and the route of administration utilized.

[0177] The therapeutically effective dosage of a rAAV vector for a patient is typically about 1×10 9 ~Approx. 1×1 0 16 Genomic rAAV vector, or approximately 1 × 10 10 ~Approx. 1×10 15 , about 1×10 1 2 ~Approx. 1×10 16 , or approximately 1 × 10 14 ~Approx. 1×10 16 Concentration of AAV genome The level of expression of the introduced gene varies depending on the dosage, Can be monitored to determine / adjust frequency, scheduling, etc.

[0178] Treatment of a subject with a therapeutically or prophylactically effective amount of an agent of the invention may include a single treatment. For example, a pharmaceutical composition containing an agent of the present invention may be administered once a day. In some embodiments, the agent may be administered once daily, twice daily, or three times daily. , every other day, once a week, twice a week, once every two weeks, once a month, once every six weeks, once every two months, The effective dosage of a given drug, e.g., the dual-dose regimen of the present invention, may be administered twice a year or once a year. It is also understood that the effective dosage of a drug comprising an antigen-binding molecule may increase or decrease during the course of treatment. It is understood.

[0179] In some embodiments, the ongoing treatment is the ongoing treatment of a chronic disease or disorder and / or or management, for example, on a long-term basis. In an embodiment, the agent of the present invention is administered for a period of time, for example, at least 6 months, at least 1 year. , at least 2 years, at least 5 years, at least 10 years, at least 15 years, at least It is administered for 20 years, or for the remainder of the life of the subject in need thereof.

[0180] The rAAV molecules of the invention can be administered alone or in combination with other prophylactic and / or therapeutic agents. Each prophylactic or therapeutic agent may be administered at the same or different time points sequentially and in any order. however, if not administered at the same time, they may not have the desired therapeutic or should be administered sufficiently close in time to provide a prophylactic effect. They may be administered separately in any appropriate form and by any suitable route.

[0181] In various embodiments, the different prophylactic and / or therapeutic agents are administered at intervals of less than one hour, about one hour apart. At intervals of about 1 hour to about 2 hours, at intervals of about 2 hours to about 3 hours, at intervals of about 3 hours to about At 4 hour intervals, at intervals of about 4 to 5 hours, at intervals of about 5 to 6 hours, at intervals of about 6 hours ~ about 7 hour intervals, about 7 hour to about 8 hour intervals, about 8 hour to about 9 hour intervals, about 9 hour intervals At intervals of about 10 hours to about 10 hours, at intervals of about 10 hours to about 11 hours, at intervals of about 11 hours to about 12 hours The doses are administered at intervals of 10 min, 24 h or less, or 48 h or less. In some configurations, two or more agents are administered within the same patient visit.

[0182] Methods of administering the agents of the invention include parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, intracutaneous, subcutaneous (including infusion or bolus injection), epidural, and epithelial or mucocutaneous or via mucosal linings (e.g., intranasal, oral, rectal, and intestinal mucosa) In certain embodiments, the transgene is expressed in the CNS. For example, if the vector is intended to be expressed in the mouse, the vector may be administered via lumbar puncture or intravenously. It is administered via a reservoir.

[0183] In certain embodiments, the agents of the invention are administered intravenously and in combination with other biologically active agents. may be administered to

[0184] In another particular embodiment, the agents of the present invention can be delivered in sustained release formulations, e.g., In this case, the formulation provides extended release and therefore extended half-life of the administered drug. Suitable controlled release systems for use include, but are not limited to, diffusion-controlled, solvent-controlled, and chemically-controlled. Diffusion-controlled systems include, for example, systems in which the release of molecules is limited by a diffusion barrier. The molecules of the present invention are encapsulated in a reservoir device in a manner controlled by permeation. Common reservoir devices include, for example, membranes, capsules, and microcapsules. Monolithic (matrix) devices include beads, liposomes, and hollow fibers. is another type of diffusion-controlled system, where the double antigen-binding molecule acts as a rate-controlling matrix. The agent of the present invention is dispersed or dissolved in a matrix (e.g., a polymer matrix). The release rate can be uniformly distributed throughout the matrix and the rate of release can be controlled by the amount of It is preferred for use in monolithic matrix devices. Suitable polymers include naturally occurring polymers, synthetic polymers and synthetically modified natural polymers. Polymers, as well as polymer derivatives, are included.

[0185] Any technique known to one of skill in the art can be used to produce sustained release formulations that contain one or more of the agents described herein. The compositions can be used to produce pharmaceutical preparations. Publication WO91 / 05548; PCT Publication WO96 / 20698; Ning et al. ,“Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sus tained-Release Gel,” Radiotherapy & Onco logy,39:179 189,1996;Song et al.,“Antibo dy Mediated Lung Targeting of Long-Circu lating Emulsions,” PDA Journal of Pharma Ceutical Science & Technology,50:372 397 ,1995;Cleek et al.,“Biodegradable Polyme ric Carriers for a bFGF Antibody for Car diovascular Application,” Pro.Intl.Symp. Control.Rel.Bioact.Mater.,24:853 854,199 7; and Lam et al., “Microencapsulation of Re combinant Humanized Monoclonal Antibody for Local Delivery,” Proc.Int'l.Symp.Con trol Rel.Bioact.Mater.,24:759 760,1997(So (each of which is incorporated herein by reference in its entirety). In, pumps can be used in controlled release systems (Langer, supra; Sefton,CRC Crit.Ref.Biomed.Eng.,14:20,19 87;Buchwald et al.,Surgery,88:507,1980; and Saudek et al., N. Engl. J. Med., 321:574,198 9). In another embodiment, the polymeric material comprises a double antigen-binding molecule, or an antigen-binding The fragments may be used to achieve controlled release of drugs (e.g., Medical A Applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974);Controlled Drug Bioavailability, Drug Product Design and Performance,Smol en and Ball (eds.), Wiley, NY (1984);Range r and Peppas, J., Macromol. Sci. Rev. Macromo. Chem., 23:61, 1983; see also Levy et al., Science nce,228:190,1985;During et al.,Ann.Neuro l.,25:351,1989;Howard et al.,J.Neurosurg .,7 1:105,1989);U.S. Patent No. 5,679,377;U.S. Patent No. 5, 916,597;U.S. Patent No. 5,912,015;U.S. Patent No. 5,989,463; U.S. Patent No. 5,128,326; PCT Publication No. WO99 / 15154; and PCT Publication No. (See, e.g., WO 99 / 20253). In yet another embodiment, the controlled release system comprises a It is placed in close proximity to the therapeutic target (e.g., the affected joint) and thus requires only a fraction of the systemic dose. (e.g., Goodson, in Medical Applications f Controlled Release,supra,vol.2,pp.115 (See Langer, Science, 138 (1984). Other controlled release systems are described in 249:1527 1533,1990.

[0186] rAAVs can also be used to deliver transgenes in vivo for scientific research, e.g., optogenetics. Recombinase delivery for gene knockdown with miRNA and conditional gene deletion It can be used for gene editing with CRISPR, etc.

[0187] 5.5. Pharmaceutical Compositions and Kits The present invention further provides a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and an agent of the invention. and the agent comprises an rAAV molecule of the invention. In some embodiments, the pharmaceutical composition The present invention includes an rAAV in combination with a pharma- ceutically acceptable carrier for administration to a subject. In embodiments, the term "pharmaceutical acceptable" refers to any compound that is approved by a federal or state regulatory agency. Approved or approved by the United States Pharmacopoeia or for use in animals, and more specifically in humans The term "carrier" means a carrier that is a pharmaceutical or other commonly recognized pharmaceutical product. The term "diluents, adjuvants (e.g., Freund's complete and incomplete adjuvants) with which the drug is administered may be used. Such pharmaceutical carriers refer to sterile liquids, e.g. For example, water and petroleum, animal, and plant oils, including, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. The oil may be of vegetable or synthetic origin. Water may be used when the pharmaceutical composition is administered intravenously. Saline solution and aqueous dextrose and glycerol are common carriers when used in pharmaceutical preparations. Solutions may also be used as liquid carriers, particularly injectable solutions. Suitable pharmaceutical excipients include: Starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, Silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride Thorium, dried skim milk, glycerol, propylene, glycol, water, ethanol Further examples of pharma- ceutically acceptable carriers, excipients, and stabilizers include buffers, Buffers, e.g., phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid low molecular weight polypeptides; proteins, e.g., serum albumin and gelatin; hydrophilic polypeptides polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, spargine, arginine or lysine; glucose, mannose, or dextrin including monosaccharides, disaccharides, and other carbohydrates; chelating agents, e.g., EDTA; sugar alcohols, e.g. for example, mannitol or sorbitol; a salt-forming counterion, for example, sodium; and / or or non-ionic surfactants, such as TWEEN, as known in the art. (trademark), polyethylene glycol (PEG), and PLURONICS (trademark). In addition to the above ingredients, the pharmaceutical composition of the present invention may also contain a lubricating agent. These compositions may include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents, and preservatives. The compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. It may take the form:

[0188] In certain embodiments of the invention, pharmaceutical compositions are provided for use in accordance with the methods of the invention. The pharmaceutical composition comprises a therapeutically and / or prophylactically effective amount of an agent of the invention. together with a commercially available carrier.

[0189] In certain embodiments, the agents of the invention are substantially pure (i.e., the active ingredient is a terpenic acid that inhibits its effect). (e.g., substantially free of substances that cause side effects that are limited or undesirable). In the present invention, the host or subject may be an animal, e.g., a mammal, e.g., a non-primate (e.g., bovine, pigs, horses, cats, dogs, rats, etc.) and primates (e.g. monkeys, e.g. cynomolgus In certain embodiments, the host is a human.

[0190] The present invention further provides kits that can be used in the above methods. The kit may include, for example, in one or more containers, one or more agents of the invention. In this embodiment, the kit comprises, in one or more containers, one or more other prophylactic or other agents useful for treating a condition. It further comprises a therapeutic agent.

[0191] The present invention also provides a method for producing a sealed container, such as an ampoule or sachet, indicating the amount of a drug or active agent. In one embodiment, the drug is packaged in a dry, sealed container. It may be supplied as a dry, sterile, lyophilized powder or water-free concentrate, e.g., in water or saline. The drug may be reconstituted with saline to the appropriate concentration for administration to a subject. Typically, the drug is administered at least at least 5 mg, more often at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or less Supplied as a dry, sterile, lyophilized powder in a sealed container in unit dosages of at least 75 mg. Lyophilized drugs should be stored in their original container at between 2 and 8°C and The drug should be administered within 12 hours after reconstitution, usually within 6 hours, 5 hours, 3 hours, or In an alternative embodiment, the agent of the present invention is or liquid form in a sealed container indicating the quantity and concentration of the active agent. The liquid form of the agent should have a concentration of at least 1 mg / ml and at least 2.5 mg / ml in a sealed container. l, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, It is provided at a dose of at least 15 mg / kg, or at least 25 mg / ml.

[0192] The compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions, e.g. non-pure or non-sterile compositions) and pharmaceutical compositions (i.e., suitable for administration to a subject or patient). The bulk drug composition may, for example, be a prophylactically or therapeutically effective amount of the present invention. The agents disclosed herein or combinations of those agents and a pharma- ceutically acceptable carrier are It may be used in the preparation of a unit dosage form comprising

[0193] The present invention relates to a pharmaceutical pack comprising one or more containers filled with one or more of the agents of the present invention. Also provided are one or more compounds useful for treating a target disease or disorder. Other prophylactic or therapeutic agents may also be included in the pharmaceutical pack or kit. a pharmaceutical composition comprising one or more containers filled with one or more of the ingredients of the pharmaceutical composition of the present invention. Optionally associated with such container(s) is: Established by a government authority that regulates the manufacture, use, or sale of drugs or biological products The notification may be in the form of a notice to the manufacturing, use, or marketing authority for human administration. This reflects approval by the FDA.

[0194] Typically, the components of the compositions of the invention are packaged, for example, in ampoules or containers indicating the amount of drug or active agent. As a dry, lyophilized powder or water-free concentrate in a sealed container such as a sachet The compositions are provided separately or mixed together in a unit dosage form. If administered, it should be done using an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, it can be dispensed in sterile water for injection or saline. An ampoule of the formulation may be provided so that the ingredients may be mixed prior to administration. EXAMPLES

[0195] The following example illustrates a method for identifying candidates for capsid modification via insertional mutagenesis. We report an analysis of surface-exposed loops on the AV9 capsid. The rRNA was modified to contain a 7-mer peptide designed based on the insulin heavy chain tail. The construction of AAV9 capsids is described in detail in

[11] . Briefly, it may be suitable for short peptide inserts. Three criteria were used to select potential surface loops: 1) the smallest number of adjacent loops; side chain interactions; 2) variable sequence and structure between serotypes (lack of conserved sequences); and 3) general A panel of peptide insert mutants was used to determine the potential of blocking neutralizing antibody epitopes targeted by the Construct and characterize individual mutants for viable capsid assembly, peptide surface exposure, and efficacy. The top candidates were then used as templates for insertion of the homing peptide. These peptide insertion points were used as a plate to target the rAAV vector. Further examples are described herein. We demonstrate improved transduction and tissue tropism for some of the modified AAV capsids.

[0196] 6.1. Example 1 - Analysis of AAV9 Capsids Figures 1 and 2 show the amino acid sequence of the adenosine 1A gene in comparison with the VR-IV of other AAVs (Figure 1). Analysis and protein model of the human avian-associated virus type 9 variable region 4 (AAV9 VR-IV) As can be seen, the AAV9 VR-IV has a three-fold spyware. Further analysis will reveal that VR-IV and VR- There are several side chain interactions between V and VR-IV, and the sequence and structure of VR-IV are consistent with those of A Variable among AV serotypes and, moreover, commonly targeted neutralizing antibody epitopes This indicates that there is the potential to disrupt the expression of modified capsids and thereby reduce the immunogenicity of the modified capsids. was doing.

[0197] 6.2. Example 2 - Construction of AAV9 mutants Eight AAV9 mutants were generated, each with a different insertion point in the VR-IV loop to insert a heterologous peptide. The heterologous peptide was constructed to contain pRGNX10 as shown in Table 4. FLA inserted immediately following the following residues in the vector identified as 90-1097 It was a G tag. [Table 4]

[0198] 6.3. Example 3 - Packaging Efficiency Analysis FIG. 3 shows wild-type AAV9 and eight candidate rAAV9 vectors (1090, 1091, 1 1092, 1093, 1094, 1095, 1096, and 1097) The candidate vectors showed high packaging efficiency in terms of gene copy number (GC / mL). Each contains a FLAG insertion at a different site within VR-IV of AAV9. All vectors were packaged with the luciferase transgene in 10 mL cultures. to facilitate determination of which insertion points disrupted capsid packaging; Bars represent the standard error of the mean.

[0199] As can be seen, all candidates package with high efficiency.

[0200] 6.4. Example 4 - Analysis of Surface FLAG Exposure Figure 4. Transduction vector immunoprecipitation confirmed by binding to anti-FLAG resin. In addition, eight candidate rAAV9 vectors (1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 4, 1095, 1096, and 1097), showing the surface exposure of the FLAG insert in each of these Binding to anti-FLAG induces the formation of capsids that display the peptide insert on their surface. 1 shows an insertion point that allows

[0201] The transduced cells were lysed and centrifuged. 500 μL of cell culture supernatant was added to 20 μL of SDS-PAG loaded onto agarose-FLAG beads and also directly loaded onto gels Elution was performed with E loading buffer. For negative control, 293 containing no FLAG insert was used. -ssc supernatant was used.

[0202] As can be seen, 1090 has the lowest titer and the fewest T cells of the candidate vectors. The positive control also showed extremely low titers. It appears that an insufficient amount of the positive control was loaded for visualization on PAGE.

[0203] 6.5. Example 5 - Analysis of Transduction Efficiency 5A-5B show wild-type AAV9 (9-luc) or eight candidate rAAV9 vectors. -(1090, 1091, 1092, 1093, 1094, 1095, 1096, and 1 097) as a transgene, The transduction efficiency of Lec2 cells transduced with a capsid vector carrying the gene was The activity is shown as a function of luciferase activity, taking the activity of 9-luc as 100%. as luminescents (Figure 5A) or as relative light units (R LU) (Figure 5B).

[0204] CHO-derived Lec2 cells were grown in αMEM and 10% FBS. Approximately 2×10 8 The cells were transduced with the GC vector at an MOI of approximately 10,000, and the Vira Lec2 cells were transfected with Ductin reagent at an MOI of approximately 10,000 GC / cell. Similar results were obtained for those treated with 40 μg / mL zinc chloride (ZnCl2) (Results not shown). Lec2 cells were able to tolerate the proline auxotrophy from CHO. This is a share request.

[0205] As can be seen, the transduction efficiency in vitro was significantly higher than that of wild-type AAV9 (9-luc ) is lower than that obtained using the homing pair. The introduction of the peptide is in non-target cells (such as 293, Lec2, or HeLa). may reduce in vitro gene transfer, whereas in vitro genetic have been shown to significantly increase offspring transfer (e.g., Nicklin et al. 001; and Grifman et al. 2001).

[0206] 6.6. Example 6 - Packaging efficiency as a factor of inserted peptide composition and length analysis FIG. 6A shows the SEQ ID NO:118 of AAV9 capsids of various peptide lengths and compositions. The insert immediately following 454 affected the efficiency of AAV particle production in packaging cell lines. The figure shows a bar graph illustrating the effect that the 10 peptides of various compositions and lengths can have on the protein synthesis. The peptide was inserted after S454 (between residues 454 and 455) in AAV9 VR-IV. Transfected suspension HEK29 cells 5 days after transfection. qPCR was performed on harvested supernatants of 3 cells. The results shown in the bar graph are shown in the insert. The characteristics and length of the product were confirmed by the production of AAV particles at high titers in 293 cells and the packaging (Error bars indicate a discrepancy in the y-axis.) (The standard error of the mean length of the peptides marked with arcs is shown).

[0207] A capsid containing a homing peptide of the following peptide sequence (Table 5) at the S454 insertion site: We investigated the AAV9 vector carrying the sid protein. Suspension-adapted HEK293 cells were cultured at 1 1 x 10 cells were cultured in 0 mL of medium one day before transduction. 6 cells / mL. Triple plasmid DNA transfection was performed using PEIpro® (Polypus transfection kit). Transfection was performed using a DNA:PEI ratio of 1:1.75. Five days after transfection, the cells were spun down and the supernatant was harvested and stored at -80°C. [Table 5]

[0208] Transfected suspension HEKs harvested 5 days after transfection qPCR was performed on the supernatants of 293 cells. Samples were subjected to DNase I treatment. Remove residual plasmid or cellular DNA, then heat-treat to inactivate DNase I The samples were then subjected to TaqMan Universal PCR. Master Mix, No AmpEraseUNG (ThermoFisherSc ientific) and the PolyA sequence packaged in the transgene construct. Titering was performed via qPCR using primers / probes for the columns. Standard curves were generated. , was established using the RGX-501 vector BDS.

[0209] Peptide inserts immediately following S454 ranging in length from 5 to 10 amino acids had adequate potency. While we generated AAV particles that expressed the 12 amino acid long peptide insert, There may be an upper size limit due to the significant packaging defects observed.

[0210] 6.7. Example 7 - Homing peptides, when inserted after S454, show in vivo Altering the transduction properties of AAV9 in vitro. Figures 6B-E show (Figure 6B) Lec2 cell line (sialic acid-deficient epithelial cell line) and (Figure 6C) HT -22 cell line (neuronal cell line), (Figure 6D) hCMEC / D3 cell line (brain endothelial cell line), ), and (Figure 6E) fluorescent images of cell cultures of the C2C12 cell line (a muscle cell line). The AAV9 wild-type and S454-inserted homing peptides in Table 5 containing the GFP transgene The tidocapsid was used to transduce the aforementioned cell lines.

[0211] Cell lines were incubated with 5–20 × 10 3 9 cells / well (depending on cell line) The cells were seeded in 6 wells. The cells were then transfected with the AAV9-GFP vector (with or without insert). ) at 1×10 10 Transduce with particles / well and measure expression rates according to differences in each cell line , and analyzed via Cytation5 (BioTek) 48–96 h after transduction. Lec2 cells were cultured as in Example 5, and the blood-brain barrier hCMEC / D3 (EMD M HILIPORES cells were cultured according to the manufacturer's protocol, and HT-22 and HUH C2C12 myoblasts were cultured in DMEM and 10% FBS. 1% FBS and cultured in DMEM supplemented with 2% horse serum and 0.1% insulin. The cells were then allowed to differentiate for 3 days before transfection. AAV9.S454.FLAG was used in All cell types showed low transduction levels.

[0212] The images show the S in the AAV9 capsid protein compared to the unmodified AAV9 capsid. When inserted after 454, it could alter the transduction properties of AAV9 in vitro. The results show that P7 (TfR1 peptide, HAIYPRH (SEQ ID NO: 17)) showed the highest transduction rate, followed by P9 (TfR3 peptide, R P4 (kidney 1 peptide, LPVAS (SEQ ID NO: 19)) was followed by P5 (kidney 1 peptide, LPVAS (SEQ ID NO: 20)). No. 13)) was slightly higher than that of AAV9 wild type for all cell types. The transduction rates of brain endothelial hCMECs were compared to Lec2 and HT-22 cell line cultures. In / D3 cell line and C2C12 muscle cell line cultures, P6 (muscle 1 peptide, ASSLN A higher transduction rate was observed for the P1 vector (P1A (SEQ ID NO: 14)). The ends are not included in the image due to low transduction efficiency, and the P8 vector is not included due to low titer. It was.

[0213] 6.8. Example 8 - Analysis of Human Axonemal Dynein (HAD) 7A-7M show the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. The amino acid sequences of each are shown.

[0214] 6.9. Example 9 - Analysis of AAV Capsids for Peptide Insertion Sites Figure 8 shows the initiation codon of VP2 highlighted in grey, variable region 1 (VR-I), variable region 4 (VR-F), and (VR-IV), and in or near variable region 8 (VR-VIII) of human axonemal dynein AAV1-9e, rh10, rh20, rh39, rh7 within the insertion site for peptides The alignment of the hu.4 and hu.37 capsid sequences shows the possible roles of each capsid protein. The specific insertion site within variable region 8 (VR-VIII) is indicated by the symbol "#". (after amino acid residue 588 according to the AAV9 amino acid numbering).

[0215] 6.10. Example 10 - Construction of rAAV Capsids Containing ARA290 FIG. 9 shows the ARA290 site between Q588 and A589 of the AAV9 capsid amino acid sequence. The amino acid sequence for the recombinant AAV9 vector capsid containing the peptide insert is shown. (SEQ ID NO: 153).

[0216] 6.11. Example 11 - Ratio of AAV genome copies / μg genomic DNA for various vectors comparison Figure 10 shows the AAV vectors: AAV9; AAV.PHP.eB; AAV.hDyn ( Between 588 and 589, there is a TLAAPFK ( AAV9) having SEQ ID NO:2); AAV.PHP.S; and AAV.PHP.SH ( GFP (green fluorescent protein) expressed in mouse brain cells after administration of Copies of the transgene are indicated.

[0217] AAV.PHP.B has no other amino acid modifications to the capsid sequence of AAV9 Capsid with TLAVPFK (SEQ ID NO: 27) insertion in the capsid. V.PHP.eB has two amino acid modifications in the capsid sequence upstream of the PHP.B insert. A capsid having a TLAVPFK (SEQ ID NO: 27) insertion in the AAV9 capsid. (See also Table 10). Table 6A summarizes the capsids utilized in the study. [Table 6A]

[0218] Materials and Methods AAV9, AAV.PHPeB, AAV.hDyn, A The AV.PHP.S and AAV.PHP.SH constructs were prepared and diluted in 1x PBS + The mice were formulated in 0.001% Pluronic. Female C57BL / 6 mice were cultured on day 1. Five groups of female C57BL / 6 mice were randomized into treatment groups based on body weight: According to Table 6B, AAV9.GFP, AAV.PHPeB.GFP, AAV.hDyn. GFP, AAV.PHP.S.GFP or AAV.PHP.SH.GFP were incubated with 100 μg / ml of 100 μl ... The dose volume was 10 mL / kg (0.200 mL / 20 g mouse). Mice were 8-12 weeks old on the day of initiation. On the 15th day after administration, animals were euthanized and brain Collect peripheral tissues including tissue, liver, forelimb biceps, heart, kidney, lung, ovary, and sciatic nerve. Ta. [Table 6B]

[0219] Number of vector genomes per µg of brain genomic DNA using quantitative PCR (qPCR) Brain samples from injected mice were processed and purified using Qiagen Blood Isolate genomic DNA using the and Tissue Genomic DNA kit A primer-probe combination specific for eGFP was used according to the standard curve method. and analyzed using a QuantStudio5 instrument (Life Technologies Inc.). qPCR assays were performed.

[0220] The AAV vector genome copies per μg of brain genomic DNA injected with AAV.hDyn were In mice receiving the vaccine, all other AAV serotypes: AAV9, AAV.PHPeB, PH It was at least 1 log higher than PS and PHP.SH (see Figure 10). As seen in the previous study, GC / μg genomic DNA was highest in AAV.hDyn. This is the sequence "TLAAPFK" (SEQ ID NO: 2) between residues 588-589 of the AAV9 capsid. AAV9 capsid containing a peptide insert (a peptide from human axonemal dynein) The study showed that AAV.hDyn carrying eGFP was administered systemically to five mice. demonstrated transduction in mouse brain at an average of over 1E04GC / μg transgene. However, the "TLAVPFK" (SEQ ID NO: 27) sequence (a peptide from mouse dynein) Other modified AAV9 capsids, including vectors containing AAV.PHPeB Systemic treatment resulted in phenotypic changes in mouse brain below 1E03GC / μg transgene. The implementation was demonstrated.

[0221] 6.12. Example 12 - Use of tissue-homing rAAV vectors in methods of treatment Identify disorders that can be treated / prevented by providing a nucleic acid (transgene) (Table 3 A subject having a disorder associated with a target tissue is identified. The subject is administered a first amount of The rAAV vector of the invention is administered, and the vector homes to and is delivered to the target tissue. The capsid protein includes a peptide insert carrying a transgene to be delivered to the host. In this case, a therapeutically effective amount of the transgene is delivered to the target tissue to provide a therapeutic or prophylactic benefit. A second or third dose of the vector is administered to the subject until delivery.

[0222] In some embodiments, a method is provided for administering a transgene to the retina, comprising: This allows the AAV.hDyn capsid carrying the transgene to be delivered intravenously and systemically. or intravitreally.

[0223] 6.13. Example 13 - rAAV Capsid Containing TLAAPFK (SEQ ID NO:2) construction FIG. 11A shows the amino acid sequence TLAAPF between Q588 and A589 of VR-IIIV. Amino acid sequence for recombinant AAV9 vector capsid containing peptide insert of K (SEQ ID NO:2) The amino acid sequence is shown. The inserted peptide is in bold.

[0224] FIG. 11B shows the amino acid sequence TLAAPFK between S268 and S269 of VR-III. Amino acid sequence for recombinant AAV9 vector capsid containing a peptide insert of (SEQ ID NO:2) The amino acid sequence is shown. The inserted peptide is in bold.

[0225] FIG. 11C shows the amino acid sequence TLAAPFK( Amino acid sequence for recombinant AAV9 vector capsid containing a peptide insert of SEQ ID NO:2 The sequences are shown, with the inserted peptide in bold.

[0226] 6.14. Example 14 - rAAV Capsid Containing KMQVPFQ (SEQ ID NO:1) construction FIG. 12A shows the amino acid sequence KMQVPF between Q588 and A589 of VR-VIII. Amino acid sequence for recombinant AAV9 vector capsid containing peptide insert Q (SEQ ID NO: 1) The amino acid sequence is shown. The inserted peptide is in bold.

[0227] FIG. 12B shows the amino acid sequence KMQVPFQ between S268 and S269 of VR-III. Amino acid sequence for recombinant AAV9 vector capsid containing a peptide insert of (SEQ ID NO:1) The amino acid sequence is shown. The inserted peptide is in bold.

[0228] FIG. 12C shows the amino acid sequence KMQVPFQ( Amino acid sequence for recombinant AAV9 vector capsid containing a peptide insert of SEQ ID NO:1 The sequences are shown, with the inserted peptide in bold.

[0229] 6.15. Example 15 - rAAV Capsid Containing QQAAPSF (SEQ ID NO:3) construction FIG. 13A shows the amino acid sequence QQAAPS between Q588 and A589 of VR-VIII. Amino acid sequence for recombinant AAV9 vector capsids containing a peptide insert of F (SEQ ID NO:3) The amino acid sequence is shown. The inserted peptide is in bold.

[0230] FIG. 13B shows the amino acid sequence QQAAPSF between S268 and S269 of VR-III. Amino acid sequence for recombinant AAV9 vector capsid containing a peptide insert of (SEQ ID NO:3) The amino acid sequence is shown. The inserted peptide is in bold.

[0231] FIG. 13C shows the amino acid sequence QQAAPSF( Amino acid sequence for recombinant AAV9 vector capsid containing a peptide insert of SEQ ID NO:3 The sequences are shown, with the inserted peptide in bold.

[0232] 6.16. Example 16 - rAAV Capsid Containing RYNAPFK (SEQ ID NO:4) construction FIG. 14A shows the amino acid sequence RYNAPF Amino acid sequence for recombinant AAV9 vector capsid containing a peptide insert of K (SEQ ID NO: 4) The amino acid sequence is shown. The inserted peptide is in bold.

[0233] FIG. 14B shows the amino acid sequence RYNAPFK between S268 and S269 of VR-III. Amino acid sequence for recombinant AAV9 vector capsid containing a peptide insert of (SEQ ID NO: 4) The amino acid sequence is shown. The inserted peptide is in bold.

[0234] FIG. 14C shows the amino acid sequence RYNAPFK( Amino acid sequence for recombinant AAV9 vector capsid containing a peptide insert of SEQ ID NO:4 The sequences are shown, with the inserted peptide in bold.

[0235] 6.17. Example 17 - rAAV Capsid Containing LKLPPIV (SEQ ID NO:5) construction FIG. 15A shows the amino acid sequence LKLPPI between Q588 and A589 of VR-VIII. Amino acid sequence for recombinant AAV9 vector capsids containing a peptide insert of V (SEQ ID NO:5) The amino acid sequence is shown. The inserted peptide is in bold.

[0236] FIG. 15B shows the amino acid sequence LKLPPIV between S268 and S269 of VR-III. Amino acid sequence for recombinant AAV9 vector capsid containing a peptide insert of (SEQ ID NO:5) The amino acid sequence is shown. The inserted peptide is in bold.

[0237] FIG. 15C shows the amino acid sequence LKLPPIV( Amino acid sequence for recombinant AAV9 vector capsid containing a peptide insert of SEQ ID NO:5 The sequences are shown, with the inserted peptide in bold.

[0238] 6.18. Example 18 - rAAV Capsid Containing PFIKPFE (SEQ ID NO:6) construction FIG. 16A shows the amino acid sequence PFIKPF Amino acid sequence for recombinant AAV9 vector capsids containing peptide inserts of E (SEQ ID NO: 6) The amino acid sequence is shown. The inserted peptide is in bold.

[0239] FIG. 16B shows the amino acid sequence PFIKPFE between S268 and S269 of VR-III. Amino acid sequence for recombinant AAV9 vector capsid containing a peptide insert of (SEQ ID NO:6) The amino acid sequence is shown. The inserted peptide is in bold.

[0240] FIG. 16C shows the amino acid sequence PFIKPFE( Amino acid sequence for recombinant AAV9 vector capsid containing a peptide insert of SEQ ID NO:6 The sequences are shown, with the inserted peptide in bold.

[0241] 6.19. Example 19 - rAAV Capsid Containing TLSLPWK (SEQ ID NO: 7) construction FIG. 17A shows the amino acid sequence TLSLPW between Q588 and A589 of VR-VIII. Amino acid sequence for recombinant AAV9 vector capsid containing peptide insert of K (SEQ ID NO: 7) The amino acid sequence is shown. The inserted peptide is in bold.

[0242] FIG. 17B shows the amino acid sequence TLSLPWK between S268 and S269 of VR-III. Amino acid sequence for recombinant AAV9 vector capsid containing a peptide insert of (SEQ ID NO: 7) The amino acid sequence is shown. The inserted peptide is in bold.

[0243] FIG. 17C shows the amino acid sequence TLSLPWK( Amino acid sequence for recombinant AAV9 vector capsid containing a peptide insert of SEQ ID NO:7 The sequences are shown, with the inserted peptide in bold.

[0244] 6.20. Example 20 - rAAV Capsid Containing LGETTRP (SEQ ID NO: 15) Construction FIG. 18A shows the amino acid sequence LGETTR between N590 and T591 of VR-VIII. An antigen for recombinant AAV8 vector capsids containing a peptide insert of P (SEQ ID NO: 15) The amino acid sequences are shown, with the inserted peptides in bold.

[0245] FIG. 18B shows the amino acid sequence LGETTRP between A269 and T270 of VR-III. Amino acid sequence for recombinant AAV8 vector capsid containing a peptide insert of (SEQ ID NO: 15) The amino acid sequence is shown. The inserted peptide is in bold.

[0246] FIG. 18C shows the amino acid sequence LGETTRP( Amino acid sequence for recombinant AAV8 vector capsid containing a peptide insert of SEQ ID NO: 15 The amino acid sequence is shown. The inserted peptide is in bold.

[0247] 6.21. Example 21 - rAAV Capsule Containing LALGETTRP (SEQ ID NO: 16) Sid's Construction FIG. 19A shows the amino acid sequence LALGET between N590 and T591 of VR-VIII. For recombinant AAV8 vector capsids containing a peptide insert of TRP (SEQ ID NO: 16) The amino acid sequence of the inserted peptide is in bold.

[0248] FIG. 19B shows the amino acid sequence LALGETT between A269 and T270 of VR-III. For recombinant AAV8 vector capsids containing a peptide insert of RP (SEQ ID NO: 16) The amino acid sequence is shown, with the inserted peptide in bold.

[0249] FIG. 19C shows the amino acid sequence LALGETTR between T453 and T454 of VR-IV. An antigen for recombinant AAV8 vector capsids containing a peptide insert of P (SEQ ID NO: 16) The amino acid sequences are shown, with the inserted peptides in bold.

[0250] 6.22. Example 22: Evaluation of modified capsids in vitro and in vivo The AAV capsid sequence is modified by either peptide insertion or directed mutagenesis, Pooling and packaging of barcoded libraries with a GFP expression cassette Next-generation sequencing (NGS) and quantitative PCR were then used to identify in vitro The modified vector was used in ro assays and for in vivo biodistribution in mice. AAV.hDyn was identified as a high-potency brain transduction vector from this pool. The transduction profile was determined and further evaluated in individual delivery studies in mice. We also performed immunohistochemistry of brain sections to understand the cellular tropism of this vector. A disciplinary analysis was conducted.

[0251] 6.22.1 Example 22A - In vitro studies of transduction across the blood-brain barrier The ability of the engineered capsids to cross the blood-brain barrier was examined using human CMEC / D3 BBB cells (SCC 066, Millipore-Sigma) was used to in vitro Transwell Assays were tested (see Figures 20A-20B). More specifically, Sade, H. et al.(2014 PLoS ONE 9(4):e96340)A human Blood-Brain Barrier transcytosis assay reveals Antibody Transcytosis influenced by pH-dependent Receptor Binding,April 2014, Vol. 9, Issue 4; and Zhang, X., Blood-brai n barrier shuttle peptides enhance AAV t ransduction in the brain after systemic administration,2018 Biomaterials 176:71- The assay was essentially adapted from 83. Briefly, 5 × 10 4 hCMEC / D3 cells cells / cm 2 were seeded onto collagen-coated Transwell inserts in 12-well plates. Each insert contained 500 μL of medium, and the lower chamber contained 1 mL of medium. The medium was changed every other day. The supernatant was removed 10 days after seeding (Zeta (b) Time (0). At this time, 1 x 10 9 GC vectors in the upper insert chamber Cells were transduced by adding 10 μL of the supernatant sample from the lower chamber to the Pulls were removed for testing at intervals of 0.5, 3, 6, and 23 hours after transduction. Each condition (vector) was tested in duplicate and titers were analyzed by qPCR against PolyA. The measurements were performed in triplicate.

[0252] Figures 20A-20B show the ability of AAV.hDyn (Amin) to cross the blood-brain barrier (BBB) ​​cell layer. Regarding AAV9, which has TLAAPFK (SEQ ID NO: 2) between amino acid residues 588 and 589, In vitro Transwell assays using AAV.hDyn (Figure 20A) and AAV.hDyn (Figure 20B) were performed. (indicated by an inverted triangle in the figure) earlier than AAV9 (square) and Results show that IgG1 crosses the BBB cell layer of the assay more quickly and to a greater extent than IgG1 (circle). The results are shown in Figure 20B. The developed in vitro assay demonstrated improved BBB function. Predicts translocation trafficking and similar assays to predict targeting to other organs It can also be used.

[0253] 6.22.2 Example 22B - Transduction and Biodistribution of Modified Capsids 6.22.2.1 Materials and methods after position S454 in VR-IV (Table 7) or on the VR-VIII surface of the AAV capsid By inserting various peptide sequences after position Q588 in the exposed loop, as well as At amino acid 137 (AAV4, AAV4-4, and AAV5) or at amino acid 138 (A AV1, AAV2, AAV3, AAV3-3, AAV6, AAV7, AAV8, AAV9 , AAV9e, rh.10, rh.20, rh.39, rh.74, and hu.37) ( The widely used insert is inserted after the VP2 initiation codon, which begins at 14C (Figure 8). For AAV capsids that have been used (including AAV8, AAV9, and AAVrh.10), Capsid modifications were performed (see also Table 10 for the given capsid sequences). Several amino acid mutations were also used to modify the capsid. Yost et al. al.,Structure-guided engineering of surf ace exposed loops on AAV Capsids.2019.AS GCT Annual Meeting; and Wu et al., 2000 J. Vi. See also rology (supra). Packaging efficiency is It was confirmed that there was no adverse effect after any of the capsid modifications.

[0254] rAAV with a given modified capsid was synthesized using Lec2 as described above in Example 5. Transduction was tested in vitro in Lec2 cells. The modified AAVs tested for transduction were: eB5 compared to AAV9 88 Ad, eB 588 Hep, eB 588 p79, eB 588 Rab, A AV9 588 Ad, AAV9 588 Hep, AAV9 588 p79, AAV 9 588 Rab, eB VP2 Ad, eB VP2 Hep, eB VP2 p7 9, eB VP2 Rab, AAV9 VP2 Ad, AAV9 VP2 Hep, AA V9 VP2 p79, AAV9 VP2Rab. The identity of the AAV capsid is described below. See Table 7B below.

[0255] To test biodistribution, the modified AAV was immunized with specific capsids corresponding to each individual capsid. The plasmid was packaged together with an eGFP transgene cassette containing a barcode. To improve the efficiency of screening, novel barcoded vectors were pooled and injected into mice. Ta.

[0256] To analyze the biodistribution of genetically engineered AAV vectors, we used vectors encoding GFP. Various vectors were prepared and formulated in 1x PBS + 0.0001% pluronic acid. All vectors allow for next generation sequencing (NGS) library (pool) preparation The three vectors were constructed using a cis plasmid containing a 10 bp barcode. Vector pools (Study 1, Study 2, and Study 3 vectors) were prepared from five female C Cohorts of 57Bl / 6 mice were injected intravenously. The dosing volume was 10 mL / kg ( The dose was 0.2 mL / 20 g mouse.

[0257] Mice were randomized into treatment groups based on their body weight on day 1 and their age at the start date was between 8 and 12 months. On the 15th day after administration, the animals were euthanized and the brain, kidneys, liver, sciatic nerve, and lungs were examined. Peripheral tissues including erythrocyte, heart, and muscle tissue were collected. Selected capsids from the pool were individually The same protocol was followed in individual injection studies.

[0258] DNeasy Blood and Tissue kit (695 Genomic DNA (gDNA) was isolated from tissue samples using the ELISA kit (06). The barcode region was then cloned into NG as recommended by the manufacturer (Illumina). S and Unique Dual Index (UDI) and Multiplex Sequencing The primers used for amplification were overlapping for the strategy. Illumina using Microkit v2 (MS-103-1001 / 1002) Using MiSeq, each barcoded AAV was identified for each sample collected from the mice. The relative abundance of the vectors was determined. Thus, the relative abundance of each vector in Tables 7A-C below Samples are barcoded as above to allow identification of each read, sorted, and then Final data analysis was performed. Data were normalized based on the composition of AAV in the original injected pool. q using primer-probe combinations specific for normalized and barcoded samples Total genome copy numbers obtained from PCR analysis were used to quantitate. [Table 7A] [Table 7B] [Table 7C]

[0259] In studies where selected capsids from the pool were injected individually, qPCR was used The number of vector genomes per μg of tissue genomic DNA was determined according to the standard curve method. Using eGFP-specific primer-probe combinations, QuantStudio qPCR was performed using o 5 (Life Technologies, Inc.) (Figure 22). .

[0260] From studies in which individual vectors were injected into mice for characterization, formalin-fixed mouse Brains were cut at a thickness of 40 μm using a vibrating blade microtome (VT1000S, Leica). Section and probe free-floating sections with an antibody against GFP to examine the cellular distribution of the delivered vector. Ta.

[0261] More specifically, fixed brains from AAV.hDyn-injected mice were cultured in a Vibratome. The sections were then sectioned using a microscope (Leica, VT-1000) and stained with an anti-GFP antibody (AB30 80, Millipore Sigma), Vectastain ABC kit(P K-6100, Vector Labs) and DAB Peroxidase kit ( GFP expression was assessed using a GFP-4100 vector (Vector Labs). AAV. Wide distribution of GFP-expressing cells throughout the brain in hDyn-injected mice This included distribution in the brain cortex, striatum, and hippocampus. ~23C shows images from these regions, with scale bars of 400 um ( (discussed below).

[0262] 6.22.2.2 Results The results are shown in Figure 21, Figures 22A-22H, and Figures 23A-23C.

[0263] Data from Lec2 cell transduction assays are not shown. (Peptide TILSRSTQTG (SEQ ID NO: 22) inserted after position 588 (Table 1b AAV9 carrying DLC-AS2 in mice showed significantly higher phenotypes than wild-type AAV9 Transduction (4-fold) and AAV9 VP2 Ad (peptide S inserted after position 138) AA having ITLVKSTQTV (SEQ ID NO: 21) (DLC-AS1 in Table 1b) V9), AAV9 VP2 Hep (peptide TILSRS inserted after position 138 AAV9 with TQTG (SEQ ID NO: 22) (DLC-AS2 in Table 1b), and AAV9 VP2 Rab (peptide RSSEEDKSTQ inserted after position 138) AAV9) having TT (SEQ ID NO: 26) (DLC-AS4 in Table 1b) 9 showed slightly higher transduction of Lec2 cells compared with the other AAVs assayed. , which showed lower levels of transduction than AAV9.

[0264] Figure 21 shows the results of next-generation sequencing (NGS) analysis of brain gDNA. We determined the relative abundance of the capsid pool delivered to the mouse brain after intravenous injection. The data were normalized based on the composition of AAV in the initial injected pool. , from qPCR analysis using a primer-probe combination specific for the eGFP sequence. The total genome copy number obtained was used for quantification. Data shown are from three different experiments. The dotted lines indicate which vectors were pooled together. The parental AAV9 was used as a standard and was included in each pool. The "BC" identifier is listed in Table 7A above. As shown in 7B and 7C.

[0265] Figures 22A-22H show in vivo expression of AAV.hDyn in female C57Bl / 6 mice. The results show a vo transduction profile in naïve mice or in mice transduced with AAV9 or AAV In mice injected with either AV.hDyn or IFN-α, the brain (Fig. 22A), liver (Fig. 22B), and IFN-α were significantly increased. ), heart (Figure 22C), lungs (Figure 22D), kidneys (Figure 22E), skeletal muscles (Figure 22F), ischium Copy numbers / microgram gDNA in nerve (Figure 22G) and ovary (Figure 22H) AAV.hDyn shows increased brain biodistribution compared to AAV9. The AAV vector genome copies per μg of brain genomic DNA was was at least 1 log higher in mice administered AAV.hDyn compared with control.

[0266] 23A-23C show images from the areas analyzed in the immunohistochemical analysis described above. Scale bar is 400 μm. The distribution of GFP from AV.hDyn is shown in the striatum (Figure 23A), hippocampus (Figure 23B), and ), and cortex (Fig. 23C). We demonstrated global transduction of the brain.

[0267] 6.22.2.3 Conclusion By insertion in the surface exposed loops of VR-IV and VR-VIII or by specific The AAV capsid modifications carried out either by amino acid mutations or by The results showed that the production efficiency of the 100% 15% 16% 17% 18% 20% 25% 30% 35% 40% 45% 50% 60% I was able to do it.

[0268] Intravenous administration of AAV.hDyn to mice demonstrated superior clonal expression compared with other modified AAV vectors and Results in higher relative abundance of viral genome and greater brain cell transduction than AAV9 Ta.

[0269] 6.23. Example 23 - Homing peptide kidney 1C exhibits improved traits following systemic delivery Show the introduction. The AAV capsid sequence was modified with either peptide insertions, pooled, and expressed as GFP. A barcoded library was obtained that was packaged with an expression cassette. Next-generation sequencing (NGS) and quantification of AV9 vector biodistribution profiles The in vivo assay was performed in mice using PCR. Between G455, the amino acid sequence CLPVASC (SEQ ID NO: 12) (Kidney 1C) or ASS The recombinant AAV9 vector containing the peptide insert LNIA (SEQ ID NO: 14) (Muscle 1) , demonstrating increased transduction efficiency in the kidney compared to the liver (FIG. 24).

[0270] 6.23.1 Materials and methods The VR-IV surface-exposed loop at position S454 of the AAV capsid is followed by various homing peptides. Capsid modifications to AAV9 were performed by inserting peptide sequences. The efficiency of capsid synthesis is not adversely affected after any of these capsid modifications on a small scale. The peptide sequences are shown in Table 8 below. All modified AAVs were and an eGFP transgene cassette containing specific barcodes corresponding to individual capsids. These new bars were packaged together to improve the efficiency of screening. The encoding vectors were pooled (as described above in Example 22B; (See Study 3, Table 7C).

[0271] The genetically altered AAV vectors were compared as described in Example 22B for the altered vectors in Study 3. The AAV in the initial injected pool was then intravenously injected into mice as described above. Data were normalized based on the composition of the primer-probe pairs specific for the eGFP sequence. Total genome copy number was quantified using qPCR analysis using the combined Liver tissue targeting was examined more closely.

[0272] 6.23.2 Results and Conclusions FIG. 24 shows the expression of different homing peptide inserts (Table 8) in female C57Bl / 6 mice. Figure 1 shows the kidney-to-liver ratio of in vivo transduction of AAV9 S454 vectors carrying Kidney vs liver transduction versus total kidney transduction of pools for engineered capsids was used for the calculation. AAV9 S454 kidney 1 and AAV9 S454 kidney 2 (kidney 1C) shows increased renal biodistribution compared to the parental AAV9 vector. shows increased liver transduction compared to kidney with a ratio of approximately 0.25, whereas kidney Insertion of the muscular peptide 1C (and also muscle 1) resulted in an increase in this ratio to approximately 1.0. The AAV vector genome copies per μg of kidney gDNA were Mice administered AAV9 S454 muscle 1 or AAV9 S454 spleen 1 were significantly more aggressive than all other AAV9 It was at least 5-fold higher compared to the S454 vector (see FIG. 24). [Table 8]

[0273] This was achieved by inserting different homing peptides in the surface-exposed loop VR-IV. The AAV capsid modifications described herein do not affect their packaging efficiency. Similar titers could be obtained in the above production systems.

[0274] Intravenous administration of AAV9 S454 Kidney 1 and AAV9 S454 Kidney 1C to mice , a higher viral genome than other modified AAV9 vectors tested and the parental AAV9 vector. AAV9 S454 Kidney 1 Intravenous administration of the AAV9 S454 muscle 1 vector into mice also resulted in lower liver function. This resulted in cell transduction.

[0275] 6.24. Example 24 - rAAV Capsid Containing TLAVPFK (SEQ ID NO:27) Construction FIG. 25 shows the amino acid sequence TLAVPFK (sequence number TLAVPFK) between S454 and G455 of VR-IV. Amino acid sequence for recombinant AAV9 vector capsid containing peptide insert of column number 27 The sequence is shown.

[0276] 6.25. Example 25 - Biodistribution of rAAV Vector Pools in Cynomolgus Monkeys Administration of a pool of recombinant AAV carrying modified capsids and a GFP transgene, in In vivo and postmortem observations, and biodistribution were performed in cynomolgus monkeys by single intravenous and intracerebroventricular administration. or intravitreal injection (Table 9). The pool contains multiple capsids, and these Each used next-generation sequencing (NGS) analysis after administration to cynomolgus monkeys. Cynomolgus monkeys are one of the largest animal species. As a model for studying AAV biodistribution in humans and for further translation to humans It was chosen as the test system because of its established usefulness and acceptability. All animals were naïve with respect to prior treatment. Pools were assigned to at least the animals listed in Table 9. The following recombinant AAVs having modified capsids may be included: [Table 9]

[0277] 6.25.1. Research design Nine female cynomolgus monkeys were used. Clinical symptom data and prescreening antibody Animals deemed suitable for the study based on their biosynthetic titer were selected using computer-generated random numbers. Three different routes of administration were used and relevant tissues were collected. , to evaluate biodistribution associated with different pathways (measured by NGS and PCR) Three animals were implanted with a catheter in the left lateral ventricle for intracerebroventricular (ICV) dose administration (group 1), three animals received a single intravenous injection (group 2), and three animals received a single intravitreal injection (group 3). (Group 3). Two animals served as replacement animals and were filled in as needed. Group 1 animals will undergo MRI scans to confirm proper ICV catheter placement. Determine the coordinates for the

[0278] IV infusion was administered at a rate of 3 mL / min, followed by 0.2 mL of vehicle to establish an IV catheter. Flush the dose from the catheter. Administer a single dose of pooled recombinant IgG to three animals intravenously. AAV is administered at a volume of 4 mL / kg. Total Dose (vg) and Dose Volume (mL / kg) Based on a literature review and previous studies in non-human primates, ×10 13 An IV dose of GC / kg body weight reduces the risk of infection in the CNS and peripheral tissues, including skeletal muscle, from systemic delivery. It has been determined that this is necessary to have a desired distribution in the weave.

[0279] ICV-implanted animals were given a single bolus dose of AAV-NAV-GFPbc in a volume of 1 mL. Ingestion (slow infusion, approximately 0.1 mL / min) was followed by 0.1 mL of vehicle. The ICV dose is administered by ingesting the drug and flushing the dose through the catheter system. Based on distribution data from prior non-human primate studies to support floor programs There are.

[0280] Intravitreal (IVT) injections are administered bilaterally as bolus injections with a dose volume of 50 μL. .

[0281] 6.25.2. Observation and testing Clinical signs were recorded at least once daily, beginning approximately 2 weeks before the start of dosing. The animals will be monitored for signs of clinical effects, illness, and / or mortality. At the discretion of the study director and / or technician, further observations may be made based on the condition of the animals. Further observations may be recorded.

[0282] For animals in Group 3, prior to dose administration and on days 2, 8, 15 and 22 Ophthalmic examination is performed. All animals are kept in HCl for ophthalmic examination performed after Day 1. Sedate with ketamine IM. On day 1, animals are sedated with anesthesia by injection for testing (see section (See Section 15.3.3.) Prior to the examination, the eye is dilated with 1% tropicamide. Biomicroscopy and indirect ophthalmoscopy are included. Applanation tonometry is also performed before and after dosing. Immediately after (approximately 10-15 min) and on days 2 and 22 for animals in group 3.

[0283] Approximately 2 to 3 weeks before dose administration, blood samples (approximately 3 h) were collected from a peripheral vein for neutralizing antibody analysis. mL) was collected.

[0284] 6.25.3. Biological Analysis Sample Collection From animals in Group 2 (IV) only, pre-dose, 3 hours (±10 minutes), and 6 hours after dose administration After 24 hours (±0.5 hours) and 30 minutes (±10 minutes), biological analysis (AAV capsid clearing) was performed. Collect a whole blood sample (approximately 0.5 mL) from a peripheral vein for blood sampling (perfusion). Use a needle to collect samples and transfer them into two K2EDTA tubes and record the time .

[0285] PBMC were collected before dose administration (day 1), on days 8 and 15, and before necropsy (day 22). Collect blood samples (approximately 5 mL) from a peripheral vein from fasted animals for analysis. Samples are obtained using paraffin tubes and the time is recorded.

[0286] For biological analysis, pre-dose (day 1, 2 mL) and at necropsy (day 22, 5 mL) Collect blood samples from a peripheral vein at 0 °C. Collect samples in coagulation tubes and record the time. The tube is kept at room temperature until fully solidified, then heated at approximately 2400 rpm in a room-temperature Centrifuge at room temperature for 15 min. Harvest serum and place it into a labeled vial (1 mL for autopsy samples). aliquots), freeze in liquid nitrogen, and store at -60°C or colder.

[0287] Collect CSF (approximately 1.5 mL) via cisternal spinal puncture from animals in Group 1 only prior to dose administration. Collect CSF (approximately 2 mL) via cisternal spinal puncture from all animals (groups 1-3) immediately prior to necropsy. Attempts were made to collect CSF, but due to unsuccessful spinal taps, samples were not collected from the animals. may not be collected at all intervals from the (multiple) sample. If collected, samples may not be processed or Store on ice at .

[0288] Autopsy Animals found dead or sacrificed moribund at least 21 days after treatment (day 22) A gross necropsy will be performed on all animals found dead and at any planned necropsies. All animals were sedated with 8 mg / kg ketamine HCl IM and 10 mg / kg isoflurane / acid. Maintain with a basal mixture and provide an intravenous bolus of heparin sodium (200 IU / kg). Animals were perfused via the left ventricle with 0.001% sodium nitrite in saline. Animals found dead are necropsied but not perfused.

[0289] The following tissues will be obtained from all animals (including those found dead): bone marrow, brain, Cecum, colon, dorsal nerve root and ganglion, duodenum, esophagus, eyes including optic nerve, gross lesions, heart , ileum, jejunum, kidneys, knee joints, liver, lungs including bronchi, lymph nodes, ovaries, pancreas, sciatic nerve , skeletal muscles, spinal cord, spleen, thyroid gland, trachea, and vagus nerve.

[0290] 6.25.5. Biological analysis Whole blood collected from animals in group 2 (IV) was analyzed by qPCR and next-generation sequencing (NGS) ) is used to evaluate.

[0291] PBMC samples collected from all animals were analyzed by flow cytometry, if desired. and enzyme-linked immunosorbent spot (ELISpot).

[0292] The presence of circulating neutralizing antibodies and free vector in serum and / or CSF is optionally , assessed by ELISA and cell-based assays.

[0293] Vector copy number and transcript number in tissues were examined by quantitative PCR and NGS. Try it.

[0294] 6.26. Capsid Amino Acid Sequence Table 10 lists certain modified IgG antibodies described and / or used in the studies described herein. The amino acid sequence of the capsid protein is provided. Heterologous peptides and amino acid substitutions are Shown in color shading. [Table 10] TIFF2025069170000022.tif181139TIFF2025069170000023.tif190140TIFF2025069170000024.tif186141 TIFF2025069170000025.tif187140TIFF2025069170000026.tif183141TIFF2025069170000027.tif135141

[0295] 7. Equivalents Although the invention has been described in detail with reference to specific embodiments thereof, functionally equivalent It is understood that certain variations are within the scope of the present invention. Various modifications of the invention in addition to those described above will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Those skilled in the art will recognize many equivalents to the specific embodiments of the invention described herein. Such a procedure may be performed or ascertained without more than routine experimentation. Equivalents are intended to be encompassed in the scope of the following claims.

[0296] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety. It is specifically and exclusively understood that all patents or patent applications are incorporated herein by reference in their entirety. Each of these patents is incorporated by reference herein to the same extent as if individually indicated.

[0297] The discussion herein provides a better understanding of the nature of the problems facing the art. These should not be construed as admissions of prior art in any way, and they are not intended to be included in any part of this specification. Any references in this document are hereby incorporated by reference without any prior notice, unless expressly stated otherwise. This document should not be construed as an admission that any such disclosure may be made without the prior written consent of the parties.

[0298] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety. Any publications or patents or patent applications incorporated by reference in their entirety for all purposes. to the same extent as if they were specifically and individually indicated to be Numerous modifications and variations of the present invention will become apparent to those of ordinary skill in the art. As will become apparent from the following description, this can be made without departing from the spirit and scope of the present invention. The specific embodiments described are offered by way of example only, and the present invention is not limited to such patents. The terms of the appended claims, along with the full scope of equivalents to which such claims are entitled, are intended to be interpreted as including all such terms. should be limited to only

Claims

1. At least 4 and at most 12 tandem repeats from a heterologous protein that is not an AAV protein. Recombinant adeno-associated virus (rAAV) capsid protein containing a peptide insert of the following amino acids: 8, wherein the peptide insert corresponds to the amino acid sequence of the AAV9 capsid protein of FIG. 138; or immediately following the amino acid residue corresponding to one of amino acids 451-461 When the capsid protein is packaged as an AAV particle, the peptide The recombinant adeno-associated virus (rAAV) capsid protein is a surface-exposed insert. Quality.

2. A recombinant adeno-associated virus (rAAV) capsid protein, comprising: Protein is (i) a neural tissue homing protein or domain, provided that the peptide insert is does not contain LAVPFK (SEQ ID NO:27); (ii) an axonemal or cytoplasmic dynein homing domain; (iii) a bone-homing domain; (iv) a kidney-homing domain; (v) muscle-homing domain; (vi) an endothelial cell homing domain; (vii) an integrin receptor binding domain; (viii) a transferrin receptor binding domain, provided that the peptide insert has the sequence does not contain IGPSV (SEQ ID NO: 19) or CRTIGPSVC (SEQ ID NO: 20); (ix) a tumor cell targeting domain; or (x) a retinal cell homing protein or domain, provided that the peptide insert comprises the sequence L does not contain either GETTRP (SEQ ID NO: 15) or LALGETTRP (SEQ ID NO: 16) At least four and up to four heterologous proteins or domains selected from the group consisting of containing a peptide insert of 12 consecutive amino acids at When the capsid protein is packaged into an AAV particle, the peptide The recombinant adeno-associated virus (rAAV) capsid protein, wherein the insert is surface exposed. quality.

3. The capsid protein is a member of AAV type 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6) , serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype type 9 (AAV9), serotype 9e (AAV9e), serotype rh10 (AAVrh10), blood serotype rh20 (AAVrh20), serotype hu. 37 (AVVhu.37), serotype rh 39 (AAVrh39), and serotype rh74 (AAVrh74).

3. The rAAV capsid protein of claim 1 or 2, wherein both are from the same AAV type. Stuffy.

4. The peptide insert is in the sequence shown in FIG. (a) 450-459 of the AAV1 capsid amino acid sequence (SEQ ID NO:110); (b) 449-458 of the AAV2 capsid amino acid sequence (SEQ ID NO:111); (c) 449-459 of the AAV3 capsid amino acid sequence (SEQ ID NO:112); (d) 443-453 of the AAV4 capsid amino acid sequence (SEQ ID NO:113); (e) 442-445 of the AAV5 capsid amino acid sequence (SEQ ID NO:114); (f) 450-459 of the AAV6 capsid amino acid sequence (SEQ ID NO:115); (g) 451-461 of the AAV7 capsid amino acid sequence (SEQ ID NO:116); (h) 451-461 of the AAV8 capsid amino acid sequence (SEQ ID NO:117); (i) 451-461 of the AAV9 capsid amino acid sequence (SEQ ID NO:118); (j) 452-461 of the AAV9e capsid amino acid sequence (SEQ ID NO:119); (k) 452-461 of the AAVrhlO capsid amino acid sequence (SEQ ID NO:120); (l) 452-461 of the AAVrh20 capsid amino acid sequence (SEQ ID NO:121); (m) 452-461 of the AAVhu.37 capsid amino acid sequence (SEQ ID NO:122); (n) AAVrh74 capsid amino acid sequence (SEQ ID NO: 123 or SEQ ID NO: 154). 452-461; or (o) 452-461 of the AAVrh39 capsid amino acid sequence (SEQ ID NO: 124) The r according to any one of claims 1 to 3, which is immediately followed by one of the amino acid residues in AAV capsid proteins.

5. The peptide insert is inserted between amino acids 1451, N452, G45 of the AAV9 capsid. 3, S454, G455, Q456, N457, Q458, Q459, T460, or The rAA of claim 4, which is present after an amino acid residue corresponding to one of L461. V capsid protein.

6. The heterologous protein may contain a homing domain, a neutralizing antibody epitope, or a purification tag. The rAAV capsid protein according to any one of claims 1, 3 to 5.

7. The homing domain is (i) a neural tissue-homing domain; (ii) an axonemal or cytoplasmic dynein homing domain; (iii) a bone-homing domain; (iv) a kidney-homing domain; (v) muscle-homing domain; (vi) an endothelial cell homing domain; (vii) an integrin receptor binding domain; (viii) a transferrin receptor binding domain; (ix) a tumor cell targeting domain; or (x) retinal cell-homing domain The rAAV capsid protein of claim 6,

8. The peptide insert has the amino acid sequence SITLVKSTQTV (SEQ ID NO:21), TI LSRSTQTG (SEQ ID NO: 22), VVMVGEKPITITQHSVETEG (SEQ ID NO: No. 25), RSSEEDKSTQTT (SEQ ID NO: 26), KMQVPFQ (SEQ ID NO: 1 ), LKLPPIV (SEQ ID NO: 5), PFIKPFE (SEQ ID NO: 6), TLSLPWK ( SEQ ID NO: 7), QQAAPSF (SEQ ID NO: 3), RYNAPFK (SEQ ID NO: 4), TLA VPFK (SEQ ID NO: 27), or TLAAPFK (SEQ ID NO: 2) dynein peptide Contains or consists of at least 4 consecutive amino acids, or 7 consecutive The rAAV capsid protein of claim 7, which is an amino acid.

9. The peptide insert from the transferrin receptor binding domain has the amino acid sequence At least one of RTIGPSV (SEQ ID NO: 19) or CRTIGPSVC (SEQ ID NO: 20) 8. The rA of claim 7, wherein the amino acid sequence is 4 or 7 consecutive amino acids. AV capsid protein.

10. The peptide insert from the retinal cell homing domain has the amino acid sequence TLAA PFK (SEQ ID NO: 2), LGETTRP (SEQ ID NO: 15) or LALGETTRP (SEQ ID NO: At least four consecutive amino acids of the amino acid sequence of 8. The rAAV capsid protein of claim 7.

11. The AAV capsid protein is an AAV8 capsid protein or an AAV9 capsid protein. The rAAV capsid protein of claim 10, which is a capsid protein.

12. The peptide insert from the purification tag has the amino acid sequence YPYDVPDYA (SEQ ID NO: No. 86) or the amino acid sequence DYKDDDDK (SEQ ID NO: No. 52), or at least 7 consecutive amino acids thereof. The rAAV capsid protein of claim 7 which is an amino acid.

13. The neural tissue homing protein or the retinal cell homing protein is a human The rAAV capsid of claim 2 or 3 is an axonemal dynein (HAD) heavy chain tail. Compact quality.

14. The peptide insert comprises at least four amino acids from the dimerization domain of the HAD heavy chain tail. The rAAV capsid protein of claim 13, comprising up to 15 consecutive amino acids. 。

15. The peptide insert is (shown in FIG. 7): (a) (aa1 to 1542 of DYH1_HUMAN UniProtKB-Q9P2D7 ) (SEQ ID NO:97); (b) (aa 1 to 1764 of DYH2_HUMAN UniProtKB-Q9P225 ) (SEQ ID NO:98); (c) (aa 1 to 1390 of DYH3_HUMAN UniProtKB-Q8TD57 ) (SEQ ID NO:99); (d) aa 1 to 1941 of DYH5_HUMAN UniProtKB-Q8TE73 ) (SEQ ID NO: 100); (e) (aa1 to 1433 of DYH6_HUMAN UniProtKB-Q9C0G6 ) (SEQ ID NO: 101); (f) (aa 1 to 1289 of DYH7_HUMAN UniProtKB-Q8WXX0 ) (SEQ ID NO: 102); (g) (aa1 to 1807 of DYH8_HUMAN UniProtKB-Q96JB1 ) (SEQ ID NO: 103); (h) (aa 1 to 1831 of DYH9_HUMAN UniProtKB-Q9NYC9 ) (SEQ ID NO: 104); (i) (aa 1 to 179 of DYH10_HUMAN UniProtKB-Q8IVF4) 3) (SEQ ID NO: 105); (j) (aa 1 to 185 of DYH11_HUMAN UniProtKB-Q96DT5 4) (SEQ ID NO: 106); (k) (aa1 to 121 of DYH12_HUMAN UniProtKB-Q6ZR08 4) (SEQ ID NO: 107); (l) (DYH14_HUMAN UniProtKB-Q0VDD8 aa1 to 200 ) (SEQ ID NO: 108); or (m) (aa1 to 179 of DYH17_HUMAN UniProtKB-Q9UFH2 4) (SEQ ID NO: 109) 15. The method according to claim 14, comprising at least 4 and at most 15 consecutive amino acids from the group consisting of: rAAV capsid proteins as described.

16. The peptide insert may be from residues 1-200 of any one of the dynein heavy chain sequences (Figure 7).

16. The rAA of claim 15, comprising at least 4 and at most 15 consecutive amino acids of V capsid protein.

17. The peptide insert comprises seven consecutive amino acids from any one of the dynein heavy chain sequences of FIG.

16. The rAAV capsid protein of claim 15, comprising the amino acid

18. The peptide insert may be from residues 1-200 of any one of the dynein heavy chain sequences (Figure 7). The rAAV capsid protein of claim 16, comprising seven consecutive amino acids:

19. The peptide insert comprises the peptide: (a) KMQVPFQ (SEQ ID NO: 1); (b) TLAAPFK (SEQ ID NO: 2); (c) QQAAPSF (SEQ ID NO: 3); (d) RYNAPFK (SEQ ID NO: 4); (e) LKLPPIV (SEQ ID NO:5); (f) PFIKPFE (SEQ ID NO: 6); or (g) TLSLPWK (SEQ ID NO: 7) rAA according to claim 2 or 3, which comprises at least four consecutive amino acids of one of V capsid protein.

20. The peptide insert comprises the peptide: (a) KMQVPFQ (SEQ ID NO: 1); (b) TLAAPFK (SEQ ID NO: 2); (c) QQAAPSF (SEQ ID NO: 3); (d) RYNAPFK (SEQ ID NO: 4); (e) LKLPPIV (SEQ ID NO:5); (f) PFIKPFE (SEQ ID NO: 6); or (g) TLSLPWK (SEQ ID NO: 7) 4. The rAAV capsid protein of claim 2 or 3, which consists of one of the following:

21. 2. The peptide insert is the amino acid sequence TLAAPFK (SEQ ID NO:2).

0. The rAAV capsid protein of

22. The neural tissue homing protein is the mouse axonemal dynein (MAD) heavy chain tail.

4. The rAAV capsid protein of claim 2 or 3.

23. The neural tissue homing domain binds to a natural repair receptor and is erythropoietic EPO (erythropoietin) domain or conformational analysis of said domain 4. The rAAV capsid protein of claim 2 or 3, which is an analogue.

24. The peptide insert comprises at least four amino acids from QEQLERALNSS (SEQ ID NO:8). and up to 11 consecutive amino acids. quality.

25. The peptide insert has the amino acid sequence QEQLERALNSS (SEQ ID NO:8).

25. The rAAV capsid protein of claim 24.

26. The neural tissue homing protein has an SRL (serine-arginine-lysine) motif. The rAAV capsid of claim 2 or 3 is a brain-homing domain having a Compact quality.

27. The peptide insert from the brain-homing domain has the amino acid sequence LSSRLDA (SEQ ID NO: 10) or CLSSRLDAC (SEQ ID NO: 11) or the amino acid sequence LSSRLDA (SEQ ID NO: 10) or CLSSRL 27. The rAAV capsid protein of claim 26, which is DAC (sequence number 11).

28. The axonemal or cytoplasmic dynein homing domain is a dynein light chain homing domain. The rAAV capsid protein of claim 2 or 3.

29. The peptide insert from the dynein light chain homing domain is SITLVKST QTV (SEQ ID NO: 21), TILSRSTQTG (SEQ ID NO: 22), VVMVGEKPI TITQHSVETEG (SEQ ID NO: 25), or RSSEEDKSTQTT (SEQ ID NO: 26) of at least 4 and at most 12 consecutive amino acids of one of the following:

8. The rAAV capsid protein according to claim 8.

30. The bone homing protein is a hydroxyapatite (HA) binding domain.

4. The rAAV capsid protein of claim 2 or 3.

31. The peptide insert from the hydroxyapatite (HA) binding domain has sequence D 31. The method of claim 30, which is at least six amino acid residues of DDDDDDD (SEQ ID NO: 9). The rAAV capsid proteins are shown.

32. The kidney-homing domain comprises the amino acid sequence CLPVASC (SEQ ID NO: 12).

4. The rAAV capsid protein of claim 2 or 3.

33. The peptide insert from the kidney-homing domain peptide has the amino acid sequence LP 33. The method according to claim 32, wherein the amino acid sequence is CLPVASC (SEQ ID NO: 12) or VAS (SEQ ID NO: 13). The rAAV capsid proteins are shown.

34. The peptide insert from the muscle homing domain has the amino acid sequence ASSLNI 4. The rAAV vector of claim 2 or 3, which comprises or consists of: Psid protein.

35. The peptide insert has the amino acid sequence QAVRTSL (SEQ ID NO:23) or QAVR 4. The rAA of claim 2 or 3, comprising or consisting of TSH (SEQ ID NO: 24). V capsid protein.

36. The peptide insert from the endothelial cell homing domain has the amino acid sequence SIGY rAA according to claim 2 or 3, comprising or consisting of PLP (SEQ ID NO: 28). V capsid protein.

37. The peptide insert from the integrin binding domain has the amino acid sequence CDCRG The rAAV capsid protein of claim 2 or 3 having DCFC (SEQ ID NO: 29). Quality.

38. The transferrin receptor binding domain is a transferrin domain or or a conformational analog or iron mimetic of claim 2 or 3. AV capsid protein.

39. The peptide insert from the transferrin domain has the sequence HAIYPRH (sequence At least four sequences from THRPPMWSPVWP (SEQ ID NO: 18) 39. The rAA of claim 38, wherein the amino acid sequence is one or seven consecutive amino acids. V capsid protein.

40. The peptide insert has the amino acid sequence HAIYPRH (SEQ ID NO: 17) or THRP 40. The rRNA of claim 39, comprising or consisting of PMWSPVWP (SEQ ID NO: 18). AAV capsid proteins.

41. The peptide insert from the tumor cell targeting domain comprises the amino acid sequence NGRAHA (SEQ ID NO: 30) Side protein.

42. The peptide inserts are TLAAPFK (SEQ ID NO:2), TLAVPFK (SEQ ID NO:2) 7), RTIGPSV (SEQ ID NO: 19), CRTIGPSVC (SEQ ID NO: 20), LGE TTRP (SEQ ID NO: 15), and LALGETTRP (SEQ ID NO: 16). or at least 7 consecutive amino acids thereof.

4. The rAAV capsid protein according to claim 2 or 3.

43. The peptide insert consists of the amino acid residues (shown in FIG. 8): (a) 138;262-272;4 of the AAV1 capsid amino acid sequence (SEQ ID NO:110) 50-459; or 585-593; (b) 138;262-272;4 of the AAV2 capsid amino acid sequence (SEQ ID NO:111). 49-458; or 584-592; (c) 138;262-272;4 of the AAV3 capsid amino acid sequence (SEQ ID NO:112) 49-459; or 585-593; (d) 137;256-262;4 of the AAV4 capsid amino acid sequence (SEQ ID NO:113). 43-453; or 583-591; (e) 137;252-262;4 of the AAV5 capsid amino acid sequence (SEQ ID NO:114). 42-445; or 574-582; (f) 138;262-272;4 of the AAV6 capsid amino acid sequence (SEQ ID NO:115). ５０～４５９；５８５～５９３； (g) 138;263-273;4 of the AAV7 capsid amino acid sequence (SEQ ID NO:116) ５１～４６１；５８６～５９４； (h) 138;263-274;4 of the AAV8 capsid amino acid sequence (SEQ ID NO:117) ５２～４６１；５８７～５９５； (i) 138;262-273;4 of the AAV9 capsid amino acid sequence (SEQ ID NO:118) ５２～４６１；５８５～５９３； (j) 138;262-273 of the AAV9e capsid amino acid sequence (SEQ ID NO:119); ４５２～４６１；５８５～５９３； (k) 138; 263-27 of the AAVrhlO capsid amino acid sequence (SEQ ID NO: 120) ４；４５２～４６１；５８７～５９５； (l) 138; 263-27 of the AAVrh20 capsid amino acid sequence (SEQ ID NO: 121) ４；４５２～４６１；５８７～５９５； (m) 138;263-27 of the AAVhu37 capsid amino acid sequence (SEQ ID NO:122) ４；４５２～４６１；５８７～５９５； (n) the AAVrh74 capsid amino acid sequence (SEQ ID NO: 123 or SEQ ID NO: 154) 138; 263-274; 452-461; 587-595; or (o) 138;263-27 of the AAVrh39 capsid amino acid sequence (SEQ ID NO:124) ４；４５２～４６１；５８７～５９５ The rAAV according to any one of claims 2 or 13 to 42, Capsid protein.

44. Between amino acid residues 588 and 589 of the AAV9 capsid or The amino acid sequence TL was inserted immediately after the amino acid residue corresponding to amino acid 138 (see FIG. 8).

44. The rAAV capsid protein of claim 43, comprising AAPFK (sequence number 2).

45. one of I451 to L461, S268, or Q588 of the AAV9 capsid; 43. The method of claim 42, further comprising inserting the amino acid sequence TLAAPFK (SEQ ID NO:2) after the sequence of The rAAV capsid protein described in.

46. The amino acid sequence between amino acid residues 588 and 589 of the AAV9 capsid (see FIG. 8) The rAAV capsid of claim 43, comprising QEQLERALNSS (SEQ ID NO: 8). Compact quality.

47. One or more selected from I451 to L461, or S268 of the AAV9 capsid (FIG. 8) containing the amino acid sequence QEQLERALNSS (SEQ ID NO:8) inserted at position 44. The rAAV capsid protein of claim 43.

48. The peptide insert comprises amino acid residues 262-27 of the AAV9 capsid protein.

3. The amino acid sequence TLAVPFK (SEQ ID NO: 27) immediately follows one of the sequences of claim 4.

3. The rAAV capsid protein according to claim 3.

49. The peptide insert is inserted between amino acid residues 269 and 270 of the AAV8 capsid protein.

44. The rRNA of claim 43, comprising the amino acid sequence LGETTRP (SEQ ID NO: 15) between 70 and 70. AAV capsid proteins.

50. The peptide insert has the amino acid sequence LALGETTRP (SEQ ID NO: 16).

50. The rAAV capsid protein of claim 49.

51. The peptide insert comprises amino acid residues 590 and 593 of the AAV8 capsid protein.

44. The rRNA of claim 43, comprising the amino acid sequence LGETTRP (SEQ ID NO: 15) between 91 and 92. AAV capsid proteins.

52. The peptide insert has the amino acid sequence LALGETTRP (SEQ ID NO: 16).

52. The rAAV capsid protein of claim 51.

53. The peptide insert is inserted between amino acid residues 453 and 454 of the AAV8 capsid protein. 54 immediately followed by the amino acid sequence LGETTRP (SEQ ID NO: 15).

43. The rAAV capsid protein according to claim 43.

54. The peptide insert comprises the amino acid sequence LALGETTRP (SEQ ID NO: 16).

54. The rAAV capsid protein of claim 53.

55. The capsid is AAV9 and the peptide insert is at amino acid residue 4 of AAV9. r according to any one of claims 2 or 13 to 43, which is between 54 and 455 (FIG. 8). AAV capsid proteins.

56. The peptide insertion is selected from the group consisting of I451 to L461, S268, and the like of the AAV9 capsid.

8. The amino acid residue of claim 2 or 13, which is located immediately after the acid residue corresponding to one of Q588 (FIG. 8).

44. An rAAV capsid protein according to any one of claims 1 to 43.

57. The peptide insert is located within amino acids 451-461 of the AAV9 capsid protein.

44. The rAAV capsid protein of claim 43, which is immediately preceded by one of the following:

58. The peptide insert is located in the AAV capsid variable region eight (VR-VIII). The rAAV capsid protein of any one of claims 2 or 13 to 43.

59. The capsid protein of the preceding claim is not the AAV2 capsid protein. rAAV capsid protein according to any one of claims 1 to 4.

60. Recombinant AAV capsid proteins, which are modified to wild-type or unmodified capsid proteins. wherein the rAAV capsid protein comprises one or more amino acid substitutions relative to A269. or an AAV8 capsid protein having an S amino acid substitution, S263G / S26 AAV9 capsules having 9R / A273T substitutions, or W503R or Q474A substitutions The one or more amino acid substitutions are a capsid protein, or a capsid of another AAV type. The recombinant AAV capsid protein comprising a corresponding substitution in a capsid protein.

61. 498-NNN / AAA-500 or AA for AAV8 capsid protein 496-NNN / AAA-498 for the V9 capsid protein, or another AAV 61. The method according to embodiment 60, further comprising a corresponding substitution in a capsid protein of a capsid of the type 60. The rAAV capsid proteins are shown.

62. A method for producing a rAAV capsid protein comprising the steps of: A nucleotide sequence encoding an amino acid sequence that shares at least 80% identity with Nucleic acid.

63. 62. A method for the preparation of a rAAV capsid protein comprising the steps of: The nucleic acid according to claim 1.

64. An AAV vector comprising the capsid protein encoded by the nucleotide sequence. A package capable of expressing the nucleic acid according to claim 62 or 63 to produce a target. Ging cells.

65. 62. An rAAV vector comprising a capsid protein according to any one of claims 1 to 61.

66. 66. The rAAV vector of claim 65, further comprising a transgene.

67. A drug comprising the rAAV vector of claim 65 or 66 and a pharma- ceutically acceptable carrier. chemical composition.

68. 65. A method for delivering a transgene to a cell, the method comprising:

66.

67. The rAAV vector of claim 65 or 66 for use in delivering to a cell. Thus, the cell is contacted with the vector, the rAAV vector.

69. A method for delivering a transgene to a target tissue in a subject in need thereof, said method comprising: Administering to the subject the rAAV vector of claim 65 or 66, The method of claim 1, wherein the peptide insert is a homing peptide; or 67. The method of claim 65 or 66 for use in delivering a compound according to claim 65 to a target tissue in a subject in need thereof. The rAAV vector of claim 1, wherein the vector is administered to the subject. Kutor.

70. The rAAV vectors can be administered systemically, intravenously, intrathecally, intranasally, intraperitoneally, or intravitreally.

70. The method of claim 69, wherein the administration is via lumbar puncture or via the cisterna magna. or rAAV vectors for use.

71. The target tissue is (i) neural tissue, and the vector is a vector that is a homing domain for the neural tissue. including a peptide insert, (ii) bone, the vector comprising a peptide insert from the bone-homing domain Including, (iii) the kidney, and the vector comprises a peptide insertion from the kidney-homing domain. Including the contents, (iv) muscle, and the vector comprises a peptide insertion from the muscle-homing domain. Including things, (v) an endothelial cell, wherein the vector comprises a peptide derived from the endothelial cell homing domain; Includes inserts, (vi) an integrin receptor, wherein the vector comprises an integrin receptor binding domain. containing a peptide insert from (vii) a transferrin receptor on a tumor cell, and the vector is containing a peptide insert from the ferrin receptor binding domain, (viii) a tumor cell, said vector comprising a peptide from said tumor cell targeting domain. containing a tide insert, or (ix) a retinal cell, wherein the vector comprises a peptide derived from the retinal cell homing domain. Chid insert 71. The method of claim 70, or the rAAV vector for use therewith, comprising:

72. The target tissue is a retinal cell and the peptide insert has the amino acid sequence TLAAPF K (SEQ ID NO: 2), LGETTRP (SEQ ID NO: 15) or LALGETTRP (SEQ ID NO:

72. The method of claim 71, or the rAAV vector for use therein, comprising:

73. The target tissue is a retinal cell and the peptide insert has the amino acid sequence TLAAPF 73. The rAAV vector for use according to claim 72, comprising: K (SEQ ID NO:2). -

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