Targeting tissues in the central nervous system

Abstract

This disclosure relates to targeting the central nervous system. Compositions for delivering therapeutic agents to specific tissues within the central nervous system are provided. Novel surface-modified viral capsids and recombinant viral particles containing the same are also provided. Furthermore, this disclosure relates to intermediates for preparing surface-modified viral capsids. The surface-modified viral capsids are designed for selectively and / or more efficiently delivering gene therapy. After integration into recombinant viral particles, the surface-modified viral capsids can be used to treat diseases characterized by genetic abnormalities.

Description

[0001] 1. Sequence List

[0002] This application incorporates by reference a computer-readable XML sequence list, named “37937-61963_010WO_ST26.xml”, created on March 11, 2025, with a file size of 61,324 bytes. 2. Background of the Invention

[0004] The central nervous system (CNS) is primarily composed of the brain and spinal cord. The CNS is surrounded by a protective barrier, known as the blood-brain barrier (BBB) ​​or, in the case of the spinal cord, the blood-spinal barrier (BSCB). These are highly selective, semi-permeable endothelial cell (EC) boundaries that prevent pathogens, solutes, and most molecules from non-selectively entering the CNS. While these barriers normally help protect the CNS, in disease conditions, both the BBB and BSCB act as barriers to therapeutic drugs, hindering their effective delivery to target cells or regions within the CNS. Achieving adequate BBB and BSCB penetration is a major obstacle in the development of treatments for CNS diseases.

[0005] Recombinant AAV viral particles (rAAV) are preferred viral delivery vectors for in vivo delivery of therapeutic nucleic acid payloads (cargo), including polynucleotides expressing therapeutic genes, therapeutic miRNAs, guide RNAs of nucleic acid-directed nucleases, genome editing enzymes, and RNA editing machines. They can also be used for in vivo delivery of nucleic acids that serve directly or indirectly as templates for genome insertion or repair. The aforementioned BBB (Brain-Body-Body-Body) poses a significant obstacle to the delivery of such therapeutic payloads to the central nervous system using rAAV.

[0006] One method to bypass the brain barrier (BBB) ​​is to administer rAAV directly into the cerebrospinal fluid (CSF) via intrathecal delivery (e.g., via lumbar puncture, or via intraventricular (ICV) or intracerebellomedullary cistern (ICM)). While direct administration to the CSF bypasses the BBB, this does not guarantee delivery to the brain parenchyma, as the meninges provide further entry barriers for neurons, astrocytes, and microglia within the parenchyma.

[0007] Another approach is to engineer AAV capsids to cross the blood-brain barrier through engineered interactions with various receptors known to mediate transcytosis across endothelial cells, and through empirical screening of large libraries of peptide-modified capsids in rodents and non-human primates (NHPs) without presupposing mechanisms.

[0008] Despite significant efforts, engineered capsids capable of reliably delivering rAAV and its payload to desired cell types in the brain remain scarce. There is an ongoing need to develop novel targeting strategies for rAAV and other therapeutic payloads that offer higher CNS cell reach efficiency, greater transduction specificity (tropism) for relevant CNS target cells and tissues, and remain effective even at lower titers. 3. Overview of the Invention

[0010] This disclosure provides engineered viral capsids with dual ligands, pharmaceutical compositions thereof, methods of use, and methods of preparation.

[0011] On one hand, this disclosure provides an engineered viral capsid comprising: a first targeting ligand coupled to a first site of a surface-modified viral capsid; and a second targeting ligand coupled to a second site of the surface-modified viral capsid, wherein the first targeting ligand and the second targeting ligand are different.

[0012] On the other hand, this disclosure provides a method for treating a disease or condition, comprising administering a therapeutically effective amount of an engineered viral capsid, the engineered viral capsid comprising: a first targeting ligand coupled to a first site of the surface-modified viral capsid; and a second targeting ligand coupled to a second site of the surface-modified viral capsid, wherein the first and second targeting ligands are different. In some embodiments, the disease or condition is a central nervous system disease or condition. In some embodiments, the central nervous system disease or condition is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is selected from aromatic L-amino acid decarboxylase (AADC) deficiency, Canavan disease, Parkinson's disease, Alzheimer's disease, and giant axonal neuropathy.

[0013] In some embodiments, this disclosure relates to a surface-modified viral capsid, typically an AAV viral capsid. In some embodiments, the surface modification includes a first ligand that promotes the entry of recombinant AAV (rAAV) from CSF into the brain parenchyma, optionally further including one or more additional surface-coupled ligands that promote CNS cell transduction, optionally promoting transduction of one or more desired CNS cell types. In some embodiments, the first ligand is covalently coupled to a viral capsid protein. In some embodiments of these embodiments, the first ligand is covalently coupled to the viral capsid protein via a linker comprising a crosslinking portion Q. In other embodiments, the first ligand is fused within a frame in the capsid protein sequence. Alternatively, recombinant viral particles are provided comprising the surface-modified viral capsid and a nucleic acid payload encapsulated by the capsid, typically a recombinant AAV viral particle (rAAV).

[0014] In some embodiments, a peptide ligand is inserted into a naturally occurring AAV serotype capsid protein or an engineered or synthetic AAV capsid protein. For example, the peptide ligand may be inserted into a naturally occurring AAV serotype (e.g., AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, or AAV9) capsid protein. In some embodiments, the peptide ligand is inserted into variable region 4 (VR4) of the AAV capsid protein, which corresponds to an exposed loop on the capsid surface. In other embodiments, the peptide ligand is inserted into variable region 8 (VR8) of the AAV capsid protein. The VR8 region may contain R585 and R588, which are required for HSPG binding, or may contain mutations that reduce or eliminate HSPG binding, such as R585A and R588A mutations. In some embodiments, the specific insertion site in VR4 or VR8 may vary depending on the capsid protein being modified. In some embodiments, the insertion site in VR4 may be between amino acid positions 400 and 500 of the capsid protein. In some embodiments, the insertion site in VR4 may be between amino acid positions 425 and 475 of the capsid protein. In some embodiments, the insertion site in VR4 may be between amino acid positions 440 and 460 of the capsid protein.

[0015] In some embodiments, the insertion site in VR8 may be between amino acid positions 550 and 650. In some embodiments, the insertion site in VR8 may be between amino acid positions 575 and 625. In some embodiments, the insertion site in VR8 may be between amino acid positions 580 and 600.

[0016] In some embodiments, the peptide ligand is PAR1. In some embodiments, the peptide ligand is PAR1A. In some embodiments, the peptide ligand has the sequence SFLLRN (residues 5 to 10 of SEQ ID NO: 61). In some embodiments, the peptide ligand has the sequence LDPRSFLLRN (SEQ ID NO: 61).

[0017] In some embodiments, this disclosure relates to viral capsids with surface modifications according to Formula I: …(I) in: It is a viral capsid; Y and Y' are independent connecting parts; n and n' are independently 0 or integers from 1 to 50; Sp and Sp' are independently optional spacers; L is a ligand; x is the ligand / capsid ratio, ranging from 1 to 500; and Q is selected from: or , Z is a 7- or 8-membered ring or heterocyclic structure.

[0018] In some embodiments, this disclosure relates to a method for preparing a surface-modified viral capsid, the method comprising the following steps: i) Obtaining a surface-functionalized viral capsid by reacting a viral capsid protein with a capsid reactive linker, said linker comprising a first member of a cross-linking agent reactant pair and optionally one or more spacers; ii) Coupling the surface-functionalized viral capsid to a functionalized ligand comprising a second member of a crosslinking agent reactant pair and optionally one or more spacers; The first and second members of the crosslinking agent reaction pair react to form the crosslinked portion Q.

[0019] In some embodiments, an engineered adeno-associated virus (AAV) capsid protein is provided. In some other embodiments, the capsid protein comprises a functional sequence motif. In some embodiments, the functional sequence motif has the sequence: X1X2X3X4X5X6 (SEQ ID NO:5); wherein X1, X2, X3, X4, X5, and X6 are each independently selected from any amino acid residues, and wherein X4, X5, and X6 are optional. In some embodiments, the functional sequence motif is a substrate of a sorting enzyme transaminases.

[0020] In some embodiments, this disclosure relates to an engineered adeno-associated virus (AAV) capsid protein, wherein: the capsid protein comprises: a functional sequence motif having the sequence: X1X2X3X4X5X6 (SEQ ID NO:5); wherein X1, X2, X3, X4, X5 and X6 are each independently selected from any amino acid residues, and wherein X4, X5 and X6 are optional; wherein the functional sequence motif is a substrate of a sorting enzyme transaminases.

[0021] In some embodiments, this disclosure relates to recombinant rAAV viral particles (rAAV) comprising the engineered rAAV capsid protein disclosed herein.

[0022] In some embodiments, this disclosure relates to a surface-modified rAAV viral particle, wherein the viral particle comprises an exogenous cargo polynucleotide and at least one engineered rAAV capsid protein, the capsid protein comprising at least one functional sequence motif LPX3TG (SEQ ID NO:48), wherein X3 is selected from any amino acid residue; wherein the functional sequence motif is located within variable regions 4, 8, or 4 and 8, and (a) wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form an N-terminal cleavage fragment and a C-terminal cleavage fragment; wherein the viral particle comprises a crosslinking portion covalently linking a T of the N-terminal cleavage fragment to a targeting ligand, wherein the covalent bond comprises an amide bond; and optionally (b) wherein the viral particle comprises a crosslinking portion covalently linking a lysine residue or an N-terminal amine of the capsid protein to a targeting ligand.

[0023] In some embodiments, this disclosure provides compositions for delivering therapeutic agents to specific tissues within the CNS. In some embodiments, the surface-modified viral capsid is designed to selectively and / or more efficiently deliver gene therapy. The surface-modified viral capsid, after integration into recombinant viral particles, can be used to treat diseases characterized by genetic abnormalities (e.g., neurodegenerative diseases, neuromuscular diseases, lysosomal storage diseases). 4. Description of the attached drawings

[0025] The features, aspects, and advantages of this disclosure will become better understood through the following detailed description of illustrative embodiments to which the principles of this disclosure are applied, in conjunction with the accompanying drawings, wherein: Figure 1A-1D An example is given of intrathecal injection of 3E+9 (3×10⁻⁹). 9 Transduction of the spinal cord parenchyma by recombinant viral particles (each carrying a polynucleotide payload expressing tdTomato) with vector genome (VG) control (AAV2-ΔHSPG) and TPA-modified AAV2-ΔHSPG (TPA::AAV2-ΔHSPG) was studied. Figure 1A and Figure 1C As shown, in the lumbar spinal cord ( Figure 1A ) or thoracic spinal cord ( Figure 1C In this study, a small number of tdTomato-positive (i.e., effectively transduced) neurons were observed after intrathecal injection of unmodified AAV2-ΔHSPG. In contrast, conjugation of TPA with AAV2-ΔHSPG (TPA::AAV2-ΔHSPG) increased transduction efficiency in the parenchyma at all levels. This was demonstrated in the lumbar spinal cord (…). Figure 1B ) and thoracic spinal cord ( Figure 1D ) slices.

[0026] Figures 2A-2FExamples of spinal cord transduction following intrathecal injection of 3E+10 VG (AAV2-ΔHSPG) and AAV2-ΔHSPG rAAV (PAR1PA::AAV2-ΔHSPG) with surface modification to display the PAR-1 (PAR1) agonist peptide SFLLRNPNDKC (SEQ ID NO:3) are illustrated. Figure 2B and Figure 2C An example is shown of increasing transduction efficiency in the viral capsid by conjugating the PAR1PA peptide ligand to the surface of the viral capsid, and this is further illustrated by conjugating a second ligand, wheat germ agglutinin (WGA), to the capsid surface. Figure 2D In contrast, co-injection of the unconjugated PAR1PA peptide with AAV2-ΔHSPG did not increase parenchymal penetration. Figure 2E Quantitative visualization of fluorescence signals in gray matter. Figure 2F .

[0027] Figures 3A-3D This is a heatmap of tdTomato expression in brain slices after intrathecal injection of 3E+10 VG control (AAV2-ΔHSPG) and PAR1A surface-modified AAV2-ΔHSPG (PAR1PA::AAV2-ΔHSPG). Figure 3A A small number of tdTomato-positive (effectively transduced) neurons were observed after intrathecal injection of unmodified AAV2-ΔHSPG. Figure 3B The conjugation of the PAR1PA peptide ligand shows that it increases transduction efficiency in the plasmonic system, which is further amplified by conjugating the second ligand WGA to the surface of the rAAV capsid. Figure 3C ). Figure 3D As shown, in contrast, co-injection of the unconjugated PAR1PA peptide with AAV2-ΔHSPG only slightly increased physical penetration. Figure 3E The quantitative analysis of fluorescence signals in the gray matter of the brain is illustrated.

[0028] Figures 4A-4D This study illustrates the transduction of rAAV (WGA::AAV2-LPETG-VR8) in PC12 cells in vitro, comprising (i) a surface-conjugated WGA ligand and (ii) an LPETG peptide (SEQ ID NO:12) fused within a frame in the variable region 8 of the AAV capsid protein, with the rAAV carrying a tdTomato expression construct as the payload. In these experiments, the AAV2 capsid lacked the ΔHSPG deletion in the capsid, and the LPETG peptide (SEQ ID NO:12) allowed for further targeted chemical surface modifications. Figure 4A The fluorescence of tdTomato is shown in PC12 cells transduced in vitro with unmodified AAV2-LPETG-VR8. Figure 4BThe tdTomato fluorescence in PC12 cells infected with the WGA::AAV2-LPETG-VR8 vector linked to GGG-PEG4-DBCO is shown. Figure 4C The fluorescence of tdTomato is shown in PC12 cells transduced with the WGA::AAV2-LPETG-VR8 vector linked to amine-DBCO. Figure 4D The fluorescence of tdTomato is shown in PC12 cells transduced with the WGA::AAV2-LPETG-VR8 vector linked to amine-PEG4-DBCO.

[0029] Figures 5A-5D This example illustrates the transduction of PC12 cells in culture by WGA::AAV2-LPETG-VR4, wherein the LPETG tag (SEQ ID NO:12) is inserted into the VR4 region of the capsid protein. Figure 5A tdTomato fluorescence was observed in PC12 cells infected with unmodified AAV2-LPETG-VR4; Figure 5B tdTomato fluorescence was observed in PC12 cells infected with the WGA::AAV2-LPETG-VR4 vector linked to GGG-PEG4-DBCO. Figure 5C tdTomato fluorescence was observed in PC12 cells infected with the WGA::AAV2-LPETG-VR4 vector linked to amine-DBCO. Figure 5D tdTomato fluorescence was observed in PC12 cells infected with the WGA::AAV2-LPETG-VR4 vector linked to amine-PEG4-DBCO.

[0030] Figure 6 The amino acid sequence of the VP1 capsid protein of AAV2-LPETG-VR4 (SEQ ID NO:59) is illustrated. The sorting enzyme A recognition sequence LPETG (residues 453 to 457 of SEQ ID NO:59) is shown underlined in variable region 4 of AAV2 VP1.

[0031] Figure 7 The amino acid sequence of the VP1 capsid protein of AAV2-LPETG-VR8 (SEQ ID NO:60) is illustrated. The sorting enzyme A recognition sequence LPETG (residues 586 to 590 of SEQ ID NO:60) is shown underlined in variable region 8 of AAV2 VP1.

[0032] Figures 8A-8F An example is given using 9×10 10Spinal and horizontal brain slices of mice that underwent IV injection of unmodified ΔHSPG and ΔHSPG-SFLLRNC (SEQ ID NO: 66) in animals. Figure 8A and Figure 8C An example is given using 9×10 10 Spinal and horizontal brain slices of mice that received IV injections of unmodified ΔHSPG in VG / animals. Figure 8B and Figure 8D An example is given using 9×10 10 Spinal and horizontal brain slices of mice that underwent IV injection of VG / animal ΔHSPG-SFLLRNC (SEQ ID NO:66). Figure 8E An example of a 20x magnified image of the cortex is shown, revealing no positive cells in the control (i.e., unmodified ΔHSPG). Figure 8F An example of a 20x magnified image of the cortex is shown, revealing the presence of positive cells in the experimental case (i.e., ΔHSPG-SFLLRNC (SEQ ID NO:66)). 5. Detailed Description of the Invention

[0034] 5.1 Definition

[0035] As used in this article, the term "rAAV" refers to recombinant viral particles containing recombinant nucleic acid constructs packaged within an AAV capsid.

[0036] In this article, the terms “AAV”, “adeno-associated virus”, “AAV virus”, “AAV virus particle”, “AAV virus particle”, “AAV particle”, “adeno-associated virus vector”, and “AAV vector” are used synonymously and all refer to rAAV.

[0037] The recombinant nucleic acid construct (synonymously referred to as the "recombinant viral genome") contains a multinucleotide payload (synonymously referred to as the "cargo") located between inverted terminal repeat sequences of AAV. The payload may be an expressible polynucleotide or DNA construct that provides a template for homology-directed repair. In various embodiments, the expressible polynucleotide encodes a protein (e.g., a transgene encoding a therapeutic protein), or encodes miRNA, siRNA, or guide RNA for gene editing or RNA editing machines (such as CRISPR, ADAR, and ADAT).

[0038] As used herein, “capsid binding” or “surface-modified capsid binding” to mammalian cell surface proteins, polysaccharides, or proteoglycans refers to the binding of a recombinant viral particle (typically rAAV) containing said capsid or surface-modified capsid.

[0039] The term “treatment” or “management” as used herein is used in its broadest clinically acceptable sense. This term includes, but is not limited to: reducing the signs or symptoms of disease; improving the signs or symptoms of disease; alleviating symptoms; reducing the severity of disease; stabilizing (i.e., not worsening) the disease state; delaying or slowing disease progression; improving or alleviating the disease state; remission (whether partial or complete), whether detectable or undetectable; cure; and extended survival compared to expected survival without treatment.

[0040] "Effective dose" refers to the amount of rAAV disclosed in this disclosure that is effective in treating diseases.

[0041] As used in this article, the term “prevention” when used with respect to a subject refers to the prevention of disease, typically for subjects who are at risk of developing the disease, such as those at risk due to the presence of genomic mutations.

[0042] As used herein, the term "tropism" refers to the preferential infection and / or transduction of a viral capsid on certain cells or tissues. In a preferred embodiment, the tropism of the AAV capsid is modified by endowing it with certain characteristics, such as certain affinity for receptors on the surface of target cells, which are not naturally present in the capsid.

[0043] In the context of this disclosure, the term "subject" preferably refers to a mammal, such as a mouse, rat, guinea pig, rabbit, cat, dog, or monkey, or preferably a human. The term "patient" preferably refers to a mammal, such as a mouse, rat, guinea pig, rabbit, horse, cow, dairy cow, cat, dog, or monkey, or preferably a human, such as a human patient requiring diagnosis, prognosis, or treatment. Subjects of this disclosure may be at risk of developing a disease, such as one that can be treated by intracellular delivery of the recombinant polynucleotide cargo. A more detailed description of the medical indications related to this disclosure can be found elsewhere herein.

[0044] The term "optionally substituted" means that a given chemical moiety (e.g., an alkyl group) may (but is not required to) be bonded to other substituents (e.g., heteroatoms). For example, an optionally substituted alkyl group can be a fully saturated alkyl chain (e.g., a pure hydrocarbon). Alternatively, the same optionally substituted alkyl group can have substituents other than hydrogen. For example, it can be bonded to a halogen atom, a hydroxyl group, or any other substituent described herein at any position on the chain. Thus, the term "optionally substituted" means that a given chemical moiety has the potential to contain other functional groups, but is not required to have any additional functional groups. Suitable substituents for optional substitution of the said group include, but are not limited to: halogen, oxo, -OH, -CN, -COOH, -CH2CN, -O-(C1-C6)alkyl, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -O-(C2-C6)alkenyl, -O-(C2-C6)ynyl, (C2-C6)alkenyl, (C2-C6)ynyl, -OP(O)(OH)2, - The substituents are OC(O) (C1-C6)alkyl, -C(O) (C1-C6)alkyl, -OC(O)O(C1-C6)alkyl, -NH2, -NH((C1-C6)alkyl), -N((C1-C6)alkyl)2, -NHC(O) (C1-C6)alkyl, -C(O)NH(C1-C6)alkyl, -S(O)2(C1-C6)alkyl, -S(O)NH(C1-C6)alkyl, and S(O)N((C1-C6)alkyl)2. The substituents themselves may be optionally substituted. As used herein, "optionally substituted" also means substituted or unsubstituted, as defined below. A portion containing additional substitutions is referred to herein as a "derivative" of the substituted portion. For example, an alkyl-substituted nitrone is an example of a derivative of the nitrone portion.

[0045] The term “substituted” means that the specified group or portion has one or more suitable substituents, wherein the substituents may be attached to the specified group or portion at one or more positions. For example, an aryl group substituted with a cycloalkyl group may mean that the cycloalkyl group is attached to one atom of the aryl group by a bond, or is attached by fused with the aryl group and sharing two or more common atoms.

[0046] Unless otherwise defined, "aryl" refers to a cyclic aromatic hydrocarbon group having one to three aromatic rings, including monocyclic or bicyclic groups such as phenyl, biphenyl, or naphthyl. When containing two aromatic rings (bicyclic, etc.), the aromatic rings of the aryl group are optionally linked at a single point (e.g., biphenyl) or fused (e.g., naphthyl). The aryl group is optionally substituted at any connection point with one or more substituents (e.g., one to five substituents). Exemplary substituents include, but are not limited to: halogen, oxo, -OH, -CN, -COOH, -CH2CN, -O-(C1-C6)alkyl, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -O-(C2-C6)alkenyl, -O-(C2-C6)ynyl, (C2-C6)alkenyl, (C2-C6)ynyl, -OP(O)(OH)2, -OC(O) (C1-C6)alkyl, -C(O)(C1-C6)alkyl, -OC(O)O(C1-C6)alkyl, -NH2, -NH((C1-C6)alkyl), -N((C1-C6)alkyl)2, -NHC(O)(C1-C6)alkyl, -C(O)NH(C1-C6)alkyl, -S(O)2(C1-C6)alkyl, -S(O)NH(C1-C6)alkyl, and S(O)N((C1-C6)alkyl)2. In some embodiments, the term "aryl" refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group with a fully conjugated π-electron system and containing no heteroatomic ring atoms. The aryl group can be selected from: monocyclic carbocyclic aromatic rings, such as phenyl; bicyclic systems, such as 7-12 membered or, for example, 9-10 membered bicyclic systems, wherein at least one ring is a carbocyclic and aromatic ring, such as those selected from naphthalene, indane, and 1,2,3,4-tetrahydroquinoline; and tricyclic systems, such as 10-15 membered tricyclic systems, wherein at least one ring is a carbocyclic and aromatic ring, such as fluorene. For example, the aryl group can be a 6-membered carbocyclic aromatic ring fused with a 5- to 7-membered cycloalkyl or heterocyclic group, optionally containing at least one heteroatom selected from N, O, and S, provided that: when the carbocyclic aromatic ring is fused with a heterocyclic group, the connection point is located on the carbocyclic aromatic ring; when the carbocyclic aromatic ring is fused with a cycloalkyl group, the connection point may be located on the carbocyclic aromatic ring or on the cycloalkyl group. A divalent (used interchangeably with "bivalent") group formed from a substituted benzene derivative and having a free valence on the ring atom is called a substituted phenylene. A divalent group derived from a monovalent polycyclic hydrocarbon group (names ending in "-yl") by removing a hydrogen atom from a carbon atom with a free valence is named by adding "-idene" to the corresponding monovalent group name. For example, a naphthyl group with two bonding points is called a naphthylene group.

[0047] "Divalent saturated or unsaturated, straight or branched hydrocarbon chains" refers to straight or branched divalent alkylene, alkenyl, and ynylene chains as defined herein.

[0048] "Bridged bicyclic" refers to any bicyclic system having at least one bridge, i.e., a carbocyclic or heterocyclic system, saturated or partially unsaturated. According to the IUPAC definition, a "bridge" is an unbranched chain of atoms or atoms or valence bonds connecting two bridgeheads, wherein a "bridgehead" is any skeletal atom of a ring system bonded to three or more skeletal atoms (excluding hydrogen). In some embodiments, the bridged bicyclic group has 7 to 12 ring members and 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Such bridged bicyclic groups are well known in the art and include groups listed below, wherein each group is connected to the remainder of the molecule at any substituted carbon or nitrogen atom. Unless otherwise stated, the bridged bicyclic group is optionally substituted with one or more substituents as described for aliphatic groups. Additionally or alternatively, any substituted nitrogen atom in the bridged bicyclic group is optionally substituted. Example bridged bicyclics include: "Bridged bicyclic" refers to any bicyclic system having at least one bridge, i.e., a carbocyclic or heterocyclic system, saturated or partially unsaturated. According to the IUPAC definition, a "bridge" is an unbranched chain of atoms or atoms or valence bonds connecting two bridgeheads, wherein a "bridgehead" is any skeletal atom of a ring system bonded to three or more skeletal atoms (excluding hydrogen). In some embodiments, the bridged bicyclic group has 7 to 12 ring members and 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Such bridged bicyclic groups are well known in the art and include groups listed below, wherein each group is connected to the remainder of the molecule at any substituted carbon or nitrogen atom. Unless otherwise stated, the bridged bicyclic group is optionally substituted with one or more substituents as described for aliphatic groups. Additionally or alternatively, any substituted nitrogen atom in the bridged bicyclic group is optionally substituted. Example bridged bicyclics include:

[0049]

[0050] .

[0051] Cycloalkyl, heterocycloalkyl, aryl, and heteroaryl bicyclic (fused, bridged, or spirocyclic) or polycyclic (fused, bridged, or spirocyclic) ring systems are defined according to the nature of the ring system and / or the bonding points. For example, if the entire bicyclic or polycyclic system is completely non-aromatic and contains at least one cyclic heteroatom, then the ring system is a heterocycloalkyl bicyclic or polycyclic system. If the entire bicyclic or polycyclic system is completely aromatic and contains at least one cyclic heteroatom, then the ring system is considered a heteroaryl bicyclic or polycyclic system. If the bicyclic or polycyclic system contains a mixture of non-aromatic and aromatic ring systems, the bonding points determine the nature of the ring system: if bonded to a non-aromatic cycloalkyl or heterocycloalkyl ring, it is considered a cycloalkyl or heterocycloalkyl bicyclic or polycyclic system; if bonded to an aromatic aryl or heteroaryl ring, it is considered an aryl or heteroaryl bicyclic or polycyclic system.

[0052] "Cycloalkyl" refers to a non-aromatic, saturated or unsaturated, monocyclic, bicyclic, or polycyclic ring system containing 3 to 16 ring carbon atoms, but without heteroatomic ring atoms. For example, 3-16 carbon or 3-8 carbon, referred to herein as "(C3-C8)-cycloalkyl," is derived from cycloalkanes. Cycloalkyl groups can contain any number of carbons, such as C3-C8. 3-6 C 4-6 C 5-6 C 3-8 C 4-8 C 5-8 C 6-8 C 3-9 C 3-10 C 3-11 and C 3-12 Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl rings include, for example, bicyclic [1.1.1]pentane, norbornene, [2.2.2]bicyclic octane, decahydronaphthalene, and adamantane. Cycloalkyl groups can also be partially unsaturated, having one or more double or triple bonds in the ring. Representative partially unsaturated cycloalkyl groups include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene, and norbornene. When the cycloalkyl group is a saturated monocyclic C 3-8 When cycloalkyl is used, exemplary groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. When the cycloalkyl group is a saturated monocyclic C 3-6When cycloalkyl is used, exemplary groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups may be substituted with alcohols (hydroxyl), alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, aralkyl, carbamate, carboxylic acid, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclic, ketone, nitro, oxo (carbonyl on the ring), phosphate ester, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thione. Cycloalkyl groups may be fused with other cycloalkyl groups (saturated or partially unsaturated), aryl, or heterocyclic groups to form bicyclic, tetracyclic, etc. In embodiments, the term cycloalkyl refers to a monocyclic ring. In embodiments, the term "bicyclic cycloalkyl" may refer to a cyclic system such as a 1,1'-bi(cyclohexyl)cyclic system. In the implementation plan, the term "bicyclic cycloalkyl" may refer to fused ring systems such as decahydronaphthalene ring systems: In the implementation plan, the term "bicyclic cycloalkyl" may refer to spirocyclic systems such as: or .

[0053] "Heteroaryl" refers to a monovalent aromatic group of a 5-, 6-, or 7-membered ring and comprises a fused ring system of 5-20 atoms (at least one of which is aromatic) containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur. In an embodiment, the heteroaryl is a 5-membered heteroaryl ring. In an embodiment, the heteroaryl is a 6-membered heteroaryl ring. In an embodiment, the heteroaryl is a 7-membered heteroaryl ring. In an embodiment, the heteroaryl is an 8-membered heteroaryl ring. In an embodiment, the heteroaryl is a 9-membered heteroaryl ring. In an embodiment, the heteroaryl is a 10-membered heteroaryl ring. Examples of heteroaryl groups include pyridinyl (including, for example, 2-hydroxypyridinyl), imidazole, imidazole-pyridinyl, pyrimidinyl (including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furanyl, thiophene, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrroleyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cenolinyl, indazole, inazinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purine, oxadiazolyl, triazolyl, thiazolyl, thiazolyl, furazonyl, benzofuranyl, benzothiophene, benzothiazolyl, benzooxazolyl, quinazolinyl, quinoxolinyl, naphridinyl, and furan-pyridinyl. In some embodiments, the heteroaryl ring is fused with one or more aryl, cycloaliphatic, or heterocyclic groups, wherein the group or linker is on the heteroaryl ring (or, in the case of a divalent fused heteroaryl ring system, at least one group or linker is on the heteroaryl ring). Non-limiting examples include indolyl, isoindolyl, benzothiophene, benzofuranyl, dibenzofuranyl, indazole, benzimidazolyl, benzothiazolyl, quinolinyl, isoquinolinyl, terpineyl, phthalazinyl, quinazolinyl, quinoxolinyl, 4H-quinazinyl, carbazole, acridineyl, phenazinyl, phenothiazinyl, phenotoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. The heteroaryl group can be monocyclic, bicyclic, or tricyclic. The term "heteroaryl" is used interchangeably with the terms "heteroary ring," "heteroaryl group," "heteroaromaticity," or "heteroary ring," all of which include optionally substituted rings. In some embodiments, the heteroaryl is an 8-12 membered bicyclic heteroaryl or a 10-14 membered tricyclic heteroaryl. In embodiments, the term "bicyclic heteroaryl" may refer to a cyclic system such as a 2-phenylpyridyl ring system. In the implementation plan, the term "bicyclic heteroaryl" may refer to fused ring systems such as indole ring systems: If a bicyclic or polycyclic system contains a mixture of non-aromatic and aromatic cyclic systems, the connection point determines the nature of the cyclic system: if it is connected to a non-aromatic cycloalkyl or heterocyclic alkyl ring, it is considered a cycloalkyl or heterocyclic bicyclic or polycyclic system; if it is connected to an aromatic aryl or heteroaryl ring, it is considered an aryl or heteroaryl bicyclic or polycyclic system.

[0054] When “heteroaryl, bicyclic heteroaryl and / or fused heteroaryl” (or any combination thereof) are listed together in the same embodiment, these terms are mutually exclusive and are not considered subclasses of heteroaryl. In other words, when used in the same embodiment, the term heteroaryl refers to a monocyclic system, while bicyclic or fused heteroaryl refers to a polycyclic system.

[0055] The terms "heterocyclic group," "heterocyclic," or "heterocyclic alkyl" refer to a ring structure having 3 to 14 atoms (e.g., 4 to 13 atoms), wherein one or more atoms are selected from N, O, and S, wherein the ring N atom can be oxidized to NO, the ring S atom can be oxidized to SO or SO2, and the remaining ring atoms are carbon. In embodiments, the heterocyclic alkyl is a 3- to 9-membered heterocyclic alkyl ring. In embodiments, the heterocyclic alkyl is a 4- to 9-membered heterocyclic alkyl ring. In embodiments, the heterocyclic alkyl is a 5- to 9-membered heterocyclic alkyl ring. In embodiments, the heterocyclic alkyl is a 6- to 9-membered heterocyclic alkyl ring. In embodiments, the heterocyclic alkyl is a 3-membered ring. In embodiments, the heterocyclic alkyl is a 4-membered ring. In embodiments, the heterocyclic alkyl is a 5-membered ring. In embodiments, the heterocyclic alkyl is a 6-membered ring. In embodiments, the heterocyclic alkyl is a 7-membered ring. In embodiments, the heterocyclic alkyl is an 8-membered ring. In embodiments, the heterocyclic alkyl is a 9-membered ring. In embodiments, the heterocyclic alkyl is a 10-membered ring. In one embodiment, the heterocyclic alkyl group is an 11-membered ring. In another embodiment, the heterocyclic alkyl group is a 12-membered ring. In yet another embodiment, the heterocyclic alkyl group is a 13-membered ring. In yet another embodiment, the heterocyclic alkyl group is a 14-membered ring. The heterocyclic group can be a monocyclic, bicyclic, tricyclic, spirocyclic, or bridged ring system. The heterocyclic group may be independently and optionally substituted at the nitrogen atom of the ring with an alkyl, aralkyl, or alkyl carbonyl group, or substituted at the sulfur atom with a lower alkyl group. Examples of heterocyclic groups include, but are not limited to: epoxy, aziridine, aziridinyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolylyl, pyrrolidoneyl, piperidinyl, piperazinyl, imidazoalkyl, thiazoalkyl, dithiazoalkyl, trithiazoalkyl, dioxolanecycloyl, oxazolidinoneyl, decahydroquinolinyl, piperidinoneyl, 4-piperidinoneyl, quininecycloyl, thiomorpholinyl, morpholinyl, aziridineheptyl, oxazidineheptyl, azirbiscyclohexyl, azirbiscycloheptyl, azirbiscycloheptyl, azirbiscycloheptyl, azirbiscycloheptyl, azirbiscycloheptyl, azirbiscycloheptyl, azirbiscycloheptyl (e.g., octahydroindazinyl), azirspiroheptyl, dihydro-1 H ,3 H 5 H -Oxazolo[3,4-c]oxazolyl,tetrahydro-1' H ,3' H -spiro[cyclopropane-1,2'-pyrrolizine], hexahydro-1 H -pyrrolizinyl, hexahydro-1 H-pyrrolo[2,1-c][1,4]oxazinyl, octahydroindazinyl, oxazaspironyl, oxazaspirooctyl, diazaspironyl, oxazabicycloheptyl, hexahydropyrroloazinyl 4(1 H )-oxides, tetrahydro-2H-thiamyl 1-oxides and tetrahydro-2 H -Thiaranyl 1,1-dioxide. Compounds having adjacent cyclic O and / or S atoms are explicitly excluded from the scope of this term. In embodiments, the term "bicyclic heterocyclic alkyl" may refer to cyclic systems such as 1-(piperidin-4-yl)piperazinyl cyclic systems: In the implementation plan, the term "bicyclic heterocyclic alkyl" may refer to fused ring systems such as 3,4-dihydro-2-dicyclic alkyl groups. H -1-Benzopyran: In the implementation plan, the term "bicyclic heterocyclic alkyl" may refer to heterospirocyclic systems such as: If a bicyclic or polycyclic system contains a mixture of non-aromatic and aromatic cyclic systems, the connection point determines the nature of the cyclic system: if it is connected to a non-aromatic cycloalkyl or heterocyclic alkyl ring, it is considered a cycloalkyl or heterocyclic bicyclic or polycyclic system; if it is connected to an aromatic aryl or heteroaryl ring, it is considered an aryl or heteroaryl bicyclic or polycyclic system.

[0056] When “heterocyclic alkyl, bicyclic heterocyclic alkyl, fused heterocyclic alkyl and / or heterospirocyclic alkyl” (or any combination thereof) are listed together in the same embodiment, these terms are mutually exclusive and are not considered subclasses of heterocyclic alkyl. In other words, when used in the same embodiment, the term heterocyclic alkyl refers to a monocyclic system, while bicyclic or fused heterocyclic alkyl refers to a polycyclic system.

[0057] As used in this article, "polycyclic" refers to a system containing multiple closed rings. In some implementations, polycyclic refers to 11 to 20-membered carbon rings or heterocyclic rings.

[0058] The substituents themselves are optionally substituted. Furthermore, when containing two fused rings, the aryl group optionally has an unsaturated or partially saturated ring fused to a fully saturated ring. Exemplary ring systems of these aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl, anthraceneyl, phenatenyl, phenanthyl, indanyl, indenyl, tetrahydronaphthyl, tetrahydrobenzoxenyl, etc.

[0059] "Halogen" or "halogenated" refers to fluorine, chlorine, bromine, or iodine.

[0060] "Halogenated alkyl" refers to an alkyl group that has been substituted with one or more halogens. Examples of halogenated alkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, pentafluoroethyl, trichloromethyl, etc.

[0061] "Heteroatom" refers to one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon; any basic nitrogen or quaternized form of substituted nitrogen in a heterocyclic compound, such as N (e.g., in 3,4-dihydro-2H-pyrrole), NH (e.g., in pyrrolealkyl), or NR). + (e.g., in N-substituted pyrrolidinyl alkyl groups). In one embodiment, the heteroatom is selected from oxygen, sulfur, nitrogen, and phosphorus. In another embodiment, the heteroatom is selected from oxygen, sulfur, and nitrogen. In another embodiment, the heteroatom is independently oxygen or nitrogen.

[0062] "alkyl" refers to a straight-chain or branched saturated hydrocarbon containing 1 to 12 carbon atoms. Examples of (C1-C6) alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, and isohexyl. In some embodiments, the term "alkyl" refers to a saturated straight-chain or branched hydrocarbon, such as a straight-chain or branched group with 1 to 8 carbon atoms, referred to herein as C1-C6. 1-8 Alkyl. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl. In some embodiments, "alkyl" is a straight-chain hydrocarbon. In some embodiments, "alkyl" is a branched-chain hydrocarbon. When a numerical range is listed, it is intended to encompass every value and subrange within that range. For example, "C1-C6 alkyl" is intended to encompass C1, C2, C3, C4, C5, C6, C 1-6 C 1-5 C 1-4 C 1-3 C 1-2 C 2-6 C 2-5 C 2-4 C 2-3 C 3-6 C 3-5 C 3-4 C 4-6 C 4-5 and C 5-6 alkyl.

[0063] "Aliphatic" or "aliphatic group" refers to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more unsaturated units, or a monocyclic or bicyclic hydrocarbon that is fully saturated or contains one or more unsaturated units but is not aromatic (also referred to herein as "carbocyclic," "cycloaliphatic," or "cycloalkyl"), and has a single connection point to the rest of the molecule. Unless otherwise stated, an aliphatic group contains 1-6 aliphatic carbon atoms. In some embodiments, the aliphatic group contains 1-5 aliphatic carbon atoms. In some embodiments, the aliphatic group contains 1-4 aliphatic carbon atoms. In some embodiments, the aliphatic group contains 1-3 aliphatic carbon atoms. In some embodiments, the aliphatic group contains 1-2 aliphatic carbon atoms. In some embodiments, "cycloaliphatic" (or "carbocyclic" or "cycloalkyl") refers to a fully saturated or non-aromatic monocyclic C3-C7 hydrocarbon that contains one or more unsaturated units and has a single connection point to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, straight-chain or branched, substituted or unsubstituted alkyl, alkenyl, ynyl and their hybrids, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0064] "Alkoxy" refers to a straight-chain or branched saturated hydrocarbon containing 1-12 carbon atoms, with a terminal "O", such as -O (alkyl). Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, tert-butoxy, or pentoxy. In some embodiments, the term "alkoxy" refers to a straight-chain or branched saturated hydrocarbon containing 1-12 carbon atoms, with a terminal "O", such as -O (alkyl). Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, tert-butoxy, or pentoxy.

[0065] "Alkenyl" refers to a straight-chain or branched unsaturated hydrocarbon containing 2-12 carbon atoms. An "alkenyl" contains at least one double bond in the chain. The double bond of an alkenyl can be non-conjugated or conjugated with another unsaturated group. Examples of alkenyl include vinyl, propenyl, n-butenyl, isobutenyl, pentenyl, or hexenyl. Alkenyl can be unsubstituted or substituted, and can be straight-chain or branched. In some embodiments, "alkenyl" refers to an unsaturated straight-chain or branched hydrocarbon having at least one carbon-carbon double bond, such as a straight-chain or branched group with 2-8 carbon atoms, referred to herein as (C2-C8)-alkenyl. Exemplary alkenyl includes, but is not limited to, vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl, and 4-(2-methyl-3-buten)-pentenyl.

[0066] "alkylene" refers to a divalent alkyl group. An "alkylene chain" is a polymethylene group, i.e., -(CH2). n- where n is a positive integer, such as 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 2 to 3. The substituted alkylene chain is a polymethylene group, wherein one or more methylene hydrogen atoms are replaced by substituents. Suitable substituents include those described below for substituted aliphatic groups.

[0067] "Alkenyl" refers to a divalent alkenyl group. A substituted alkenyl chain is a polymethylene group containing at least one double bond, wherein one or more hydrogen atoms are replaced by substituents. Suitable substituents include those described below for substituted aliphatic groups.

[0068] "Alynyl" refers to a straight-chain or branched unsaturated hydrocarbon containing 2 to 12 carbon atoms. An alkynyl group contains at least one triple bond in its chain. Examples of alkynyl groups include ethynyl, propynyl, n-butynyl, isobutynyl, pentyynyl, or hexynyl. The alkynyl group can be unsubstituted or substituted.

[0069] “Cycloalkyl” or “carbocyclic” refers to a monocyclic or polycyclic saturated carbocyclic ring containing 3 to 18 carbon atoms. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, norbornenyl, bicyclo[2.2.2]octyl or bicyclo[2.2.2]octenyl and their derivatives. (C3-C8) cycloalkyl is a cycloalkyl group containing 3 to 8 carbon atoms. Cycloalkyl can be fused (e.g., naphthane) or bridged (e.g., norbornane). In some embodiments, when “cycloalkyl, bicyclocycloalkyl, fused bicyclocycloalkyl and / or spirocyclic” (or any combination thereof) are listed together in the same embodiment, these terms are mutually exclusive and are not considered subclasses of cycloalkyl. In other words, when used in the same embodiment, the term cycloalkyl refers to a monocyclic system, while bicyclic or fused cycloalkyl refers to a polycyclic system.

[0070] "Halogenated alkyl" refers to an alkyl group that has been substituted with one or more halogens. Examples of halogenated alkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, pentafluoroethyl, trichloromethyl, etc.

[0071] "Haloalkoxy" refers to an alkoxy group that has been substituted with one or more halogens. Examples of haloalkoxy groups include, but are not limited to, trifluoromethoxy, difluoromethoxy, pentafluoroethoxy, and trichloromethoxy.

[0072] As used herein and unless otherwise stated, the suffix "-alkyl" is used to describe divalent groups. Therefore, any of the above terms may be prefixed with "-alkyl" to describe the divalent form of that part. For example, a divalent carbocyclic ring is "alkylenemonene", a divalent aromatic ring is "arylenemonene", a divalent benzene ring is "phenylenemonene", a divalent heterocyclic ring is "heterocyclic", a divalent heteroaromatic ring is "heteroarylene", a divalent alkyl chain is "alkylenemonene", a divalent alkenyl chain is "alkenylene", a divalent alkynyl chain is "alkynylene", and so on.

[0073] As used herein, the term "pharmaceuticalally acceptable" means a molecular entity and composition that, when administered to humans, is physiologically tolerable and generally does not produce toxicity, allergic reactions, or similar adverse reactions (such as stomach upset, dizziness, etc.). Preferably, as used herein, the term "pharmaceuticalally acceptable" means approved by federal or state regulatory agencies, or listed in the United States Pharmacopeia or other recognized pharmacopoeia for use in animals, and more particularly for use in humans.

[0074] As used in this article, the "therapeutic index" is a parameter that indicates the therapeutic efficacy of an active pharmaceutical ingredient. For example, a low therapeutic index occurs when a high concentration of the active substance is required to achieve therapeutic efficacy, or when the dose required to achieve therapeutic efficacy triggers toxicity. Conversely, a high therapeutic index means that a lower dose of the active substance is required to provide therapeutic efficacy, and / or that the active pharmaceutical ingredient has lower toxicity.

[0075] A "linker" is a fragment that connects a ligand to a viral capsid protein and includes a cross-linking portion.

[0076] "Crosslinking" refers to the process of chemically linking two or more molecules through covalent bonds. In some embodiments, crosslinking refers to the reaction between a first member of a crosslinking agent reactant pair and a second member of the crosslinking agent reactant pair, wherein the first and second members of the crosslinking agent react to form a crosslinked portion.

[0077] "Copper-free click chemistry" refers to bioorthogonal reactions that do not use copper catalysts. In some embodiments, the process refers to reactions carried out by substrates (or crosslinking agents) without the need for exogenous catalysts (such as copper).

[0078] "Cross-linked portion" refers to the portion formed by the reaction between cross-linking agent reaction pairs.

[0079] "Enzyme cleavage" refers to the breaking of peptide bonds between two amino acids that an enzyme recognizes through its secondary structure.

[0080] "Ligand" refers to a molecule that can couple to the surface of a viral capsid and bind to receptors (protein molecules) in the cell.

[0081] "Spacer" refers to the portion that connects the surface-functionalized viral capsid to the capsid reactive linker and / or connects the ligand to the ligand reactive linker.

[0082] "Cell type specific ligands" refer to ligands that can bind to specific receptors on the cell surface.

[0083] 5.2 Other Interpretive Agreements

[0084] Although similar or equivalent methods and materials may be used in implementing or testing the methods and compositions described herein, suitable methods and materials are described below. Furthermore, the materials, methods, and examples described are for illustrative purposes only and are not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety.

[0085] It must be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “an antibody or antigen-binding fragment” includes a plurality of such antibodies and antigen-binding fragments; reference to “the recombinant adeno-associated virus” includes one or more recombinant adeno-associated viruses and their equivalents known to those skilled in the art, and so on. It should also be noted that the claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a prior basis for the use of exclusive terms such as “only” or “unique” in relation to the recitation of claim elements, or for the use of negative limiting terms.

[0086] It should be understood that certain features of this disclosure—described in the context of individual embodiments for clarity—may also be provided in combination within a single embodiment. Conversely, various features of this disclosure, described in the context of individual embodiments for brevity, may also be provided individually or in any suitable sub-combination. This disclosure specifically covers all combinations of embodiments relating to this disclosure and discloses them herein as if each combination were disclosed individually and explicitly. Furthermore, all sub-combinations of the various embodiments and their elements are also specifically covered by this disclosure and disclosed herein as if each such sub-combination were disclosed individually and explicitly herein.

[0087] The publications discussed herein are provided only because they were published prior to the filing date of this application. The publication dates provided may differ from the actual publication dates and may require independent verification.

[0088] Where a numerical range is provided, it should be understood that the endpoints of the range are included. Furthermore, every intermediate value between the upper and lower limits of the range (accurate to one-tenth of the lower limit unit unless the context explicitly indicates otherwise), as well as any other specified value or intermediate value within the specified range, is included in this disclosure. The upper and lower limits of these smaller ranges may be independently included within the smaller range and are also covered within this disclosure, but are subject to any explicitly excluded limits within the specified range. When a specified range includes one or two limits, the range excluding one or both of the included limits is also included in this disclosure.

[0089] The numerical ranges described herein should be understood as abbreviated forms of all numerical values ​​within that range, including the endpoints. For example, the range 1 to 50 should be understood as including any number, combination of numbers, or subrange consisting of the following values: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50, including subranges such as 11 to 48 or 39 to 41.

[0090] The term “about” as used in this document allows for a certain degree of variation in the value or range, for example, within 10% of the specified value or range limit.

[0091] 5.3 Surface-modified viral capsids

[0092] On one hand, a surface-modified viral capsid is provided, wherein the surface-modified viral capsid comprises one or more ligands covalently coupled to a viral capsid protein via a linker, the linker comprising a crosslinking portion, wherein the crosslinking portion is formed by a reaction between a first member and a second member of a crosslinking agent reaction pair; and includes optionally one or more spacers.

[0093] According to this disclosure, in a first aspect, a surface-modified viral capsid, typically an AAV viral capsid, is provided. The surface modification includes a first ligand that facilitates rAAV entry from CSF into the brain parenchyma, and optionally also includes one or more additional surface-coupled ligands that facilitate CNS cell transduction, optionally facilitating transduction of one or more desired CNS cell types. In some embodiments, the first ligand is covalently coupled to a viral capsid protein. In some embodiments of these embodiments, the first ligand is covalently coupled to the viral capsid protein via a linker comprising a crosslinking portion Q. In other embodiments, the first ligand is fused within a capsid protein sequence. In another aspect, recombinant viral particles, typically recombinant AAV viral particles (rAAV), are provided, comprising the surface-modified viral capsid and a nucleic acid payload encapsulated within the capsid.

[0094] 5.3.1 Direct Surface Modification via Ligand Coupling

[0095] According to this disclosure, a surface-functionalized viral capsid is provided, comprising a first member of a crosslinking agent reactant pair. A functionalized ligand is also provided, comprising a second member of the crosslinking agent reactant pair, wherein the first and second members of the crosslinking agent reactant pair react to form a crosslinked portion Q. The surface-functionalized viral capsid is capable of crosslinking, i.e., coupling, with a ligand having a complementary member of the crosslinking agent reactant pair.

[0096] In some embodiments, the surface-modified viral capsid in the composition comprises x conjugated ligands, where x is the average number of ligands conjugated to each capsid in the composition, also referred to herein as the ligand / capsid ratio or LCR. In some embodiments, x is 1 to 500. In some embodiments, x is 1 to 300. In some embodiments, x is 100 to 200. In some embodiments, x is 110 to 190. In some embodiments, x is 130 to 170. In some implementations, x is 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300, or a range defined by any two of the aforementioned numbers. In some embodiments, x is about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, or about 180. In some embodiments, x is about 55 to about 85. In some embodiments, x is about 140 to about 160. In some embodiments, x is about 135 to about 165. In some embodiments, x is about 130 to about 170. In some embodiments, x is about 150. In some embodiments, x falls within the range between any two numbers provided above.

[0097] This disclosure also provides a capsid reactive linker comprising (i) a capsid reactive portion capable of covalently linking to a viral capsid protein, and (ii) a member of a crosslinking agent reactant pair.

[0098] This disclosure also provides a recognition sequence reactive linker comprising (i) an enzyme substrate reactive portion capable of participating in an enzymatic linking to a recognition sequence in a viral capsid protein, and (ii) a member of a crosslinking agent reaction pair.

[0099] In embodiments of this disclosure, the surface-modified viral capsid according to this disclosure is prepared by the following steps: A surface-functionalized viral capsid is obtained by reacting viral capsid proteins with a capsid reactive linker, said linker comprising a first member of a cross-linking agent reaction pair and optionally one or more spacers; and The surface-functionalized viral capsid is coupled with a functionalized ligand containing a cross-linking agent to react with a second member. The first and second members of the crosslinking agent reaction pair react to form the crosslinked portion Q; and The surface-modified viral capsid is obtained.

[0100] In other embodiments, the method for preparing a surface-modified viral capsid according to this disclosure includes the following steps: A surface-functionalized viral capsid is obtained through the enzymatic ligation between the following two components: The recognition sequence located in the circular region on the surface of the viral capsid protein; and An enzyme-reactive linker, wherein the linker comprises a first member of an enzyme substrate and cross-linking agent reaction pair; and The obtained surface-functionalized viral capsid is reacted with a functionalized ligand containing a cross-linking agent to react with a second member.

[0101] In some embodiments, the method further includes the step of generating an AAV capsid comprising at least one viral capsid protein, said viral capsid protein containing at least one recognition sequence. Optionally, the recognition sequence is located in a surface loop region, such as variable region 4 or 8.

[0102] In other embodiments, the method for preparing a surface-modified viral capsid according to this disclosure includes the following steps: A surface-functionalized viral capsid is obtained through the enzymatic ligation between the following two components: The recognition sequence located in the circular region on the surface of the viral capsid protein; and Functionalized ligands containing enzyme substrates; and The obtained surface-functionalized viral capsid is reacted with functionalized ligands in the presence of the enzyme.

[0103] In one aspect of this disclosure, the methods described herein are contemplated to be performed sequentially to produce a surface-modified virus comprising two or more different ligands attached to the surface of the virus, for example using different surface functionalization strategies described herein, or repeating the same strategy using different ligands. That is, surface-modified viral particles can be used as starting materials in any of the above methods to modify the surface of the viral particles with a second (and third, etc.) type of ligand.

[0104] 5.3.1.1 The effect of crosslinking agent reaction

[0105] Exemplary crosslinking agent reaction pairs available according to certain embodiments include, for example, those disclosed in WO 2022 / 101363, the disclosure of which is expressly incorporated herein by reference in its entirety.

[0106] In embodiments of this disclosure, to achieve covalent bonding between the ligand and the surface of the viral capsid, thereby generating a surface-modified viral capsid, the recombinant viral particles are first surface-functionalized to generate a surface-functionalized viral capsid, which is then crosslinked with the functionalized ligand. Both the surface-functionalized capsid and the functionalized ligand contain members of a crosslinking agent reaction pair. The crosslinking agent reaction pair members react to form a Q portion, which covalently crosslinks the viral capsid with the ligand.

[0107] In some embodiments, the first and second members of the crosslinking agent reaction pair participate in reactions selected from: Cu(I)-catalyzed azido-yne cycloaddition (CuAAC), strain-promoted azido-yne cycloaddition (SPAAC), strain-promoted yne-nitroketone cycloaddition (SPANC), anti-electron-demanding Diels-Alder (IEDDA) reactions, and Staudinger linkage and [4+1] cycloaddition reactions. In some embodiments, the first and second members of the crosslinking agent reaction pair participate in Cu(I)-catalyzed azido-yne cycloaddition (CuAAC). In some embodiments, the first and second members of the crosslinking agent reaction pair participate in strain-promoted azido-yne cycloaddition (SPAAC). In some embodiments, the first and second members of the crosslinking agent reaction pair participate in strain-promoted yne-nitroketone cycloaddition (SPANC). In some embodiments, the first and second members of the crosslinking agent reaction pair participate in anti-electron-demanding Diels-Alder (IEDDA) reactions. In some embodiments, the first and second members of the crosslinker reaction pair participate in Staudinger linkage and [4+1] cycloaddition reactions.

[0108] In some embodiments, the first and second members of the crosslinking agent reaction pair participate in a reaction selected from: strain-promoted azido-acetylene cycloaddition (SPAAC), strain-promoted acetylene-nitroketone cycloaddition (SPANC), anti-electron-demanding Diels-Alder (IEDDA) reaction, and Staudinger linkage and [4+1] cycloaddition reaction.

[0109] In a typical implementation, the crosslinking agent reaction pair members are bioorthogonal. As used herein, the term bioorthogonal chemistry refers to any chemical process that can occur within a living system without interfering with natural biochemical processes, or that can occur in vitro without interfering with the biochemical / biological activity of the reaction products. Many chemical coupling strategies that meet the bioorthogonality requirement have been developed, including 1,3-dipolar cycloaddition between azides and cyclooctyne (also known as copper-free click chemistry), 1,3-dipolar cycloaddition between nitrones and cyclooctyne, oxime / hydrazone formation from aldehydes and ketones, tetrazine linkages, such as cycloaddition of S-tetrazines to trans-cyclooctene derivatives or isonitrile-based click reactions, and more recently, tetracycloalkane linkages.

[0110] 5.3.1.1.1 CuAAC

[0111] In some embodiments, the crosslinking agent reaction pair is selected from chemical moieties involved in copper(I)-catalyzed azido-yne cycloaddition reactions (CuAAC). In some embodiments, the crosslinking agent reaction pair comprises an azide group and an alkynyl group. Derivatives of these moieties that retain the desired chemical reactivity are also covered herein. In some embodiments, the crosslinking moiety Q comprises a five-membered heteroatom ring. In some embodiments, the crosslinking moiety Q comprises a 1,4-triazole.

[0112] 5.3.1.1.2 SPAAC and SPANC

[0113] Unlike CuAAC, copper-free click chemistry has been modified to be bioorthogonal, enabling rapid reactions without producing live cytotoxicity by eliminating cytotoxic copper catalysts. The reaction does not use copper, but instead employs a strain-promoted alkyne-azide cycloaddition reaction (SPAAC). Copper-free click chemistry has been adapted to use nitrones instead of azides as 1,3-dipolar bodies and has been applied to peptide modification.

[0114] In some embodiments, the crosslinking agent reaction pair is selected from chemical moieties involved in strain-promoted acetylene-nitroketone cycloaddition reactions (SPANC). In some embodiments, the crosslinking agent reaction pair comprises an azide group and a nitroketone group. Derivatives of these moieties that retain the desired chemical reactivity are also covered herein. In some embodiments, the crosslinking moiety Q comprises isoxazoline.

[0115] In some embodiments, the crosslinking agent reaction pair is an azide group and a nitrone group, as shown below, where the R group represents a connection point with a capping reactive linker or functionalized ligand. This document also covers derivatives that retain these parts while retaining the desired chemical reactivity. For example, substitution can occur at both the carbon and nitrogen atoms of the nitrone dipole, and both acyclic and intracyclic nitrones are acceptable.

[0116]

[0117] In some embodiments, the crosslinking agent reaction pair comprises a cyclooctyn analog. In some embodiments, the crosslinking agent reaction pair comprises a cyclooctyn analog, such as the analog shown in the figure below, wherein the R group represents a connection site with a capsid reactive linker or functionalized ligand. Derivatives that retain these portions of the desired chemical reactivity are also covered herein.

[0118]

[0119]

[0120]

[0121] In some embodiments, the crosslinking agent reaction pair comprises dibenzylcyclooctyne analogs selected from the group consisting of dibenzylcyclooctyne (DIBO), dibenzozazepine octyne (DIBAC or DBCO), and biarylazazepine octyneone (BARAC). Derivatives of these moieties that retain the desired chemical reactivity are also covered herein.

[0122] In some embodiments, the crosslinking agent reaction pair comprises a nitroketone having the following structure, wherein R 1 The group represents the connection point with a capsid reactive linker or functionalized ligand. R 2 and R 3 There are no particular restrictions. In some implementations, R 2 and R 3 Independently selected from hydrogen and C1-C4 alkyl groups, such as methyl, ethyl, propyl, and butyl. This article also covers derivatives of these moieties that retain the desired chemical reactivity.

[0123]

[0124] In some embodiments, the crosslinking agent reaction pair comprises a dibenzylcyclooctyne analogue as identified above, and a 1,3-nitroketone or azide. In some embodiments, the crosslinking agent reaction pair comprises dibenzylcyclooctyne (or an analogue thereof) and a 1,3-nitroketone or azide, as shown below, wherein R 1 The group represents the connection point with the viral capsid or capsid reactive linker, and the R on the azide group or nitro group 2 The group represents the connection site with the functionalized ligand. In another embodiment, the crosslinking agent reaction pair comprises dibenzylcyclooctyne (or an analogue thereof) and 1,3-nitroketone or azide, as shown below, wherein R 1 The group represents the binding site with the ligand, and the R on the azide group or nitro group 2 The group represents the connection point with the surface-functionalized viral capsid or capsid reactive linker.

[0125]

[0126] In some embodiments, the crosslinked portion Q comprises a ring-shaped portion as shown in any of the following figures, wherein R 1 and R 2 Represents the connection point with the viral capsid. R 3 and R 4 It can be H or any substituent described herein, provided that the substituted derivative retains the desired chemical reactivity, and such derivatives are also covered herein.

[0127]

[0128] IEDDA

[0129] In some embodiments, the crosslinking agent reaction pair comprises a chemical moiety that participates in the anti-electron-demanding Diels-Alder (IEDDA) reaction. In some embodiments, the crosslinking agent reaction pair comprises an electron-deficient diene and an electron-rich dienophile. Examples of such groups are known in the art and described in other literature, such as F. Thalhammer, et al., Tetrahedron Lett., 1990, 31, 6851-6854; and BL Oliveira, Chem. Soc. Rev., 2017, 46, 4895-4950. In some embodiments, the electron-deficient diene is substituted with an electron-withdrawing group on the diene, as illustrated below. In some embodiments, the electron-rich dienophile is substituted with an electron-donating group on the dienophile, as illustrated below.

[0130]

[0131] In some embodiments, the crosslinking agent reaction involves the chemical moiety involved in the Diels-Alder [4+2] cycloaddition reaction, namely the reaction between the diene and the dienophile, through a face-to-face / face-to-face interaction between the 4π electrons of the diene and the 2π electrons of the dienophile, forming a six-membered ring in a π4s + π2s manner. In contrast to the normal electron-demanding Diels-Alder reaction (reaction of an electron-rich diene with an electron-deficient dienophile), in the anti-electron-demanding Diels-Alder reaction (IEDDA), the electron-rich dienophile reacts with the electron-deficient diene. The alkynyl dienophile directly generates the corresponding pyridazine after the reaction.

[0132] In some embodiments, the crosslinking agent reaction pair comprises a triazine (e.g., 1,2,4-triazine), a tetrazine (Tz) (e.g., 1,2,4,5-tetraazine, also known as s-tetraazine), or a strained dienophile such as norbornene, trans-cyclooctene (TCO), cyclopropene, or N-acylazirhexacyclobutene. In some embodiments, the crosslinking agent reaction pair comprises a crosslinking agent reactive moiety as illustrated below, wherein the R group represents a connection point with a capsid reactive linker, a surface-functionalized viral capsid, or a functionalized ligand of the present disclosure. Derivatives of these moiety retaining the desired chemical reactivity are also covered herein. In some embodiments, the crosslinking agent reaction pair comprises TCO and a tetrazine.

[0133]

[0134] 5.3.1.1.1 Staudinger Connection

[0135] In some embodiments, the crosslinking agent reaction pair is selected from the reactive portions of the crosslinking agent involved in the Staudinger reaction, such as azides, phosphine (PPh2), or phosphites, which are capable of reacting to generate iminophosphine.

[0136] In some embodiments, the reactive portion of the crosslinking agent is triphenylphosphine, as shown below, wherein the R group represents a connection point with the capsid reactive linker of this disclosure. Derivatives of this reactive portion of the crosslinking agent that retain the desired chemical reactivity are also covered herein.

[0137]

[0138] Reactive part of crosslinking agent

[0139] 5.3.1.1.2 [4+1] Cycloaddition Reaction

[0140] In some embodiments, the crosslinking agent reactive pair is selected from the reactive portion of the crosslinking agent that participates in the [4+1] cycloaddition reaction and subsequently eliminates N2 via the reverse Diels Alder reaction, such as isocyanates or 1,2,4,5-tetraazine.

[0141] In some embodiments, the reactive portion of the crosslinking agent is an isocyanate as shown below, wherein R 1 The group represents a connection point, such as a connection point with a capsid reactive linker. In some embodiments, the reactive portion of the crosslinking agent is a 1,2,4,5-tetraazine as shown below, wherein R 2 Groups represent connection sites, such as those with ligands. This article also covers derivatives that retain these parts to maintain the desired chemical reactivity.

[0142]

[0143] 5.3.1.1.1 Labeling Response

[0144] In some embodiments, the reactive portion of the crosslinking agent is a bioorthogonal tag known in the art, such as a SNAP-tag, CLIP-tag, Halo-tag, or LUMIO-tag, or a chemical group that reacts with these tags, such as a benzylguanine group, a benzylcytosine group, or a chloroalkyl group. In some embodiments, one member of the reactive portion of the crosslinking agent contains a SNAP-tag, while another member of the reactive portion of the crosslinking agent contains a benzylguanine group.

[0145] 5.3.1.2 Crosslinking Part - Q

[0146] In one aspect of this disclosure, the surface-modified viral capsid includes a portion Q, which is formed by a reaction between the crosslinking agent reaction pairs described herein. In some embodiments, the first and second members of the crosslinking agent reaction pair participate in a reaction selected from: Cu(I)-catalyzed azido-acetylene cycloaddition (CuAAC), strain-promoted acetylene-azido cycloaddition (SPAAC), strain-promoted acetylene-nitroketone cycloaddition (SPANC), anti-electron-demanding Diels-Alder (IEDDA) reaction, and Staudinger linkage and [4+1] cycloaddition reactions. In some embodiments, the first and second members of the crosslinking agent reaction pair participate in a Cu(I)-catalyzed azido-acetylene cycloaddition (CuAAC). In some embodiments, the first and second members of the crosslinking agent reaction pair participate in a strain-promoted acetylene-azido cycloaddition (SPAAC). In some embodiments, the first and second members of the crosslinking agent reaction pair participate in a strain-promoted acetylene-nitroketone cycloaddition (SPANC). In some embodiments, the first and second members of the crosslinking agent reactant pair participate in the anti-electron demand Diels-Alder (IEDDA) reaction. In some embodiments, the first and second members of the crosslinking agent reactant pair participate in Staudinger linkage and [4+1] cycloaddition reactions.

[0147] In some embodiments, Q comprises the product of the CuAAC reaction. In some embodiments, Q comprises the product of the SPAAC reaction. In some embodiments, Q is the product of the SPANC reaction. In some embodiments, Q comprises the product of the IEDDA reaction. In some embodiments, Q comprises a Staudinger-linked product. In some embodiments, Q comprises the product of a [4+1] cycloaddition reaction. In some embodiments, Q comprises a product of a strain-promoted reaction, such as SPAAC, SPANC, and IEDDA.

[0148] In some embodiments, the crosslinked portion comprises at least one of an eight-membered ring and a triazole ring.

[0149] In some embodiments, Q comprises a cyclic group. In some embodiments, Q comprises a bicyclic group. In some embodiments, Q comprises a tricyclic group. In some embodiments, Q is a fused polycyclic ring. In some embodiments, Q comprises a 5-8 membered carbon ring containing 0 to 3 heteroatoms selected from O, S, or N. In some embodiments, Q comprises an 8-membered ring containing 0 to 1 heteroatoms selected from O and N. In some embodiments, Q comprises a 5-membered ring containing 0 to 3 heteroatoms selected from O and N. In some embodiments, Q is a triazole ring. In some embodiments, Q comprises a 6-membered ring containing 0-3 heteroatoms selected from O and N. In some embodiments, Q comprises a 6-membered ring containing 2 N heteroatoms.

[0150] In some embodiments, the crosslinking agent reaction pair comprises cyclooctylene and azide.

[0151] In some embodiments, the crosslinking agent reaction pair comprises optionally substituted cyclooctylene and azide.

[0152] In some embodiments, the crosslinking agent reaction pair comprises optionally substituted cyclooctyne and nitroketone.

[0153] In some embodiments, the cyclooctyne is selected from dibenzylcyclooctyne (DIBO), dibenzozacyclooctyne (DBCO), and biarylazacyclooctynone (BARAC), or derivatives thereof. In some embodiments, the cyclooctyne is dibenzylcyclooctyne (DIBO). In some embodiments, the cyclooctyne is dibenzozacyclooctyne (DBCO). In some embodiments, the cyclooctyne is biarylazacyclooctynone (BARAC).

[0154] In some embodiments, Q comprises a cyclic group having the structure shown below, wherein Z is a 7- or 8-membered carbon ring containing 0-3 heteroatoms selected from O or N. In some embodiments, Z is optionally substituted with one or more arylene groups. In some embodiments, Z is not substituted. In some embodiments, Z is substituted with one arylene group. In some embodiments, Z is substituted with two arylene groups. In some embodiments, the arylene groups are independently selected. In some embodiments, the arylene groups are identical.

[0155]

[0156] In some embodiments, Q comprises the reaction products of the cyclooctylene reaction moiety and the azide reaction moiety.

[0157] In some embodiments, the crosslinked portion comprises the following structure:

[0158] Where R 1 and R 2 Indicates the connection point with the connector.

[0159] In some implementations, Q includes one of the following structures: and .

[0160] In some embodiments, the crosslinking agent reaction pair comprises trans-cyclooctene and tetrazine.

[0161] In some embodiments, the crosslinked portion (Q) comprises the following structure:

[0162] Where R 1 and R 2 Indicates the connection point with the connector.

[0163] In some embodiments, the crosslinked portion (Q) comprises a phosphine oxide derivative having the following structure: or , Where R 1 and R 2 Indicates the connection point with the connector.

[0164] In some embodiments, the crosslinked portion (Q) comprises a 4H-pyrazole-4-imine derivative having the following structure: , Where R 1 and R 2 Indicates the connection point with the connector.

[0165] In some implementations, Q includes the following structure: .

[0166] 5.3.1.3 Surface-functionalized capsid

[0167] In one aspect of this disclosure, a surface-functionalized capping shell is provided, wherein the surface of the capping shell is functionalized to include a member of a crosslinking agent reaction pair, namely a crosslinking agent reactive portion.

[0168] In some embodiments, amino acids in the primary sequence of the capsid protein are functionalized by reacting with capsid reactive linkers provided in this disclosure. In some embodiments, genetically introduced in-frame enzyme recognition sequences (e.g., bioorthogonal tags) in the primary sequence of the capsid protein are functionalized by an enzymatic reaction with enzyme reactive linkers (recognition sequence reactive linkers) provided in this disclosure. In some embodiments, the primary sequence of the capsid protein comprises non-natural amino acids, wherein said non-natural amino acids comprise cross-linking agent reactive moieties.

[0169] 5.3.1.3.1 Capsule Reactive Connectors

[0170] Exemplary capsid reactive connectors available according to certain embodiments include, for example, those disclosed in patent publication WO 2022 / 101363, which is expressly incorporated herein by reference in its entirety.

[0171] The capsid reactive linker according to this disclosure comprises: i) a capsid surface reactive portion, which can be used to form a covalent link with the capsid surface described herein, and ii) a member or ligand of a crosslinker reactive pair. In some embodiments, the first member of the crosslinker reactive pair is selected to react with a ligand functionalized with a second member of the crosslinker reactive pair as described herein.

[0172] 5.3.1.3.2 Spacers

[0173] The capping reactive linker optionally further comprises one or more spacer portions. The spacer portions are not particularly limited and can be any spacer known in the art. According to this disclosure, the spacers are not particularly limited and include any divalent portion capable of covalently attaching to the linker. In some embodiments, the divalent portion includes alkylene, alkenylene, ynylene, arylene, heteroarylene, fused arylene, fused heteroarylene, cycloalkylene, heterocyclic, fused cycloalkylene, or fused heterocyclic.

[0174] In some embodiments, the spacer comprises one or more ethylene glycol monomers, namely polyethylene glycol, -(O-CH2-CH2). n - or [PEG] n Also known as "discrete polyethylene glycol" or "dPEG". n The expression “n” represents the number of ethylene oxide (or “ethylene glycol”) units. In some embodiments, n is 0. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, or 100. In some embodiments, the spacer is [PEG]. n, where n is 4, 8, or 12. In some implementations, the spacer is [PEG]. n , where n is 4, and should be understood to include four -(O-CH2-CH2)- groups, without restricting the relative positions of the ether oxygen.

[0175] In some embodiments, the spacer comprises one or more divalent groups, such as -CH2- (methylene), -O- (ether), -C(=O)- (carbonyl), and -N(R)- (amine, wherein R is H or C). 1-3 alkyl).

[0176] In some embodiments, the spacer comprises one or more -YC(O)-, -YC(O)O-, -Y-NHC(O)-, -Y-NHC(S)-, or -YC(O)-, wherein Y is a bond or one or more ethylene glycol monomers.

[0177] In some implementations, the spacer comprises one or more -([PEG]) n )-C(O)-, where n is 1 to 100, for example 1 to 25, 1 to 20, 1 to 15, 1 to 10, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 4 to 15 or 4 to 10.

[0178] In some embodiments, the spacer comprises one or more -(CH2) groups. n - where n is 1 to 100, for example 1 to 25, 1 to 20, 1 to 15, 1 to 10, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 4 to 15, or 4 to 10. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, 100.

[0179] 5.3.1.3.3 Reactive components on the capsid surface

[0180] According to this disclosure, the reactive portion of the capping surface is not particularly limited, including any portion that can be covalently attached to the desired capping surface.

[0181] In some embodiments, the surface reactive portion of the capsid is covalently linked to surface-exposed amino acid residues in the primary sequence of the capsid protein using known techniques in residue-specific protein labeling.

[0182] In some embodiments, the amino acid residues are present in the wild-type capsid protein. In some embodiments, the capsid protein is a known variant of the wild-type capsid protein.

[0183] In some embodiments, the amino acid residues are naturally occurring residues. In these specific embodiments, the primary sequence of the capsid protein is a wild-type sequence or includes genetically engineered regions.

[0184] In other embodiments, the amino acid residues are engineered into the primary amino acid sequence of the capsid.

[0185] i. Primary amines on the capsid surface

[0186] In some embodiments, the reactive portion of the capsid surface comprises a chemical group that reacts with a primary amine (-NH2). The primary amine is present at the N-terminus of each capsid protein and at the side chain (ε-position) of the lysine (Lys, K) amino acid residue in the capsid protein sequence.

[0187] Exemplary chemical groups that react with primary amines include isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxal, epoxides, ethylene oxides, carbonates, aryl halides, imine esters, carbodiimides, acid anhydrides, and fluorophenyl esters. Most of these are coupled to the amine via acylation or alkylation. In some embodiments, the reactive portion of the capsid surface comprises an NHS ester or an imine ester, such as those shown below, wherein the R group represents a connection point with the remainder of the capsid reactive linker (further comprising a member of a crosslinker reaction pair as defined in this disclosure or its reaction product).

[0188]

[0189] In some embodiments, the capsid surface-reactive portion is covalently linked to surface-exposed lysine residues of the primary sequence of the capsid protein. In some of these embodiments, the capsid surface-reactive portion comprises an NHS-ester, a fluorophenyl ester (e.g., TFP), an isocyanate, an isothiocyanate, or benzoyl fluoride, as shown below, wherein the R group represents a connection point with the remainder of the capsid reactive linker (further comprising a member of a crosslinking agent reaction pair as defined herein or its reaction product), and the symbol is... This indicates the connection point of lysine residues within the capsid protein sequence.

[0190]

[0191] In some embodiments, the capsid reactive linker comprises an N-hydroxysuccinimide ester (NHS ester). The NHS ester is a reactive group formed by activating a carboxylic acid molecule with a carbodiimide. The NHS ester-activated capsid reactive linker reacts with a primary amine under physiological to slightly alkaline conditions (pH 7.2 to 9) to form a stable amide bond. This reaction releases N-hydroxysuccinimide (NHS).

[0192] In some embodiments, the capping reactive linker comprises a fluorophenyl ester, such as tetrafluorophenyl ester (TFP) or pentafluorophenyl ester (PFP). The fluorophenyl ester is a reactive group formed by activating a carboxylic acid molecule with a carbodiimide. The fluorophenyl ester of the carboxylic acid reacts with a primary amine to form a covalent amide bond, which is the same as the amide bond formed by the reaction of a primary amine with an NHS ester.

[0193] ii. Thiol groups on the capsid surface

[0194] In some embodiments, the capsid surface-reactive portion is covalently linked to surface-exposed thiol groups. In some embodiments, the capsid surface-reactive portion is covalently linked to surface-exposed cysteine ​​residues in the primary sequence of the capsid protein.

[0195] In some of these embodiments, the reactive portion of the capsid surface comprises maleimide, iodoacetamide, 2-thiopyridine, or 3-arylpropyne nitrile, as shown in the following examples, where the R group represents a connection point with the capsid reactive linker, and the symbol is... This indicates the cysteine ​​residue linking point in the capsid protein sequence.

[0196]

[0197] In some embodiments, the capsid reactive linker comprises maleimide. Maleimide and its derivatives are prepared by amine treatment of maleic anhydride followed by dehydration. When the pH of the reaction mixture is between 6.5 and 7.5, the maleimide group reacts specifically with the thiol group; the result is the formation of a stable, irreversible thioether bond.

[0198] iii. Non-natural amino acids

[0199] In some embodiments, the surface of the viral capsid contains one or more proteins having non-natural amino acids comprising a cross-linking agent reactive portion.

[0200] In some embodiments, the non-natural amino acid is selected from: 1: 3-(6-acetylnaphthyl-2-ylamino)-2-aminopropionic acid (Anap), 2: (S)-1-carboxy-3-(7-hydroxy-2-oxo-2H-chromen-4-yl)prop-1-ammonium (CouAA), 3: 3-(5-(dimethylamino)naphthyl-1-sulfonamido)propionic acid (Dansylalanine), 4: - p-Azide-benzyloxycarbonyl lysine (PABK), 5: propargyl-L-lysine (PrK), 6: -(1-Methylcyclopropionic-2-encarbamoyl)lysine (CpK), 7: -Acryloyllysine (AcrK), 8: 9: 9: Bicyclo[6.1.0]non-4-yn-9-ylmethanol lysine (BCNK), 10: trans-cyclooctyl-2-enyllysine (2'-TCOK), 11: trans-cyclooctyl-4-enyllysine (4'-TCOK), 12: dioxo-TCO lysine (DOTCOK), 13: 3-(2-cyclobuten-1-yl)propionic acid (CbK), 14: -5-norbornen-2-yloxycarbonyl-L-lysine (NBOK), 15: cyclooctylenyllysine (SCOK), 16: 5-norbornen-2-oltyrosine (NOR), 17: cyclooctyl-2-ynyltyrosine (COY), 18: (E)-2-(cyclooct-4-en-1-yloxy)ethanoltyrosine (DS1 / 2), 19: azoxyhoalanine (AHA), 20: high-alkyneglycine (HPG), 21: azoxynorleucine (ANL), and 22: -2-Azideethoxycarbonyl-L-lysine (NEAK), as shown below.

[0201]

[0202]

[0203]

[0204]

[0205] 5.3.2 Surface modification by recognizing sequence-reactive linkers (chemogenetic capsid modification)

[0206] According to this disclosure, the identifying sequence reactive linker comprises: i) an enzyme substrate moiety, and ii) a member of the cross-linking agent reaction pair, namely, the cross-linking agent reactive moiety. The member of the cross-linking agent reactive moiety is preferably selected to react with the cross-linking agent reactive moiety on the functionalized ligand of this disclosure.

[0207] 5.3.2.1.1 Connector

[0208] In some embodiments, the "linker" or linker portion is derived from a molecule having a reactive end, for example, suitable for coupling with a target protein. In some cases, the reactive end of the linker precursor comprises a chemiselective linker group capable of coupling to amino acid residues of a polypeptide. In some cases, the chemiselective linker group is coupled to an accessible cysteine ​​thiol group or a lysine side-chain amino group of the polypeptide. A variety of coupling chemistry (e.g., described herein) can be utilized in the conjugates of this disclosure. In some embodiments, the chemiselective linker group is a thiol-reactive group, such as maleimide or dibromomaleimide. In some embodiments, the chemiselective linker group is an amine-reactive group, such as an active ester, for example, pentafluorophenyl ester or tetrafluorophenyl ester, or N-hydroxysuccinimide ester (NHS) or sulfonated NHS, or as defined in this disclosure.

[0209] It should be understood that a variety of substituents and chemical methods can be used to connect a specific X-ligand moiety (such as those described in this disclosure) to an adjacent linker. In some embodiments, the linker portion comprises a triazole group derived from click chemistry coupling.

[0210] i. Chemically selective linking groups

[0211] A chemoselective linker is a group with reactive functionalities or groups that can couple with a compatible group of a second part (e.g., a polypeptide or a chemically modified polypeptide). For example, a chemoselective linker (or its precursor) can be one of a pair of groups associated with coupling chemistry, such as azide-yne ​​click chemistry, copper-free click chemistry, Staudinger linkage, tetrazine linkage, hydrazine-iso-Pictet-Spengler (HIPS) linkage, cysteine ​​reactive linking chemistry (e.g., thiol-maleimide, thiol-haloacetamide, or alkyne hydrothiolation), lysine reactive linking chemistry (e.g., amine-active ester coupling, such as PFP ester or NHS ester), tyrosine-specific coupling chemistry (e.g., eY-CLICK), methionine-specific coupling chemistry (e.g., based on oxazacyclopropane or ReACT chemistry), reductive amination, dialkyl squaric acid ester chemistry, etc.

[0212] Chemically selective linking groups that can be used to connect two parts include, but are not limited to, amino groups (e.g., the N-terminal amino group or lysine side chain group of a polypeptide), azide groups, aryl azides, alkynyl groups (e.g., ethynyl or cyclooctyne or their derivatives), active esters (e.g., N-hydroxysuccinimide (NHS) esters, sulfonyl-NHS esters, pentafluorophenyl (PFP) esters, tetrafluorophenyl (TFP) esters, or thioesters), haloacetamides (e.g., iodoacetamide or bromoacetamide), chloroacetyl groups, bromoacetyl groups, acylhydrazides, maleimides, vinyl sulfones, 2-sulfonylpyridine, cyano-alkynyls, thiols (e.g., cysteine ​​residues), disulfides or protected thiols, isocyanates, isothiocyanates, aldehydes, ketones, alkoxyamines, acylhydrazides, aminooxy groups, phosphine, HIPS hydrazyl-indole groups, or aza-HIPS hydrazyl-pyrrolo-pyridine groups, tetrazines, cyclooctene, squaric acid esters, etc.

[0213] In some embodiments, when the two groups come into contact under suitable conditions (e.g., copper-free click chemistry conditions), the chemoselective linker can spontaneously couple with the compatible chemical group. In some cases, when the two groups come into contact under suitable conditions in the presence of a catalyst or other reagent (e.g., copper-catalyzed click chemistry conditions), the chemoselective linker can couple with the compatible chemical group.

[0214] In some embodiments, the chemically selective linker is a photoactive linker. For example, under ultraviolet light irradiation, the diaziridine group can form reactive carbenes that can insert into the second part of the CH, NH, and OH bonds.

[0215] In some embodiments, the terminus of the linker precursor includes a reactive functional or functional group that can couple (e.g., form a covalent bond) to a compatible group of the polypeptide (e.g., a compatible amino acid side chain of the polypeptide). The reactive portion may be referred to as a chemiselective linker. For example, carboxylic acids are precursors to chemiselective linkers of active esters.

[0216] In some embodiments, the linker precursor is terminated by a thiol-reactive chemoselective linker (e.g., as described herein). The residue portion Z (e.g., of formula (Ia)) may be derived from the covalent linkage of the thiol-reactive chemoselective linker to one or more cysteine ​​residues (Cys) of a protein (e.g., a carrier protein and / or a MuSK peptide). In some embodiments, the Cys-reactive chemoselective linker is a maleimide derivative, as described herein. In some cases, the Cys-reactive chemoselective linker is maleimide.

[0217] In some embodiments, the linker precursor is terminated by an amine-reactive chemoselective linker group (e.g., as described in this disclosure). The residue moiety Z (e.g., a residue of formula (Ia)) may be derived from the covalent linkage of the amine-reactive chemoselective linker group to one or more lysine residues (Lys) of a protein (e.g., a carrier protein and / or a MuSK polypeptide). For amine-active ester coupling, the residue moiety Z is an amide bond. In some embodiments, the Lys-reactive chemoselective linker group is a PFP ester. In some embodiments, the Lys-reactive chemoselective linker group is a TFP ester. In some embodiments, the Lys-reactive chemoselective linker group is an NHS or sulfonyl-NHS ester.

[0218] 5.3.2.1.2 Spacers

[0219] The receptive sequence reactive linker optionally further comprises one or more spacer portions. The spacer portions are not particularly limited and can be any spacer known in the art. According to this disclosure, the spacers are not particularly limited and include any divalent portion. In some embodiments, the divalent portion includes alkylene, alkenylene, ynylene, arylene, heteroarylene, fused arylene, fused heteroarylene, cycloalkylene, heterocyclic, fused cycloalkylene, or fused heterocyclic.

[0220] In some embodiments, the spacer comprises one or more ethylene glycol monomers, namely polyethylene glycol, -(O-CH2-CH2). n - or [PEG] n Also known as "discrete polyethylene glycol" or "dPEG". n The expression “n” represents the number of ethylene oxide (or “ethylene glycol”) units. In some embodiments, n is 0. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, or 100. In some embodiments, the spacer is [PEG]. n , where n is 4, 8, or 12. In some implementations, the spacer is [PEG]. n , where n is 4, and should be understood as containing four -(O-CH2-CH2)- groups.

[0221] In some embodiments, the surface-modified viral capsid comprises one or more spacers. In some embodiments, the one or more spacers comprise 1 to 20 ethylene glycol monomers. In some embodiments, the one or more spacers comprise 2 to 8 ethylene glycol monomers. In some embodiments, at least one of the one or more spacers comprises 4 ethylene glycol monomers. In some embodiments, the surface-modified viral capsid comprises two spacers, each comprising 4 ethylene glycol monomers.

[0222] In some embodiments, the spacer comprises one or more divalent groups, such as -CH2- (methylene), -O- (ether), -C(=O)- (carbonyl), and -N(R)- (amine, wherein R is H or C). 1-3 alkyl).

[0223] In some embodiments, the spacer comprises one or more -YC(O)-, -YC(O)O-, -Y-NHC(O)-, -Y-NHC(S)-, or -YC(O)-, wherein Y is a bond or one or more ethylene glycol monomers.

[0224] In some implementations, the spacer comprises one or more -([PEG]) n )-C(O)-, where n is 1 to 100, for example 1 to 25, 1 to 20, 1 to 15, 1 to 10, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 4 to 15 or 4 to 10.

[0225] In some embodiments, the spacer comprises one or more -(CH2) groups. n - where n is 1 to 100, for example 1 to 25, 1 to 20, 1 to 15, 1 to 10, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 4 to 15, or 4 to 10. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, 100.

[0226] 5.3.2.1.3 Enzyme substrate portion

[0227] The enzyme substrate moiety described in this article is a chemical or biological functional group that is recognized as a substrate by the relevant enzyme and participates in the enzymatic linking (formation of covalent linkage) between the recognition sequence and the enzyme substrate moiety.

[0228] In some embodiments, the enzyme substrate is selected from oligoglycine (i.e., an oligopeptide containing at least two glycine residues such as GG, GGG, GGGG, etc.) or a primary amine.

[0229] 5.3.2.1.4 Sequence Recognition

[0230] According to this disclosure, the recognition sequence is (i) engineered into the capsid protein, (ii) recognized by the relevant enzyme, and (iii) involved in the enzyme-catalyzed enzymatic linkage, i.e., the formation of a covalent link between the recognition sequence and the enzyme substrate portion.

[0231] In some embodiments, the enzyme cleaves between amino acids of the recognition sequence, enabling the formation of covalent bonds between the enzyme substrate and the remaining (i.e., residual) amino acids in the recognition sequence, as described in more detail herein.

[0232] In embodiments of this disclosure, the recognition sequence is longer than three amino acids, for example, an oligopeptide. In some embodiments, the recognition sequence is shorter than fifteen amino acids. In some embodiments, the recognition sequence is three amino acids. In some embodiments, the recognition sequence is four amino acids. In some embodiments, the recognition sequence is five amino acids. In some embodiments, the recognition sequence is six amino acids. In some embodiments, the recognition sequence is seven amino acids. In some embodiments, the recognition sequence is eight amino acids. In some embodiments, the recognition sequence is nine amino acids. In some embodiments, the recognition sequence is ten amino acids. In some embodiments, the recognition sequence is eleven amino acids. In some embodiments, the recognition sequence is twelve amino acids. In some embodiments, the recognition sequence is thirteen amino acids. In some embodiments, the recognition sequence is fourteen amino acids. In some embodiments, the recognition sequence is fifteen amino acids.

[0233] In some embodiments, the recognition sequence is selected from LPXTA (SEQ ID NO:15), LPTUG (SEQ ID NO:45), NXXTN (SEQ ID NO:67), PGF, VPXXXP (SEQ ID NO:68), PEF, or PEP, wherein X can be any amino acid. In one particular embodiment, the recognition sequence is LPTUG (SEQ ID NO:45). In some embodiments, the recognition sequence is LPETG (SEQ ID NO:12).

[0234] 5.3.2.1.5 Residual recognition sequence

[0235] The residual recognition sequence refers to the amino acids of the recognition sequence that are retained after enzyme cleavage of the recognition sequence during the enzymatic ligation process.

[0236] In some embodiments, the residual recognition sequence is a recognition sequence with the last amino acid removed. In embodiments where the recognition sequence is selected from LPXTA (SEQ ID NO:15), LPTUG (SEQ ID NO:45), NXXTN (SEQ ID NO:67), PGF, VPXXXP (SEQ ID NO:68), PEF, or PEP, in some embodiments, the corresponding residual recognition sequences are LPXT (positions 1-4 of SEQ ID NO:15), LPXT (positions 1-4 of SEQ ID NO:45), NXXT (positions 1-4 of SEQ ID NO:67), PG, VPXXX (positions 1-5 of SEQ ID NO:68), PE, or PE, where X is any amino acid. In a particular embodiment, the recognition sequence is LPTUG (SEQ ID NO:45), and the residual recognition sequence is LPXT (positions 1-4 of SEQ ID NO:15). For example, when the recognition sequence is LPETG (SEQ ID NO:12), the residual recognition sequence is LPET (positions 1-4 of SEQ ID NO:12).

[0237] In some embodiments, one amino acid is cleaved from the recognition sequence to form the residual recognition sequence. In some embodiments, two amino acids are cleaved from the recognition sequence to form the residual recognition sequence. In some embodiments, three amino acids are cleaved from the recognition sequence to form the residual recognition sequence. In some embodiments, four amino acids are cleaved from the recognition sequence to form the residual recognition sequence. In some embodiments, five amino acids are cleaved from the recognition sequence to form the residual recognition sequence.

[0238] 5.3.2.1.6 Enzymes

[0239] The enzymes applicable to this disclosure are capable of catalyzing site-specific enzymatic ligation between an enzyme substrate and a corresponding recognition sequence engineered into the capsid protein, thereby forming an enzymatically ligated product. Exemplary enzymes applicable to this disclosure are not particularly limited, including transpeptidases such as sortase A, sortase B, archaeosorzyme A, exosorzyme A, rhombic sortase, and PorU, and ligases such as Spyligase and Snoopligase, as well as engineered variants of any of the foregoing enzymes.

[0240] i. transpeptidase

[0241] Transpeptidases are enzymes that catalyze nucleophilic carbonyl substitution reactions. In some embodiments, the transpeptidase first binds to a recognition sequence, then cleaves the recognition sequence, and subsequently catalyzes nucleophilic ligation with a linker containing the enzyme substrate moiety.

[0242] Examples of transpeptidases applicable to this disclosure include: sortase A, sortase B, archaeosortase A, archaeosortase B, archaeosortase C, exosortase A, rhombic sortase and PorU, and their functional mutants.

[0243] ii. Sorting enzymes

[0244] In some embodiments, the enzyme that catalyzes the enzymatic linker to the recognition sequence is a sorting enzyme. In some embodiments, the sorting enzyme, the recognition sequence (or "motif"), and the enzyme substrate motif are as described in Bradshaw, WJ, et al. (2015), Molecular features of the sortase enzyme family. FEBS J,282: 2097-2114, which is incorporated herein by reference in its entirety.

[0245] SrtA is a thiol transpeptidase that cleaves a protein substrate between the threonine and glycine residues of LPXTG (SEQ ID NO:45) and then links the protein to the enzyme substrate moiety. SrtA catalyzes the formation of an amide bond (i.e., a biocoupled moiety) between the carboxyl group of threonine in LPXT (positions 1 to 4 of SEQ ID NO:45) and the amino group of the enzyme substrate moiety.

[0246] In some embodiments, the enzyme that bioconjugates the enzyme reactive linker to the recognition sequence is sorting enzyme A (Sa-SrtA) from Staphylococcus aureus, the recognition sequence is LPXTG (SEQ ID NO:45), the residual recognition sequence is LPXT (positions 1 to 4 of SEQ ID NO:45), and the enzyme substrate moiety is a primary amine, including glycine or oligoglycine.

[0247] The transpeptidase may also be mutated from the natural sequence to include mutations. In some embodiments, the enzyme is a transpeptidase carrying a mutation that improves cleavage efficiency and thus improves overall reaction efficiency. Mutant sorting enzymes identified in J. Biol. Chem. (2020) 295:9, 2664-2675 and Scientific reports 2016 (6:31899) are incorporated herein by reference. In some embodiments, the sorting enzyme used in this disclosure carries one or more of the following mutations: P94R, E105K, E108A, D124G, D160N, D165A, D186G, Y187L, E189R, K190E, and K196T. In some embodiments, the transpeptidase is a seven-mutant Staphylococcus aureus sorting enzyme A (SrtA7m).

[0248] In some embodiments, the recognition sequence, the corresponding enzyme, and the enzyme substrate portion used to functionalize the ligand are selected from those described in the “Peptide Linking” table below.

[0249]

[0250] 5.3.2.1.7 Functionalized ligands

[0251] In one aspect of this disclosure, a functionalized ligand is provided, wherein the ligand comprises a member of a cross-linker reactive pair or an enzyme substrate. In some embodiments, the ligand is functionalized by reacting with a ligand-reactive linker. In some embodiments, the functionalized ligand is a polypeptide mutated to comprise a non-natural amino acid containing a cross-linker reactive moiety, an enzyme substrate moiety, or a bioorthogonal tag. In some of these embodiments, the enzyme substrate moiety is an oligoglycinase substrate moiety or an amine moiety.

[0252] 5.3.2.2 Ligand-Reactive Linkers

[0253] Examples of ligand reactivity pairs available according to certain embodiments include, for example, those disclosed in WO 2022 / 101363, which is expressly incorporated herein by reference in its entirety.

[0254] The ligand-reactive linker according to this disclosure comprises: (i) a ligand-reactive portion which can be used to form a covalent link with a ligand, and (ii) a member of a crosslinking agent reactant pair which can be used to crosslink with the surface-functionalized viral capsid of this disclosure.

[0255] 5.3.2.2.1 Connector

[0256] In some embodiments, the "linker" or linker portion is derived from a molecule having a reactive end, for example, suitable for coupling with a target protein. In some cases, the reactive end of the linker precursor comprises a chemiselective linker group capable of coupling to amino acid residues of a polypeptide. In some cases, the chemiselective linker group is coupled to an accessible cysteine ​​thiol group or a lysine side-chain amino group of the polypeptide. A variety of coupling chemistry (e.g., described herein) can be utilized in the conjugates disclosed herein. In some embodiments, the chemiselective linker group is a thiol reactive group, such as maleimide or dibromomaleimide. In some embodiments, the chemiselective linker group is an amine reactive group, such as an active ester, such as pentafluorophenyl ester or tetrafluorophenyl ester, or N-hydroxysuccinimide ester (NHS) or sulfonated NHS, or as defined herein.

[0257] It should be understood that a variety of substituents and chemical methods can be used to connect a specific X-ligand moiety (such as those described herein) to an adjacent linker. In some embodiments, the linker moiety comprises a triazole group derived from click chemistry coupling.

[0258] 5.3.2.2.2 Spacers

[0259] The ligand-reactive linker optionally further comprises at least one spacer portion. The spacer portion is not particularly limited and can be any spacer known in the art. In some embodiments, the spacer comprises an ethylene glycol monomer, namely polyethylene glycol, -(O-CH2-CH2). n - or [PEG] n Also known as "discrete polyethylene glycol" or "dPEG". n The expression “n” represents the number of ethylene oxide (or “ethylene glycol”) units. In some embodiments, n is 0. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, or 100. In some embodiments, n is 4, 8, 12, or 16. In some embodiments, the spacer is [PEG]. n , where n is 4, and should be understood as containing four -(O-CH2-CH2)- groups.

[0260] In some embodiments, the surface-modified viral capsid comprises one or more spacers. In some embodiments, the one or more spacers comprise 1 to 20 ethylene glycol monomers. In some embodiments, the one or more spacers comprise 2 to 8 ethylene glycol monomers. In some embodiments, at least one of the one or more spacers comprises 4 ethylene glycol monomers. In some embodiments, the surface-modified viral capsid comprises two spacers, each comprising 4 ethylene glycol monomers.

[0261] In some embodiments, the spacer comprises one or more divalent groups, such as -CH2- (methylene), -O- (ether), -C(=O)- (carbonyl), and -N(R)- (amine, wherein R is H or C). 1-3 alkyl).

[0262] In some embodiments, the spacer comprises one or more -YC(O)-, -YC(O)O-, -Y-NHC(O)-, -Y-NHC(S)-, or -YC(O)-, wherein Y is a bond or one or more ethylene glycol monomers.

[0263] In some implementations, the spacer comprises one or more -([PEG])n )-C(O)-, where n is 1 to 100, for example 1 to 25, 1 to 20, 1 to 15, 1 to 10, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 4 to 15 or 4 to 10.

[0264] In some embodiments, the spacer comprises one or more -(CH2) groups. n - where n is 1 to 100, for example 1 to 25, 1 to 20, 1 to 15, 1 to 10, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 4 to 15, or 4 to 10. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, 100.

[0265] 5.3.2.2.3 Ligand Reactivity

[0266] According to this disclosure, the ligand reactive portion is not particularly limited and includes any portion capable of covalently attaching to the desired ligand.

[0267] In some embodiments, the ligand is a peptide, oligopeptide, or polypeptide, and the reactive portion of the ligand is linked to an amino acid in the primary sequence of the ligand protein using, for example, a known residue-specific protein labeling technique (via covalent bonding). In a further embodiment, the amino acid residue is present in the wild-type ligand protein sequence. In other embodiments, the amino acid is engineered into the primary amino acid sequence of the ligand.

[0268] i. Ligand primary amine

[0269] In some embodiments, the ligand reactive portion comprises a chemical group that reacts with a primary amine (-NH2). In embodiments where the ligand is a polypeptide, the primary amine is present at the N-terminus of each ligand protein and on the side chain of a lysine (Lys, K) amino acid residue in the ligand protein sequence. Exemplary chemical groups that react with primary amines include isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxal, epoxides, ethylene oxides, carbonates, aryl halides, imine esters, carbodiimides, acid anhydrides, and fluorophenyl esters. Most of these chemical groups are coupled to the amine via acylation or alkylation. In some embodiments, the ligand surface reactive portion comprises an NHS ester or an imine ester, such as those shown below, wherein the R group represents a connection point with a ligand reactive linker.

[0270]

[0271] In some embodiments where the ligand is a polypeptide, the ligand reactive portion is covalently linked to surface-exposed lysine residues of the ligand protein's primary sequence. In some embodiments of these embodiments, the ligand surface reactive portion comprises an NHS-ester, a TFP-ester, an isocyanate, an isothiocyanate, or benzoyl fluoride, as shown below, where the R group represents the connection point with the ligand reactive linker, and the symbol is... This indicates the connection point of the lysine residue in the ligand protein sequence.

[0272]

[0273] In some embodiments, the ligand-reactive linker comprises an N-hydroxysuccinimide ester (NHS ester). The NHS ester is a reactive group formed by activating a carboxylic acid molecule with a carbodiimide. The NHS ester-activated ligand-reactive linker reacts with a primary amine under physiological to slightly alkaline conditions (pH 7.2 to 9) to form a stable amide bond. This reaction releases N-hydroxysuccinimide (NHS).

[0274] In some embodiments, the capping reactive linker comprises a fluorophenyl ester, such as tetrafluorophenyl (TFP) ester or pentafluorophenyl (PFP) ester. The fluorophenyl ester is a reactive group formed by activating a carboxylic acid molecule with a carbodiimide. The fluorophenyl ester of the carboxylic acid reacts with a primary amine to form a covalent amide bond, which is the same as the amide bond formed by the reaction of a primary amine and an NHS ester.

[0275] ii. Ligand thiol group

[0276] In some embodiments, the ligand reactive portion is covalently linked to a surface-exposed thiol group. In some embodiments where the ligand is a polypeptide, the ligand reactive portion is covalently linked to a cysteine ​​residue in the primary sequence of the ligand protein.

[0277] In some embodiments of these implementations, the ligand reactive portion comprises maleimide, iodoacetamide, 2-thiopyridine, or 3-arylpropyne nitrile, as shown in the following example, where the R group represents the connection point with the ligand reactive linker, and the symbol is... This indicates the connection point of the cysteine ​​residue in the ligand protein sequence.

[0278]

[0279] In some embodiments, the ligand-reactive linker comprises maleimide. Maleimide and its derivatives are prepared by amine treatment of maleic anhydride followed by dehydration. When the pH of the reaction mixture is between 6.5 and 7.5, the maleimide group reacts specifically with the thiol group; the result is the formation of a stable, irreversible thioether bond.

[0280] iii. Non-natural amino acids

[0281] In some embodiments, the ligand is a polypeptide that has been mutated to include non-natural amino acids containing a cross-linking agent reactive portion.

[0282] In some embodiments, the ligand polypeptide is mutated to include one or more non-natural amino acids selected from the following: 1: 3-(6-acetylnaphthyl-2-ylamino)-2-aminopropionic acid (Anap), 2: (S)-1-carboxy-3-(7-hydroxy-2-oxo-2H-chromen-4-yl)prop-1-ammonium (CouAA), 3: 3-(5-(dimethylamino)naphthyl-1-sulfonamido)propionic acid (Dansylalanine), 4: - p-Azide-benzyloxycarbonyl lysine (PABK), 5: propargyl-L-lysine (PrK), 6: -(1-Methylcyclopropionic-2-encarbamoyl)lysine (CpK), 7: -Acryloyllysine (AcrK), 8: 9: 9: Bicyclo[6.1.0]non-4-yn-9-ylmethanol lysine (BCNK), 10: trans-cyclooctyl-2-enyllysine (2'-TCOK), 11: trans-cyclooctyl-4-enyllysine (4'-TCOK), 12: dioxo-TCO lysine (DOTCOK), 13: 3-(2-cyclobuten-1-yl)propionic acid (CbK), 14: -5-norbornen-2-yloxycarbonyl-L-lysine (NBOK), 15: cyclooctylenyllysine (SCOK), 16: 5-norbornen-2-oltyrosine (NOR), 17: cyclooctyl-2-ynyltyrosine (COY), 18: (E)-2-(cyclooct-4-en-1-yloxy)ethanoltyrosine (DS1 / 2), 19: azoxyhoalanine (AHA), 20: high-alkyneglycine (HPG), 21: azoxynorleucine (ANL), and 22: -2-Azideethoxycarbonyl-L-lysine (NEAK).

[0283]

[0284]

[0285]

[0286]

[0287] iv. Fusion proteins with tagged reactive molecules

[0288] In embodiments of this disclosure, the ligand is a fusion protein containing a tag capable of binding with its corresponding pair at high affinity, such as a SNAP-tag, CLIP-tag, Halo Tag, Lumio Tag, and other tags known to those skilled in the art.

[0289] Benzylguanine, benzylcytosine, and chloroalkanes are recognized by "suicide" enzymes such as SNAP. In the context of this disclosure, benzylguanine or benzylcytosine may optionally be substituted to form derivatives of benzylguanine or benzylcytosine. Benzylguanine derivatives or benzylcytosine derivatives should be understood as modified benzylguanine or benzylcytosine groups that are still recognized by suicide enzymes.

[0290] The tag molecule can be any molecule or biomolecule capable of specifically binding to another molecule. Examples may include SNAP-tag, CLIP-tag, Lumio-Tag, or Halo-Tag. For example, an affinity tag can be a SNAP-tag, which is a mutant of alkylguanine-DNA alkyltransferase. Importantly, one of the substrates of a SNAP-tag is benzylguanine. Commercially available products that can be used with this disclosure include, for example, HaloTag from Promega, Lumio Tag from Life Technologies, and SNAP / CLIP Tags from NEB.

[0291] Self-labeled protein tags are commercially available in various expression vectors. SNAP-tag is a 182-residue polypeptide (19.4 kDa) that can be fused to any target protein and further specifically and covalently labeled with suitable ligands such as fluorescent dyes. The SNAP-tag protein is an engineered version of the ubiquitous mammalian enzyme AGT, encoded in humans by the O-6-methylguanine-DNA methyltransferase (MGMT) gene. SNAP-tag was obtained through a directed evolution strategy, resulting in an hAGT variant that accepts an O6-benzylguanine derivative as a substrate, rather than alkylated guanine derivatives for repairing damaged DNA.

[0292] CLIP-tag is a further engineering modification of SNAP-tag, which accepts O2-benzylcytosine derivatives instead of O6-benzylguanine as substrates. Later, a split SNAP-tag version was developed for protein complementation assays and protein-protein interaction studies.

[0293] HaloTag is a self-tagged protein tag. It is a 297-residue peptide (33 kDa) derived from a bacterial enzyme, designed for covalent binding to a synthetic ligand. HaloTag is a hydrolase with a genetically modified active site that specifically binds to a reactive chloroalkane linker and exhibits increased ligand binding. Under physiological conditions, the reaction in which the protein tag forms a bond with the chloroalkane linker is rapid and substantially irreversible due to the terminal chlorine atom of the linker moiety. In this reaction, nucleophilic attack on the reactive chloroalkane linker leads to the substitution of a halogen with an amino acid residue, forming a covalently alkyl-enzyme intermediate. This intermediate is subsequently hydrolyzed by the amino acid residues within the wild-type hydrolase. This results in the regeneration of the enzyme after the reaction. However, in the modified haloalkane dehalogenase (HaloTag), the intermediate cannot proceed to the subsequent reaction because it cannot be hydrolyzed due to a mutation in the enzyme. This results in the intermediate persisting as a stable covalent adduct, and no associated reverse reaction occurs.

[0294] This system involves two steps: cloning and expressing the target protein as a SNAP-tag® fusion protein, and labeling the fusion protein with a selected SNAP-tag substrate. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. In this case, the SNAP-tag substrate is a guanine leaving group linked to a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag.

[0295] The SNAP-tag protein labeling system can specifically covalently link almost any molecule to a target protein.

[0296] 5.3.2.3 Examples of reactive linkers

[0297] The following reactive linkers are examples suitable for use as capsid reactive linkers or ligand reactive linkers according to various embodiments of this disclosure. Those skilled in the art will understand that the number of PEG monomers (i.e., -(O-CH2-CH2)- groups) in the following examples can be easily adjusted to reflect the number of PEG monomers described herein. In other embodiments of the linkers in the following examples, the number of PEG monomers is 4, 8, 12, or 16.

[0298] 5.3.2.3.1 TCO-PEG4-NHS

[0299] Synonyms: trans-cyclooctene-PEG4-NHS; Empirical formula (Hill representation): C24 H 38 N2O 10 Molecular weight: 514.57.

[0300] 5.3.2.3.2 Tetraazine-PEG5-NHS

[0301] Tetraazine-PEG5-NHS ester is an amine-reactive linker commonly used to modify oligonucleotides of proteins, peptides, or amines via the tetraazine moiety.

[0302] 5.3.2.3.3 Azide-PEG4-NHS

[0303] In this article, it is also referred to as "azide-PEG4-NHS".

[0304] 5.3.2.3.4 Phosphine-NHS

[0305] Molecular weight: 461.40.

[0306] 5.3.2.3.5 DBCO-PEGn-NHS

[0307] Where y is an integer from 1 to 4; n is an integer from 1 to 10, such as 4, 5, 6, 7 or 8.

[0308] i. DBCO-PEG4-NHS

[0309] Molecular weight: 649.26.

[0310] 5.3.2.3.6 DBCO-PEGn-TFP ester

[0311] i. DBCO-PEG4-TFP ester

[0312] 5.3.2.3.7 DBCO-PEGn-amine

[0313] 5.3.2.3.8 Maleimide-PEGn-azide

[0314] 5.3.2.3.9 DBCO-amine

[0315] 5.3.2.3.10 Maleimide-PEGn-succinimide ester

[0316] 5.3.2.3.11 DBCO-PEG8-succinimide ester

[0317] Maleimide PEG8 succinimide ester, 31-(2,5-dihydro-2,5-dioxo-1H-pyrrolo-1-yl)-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-aza-triacontane 2,5-dioxo-1-pyrroloalkyl ester, maleimide-PEG8-NHS ester.

[0318] 5.3.2.3.12 N3-PEGn-maleimide

[0319] 5.3.2.3.12 Dibenzocyclooctyne-TFP

[0320] In some embodiments, the capsid reactive linker has the following structure: , SP 1 It is a bond or spacer; and “a” is an integer from 1 to 5, or from 2 to 4, such as 1, 2, 3, 4 or 5.

[0321] In some implementations, a is 4.

[0322] 5.3.3 Surface modification via peptide ligand insertion

[0323] In various embodiments, the surface-modified viral capsid comprises a peptide inserted within a frame into a capsid protein. In some embodiments, the inserted peptide is a ligand that promotes rAAV translocation from CSF into the brain parenchyma. In certain embodiments, flanking regions of the inserted peptide must be enzymatically cleaved to activate the ligand. In various embodiments, the capsid is optionally modified with one or more additional surface-coupled ligands that promote CNS cell transduction, and optionally promote transduction of one or more desired CNS cell types.

[0324] In some embodiments, the peptide ligand is inserted into the capsid protein of a naturally occurring AAV serotype or an engineered or synthetic AAV capsid protein. For example, the peptide ligand may be inserted into the capsid protein of a naturally occurring human AAV serotype (such as AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, AAV9, or hu68) or into the capsid protein of a naturally occurring rhesus monkey serotype (such as rh34). In other embodiments, the peptide ligand is inserted into an artificial AAV capsid sequence, such as the bioinformatics-predicted ancestral capsid described in U.S. Patent Nos. 9,695,220 and 10,738,087, the disclosure of which is incorporated herein by reference in its entirety.

[0325] In some embodiments, the peptide ligand is inserted into variable region 4 (VR4) of the AAV capsid protein, corresponding to an exposed loop on the capsid surface. In other embodiments, the peptide ligand is inserted into variable region 8 (VR8) of the AAV capsid protein. The VR8 region may contain R585 and R588 required for HSPG binding, or may have further mutations to reduce or eliminate binding to HSPG.

[0326] The specific insertion site in VR4 or VR8 can vary depending on the engineered capsid protein.

[0327] In some embodiments, the insertion site in VR4 is between amino acid positions 400 and 500 of the capsid protein. In some embodiments, the insertion site in VR4 is between amino acid positions 425 and 475 of the capsid protein. In some embodiments, the insertion site in VR4 is between amino acid positions 440 and 460 of the capsid protein.

[0328] In some embodiments, the insertion site in VR8 is between amino acid positions 550 and 650. In some embodiments, the insertion site in VR8 can be between amino acid positions 575 and 625. In some embodiments, the insertion site in VR8 can be between amino acid positions 580 and 600.

[0329] Exemplary capsid insertion sites for naturally occurring AAV serotypes AAV1 to AAV9 are provided in Table 1. For other (e.g., modified) capsids, the insertion site can be determined by sequence alignment to identify the amino acid position corresponding to the insertion sites listed in Table 1. For example, when the sequence is aligned with the AAV1 capsid protein, the insertion site may be the amino acid position corresponding to the position following serine (S) at position 443. In another example, when the sequence is aligned with the AAV2 capsid protein, the insertion site is the amino acid position corresponding to the position following serine (S) at position 442.

[0330]

[0331] In some embodiments, the surface-modified viral capsid protein comprises the capsid protein of AAV1 with a peptide insertion after serine (S) at position 443. In some embodiments, the surface-modified viral capsid protein comprises the capsid protein of AAV2 with a peptide insertion after serine (S) at position 442. In some embodiments, the surface-modified viral capsid protein comprises the capsid protein of AAV6 with a peptide insertion after serine (S) at position 443. In some embodiments, the surface-modified viral capsid protein comprises the capsid protein of AAV7 with a peptide insertion after proline (P) at position 454. In some embodiments, the surface-modified viral capsid protein comprises the capsid protein of AAV8 with a peptide insertion after threonine (T) at position 454. In some embodiments, the surface-modified viral capsid protein comprises the capsid protein of AAV9 with a peptide insertion after serine (S) at position 454.

[0332] In some embodiments, the surface-modified viral capsid protein comprises the capsid protein of AAV2, with a peptide insertion following arginine (R) at position 589. In some embodiments, the surface-modified viral capsid protein comprises the capsid protein of AAV5, with a peptide insertion following serine (S) at position 576. In some embodiments, the surface-modified viral capsid protein comprises the capsid protein of AAV6, with a peptide insertion following serine (S) at position 591. In some embodiments, the surface-modified viral capsid protein comprises the capsid protein of AAV7, with a peptide insertion following alanine (A) at position 591. In some embodiments, the surface-modified viral capsid protein comprises the capsid protein of AAV8, with a peptide insertion following glutamine (Q) at position 593.

[0333] In some embodiments, the peptide ligand is a peptide agonist of the PAR1 receptor. In some embodiments, the peptide ligand is the PAR1A peptide. In some embodiments, the peptide ligand has the sequence SFLLRN (residues 5 to 10 of SEQ ID NO: 61). In some embodiments, the peptide ligand has the sequence LDPRSFLLRN (SEQ ID NO: 61).

[0334] In some embodiments, the PAR1A peptide is inserted into VR-IV (VR-4) of the AAV capsid protein sequence.

[0335] In some embodiments, the PAR1A peptide may be inserted into VR-VIII (VR-8) of the AAV capsid protein sequence.

[0336] 5.3.4 Viral capsids with surface modifications according to Formula I

[0337] In some embodiments, the surface-modified viral capsid comprises a ligand and a linker according to Formula I: …(I) in: It is a viral capsid; Y is the connecting part; Y' is the connecting part; Q represents the cross-linked portion; PEG is an ethylene glycol monomer; n and n' are independent integers from 0 to 100; Sp and Sp' are independently optional spacers; L is a ligand, and There is no particular restriction on x. In some implementations, x is an integer from 1 to 300, 100 to 200, 120 to 180, or about 150.

[0338] In some implementations, x ranges from 100 to 200. In other implementations, x ranges from 130 to 170.

[0339] In some embodiments, the linker Y is formed by a reaction between a capsid reactive moiety and a capsid protein. In some embodiments, the linker Y is formed by a reaction between an NHS or TFP ester and a primary amino group of a capsid protein amino acid. In some embodiments, the amino group is a lysine side chain present in the primary sequence of the capsid protein. In some embodiments, the amino group is a lysine present in the primary sequence of the AAV capsid protein.

[0340] In some embodiments, the linking portion Y' is formed by any known chemical method of coupling reaction between the ligand reactive portion and the ligand. In some embodiments, the linking portion Y' is formed by the reaction between an ester (e.g., NHS or TFP) and the amino group of the ligand.

[0341] In some embodiments, Q is a product formed by the reaction between members of a crosslinking agent reactant pair. In some embodiments, Q is a crosslinked portion formed by the reaction of DBCO with an azide group.

[0342] In some implementations, Q is selected from: or , Z is a 7- or 8-membered cyclic or heterocyclic structure. In some embodiments, Z is optionally substituted with one or more arylene groups. In some embodiments, Z is unsubstituted. In some embodiments, Z is substituted with one arylene group. In some embodiments, Z is substituted with two arylene groups. In some embodiments, the arylene groups are independently selected. In some embodiments, the arylene groups are identical.

[0343] In some embodiments, the surface-modified viral capsid is as shown in Formula I-1: (I-1) in: It is a viral capsid; n and n' are independent integers selected from 0 to 30; L is a ligand; and x is an integer between 50 and 250.

[0344] In some embodiments, x ranges from 100 to 200. In some embodiments, the ligand / capsule ratio (x) is in the range of 130 to 170.

[0345] In some implementations, n is an integer selected from 0 to 100. In some implementations, n is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, and 100.

[0346] In some implementations, n' is an integer selected from 0 to 100. In some implementations, n' is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, and 100.

[0347] 5.3.5 Engineered Viral Capsids

[0348] On one hand, an engineered viral capsid is provided, comprising: a first targeting ligand coupled to a first site of a surface-modified viral capsid; and a second targeting ligand coupled to a second site of the surface-modified viral capsid, wherein the first targeting ligand and the second targeting ligand are different.

[0349] In some embodiments, the first targeting ligand promotes crossing the blood-brain barrier (BBB), and the second targeting ligand is a cell-targeting ligand. In some embodiments, the first targeting ligand is a blood-brain barrier (BBB) ​​transpeptide or a lectin ligand.

[0350] In some embodiments, one of the first and second targeting ligands is covalently linked (e.g., coupled) to a lysine or cysteine ​​residue of the viral capsid protein via a linker. In some embodiments, the other of the first and second targeting ligands is covalently linked (e.g., coupled) to a recognition sequence motif residue via a linker.

[0351] In some embodiments, the viral capsid is an adeno-associated virus (AAV) capsid. In some embodiments, the first or second targeting ligand is a peptide, such as a PAR-1 agonist.

[0352] In some embodiments, the first or second targeting ligand is a peptide or a portion thereof as shown in SEQ ID NO:3. In some embodiments, the first or second targeting ligand is nerve growth factor. In some embodiments, the first or second targeting ligand binds to brain microvascular endothelial cells (BMECs). In some embodiments, the first or second targeting ligand binds to neuronal membranes, such as wheat germ lectin (WGA).

[0353] In some embodiments, the engineered viral capsid coupled to the first and second targeting ligands further comprises: (a) a modified viral capsid containing a plurality of recognition sequence motif residues; and (b) a first targeting ligand coupled to the modified viral capsid via a first linker covalently linked to the recognition sequence motif residues, comprising: b1. residues covalently linked to amide-C(O)NH-; b2. a first spacer (Sp)n; and b3. a divalent fused polycyclic 11- to 20-membered heterocycle, a phosphine oxide derivative, or a 4H-pyrazole-4-imine derivative. b4. Second spacer (Sp')n'; b5. First targeting ligand; and (c) a second targeting ligand coupled to the modified viral capsid via a second linker covalently linked to a primary amine group or thiol group of the primary sequence of the modified viral capsid, comprising: c1. amide-NHC(O)-; c2. First spacer (Sp)n; c3. a divalent fused polycyclic 11 to 20-membered heterocycle, phosphine oxide derivative, or 4H-pyrazole-4-imine derivative; c4. Second spacer (Sp')n'; and c5. Second targeting ligand; wherein n and n' are each independently an integer from 0 to 20.

[0354] In some embodiments, the recognition sequence motif residues are selected from any peptide sequence or portion thereof shown in SEQ ID NO:1-58 or 61-75. In some embodiments, the recognition sequence motif residues comprise at least four consecutive amino acids selected from the peptide sequences shown in SEQ ID NO:8-33, 35-44, 62-64, and 65. In some embodiments, the recognition sequence motif residues comprise at least six consecutive amino acids shown in SEQ ID NO:61 or SEQ ID NO:66.

[0355] In some embodiments, the engineered viral capsid coupled to the first and second targeting ligands further comprises: (a) a modified viral capsid; and (b) a first targeting ligand coupled to the modified viral capsid via a first linker covalently linked to a primary amine or thiol group of the primary sequence of the modified viral capsid, comprising: b1. a residue covalently linked to an amide-C(O)NH-; b2. a first spacer (Sp)n; b3. a divalent fused polycyclic 11- to 20-membered heterocycle, a phosphine oxide derivative, or a 4H-pyrazole-4-imine derivative; and b4. a second spacer. (Sp')n'; b5. First targeting ligand; and (c) a second targeting ligand coupled to the modified viral capsid via a second linker covalently linked to a primary amine group or thiol group of the primary sequence of the modified viral capsid, comprising: c1. amide-NHC(O)-; c2. first spacer (Sp)n; c3. divalent fused polycyclic 11 to 20-membered heterocycle, phosphine oxide derivative, or 4H-pyrazole-4-imine derivative; c4. second spacer (Sp')n'; and c5. second targeting ligand; wherein n and n' are each independently an integer from 0 to 20.

[0356] In some embodiments, the primary amine group is selected from N-terminal amino groups, lysine ε-amino groups, and arginine amino groups.

[0357] In some embodiments, the divalent fused polycyclic 11- to 20-membered heterocyclic ring is: or , Z is an optionally substituted 7- to 9-membered carbon ring or heterocycle.

[0358] In some implementations, Z is replaced by one or more aryl groups.

[0359] In some embodiments, the phosphine oxide derivative is: .

[0360] In some embodiments, the 4H-pyrazole-4-imine derivative is: .

[0361] 5.3.6 Ligands

[0362] According to this disclosure, a surface-modified viral capsid, typically an AAV viral capsid, is provided. The surface modification includes a first ligand that facilitates the entry of rAAV from CSF into the brain parenchyma.

[0363] Optionally, the surface-modified viral capsid comprises one or more additional surface-coupled ligands. In a particular embodiment, at least one additional surface-coupled ligand promotes transduction to CNS cells, optionally promoting transduction to one or more desired CNS cell types. In various embodiments, the optional ligand is not particularly limited, provided that the ligand is suitable for coupling to the surface of the viral capsid as described herein. In some embodiments, the ligand is selected from protein ligands having homologs located on the surface of mammalian cells, such as receptors. In some of these embodiments, the homologous protein is involved in the transduction of the surface-modified viral capsid. In some embodiments, the ligand is a protein known to promote the permeability of tissue barriers (such as the meninges, including the pia mater) in vivo.

[0364] In some embodiments, the surface-modified viral capsid contains a ligand. In some embodiments, the ligand is a cell type-specific ligand.

[0365] In some embodiments, the ligand is a cell-type or receptor-specific ligand. In some embodiments, the ligand is selected from peptides, proteins, monosaccharides or polysaccharides, steroid hormones, RGD motif peptides, vitamins, small molecules, or targeting peptides. Also considered are antibodies (e.g., single-chain antibodies) and nanobodies; enzymes such as proteases, glycosidases, lipases, and peptidases; immunoglobulins such as CD47 (“Don’t eat me” signal); IgG proteases such as IdeZ and IdeS; and protein- and small-molecule-based adjuvants for vaccination.

[0366] According to one implementation, cell type-specific ligands are derived from proteins such as transferrin, epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF).

[0367] According to one implementation, the cell type-specific ligand is derived from monosaccharides or polysaccharides, such as galactose, N-acetylgalactosamine, and mannose.

[0368] According to one implementation scheme, cell type-specific ligands are derived from vitamins, such as folate.

[0369] According to one implementation, the cell type-specific ligand is derived from small molecules, including naproxen, ibuprofen, or other known protein-binding molecules.

[0370] In some embodiments, the ligand is selected from protein ligands, such as growth factors or cytokines; toxin subunits, such as cholera toxin B subunits; lectins, such as isoglucosinolate B4 or wheat germ lectin; adhesion factors, such as lactobacin; antibodies or single-chain variable fragments thereof, such as anti-CD34 antibodies; more specifically, E. coli recombinant scFv CD34 antibody fragments; peptides, such as delta-opioid receptor ligands; and gene-editing nucleases, such as Cas9. In some embodiments, the ligand is a cytokine. In some embodiments, the ligand is a growth factor. In some embodiments, the ligand is a lectin. In some embodiments, the ligand is a toxin. In some embodiments, the ligand is a single-chain antibody. In some embodiments, the ligand is a multi-chain antibody. In some embodiments, the ligand is an antigen-binding antibody fragment. In some embodiments, the ligand is a peptide.

[0371] In some embodiments, the ligand is selected from agents known to promote tissue barrier permeability, such as tissue plasminogen activator (TPA), ligands, nanobodies, single-chain antibodies, metalloproteinases, kallikrein (serine protease subgroup), bacterial proteases (e.g., from bacteria crossing the blood-brain barrier), occluder toxin / δG, EphA2 / EphA1 agonists / ligands (known to regulate astrocytes / pericytes), and phage-derived peptides. In some embodiments, the ligand is tissue plasminogen activator (TPA).

[0372] In some embodiments, the ligand is selected from agents known to target receptors highly expressed on endothelial cells that constitute the blood-brain barrier. In some embodiments, the receptor is a receptor-mediated transport protein. These include the insulin receptor, transferrin receptor, LDL receptor and their associated proteins, as well as other receptors known in the art.

[0373] In some implementations, the ligand is a protein known to promote the permeability of tissue barriers (such as the meninges, including the pia mater) in vivo.

[0374] In some embodiments, the ligand is known to bind to endogenous receptors on brain microvascular endothelial cells (BMECs). In some embodiments, the receptor is a protease-activated receptor (PAR). In some embodiments, the receptor is endothelial protease-activated receptor 1 (PAR1). In some embodiments, the ligand binds to PAR1. In some embodiments, the ligand is a PAR-1 activating peptide, such as thrombin or a derivative thereof. In some embodiments, the ligand binds to thrombin or a derivative thereof. In some embodiments, the ligand is a peptide containing the sequence SFLLR (residues 1 to 5 of SEQ ID NO:3). In some embodiments, the ligand is a peptide having the sequence SFLLRNPNDKC (SEQ ID NO:3).

[0375] In some embodiments, the ligand is selected from agents known to promote tissue barrier permeability, such as tissue plasminogen activator (TPA), ligands, nanobodies, single-chain antibodies, metalloproteinases, kallikrein (serine protease subgroup), bacterial proteases (e.g., from bacteria crossing the blood-brain barrier), occluder toxin / δG, EphA2 / EphA1 agonists / ligands (known to regulate astrocytes / pericytes), and phage-derived peptides. In some embodiments, the ligand is tissue plasminogen activator (TPA).

[0376] In some embodiments, the ligand is a lectin. In some embodiments, the ligand is wheat germ lectin (WGA). In some embodiments, the ligand is selected from WGA1, WGA2, and WGA3. In some embodiments, the ligand is WGA1. In some embodiments, the ligand is WGA2. In some embodiments, the ligand is WGA3. In some embodiments, the ligand binds to PAR1, or the ligand is WGA. In some embodiments, the ligand binds to PAR1. In some embodiments, the ligand is WGA. In some embodiments, the capsid comprises a first ligand that binds to PAR1 and a second ligand that comprises WGA.

[0377] 5.3.7 Viral Capsid

[0378] In embodiments of this disclosure, the viral capsid (hereinafter referred to as the capsid) is not particularly limited. In some embodiments, the capsid is selected from non-enveloped viruses, such as adenoviruses or adeno-associated viruses. In some embodiments, the capsid is selected from enveloped viruses, such as retroviruses, lentiviruses, herpes simplex viruses, and baculoviruses. Embodiments include non-naturally occurring capsids and include biological or chemical alterations or variations to naturally occurring capsid proteins, other than changes to the primary amino acid sequence.

[0379] In embodiments of this disclosure, the capsid comprises an engineered capsid protein, for example prepared by site-directed mutagenesis, wherein the engineered capsid protein comprises at least one recognition sequence, as described elsewhere herein.

[0380] In some embodiments, the surface-modified viral capsid contains a first ligand that binds PAR1 and a second ligand that contains WGA.

[0381] In some embodiments, the surface-modified viral capsid includes a linker. In some embodiments, the linker is covalently linked to a primary amine group of the capsid protein's primary sequence.

[0382] In some embodiments, the primary amine group is selected from N-terminal amino groups, lysine ε-amino groups, and arginine amino acid groups. In some embodiments, the primary amine group is an N-terminal amino group. In some embodiments, the primary amine group is an ε-amino group of a lysine amino acid residue. In some embodiments, the primary amine group is an arginine amino acid group.

[0383] In some embodiments, the surface-modified viral capsid includes a linker, wherein the linker is covalently linked to the ligand via a primary amine group of the ligand.

[0384] In some implementations, the linker is covalently linked to the target ligand via non-natural amino acid residues in the primary sequence of the target ligand.

[0385] In some embodiments, the non-natural amino acid residue comprises a member of a crosslinking agent reaction pair participating in a reaction selected from: Cu(I)-catalyzed azido-alkyne cycloaddition (CuAAC), strain-promoted alkyne-azido cycloaddition (SPAAC), strain-promoted alkyne-nitroketone cycloaddition (SPANC), anti-electron-demanding Diels-Alder (IEDDA) reaction, Staudinger linkage, and [4+1] cycloaddition reactions. In some embodiments, the non-natural amino acid residue comprises a member of a crosslinking agent reaction pair participating in a Cu(I)-catalyzed azido-alkyne cycloaddition (CuAAC). In some embodiments, the non-natural amino acid residue comprises a member of a crosslinking agent reaction pair participating in a strain-promoted alkyne-azido cycloaddition (SPAAC). In some embodiments, the non-natural amino acid residue comprises a member of a crosslinking agent reaction pair participating in a strain-promoted alkyne-nitroketone cycloaddition (SPANC). In some embodiments, the non-natural amino acid residue comprises a member of a cross-linking agent reaction pair that participates in an anti-electron-demanding Diels-Alder (IEDDA) reaction. In some embodiments, the non-natural amino acid residue comprises a member of a cross-linking agent reaction pair that participates in Staudinger linkage and [4+1] cycloaddition reactions.

[0386] In some embodiments, the non-natural amino acid residue comprises a member of a crosslinking agent reaction pair that participates in a reaction selected from: Cu(I)-catalyzed azido-alkynyl cycloaddition (CuAAC), strain-promoted alkynyl-azido cycloaddition (SPAAC), strain-promoted alkynyl-nitroketone cycloaddition (SPANC), anti-electron-demanding Diels-Alder (IEDDA) reaction, Staudinger linkage, and [4+1] cycloaddition.

[0387] In some embodiments, the surface-modified viral capsid comprises a crosslinking agent reaction pair. In some embodiments, the crosslinking agent reaction pair comprises an azide, cyclooctyne, cyclooctene, or 1,2,4,5-tetraazine moiety.

[0388] In some embodiments, the surface-modified viral capsid contains protein sequences. In some embodiments, the protein sequences of the viral capsid have been mutated to weaken or eliminate the binding of the capsid to mammalian cellular polysaccharides or proteoglycans.

[0389] In some implementations, the surface-modified viral capsid is characterized by increased infectivity compared to an unmodified viral capsid having the same capsid protein sequence.

[0390] In some embodiments, the viral capsid is selected from adenovirus capsids, adeno-associated virus capsids, retrovirus capsids, lentivirus capsids, herpes simplex virus capsids, and baculovirus capsids. In some embodiments, the viral capsid is an adenovirus capsid. In some embodiments, the viral capsid is an adeno-associated virus capsid. In some embodiments, the viral capsid is a retrovirus capsid. In some embodiments, the viral capsid is a herpes simplex virus capsid. In some embodiments, the viral capsid is a baculovirus capsid.

[0391] In some embodiments, the surface-modified viral capsid contains at least one of the arginine residues at positions 585 and 588 of the mutated VP1 or at similar positions in VP2 or VP3.

[0392] In some implementations, the surface-modified viral capsid contains arginine residues at positions 585 and 588 of VP1, which have been mutated to alanine residues.

[0393] In some implementations, the surface-modified viral capsid is characterized by altered tropism compared to the unmodified viral capsid.

[0394] In some implementations, the surface-modified viral capsid exhibits the ability to evade pre-existing neutralizing antibodies.

[0395] 5.3.7.1 AAV

[0396] All recombinant adeno-associated viruses (rAAV, used interchangeably with AAV herein) can be implemented within the framework of this disclosure. Such AAV particles are capable of transducing a variety of post-mitotic cells in mammals in vivo, including, but not limited to, myocytes, hepatocytes, and neurons.

[0397] In some embodiments, the AAV capsid comprises naturally occurring AAV serotypes of VP1, VP2, and / or VP3 capsid proteins. In some embodiments, the AAV comprises one or more non-naturally occurring VP1, VP2, and / or VP3 capsid proteins. In some of these embodiments, the non-naturally occurring VP1, VP2, or VP3 capsid protein differs from the naturally occurring capsid in its primary amino acid sequence. In some embodiments, the non-naturally occurring capsid includes biological or chemical alterations or variations to the naturally occurring AAV capsid protein, other than changes to the primary amino acid sequence.

[0398] In various embodiments, the capsid protein is a naturally occurring AAV serotype of AAV, such as AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, or AAV9. In various embodiments, the capsid protein is selected from those disclosed in PCT / US2014 / 060163, USP9695220, PCT / US2016 / 044819, PCT / US2018 / 032166, PCT / US2019 / 031851, and PCT / US2019 / 047546, which are incorporated herein by reference in their entirety.

[0399] The adeno-associated virus capsid can be selected from all identified natural serotypes, particularly AAV2, AAV3b, AAV5, AAV8, AAV9 and AAV10, and more particularly AAV2.

[0400] Furthermore, the adeno-associated virus can be selected from synthetic serotypes produced by non-natural methods, such as, but not limited to: capsid mutagenesis, peptide insertion into or deletion of the capsid sequence, capsid tampering from various serotypes, or ancestral reconstruction.

[0401] The AAV capsid used in this disclosure can be produced by any method known in the art, without limitation. For example, the AAV capsid can be produced by several methods, including: transient transfection of HEK293 cells, stable cell lines infected with Ad or HSV, mammalian cells infected with Ad or HSV (expressing rep-cap and transgenes), or insect cells infected with a baculovirus vector (expressing rep-cap and transgenes). AAV capsids produced by any of these methods can be used to produce the surface-functionalized and surface-modified viral capsids described herein. In some embodiments, the vector is produced by transient transfection of HEK293 cells with two plasmids via the calcium phosphate-HeBS method: pHelper, PDP2-KANA encoding AAV Rep2-Cap2 and adenovirus accessory genes (E2A, VARNA, and E4), and pVector ss-CAG-eGFP, as shown in the provided examples.

[0402] In some implementations, the AAV capsid of this disclosure may, as needed, contain one or more sequences from sources other than viruses.

[0403] In some embodiments, the capsid of the AAV is composed of three overlapping capsid proteins (VP1, VP2, VP3), containing a unique VP1 N-terminus, a common portion of VP1 / VP2, and a portion common to VP1, VP2, and VP3.

[0404] In some embodiments, one or more capsid proteins comprise naturally occurring amino groups, i.e., primary sequences corresponding to wild-type capsid proteins. In other embodiments, the primary sequence of one or more capsid proteins comprises amino acids engineered into the wild-type capsid protein sequence. In some embodiments of these embodiments, the engineered amino acids comprise one or more amino groups present on the capsid surface and participate in the surface functionalization of one or more capsid proteins. In some embodiments, the naturally occurring or engineered amino groups participating in the capsid surface functionalization are selected from lysine, arginine, and cysteine. In some embodiments, the amino acid is lysine.

[0405] According to certain embodiments, the AAV capsid comprises one or more wild-type capsid proteins derived from naturally occurring serotypes.

[0406] According to some embodiments, the AAV capsid comprises a genetically modified capsid protein. In some embodiments, the genetically modified capsid protein is an naturally occurring serotype engineered to include one or more genetic modifications (mutations, insertions, or deletions). In other embodiments, the rAAV capsid is composed of one or more synthetic capsid proteins. In some embodiments, the AAV capsid is engineered to modify its natural tropism, for example, to reduce heparin binding.

[0407] Within the framework of this disclosure, synthetic capsids include any combination of capsid proteins from natural, genetically modified, and artificially created (random mutations, sequence shuffling, computer design, etc.) serotypes that are capable of assembling and producing novel AAV viral capsids not found in nature.

[0408] Currently, over 100 AAV serotypes have been identified, differing in their ability to bind capsid proteins to specific cell surface receptors (which can transduce different cell types). AAV2 was the first serotype cloned into a bacterial plasmid and has since been used as a comparison for identifying other serotypes. Twelve serotypes (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12) have been thoroughly tested for their ability to transduce specific cell types, and capsid protein motifs that bind to specific cell surface receptors for cell attachment have been differentiated. In the context of this disclosure, AAV capsids are preferably selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. However, it should be understood that any other AAV capsid may be used in the context of this disclosure.

[0409] In one embodiment, the adeno-associated virus (AAV) particles of this disclosure are selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. The most commonly used gene transfer systems to date are viral derivatives, such as adeno-associated virus type 2 (AAV2), AAV9, and AAV8. In some embodiments, the rAAV capsid is AAV-2 or AAV-9, wherein the capsid protein is optionally further engineered to reduce or modify natural tropism, for example, to reduce heparin binding.

[0410] 5.3.7.2 Removal of naturally bound portions

[0411] In some embodiments of the adeno-associated virus (rAAV) capsid disclosed herein, the rAAV is selected from naturally occurring serotypes having natural cell-binding sites that have been removed to bind heparan sulfate proteoglycans.

[0412] In some embodiments, the removal of heparin binding has been engineered by replacing arginine at position 585 or 588 of VP1 and / or at least one of the arginine analogues in VP2 or VP3 with a different amino acid (such as alanine). In some embodiments, at least one of the arginines at positions 448 and 451 of VP2 or at positions 383 and 386 of VP3 is altered.

[0413] In some embodiments, the adeno-associated virus (AAV) capsid of this disclosure is composed of at least one protein mutated from the wild type, for example, wherein the engineered / mutated protein is selected from wild-type proteins VP1, VP2, and / or VP3. Alternatively, two of the VP1, VP2, and / or VP3 proteins in the capsid are mutated, or all three proteins VP1, VP2, and VP3 in the capsid are modified. In some embodiments, at least one portion (e.g., one amino acid) of at least one protein to be modified in the capsid is mutated (replaced, inserted, or deleted). However, multiple portions of the proteins VP1, VP2, and VP3 in the capsid may also be mutated, for example, multiple amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or any other number of portions or amino acids. In some embodiments, arginines at positions 484, 487, 585, and 588 of VP1 and lysine at position 532 and / or at least one of the arginine analogues in VP2 or VP3 are removed by replacing them with a different amino acid (e.g., alanine).

[0414] 5.3.7.3 PEG Immunomasking

[0415] According to certain embodiments, the surface of the viral capsid may be modified according to methods known in the art to include a spatial shielding agent for preventing interaction with neutralizing antibodies. In some embodiments, the spatial shielding agent is derived from a synthetic polymer, such as polyethylene glycol (PEG) or pHPMA. PEG polymers are prepared by a polymerization process and comprise a heterogeneous mixture of sizes and molecular weights, characterized by a Poisson distribution of chain lengths and molecular weights, also known as the polydispersity index (PDI), dispersion index, or simply dispersion (symbolized by "..."). (This indicates that the reported molecular weight is the average molecular weight). (Or PDI) indicates the molecular weight range in the sample.

[0416] 5.4 Engineered capsid proteins

[0417] In certain aspects of this disclosure, an engineered adeno-associated virus (AAV) capsid protein is provided, wherein the capsid protein comprises: a functional sequence motif having the sequence X1X2X3X4X5X6 (SEQ ID NO:5); wherein X1, X2, X3, X4, X5 and X6 are each independently selected from any amino acid residues, and wherein X4, X5 and X6 are optional; wherein the functional sequence motif is a substrate of a transaminases.

[0418] In some embodiments, the enzyme is selected from sorting enzyme A, sorting enzyme B, ancient sorting enzyme A, external sorting enzyme A, rhombic sorting enzyme, and PorU. In some embodiments, the enzyme is sorting enzyme A. In some embodiments, the enzyme is sorting enzyme B. In some embodiments, the enzyme is ancient sorting enzyme A. In some embodiments, the enzyme is external sorting enzyme A. In some embodiments, the enzyme is rhombic sorting enzyme. In some embodiments, the enzyme is PorU.

[0419] In some embodiments, the engineered rAAV capsid protein is a seven-mutant (SrtA7M) or a five-mutant (SrtA5M). In some embodiments, the engineered rAAV capsid protein is a seven-mutant (SrtA7M). In some embodiments, the engineered rAAV capsid protein is a five-mutant (SrtA5M).

[0420] In some embodiments, the enzyme is a seven-mutant Staphylococcus aureus sorting enzyme A (SrtA7m).

[0421] In some embodiments, the engineered AAV capsid protein of this disclosure comprises one or more sequence alterations. In some embodiments, the sequence alteration is selected from insertions, deletions, or substitutions compared to a known wild-type protein. A single insertion, deletion, or mutation comprises one or more consecutive amino acid residues. These engineered sequence alterations are used to introduce a functional sequence motif into the capsid protein. In some of these embodiments, the sequence alteration comprises the insertion of at least one insert sequence that forms a functional sequence motif.

[0422] 5.4.1 Transaminases

[0423] In some embodiments, the functional sequence motifs of this disclosure include any known transaminases' recognition sequences. Recognition sequences are specific amino acid sequences present in proteins that are recognized and targeted by transaminases such as transpeptidases. Transpeptidases, such as sorting enzymes, are a class of enzymes that play a crucial role in protein cross-linking processes, particularly in bacterial cell wall formation. Transaminases' recognition sequences typically consist of a specific arrangement of amino acids, such as a sequence of three to five amino acids. The exact composition and arrangement of amino acids vary depending on the bacterial species.

[0424] The transaminases used in this disclosure may also be selected from enzymes derived from natural sequences and containing mutations. In some embodiments, the enzyme is a transaminas carrying a mutation that improves cleavage efficiency and thus overall reaction efficiency. Exemplary mutant sorting enzymes used in this disclosure are those identified in J. Biol. Chem. (2020) 295:9, 2664-2675 and Scientific reports 2016 (6:31899), which are incorporated herein by reference. In some embodiments, the sorting enzymes used in this disclosure carry one or more of the following mutations: P94R, E105K, E108A, D124G, D160N, D165A, D186G, Y187L, E189R, K190E, and K196T. In a particular embodiment, the transaminas is a seven-mutant Staphylococcus aureus sorting enzyme A (SrtA7m).

[0425] In some embodiments, the functional sequence motif, the corresponding enzyme for ligand functionalization, and the enzyme substrate portion are selected from those described in the “Peptide Linking” table below.

[0426] peptide linker

[0427] 5.4.1.1 Sorting enzymes and transaminases

[0428] As used herein, the term "soramiase" refers to an enzyme capable of transpeptidation via transamidation, for example, coupling the C-terminus of a protein to its N-terminus. Soramiases, also known as transaminases, typically possess both protease and transpeptidase activity. A variety of sortases from prokaryotes have been identified. For example, some sortases from Gram-positive bacteria cleave proteins and translocate them to the proteoglycan portion of the intact cell wall. Soramiases isolated from Staphylococcus aureus include sortase A (Srt A) and sortase B (Srt B). Thus, in some embodiments, the transaminas used according to this disclosure is sortase A, such as sortase A from Staphylococcus aureus, also referred to herein as SrtAaureus. In some embodiments, the transaminas is sortase B, such as sortase B from Staphylococcus aureus, also referred to herein as SrtBaureus.

[0429] Based on sequence alignment and phylogenetic analysis of 61 sorting enzymes from the genomes of Gram-positive bacteria, sorting enzymes have been classified into four classes, named A, B, C, and D, namely sorting enzyme A, sorting enzyme B, sorting enzyme C, and sorting enzyme D (Dramsi S., Trieu-Cuot P., Bierne H., Sorting sortases: a nomenclature proposal for the various sortases of Gram-positive bacteria. Res Microbiol. 156(3):289-97, 2005; the entire contents of which are incorporated herein by reference). These categories correspond to the following subfamilies into which sorting enzymes are also classified by Comfort and Clubb (Comfort D., Clubb RT A comparative genome analysis identifies distinct sorting pathways in gram-positive bacteria. Infect Immun., 72(5):2710-22, 2004; the entire contents of which are incorporated herein by reference): Class A (subfamily 1), Class B (subfamily 2), Class C (subfamily 3), and Class D (subfamilies 4 and 5). The above references disclose a large number of sorting enzymes and recognition motifs. See also Pallen, MJ; Lam, AC; Antonio, M.; Dunbar, K. Trends in Microbiology, 2001, 9(3), 97-101; the entire contents of which are incorporated herein by reference. Those skilled in the art will readily classify sorting enzymes into the correct categories based on their sequence and / or other characteristics (such as those described above by Dramsi et al.). As used herein, the term "sorting enzyme A" refers to a class A sorting enzyme, typically named SrtA in any particular bacterial species, such as SrtA from Staphylococcus aureus. Similarly, "sorting enzyme B" is used herein to refer to a class B sorting enzyme, typically named SrtB in any particular bacterial species, such as SrtB from Staphylococcus aureus. This disclosure covers embodiments involving sorting enzyme A from any bacterial species or strain. This disclosure covers embodiments involving sorting enzyme B from any bacterial species or strain. This disclosure covers embodiments involving class C sorting enzymes from any bacterial species or strain. This disclosure covers embodiments involving class D sorting enzymes from any bacterial species or strain.

[0430] The amino acid sequences of Srt A and Srt B, and the nucleotide sequences encoding them, are known to those skilled in the art and are disclosed in numerous references cited herein, the entire contents of which are incorporated herein by reference. The amino acid sequences of Staphylococcus aureus (S. aureus) SrtA and SrtB are homologous, sharing, for example, 22% sequence identity and 37% sequence similarity. The amino acid sequences of sorting enzymes—transaminases—from Staphylococcus aureus are also substantially homologous to enzyme sequences from other Gram-positive bacteria, and such transaminases can be used in the ligation process described herein. For example, for SrtA, the best alignment with the entire sequencing region of the open reading frame of Streptococcus pyogenes (S. pyogenes) shows approximately 31% sequence identity (and approximately 44% sequence similarity). The best alignment with the entire sequencing region of the open reading frame of Actinomyces naeslundii (A. naeslundii) shows approximately 28% sequence identity. It should be understood that different bacterial strains may have differences in the sequence of a specific polypeptide, and the sequence described in this article is exemplary.

[0431] 5.4.2 Functional sequence motifs

[0432] In some embodiments, the engineered rAAV capsid protein includes a functional sequence motif located within a surface-accessible variable region (VR) of the capsid primary sequence.

[0433] In some embodiments, the functional sequence motif has the sequence X1X2X3 (SEQ ID NO:6); wherein X1, X2 and X3 are each independently selected from any amino acid residues.

[0434] In some embodiments, the functional sequence motif has the sequence X1X2X3 (SEQ ID NO:7), wherein two or more of X1, X2 and X3 are selected from: X1 is valine (V); X2 is proline (P); and X3 is proline (P).

[0435] In some embodiments, X1, X2 and X3 are selected from: X1 is proline (P); X2 is selected from glycine (G) and glutamic acid (E); and X3 is selected from proline (P) and phenylalanine (F).

[0436] In some embodiments, the functional sequence motif has a sequence selected from: PEF (SEQ ID NO:50), PGF (SEQ ID NO:51), or PEP (SEQ ID NO:52). In some embodiments, the functional sequence motif has the sequence PEF (SEQ ID NO:50). In some embodiments, the functional sequence motif has the sequence PGF (SEQ ID NO:51). In some embodiments, the functional sequence motif has the sequence PEP (SEQ ID NO:52).

[0437] In some embodiments, the functional sequence motif has the sequence X1X2X3X4 (SEQ ID NO:53); wherein X1, X2, X3 and X4 are each independently selected from any amino acid residues.

[0438] In some embodiments, the functional sequence motif has the sequence X1X2X3X4X5 (SEQ ID NO:54); wherein X1, X2, X3, X4 and X5 are each independently selected from any amino acid residues.

[0439] In some embodiments, the functional sequence motif has the sequence X1X2X3X4X5 (SEQ ID NO:54), wherein: X1 is independently selected from lysine (L), valine (V), isoleucine (I), and asparagine (N); X2 is independently selected from proline (P); X3 is independently selected from any amino acid residue; X4 is independently selected from threonine (T); and X5 is independently selected from alanine (A), glycine (G), and asparagine (N).

[0440] In some embodiments, two or more of X1, X2, X3, X4 and X5 are selected from: X1 is independently selected from lysine (L); X2 is independently selected from proline (P); X3 is independently selected from any amino acid residue; X4 is independently selected from threonine (T); and X5 is independently selected from alanine (A), glycine (G) and asparagine (N).

[0441] In some embodiments, three or more of X1, X2, X3, X4 and X5 are selected from: X1 is independently selected from lysine (L), valine (V), isoleucine (I) and asparagine (N); X2 is independently selected from proline (P); X3 is independently selected from any amino acid residue; X4 is independently selected from threonine (T); and X5 is independently selected from alanine (A) and glycine (G).

[0442] In some embodiments, X1 is lysine (K); X2 is proline (P); X3 is glutamic acid (E), serine (S) or alanine (A); X4 is threonine (T); and X5 is alanine (A) or glycine (G).

[0443] In some embodiments, the functional sequence motif comprises a sequence selected from: LPX3TG (SEQ ID NO: 48), LPX3TA (SEQ ID NO: 55), LPETG (SEQ ID NO: 12), and LPETA (SEQ ID NO: 56). In some embodiments, the functional sequence motif comprises the sequence LPX3TG (SEQ ID NO: 48). In some embodiments, the functional sequence motif comprises the sequence LPX3TA (SEQ ID NO: 55). In some embodiments, the functional sequence motif comprises the sequence LPETG (SEQ ID NO: 12). In some embodiments, the functional sequence motif comprises the sequence LPETA (SEQ ID NO: 56).

[0444] In some embodiments, the functional sequence motif has the sequence X1X2X3X4X5X6 (SEQ ID NO:5); wherein X1, X2, X3, X4, X5 and X6 are each independently selected from any amino acid residues.

[0445] In some embodiments, the functional sequence motif has the sequence X1X2X3X4X5X6 (SEQ ID NO:5); wherein two or more of X1, X2, X3, X4, X5 and X6 are: X1 is independently asparagine (N); X4 is independently threonine (T); X5 is independently asparagine (N); and X6 is independently proline (P).

[0446] In some embodiments, the functional sequence motif has the sequence X1X2X3X4X5X6 (SEQ ID NO:5); wherein at least three of X1, X2, X3, X4, X5 and X6 are: X1 is independently asparagine (N); X4 is independently threonine (T); X5 is independently asparagine (N); and X6 is independently proline (P).

[0447] In some embodiments, the functional sequence motif comprises a sequence selected from NX2X3TNX6 (SEQ ID NO: 57) and VPX3X4X5P (SEQ ID NO: 58). In some embodiments, the functional sequence motif comprises the sequence NX2X3TNX6 (SEQ ID NO: 57). In some embodiments, the functional sequence motif comprises the sequence VPX3X4X5P (SEQ ID NO: 58).

[0448] In some embodiments, the engineered rAAV capsid protein includes one or more sequence alterations compared to the wild-type protein, said sequence alterations being selected from insertions, deletions, or substitutions, used to introduce a functional sequence motif. In some embodiments, the sequence alteration is an insertion. In some embodiments, the sequence alteration is a deletion. In some embodiments, the sequence alteration is a substitution.

[0449] In some embodiments, the engineered rAAV capsid protein contains at least one insertion mutation, wherein the insertion forms a functional sequence motif.

[0450] In some embodiments, the insertion mutation is an insertion of a foreign peptide selected from LPET (SEQ ID NO:49) and LPETG (SEQ ID NO:12). In some embodiments, the insertion mutation is LPET (SEQ ID NO:49). In some embodiments, the insertion mutation is LPETG (SEQ ID NO:12).

[0451] In some embodiments, the rAAV capsid protein is selected from one or more of VP1, VP2, and VP3. In some embodiments, the rAAV capsid protein is VP1. In some embodiments, the rAAV capsid protein is VP2. In some embodiments, the rAAV capsid protein is VP3.

[0452] In some embodiments, the engineered rAAV capsid protein comprises a functional sequence, wherein the functional sequence motif is located within a surface loop region selected from the following: AB, BC, CD, DE, EF, FG, GH, and HI loops. In some embodiments, the functional sequence motif is located within the AB surface loop region. In some embodiments, the functional sequence motif is located within the BC surface loop region. In some embodiments, the functional sequence motif is located within the CD surface loop region. In some embodiments, the functional sequence motif is located within the DE surface loop region. In some embodiments, the functional sequence motif is located within the EF surface loop region. In some embodiments, the functional sequence motif is located within the FG surface loop region. In some embodiments, the functional sequence motif is located within the GH surface loop region. In some embodiments, the functional sequence motif is located within the HI surface loop region.

[0453] In embodiments of this disclosure, an engineered adeno-associated virus (AAV) capsid protein is provided, comprising one or more functional sequence motifs, wherein the functional sequence motifs include sorting enzyme recognition motifs.

[0454] In some embodiments, the functional sequence motif is located within one or more variable regions selected from VR1, VR2, VR3, VR4, VR5, VR6, VR7, VR8, and VR9. In some embodiments, the functional sequence motif is located within VR1. In some embodiments, the functional sequence motif is located within VR2. In some embodiments, the functional sequence motif is located within VR3. In some embodiments, the functional sequence motif is located within VR4. In some embodiments, the functional sequence motif is located within VR5. In some embodiments, the functional sequence motif is located within VR6. In some embodiments, the functional sequence motif is located within VR7. In some embodiments, the functional sequence motif is located within VR8. In some embodiments, the functional sequence motif is located within VR9.

[0455] In some embodiments, the engineered rAAV capsid protein contains a functional sequence motif located within variable region IV or VIII.

[0456] In some embodiments, the engineered rAAV capsid protein contains a functional sequence motif located within variable region VIII.

[0457] In some embodiments, the engineered rAAV capsid protein comprises functional sequence motifs located within variable regions IV and VIII, wherein each functional sequence motif is independently selected.

[0458] In some implementations, each functional sequence motif is identical.

[0459] In some embodiments, the rAAV capsid protein is selected from serotypes AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9. In some embodiments, the rAAV capsid protein is AAV1. In some embodiments, the rAAV capsid protein is AAV2. In some embodiments, the rAAV capsid protein is AAV4. In some embodiments, the rAAV capsid protein is AAV5. In some embodiments, the rAAV capsid protein is AAV8. In some embodiments, the rAAV capsid protein is AAV9.

[0460] In some embodiments, the functional sequence motif begins after: S453 in AAV1 capsid serotype VR4; after S452 in AAV2 capsid serotype VR4 or R585 in VR8; after S576 in AAV5 capsid serotype VR8; after S453 in AAV6 capsid serotype VR4 or S587 in VR8; after P454 in AAV7 capsid serotype VR4 or A587 in VR8; after T454 in AAV8 capsid serotype VR4 or Q589 in VR8; and after S454 in AAV9 capsid serotype VR8.

[0461] In some embodiments, the functional sequence motif begins after S452 in AAV2 capsid serotype VR4 or after R585 in VR8. In some embodiments, the functional sequence motif begins after S452 in VR4. In some embodiments, the functional sequence motif begins after R585 in VR8.

[0462] In some implementations, LPET (SEQ ID NO:49) is inserted after S452 to form a functional sequence motif.

[0463] In some implementations, the engineered rAAV capsid protein does not contain additional mutations compared to the wild-type capsid protein.

[0464] On the one hand, it provides a polynucleotide encoding the engineered rAAV capsid protein disclosed herein.

[0465] On the one hand, a vector containing the polynucleotides disclosed herein is provided.

[0466] In some embodiments, the vector contains a promoter operatively linked to the polynucleotide.

[0467] On the one hand, a host cell containing the engineered rAAV capsid protein, polynucleotide, or vector disclosed herein is provided.

[0468] On the one hand, recombinant rAAV viral particles (rAAV) containing the engineered rAAV capsid protein of this disclosure are provided.

[0469] In some embodiments, the rAAV virion also includes exogenous cargo polynucleotides.

[0470] In some implementations, the exogenous cargo polynucleotide contains a template for homology-directed repair.

[0471] In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic tRNA, miRNA, gene-editing guide RNA, or RNA-editing guide RNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic tRNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a miRNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a gene-editing guide RNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding an RNA-editing guide RNA.

[0472] In some implementations, the exogenous polynucleotide comprises an expressible polynucleotide encoding a therapeutic protein.

[0473] In some embodiments, the rAAV virion includes a functional sequence motif, wherein the functional sequence motif weakens or eliminates the binding of the virion to mammalian cytopolysaccharides or proteoglycans compared to a reference virion having the same capsid protein sequence but lacking the functional sequence motif. In some embodiments, the functional sequence motif weakens or eliminates the binding of the virion to mammalian cytopolysaccharides compared to a reference virion having the same capsid protein sequence but lacking the functional sequence motif. In some embodiments, the functional sequence motif weakens or eliminates the binding of the virion to mammalian cytoglycans compared to a reference virion having the same capsid protein sequence but lacking the functional sequence motif.

[0474] In some embodiments, the rAAV virion includes at least one cross-linking reactive motif covalently linked to an amino acid residue in the engineered rAAV capsid protein, wherein the amino acid residue is not within the functional sequence motif, and includes exogenous cargo polynucleotides.

[0475] In some embodiments, the amino acid residue is lysine. In some embodiments, the amino acid residue in the viral capsid protein is an N-terminal amine. In some embodiments, the amino acid residue in the viral capsid protein is a non-natural amino acid residue.

[0476] On one hand, a surface-functionalized rAAV virion is provided, wherein the virion comprises at least one engineered rAAV capsid protein of the present disclosure and an exogenous cargo polynucleotide, wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form (i) an N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and at least one crosslinking reactive portion covalently linked to a C-terminal amino acid of the N-terminal cleavage fragment.

[0477] In some embodiments, the covalent link comprises an amide bond. In some embodiments, the C-terminal amino acid of the N-terminal cleavage fragment is threonine.

[0478] On one hand, a surface-functionalized rAAV virion is provided, wherein the virion comprises at least one engineered rAAV capsid protein according to the present disclosure and an exogenous cargo polynucleotide, and wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form (i) an N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and wherein at least one cross-linking reactive portion is covalently linked to a C-terminal amino acid of the N-terminal cleavage fragment; and optionally at least one cross-linking reactive portion is covalently linked to an amino acid residue in the engineered rAAV capsid protein, wherein the amino acid residue is not within the functional sequence motif.

[0479] In some embodiments, the surface-functionalized rAAV virion comprises at least one crosslinking agent portion, wherein each crosslinking agent reactive portion is independently selected from crosslinking agent reactive portions participating in reactions selected from: Cu(I)-catalyzed azido-acetylene cycloaddition (CuAAC), strain-promoted acetylene-azido cycloaddition (SPAAC), strain-promoted acetylene-nitroketone cycloaddition (SPANC), anti-electron-demanding Diels-Alder (IEDDA) reactions, Staudinger linkage, and [4+1] cycloaddition reactions. In some embodiments, the crosslinking agent reactive portion participates in the Cu(I)-catalyzed azido-acetylene cycloaddition (CuAAC). In some embodiments, the crosslinking agent reactive portion participates in the strain-promoted acetylene-nitroketone cycloaddition (SPANC) reaction. In some embodiments, the crosslinking agent reactive portion participates in the anti-electron-demanding Diels-Alder (IEDDA) reaction. In some implementations, the reactive portion of the crosslinker participates in Staudinger linkage and [4+1] cycloaddition reactions.

[0480] In some embodiments, the reactive portion of the crosslinking agent comprises at least one of an eight-membered ring and a triazole ring. In some embodiments, the reactive portion of the crosslinking agent is selected from cyclooctyne and azides.

[0481] In some embodiments, the cyclooctyne is selected from dibenzylcyclooctyne (DIBO), dibenzozacyclooctyne (DBCO), and biarylazacyclooctynone (BARAC), or derivatives thereof. In some embodiments, the cyclooctyne is dibenzylcyclooctyne (DIBO). In some embodiments, the cyclooctyne is dibenzozacyclooctyne (DBCO). In some embodiments, the cyclooctyne is biarylazacyclooctynone (BARAC).

[0482] In some embodiments of this disclosure, the sorting enzyme is sorting enzyme A (SrtA). SrtA recognizes the motif LPX1TX2 (SEQ ID NO:8); wherein each occurrence of X1 and X2 independently represents any amino acid residue, and common recognition motifs include, for example, LPKTG (SEQ ID NO:9), LPATG (SEQ ID NO:10), and LPNTG (SEQ ID NO:11). In some embodiments, LPETG (SEQ ID NO:12) is used as the sorting enzyme recognition motif. However, motifs falling outside this common sequence can also be recognized. For example, in some embodiments, the motif contains "A" instead of "T" at position 4, such as LPXAG (SEQ ID NO:13) and LPNAG (SEQ ID NO:14). In some embodiments, the motif contains "A" instead of "G" at position 5, such as LPXTA (SEQ ID NO:15) and LPNTA (SEQ ID NO:16). In some embodiments, the motif contains a "G" instead of a "P" at the second position, such as LGXTG (SEQ ID NO:17) or LGATG (SEQ ID NO:18). In some embodiments, the motif contains an "I" instead of an "L" at the first position, such as IPXTG (SEQ ID NO:19), IPNTG (SEQ ID NO:20), or IPETG (SEQ ID NO:21). Other suitable sorting enzyme recognition motifs will be apparent to those skilled in the art, and this disclosure is not limited to this aspect. It should be understood that the terms "recognition motif" and "recognition sequence" are used interchangeably with respect to sequences recognized by transaminases, transpeptidases, or sorting enzymes.

[0483] In some embodiments of this disclosure, the term "recognition sequence motif residue" will be apparent to those skilled in the art. In these embodiments, the recognition sequence motif residue refers to a portion of the enzyme substrate that has been cleaved and then functionalized. Functionalization may refer to the conversion of a free carboxylic acid into an activated ester, such as a thioester, and its reaction with a nucleophile (such as an amine) to form a peptide bond. For example, LPETG (SEQ ID NO:12) is a functional sequence motif recognized by sorting enzyme A. Sorting enzyme A recognizes a substrate such as SEQ ID NO:12 and cleaves the peptide bond between threonine and glycine to generate an intermediate (e.g., a thioester-linked acylase intermediate), which it then links (or functionalizes). Therefore, in this example, the recognition sequence motif residue refers to LPET (residues 1 to 4 of SEQ ID NO:12) or (SEQ ID NO:49).

[0484] In some embodiments of this disclosure, the sorting enzyme is sorting enzyme B (SrtB), such as sorting enzyme B of Staphylococcus aureus, Bacillus anthracis, or Listeria monocytogenes. Class B sorting enzymes (SrtB) recognize motifs that typically belong to the common sequence NPXTX (SEQ ID NO:74), such as NP[Q / K]-T[sH / N / G / sG], like NPQTN (SEQ ID NO:22) or NPKTG (SEQ ID NO:23). For example, sorting enzyme B of Staphylococcus aureus or Bacillus anthracis cleaves the NPQTN (SEQ ID NO:24) or NPKTG (SEQ ID NO:25) motif of IsdC in their respective bacteria (see, for example, Marraffini, L. and Schneewind, O., Journal of Bacteriology, 189(17), p. 6425-6436, 2007). Other recognition motifs found in the presumed substrates of class B sortases include NSKTA (SEQ ID NO:28), NPQTG (SEQ ID NO:29), NAKTN (SEQ ID NO:26), and NPQSS (SEQ ID NO:27). For example, SrtB from Listeria monocytogenes recognizes certain motifs lacking P at position 2 and / or Q or K at position 3, such as NAKTN (SEQ ID NO:30) and NPQSS (SEQ ID NO:31) (Mariscotti JF, García-Del Portillo F., Pucciarelli MG The listeria monocytogenes sortase-B recognizes varied amino acids atposition two of the sorting motif. J. Biol. Chem., 2009).

[0485] In some embodiments, the sorting enzyme, functional sequence motif (or “motif”), and enzyme substrate portion are as described in Bradshaw WJ, et al. (2015), Molecular Features of the Sortase Enzyme Family. FEBS J, 282: 2097-2114, which is incorporated herein by reference in its entirety.

[0486] In some embodiments, the sorting enzyme is sorting enzyme C (Srt C). Sorting enzyme C may utilize LPXTX (SEQ ID NO: 8) as a recognition motif, where each occurrence of X independently represents any amino acid residue.

[0487] In some embodiments, the sorting enzyme is sorting enzyme D (Srt D). This class of sorting enzymes is predicted to recognize motifs with the shared sequence NA-[E / A / S / H]-TG (SEQ ID NO:75) (Comfort D, ibid.). Sorting enzyme D has been found in, for example, *Streptomyces* spp., *Corynebacterium* spp., *Tropheryma whipplei*, *Thermobifida fusca*, and *Bifidobacterium longhum*.

[0488] LPXTA (SEQ ID NO:15) or LAXTG (SEQ ID NO:32) can serve as recognition sequences for sorting enzymes D, such as subfamilies 4 and 5, which process the LPXTA (SEQ ID NO:15) and LAXTG (SEQ ID NO:32) motifs, respectively. For example, Bacillus anthracis sorting enzyme C has been shown to specifically cleave the LPNTA (SEQ ID NO:16) motif in Bacillus anthracis BasI and BasH (see Marrafini, ibid.).

[0489] Barnett and Scott describe a sorting enzyme that recognizes the QVPTGV (SEQ ID NO:33) motif (Barnett, TC and Scott, JR, Differential Recognition of Surface Proteins in Streptococcus pyogenes by Two Sortase Gene Homologs. Journal of Bacteriology, 184, 8, p. 2181-2191, 2002; the entire contents of which are incorporated herein by reference). Other sorting enzymes, including but not limited to those that recognize motifs recognized by other sorting enzymes, are also applicable to some embodiments of this disclosure. For example, a sorting enzyme is described in Chen I., Dorr BM, and Liu DR, A general strategy for the evolution of bond-forming enzymes using yeast display. Proc. Natl. Acad. Sci. USA. 2011 Jul.12; 108(28):11399, the entire contents of which are incorporated herein by reference.

[0490] In the context of some embodiments of this disclosure, the use of sorting enzymes found in any Gram-positive organisms such as those mentioned herein and / or referenced in databases is contemplated. The use of sorting enzymes found in Gram-negative bacteria, such as *Colwellia psychrerythraea*, *Microbulbifer degradans*, *Bradyrhizobium japonicum*, *Shewanella oneidensis*, and *Shewanella putrefaciens*, is also contemplated. Such sorting enzymes recognize sequence motifs other than the LPXTX (SEQ ID NO:8) common sequence, such as LP[Q / K]T[A / S]T (SEQ ID NO:34), such as LPQTAT (SEQ ID NO:62), LPKTAT (SEQ ID NO:63), LPQTST (SEQ ID NO:64), or LPKTST (SEQ ID NO:65). Consistent with the allowable variation at position 3 of the Gram-positive bacterial sorting enzyme, the sequence motif LPXT[A / S] (SEQ ID NO:8), such as LPXTA (SEQ ID NO:15) or LPSTS (SEQ ID NO:35), can be used.

[0491] Those skilled in the art will understand that any sorting enzyme recognition motif known in the art can be used in some embodiments of this disclosure, and this disclosure is not limited thereto. For example, in some embodiments, the sorting enzyme recognition motif is selected from: LPKTG (SEQ ID NO:9), LPITG (SEQ ID NO:36), LPDTA (SEQ ID NO:37), SPKTG (SEQ ID NO:38), LAETG (SEQ ID NO:39), LAATG (SEQ ID NO:40), LATTG (SEQ ID NO:41), LASTG (SEQ ID NO:42), LPLTG (SEQ ID NO:43), LSRTG (SEQ ID NO:44), LPETG (SEQ ID NO:12). This disclosure covers embodiments in which “X” in any sorting enzyme recognition motif disclosed herein or known in the art is an amino acid, such as any naturally occurring or non-naturally occurring amino acid. In some embodiments, X is selected from the 20 most common standard amino acids found in proteins in living organisms. In some embodiments, for example, the recognition motif is LPXTG (SEQ ID NO:45) or LPXT, where X is D, E, A, N, Q, K, or R. In some embodiments, X in a particular recognition motif is selected from the 3rd naturally occurring amino acid in a naturally occurring sorting enzyme substrate. For example, in some embodiments, X in LPXTG (SEQ ID NO:45) or the LPXT motif (residues 1 to 4 of SEQ ID NO:45) is selected from K, E, N, Q, or A, wherein the sorting enzyme is sorting enzyme A. In some embodiments, X in LPXTG (SEQ ID NO:45) or the LPXT motif (residues 1 to 4 of SEQ ID NO:45) is selected from K, S, E, L, A, or N, and a class C sorting enzyme is used.

[0492] In some embodiments of these implementations, the functional sequence motif is LPETG (SEQ ID NO: 12). In some embodiments, the functional sequence motif has a sequence selected from PEF, PGF, or PEP. In some embodiments, the functional sequence motif has the sequence X1X2X3X4 (SEQ ID NO: 53); wherein X1, X2, X3, and X4 are each independently selected from any amino acid residues. In some embodiments, the functional sequence motif has the sequence X1X2X3X4X5 (SEQ ID NO: 54), wherein X1, X2, X3, X4, and X5 are each independently selected from any amino acid residues. In some embodiments, the functional sequence motif comprises a sequence selected from LPX3TG (SEQ ID NO: 48), LPX3TA (SEQ ID NO: 55), LPETG (SEQ ID NO: 12), and LPETA (SEQ ID NO: 56). In some embodiments, the functional sequence motif comprises a sequence selected from the following: NX2X3TNX6 (SEQ ID NO:57) and VPX3X4X5P (SEQ ID NO:58).

[0493] On one hand, a surface-modified rAAV virion is provided, wherein the virion comprises an exogenous cargo polynucleotide and at least one engineered rAAV capsid protein, the capsid protein comprising at least one functional sequence motif LPX3TG (SEQ ID NO:48), wherein X3 is selected from any amino acid residue; wherein the functional sequence motif is located within variable regions 4, 8, or 4 and 8, and (a) wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form an N-terminal cleavage fragment and a C-terminal cleavage fragment; wherein the virion comprises a cross-linked portion covalently linking a T of the N-terminal cleavage fragment to a targeting ligand, wherein the covalent link comprises an amide bond; and optionally (b) wherein the virion comprises a cross-linked portion covalently linking a lysine residue or an N-terminal amine of the capsid protein to a targeting ligand.

[0494] In some embodiments, the functional sequence motif is selected from one of SEQ ID NO:1-58.

[0495] In some embodiments, the surface-modified rAAV virion is characterized by increased infectivity compared to a reference virion having the same capsid protein sequence but lacking the functional sequence motif. In some embodiments, the increased infectivity is specific to at least one cell or tissue type.

[0496] In some embodiments, the cell or tissue type is selected from the heart, nerves, central and peripheral nervous systems, hematopoietic system, liver, muscle, connective tissue, lungs, kidneys, and retina. In some embodiments, the cell or tissue type is the heart. In some embodiments, the cell or tissue type is nerves. In some embodiments, the cell or tissue type is the central and peripheral nervous system. In some embodiments, the cell or tissue type is the hematopoietic system. In some embodiments, the cell or tissue type is the liver. In some embodiments, the cell or tissue type is connective tissue. In some embodiments, the cell or tissue type is the lungs. In some embodiments, the cell or tissue type is the kidneys and retina.

[0497] As used herein, the term "sorting enzyme substrate" refers to any molecule recognized by a sorting enzyme, such as any molecule that can participate in a sorting enzyme-mediated transpeptidation reaction. A typical sorting enzyme-mediated transpeptidation reaction involves a substrate containing a C-terminal sorting enzyme recognition motif, such as the LPXTX motif (SEQ ID NO:8), and a second substrate containing an N-terminal sorting enzyme recognition motif, such as an N-terminal polyglycine or polyalanine. Sorting enzyme substrates can be peptides or proteins, such as target proteins on the surface of viruses, or peptides containing a sorting enzyme recognition motif, such as the LPXTX motif (SEQ ID NO:8 or polyglycine or polyalanine), wherein the peptide is conjugated to a reagent (e.g., a small molecule, a binding agent, or a fluorophore). Therefore, both protein and non-protein molecules can be sorting enzyme substrates as long as they contain a sorting enzyme recognition motif. Some examples of sorting enzyme substrates are described in more detail elsewhere herein, and other suitable sorting enzyme substrates will be apparent to those skilled in the art. This disclosure is not limited to this aspect.

[0498] As used herein, the term "sorting enzyme labeling" refers to the process of functionalizing the surface of AAV virions with a reactive moiety, tag, or ligand (e.g., protein, peptide, detectable label, binding agent, or click chemical handle) via a sorting enzyme-mediated transpeptidation reaction. Examples of other suitable tags or ligands include, but are not limited to, amino acids, nucleic acids, polynucleotides, sugars, carbohydrates, polymers, lipids, fatty acids, and small molecules. Other suitable tags or ligands will be apparent to those skilled in the art, and this disclosure is not limited to this. In some embodiments, the tag comprises a sequence that can be used to purify, express, solubilize, and / or detect the peptide. In some embodiments, the tag may have multiple functions. In some embodiments, the tag is relatively small, for example, ranging in length from a few amino acids to about 100 amino acids. In some embodiments, the tag is longer than 100 amino acids, for example, up to about 500 amino acids or longer.

[0499] In the context of sorting enzyme-mediated viral particle modification, the term "target protein" refers to a capsid protein constituting the surface of a virus that is the target of sorting enzyme-mediated coupling. For example, in an embodiment where AAV2 is modified by sorting enzyme labeling, VP1 of AAV2 is the target protein, for instance, by adding a targeting ligand to VR4 on the surface of the AAV2 particle. The term "target protein" can refer to the wild-type or naturally occurring form of the corresponding protein, or to an engineered form, such as a recombinant protein variant containing a sorting enzyme recognition motif not present in the wild-type protein form. In the context of sorting enzyme-mediated protein modification, the term "modified target protein" refers to the process of altering a target protein containing a sorting enzyme recognition motif through a sorting enzyme-mediated transpeptidation reaction. Typically, the modification results in the coupling of the target protein with a ligand (e.g., peptide, protein, binding agent, detectable label, or small molecule).

[0500] 5.4.3 AAV sequence alteration

[0501] In some embodiments, the engineered rAAV capsid protein comprises one or more sequence alterations as described above. In some embodiments, the sequence alteration comprises the insertion of a foreign peptide (insertion peptide) selected from LPET, LPETX (SEQ ID NO:46), and LPETG (SEQ ID NO:12).

[0502] The sequence alteration is present in one or more of VP1, VP2, and VP3, which constitute the viral capsid of this disclosure.

[0503] In embodiments of this disclosure, the functional sequence motif is located within a surface ring region selected from the following: AB, BC, CD, DE, EF, FG, GH, and HI rings. For example, in some embodiments, the functional sequence motif is located within the GH surface ring region.

[0504] In embodiments of this disclosure, the functional sequence motif is located within one or more variable regions (VRs) selected from VR1, VR2, VR3, VR4, VR5, VR6, VR7, VR8, and VR9. In some embodiments of these embodiments, the functional sequence motif is located within a surface-accessible variable region of the capsid primary sequence. In some of these embodiments, the engineered rAAV capsid protein comprises a functional sequence motif located within variable region IV or VIII. For example, in some embodiments, the engineered rAAV capsid protein comprises a functional sequence motif located within variable region VIII. In some embodiments, the engineered rAAV capsid protein comprises a functional sequence motif located within variable region IV. In other embodiments, the engineered rAAV capsid protein comprises functional sequence motifs located within variable regions IV and VIII. In some of these embodiments having more than one functional sequence, each functional sequence motif is selected independently.

[0505] In some embodiments, the engineered rAAV capsid protein comprises more than one functional sequence motif. In some embodiments, the rAAV virion of this disclosure comprises more than one engineered rAAV capsid protein. In some embodiments, each functional sequence motif (in the same or different capsid proteins) is identical. In other embodiments, each functional sequence motif is distinct.

[0506] 5.4.4 AAV serotype and insertion site

[0507] Within the framework of this disclosure, the AAV capsid comprises any combination of capsid proteins from natural, genetically modified, and artificially created (random mutations, sequence shuffling, computer design, etc.) serotypes that are capable of assembling and producing novel AAV viral capsids that are not known to exist in nature.

[0508] Currently, over 100 AAV serotypes capable of transducing different cell types have been identified, differing in their ability to bind capsid proteins to specific cell surface receptors. AAV2 was the first serotype cloned into a bacterial plasmid and has since been used as a comparison to identify other serotypes. Twelve serotypes (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12) have been thoroughly tested for their ability to transduce specific cell types and differentiated among capsid protein motifs that bind to specific cell surface receptors for cell-cell junctioning. In the context of this disclosure, rAAV capsid proteins are preferably selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. However, any other AAV capsid protein may be used in the context of this disclosure.

[0509] In one embodiment, the AAV capsid protein of this disclosure comprises a capsid protein selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In particular, the capsid of this disclosure comprises a capsid protein containing AAV2, AAV9, and AAV8.

[0510] In some embodiments, the engineered AAV capsid protein is selected from AAV1, AAV2, AAV4, AAV5, AAV8 and AAV9 serotypes.

[0511] In a specific embodiment of this disclosure, the AAV serotype, variable region, insertion site, and insertion sequence constituting a functional sequence motif are shown in the table below: Table 2. AAV serotypes, variable regions, insertion sites, and insertion sequences constituting functional sequence motifs.

[0512] In some embodiments, the capsid is the AAV2 subtype, and the functional sequence motif begins after S452 or R585, or both S452 and R585. In some embodiments of these embodiments, LPET (SEQ ID NO: 49) is inserted to form the functional sequence motif. In some embodiments of these embodiments, LPETG (SEQ ID NO: 12) is inserted to form the functional sequence motif.

[0513] In some embodiments, the “VR4 mutant” comprises a functional sequence motif formed by inserting LPET, namely CTGCCCGAGACC (SEQ ID NO:47), between S452 and G453 of VP1 of AAV2 (in variable region 4). In this case, this portion of the engineered capsid protein sequence becomes S(452)-L(453)-P(454)-E(455)-T(456)-G(457), with the remaining amino acid residues numbered sequentially according to the number of inserted amino acid residues (four in this case).

[0514] In some embodiments, the “VR8 mutant” comprises a functional sequence motif formed by inserting LPET, namely CTGCCCGAGACC (SEQ ID NO:47), between R585 and G586 of VP1 of AAV2 (in variable region 8). In this case, this portion of the engineered capsid protein sequence becomes R(585)-L(586)-P(587)-E(589)-T(590)-G(591), etc.

[0515] In some embodiments, the “VR4 / VR8 mutant” comprises a functional sequence motif formed by inserting LPET, namely CTGCCCGAGACC (SEQ ID NO:47), between S452 and G453 of VP1 of AAV2 (in variable region 4), and a functional sequence motif formed by inserting LPET, namely CTGCCCGAGACC (SEQ ID NO:47), between R585 and G586 of VP1 of AAV2 (in variable region 8). In this case, this portion of the engineered capsid protein sequence becomes R589-L590-P591-E592-T593-G594.

[0516] In some embodiments, the engineered rAAV capsid protein does not contain any additional mutations other than sequence alterations that form functional sequence motifs, compared to the wild-type capsid protein.

[0517] 5.5 rAAV cargo

[0518] The nucleic acid cargo packaged inside the surface-modified rAAV capsid of this disclosure can be any type of nucleic acid molecule that can be efficiently transduced into cells via rAAV.

[0519] In some embodiments, the payload or cargo of the rAAV capsid is an expressible polynucleotide. In some embodiments, the expressible polynucleotide encodes a protein (e.g., a therapeutic protein). In some embodiments, the expressible polynucleotide encodes a transgene. In some embodiments, the expressible polynucleotide can be transcribed to provide guide RNA, trans-activating CRISPR RNA (tracrRNA), messenger RNA (mRNA), microRNA (miRNA), or shRNA.

[0520] In some implementations, the payload provides a DNA homology construct for homology-directed repair.

[0521] In some embodiments, the nucleic acid molecule encodes an intracellular antibody (e.g., for neutralizing certain proteins within the cell), a nucleic acid molecule encoding a peptide toxin (e.g., for blocking ion channels in pain pathways), a nucleic acid molecule encoding an optogenetic effector (e.g., for using light to turn neuronal activity on or off), a nucleic acid molecule encoding a pharmacogenetic tool (e.g., for using chemical ligands with no interfering pharmacological effects to turn neuronal signaling on or off), a nucleic acid molecule encoding a CRISPR-based editor for precise gene editing, a nucleic acid molecule encoding a CRISPR epigenetic tool for regulating gene expression, and / or a nucleic acid molecule encoding a suicide gene that induces cell death.

[0522] Preferably, when the cargo contains a gene-editing nuclease such as Cas9, the cargo also contains nucleic acid molecules such as gRNA and / or specific DNA to be inserted into the host genome. In some embodiments of these implementations, the cargo contains transgenes known to be associated with genetic conditions.

[0523] Those skilled in the art are familiar with other gene-editing nucleases besides Cas9, such as Cpf1, TALEN, ZFN, or homing endonucleases. Furthermore, engineering using a DNA-guided Argonaute interference system (DAIS) may also be convenient. Essentially, the Argonaute (Ago) protein is heterologously expressed by a polynucleotide introduced into the cell, providing cleavage specificity for a preselected locus in the presence of at least one exogenous oligonucleotide (DNA guide). The TALEN and Cas9 systems are described in WO 2013 / 176915 and WO 2014 / 191128, respectively. Zinc finger nucleases (ZFNs) were originally described in Kim, YG; Cha, J.; Chandrasegaran, S. (“Hybrid restriction enzymes: zinc fingerfusions to Fok I cleavage domain” (1996), Proc. Natl. Acad. Sci. USA 93 (3):1156-60). Cpf1 is a Class 2 CRISPR-Cas system described by Zhang et al. (Cpfl is a single RNA-guided Endonuclease of a Class 2 CRIPR-Cas System. (2015), Cell; 163:759-771). The Argonaute (AGO) gene family was first described by Guo S, Kemphues KJ. (Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser / Thrkinase that is asymmetrically distributed. (1995), Cell; 81(4):611-20).

[0524] In some embodiments, the exogenous cargo polynucleotide comprises a template for homology-directed repair. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic tRNA, miRNA, gene-editing guide RNA, or RNA-editing guide RNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic tRNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a miRNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a gene-editing guide RNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding an RNA-editing guide RNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic protein.

[0525] 5.6 Method for preparing surface-modified rAAV viral particles

[0526] Another aspect of this disclosure relates to a method for producing surface-modified recombinant AAV virus particles.

[0527] The method includes the step of covalently coupling a ligand to an engineered viral capsid protein via a linker containing a crosslinking portion Q. In some embodiments, the ligand introduces at least one mammalian cell surface target binding site into the capsid; optionally, the native cell surface target binding sites in the capsid are removed, as previously removed.

[0528] In one embodiment, the method for preparing a surface-modified viral capsid described herein includes the following steps: By coupling the engineered viral capsid protein described herein with the bifunctional linker described herein, a surface-functionalized viral capsid is obtained. The surface-functionalized viral capsid is coupled with a functionalized ligand to form a cross-linked portion Q; and The surface-modified viral capsid is obtained.

[0529] In one embodiment, the method for preparing the surface-modified viral capsid described herein includes the following steps: The surface-functionalized viral capsid is obtained through the following enzymatic coupling between the two: Functional motifs in viral capsid proteins; and Enzyme-reactive linkers; wherein the linker comprises an enzyme substrate portion and a cross-linking agent reactive portion; and The resulting surface-functionalized viral capsid is reacted with a functionalized ligand containing a second member of the cross-linking agent reactive portion.

[0530] In some embodiments, the method further includes the step of generating an AAV capsid comprising at least one viral capsid protein containing at least one recognition sequence. Optionally, the recognition sequence is located in a surface loop region, such as variable region 4 or 8, or both 4 and 8.

[0531] 5.7 Method for preparing surface-modified viral capsids

[0532] On one hand, a method for preparing a surface-modified viral capsid is provided, the method comprising the following steps: 1) obtaining a surface-functionalized viral capsid by reacting a viral capsid protein with a capsid reactive linker, wherein the linker comprises a first member of a crosslinking agent reaction pair and optionally one or more spacers; 2) coupling the surface-functionalized viral capsid with a functionalized ligand comprising a second member of a crosslinking agent reaction pair and optionally one or more spacers; wherein the first and second members of the crosslinking agent reaction pair react to form a crosslinked portion Q.

[0533] In some embodiments, a method for preparing the surface-modified viral capsid of this disclosure is provided.

[0534] Another aspect of this disclosure relates to a method for producing a surface-modified recombinant viral capsid. In some embodiments, the provided capsid is used for transducing nucleic acids into eukaryotic cells, typically mammalian, and particularly human cells. In some embodiments, the surface-modified viral capsid is a recombinant adenovirus particle. In some embodiments, the surface-modified viral capsid is a recombinant AAV particle.

[0535] The method includes a crosslinking step, in which the ligand is covalently coupled to the viral capsid protein via a linker containing a crosslinking portion Q. Preferably, the ligand introduces at least one mammalian cell surface target binding site into the capsid, optionally the native cell surface target binding sites in the capsid are removed, as previously removed.

[0536] In one embodiment, the method for preparing the surface-modified viral capsid described herein includes the following steps: A surface-functionalized viral capsid is obtained by reacting viral capsid proteins with a capsid reactive linker containing a first member and one or more optional spacers. The surface-functionalized viral capsid is coupled with a functionalized ligand comprising a second member of a crosslinking agent reaction pair and optionally one or more spacers. The first and second members of the crosslinking agent reaction pair react to form the crosslinked portion Q; and The surface-modified viral capsid is obtained.

[0537] In another embodiment, the method for preparing the surface-modified viral capsid described herein includes the following steps: The surface-functionalized viral capsid is obtained through the following enzymatic coupling between the two: Recognition sequences located in the surface loop region of viral capsid proteins; and Enzyme-reactive linkers; wherein the linker comprises a first member of an enzyme substrate and cross-linking agent reaction pair; and The resulting surface-functionalized viral capsid is reacted with a functionalized ligand containing a cross-linking agent to react with a second member.

[0538] In some embodiments, the method further includes the step of generating an AAV capsid comprising at least one viral capsid protein containing at least one recognition sequence. Optionally, the recognition sequence is located in a surface loop region, such as variable region 4 or 8.

[0539] As described above, if the capsid contains a natural mammalian cell surface target binding site that is not removed, and the capsid is surface-modified to include at least one ligand according to this disclosure, the provided surface-modified viral capsid has a higher infection rate (i.e., improved transduction, higher or similar efficiency at lower titers) compared to a capsid without the surface modification described herein. In another embodiment, if the natural mammalian cell surface target binding site in the capsid is removed (e.g., genetically modified for a known heparin binding site) before surface modification to include the ligand, the provided surface-modified capsid has one or more of the following characteristics compared to a capsid without the surface modification described herein: i) modification tropism and ii) improved transduction.

[0540] The adeno-associated virus (AAV) particles produced by the above method are preferably selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, hu68, rh.10, and mixtures thereof.

[0541] Any protein in the adeno-associated virus (AAV) particles produced by the above method can be modified. Preferably, at least one of the VP1, VP2, or VP3 proteins in the capsid is modified in the above method. Alternatively, two of the VP1, VP2, and / or VP3 proteins in the capsid are modified, or all three proteins of the capsid are modified. Preferably, at least a portion of the at least one protein to be modified in the capsid is modified, for example, at least one amino acid. However, multiple portions of the VP1, VP2, and VP3 proteins in the capsid can also be modified, for example, multiple amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or any other number of portions or amino acids. Preferably, this is achieved by replacing at least one amino acid in VP1 (arginine at positions 484, 487, 585, and 588) and VP1 (lysine at position 532) and / or VP2 or VP3 (arginine analogues) with a different amino acid, such as alanine.

[0542] Capsid proteins derived from natural AAV serotypes (which may be further genetically modified if necessary), such as VP1, VP2, or VP3, may be chemically modified at specific amino acids. Examples of such modifications are well known in the art and are summarized, for example, in R. Lundblad, Chemical Reagents for Protein Modification, 3rd ed. CRCPress, 2005, which is incorporated herein by reference. Chemical modifications of amino acids include, but are not limited to: acylation, amidation, pyridoxalization of lysine, reductive alkylation, trinitrophenylization of the amino group with 2,4,6-trinitrobenzenesulfonic acid (TNBS), amide modification of the carboxyl group, thiol modification by performic acid oxidation of cysteine ​​to sulfoalanine, mercury derivative formation, mixed disulfide formation with other thiols, reaction with maleimide, carboxymethylation with iodoacetic acid or iodoacetamide at alkaline pH, and carbamylation with cyanate, but are not limited to these. In this regard, those skilled in the art may refer to Chapter 15 of Current Protocols in Protein Science, Eds. Colligan et al. (John Wiley & Sons NY 1995-2000, the entire contents of which are expressly incorporated herein by reference) for a broader range of approaches to the chemical modification of proteins.

[0543] In some embodiments of the above-described method for generating modified adeno-associated virus (AAV), the capsid modification includes both the removal of natural binding sites and the introduction of ligand binding sites, for example, by functionalizing the capsid surface with reactive portions. In some other embodiments, the natural binding sites of the AAV capsid remain unchanged, i.e., are not removed, and at least one ligand binding site or ligand is introduced according to this disclosure.

[0544] In some embodiments, the natural binding site, which is capable of binding heparan sulfate proteoglycan, is removed by the method described above for generating modified adeno-associated virus (AAV) particles. In some embodiments of these embodiments, the natural binding site is removed by replacing at least one amino acid in arginines at positions 585 and 588 of VP1 and / or in arginine analogues of VP2 or VP3 with a different amino acid such as alanine.

[0545] In some embodiments, the ligand-binding site introduced according to this disclosure is a site capable of covalently attaching a ligand. In some of these embodiments, the ligand-binding site is selected from a benzylguanine group attached to a usable lysine residue, more preferably by reacting the capsid with benzylguanine N-hydroxysuccinimide ester (BG-NHS) and / or benzylcytosine N-hydroxysuccinimide ester (BC-NHS).

[0546] This disclosure preferably utilizes tags capable of binding their specific ligands with high affinity, such as SNAP-tags, CLIP-tags, Halo-tags, Lumio-tags, etc. The tag molecule introduced in the above methods can be any molecule or biomolecule capable of specifically binding to another molecule. Examples may include SNAP-tags, CLIP-tags, Lumio-tags, or Halo-tags. For example, the affinity tag can be a SNAP-tag, a mutant of alkylguanine-DNA alkyltransferase. Importantly, one of the substrates of the SNAP-tag is benzylguanine. Commercially available products that can be used in this disclosure include, for example, Promega's HaloTag, Life Technologies' Lumio Tag, and NEB's SNAP / CLIP Tags. The introduced ligand binding site is preferably linked to an ε-amino or primary amine of the available lysine residue.

[0547] Therefore, the above-described method for generating modified adeno-associated virus (AAV) particles is further preferred, wherein the method further includes the step of linking a ligand to the benzylguanine and / or the benzylcytosine group, particularly HaloTag™, SNAP-tag™, or CLIP-tag™.

[0548] The ligand to be linked can be any type of ligand, but is preferably selected from protein ligands, such as growth factors or cytokines; toxin subunits, such as cholera toxin B subunit; lectins, such as isoglucosinolate B4 or wheat germ lectin; adhesion factors, such as lactobacillus; antibodies, such as anti-CD-34 antibodies; peptides, such as delta-opioid receptor ligands; and gene-editing nucleases, such as Cas9.

[0549] 5.8 Formulation

[0550] Another embodiment of this disclosure relates to the above-mentioned surface-modified viral capsid for treating diseases, wherein the AAV is applied to a subject in liquid, dry, or semi-solid form, such as tablets, coated tablets, effervescent tablets, capsules, powders, granules, sugar-coated tablets, lozenges, pills, ampoules, drops, suppositories, emulsions, ointments, gels, tinctures, pastes, creams, wet dressings, mouthwashes, plant extracts, nasal preparations, inhalation mixtures, aerosols, mouthwashes, oral sprays, nasal sprays, or room sprays.

[0551] In some embodiments, a pharmaceutical composition comprising recombinant viral particles containing a surface-modified viral capsid as described herein and recombinant nucleic acid cargo therein, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier, diluent, solubilizer, filler, preservative, and / or excipient. Such pharmaceutically acceptable carriers, diluents, solubilizers, fillers, preservatives, and / or excipients are described, for example, in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins, 2000.

[0552] Another aspect of this disclosure relates to a pharmaceutical composition comprising a surface-modified viral capsid of this disclosure and at least one pharmaceutically acceptable carrier and / or diluent, i.e., in combination with pharmaceutically acceptable additives, carriers, diluents, solvents, fillers, lubricants, excipients, binders, or stabilizers. Preferably, the composition is administered to the subject in the form of a spray, coating, foam, lotion, gel, mouthwash, oral formulation, or injection. The composition may be administered to the subject systemically, orally, or by any other clinically / medically acceptable method.

[0553] On one hand, a composition is provided for treating a disease that can be treated by intracellular delivery of a recombinant polynucleotide cargo, the composition comprising a therapeutically effective amount of a pharmaceutical composition according to the present disclosure, wherein the recombinant polynucleotide cargo is capable of treating the disease.

[0554] Another aspect of this disclosure relates to a kit comprising: a) a surface-modified viral capsid as described and / or used according to this disclosure, or a pharmaceutical composition comprising a surface-modified viral capsid as described in this disclosure; b) written instructions on applying the surface-modified viral capsid or the pharmaceutical composition to a target; and optionally, a container containing the surface-modified viral capsid or the composition and the written instructions.

[0555] Another aspect of this disclosure relates to the use of the above-described kit in the prevention, treatment, and / or inhibition of viral infection in subjects requiring said treatment.

[0556] 5.9 Methods of Treating Diseases

[0557] This disclosure also includes a method for treating a subject at risk of developing and / or progressing a disease (including monogenic or polygenic inherited diseases), wherein the patient is administered a therapeutically effective amount of AAV particles provided in this disclosure. In this context, a "therapeutically effective amount" refers to an amount of AAV particles sufficient to treat a disease, such as a genetic disease, by symptom relief. A "therapeutically effective amount" can also refer to an amount sufficient to prevent the occurrence of symptoms of a disease, such as a genetic disease. The presence of such disease risk may be due to, for example, genetic and / or phenotypic symptoms that predispose the patient to the disease. In some embodiments, patients identified as being at risk of a genetic disease have carried a gene associated with the genetic disease or lack that gene.

[0558] On one hand, a method is provided for treating a disease that can be treated by intracellular delivery of a recombinant polynucleotide cargo, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of the present disclosure, wherein the recombinant polynucleotide cargo is capable of treating the disease.

[0559] On one hand, a method for treating a disease or condition is provided, the method comprising administering a therapeutically effective amount of the engineered viral capsid of this disclosure. In some embodiments, the disease or condition is a central nervous system disease or condition, such as a neurodegenerative disease, more specifically aromatic L-amino acid decarboxylase (AADC) deficiency, Canavan disease, Parkinson's disease, Alzheimer's disease, and giant axonal neuropathy. In some embodiments, the disease or condition is aromatic L-amino acid decarboxylase (AADC) deficiency. In some embodiments, the disease or condition is Canavan disease. In some embodiments, the disease or condition is Parkinson's disease. In some embodiments, the disease or condition is Alzheimer's disease. In some embodiments, the disease or condition is giant axonal neuropathy.

[0560] Another aspect of this disclosure relates to a method for treating a disease that can be treated with gene therapy, the method comprising administering a surface-modified viral capsid of this disclosure to a subject in need.

[0561] Cells and / or subjects treated with the surface-modified viral capsid of this disclosure are preferably of mammalian origin, such as human origin. However, based on the similarity of the mechanisms of cell entry, this disclosure can also be advantageously used in veterinary medicine, cell culture procedures, and even plant cell diseases. In some embodiments, the cells to be treated are mammalian cells, prokaryotic cells, or plant cells. In a particular embodiment, the cells to be treated are human cells.

[0562] Another embodiment of this disclosure relates to a method for treating the aforementioned disease, comprising applying the surface-modified viral capsid of this disclosure to a subject in need, wherein the surface-modified viral capsid is applied to the subject in liquid, dry, or semi-solid form, such as tablets, coated tablets, effervescent tablets, capsules, powders, granules, sugar-coated tablets, lozenges, pills, ampoules, drops, suppositories, emulsions, ointments, gels, tinctures, pastes, creams, wet dressings, mouthwashes, plant extracts, nasal preparations, inhalation mixtures, aerosols, mouthwashes, oral sprays, nasal sprays, or room sprays.

[0563] Treating a disease using the methods described above involves administering the surface-modified viral capsid to a subject. In some embodiments, the disease is selected from cancer, hereditary single-gene disorders such as hereditary retinal diseases, hereditary skin diseases such as Olmsted syndrome or familial primary localized cutaneous amyloidosis, infectious diseases, adrenoleukodystrophy, α-1 antitrypsin deficiency, aromatic L-amino acid deficiency, Batten's disease, Becker's muscular dystrophy, β-thalassemia, Canavan's disease, chronic granulomatous disease, Crigler-Najjar syndrome, cystic fibrosis, Duchenne muscular dystrophy, Fabry disease, familial adenomatous polyposis, familial hypercholesterolemia, familial lecithin cholesterol acyltransferase deficiency, Fanconi anemia, galactosialidosis, Gaucher's disease, and cycloplegic retinopathy. Hemophilia A, Hemophilia B, Hurler syndrome (mucopolysaccharidosis type I), Hunter syndrome (mucopolysaccharidosis type II), Huntington's disease, junctional epidermolysis bullosa, late-stage infantile neuronal ceroid lipofuscin deposition, leukocyte adhesion deficiency, limb-girdle muscular dystrophy, lipoprotein lipase deficiency, metachromatic leukodystrophy, Sly syndrome (mucopolysaccharidosis type VII), Netherton syndrome, ornithine carbamoyltransferase deficiency, Pompe disease, purine nucleoside phosphorylase deficiency, latent dystrophic epidermolysis bullosa, St. Philip's disease type A (mucopolysaccharidosis type IIIA), St. Philip's disease type B (mucopolysaccharidosis type IIIB), sickle cell disease, severe combined immunodeficiency, spinal muscular atrophy, TaySachs disease, Wiskott-Aldrich syndrome, von Gilke's disease (glycogen storage disease type Ia), X-linked myotube myopathy, end-stage renal anemia, angina pectoris (stable, unstable, refractory), coronary artery stenosis, severe limb ischemia, heart failure, intermittent claudication, myocardial ischemia, peripheral vascular disease, pulmonary hypertension, venous ulcer, adenovirus infection, cytomegalovirus infection, Epstein-Barr virus infection, hepatitis B infection, hepatitis C infection, HIV / AIDS, influenza, Japanese encephalitis, malaria, childhood respiratory diseases, respiratory tract infections. Cellular viruses, tetanus, tuberculosis, gynecological cancers, breast cancer, ovarian cancer, cervical cancer, vulvar cancer, nervous system cancers, glioblastoma, leptomeningeal carcinoma, glioma, astrocytoma, neuroblastoma, retinoblastoma, gastrointestinal cancer, colon cancer, colorectal cancer, liver metastases, post-hepatitis liver cancer, pancreatic cancer, gallbladder cancer, hepatocellular carcinoma, genitourinary cancers, prostate cancer, kidney cancer, bladder cancer, anogenital tumors, skin cancer, melanoma (malignant / metastatic), head and neck cancer, nasopharyngeal carcinoma, squamous cell carcinoma, esophageal cancer, lung cancer, adenocarcinoma, small cell / non-small cell lung cancer. Cancer, mesothelioma, hematologic malignancies, leukemia, lymphoma, multiple myeloma, sarcoma, germ cell cancer, Li-Fraumeni syndrome, thyroid cancer, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), carpal tunnel syndrome, chronic traumatic brain injury, cubital tunnel syndrome, diabetic neuropathy, epilepsy, giant axonal neuropathy, advanced infantile neuronal cerebrospinal lipofuscinosis, multiple sclerosis, myasthenia gravis, pain, Parkinson's disease, peripheral neuropathy, spinal muscular atrophy type 2, achromatopsia, age-related macular degeneration, choroidal agenesis, diabetic macular edema Glaucoma, Leber congenital amaurosis, macular telangiectasia type 2, retinitis pigmentosa, superficial corneal opacity, X-linked retinoschisis, arthritis (rheumatoid, inflammatory, degenerative), degenerative arthropathy, severe inflammatory rectal disease, ulcerative colitis, chronic kidney disease, diabetic ulcers, foot ulcers, detrusor overactivity, erectile dysfunction, fractures, hearing loss, hereditary inclusion body myopathy, graft-versus-host disease / transplantation patients, stomatitis, parotid hyposalicytosis, systemic scleroderma, type I diabetes, and wound healing, or combinations thereof.

[0564] A method of treating a disease is also provided, comprising administering a surface-modified viral capsid of the present disclosure to a subject in need, wherein the surface-modified viral capsid is administered to the subject or cells in the form of a pharmaceutical composition, such as in combination with pharmaceutically acceptable additives, carriers, diluents, solvents, fillers, lubricants, excipients, binders, or stabilizers. In some embodiments, the composition is administered to the subject in the form of a spray, coating, foam, lotion, gel, mouthwash, oral formulation, or injection. The composition may be administered to the subject systemically, orally, or by any other clinically / medically acceptable method.

[0565] Another aspect of this disclosure relates to the use of the surface-modified viral capsids of this disclosure in cell transfection, such as as a gene delivery tool in research. This use can also be for cosmetic purposes, and this disclosure includes cosmetic treatments similar to the medical treatments disclosed herein. For this purpose, the application of the surface-modified viral capsids of this disclosure to a subject or cells can also be achieved in the form of cosmetic compositions, such as in combination with cosmetically safe and acceptable additives, carriers, diluents, solvents, fillers, lubricants, excipients, adhesives, or stabilizers. In some embodiments, the composition is applied to the subject in the form of a spray, coating, foam, lotion, gel, mouthwash, oral formulation, or injection. The composition can be applied to the subject systemically, orally, or by any other clinically / cosmetically acceptable method.

[0566] Those skilled in the art are familiar with methods for transferring genes in vitro and in vivo using vectors derived from AAV, such as those described in WO93 / 09239, US4797368, US 5139941, and EP 488 528.

[0567] On one hand, a composition is provided for treating a disease that can be treated by intracellular delivery of a recombinant polynucleotide cargo, the composition comprising a therapeutically effective amount of the pharmaceutical composition of the present disclosure, wherein the recombinant polynucleotide cargo is capable of treating the disease.

[0568] On the one hand, the use of the surface-modified viral capsid of this disclosure in the preparation of medicaments for treating diseases that can be treated by intracellular delivery of recombinant polynucleotide cargoes is provided.

[0569] On one hand, a surface-functionalized viral capsid is provided, wherein the surface-functionalized viral capsid comprises a first member of a crosslinking agent reaction pair and optionally one or more spacers. In some embodiments, the surface-functionalized viral capsid is adapted to react with a functionalized ligand comprising a second member of a crosslinking agent reaction pair and optionally one or more spacers. In some embodiments, the member of the crosslinking agent reaction pair participates in a reaction selected from: Cu(I)-catalyzed azido-acetylene cycloaddition (CuAAC) reaction, strain-promoted acetylene-azido cycloaddition (SPAAC) reaction, strain-promoted acetylene-nitroketone cycloaddition (SPANC) reaction, anti-electron-demanding Diels-Alder (IEDDA) reaction, and Staudinger linkage and [4+1] cycloaddition reactions. In some embodiments, the member of the crosslinking agent reaction pair participates in a Cu(I)-catalyzed azido-acetylene cycloaddition (CuAAC) reaction. In some embodiments, the member of the crosslinking agent reaction pair participates in a strain-promoted acetylene-azido cycloaddition (SPAAC) reaction. In some embodiments, a member of the crosslinking agent reactant pair participates in a strain-promoted acetylene-nitroketone cycloaddition (SPANC) reaction. In some embodiments, a member of the crosslinking agent reactant pair participates in an anti-electron-demanding Diels-Alder (IEDDA) reaction. In some embodiments, a member of the crosslinking agent reactant pair participates in Staudinger linkage and [4+1] cycloaddition reactions.

[0570] Another aspect of this disclosure relates to a kit comprising: a) the surface-modified viral capsid for cell transfection; b) written instructions for cell transfection using the surface-modified viral capsid; and optionally a container for containing the surface-modified viral capsid and the written instructions.

[0571] 5.9.1 Indications

[0572] Another aspect of this disclosure relates to recombinant viral particles comprising a viral capsid with the surface modifications of this disclosure for treating a disease, and a method of treating a disease by administering an effective amount of recombinant viral particles comprising the surface-modified capsid described herein. In some embodiments, the compositions provided herein are used to treat a disease in a patient that can be treated by intracellular delivery of a recombinant polynucleotide cargo. In some embodiments, the compositions provided herein are used for treatment comprising gene therapy. Furthermore, this disclosure provides the use of surface-modified viral capsid compositions in the preparation of medicaments for gene therapy. Additionally, this disclosure provides a treatment method comprising gene therapy, wherein the method includes administering an rAAV composition comprising the surface-modified capsid.

[0573] In a preferred embodiment, the disease is one affecting the central nervous system. See, for example, Hocquemiller et al. Hum. Gene Ther . 27(7):478-496 (2016) and Daci and Flotte, Int. J. Mol. Sci. 25(2):1050 (2024), the disclosure of the above-mentioned document is incorporated herein by reference in its entirety. In some of these embodiments, the disease is frontotemporal dementia (FTD), ALS, Huntington's disease, Krabbe disease, Alzheimer's disease, metachromatic leukodystrophy, or GM1 ganglioside storage disease.

[0574] There are no particular limitations on the types of diseases that can be treated or prevented by applying the rAAV disclosed herein. Diseases include those treatable with gene therapy, such as cancer, hereditary single-gene disorders such as hereditary retinal diseases, hereditary skin diseases such as Olmsted syndrome or familial primary localized cutaneous amyloidosis, infectious diseases, ataxia, adrenoleukodystrophy, α-1 antitrypsin deficiency, aromatic L-amino acid deficiency, Batten's disease, Becker's muscular dystrophy, β-thalassemia, Canavan disease, chronic granulomatous disease, Crigler-Najjar syndrome, cystic fibrosis, Duchenne muscular dystrophy, Fabry disease, familial adenomatous polyposis, familial hypercholesterolemia, familial lecithin cholesterol acyltransferase deficiency, Fanconi anemia, galactosialidosis, Gaucher's disease, and circumflex arthritis. Choroidal retinal atrophy, hemophilia A and B, Hurler syndrome (mucopolysaccharidosis type I), Hunter syndrome (mucopolysaccharidosis type II), Huntington's disease, junctional epidermolysis bullosa, late-stage infantile neuronal ceroid lipofuscinosis, leukocyte adhesion deficiency, limb-girdle muscular dystrophy, lipoprotein lipase deficiency, metachromatic leukodystrophy, Sly syndrome (mucopolysaccharidosis type VII), Netherton syndrome, ornithine carbamoyltransferase deficiency, Pompe disease, purine nucleoside phosphorylase deficiency, latent dystrophic epidermolysis bullosa, St. Philip's disease type A (mucopolysaccharidosis type IIIA), St. Philip's disease type B (mucopolysaccharidosis type IIIB), sickle cell disease, severe combined immunodeficiency, spinal muscular atrophy, TaySachs disease, Wiskott-Aldrich syndrome, von Gilke's disease (glycogen storage disease type Ia), X-linked myotube myopathy, end-stage renal anemia, angina pectoris (stable, unstable, refractory), coronary artery stenosis, severe limb ischemia, heart failure, intermittent claudication, myocardial ischemia, peripheral vascular disease, pulmonary hypertension, venous ulcer, adenovirus infection, cytomegalovirus infection, Epstein-Barr virus infection, hepatitis B infection, hepatitis C infection, HIV / AIDS, influenza, Japanese encephalitis, malaria, childhood respiratory diseases, respiratory tract infections. Cellular viruses, tetanus, tuberculosis, gynecological cancers, breast cancer, ovarian cancer, cervical cancer, vulvar cancer, nervous system cancers, glioblastoma, leptomeningeal carcinoma, glioma, astrocytoma, neuroblastoma, retinoblastoma, gastrointestinal cancer, colon cancer, colorectal cancer, liver metastases, post-hepatitis liver cancer, pancreatic cancer, gallbladder cancer, hepatocellular carcinoma, genitourinary cancers, prostate cancer, kidney cancer, bladder cancer, anogenital tumors, skin cancer, melanoma (malignant / metastatic), head and neck cancer, nasopharyngeal carcinoma, squamous cell carcinoma, esophageal cancer, lung cancer, adenocarcinoma, small cell / non-small cell lung cancer. Lung cancer, mesothelioma, hematologic malignancies, leukemia, lymphoma, multiple myeloma, sarcoma, germ cell cancer, Li-Fraumeni syndrome, thyroid cancer, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), carpal tunnel syndrome, chronic traumatic brain injury, cubital tunnel syndrome, diabetic neuropathy, epilepsy, giant axonal neuropathy, advanced infantile neuronal cerebrospinal fluid deposition, multiple sclerosis, myasthenia gravis, pain, Parkinson's disease, peripheral neuropathy, spinal muscular atrophy type 2, achromatopsia, age-related macular degeneration, choroidal agenesis, diabetic macular degeneration. Edema, glaucoma, Leber congenital amaurosis, macular telangiectasia type 2, retinitis pigmentosa, superficial corneal opacity, X-linked retinoschisis, arthritis (rheumatoid, inflammatory, degenerative), degenerative arthropathy, severe inflammatory rectal disease, ulcerative colitis, chronic kidney disease, diabetic ulcers / foot ulcers, detrusor overactivity, erectile dysfunction, fractures, hearing loss, hereditary inclusion body myopathy, graft-versus-host disease / transplant patients, stomatitis, parotid hyposaliva secretion, systemic scleroderma, type I diabetes, and / or wound healing.

[0575] In some embodiments, the condition to be treated according to this disclosure is a CNS condition or disease. In some embodiments, the CNS condition or disease is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is selected from aromatic L-amino acid decarboxylase (AADC) deficiency, Canavan disease, Parkinson's disease, Alzheimer's disease, and giant axonal neuropathy.

[0576] In some embodiments, the ataxia to be treated according to this disclosure is ataxia associated with a hereditary disease, including degenerative changes in the cerebellum or spinal cord, and may even manifest as overlapping cerebellar and sensory ataxia. Hereditary diseases causing ataxia include autosomal dominant disorders such as spinocerebellar ataxia, episodic ataxia, and Lewy body atrophy of the dentate nucleus-rubra-pallidus nucleus, as well as autosomal recessive disorders such as Friedreich's ataxia (sensory and cerebellar, predominantly the former) and Niemann Pick's disease, ataxia-telangiectasia (sensory and cerebellar, predominantly the latter), and abetalipoproteinemia. An example of an X-linked ataxia is the rare fragile X-related tremor / ataxia syndrome, or FXTAS.

[0577] In some implementations, the indications for treatment are lipoprotein lipase deficiency, large B-cell lymphoma, β-thalassemia, mantle cell lymphoma, vascular endothelial growth factor peripheral artery disease, squamous cell carcinoma of the head and neck, spinal muscular atrophy, adenosine deaminase deficiency (ADA-SCID), melanoma in patients with recurrent skin lesions, B-cell lymphoblastic leukemia, or Leber congenital amaurosis.

[0578] In some embodiments, the condition to be treated according to this disclosure is a neuromuscular disease. In some embodiments, the neuromuscular disease is spinal muscular atrophy or amyotrophic lateral sclerosis (ALS).

[0579] In some embodiments, the condition to be treated according to this disclosure is a lysosomal storage disease. In some embodiments, the lysosomal storage disease is selected from Pompe disease, Tay-Sachs disease, mucopolysaccharidosis, Batten disease, metachromatic leukodystrophy, and GM1 ganglioside storage disease.

[0580] In some implementations, the indications for treatment include peroneal muscular atrophy (Charcot-Marie-Tooth, all types), ganglioside storage disorders (all types), hereditary epilepsy (such as Dravet syndrome), tuberous sclerosis complex, spinal cord injury, all demyelinating hereditary motor and sensory neuropathies (HMSN), Krabbe disease, progressive ossifying fibrodysplasia, neurofibromatosis type 1 and 2, essential tremor, fragile X syndrome, Lesch-Nyhan syndrome, myotonic dystrophy, multiple system atrophy (MSA), Zellweger syndrome, neuromyelitis optica, or Devic's disease, central pontine myelinolysis, myelopathy such as tabes dorsalis (syphilitic myelopathy), leukoencephalopathy such as progressive multifocal leukoencephalopathy, leukodystrophy, and Guillain-Barré syndrome and its chronic counterpart, chronic inflammatory demyelinating polyneuropathy.

[0581] In some implementations, the indication for treatment is anti-MAG peripheral neuropathy, or copper deficiency-related conditions (peripheral neuropathy, myelopathy, and in rare cases, optic neuropathy), or progressive inflammatory neuropathy.

[0582] 5.9.2 Application method

[0583] Another aspect of this disclosure relates to the method of application of surface-modified rAAV according to this disclosure in the treatment of diseases.

[0584] In some embodiments, the rAAV is administered directly to the cerebrospinal fluid (CSF) via intrathecal application, for example, by injection into the spinal canal, intraventricular (ICV) administration, or intracerebellomedullary cistern (ICM) administration.

[0585] In some embodiments, the surface-modified viral capsid according to this disclosure can be applied directly or indirectly by suitable means known in the art. Methods and uses of this disclosure include systemic, regional, or local delivery and application of the surface-modified viral capsid composition of this disclosure, or delivery and application via any route such as injection or infusion. Exemplary routes of application and delivery include intravenous (iv), intra-articular, intraperitoneal (ip), intra-arterial, intramuscular, parenteral, subcutaneous, intrapleural, local, dermal, intradermal, percutaneous, parenteral (e.g., via mucosa), intracranial, intraspinal, oral (gastrointestinal), mucosal, respiratory, intranasal, intubated, intrapulmonary, intrapulmonary infusion, buccal, sublingual, intravascular, intrathecal, intracavitary, iontophoresis, intraocular, ophthalmic, optometric, intraglandular, intraorganic, intralymphatic, intrathecal, cerebellomedullary cistern, etc.

[0586] In some embodiments, the administration method is systemic. Systemic administration includes systemic injection routes, such as intracardiac, intramuscular, intravenous, and intravascular injection. In some embodiments, the administration method is intracardiac injection. In some embodiments, the administration method is intramuscular injection. In some embodiments, the administration method is intravenous injection. In some embodiments, the administration method is intravascular injection. In some embodiments, the administration method is intravascular injection. In some embodiments, the surface-modified viral capsid contains an enzyme ligand (such as a tissue-type plasminogen activator) capable of penetrating the blood-brain barrier (e.g., the pia mater), thereby allowing transduction to the spinal cord and brain parenchyma after, for example, intrathecal injection. In embodiments, the surface-modified viral capsid also contains a cell-targeting ligand.

[0587] In light of the progress made to date, it is foreseeable that the means of providing the surface-modified viral capsid compositions of this disclosure to an individual or the cells, tissues, or organs of said individual will be further improved. These future improvements can, of course, be incorporated to achieve the effects described in this disclosure.

[0588] In some embodiments, during the step of applying the surface-modified viral capsid of this disclosure, the capsid composition is dissolved in a solution compatible with the delivery method. In some embodiments, for formulations administered intravenously, subcutaneously, intramuscularly, intrathecally, intra-articularly, and / or intraventricularly, the capsid composition is formulated as an aqueous saline solution.

[0589] In some embodiments, the surface-modified rAAV is administered intrathecally, while IVIG is administered intravenously. Exemplary intrathecal administration of surface-modified rAAV and intravenous IVIG administration available according to certain embodiments include, for example, those disclosed in WO2022 / 173847, the disclosure of which is expressly incorporated herein by reference in its entirety. In typical embodiments, IVIG is administered at a time and dose that effectively reduces the transduction of rAAV to extracellular cells in the CNS by peripheral migration. In some embodiments of these embodiments, the surface-modified rAAV is administered via lumbar puncture. In some embodiments of these embodiments, the surface-modified rAAV is administered intraventricularly (ICV). In some embodiments of these embodiments, the surface-modified rAAV is administered via intracerebellomedullary cistern (ICM) injection into the cerebellomedullary cistern.

[0590] In some embodiments, this disclosure provides a modified version of the Par1 agonist SFLLRN (residues 1 to 6 of SEQ ID NO: 66) wherein an additional cysteine ​​(C) residue is included at its amidated C-terminus. In some embodiments, the resulting peptide SFLLRNC (SEQ ID NO: 66) is subsequently modified with a linker (e.g., a maleimide linker) to directly attach an azide group to its additional C residue. In some embodiments, ΔHSPG-SFLLRNC (SEQ ID NO: 66) is efficiently transported to different regions of the CNS via the bloodstream. In some embodiments, the modified vector (e.g., the ΔHSPG-SFLLRNC vector (SEQ ID NO: 66)) is delivered systemically via intravenous (IV) injection. In some embodiments, the ΔHSPG-SFLLRNC vector (SEQ ID NO: 66) is administered at 9 × 10 10 The virus is administered intravenously at a dose of VG per animal. In some embodiments, the virus is delivered via the bloodstream, and its distribution is not limited to a specific region but is uniformly distributed throughout the nervous system. In some embodiments, the presence of positive neurons in different regions of the brain of subjects (e.g., mice) injected with ΔHSPG-SFLLRNC (SEQ ID NO:66) may indicate that the Par1 agonist SFLLRNC (SEQ ID NO:66) confers an effective ability to enhance the permeability of the ΔHSPG vector to the BBB.

[0591] 5.10 Additional Implementation Plan

[0592] 1. A surface-modified viral capsid comprising one or more of the following: The ligand is covalently conjugated to the viral capsid protein via a linker. The connector includes: The crosslinked portion, wherein the crosslinked portion is formed by a reaction between a first member and a second member of a crosslinking agent reaction pair; and Optionally one or more spacers.

[0593] 2. The surface-modified viral capsid according to embodiment 1, wherein the first and second members of the crosslinking agent reaction pair participate in a reaction selected from: Cu(I)-catalyzed azido-acetylene cycloaddition (CuAAC), strain-promoted acetylene-azido cycloaddition (SPAAC), strain-promoted acetylene-nitroketone cycloaddition (SPANC), anti-electron-demanding Diels-Alder (IEDDA) reaction, and Staudinger linkage and [4+1] cycloaddition reactions. In some embodiments, the first and second members of the crosslinking agent reaction pair participate in the Cu(I)-catalyzed azido-acetylene cycloaddition (CuAAC). In some embodiments, the first and second members of the crosslinking agent reaction pair participate in the strain-promoted acetylene-azido cycloaddition (SPAAC). In some embodiments, the first and second members of the crosslinking agent reaction pair participate in the strain-promoted acetylene-nitroketone cycloaddition (SPANC) reaction. In some embodiments, the first and second members of the crosslinking agent reaction pair participate in the anti-electron-demanding Diels-Alder (IEDDA) reaction. In some embodiments, the first and second members of the crosslinking agent reaction pair participate in Staudinger linkage and [4+1] cycloaddition reactions.

[0594] 3. The surface-modified viral capsid according to any one of the foregoing embodiments, wherein the crosslinked portion comprises at least one of an eight-membered ring and a triazole ring.

[0595] 4. The surface-modified viral capsid according to embodiment 2, wherein the reaction is a strain-promoted acetylene-azidocycloaddition (SPAAC) reaction.

[0596] 5. A surface-modified viral capsid according to any one of the foregoing embodiments, wherein the crosslinking agent reaction pair comprises cyclooctylene and azide.

[0597] 6. The surface-modified viral capsid according to embodiment 1, wherein the cyclooctyne is selected from dibenzylcyclooctyne (DIBO), dibenzo-azacyclooctyne (DBCO), and biarylazacyclooctynone (BARAC), or derivatives thereof. In some embodiments, the cyclooctyne is dibenzo-azacyclooctyne (DBCO). In some embodiments, the cyclooctyne is biarylazacyclooctynone (BARAC).

[0598] 7. The surface-modified viral capsid according to embodiment 1, wherein the cyclooctylene is DBCO.

[0599] 8. The surface-modified viral capsid according to any one of the foregoing embodiments, wherein the cross-linked portion comprises the following structure:

[0600] Where R 1 and R 2 This indicates the connection point with the connector.

[0601] 9. The surface-modified viral capsid according to embodiment 2, wherein the reaction is an anti-electron demand Diels-Alder (IEDDA) reaction.

[0602] 10. A surface-modified viral capsid according to any one of embodiments 1, 2 and 9, wherein the crosslinking agent reaction pair comprises trans-cyclooctene and tetrazine.

[0603] 11. A surface-modified viral capsid according to any one of embodiments 1, 2, 9, and 10, wherein the crosslinked portion comprises the following structure:

[0604] Where R 1 and R 2 This indicates the connection point with the connector.

[0605] 12. A surface-modified viral capsid according to any one of the foregoing embodiments, wherein the connector comprises one or more spacers.

[0606] 13. The surface-modified viral capsid according to embodiment 12, wherein the one or more spacers comprise 1 to 20 polyethylene glycol monomers. In some embodiments, the one or more spacers comprise 1 to 20 ethylene glycol monomers.

[0607] 14. The surface-modified viral capsid according to embodiment 13, wherein the one or more spacers comprise 2 to 8 polyethylene glycol monomers. In some embodiments, the one or more spacers comprise 2 to 8 ethylene glycol monomers.

[0608] 15. The surface-modified viral capsid according to embodiment 14, wherein at least one of the one or more spacers comprises four polyethylene glycol monomers. In some embodiments, at least one of the one or more spacers comprises four ethylene glycol monomers.

[0609] 16. The surface-modified viral capsid according to embodiment 15, comprising two spacers, each spacer comprising four polyethylene glycol monomers. In some embodiments, the surface-modified viral capsid comprises two spacers, each spacer comprising four polyethylene glycol monomers.

[0610] 17. A surface-modified viral capsid according to any one of the foregoing embodiments, wherein the ligand is a cell type-specific ligand.

[0611] 18. A surface-modified viral capsid according to any one of the foregoing embodiments, wherein the ligand is selected from cytokines, growth factors, lectins, toxins, single-chain antibodies, multi-chain antibodies or antigen-binding antibody fragments, peptides, and combinations thereof.

[0612] 19. In some embodiments, the ligand is known to bind to endogenous receptors in brain microvascular endothelial cells (BMECs).

[0613] 20. In some embodiments, the surface-modified viral capsid contains a receptor. In some embodiments, the receptor is a protease-activated receptor (PAR).

[0614] 21. In some embodiments, the receptor is endothelial protease activation receptor 1 (PAR1).

[0615] 22. In some embodiments, the ligand binds to PAR1.

[0616] 23. In some embodiments, the ligand is a PAR-1 activating peptide. In some embodiments, the ligand is thrombin or a derivative thereof.

[0617] 24. In some embodiments, the ligand is a peptide containing the sequence SFLLR (SEQ ID NO:2).

[0618] 25. In some embodiments, the ligand comprises a peptide having the sequence SFLLRNPNDKC (SEQ ID NO:3).

[0619] 26. In some embodiments, the ligand comprises wheat germ lectin (WGA).

[0620] 27. In some embodiments, the ligand is selected from WGA1, WGA2 and WGA3.

[0621] 28. In some embodiments, the ligand binds to PAR1, or the ligand is a WGA. In some embodiments, the ligand binds to PAR1. In some embodiments, the ligand is a WGA.

[0622] 29. In some embodiments, the surface-modified viral capsid contains a first ligand that binds to PAR1 and a second ligand that contains WGA.

[0623] 30. A surface-modified viral capsid according to any one of the foregoing embodiments, wherein the linker is covalently linked to a primary amino group of the primary sequence of the capsid protein.

[0624] 31. In some embodiments, the primary amino group is selected from N-terminal amino groups, lysine ε-amino groups, and arginine amino acid groups.

[0625] 32. In some embodiments, the primary amino group is the ε-amino group of a lysine amino acid residue.

[0626] 33. A surface-modified viral capsid according to any one of the foregoing embodiments, wherein the connector is covalently linked to the ligand via a primary amino group of the ligand.

[0627] 34. A surface-modified viral capsid according to any one of the foregoing embodiments, wherein the linker is covalently linked to the target ligand via non-natural amino acid residues of the primary sequence of the target ligand.

[0628] 35. The surface-modified viral capsid according to embodiment 23, wherein the non-natural amino acid residue comprises a member of the crosslinking agent reaction pair, the member participating in a reaction selected from: Cu(I)-catalyzed azido-acetylene cycloaddition (CuAAC), strain-promoted acetylene-azido cycloaddition (SPAAC), strain-promoted acetylene-nitroketone cycloaddition (SPANC), anti-electron-demanding Diels-Alder (IEEDD) reaction, Staudinger linkage, and [4+1] cycloaddition reaction.

[0629] 36. The surface-modified viral capsid according to embodiment 24, wherein the crosslinking agent reaction pair comprises an azide, cyclooctyne, cyclooctene, or 1,2,4,5-tetraazine moiety.

[0630] 37. A surface-modified viral capsid according to any one of embodiments 1 to 25, wherein the protein sequence of the viral capsid has been mutated to weaken or eliminate the binding of the capsid to mammalian cell polysaccharides or proteoglycans.

[0631] 38. A surface-modified viral capsid according to any one of embodiments 1 to 25, wherein the protein sequence of the viral capsid is not mutated to weaken or eliminate the binding of the capsid protein to mammalian cell polysaccharides or proteoglycans.

[0632] 39. The surface-modified viral capsid according to embodiment 1, characterized in that it has increased infectivity compared to an unmodified viral capsid having the same capsid protein sequence.

[0633] 40. A surface-modified viral capsid according to any one of the foregoing embodiments, wherein the viral capsid is selected from adenovirus capsids, adeno-associated virus capsids, retrovirus capsids, lentivirus capsids, herpes simplex virus capsids, and baculovirus capsids. In some embodiments, the viral capsid is an adenovirus capsid. In some embodiments, the viral capsid is an adeno-associated virus capsid. In some embodiments, the viral capsid is a retrovirus capsid. In some embodiments, the viral capsid is a herpes simplex virus capsid. In some embodiments, the viral capsid is a baculovirus capsid.

[0634] 41. The surface-modified viral capsid according to embodiment 1, wherein the viral capsid is an adeno-associated virus (AAV) capsid.

[0635] 42. The surface-modified viral capsid according to embodiment 1, wherein at least one of the arginine residues at positions 585 and 588 of VP1 or a similar position in VP2 or VP3 has been mutated.

[0636] 43. The surface-modified viral capsid according to embodiment 31, wherein the arginine residues at positions 585 and 588 of VP1 have been mutated to alanine residues.

[0637] 44. A surface-modified viral capsid according to embodiment 31 or 32, wherein the tropism is altered compared to an unmodified viral capsid.

[0638] 45. The surface-modified viral capsid according to any one of the foregoing embodiments, characterized in that the surface-modified viral capsid exhibits the ability to evade previously present neutralizing antibodies.

[0639] 46. ​​A viral capsid with surface modification according to Formula I: …(I) in: It is a viral capsid; Y and Y' are independent connecting parts; n and n' are independently 0 or integers from 1 to 50; Sp and Sp' are optional spacers, independently; L is a ligand; x is the ligand / capsid ratio, ranging from 1 to 500; and Q is selected from: or , Z is a 7- or 8-membered cyclic or heterocyclic structure.

[0640] 47. The surface-modified viral capsid according to embodiment 35, wherein x ranges from 100 to 200.

[0641] 48. The surface-modified viral capsid according to embodiment 35, wherein x ranges from 130 to 170.

[0642] 49. A viral capsid with surface modification according to Formula I-1: (I-1) in: It is a viral capsid; n and n' are independently selected from integers from 0 to 30; T is a ligand; and x is an integer from 1 to 500.

[0643] 50. The surface-modified viral capsid according to embodiment 38, wherein x ranges from 100 to 200.

[0644] 51. A surface-modified viral capsid according to any one of embodiments 1 to 38, wherein the ligand / capsid ratio (x) is in the range of 130-170.

[0645] 52. In some embodiments, the ligand is known to bind to endogenous receptors in brain microvascular endothelial cells (BMECs).

[0646] 53. In some embodiments, the receptor is a protease-activated receptor (PAR).

[0647] 54. In some embodiments, the receptor is endothelial protease activation receptor 1 (PAR1).

[0648] 55. In some embodiments, the ligand binds to PAR1.

[0649] 56. In some embodiments, the ligand is a PAR-1 activating peptide. In some embodiments, the ligand is thrombin or a derivative thereof.

[0650] 57. In some embodiments, the ligand is a peptide containing the sequence SFLLR (SEQ ID NO:2).

[0651] 58. In some embodiments, the ligand comprises a peptide having the sequence SFLLRNPNDKC (SEQ ID NO:3).

[0652] 59. In some embodiments, the ligand comprises wheat germ lectin (WGA).

[0653] 60. In some embodiments, the ligand is selected from WGA1, WGA2, and WGA3. In some embodiments, the ligand is WGA1. In some embodiments, the ligand is WGA2. In some embodiments, the ligand is WGA3.

[0654] 61. In some embodiments, the ligand binds to PAR1, or the ligand comprises WGA.

[0655] 62. In some embodiments, the capsid comprises a first ligand that binds to PAR1 and a second ligand that comprises WGA.

[0656] 63. A pharmaceutical composition comprising at least one recombinant viral particle, said recombinant viral particle comprising a surface-modified viral capsid and recombinant polynucleotide cargo according to the present disclosure, and further comprising a pharmaceutically acceptable carrier, diluent, solubilizer, filler, preservative, excipient, or combination thereof.

[0657] 64. A method of treating a patient suffering from a disease that can be treated by intracellular delivery of a recombinant polynucleotide cargo, the method comprising: Administer a therapeutically effective amount of the pharmaceutical composition according to embodiment 41. The recombinant polynucleotide cargo is capable of treating the disease.

[0658] 65. A composition for treating a disease that can be treated by intracellular delivery of a recombinant polynucleotide cargo, the composition comprising: The therapeutically effective amount is the pharmaceutical composition according to embodiment 41. The recombinant polynucleotide cargo is capable of treating the disease.

[0659] 66. Use of the surface-modified viral capsid according to any one of embodiments 1 to 39 in the preparation of a medicament for treating diseases that can be treated by intracellular delivery of recombinant polynucleotide cargoes.

[0660] 67. A surface-functionalized viral capsid comprising a first member of a crosslinking agent reaction pair and optionally one or more spacers, wherein the surface-functionalized viral capsid is adapted to react with a functionalized ligand comprising a second member of the crosslinking agent reaction pair and optionally one or more spacers, wherein the member of the crosslinking agent reaction pair participates in a reaction selected from: Cu(I)-catalyzed azido-acetylene cycloaddition (CuAAC), strain-promoted acetylene-azido cycloaddition (SPAAC), strain-promoted acetylene-nitroketone cycloaddition (SPANC), anti-electron-demanding Diels-Alder (IEEDD) reaction, and Staudinger linkage and [4+1] cycloaddition reactions.

[0661] 68. A method for preparing a surface-modified viral capsid, the method comprising the following steps: A surface-functionalized viral capsid is obtained by reacting viral capsid proteins with a capsid reactive linker, the linker comprising a first member of a crosslinking agent reactant pair and optionally one or more spacers. The surface-functionalized viral capsid is conjugated with a functionalized ligand comprising a second member of the crosslinking agent reactant pair and optionally one or more spacers; The first and second members of the crosslinking agent reaction pair react to form the crosslinked portion Q.

[0662] 69. The method according to embodiment 46, wherein the surface-modified viral capsid is according to any one of embodiments 1 to 39.

[0663] 70. A recombinant viral particle comprising a surface-modified capsid according to any one of embodiments 1 to 39.

[0664] 71. The recombinant viral particle according to embodiment 48, wherein the recombinant viral particle is rAAV.

[0665] 5.11 Further Implementation Scheme

[0666] 72. In one aspect, an engineered adeno-associated virus (AAV) capsid protein is provided, wherein the capsid protein comprises a functional sequence motif having the sequence: X1X2X3X4X5X6 (SEQ ID NO:5); wherein X1, X2, X3, X4, X5 and X6 are each independently selected from any amino acid residues, and wherein X4, X5 and X6 are optional; wherein the functional sequence motif is a substrate of a sorting enzyme transaminasase.

[0667] 73. In some embodiments, the enzyme is selected from sorting enzyme A, sorting enzyme B, archaea sorting enzyme A, external sorting enzyme A, rhomboid sorting enzyme, and PorU. In some embodiments, the enzyme is sorting enzyme A. In some embodiments, the enzyme is sorting enzyme B. In some embodiments, the enzyme is archaea sorting enzyme A. In some embodiments, the enzyme is external sorting enzyme A. In some embodiments, the enzyme is rhomboid sorting enzyme. In some embodiments, the enzyme is PorU.

[0668] 74. In some embodiments, the enzyme is sorting enzyme A.

[0669] 75. In some embodiments, the engineered rAAV capsid protein is a seven-mutant (SrtA7M) or a five-mutant (SrtA5M). In some embodiments, the engineered rAAV capsid protein is a seven-mutant (SrtA7M). In some embodiments, the engineered rAAV capsid protein is a five-mutant (SrtA5M).

[0670] 76. In some embodiments, the enzyme is a seven-mutant Staphylococcus aureus sorting enzyme A (SrtA7m).

[0671] 77. In some embodiments, the engineered rAAV capsid protein comprises a functional sequence motif located within a surface-accessible variable region (VR) of the primary capsid sequence.

[0672] 78. In some embodiments, the functional sequence motif has the sequence X1X2X3 (SEQ ID NO:6); wherein X1, X2 and X3 are each independently selected from any amino acid residue.

[0673] 79. In some embodiments, the functional sequence motif has the sequence X1X2X3 (SEQ ID NO:7), wherein two or more of X1, X2 and X3 are selected from: X1 is valine (V); X2 is proline (P); and X3 is proline (P).

[0674] 80. In some embodiments, X1, X2 and X3 are selected from: X1 is proline (P); X2 is selected from glycine (G) and glutamic acid (E); and X3 is selected from proline (P) and phenylalanine (F).

[0675] 81. In some embodiments, the functional sequence motif has a sequence selected from: PEF (SEQ ID NO:50), PGF (SEQ ID NO:51), or PEP (SEQ ID NO:52). In some embodiments, the functional sequence motif has the sequence PEF (SEQ ID NO:50). In some embodiments, the functional sequence motif has the sequence PGF (SEQ ID NO:51). In some embodiments, the functional sequence motif has the sequence PEP (SEQ ID NO:52).

[0676] 82. In some embodiments, the functional sequence motif has the sequence X1X2X3X4 (SEQ ID NO:53); wherein X1, X2, X3 and X4 are each independently selected from any amino acid residue.

[0677] 83. In some embodiments, the functional sequence motif has the sequence X1X2X3X4X5 (SEQ ID NO:54); wherein X1, X2, X3, X4, and X5 are each independently selected from any amino acid residue.

[0678] 84. In some embodiments, the functional sequence motif has the sequence X1X2X3X4X5 (SEQ ID NO:54), wherein: X1 is independently selected from lysine (L), valine (V), (I), and asparagine (N); X2 is independently selected from proline (P); X3 is independently selected from any amino acid residue; X4 is independently selected from threonine (T); and X5 is independently selected from alanine (A), glycine (G), and asparagine (N).

[0679] 85. In some embodiments, two or more of X1, X2, X3, X4 and X5 are selected from: X1 is independently selected from lysine (L); X2 is independently selected from proline (P); X3 is independently selected from any amino acid residue; X4 is independently selected from threonine (T); and X5 is independently selected from alanine (A), glycine (G) and asparagine (N).

[0680] 86. In some embodiments, three or more of X1, X2, X3, X4 and X5 are selected from: X1 is independently selected from lysine (L), valine (V), (I) and asparagine (N); X2 is independently selected from proline (P); X3 is independently selected from any amino acid residue; X4 is independently selected from threonine (T); and X5 is independently selected from alanine (A) and glycine (G).

[0681] 87. In some embodiments, X1 is lysine (L); X2 is proline (P); X3 is glutamic acid (E), serine (S) or alanine (A); X4 is threonine (T); and X5 is alanine (A) or glycine (G).

[0682] 88. In some embodiments, the functional sequence motif comprises a sequence selected from: LPX3TG (SEQ ID NO:48), LPX3TA (SEQ ID NO:55), LPETG (SEQ ID NO:12), and LPETA (SEQ ID NO:56). In some embodiments, the functional sequence motif comprises a sequence of LPX3TG (SEQ ID NO:48). In some embodiments, the functional sequence motif comprises a sequence of LPX3TA (SEQ ID NO:55). In some embodiments, the functional sequence motif comprises a sequence of LPETG (SEQ ID NO:12). In some embodiments, the functional sequence motif comprises a sequence of LPETA (SEQ ID NO:56).

[0683] 89. In some embodiments, the functional sequence motif has the sequence X1X2X3X4X5X6 (SEQ ID NO:5); wherein X1, X2, X3, X4, X5 and X6 are each independently selected from any amino acid residue.

[0684] 90. In some embodiments, the functional sequence motif has X1X2X3X4X5X6 (SEQ ID NO:5); wherein two or more of X1, X2, X3, X4, X5 and X6 are: X1 independently is asparagine (N); X4 independently is threonine (T); X5 independently is asparagine (N); and X6 independently is proline (P).

[0685] 91. In some embodiments, the functional sequence motif has X1X2X3X4X5X6 (SEQ ID NO:5); wherein at least three of X1, X2, X3, X4, X5 and X6 are: X1 independently is asparagine (N); X4 independently is threonine (T); X5 independently is asparagine (N); and X6 independently is proline (P).

[0686] 92. In some embodiments, the functional sequence motif comprises a sequence selected from: NX2X3TNX6 (SEQ ID NO: 57) and VPX3X4X5P (SEQ ID NO: 58). In some embodiments, the functional sequence motif comprises the sequence NX2X3TNX6 (SEQ ID NO: 57). In some embodiments, the functional sequence motif comprises the sequence VPX3X4X5P (SEQ ID NO: 58).

[0687] 93. In some embodiments, the engineered rAAV capsid protein comprises one or more sequence alterations selected from insertion, deletion, or substitution, compared to the wild-type protein, said alterations being used to introduce the functional sequence motif. In some embodiments, the sequence alteration is an insertion. In some embodiments, the sequence alteration is a deletion. In some embodiments, the sequence alteration is a substitution.

[0688] 94. In some embodiments, the engineered rAAV capsid protein contains at least one insertion mutation, wherein the insertion forms the functional sequence motif.

[0689] 95. In some embodiments, the insertion mutation is the insertion of a foreign peptide selected from LPET (SEQ ID NO:49) and LPETG (SEQ ID NO:12). In some embodiments, the insertion mutation is LPET (SEQ ID NO:49). In some embodiments, the insertion mutation is LPETG (SEQ ID NO:12).

[0690] 96. In some embodiments, the rAAV capsid protein is selected from one or more of VP1, VP2, and VP3. In some embodiments, the rAAV capsid protein is VP1. In some embodiments, the rAAV capsid protein is VP2. In some embodiments, the rAAV capsid protein is VP3.

[0691] 97. In some embodiments, the engineered rAAV capsid protein comprises a functional sequence, wherein the functional sequence motif is located within a surface loop region selected from the following: AB, BC, CD, DE, EF, FG, GH, and HI loops. In some embodiments, the functional sequence motif is located within the AB surface loop region. In some embodiments, the functional sequence motif is located within the BC surface loop region. In some embodiments, the functional sequence motif is located within the CD surface loop region. In some embodiments, the functional sequence motif is located within the DE surface loop region. In some embodiments, the functional sequence motif is located within the EF surface loop region. In some embodiments, the functional sequence motif is located within the FG surface loop region. In some embodiments, the functional sequence motif is located within the GH surface loop region. In some embodiments, the functional sequence motif is located within the HI surface loop region.

[0692] 98. In some embodiments, the functional sequence motif is located within the GH surface ring region.

[0693] 99. In some embodiments, the functional sequence motif is located within one or more variable regions selected from VR1, VR2, VR3, VR4, VR5, VR6, VR7, VR8, and VR9. In some embodiments, the functional sequence motif is located within VR1. In some embodiments, the functional sequence motif is located within VR2. In some embodiments, the functional sequence motif is located within VR3. In some embodiments, the functional sequence motif is located within VR4. In some embodiments, the functional sequence motif is located within VR5. In some embodiments, the functional sequence motif is located within VR6. In some embodiments, the functional sequence motif is located within VR7. In some embodiments, the functional sequence motif is located within VR8. In some embodiments, the functional sequence motif is located within VR9.

[0694] 100. In some embodiments, the engineered rAAV capsid protein comprises a functional sequence motif located within variable region IV or VIII.

[0695] 101. In some embodiments, the engineered rAAV capsid protein comprises a functional sequence motif located within variable region VIII.

[0696] 102. In some embodiments, the engineered rAAV capsid protein comprises functional sequence motifs located within variable regions IV and VIII, wherein each functional sequence motif is independently selected.

[0697] 103. In some implementations, each functional sequence motif is identical.

[0698] 104. In some embodiments, the rAAV capsid protein is selected from serotypes AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9. In some embodiments, the rAAV capsid protein is AAV1. In some embodiments, the rAAV capsid protein is AAV2. In some embodiments, the rAAV capsid protein is AAV4. In some embodiments, the rAAV capsid protein is AAV5. In some embodiments, the rAAV capsid protein is AAV8. In some embodiments, the rAAV capsid protein is AAV9.

[0699] 105. In some embodiments, the functional sequence motif begins after: S453 in AAV1 capsid serotype VR4; S452 in AAV2 capsid serotype VR4 or R585 in VR8; S576 in AAV5 capsid serotype VR8; S453 in AAV6 capsid serotype VR4 or S587 in VR8; P454 in AAV7 capsid serotype VR4 or A587 in VR8; T454 in AAV8 capsid serotype VR4 or Q589 in VR8; and S454 in AAV9 capsid serotype VR8.

[0700] 106. In some embodiments, the functional sequence motif begins after S452 in AAV2 capsid serotype VR4 or R585 in VR8. In some embodiments, the functional sequence motif begins after S452 in VR4. In some embodiments, the functional sequence motif begins after R585 in VR8.

[0701] 107. In some embodiments, LPET (SEQ ID NO:49) is inserted after S452 to form the functional sequence motif.

[0702] 108. In some embodiments, the engineered rAAV capsid protein does not contain additional mutations compared to the wild-type capsid protein.

[0703] 109. On the one hand, a polynucleotide encoding the engineered rAAV capsid protein disclosed herein is provided.

[0704] 110. On the one hand, a vector comprising the polynucleotides disclosed herein is provided.

[0705] 111. In some embodiments, the vector comprises a promoter operatively linked to the polynucleotide.

[0706] 112. On the one hand, a host cell is provided that contains the engineered rAAV capsid protein, polynucleotide, or vector disclosed herein.

[0707] 113. In one aspect, a recombinant rAAV viral particle (rAAV) is provided, which contains the engineered rAAV capsid protein of this disclosure.

[0708] 114. In some embodiments, the rAAV viral particles further comprise exogenous cargo polynucleotides.

[0709] 115. In some embodiments, the exogenous cargo polynucleotide contains a template for homology-directed repair.

[0710] 116. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic tRNA, miRNA, gene-editing guide RNA, or RNA-editing guide RNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic tRNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a miRNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a gene-editing guide RNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding an RNA-editing guide RNA.

[0711] 117. In some embodiments, the exogenous polynucleotide comprises an expressible polynucleotide encoding a therapeutic protein.

[0712] 118. In some embodiments, the rAAV viral particle comprises a functional sequence motif, wherein the functional sequence motif weakens or eliminates the binding of the viral particle to mammalian cytopolysaccharides or proteoglycans compared to a reference viral particle having the same capsid protein sequence but lacking the functional sequence motif. In some embodiments, the functional sequence motif weakens or eliminates the binding of the viral particle to mammalian cytopolysaccharides compared to a reference viral particle having the same capsid protein sequence but lacking the functional sequence motif. In some embodiments, the functional sequence motif weakens or eliminates the binding of the viral particle to mammalian cytopolysaccharides compared to a reference viral particle having the same capsid protein sequence but lacking the functional sequence motif.

[0713] 119. In some embodiments, the rAAV viral particle comprises at least one cross-linking reactive moiety covalently linked to an amino acid residue in the engineered rAAV capsid protein, wherein the amino acid residue is not within the functional sequence motif, and an exogenous cargo polynucleotide.

[0714] 120. In some embodiments, the amino acid residue is lysine.

[0715] 121. In some embodiments, the amino acid residues in the viral capsid protein are N-terminal amines.

[0716] 122. In some embodiments, the amino acid residues in the viral capsid protein are non-natural amino acid residues.

[0717] 123. In one aspect, a surface-functionalized rAAV viral particle is provided, wherein the viral particle comprises at least one engineered rAAV capsid protein of the present disclosure and an exogenous cargo polynucleotide, wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form (i) an N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and at least one cross-linking reactive portion covalently linked to a C-terminal amino acid of the N-terminal cleavage fragment.

[0718] 124. In some embodiments, the covalent connection comprises an amide bond.

[0719] 125. In some embodiments, the C-terminal amino acid of the N-terminal cleavage fragment is threonine.

[0720] 126. In one aspect, a surface-functionalized rAAV viral particle is provided, wherein the viral particle comprises at least one engineered rAAV capsid protein according to the present disclosure and an exogenous cargo polynucleotide, wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form (i) an N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and wherein at least one cross-linking reactive portion is covalently linked to a C-terminal amino acid of the N-terminal cleavage fragment; and optionally at least one cross-linking reactive portion is covalently linked to an amino acid residue in the engineered rAAV capsid protein, wherein the amino acid residue is not within the functional sequence motif.

[0721] 127. In some embodiments, the surface-functionalized rAAV virus particle comprises at least one crosslinking agent portion, wherein each crosslinking agent reactive portion is independently selected from crosslinking agent reactive portions participating in reactions selected from: Cu(I)-catalyzed azido-acetylene cycloaddition (CuAAC), strain-promoted acetylene-azido cycloaddition (SPAAC), strain-promoted acetylene-nitroketone cycloaddition (SPANC), anti-electron-demanding Diels-Alder (IEDDA) reactions, and Staudinger linkage and [4+1] cycloaddition reactions. In some embodiments, the crosslinking agent reactive portion participates in the Cu(I)-catalyzed azido-acetylene cycloaddition (CuAAC) reaction. In some embodiments, the crosslinking agent reactive portion participates in the strain-promoted acetylene-nitroketone cycloaddition (SPANC) reaction. In some embodiments, the crosslinking agent reactive portion participates in the anti-electron-demanding Diels-Alder (IEDDA) reaction. In some implementations, the reactive portion of the crosslinker participates in Staudinger linkage and [4+1] cycloaddition reactions.

[0722] 128. In some embodiments, the reactive portion of the crosslinking agent comprises at least one of an eight-membered ring and a triazole ring.

[0723] 129. In some embodiments, the reaction is a strain-promoted acetylene-azidocycloaddition (SPAAC) reaction.

[0724] 130. In some embodiments, the reactive portion of the crosslinking agent is selected from cyclooctyne and azides.

[0725] 131. In some embodiments, the cyclooctyne is selected from dibenzylcyclooctyne (DIBO), dibenzozacyclooctyne (DBCO), and biarylazacyclooctynone (BARAC), or derivatives thereof. In some embodiments, the cyclooctyne is dibenzylcyclooctyne (DIBO). In some embodiments, the cyclooctyne is dibenzozacyclooctyne (DBCO). In some embodiments, the cyclooctyne is biarylazacyclooctynone (BARAC).

[0726] 132. In some embodiments, the cyclooctylene is DBCO.

[0727] 133. In some embodiments, the reaction is an anti-electron demand Diels-Alder (IEDDA) reaction.

[0728] 134. In some embodiments, the reactive portion of the crosslinking agent is selected from trans-cyclooctene and tetrazine.

[0729] 135. In one aspect, a surface-modified rAAV viral particle is provided, wherein the viral particle comprises at least one engineered rAAV capsid protein according to the present disclosure and an exogenous cargo polynucleotide, and wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form (i) an N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and wherein the viral particle comprises a crosslinked portion of the C-terminal amino acid of the N-terminal cleavage fragment covalently linked to a targeting ligand.

[0730] 136. In some embodiments, the covalent connection comprises an amide bond.

[0731] 137. In some embodiments, the C-terminal amino acid of the N-terminal cleavage fragment is threonine.

[0732] 138. In some embodiments, the crosslinking portion comprises a product selected from the following reactions: CuAAC reaction, SPAAC reaction, SPANC reaction, IEDDA reaction, Staudinger linkage, and [4+1] cycloaddition reaction. In some embodiments, the crosslinking portion comprises a product of the CuAAC reaction. In some embodiments, the crosslinking portion comprises a product of the SPAAC reaction. In some embodiments, the crosslinking portion comprises a product of the SPANC reaction. In some embodiments, the crosslinking portion comprises a product of the IEDDA reaction. In some embodiments, the crosslinking portion comprises a Staudinger-linked product. In some embodiments, the crosslinking portion comprises a product of the [4+1] cycloaddition reaction.

[0733] 139. In some embodiments, the reaction is selected from SPAAC, SPANC, and IEDDA reactions.

[0734] 140. In some embodiments, the crosslinked portion comprises a 7- or 8-membered carbon ring containing 0 to 3 heteroatoms selected from O or N.

[0735] 141. In some embodiments, the crosslinking portion comprises or .

[0736] 142. In some embodiments, the functional sequence motif is selected from one of SEQ ID NO:1-58.

[0737] 143. In some embodiments, the functional sequence motif is LPETG (SEQ ID NO:12).

[0738] 144. In one aspect, a surface-modified rAAV viral particle is provided, wherein the viral particle comprises an exogenous cargo polynucleotide and at least one engineered rAAV capsid protein, the capsid protein comprising at least one functional sequence motif LPX3TG (SEQ ID NO:48), wherein X3 is selected from any amino acid residue; wherein the functional sequence motif is located within variable regions 4, 8, or 4 and 8, and (a) wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form an N-terminal cleavage fragment and a C-terminal cleavage fragment; wherein the viral particle comprises a cross-linked portion of the T of the N-terminal cleavage fragment covalently linked to a targeting ligand, wherein the covalent link comprises an amide bond; and optionally (b) wherein the viral particle comprises a cross-linked portion of the lysine residue or the N-terminal amine of the capsid protein covalently linked to a targeting ligand.

[0739] 145. In some embodiments, the crosslinked portion comprises or .

[0740] 146. In some embodiments, the surface-modified rAAV viral particles contain a targeting ligand, wherein the targeting ligand is a cell type-specific ligand.

[0741] 147. In some embodiments, the targeting ligand is selected from enzymes, peptide mimics, ligands of endogenous receptors, lipid-binding proteins, toxins, phage peptidases, nucleotide- and RNA / DNA-based ligands, aptamers, viral vectors, DNA or RNA editing proteins, nanoparticles, lipids, small molecules, and combinations thereof. In some embodiments, the targeting ligand is an enzyme. In some embodiments, the targeting ligand is a peptide mimic. In some embodiments, the targeting ligand is a ligand of an endogenous receptor. In some embodiments, the targeting ligand is a lipid-binding protein. In some embodiments, the targeting ligand is a toxin. In some embodiments, the targeting ligand is a phage peptidase. In some embodiments, the targeting ligand is a nucleotide- and RNA / DNA-based ligand. In some embodiments, the targeting ligand is an aptamer. In some embodiments, the targeting ligand is a viral vector. In some embodiments, the targeting ligand is a DNA or RNA editing protein. In some embodiments, the targeting ligand is a nanoparticle. In some embodiments, the targeting ligand is a lipid. In some embodiments, the targeting ligand is a small molecule.

[0742] 148. In some embodiments, the targeting ligand binds to receptor-mediated transporters expressed on endothelial cells that form the blood-brain barrier (BBB).

[0743] 149. In some embodiments, the receptor is selected from insulin receptors, transferrin receptors, choline transporters, amino acid transporters (e.g., large neutral amino acid carriers), and LDL receptors. In some embodiments, the receptor is an insulin receptor. In some embodiments, the receptor is a transferrin receptor. In some embodiments, the receptor is a choline transporter. In some embodiments, the receptor is an amino acid transporter. In some embodiments, the receptor is an LDL receptor.

[0744] 150. In some embodiments, the ligand is known to bind to endogenous receptors in brain microvascular endothelial cells (BMECs).

[0745] 151. In some embodiments, the receptor is a protease-activated receptor (PAR).

[0746] 152. In some embodiments, the receptor is endothelial protease activation receptor 1 (PAR1).

[0747] 153. In some embodiments, the ligand binds to PAR1.

[0748] 154. In some embodiments, the ligand is a PAR-1 activating peptide. In some embodiments, the ligand is thrombin or a derivative thereof.

[0749] 155. In some embodiments, the ligand is a peptide comprising the sequence SFLLR (SEQ ID NO:2).

[0750] 156. In some embodiments, the ligand comprises a peptide having the sequence SFLLRNPNDKC (SEQ ID NO:3).

[0751] 157. In some embodiments, the ligand comprises wheat germ lectin (WGA).

[0752] 158. In some embodiments, the ligand is selected from WGA1, WGA2, and WGA3. In some embodiments, the ligand is WGA1. In some embodiments, the ligand is WGA2. In some embodiments, the ligand is WGA3.

[0753] 159. In some embodiments, the ligand binds to PAR1 or the ligand comprises WGA. In some embodiments, the ligand binds to PAR1. In some embodiments, the ligand comprises WGA.

[0754] 160. In some embodiments, the capsid protein comprises a first ligand that binds to PAR1 and a second ligand comprising WGA.

[0755] 161. In some embodiments, the surface-modified rAAV viral particles comprise a functional sequence motif, wherein the functional sequence motif weakens or eliminates the binding of the viral particles to mammalian cytopolysaccharides or proteoglycans compared to reference viral particles having the same capsid protein sequence but lacking the functional sequence motif. In some embodiments, the functional sequence motif weakens or eliminates the binding of the viral particles to mammalian cytopolysaccharides compared to reference viral particles having the same capsid protein sequence but lacking the functional sequence motif. In some embodiments, the functional sequence motif weakens or eliminates the binding of the viral particles to mammalian cytopolysaccharides compared to reference viral particles having the same capsid protein sequence but lacking the functional sequence motif.

[0756] 162. In some embodiments, the surface-modified rAAV viral particles are characterized by increased infectivity compared to reference viral particles having the same capsid protein sequence but lacking the said functional sequence motif.

[0757] 163. In some embodiments, the increased infectivity is relative to at least one cell or tissue type.

[0758] 164. In some embodiments, the cell or tissue type is selected from the heart, nerves, central and peripheral nervous systems, hematopoietic system, liver, muscle, connective tissue, lungs, kidneys, and retina. In some embodiments, the cell or tissue type is the heart. In some embodiments, the cell or tissue type is nerves. In some embodiments, the cell or tissue type is the central and peripheral nervous system. In some embodiments, the cell or tissue type is the hematopoietic system. In some embodiments, the cell or tissue type is the liver. In some embodiments, the cell or tissue type is connective tissue. In some embodiments, the cell or tissue type is the lungs. In some embodiments, the cell or tissue type is the kidneys and retina.

[0759] 165. In some embodiments, the exogenous cargo polynucleotide contains a template for homology-directed repair.

[0760] 166. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic tRNA, miRNA, gene-editing guide RNA, or RNA-editing guide RNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic tRNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a miRNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a gene-editing guide RNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding an RNA-editing guide RNA.

[0761] 167. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic protein.

[0762] 168. In one aspect, a pharmaceutical composition is provided comprising a plurality of surface-modified rAAV viral particles according to the present disclosure, said composition further comprising a pharmaceutically acceptable carrier, diluent, solubilizer, filler, preservative, excipient, or combination thereof.

[0763] 169. In one aspect, a method is provided for treating a patient suffering from a disease that can be treated by intracellular delivery of a recombinant polynucleotide cargo, the method comprising administering a therapeutically effective amount of a pharmaceutical composition according to the present disclosure, wherein the recombinant polynucleotide cargo is capable of treating the disease.

[0764] 170. In one aspect, a composition is provided for treating a disease that can be treated by intracellular delivery of a recombinant polynucleotide cargo, the composition comprising a therapeutically effective amount of a pharmaceutical composition according to the present disclosure, wherein the recombinant polynucleotide cargo is capable of treating the disease.

[0765] 171. In one aspect, the use of rAAV viral particles according to this disclosure in the preparation of medicaments for treating diseases that can be treated by intracellular delivery of recombinant polynucleotide cargoes is provided.

[0766] 172. In one aspect, an engineered viral capsid is provided, comprising: a first targeting ligand conjugated to a first site of a surface-modified viral capsid; and a second targeting ligand conjugated to a second site of the surface-modified viral capsid, wherein the first targeting ligand and the second targeting ligand are different.

[0767] 173. In some embodiments, the first targeting ligand promotes crossing the blood-brain barrier (BBB), and the second targeting ligand is a cell-targeting ligand.

[0768] 174. In some embodiments, the first targeting ligand is a blood-brain barrier (BBB) ​​transpeptide or a lectin ligand.

[0769] 175. In some embodiments, one of the first and second targeting ligands is covalently linked (e.g., conjugated) to a lysine or cysteine ​​residue of the viral capsid protein via a linker.

[0770] 176. In some embodiments, another of the first and second targeting ligands is covalently linked (e.g., conjugated) to a recognition sequence motif residue via a linker.

[0771] 177. In some embodiments, the viral capsid is an adeno-associated virus (AAV) capsid.

[0772] 178. In some embodiments, the first or second targeting ligand is a peptide, such as a PAR-1 agonist.

[0773] 179. In some embodiments, the first or second targeting ligand is a peptide or a portion thereof as described in SEQ ID NO:3.

[0774] 180. In some embodiments, the first or second targeting ligand is nerve growth factor.

[0775] 181. In some embodiments, the first or second targeting ligand binds to brain microvascular endothelial cells (BMECs).

[0776] 182. In some embodiments, the first or second targeting ligand binds to the neuronal membrane, such as wheat germ lectin (WGA).

[0777] 183. In some embodiments, the engineered viral capsid conjugated to the first and second targeting ligands further comprises: (a) a modified viral capsid containing a plurality of recognition sequence motif residues; (b) a first targeting ligand conjugated to the modified viral capsid via a first linker covalently linked to the recognition sequence motif residues, comprising: b1. residues covalently linked to amide-C(O)NH-; b2. a first spacer (Sp)n; b3. a divalent fused polycyclic 11- to 20-membered heterocycle, phosphine oxide derivative, or 4H-pyrazole-4-imine derivative; b4. a second b5. A first targeting ligand; and c6. A second targeting ligand, which is conjugated to the modified viral capsid via a second linker covalently linked to a primary amino or thiol group of the primary sequence of the modified viral capsid, comprising: c1. an amide-NHC(O)-; c2. a first spacer (Sp)n; c3. a divalent fused polycyclic 11 to 20-membered heterocycle, a phosphine oxide derivative, or a 4H-pyrazole-4-imine derivative; c4. a second spacer (Sp')n'; and c5. a second targeting ligand; wherein n and n' are each independently an integer from 0 to 20.

[0778] 184. In some embodiments, the recognition sequence motif residue is selected from any peptide sequence or a portion thereof shown in SEQ ID NO:1-58 or 61-75.

[0779] 185. In some embodiments, the recognition sequence motif residues comprise at least four consecutive amino acids selected from the peptide sequences shown in SEQ ID NO:8-33, 35-44, 62-64 and 65.

[0780] 186. In some embodiments, the recognition sequence motif residues comprise at least six consecutive amino acids as shown in SEQ ID NO:61 or SEQ ID NO:66.

[0781] 187. In some embodiments, the engineered viral capsid conjugated to the first and second targeting ligands further comprises: (a) a modified viral capsid; (b) a first targeting ligand conjugated to the modified viral capsid via a first linker covalently linked to a primary amino or thiol group of the primary sequence of the modified viral capsid, comprising: b1. a residue covalently linked to an amide-C(O)NH-; b2. a first spacer (Sp)n; b3. a divalent fused polycyclic 11- to 20-membered heterocycle, a phosphine oxide derivative, or a 4H-pyrazole-4-imine derivative; b4. a second spacer. b5. A first targeting ligand; and c6. A second targeting ligand, which is conjugated to the modified viral capsid via a second linker covalently linked to a primary amino or thiol group of the primary sequence of the modified viral capsid, comprising: c1. an amide-NHC(O)-; c2. a first spacer (Sp)n; c3. a divalent fused polycyclic 11 to 20-membered heterocycle, a phosphine oxide derivative, or a 4H-pyrazole-4-imine derivative; c4. a second spacer (Sp')n'; and c5. a second targeting ligand; wherein n and n' are each independently an integer from 0 to 20.

[0782] 188. In some embodiments, the primary amino group is selected from N-terminal amino groups, lysine ε-amino groups, and arginine amino acid groups.

[0783] 189. In some embodiments, the thiol group is a cysteine ​​amino acid group.

[0784] 190. In some embodiments, each first spacer is selected from -(CH2)n-, -(CH2CH2O)n-, -C(O)-, -C(O)NH-, 5-membered heterocycles, and combinations thereof, wherein n is 0 to 20, for example n is 1 to 20, for example n is 1 to 8, for example n is 2 to 8, for example n is 1 to 4, for example n is 2 to 4, for example n is 4.

[0785] 191. In some embodiments, each second spacer is selected from -(CH2)n'-, -(CH2CH2O)n'-, -C(O)-, -C(O)NH-, 5-membered heterocycles and combinations thereof, wherein n' is 0 to 20, for example n' is 1 to 20, for example n' is 1 to 8, for example n' is 2 to 8, for example n' is 1 to 4, for example n' is 2 to 4, for example n' is 4.

[0786] 192. In some embodiments, the divalent fused polycyclic 11- to 20-membered heterocyclic ring is: or , Z is an optionally substituted 7- to 9-membered carbon ring or heterocycle.

[0787] 193. In some embodiments, Z is substituted with one or more arylene groups.

[0788] 194. In some embodiments, the phosphine oxide derivative is: .

[0789] 195. In some embodiments, the 4H-pyrazole-4-imine derivative is: .

[0790] 196. In one aspect, a method for treating a disease or symptom is provided, the method comprising applying a therapeutically effective amount of an engineered viral capsid according to the present disclosure.

[0791] 197. In some embodiments, the disease or condition is a central nervous system disease or condition, such as a neurodegenerative disease, more specifically aromatic L-amino acid decarboxylase (AADC) deficiency, Canavan disease, Parkinson's disease, Alzheimer's disease, and giant axonal neuropathy. In some embodiments, the disease or condition is aromatic L-amino acid decarboxylase (AADC) deficiency. In some embodiments, the disease or condition is Canavan disease. In some embodiments, the disease or condition is Parkinson's disease. In some embodiments, the disease or condition is Alzheimer's disease. In some embodiments, the disease or condition is giant axonal neuropathy.

[0792] 5.12 Example

[0793] Surface modification of viral capsid

[0794] Recombinant adeno-associated virus (rAAV) has become the preferred in vivo gene delivery vector for both basic research and clinical applications. Recombinant AAV vectors do not integrate site-specifically into the host genome, and their moderate immunogenicity makes them one of the safest gene therapy strategies (Naso MF, Tomkowicz B., Perry WL, 3rd, Str...

Claims

1. An engineered viral capsid comprising: The first targeting ligand conjugates to the first site of the surface-modified viral capsid; and A second targeting ligand conjugated to a second site on the surface-modified viral capsid; The first and second targeting ligands are different.

2. The engineered viral capsid according to claim 1, wherein: The first targeting ligand promotes crossing the blood-brain barrier (BBB), and The second targeting ligand is a cell-targeting ligand.

3. The engineered viral capsid according to claim 1 or 2, wherein the first targeting ligand is a blood-brain barrier (BBB) ​​transpeptide or a lectin ligand.

4. The engineered viral capsid according to any one of claims 1 to 3, wherein: One of the first and second targeting ligands is covalently linked (e.g., conjugated) to a lysine or cysteine ​​residue of the viral capsid protein via a linker.

5. The engineered viral capsid according to any one of claims 1 to 3, wherein: The other of the first and second targeting ligands is covalently linked (e.g., conjugated) to a recognition sequence motif residue via a linker.

6. The engineered viral capsid according to claim 1 or 5, wherein the viral capsid is an adeno-associated virus (AAV) capsid.

7. The engineered viral capsid according to any one of claims 1 to 6, wherein the first or second targeting ligand is a peptide, such as a PAR-1 agonist.

8. The engineered viral capsid according to any one of claims 1 to 7, wherein the first or second targeting ligand is a peptide or a portion thereof as shown in SEQ ID NO:

3.

9. The engineered viral capsid according to any one of claims 1 to 8, wherein the first or second targeting ligand is nerve growth factor.

10. The engineered viral capsid according to any one of claims 1 to 9, wherein the first or second targeting ligand binds to brain microvascular endothelial cells (BMECs).

11. The engineered viral capsid according to any one of claims 1 to 10, wherein the first or second targeting ligand binds to the neuronal membrane, such as wheat germ agglutinin (WGA).

12. The engineered viral capsid conjugated to the first and second targeting ligands according to any one of claims 1 to 11, further comprising: (a) A modified viral capsid containing multiple recognition sequence motif residues; (b) A first targeting ligand conjugated to the modified viral capsid via a first adapter, wherein the first adapter is covalently linked to the recognition sequence motif residue: b1. Residues covalently linked to amide -C(O)NH-; b2. First spacer (Sp)n; b3. Divalent fused polycyclic heterocycles of 11 to 20 members, phosphine oxide derivatives, or 4H-pyrazole-4-imine derivatives; b4. Second spacer (Sp')n'; b5. First-targeting ligand; and (c) A second targeting ligand conjugated to the modified viral capsid via a second connector, wherein the second connector is covalently linked to a primary amino or thiol group of the primary sequence of the modified viral capsid: c1. Amide-NHC(O)-; c2. First spacer (Sp)n; c3. Divalent fused polycyclic heterocycles of 11 to 20 members, phosphine oxide derivatives, or 4H-pyrazole-4-imine derivatives; c4. Second spacer (Sp')n'; and c5. Second targeting ligand; Where n and n' are each an independent integer from 0 to 20.

13. The engineered viral capsid according to any one of claims 1 to 12, wherein the recognition sequence motif residue is selected from any one or a portion thereof of the peptide sequences shown in SEQ ID NO: 1-58 or 61-75.

14. The engineered viral capsid according to any one of claims 1 to 13, wherein the recognition sequence motif residue comprises at least four consecutive amino acids selected from the peptide sequences shown in SEQ ID NO: 8-33, 35-44, 62-64 and 65.

15. The engineered viral capsid according to any one of claims 1 to 14, wherein the recognition sequence motif residues comprise at least six consecutive amino acids as shown in SEQ ID NO:61 or SEQ ID NO:

66.

16. The engineered viral capsid conjugated to the first and second targeting ligands according to any one of claims 1 to 11, further comprising: (a) Modified viral capsid; (b) A first targeting ligand conjugated to the modified viral capsid via a first adapter, wherein the first adapter is covalently linked to a primary amino or thiol group of the primary sequence of the modified viral capsid: b1. Residues covalently linked to amide -C(O)NH-; b2. First spacer (Sp)n; b3. Divalent fused polycyclic heterocycles of 11 to 20 members, phosphine oxide derivatives, or 4H-pyrazole-4-imine derivatives; b4. Second spacer (Sp')n'; b5. First-targeting ligand; and (c) A second targeting ligand conjugated to the modified viral capsid via a second connector, wherein the second connector is covalently linked to a primary amino or thiol group of the primary sequence of the modified viral capsid: c1. Amide-NHC(O)-; c2. First spacer (Sp)n; c3. Divalent fused polycyclic heterocycles of 11 to 20 members, phosphine oxide derivatives, or 4H-pyrazole-4-imine derivatives; c4. Second spacer (Sp')n'; and c5. Second targeting ligand; Where n and n' are each an independent integer from 0 to 20.

17. The engineered viral capsid according to any one of claims 1 to 16, wherein the primary amino group is selected from N-terminal amino groups, lysine ε-amino groups, and arginine amino acid groups.

18. The engineered viral capsid according to any one of claims 1 to 17, wherein the thiol group is a cysteine ​​amino acid group.

19. The engineered viral capsid according to any one of claims 1 to 18, wherein each first spacer is selected from -(CH2). n -、-(CH2CH2O) n -, -C(O)-, -C(O)NH-, 5-membered heterocycles, and combinations thereof, where n is 0 to 20, for example n is 1 to 20, for example n is 1 to 8, for example n is 2 to 8, for example n is 1 to 4, for example n is 2 to 4, for example n is 4.

20. The engineered viral capsid according to any one of claims 1 to 19, wherein each second spacer is selected from -(CH2). n’ -、-(CH2CH2O) n’ -, -C(O)-, -C(O)NH-, 5-membered heterocycles, and combinations thereof, wherein n' is 0 to 20, for example n' is 1 to 20, for example n' is 1 to 8, for example n' is 2 to 8, for example n' is 1 to 4, for example n' is 2 to 4, for example n' is 4.

21. The engineered viral capsid according to any one of claims 1 to 20, wherein the divalent fused polycyclic 11- to 20-membered heterocyclic ring is: or , Z represents an optional substituted 7- to 9-membered carbon ring or heterocycle.

22. The engineered viral capsid of claim 21, wherein Z is replaced by one or more arylene groups.

23. The engineered viral capsid according to any one of claims 1 to 20, wherein the phosphine oxide derivative is: 。 24. The engineered viral capsid according to any one of claims 1 to 20, wherein the 4H-pyrazole-4-imine derivative is: 。 25. A method of treating a disease or ailment, comprising administering a therapeutically effective amount of an engineered viral capsid according to any one of claims 1 to 24.

26. The method of treating a disease or condition according to claim 25, wherein the disease or condition is a central nervous system disease or condition, such as a neurodegenerative disease, more specifically aromatic amino acid decarboxylase (AADC) deficiency, canavan disease, Parkinson's disease, Alzheimer's disease, and giant axonal neuropathy.

27. A viral capsid with surface modification according to Formula I: …(I) in: It is a viral capsid; Y and Y' are independent connecting parts; n and n' are independently 0 or integers from 1 to 50; Sp and Sp' are optional spacers, independently; L is a ligand; x is the ligand / capsid ratio, ranging from 1 to 500; and Q is selected from: or Z is a 7- or 8-membered ring or heterocyclic structure.

28. The surface-modified viral capsid of claim 27, wherein x ranges from 100 to 200.

29. The surface-modified viral capsid of claim 28, wherein x ranges from 130 to 170.

30. The surface-modified viral capsid according to claim 27, having a structure according to formula (I-1): (I-1) in: It is a viral capsid; n and n' are independent integers selected from 0 to 30; T is a ligand; and x is an integer from 1 to 500.

31. The surface-modified viral capsid according to claim 29, wherein x ranges from 100 to 200.

32. The surface-modified viral capsid according to any one of claims 27 to 31, wherein the ligand / capsid ratio (x) ranges from 130 to 170.

33. The surface-modified viral capsid according to any one of claims 27 to 32, wherein the ligand is known to bind to endogenous receptors in brain microvascular endothelial cells (BMECs).

34. The surface-modified viral capsid of claim 33, wherein the receptor is a protease-activated receptor (PAR).

35. The surface-modified viral capsid of claim 34, wherein the receptor is endothelial protease activation receptor 1 (PAR1).

36. The surface-modified viral capsid of claim 31, wherein the ligand binds to PAR1.

37. The surface-modified viral capsid of claim 36, wherein the ligand is a PAR-1 activating peptide, such as thrombin or a derivative thereof.

38. The surface-modified viral capsid of claim 37, wherein the ligand is a peptide comprising the sequence SFLLR (SEQ ID NO:2).

39. The surface-modified viral capsid of claim 38, wherein the ligand comprises a peptide having the sequence SFLLRNPNDKC (SEQ ID NO:3).

40. The surface-modified viral capsid of claim 33, wherein the ligand comprises wheat germ agglutinin (WGA).

41. The surface-modified viral capsid of claim 40, wherein the ligand is selected from WGA1, WGA2 and WGA3.

42. The surface-modified viral capsid according to any one of claims 27 to 41, wherein the ligand binds to PAR1 or wherein the ligand comprises WGA.

43. The surface-modified viral capsid according to any one of claims 27 to 41, wherein the capsid comprises a first ligand binding PAR1 and a second ligand comprising WGA.

44. A pharmaceutical composition comprising at least one recombinant viral particle, said recombinant viral particle comprising a surface-modified viral capsid and recombinant polynucleotide cargo according to any one of claims 27 to 43, and further comprising a pharmaceutically acceptable carrier, diluent, solubilizer, filler, preservative, excipient, or combination thereof.

45. A method for treating a patient suffering from a disease, said disease being treatable by intracellular delivery of a recombinant polynucleotide cargo, the method comprising: Administer a therapeutically effective amount of the pharmaceutical composition according to claim 44. The recombinant polynucleotide cargo is capable of treating the disease.

46. ​​A composition for treating a disease, said disease being treatable by intracellular delivery of a recombinant polynucleotide cargo, said composition comprising: A therapeutically effective amount of the pharmaceutical composition according to claim 44, The recombinant polynucleotide cargo is capable of treating the disease.

47. Use of the surface-modified viral capsid according to any one of claims 27 to 43 in the preparation of a medicament for treating a disease, said disease being treatable by intracellular delivery of a recombinant polynucleotide cargo.

48. A surface-functionalized viral capsid comprising a first member of a crosslinking agent reaction pair and optionally one or more spacers, wherein the surface-functionalized viral capsid is adapted to react with a functionalized ligand comprising a second member of the crosslinking agent reaction pair and optionally one or more spacers, wherein the member of the crosslinking agent reaction pair participates in a reaction selected from: Cu(I)-catalyzed azido-acetylene cycloaddition (CuAAC), strain-promoted acetylene-azido cycloaddition (SPAAC), strain-promoted acetylene-nitroketone cycloaddition (SPANC), anti-electron-demanding Diels-Alder (IEEDD) reaction, and Staudinger linkage and [4+1] cycloaddition reactions.

49. A method for preparing a surface-modified viral capsid, the method comprising the following steps: A surface-functionalized viral capsid is obtained by reacting viral capsid proteins with a capsid reactive linker, the linker comprising a first member of a crosslinking agent reactant pair and optionally one or more spacers. The surface-functionalized viral capsid is conjugated with a functionalized ligand comprising a second member of the crosslinking agent reactant pair and optionally one or more spacers; The first and second members of the crosslinking agent reaction pair react to form the crosslinked portion Q.

50. The method of claim 68, wherein the surface-modified viral capsid is a viral capsid according to any one of claims 27 to 43.

51. A recombinant viral particle comprising a capsid with a surface modification according to any one of claims 27 to 43.

52. The recombinant viral particle of claim 51, wherein the recombinant viral particle is rAAV.

53. An engineered adeno-associated virus (AAV) capsid protein, wherein: The capsid protein contains: a functional sequence motif. The functional sequence motif described therein has the following sequence: X1X2X3X4X5X6 (SEQ ID NO:5); wherein X1, X2, X3, X4, X5 and X6 are each independently selected from any amino acid residue, and wherein X4, X5 and X6 are optional; The functional sequence motifs mentioned above are substrates for the sorting enzyme transaminases.

54. The engineered rAAV capsid protein according to claim 53, wherein the enzyme is selected from sorting enzyme A, sorting enzyme B, archaeal sorting enzyme A, exosorting enzyme A, rhombic sorting enzyme and PorU.

55. The engineered rAAV capsid protein according to claim 54, wherein the enzyme is sorting enzyme A.

56. The engineered rAAV capsid protein according to claim 55, a seven-mutant (SrtA7M) or a five-mutant (SrtA5M).

57. The engineered rAAV capsid protein according to claim 55, wherein the enzyme is a seven-mutant Staphylococcus aureus sorting enzyme A (SrtA7m).

58. The engineered rAAV capsid protein according to any one of claims 27 to 57, wherein the functional sequence motif is located within the surface-accessible variable region (VR) of the capsid primary sequence.

59. The engineered rAAV capsid protein according to any one of claims 27 to 58, wherein the functional sequence motif has a sequence of X1X2X3 (SEQ ID NO:6); wherein X1, X2 and X3 are each independently selected from any amino acid residue.

60. The engineered rAAV capsid protein of claim 59, wherein the functional sequence motif has an X1X2X3 (SEQ ID NO:7) sequence, and wherein two or more of X1, X2, and X3 are selected from: X1 is valine (V); X2 is proline (P); and X3 is proline (P).

61. The engineered rAAV capsid protein according to claim 59, wherein X1, X2, and X3 are selected from: X1 is proline (P) independently; X2 is independently selected from glycine (G) and glutamic acid (E); and X3 is independently selected from proline (P) and phenylalanine (F).

62. The engineered rAAV capsid protein according to claim 61, wherein the functional sequence motif has a sequence selected from: PEF (SEQ ID NO:50), PGF (SEQ ID NO:51), or PEP (SEQ ID NO:52).

63. The engineered rAAV capsid protein according to any one of claims 27 to 58, wherein the functional sequence motif has a sequence of X1X2X3X4 (SEQ ID NO:53); wherein X1, X2, X3 and X4 are each independently selected from any amino acid residue.

64. The engineered rAAV capsid protein according to any one of claims 27 to 58, wherein the functional sequence motif has a sequence of X1X2X3X4X5 (SEQ ID NO:54); wherein X1, X2, X3, X4 and X5 are each independently selected from any amino acid residue.

65. The engineered rAAV capsid protein according to claim 64, wherein the functional sequence motif has an X1X2X3X4X5 (SEQ ID NO:54) sequence, and wherein: X1 is independently selected from lysine (L), valine (V), (I) and asparagine (N); X2 is proline (P) independently; X3 is independently selected from any amino acid residue; X4 is independently threonine (T); and X5 is independently selected from alanine (A), glycine (G) and asparagine (N).

66. The engineered rAAV capsid protein according to claim 65, wherein two or more of X1, X2, X3, X4 and X5 are selected from: X1 is independently selected from lysine (L); X2 is proline (P) independently; X3 is independently selected from any amino acid residue; X4 is independently threonine (T); and X5 is independently selected from alanine (A), glycine (G) and asparagine (N).

67. The engineered rAAV capsid protein of claim 66, wherein three or more of X1, X2, X3, X4 and X5 are selected from: X1 is independently selected from lysine (L), valine (V), (I) and asparagine (N); X2 is proline (P) independently; X3 is independently selected from any amino acid residue; X4 is independently threonine (T); and X5 is independently selected from alanine (A) and glycine (G).

68. The engineered rAAV capsid protein according to claim 67, wherein... X1 is lysine (L); X2 is proline (P); X3 is glutamic acid (E), serine (S), or alanine (A); X4 is threonine (T); and X5 is either alanine (A) or glycine (G).

69. The engineered rAAV capsid protein according to any one of claims 64 to 68, wherein the functional sequence motif comprises a sequence selected from the group consisting of LPX3TG (SEQ ID NO:48), LPX3TA (SEQ ID NO:55), LPETG (SEQ ID NO:12), and LPETA (SEQ ID NO:56).

70. The engineered rAAV capsid protein according to any one of claims 1 to 77, wherein the functional sequence motif has a sequence of X1X2X3X4X5X6 (SEQ ID NO:5); wherein X1, X2, X3, X4, X5 and X6 are each independently selected from any amino acid residue.

71. The engineered rAAV capsid protein according to claim 70, wherein the functional sequence motif has X1X2X3X4X5X6 (SEQ ID NO:5); wherein two or more of X1, X2, X3, X4, X5 and X6 are: X1 is independently asparagine (N); X4 is independently threonine (T); X5 is independently asparagine (N); and X6 is proline (P) independently.

72. The engineered rAAV capsid protein according to claim 71, wherein the functional sequence motif has X1X2X3X4X5X6 (SEQ ID NO:5); wherein at least three of X1, X2, X3, X4, X5 and X6 are: X1 is independently asparagine (N); X4 is independently threonine (T); X5 is independently asparagine (N); and X6 is proline (P) independently.

73. The engineered rAAV capsid protein according to claim 72, wherein the functional sequence motif comprises a sequence selected from the following: NX2X3TNX6 (SEQ ID NO:57) and VPX3X4X5P (SEQ ID NO:58).

74. The engineered rAAV capsid protein according to any one of claims 27 to 73, wherein the engineered rAAV capsid protein comprises one or more sequence alterations selected from insertion, deletion, or substitution compared to the wild-type protein, the sequence alterations being used to introduce the functional sequence motif.

75. The engineered rAAV capsid protein of claim 74, comprising at least one insertion mutation, wherein the insertion forms the functional sequence motif.

76. The engineered rAAV capsid protein according to claim 75, wherein the insertion mutation is the insertion of an exogenous peptide selected from LPET (SEQ ID NO:49) and LPETG (SEQ ID NO:12).

77. The engineered rAAV capsid protein according to any one of claims 27 to 76, wherein the rAAV capsid protein is selected from one or more of VP1, VP2 and VP3.

78. The engineered rAAV capsid protein of claim 77, wherein the functional sequence motif is located within a surface loop region selected from the following: AB, BC, CD, DE, EF, FG, GH, and HI loops.

79. The engineered rAAV capsid protein of claim 78, wherein the functional sequence motif is located within the GH surface loop region.

80. The engineered rAAV capsid protein according to any one of claims 27 to 79, wherein the functional sequence motif is located in one or more variable regions selected from VR1, VR2, VR3, VR4, VR5, VR6, VR7, VR8, and VR9.

81. The engineered rAAV capsid protein of claim 80, wherein the engineered rAAV capsid protein comprises a functional sequence motif within variable region IV or VIII.

82. The engineered rAAV capsid protein of claim 80, wherein the engineered rAAV capsid protein comprises a functional sequence motif within variable region VIII.

83. The engineered rAAV capsid protein of claim 80, wherein the engineered rAAV capsid protein comprises functional sequence motifs within variable regions IV and VIII; and wherein each functional sequence motif is independently selected.

84. The engineered rAAV capsid protein according to any one of claims 27 to 83, wherein each functional sequence motif is identical.

85. The engineered rAAV capsid protein according to any one of claims 27 to 84, wherein the rAAV capsid protein is selected from serotypes AAV1, AAV2, AAV4, AAV5, AAV8 and AAV9.

86. The engineered rAAV capsid protein of claim 85, wherein the functional sequence motif begins after: S453 in AAV1 capsid serotype VR4; S452 in VR4 or R585 in VR8 of AAV2 capsid serotype; S576 in AAV5 capsid serotype VR8; S453 in VR4 or S587 in VR8 of AAV6 capsid serotype; AAV7 capsid serotype VR4 P454 or VR8 A587; AAV8 capsid serotype VR4 contains T454 or VR8 contains Q589; as well as S454 in AAV9 capsid serotype VR8.

87. The engineered rAAV capsid protein of claim 86, wherein the functional sequence motif begins after S452 in AAV2 capsid serotype VR4 or after R585 in VR8.

88. The engineered rAAV capsid protein according to claim 87, wherein LPET (SEQ ID NO: 49) is inserted after S452 to form the functional sequence motif.

89. The engineered rAAV capsid protein according to any one of claims 53 to 88, wherein the engineered rAAV capsid protein does not contain additional mutations compared to the wild-type capsid protein.

90. A polynucleotide encoding an engineered rAAV capsid protein according to any one of claims 27 to 89.

91. A vector comprising the polynucleotide of claim 90.

92. The vector of claim 91 further comprises a promoter operatively linked to the polynucleotide.

93. A host cell comprising an engineered rAAV capsid protein according to any one of claims 1 to 37, a polynucleotide according to claim 90, or a vector according to claim 91 or 92.

94. A recombinant rAAV viral particle (rAAV) comprising the engineered rAAV capsid protein according to any one of claims 53 to 89.

95. The rAAV viral particle according to claim 94 further comprises exogenous cargo polynucleotides.

96. The rAAV viral particle of claim 94, wherein the exogenous cargo polynucleotide comprises a template for homology-directed repair.

97. The rAAV viral particle of claim 94, wherein the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic tRNA, miRNA, gene editing guide RNA, or RNA editing guide RNA.

98. The rAAV viral particle of claim 94, wherein the exogenous polynucleotide comprises an expressible polynucleotide encoding a therapeutic protein.

99. The rAAV viral particle according to any one of claims 94 to 98, wherein the functional sequence motif weakens or eliminates the binding of the viral particle to mammalian cellular polysaccharides or proteoglycans compared to a reference viral particle having the same capsid protein sequence but lacking the functional sequence motif.

100. The surface-functionalized rAAV viral particle according to any one of claims 94 to 99, wherein the rAAV viral particle comprises: at least one cross-linking reactive moiety covalently linked to amino acid residues in the engineered rAAV capsid protein and an exogenous cargo polynucleotide, wherein the amino acid residues are not within the functional sequence motif.

101. The surface-functionalized rAAV virus particle according to claim 100, wherein the amino acid residue is lysine.

102. The surface-functionalized rAAV virus particle according to claim 100, wherein the amino acid residues in the viral capsid protein are N-terminal amines.

103. The surface-functionalized rAAV virus particle according to claim 100, wherein the amino acid residues in the viral capsid protein are non-natural amino acid residues.

104. A surface-functionalized rAAV viral particle, wherein the viral particle comprises at least one engineered rAAV capsid protein according to any one of claims 53 to 89 and an exogenous cargo polynucleotide, wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form (i) an N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and at least one cross-linking reactive portion covalently linked to a C-terminal amino acid of the N-terminal cleavage fragment.

105. The surface-functionalized rAAV virus particle of claim 104, wherein the covalent link comprises an amide bond.

106. The surface-functionalized rAAV virus particle according to claim 105, wherein the C-terminal amino acid of the N-terminal cleavage fragment is threonine.

107. A surface-functionalized rAAV viral particle, wherein the viral particle comprises at least one engineered rAAV capsid protein according to any one of claims 53 to 89 and an exogenous cargo polynucleotide, and The engineered rAAV capsid protein is cleaved within the functional sequence motif to form (i) an N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and At least one reactive portion of the crosslinking agent is covalently linked to the C-terminal amino acid of the N-terminal cleavage fragment; and Optionally, at least one cross-linking agent reactive portion is covalently linked to an amino acid residue in the engineered rAAV capsid protein, wherein the amino acid residue is not within the functional sequence motif.

108. The surface-functionalized rAAV virus particle according to any one of claims 100 to 107, wherein each crosslinking agent reactive portion is independently selected from crosslinking agent reactive portions participating in reactions selected from: Cu(I)-catalyzed azido-acetylene cycloaddition (CuAAC) reaction, strain-promoted acetylene-azido cycloaddition (SPAAC) reaction, strain-promoted acetylene-nitroketone cycloaddition (SPANC) reaction, anti-electron-demanding Diels-Alder (IEEDD) reaction, and Staudinger linkage and [4+1] cycloaddition reaction.

109. The surface-functionalized rAAV virus particle according to claim 108, wherein the reactive portion of the crosslinking agent comprises at least one of an eight-membered ring and a triazole ring.

110. The surface-functionalized rAAV virus particle according to claim 108, wherein the reaction is a strain-promoted acetylation-azidocycloaddition (SPAAC) reaction.

111. The surface-functionalized rAAV virus particle according to claim 108, wherein the reactive portion of the crosslinking agent is selected from cyclooctylene and azide.

112. The surface-functionalized rAAV virus particle according to claim 108, wherein the cyclooctyne is selected from dibenzylcyclooctyne (DIBO), dibenzozacyclooctyne (DBCO), and biarylazacyclooctyne one (BARAC) or derivatives thereof.

113. The surface-functionalized rAAV virus particle according to claim 112, wherein the cyclooctyne is DBCO.

114. The surface-functionalized rAAV virus particle according to claim 108, wherein the reaction is an anti-electron demand Diels-Alder (IEEDD) reaction.

115. The surface-functionalized rAAV virus particle according to claim 108, wherein the reactive portion of the crosslinking agent is selected from trans-cyclooctene and tetrazine.

116. A surface-modified rAAV viral particle, wherein the viral particle comprises at least one engineered rAAV capsid protein according to any one of claims 53 to 89 and an exogenous cargo polynucleotide, wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form (i) an N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and wherein the viral particle comprises a crosslinked portion of the C-terminal amino acid of the N-terminal cleavage fragment covalently linked to a targeting ligand.

117. The surface-modified rAAV virus particle of claim 116, wherein the covalent linkage comprises an amide bond.

118. The surface-modified rAAV viral particle according to claim 116 or 117, wherein the C-terminal amino acid of the N-terminal cleavage fragment is threonine.

119. The surface-modified rAAV virus particle according to any one of claims 116 to 118, wherein the crosslinked portion comprises a product selected from the following reactions: CuAAC reaction, SPAAC reaction, SPANC reaction, IEEDD reaction, Staudinger linkage, [4+1] cycloaddition reaction.

120. The surface-modified rAAV virus particle according to claim 119, wherein the reaction is selected from: SPAAC, SPANC, and IEEDD reactions.

121. The surface-modified rAAV virus particle according to any one of claims 116 to 120, wherein the crosslinked portion comprises a 7- or 8-membered carbon ring containing 0 to 3 heteroatoms selected from O or N.

122. The surface-modified rAAV virus particle of claim 121, wherein the crosslinked portion comprises or .

123. The surface-modified rAAV viral particle according to any one of claims 116 to 122, wherein the functional sequence motif is selected from one of SEQ ID NO: 1-58.

124. The surface-modified rAAV viral particle according to claim 123, wherein the functional sequence motif is LPETG (SEQ ID NO: 12).

125. A surface-modified rAAV viral particle, wherein the viral particle comprises an exogenous cargo polynucleotide and at least one engineered rAAV capsid protein, the engineered rAAV capsid protein comprising at least one functional sequence motif LPX3TG (SEQ ID NO:48), wherein X3 is selected from any amino acid residue; wherein the functional sequence motif is located within variable regions 4, 8, or 4 and 8, and (a) wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form an N-terminal cleavage fragment and a C-terminal cleavage fragment; wherein the viral particle comprises a cross-linked portion of the T-terminal cleavage fragment covalently linked to a targeting ligand, wherein the covalent bond comprises an amide bond; and optionally (b) wherein the viral particle comprises a cross-linked portion of the capsid protein covalently linked to a lysine residue or an N-terminal amine of the capsid protein and a targeting ligand.

126. The surface-modified rAAV viral particle of claim 125, wherein the crosslinked portion comprises or .

127. The surface-modified rAAV viral particle according to any one of claims 116 to 126, wherein the targeting ligand is a cell type-specific ligand.

128. The surface-modified rAAV viral particle of claim 127, wherein the targeting ligand is selected from enzymes, peptide mimics, ligands of endogenous receptors, lipid-binding proteins, toxins, phage peptidases, nucleotide- and RNA / DNA-based ligands, aptamers, viral vectors, DNA or RNA editing proteins, nanoparticles, lipids, small molecules, and combinations thereof.

129. The surface-modified rAAV viral particle according to any one of claims 116 to 128, wherein the targeting ligand binds to a receptor-mediated transporter expressed on endothelial cells forming the blood-brain barrier (BBB).

130. The surface-modified rAAV viral particle of claim 129, wherein the receptor is selected from insulin receptors, transferrin receptors, choline transporters, amino acid transporters (e.g., large neutral amino acid carriers) and LDL receptors.

131. A surface-modified viral capsid according to any one of the preceding claims, wherein the ligand is known to bind to endogenous receptors in brain microvascular endothelial cells (BMECs).

132. The surface-modified viral capsid of claim 131, wherein the receptor is a protease-activated receptor (PAR).

133. The surface-modified viral capsid of claim 132, wherein the receptor is endothelial protease activation receptor 1 (PAR1).

134. The surface-modified viral capsid of claim 133, wherein the ligand binds to PAR1.

135. The surface-modified viral capsid of claim 134, wherein the ligand is a PAR-1 activating peptide, such as thrombin or a derivative thereof.

136. The surface-modified viral capsid of claim 131, wherein the ligand is a peptide comprising the sequence SFLLR (SEQ ID NO:2).

137. The surface-modified viral capsid of claim 136, wherein the ligand comprises a peptide having the sequence SFLLRNPNDKC (SEQ ID NO:3).

138. The surface-modified viral capsid of claim 131, wherein the ligand comprises wheat germ agglutinin (WGA).

139. The surface-modified viral capsid of claim 138, wherein the ligand is selected from WGA1, WGA2 and WGA3.

140. The surface-modified viral capsid according to any one of claims 116 to 128, wherein the ligand binds to PAR1 or wherein the ligand comprises WGA.

141. The surface-modified viral capsid according to any one of claims 116 to 128, wherein the capsid comprises a first ligand binding PAR1 and a second ligand comprising WGA.

142. The surface-modified rAAV viral particle according to any one of claims 116 to 141, wherein the functional sequence motif weakens or eliminates the binding of the viral particle to mammalian cell polysaccharides or proteoglycans compared to a reference viral particle having the same capsid protein sequence but lacking the functional sequence motif.

143. The surface-modified rAAV virus particles according to any one of claims 116 to 141, characterized in that... Compared to a reference viral particle with the same capsid protein sequence but lacking the aforementioned functional sequence motif, infectivity is increased.

144. The surface-modified rAAV viral particles of claim 143, wherein the increased infectivity is relative to at least one cell or tissue type.

145. The surface-modified rAAV viral particle of claim 143, wherein the cell or tissue type is selected from the heart, nerves, central and peripheral nervous system, hematopoietic system, liver, muscle, connective tissue, lung, kidney and retina.

146. The surface-modified rAAV viral particle according to any one of claims 116 to 145, wherein the exogenous cargo polynucleotide comprises a template for homology-directed repair.

147. The surface-modified rAAV viral particle of claim 146, wherein the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic tRNA, miRNA, gene-editing guide RNA, or RNA-editing guide RNA.

148. The surface-modified rAAV viral particle of claim 146, wherein the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic protein.

149. A pharmaceutical composition comprising a plurality of surface-modified rAAV viral particles according to any one of claims 116 to 148, the composition further comprising a pharmaceutically acceptable carrier, diluent, solubilizer, filler, preservative, excipient, or combination thereof.

150. A method for treating a patient suffering from a disease, said disease being treatable by intracellular delivery of a recombinant polynucleotide cargo, said method comprising: Administer a therapeutically effective amount of the pharmaceutical composition according to claim 149. The recombinant polynucleotide cargo is capable of treating the disease.

151. A composition for treating a disease, said disease being treatable by intracellular delivery of a recombinant polynucleotide cargo, said composition comprising: a therapeutically effective amount of the pharmaceutical composition of claim 149, wherein said recombinant polynucleotide cargo is capable of treating said disease.

152. Use of the rAAV virus particles according to any one of claims 116 to 148 in the preparation of a medicament for treating a disease, wherein the disease is treatable by intracellular delivery of recombinant polynucleotide cargo.

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