Targeting tissues in the CNS

Abstract

This disclosure relates to targeting the central nervous system. Provided are compositions for delivering therapeutics to certain tissue within the CNS. Also provided are novel surface modified viral capsids and recombinant virions comprising the same. Furthermore, this disclosure concerns intermediates for the preparation of surface modified viral capsids. The surface modified viral capsids are designed to selectively and / or more efficiently deliver gene therapy. The surface modified viral capsids, when incorporated into a recombinant virion, can be used to treat an illness that is characterized by genetic abnormality.

Description

TARGETING TISSUES IN THE CNS1. SEQUENCE LISTING

[0001] The instant application incorporates by reference a computer readable sequence listing in XML format, entitled “37937-61963_010WO_ST26.xml” created on March 11, 2025 and is 61,324 bytes in size.2. BACKGROUND

[0002] The central nervous system (CNS) is comprised primarily of the brain and spinal cord. The CNS is surrounded by a protective barrier, termed the blood-brain barrier (BBB) — or blood-spinal cord barrier (BSCB) in the case of the spinal cord — a highly selective, semipermeable, border of endothelial cells (EC) that prevents pathogens, solutes and most molecules from non-selectively crossing into the CNS. While normally helping to protect the CNS, in the case of disease the BBB and BSCB also function as a “barrier” to therapies and can prevent effective delivery of therapeutics to target cells or regions within the CNS. In the development of therapeutics for disorders of the CNS, achieving sufficient penetration of the BBB and BSCB is a major hurdle.

[0003] Recombinant AAV virions (rAAV) are favored viral delivery vehicles for in vivo delivery of therapeutic nucleic acid payloads (cargoes), including polynucleotides that express therapeutic genes, therapeutic miRNA, guide RNAs for nucleic acid-guided nucleases, genome editing enzymatic machinery, and RNA editing machinery, and for in vivo delivery of nucleic acids that act directly or indirectly as templates for genomic insertion or repair. The BBB presents a formidable barrier to use of rAAV to deliver such therapeutics payloads to the central nervous system.

[0004] One approach to circumventing the BBB is to administer rAAV directly to the cerebrospinal fluid (CSF) via intrathecal delivery, such as administration via lumbar puncture or via intracerebroventricular (ICV) or intra-ci sterna magna (ICM) administration. While direct administration to the CSF circumvents the BBB, it does not ensure delivery to the brain parenchyma because the meningeal membranes provide a further barrier to access to neurons, astrocytes, and microglia in the parenchyma.

[0005] Another approach is to engineer AAV capsids to cross the blood brain barrier through engineered interaction with various receptors known to mediate transcytosis across ECs, andthrough mechanism-agnostic empiric interrogation of large libraries of peptide-modified capsids in rodents and non-human primates (NHPs).

[0006] Despite substantial efforts, there are still few engineered capsids that reliably deliver rAAV, and their payloads, to desired cell types in the brain. There is a continuing need to develop new targeting strategies for rAAV and for other therapeutic payloads that have a higher efficiency for reaching cells in the CNS and that have higher transduction specificity (tropism) for relevant CNS target cells and tissues, and that can be efficacious when delivered at a lower titer.3. SUMMARY OF THE DISCLOSURE

[0007] The present disclosure provides herein engineered viral capsids with dual ligands, pharmaceutical compositions thereof, methods of use, and methods of making.

[0008] In one aspect, provided herein is an engineered viral capsid 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 second targeting ligand are distinct.

[0009] In another aspect, provided herein is a method of treating a disease or disorder comprising administering a therapeutically effective amount of an engineered viral capsid comprising: 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 second targeting ligand are distinct. In some embodiments, the disease or disorder is a central nervous system disease or disorder. In some embodiments., the central nervous disease or disorder is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is selected from aromatic amino acid decarboxylase (AADC) deficiency, Canavan disease, Parkinson’s disease, Alzheimer’s disease, and giant axonal neuropathy.

[0010] The present disclosure is directed, in some embodiments, to a surface-modified viral capsid, typically an AAV viral capsid. In some embodiments, the surface modification comprises a first ligand that facilitates passage of the recombinant AAV (rAAV) from CSF into the brain parenchyma, optionally with one or more additional surface-conjugated ligands that facilitate transduction of CNS cells, optionally transduction of one or more desired CNScell types. In some embodiments, the first ligand is covalently conjugated to a viral capsid protein. In certain of these embodiments, the first ligand is covalently conjugated to the viral capsid protein via a linker comprising a crosslinked moiety, Q. In other embodiments, the first ligand is fused in-frame in the capsid protein sequence. In another aspect, recombinant virions, typically recombinant AAV virions (rAAV), that comprise the surface-modified viral capsid and an encapsidated nucleic acid payload are provided.

[0011] In some embodiments, a peptide ligand is inserted into the capsid protein of a naturally occurring AAV serotype or an engineered or synthesized AAV capsid protein. For example, the peptide ligand can be inserted into a capsid protein of a naturally occurring AAV serotype, e.g., AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, or AAV9. In some embodiments, the peptide ligand is inserted into the 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 the variable region 8 (VR8) of the AAV capsid protein. The VR8 region may contain R585 and R588 required for HSPG binding, or may comprise mutations that reduce or abrogate binding to HSPG, such as R585A and R588A mutations. In some embodiments, the specific insertion site in VR4 or VR8 can be different depending on the capsid protein used for the modification. In some embodiments, the insertion site in VR4 can be between amino acid positions 400 and 500 of the capsid protein. In some embodiments, the insertion site in VR4 can be between amino acid positions 425 and 475 of the capsid protein. In some embodiments, the insertion site in VR4 can be between amino acid positions 440 and 460 of the capsid protein.

[0012] In some embodiments, the insertion site in VR8 can be 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.

[0013] In some embodiments, the peptide ligand is PARI. In some embodiments, the peptide ligand is PARI A. In some embodiments, the peptide ligand has a sequence of SFLLRN (residues 5 to 10 of SEQ ID NO: 61). In some embodiments, the peptide ligand has a sequence of LDPRSFLLRN (SEQ ID NO: 61).

[0014] In some embodiments, the disclosure is directed to a surface modified viral capsid according to Formula I:wherein:viral capsid;Y and Y’ are independently an attachment moiety; n and n’ are independently 0 or an integer from 1 to 50;Sp and Sp’ are independently an optional spacer;L is a ligand; x is the ligand per capsid ratio that is in a range from 1 to 500; and Q is selected from:wherein, Z is a 7 or 8 membered cyclic or heterocyclic structure.

[0015] In some embodiments, the disclosure is directed to a method of a making a surface modified viral capsid, the method comprising the steps: i) obtaining a surface functionalized viral capsid by reacting a viral capsid protein with a capsid-reactive linker, the linker comprising a first member of a crosslinker reactive pair and optionally one or more of a spacer; ii) conjugating the surface functionalized viral capsid with a functionalized ligand comprising a second member of the crosslinker reactive pair and optionally one or more of a spacer; wherein the first and second members of the crosslinker reactive pair react to form a crosslinked moiety, Q.

[0016] In some embodiments, an engineered adeno-associated virus (AAV) capsid protein is provided. In some other embodiment, the capsid protein comprises a functional sequence motif. In some embodiments, the functional sequence motif has the sequence:X1X2X3X4X5X6 (SEQ ID NO: 5); wherein Xi, X2, X3, X4, X5, and Xe are each independently selected from any amino acid residue and wherein X4, X5, and Xe are optional. In some embodiments, the functional sequence motif is a substrate for a sortase transamidase enzyme.

[0017] In some embodiments, the disclosure is directed to an engineered adeno-associated virus (AAV) capsid protein, wherein: the capsid protein comprises: a functional sequence motif, wherein the functional sequence motif has the sequence:X1X2X3X4X5X6 (SEQ ID NO: 5); wherein Xi, X2, X3, X4, X5, and X6are each independently selected from any amino acid residue and wherein X4, X5, and Xe are optional; wherein the functional sequence motif is a substrate for a sortase transamidase enzyme.

[0018] In some embodiments, the disclosure is directed to a recombinant rAAV virion (rAAV) comprising the engineered rAAV capsid protein disclosed herein.

[0019] In some embodiments, the disclosure is directed a surface modified rAAV virion, wherein the virion comprises an exogenous cargo polynucleotide and at least one 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 region 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 crosslinked moiety that covalently connects the T of the N-terminal cleavage fragment and a targeting ligand wherein the covalent linkage comprises amide bond; and optionally (b) wherein the virion comprises a crosslinked moiety that covalently connects a lysine residue or a N terminal amine of a capsid protein and a targeting ligand.

[0020] In some embodiments, the disclosure provides compositions for delivering therapeutics to certain tissue within the CNS. In some embodiments, the surface modified viral capsids are designed to selectively and / or more efficiently deliver gene therapy. The surface modified viral capsids, when incorporated into a recombinant virion, can be used to treat an illness that is characterized by genetic abnormality (e.g., neurodegenerative diseases, neuromuscular diseases, lysosomal storage diseases).4. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] A better understanding of the features, aspects, and advantages of the present disclosure will become better understood with regard to the following detailed descriptionwhich sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and accompanying drawings of which:

[0022] FIGs. 1A-1D illustrate transduction of spinal cord parenchyma following intrathecal injection of 3E+9 (3 x 109) vector genomes (VG) of control (AAV2-AHSPG) and TPA- modified AAV2-AHSPG (TPA::AAV2-AHSPG) recombinant virions, each carrying a tdTomato-expressing polynucleotide payload. As shown in FIG. 1A and FIG. 1C, few tdTomato-positive (that is, effectively transduced) neurons are observed upon intrathecal injection of unmodified AAV2-AHSPG in lumbar spinal cord (FIG. 1A) or thoracic spinal cord (FIG. 1C). In contrast, conjugation of TPA to AAV2-AHSPG (TPA: : AAV2-AHSPG) increased transduction efficiency in the parenchyma at all levels. Sections are shown of lumbar (FIG. IB) and thoracic (FIG. ID) spinal cord.

[0023] FIGs. 2A-2F illustrate transduction of spinal cord following intrathecal injection of 3E+10 VG of control (AAV2- AHSPG) and AAV2-AHSPG rAAV surface-modified to display a PAR-1 (PARI) agonist peptide, SFLLRNPNDKC (SEQ ID NO: 3) (PAR1PA::AAV2-AHSPG). FIG. 2B and FIG. 2C illustrate that conjugation of the PAR1PA peptide ligand to the viral capsid surface increases transduction efficiency in the parenchyma and further illustrate that this is further amplified through conjugation of a second ligand, wheat germ agglutinin (WGA), to the capsid surface (FIG. 2D). In contrast, unconjugated PAR1PA peptide injected at the same time as AAV2-AHSPG does not increase parenchymal penetrance (FIG. 2E). Quantification of fluorescent signal in the grey matter is shown in FIG. 2F.

[0024] FIGs. 3A-3D are heat maps of tdTomato expression in brain sections following intrathecal injection of 3E+10 VG control (AAV2-AHSPG) and PARI A surface-modified AAV2-AHSPG (PAR1PA: : AAV2-AHSPG). FIG. 3A shows that few tdTomato positive (effectively transduced) neurons are observed upon intrathecal injection of unmodified AAV2-AHSPG. FIG. 3B shows that conjugation of the PAR1PA peptide ligand increases transduction efficiency in the parenchyma, which is further amplified through conjugation of a second ligand, WGA, to the rAAV capsid surface (FIG. 3C). FIG. 3D shows that, in contrast, unconjugated PAR1PA peptide injected at the same time as AAV2-AHSPG increased parenchymal penetrance only slightly. FIG. 3E illustrates the quantification of fluorescent signal in brain gray matter.

[0025] FIGs. 4A-4D illustrate transduction of PC 12 cells in in vitro culture by rAAV having (i) surface-conjugated WGA ligand and (ii) an LPETG peptide (SEQ ID NO: 12) fused inframe in variable region 8 of the AAV capsid proteins (WGA::AAV2-LPETG-VR8), carrying a tdTomato expression construct as payload. In these experiments, the AAV2 capsid does not have the AHSPG deletion in the capsid and the LPETG peptide (SEQ ID NO: 12) permits further, directed, chemical surface modifications. FIG. 4A shows tdTomato fluorescence in PC 12 cells transduced in vitro with unmodified AAV2-LPETG-VR8. FIG. 4B shows tdTomato fluorescence in PC12 infected with WGA::AAV2-LPETG-VR8 vector ligated to GGG-PEG4-DBCO. FIG. 4C shows tdTomato fluorescence in PC12 cells transduced with WGA::AAV2-LPETG-VR8 vector ligated to amine DBCO. FIG. 4D shows tdTomato fluorescence in PC12 cells transduced with WGA::AAV2-LPETG-VR8 vector ligated to Amine-PEG4-DBCO.

[0026] FIGs. 5A-5D illustrate transduction of PC12 cells in culture by WGA::AAV2- LPETG-VR4, in which the LPETG tag (SEQ ID NO: 12) is inserted in frame into the VR4 region of the capsid protein. FIG. 5A presents tdTomato fluorescence in PC12 cells infected with unmodified AAV2-LPETG-VR4; FIG. 5B presents tdTomato fluorescence in PC 12 infected with WGA::AAV2-LPETG-VR4 vector ligated to GGG-PEG4-DBCO; FIG. 5C presents tdTomato fluorescence in PC 12 cells infected with WGA::AAV2-LPETG-VR4 vector ligated to amine DBCO; and FIG. 5D presents tdTomato fluorescence in PC12 cells infected with WGA::AAV2-LPETG-VR4 vector ligated to amine-PEG4-DBCO.

[0027] FIG. 6 illustrates the amino acid sequence (SEQ ID NO: 59) of the VP1 capsid protein of AAV2-LPETG-VR4. The Sortase A recognition sequence LPETG (residues 453 to 457 of SEQ ID NO: 59) is shown underlined in variable region 4 of AAV2 VP1.

[0028] FIG. 7 illustrates the amino acid sequence (SEQ ID NO: 60) of the VP1 capsid protein of AAV2-LPETG-VR8. The Sortase A recognition sequence LPETG (residues 586 to 590 of SEQ ID NO: 60) is shown underlined in variable region 8 of AAV2 VP1.

[0029] FIGs. 8A-8F illustrate spinal cord and horizontal brain sections of mice injected IV with 9xlO10VG / animal of unmodified AHSPG and AHSPG-SFLLRNC (SEQ ID NO: 66). FIG. 8 A and FIG. 8C illustrate spinal cord and horizontal brain sections of mice injected IV with 9xlO10VG / animal of unmodified AHSPG. FIG. 8B and FIG. 8D illustrate spinal cord and horizontal brain sections of mice injected IV with 9xlO10VG / animal of AHSPG-SFLLRNC (SEQ ID NO: 66). FIG. 8E illustrates 20x magnification on the cortex shows no positive cells in the control (i.e., unmodified AHSPG). FIG. 8F illustrates 20x magnification on the cortex reveals presence of positive cells in the experimental condition (i.e., AHSPG- SFLLRNC (SEQ ID NO: 66)).5. DETAILED DESCRIPTION5.1. Definitions

[0030] The term “rAAV” as used herein refers to a recombinant virion comprising a recombinant nucleic acid construct packaged within an AAV capsid.

[0031] The terms “AAV”, “adeno-associated virus”, “AAV virus”, “AAV virion”, “AAV viral particle”, “AAV particle”, “adeno-associated viral vector”, and “AAV vector” are used synonymously herein for rAAV.

[0032] The recombinant nucleic acid construct (synonymously, “recombinant viral genome”) comprises a polynucleotide payload (synonymously, “cargo”) positioned between AAV inverted terminal repeats. The payload can be an expressible polynucleotide or a 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 an miRNA, siRNA, or a guide RNA for gene editing or RNA editing machinery such as CRISPR, ADAR, and AD AT.

[0033] As used herein, “binding of a capsid” or “binding of a surface-modified capsid” to a mammalian cell surface protein, polysaccharide, or proteoglycan intends binding of a recombinant virion, typically rAAV, that comprises said capsid or surface-modified capsid.

[0034] As used herein, the terms “treat” or “treatment” are used in their broadest accepted clinical sense. The terms include, without limitation, lessening a sign or symptom of disease; improving a sign or symptom of disease; alleviation of symptoms; diminishment of extent of disease; stabilization (i.e., not worsening) of the state of disease; delay or slowing of disease progression; amelioration or palliation of the disease state; remission (whether partial or total), whether detectable or undetectable; cure; prolonging survival as compared to expected survival if not receiving treatment.

[0035] An “effective amount” is an amount of the rAAV of the present disclosure effective to treat a disease.

[0036] As used herein, the term “prevention” or “preventing” when used in the context of a subject refers to prophylaxis of a disease, typically in a subject at risk for developing the disease, for example by presence of a genomic mutation.

[0037] As used herein the term “tropism” refers to preferential infection and / or transduction by a viral capsid of certain cells or tissues. In a preferred embodiment, to modify an AAV capsid’s tropism, the capsids are being given certain features such as certain affinities to receptors on the target cell's surface which they do not possess by nature.

[0038] In the context of the present disclosure, the term “subject”, as used in certain embodiments, preferably refers to a mammal, such as a mouse, rat, guinea pig, rabbit, cat, dog, monkey, or preferably a human. The term “patient” preferably refers to a mammal, such as a mouse, rat, guinea pig, rabbit, horse, cattle, cow, cat, dog, monkey, or preferably a human, for example a human patient, for whom diagnosis, prognosis, or therapy is desired. The subject of the disclosure may be at risk of suffering from a disease, such as a having a disease treatable by intracellular delivery of a recombinant polynucleotide cargo. A more detailed description of medical indications relevant in the context of this disclosure is provided herein elsewhere.

[0039] The term “optionally substituted” means that a given chemical moiety (e.g., an alkyl group) can (but is not required to) be bonded other substituents (e.g., heteroatoms). For instance, an alkyl group that is optionally substituted can be a fully saturated alkyl chain (e.g., a pure hydrocarbon). Alternatively, the same optionally substituted alkyl group can have substituents different from hydrogen. For instance, it can, at any point along the chain be bounded to a halogen atom, a hydroxyl group, or any other substituent described herein. Thus, the term “optionally substituted” means that a given chemical moiety has the potential to contain other functional groups but does not necessarily have any further functional groups. Suitable substituents used in the optional substitution of the described groups include, without limitation, halogen, oxo, — OH, — CN, — COOH, — CH2CN, — O — (Ci-Ce)alkyl, (Ci- Ce)alkyl, (Ci-Ce)alkoxy, (Ci-Ce)haloalkyl, (Ci-Ce)haloalkoxy, — O — (C2-Ce)alkenyl, — O— ( C2-C6)alkynyl, (C2-C6)alkenyl, (C2-C6)alkynyl, — OP(O)(OH)2,— OC(O)(Ci-C6)alkyl, — C(O)( Ci-C6)alkyl, — OC(O)O(Ci-C6)alkyl,— NH2, — NH((Ci-C6)alkyl), — N((Ci-C6)alkyl)2, — NHC(O)( Ci-C6)alkyl,— C(O)NH(Ci-C6)alkyl, — S(O)2(Ci-C6)alkyl, — S(O)NH(Ci-C6)alkyl, andS(O)N((Ci-Ce)alkyl)2. The substituents can themselves be optionally substituted. “Optionally substituted” as used herein also refers to substituted or unsubstituted whose meaning isdescribed below. A moiety that includes additional substitution is referred to herein as a “derivative” of the substituted moiety. For example, an alkyl substituted nitrone is an example of a derivative of a nitrone moiety.

[0040] The term “substituted” means that the specified group or moiety bears one or more suitable substituents wherein the substituents may connect to the specified group or moiety at one or more positions. For example, an aryl substituted with a cycloalkyl may indicate that the cycloalkyl connects to one atom of the aryl with a bond or by fusing with the aryl and sharing two or more common atoms.

[0041] Unless otherwise specifically defined, “aryl” means a cyclic, aromatic hydrocarbon group having 1 to 3 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 joined at a single point (e.g., biphenyl), or fused (e.g., naphthyl). The aryl group is optionally substituted by one or more substituents, e.g., 1 to 5 substituents, at any point of attachment. Exemplary substituents include, but are not limited to, -halogen, oxo, —OH, — CN, — COOH, — CH2CN, — O— (Ci-Ce)alkyl,(Ci-Ce)alkyl, (Ci-Ce)alkoxy, (Ci-Ce)haloalkyl, (Ci-Ce)haloalkoxy, — O — (C2-Ce)alkenyl, — O— ( C2-C6)alkynyl, (C2-C6)alkenyl, (C2-C6)alkynyl, — OP(O)(OH)2,— OC(O)(Ci-C6)alkyl, — C(O)( Ci-C6)alkyl, — OC(O)O(Ci-C6)alkyl ,— NH2,— NH((Ci-C6)alkyl), — N((Ci-C6)alkyl)2, — NHC(O)(Ci-C6)alkyl,— C(O)NH(Ci-C6)alkyl, — S(O)2(Ci-C6)alkyl, — S(O)NH(Ci-C6)alkyl, and S(O)N((Ci-Ce)alkyl)2. In some embodiments, the term “aryl” refers to an all carbon monocyclic or fused- ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system without any heteroatom ring atoms. An aryl group may be selected from: monocyclic carbocyclic aromatic rings, for example, phenyl; bicyclic ring systems such as 7-12 membered, e.g., 9-10 membered, bicyclic ring systems wherein at least one ring is carbocyclic and aromatic, selected, for example, from naphthalene, indane, and 1,2,3,4-tetrahydroquinoline; and tricyclic ring systems such as 10-15 membered tricyclic ring systems wherein at least one ring is carbocyclic and aromatic, for example, fluorene. For example, the aryl group may be a 6-membered carbocyclic aromatic ring fused to a 5- to 7- membered cycloalkyl or heterocyclic ring optionally comprising at least one heteroatom selected from N, O, and S, provided that the point of attachment is at the carbocyclic aromatic ring when the carbocyclic aromatic ring is fused with a heterocyclic ring, and the point ofattachment can be at the carbocyclic aromatic ring or at the cycloalkyl group when the carbocyclic aromatic ring is fused with a cycloalkyl group. Divalent (used interchangeably with “bivalent”) radicals formed from substituted benzene derivatives and having the free valences at ring atoms are named as substituted phenylene radicals. Divalent radicals derived from univalent polycyclic hydrocarbon radicals whose names end in “-yl” by removal of one hydrogen atom from the carbon atom with the free valence are named by adding “-idene” to the name of the corresponding univalent radical, e.g., a naphthyl group with two points of attachment is termed naphthylidene.

[0042] “Bivalent saturated or unsaturated, straight or branched, hydrocarbon chain,” refers to bivalent alkylene, alkenylene, and alkynylene chains that are straight or branched as defined herein.

[0043] “Bridged bicyclic” refers to any bicyclic ring system, i.e. carbocyclic or heterocyclic, saturated or partially unsaturated, having at least one bridge. As defined by IUPAC, a “bridge” is an unbranched chain of atoms or an atom or a valence bond connecting two bridgeheads, where a “bridgehead” is any skeletal atom of the ring system which is bonded to three or more skeletal atoms (excluding hydrogen). In some embodiments, a 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 those groups set forth below where each group is attached to the rest of the molecule at any substitutable carbon or nitrogen atom. Unless otherwise specified, a bridged bicyclic group is optionally substituted with one or more substituents as set forth for aliphatic groups.Additionally or alternatively, any substitutable nitrogen of a bridged bicyclic group is optionally substituted. Exemplary bridged bicyclics include:

[0044] “Bridged bicyclic” refers to any bicyclic ring system, i.e. carbocyclic or heterocyclic, saturated or partially unsaturated, having at least one bridge. As defined by IUPAC, a “bridge” is an unbranched chain of atoms or an atom or a valence bond connecting two bridgeheads, where a “bridgehead” is any skeletal atom of the ring system which is bonded to three or more skeletal atoms (excluding hydrogen). In some embodiments, a 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 those groups set forth below where each group is attached to the rest of the molecule at any substitutable carbon or nitrogen atom. Unless otherwise specified, a bridged bicyclic group isoptionally substituted with one or more substituents as set forth for aliphatic groups. Additionally or alternatively, any substitutable nitrogen of a bridged bicyclic group is optionally substituted. Exemplary bridged bicyclics include:

[0045] Cycloalkyl, heterocycloalkyl, aryl, and heteroaryl bicyclic (fused, bridged, or spirocyclic) or polycyclic (fused, bridged, or spirocyclic) ring systems are defined based on the nature of the ring system and / or point of attachment. For example, if the entire bicyclic or polycyclic ring system is fully non-aromatic and contains at least one ring heteroatom, then the ring system is a heterocycloalkyl bicyclic or polycyclic ring system. If the entire bicyclic or polycyclic ring system is fully aromatic and contains at least one ring heteroatom, then the ring system is considered a heteroaryl bicyclic or polycyclic ring system. If the bicyclic or polycyclic ring system contains a mix of non-aromatic and aromatic ring systems, then it is the point of attachment that dictates the nature of the ring system: if attached to the non- aromatic cycloalkyl or heterocycloalkyl ring, it is considered a cycloalkyl or heterocycloalkylbicyclic or polycyclic ring system; if it is attached to the aromatic aryl or heteroaryl ring, it is considered an aryl or heteroaryl bicyclic or polycyclic ring system.

[0046] “Cycloalkyl” refers to a non-aromatic saturated or unsaturated, monocyclic, bicyclic, or polycyclic ring system containing 3 to 16 ring carbon atoms, but no heteroatom ring atoms. For example, 3-16 carbons, or 3-8 carbons, referred to herein as “(C3-C8)-cycloalkyl,” derived from a cycloalkane. Cycloalkyl can include any number of carbons, such as C3-6, C4-6, C5-6, C3-8, C4-8, C5-8, Ce-8, C3-9, C3-10, C3-11, and C3-12. Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl rings include, for example, bicyclo[l. l.l]pentane, norbornane, [2.2.2] bicyclooctane, decahydronaphthalene and adamantane. Cycloalkyl groups can also be partially unsaturated, having one or more double or triple bonds in the ring. Representative cycloalkyl groups that are partially unsaturated 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 norbomadiene. When cycloalkyl is a saturated monocyclic C3-8 cycloalkyl, exemplary groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. When cycloalkyl is a saturated monocyclic C3-6 cycloalkyl, exemplary groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups may be substituted with an alcohol (hydroxy), alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxylic acid, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, ketone, nitro, oxo (a carbonyl on the ring), phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide and thioketone. Cycloalkyl groups can be fused to other cycloalkyl (saturated or partially unsaturated), aryl, or heterocyclyl groups, to form a bicycle, tetracycle, etc. In embodiments, the term cycloalkyl is a monocyclic ring. In embodiments, the term “bicyclic cycloalkyl” may refer to a ring system such as a 1,1'- bi(cyclohexyl) ring system:. In embodiments, the term “bicyclic cycloalkyl” may refer to a fused ring system such as a decahydronaphthalene ring system:embodiments, the term “bicyclic cycloalkyl” may refer to a spirocyclic ring system such as:

[0047] “Heteroaryl” refers to a monovalent aromatic radical of 5-, 6-, or 7- membered rings, and includes fused ring systems (at least one of which is aromatic) of 5-20 atoms, containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur. In embodiments, the heteroaryl is a 5-membered heteroaromatic ring. In embodiments, the heteroaryl is a 6-membered heteroaromatic ring. In embodiments, the heteroaryl is a 7- membered heteroaromatic ring. In embodiments, the heteroaryl is a 8-membered heteroaromatic ring. In embodiments, the heteroaryl is a 9-membered heteroaromatic ring. In embodiments, the heteroaryl is a 10-membered heteroaromatic ring. Examples of heteroaryl groups are pyridinyl (including, for example, 2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl (including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, triazolyl, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. In some embodiments, a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring (or in the case of a divalent fused heteroarylene ring system, at least one radical or point of attachment is on a heteroaromatic ring). Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbozolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydrquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3- b]-l,4-oxazin-3(4H)-one. A heteroaryl group may be mono-, bicyclic, or tricyclic. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring,” “heteroarylgroup,” “heteroaromatic,” or “heteroaromatic ring” any of which terms include rings that are optionally substituted. 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 ring system such as a 2-phenylpyridinyl ring system:embodiments, the term “bicyclic heteroaryl” may refer to a fused ring system such as an idolyl ring system:If the bicyclic or polycyclic ring system contains a mix of non-aromatic and aromatic ring systems, then it is the point of attachment that dictates the nature of the ring system: if attached to the non-aromatic cycloalkyl or heterocycloalkyl ring, it is considered a cycloalkyl or heterocycloalkyl bicyclic or polycyclic ring system; if it is attached to the aromatic aryl or heteroaryl ring, it is considered an aryl or heteroaryl bicyclic or polycyclic ring system.

[0048] “Heteroaryl, bicyclic heteroaryl, and / or fused heteroaryl” (or any combination of the preceding) are used alongside in the same embodiment, the terms are mutually exclusive of each other and not considered subgenera of heteroaryl. In other words, when used in the same embodiment, the term heteroaryl refers to a monocyclic ring system whereas a bicyclic or fused heteroaryl refer to polycyclic ring systems.

[0049] “Heterocyclyl,” “heterocyclic,” or “heterocycloalkyl” group refers to a ring structure having from 3 to 14 atoms, for example 4 to 13 atoms, wherein one or more atoms are selected from the group consisting of N, O, and S wherein the ring N atom may be oxidized to N-O, and the ring S atom may be oxidized to SO or SO2, the remainder of the ring atoms being carbon. In embodiments, the heterocycloalkyl is a 3- to 9-membered heterocycloalkyl ring. In embodiments, the heterocycloalkyl is a 4- to 9-membered heterocycloalkyl ring. In embodiments, the heterocycloalkyl is a 5- to 9-membered heterocycloalkyl ring. In embodiments, the heterocycloalkyl is a 6- to 9-membered heterocycloalkyl ring. In embodiments, the heterocycloalkyl group is a 3 -membered ring. In embodiments, the heterocycloalkyl group is a 4-membered ring. In embodiments, the heterocycloalkyl group is a 5-membered ring. In embodiments, the heterocycloalkyl group is a 6-membered ring. Inembodiments, the heterocycloalkyl group is a 7-membered ring. In embodiments, the heterocycloalkyl group is an 8-membered ring. In embodiments, the heterocycloalkyl group is a 9-membered ring. In embodiments, the heterocycloalkyl group is a 10-membered ring. In embodiments, the heterocycloalkyl group is a 11 -membered ring. In embodiments, the heterocycloalkyl group is a 12-membered ring. In embodiments, the heterocycloalkyl group is a 13-membered ring. In embodiments, the heterocycloalkyl group is a 14-membered ring. The heterocyclyl may be a monocyclic, a bicyclic, a tricyclic, a spirocyclic, or a bridged ring system. The heterocyclic group is independently optionally substituted on a ring nitrogen atom with alkyl, aralkyl, alkylcarbonyl, or on sulfur with lower alkyl. Examples of heterocyclic groups include, without limitation, epoxy, azetidinyl, aziridinyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, pyrrolidinonyl, piperidinyl, piperazinyl, imidazolidinyl, thiazolidinyl, dithianyl, trithianyl, dioxolanyl, oxazolidinyl, oxazolidinonyl, decahydroquinolinyl, piperidonyl, 4-piperidinonyl, quinuclidinyl, thiomorpholinyl, morpholinyl, azepanyl, oxazepanyl, azabicyclohexanyls, azabicycloheptanyl, azabicyclooctanyls, azabicyclononanyls (e.g., octahydroindolizinyl), azaspiroheptanyls, dihydro- 1 H,3 H.5 / / -oxazolo[3 ,4-c]oxazolyl, tetrahydro- 1spirofcyclopropane- 1 ,2'- pyrrolizine], hexahydro- I / / -pyrrol izinyl, hexahydro- l / / -pyrrolo[2, l - c][l,4]oxazinyl, octahydroindolizinyl, oxaazaspirononanyls, oxaazaspirooctanyls, diazaspirononanyls, oxaazabiocycloheptanyls, hexahydropyrrolizinyl 4(l / 7)-oxide, tetrahydro- 2H-thiopyranyl 1- oxide and tetrahydro-2 / / -thiopyranyl 1,1 -di oxide. Specifically excluded from the scope of this term are compounds having adjacent annular O and / or S atoms. In embodiments, the term “bicyclic heterocycloalkyl” may refer to a ring system such as a l-(piperidin-4- yljpiperazinyl ring system:embodiments, the term “bicyclic heterocycloalkyl” may refer to a fused ring system such as a 3, 4-dihydro-2 / / - l -benzopyran:embodiments, the term “bicyclic heterocycloalkyl” may refer to a heterospirocyclic ring system such as:f the bicyclic or polycyclic ring system contains a mix of non-aromatic and aromatic ring systems, then it is the point of attachment that dictates the nature of the ring system: if attached to the non-aromatic cycloalkyl or heterocycloalkyl ring, it is considered a cycloalkyl or heterocycloalkyl bicyclic or polycyclic ring system; if it is attached to the aromatic aryl or heteroaryl ring, it is considered an aryl or heteroaryl bicyclic or polycyclic ring system.

[0050] “Heterocycloalkyl, bicyclic heterocycloalkyl, fused heterocycloalkyl, and / or hetero- spirocyclyl” (or any combination of the preceding) are used alongside in the same embodiment, the terms are mutually exclusive of each other and not considered subgenera of heterocycloalkyl. In other words, when used in the same embodiment, the term heterocycloalkyl refers to a monocyclic ring system whereas a bicyclic or fused heterocycloalkyl refer to polycyclic ring systems.

[0051] “Polycyclic,” as used herein, refers to a system comprising several closed rings. In some embodiments, polycyclic refers to a 11- to 20-membered carbocyclic or heterocyclic ring.

[0052] The substituents are themselves optionally substituted. Furthermore, when containing two fused rings, the aryl groups optionally have an unsaturated or partially saturated ring fused with a fully saturated ring. Exemplary ring systems of these aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl, anthracenyl, phenalenyl, phenanthrenyl, indanyl, indenyl, tetrahydronaphthalenyl, tetrahydrobenzoannulenyl, and the like.

[0053] Halogen or “halo” mean fluorine, chlorine, bromine, or iodine.

[0054] “Haloalkyl” refers to an alkyl group substituted with one or more halogens. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, pentafluoroethyl, trichloromethyl, etc.

[0055] ‘ ‘Heteroatom” means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quatemized form of any basic nitrogen or a substituted nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR+ (as in N-substituted pyrrolidinyl)). In embodiments, the heteroatom is selected from oxygen, sulfur, nitrogen, andphosphorus. In another embodiment, the heteroatom is selected from oxygen, sulfur, and nitrogen. In embodiments, the heteroatom is independently oxygen or nitrogen.

[0056] “Alkyl” means a straight or branched chain saturated hydrocarbon containing 1-12 carbon atoms. Examples of a (Ci-Ce) alkyl group 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 or branched hydrocarbon, such as a straight or branched group of 1 to 8 carbon atoms, referred to herein as Ci-8 alkyl. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-l -propyl, 2-methyl-2-propyl, 2-methyl-l-butyl, 3 methyl- 1 -butyl, 2- methyl-3 -butyl, 2,2-dimethyl-l -propyl, 2-methyl-l -pentyl, 3 methyl- 1 -pentyl, 4-methyl-l- pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4 methyl-2-pentyl, 2,2-dimethyl-l -butyl, 3,3- dimethyl-1 -butyl, 2-ethyl-l -butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl. In some embodiments, “alkyl” is a straight-chain hydrocarbon. In some embodiments, “alkyl” is a branched hydrocarbon. When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example, “Ci-Ce alkyl” is intended to encompass Ci, C2, C3, C4, Cs, Ce, C1-6, C1-5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6 alkyl.

[0057] “Aliphatic” or “aliphatic group” refers to straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocyclyl,” “cycloaliphatic,” or “cycloalkyl”), that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. In some embodiments, “cycloaliphatic” (or “carbocyclyl” or “cycloalkyl”) refers to a monocyclic C3-C7 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to,linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0058] “Alkoxy” means a straight or branched chain saturated hydrocarbon containing 1-12 carbon atoms containing a terminal “O” in the chain, e.g., — O(alkyl). Examples of alkoxy groups include, without limitation, methoxy, ethoxy, propoxy, butoxy, t-butoxy, or pentoxy groups. In some embodiments, the term “alkoxy” refers to a straight or branched chain saturated hydrocarbon containing 1-12 carbon atoms containing a terminal “O” in the chain, e.g., -O(alkyl). Examples of alkoxy groups include, without limitation, methoxy, ethoxy, propoxy, butoxy, t-butoxy, or pentoxy groups.

[0059] “Alkenyl” means a straight or branched chain unsaturated hydrocarbon containing 2- 12 carbon atoms. The “alkenyl” group contains at least one double bond in the chain. The double bond of an alkenyl group can be unconjugated or conjugated to another unsaturated group. Examples of alkenyl groups include ethenyl, propenyl, n-butenyl, isobutenyl, pentenyl, or hexenyl. An alkenyl group can be unsubstituted or substituted and may be straight or branched. In some embodiments, “alkenyl” refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond, such as a straight or branched group of 2-8 carbon atoms, referred to herein as (C2-C8)-alkenyl. Exemplary alkenyl groups include, but are not limited to, vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl, and 4-(2-methyl-3-butene)-pentenyl.

[0060] “Alkylene” refers to a divalent alkyl group. An “alkylene chain” is a polymethylene group, i.e., -(CH2)n-, wherein n is a positive integer, for example, from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.

[0061] “Alkenylene” refers to a bivalent alkenyl group. A substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.

[0062] “Alkynyl” means a straight or branched chain unsaturated hydrocarbon containing 2-12 carbon atoms. The “alkynyl” group contains at least one triple bond in the chain. Examplesof alkenyl groups include ethynyl, propargyl, n-butynyl, isobutynyl, pentynyl, or hexynyl. An alkynyl group can be unsubstituted or substituted.

[0063] “Cycloalkyl” or “carbocyclyl” means a monocyclic or polycyclic saturated carbon ring containing 3-18 carbon atoms. Examples of cycloalkyl groups include, without limitations, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptanyl, cyclooctanyl, norboranyl, norborenyl, bicyclo[2.2.2]octanyl, or bicyclo[2.2.2]octenyl and derivatives thereof. A (Cs-Cs) cycloalkyl is a cycloalkyl group containing between 3 and 8 carbon atoms. A cycloalkyl group can be fused (e.g., decalin) or bridged (e.g., norbomane). In some embodiments, “cycloalkyl, bicyclic cycloalkyl, fused bicyclic cycloalkyl, and / or spirocyclyl” (or any combination of the preceding) are used alongside in the same embodiment, the terms are mutually exclusive of each other and not considered subgenera of cycloalkyl. In other words, when used in the same embodiment, the term cycloalkyl refers to a monocyclic ring system whereas a bicyclic or fused cycloalkyl refer to polycyclic ring systems.

[0064] “Haloalkyl” means an alkyl group substituted with one or more halogens. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, pentafluoroethyl, tri chloromethyl, etc.

[0065] “Haloalkoxy” means an alkoxy group substituted with one or more halogens. Examples of haloalkyl groups include, but are not limited to, trifluoromethoxy, difluoromethoxy, pentafluoroethoxy, trichloromethoxy, etc.

[0066] As used herein and unless otherwise specified, the suffix “-ene” is used to describe a divalent group. Thus, any of the terms above can be modified with the suffix “-ene” to describe a divalent version of that moiety. For example, a divalent carbocycle is “carbocyclylene,” a divalent aryl ring is “arylene,” a divalent benzene ring is “phenylene,” a divalent heterocycle is “heterocyclylene,” a divalent heteroaryl ring is “heteroarylene,” a divalent alkyl chain is “alkylene,” a divalent alkenyl chain is “alkylene,” a divalent alkynyl chain is “alkynylene,” and so forth.

[0067] The term “pharmaceutically acceptable” as used herein refers to molecular entities and compositions that are physiologically tolerable and do not typically produce toxicity or an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0068] As used herein the “therapeutic index” is a parameter expressing the therapeutic efficiency of the active drug. It is for example low when implying that high concentration of the active substance is needed to achieve therapeutic efficacy or when the dose required obtaining efficacy induce toxicity. On the contrary, high therapeutic index implies that the dose required of the active substance to provide therapeutic efficacy is low and / or when toxicity of the active drug is low.

[0069] “Linker” refers to a fragment connecting a ligand to a viral capsid protein, comprising a crosslinked moiety.

[0070] “Crosslinking” refers to a process of chemically joining two or more molecules by a covalent bond. In some embodiments, crosslinking refers to a reaction between a first member of a crosslinker reactive pair and a second member of a crosslinker reactive pair, wherein the first and second members of the crosslinker reactive pair react to form a crosslinked moiety.

[0071] “Copper-free click chemistry” refers to a biorthogonal reaction, which does not use copper catalysts. In some embodiments, the process refers to reactions that proceed through a substrate (or crosslinker reactive pair member) that overcomes the need for an exogenous catalyst such as copper.

[0072] ‘ ‘Crosslinked moiety” refers to a moiety formed by the reaction between a crosslinker reactive pair.

[0073] “Enzymatically cleaved” refers to a peptide bond being broken between two amino acids that the enzyme recognizes by its secondary structure.

[0074] “Ligand” refers to a molecule that is capable of conjugation to the viral capsid surface, which binds to a receptor (a protein molecule) in a cell.

[0075] “Spacer” refers to a moiety connecting a surface functionalized viral capsid with a capsid-reactive linker and / or a ligand with a ligand-reactive linker.

[0076] ‘ ‘Cell-type specific ligand” refers to a ligand capable of binding to a specific receptor on the cell surface.5.2. Other interpretational conventions

[0077] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0078] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an antibody or antigen binding fragment” includes a plurality of such antibodies and antigen binding fragments and reference to “the recombinant adeno-associated virus” includes reference to one or more recombinant adeno-associated viruses and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation.

[0079] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0080] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. The dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0081] Where a range of values is provided, it is understood that the recited endpoints of the range are included. In addition, each intervening value, to the tenth of the unit of the lowerlimit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0082] Ranges recited herein are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 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 from 11 to 48 or 39 to 41.

[0083] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10% of a stated value or of a stated limit of a range.5.3. Surface-Modified Viral Capsid

[0084] In one aspect, a surface modified viral capsid is provided, wherein the surface modified viral capsid comprises one or more of a ligand covalently conjugated to a viral capsid protein via a linker, the linker comprising a crosslinked moiety, wherein the crosslinked moiety is formed by a reaction between first and second members of a crosslinker reactive pair; and optionally one or more spacers.

[0085] In accordance with the present disclosure, in a first aspect a surface-modified viral capsid, typically an AAV viral capsid, is provided. The surface modification comprises a first ligand that facilitates passage of the rAAV from CSF into the brain parenchyma, optionally with one or more additional surface-conjugated ligands that facilitate transduction of CNS cells, optionally transduction of one or more desired CNS cell types. In some embodiments, the first ligand is covalently conjugated to a viral capsid protein. In certain of these embodiments, the first ligand is covalently conjugated to the viral capsid protein via a linker comprising a crosslinked moiety, Q. In other embodiments, the first ligand is fused inframe in the capsid protein sequence. In another aspect, recombinant virions, typicallyrecombinant AAV virions (rAAV), that comprise the surface-modified viral capsid and an encapsidated nucleic acid payload are provided.5.3.1. Direct surface-modification via ligand conjugation

[0086] In accordance with the present disclosure, a surface functionalized viral capsid is provided comprising a first member of a crosslinker reactive pair. Also provided is a functionalized ligand comprising a second member of a crosslinker reactive pair, wherein the first and second members of the crosslinker reactive pair react to form a crosslinked moiety, Q. The surface functionalized viral capsid is capable of being crosslinked, i.e., conjugated, to a ligand having a complementary member of a crosslinker reactive pair.

[0087] In some embodiments, the surface modified viral capsid in a composition comprises x conjugated ligands where x is the average number of ligands conjugated per capsid in a composition, also referred to herein as the ligand per capsid ratio or LCR. In some embodiments, x is from 1 to 500. In certain embodiments x is from 1 to 300. In certain embodiments x is from 100 to 200. In certain embodiments x is from 110 to 190. In certain embodiments x is from 130 to 170. In some embodiments, 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 preceding numbers. In certain embodiments, x is about 1350, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175 or about 180. In certain embodiments, x is from about 55 to about 85. In certain embodiments, x is from about 140 to about 160. In certain embodiments, x is from about 135 to about 165. In certain embodiments, x is from about 130 to about 170. In certain embodiments, x is about 150. In certain embodiments, x is in a range between any two of numbers the provided above.

[0088] Also provided by the present disclosure are capsid-reactive linkers comprising (i) a capsid reactive moiety that is capable of covalent attachment to a viral capsid protein, and (ii) a member of a crosslinker reactive pair.

[0089] Also provided by the present disclosure are recognition sequence-reactive linkers comprising (i) an enzyme substrate reactive moiety that is capable of participating in an enzymatic ligation to a recognition sequence in a viral capsid protein, and (ii) a member of a crosslinker reactive pair.

[0090] In embodiments of the present disclosure, a surface modified viral capsid according to the present disclosure is produced by the steps of: obtaining a surface functionalized viral capsid by reacting a viral capsid protein with a capsid-reactive linker comprising a first member of a crosslinker reactive pair and optionally one or more of a spacer; and conjugating the surface functionalized viral capsid with a functionalized ligand comprising a second member of the crosslinker reactive pair, wherein the first and second members of the crosslinker reactive pair react to form a crosslinked moiety, Q; and obtaining the surface modified viral capsid.

[0091] In other embodiments, a method of making a surface modified viral capsid according to the present disclosure comprises the steps: obtaining a surface functionalized viral capsid by enzymatic ligation between: a recognition sequence located in a surface loop region in a viral capsid protein; and an enzyme-reactive linker; wherein the linker comprises an enzyme substrate and a first member of a crosslinker reactive pair; and reacting the obtained surface functionalized viral capsid with a functionalized ligand comprising a second member of the crosslinker reactive pair.

[0092] In certain embodiments, the method further comprises the step of producing an AAV capsid comprising at least one viral capsid protein comprising at least one recognition sequence. Optionally, the recognition sequence is located in a surface loop region, such as variable region 4 or 8.

[0093] In other embodiments, a method of making a surface modified viral capsid according to the present disclosure comprises the steps: obtaining a surface functionalized viral capsid by enzymatic ligation between:a recognition sequence located in a surface loop region in a viral capsid protein; and a functionalized ligand comprising an enzyme substrate; and reacting the obtained surface functionalized viral capsid with a functionalized ligand in the presence of the enzyme.

[0094] In an aspect of the present disclosure, it is contemplated that the methods described herein are conducted in sequence to produce a surface modified virus comprising two or more different ligands attached to the surface of the virus, e.g., using different surface functionalization strategies as described herein or repeating the same strategy with different ligands. That is to say that a surface modified viral particle can be used as a starting material in any of the above methods to modify the surface of the viral particle with a second (and third and so on) type of ligand.5.3.1.1 Crosslinker Reactive Pair

[0095] Exemplary crosslinker reactive pairs useful according to certain embodiments, include, e.g., those as disclosed in WO 2022 / 101363, the disclosure of which is herein expressly incorporated by reference in its entirety.

[0096] In embodiments of the present disclosure, to effect covalent attachment of a ligand to a viral capsid surface and thereby produce a surface modified viral capsid, recombinant virions are first surface-functionalized to produce surface functionalized viral capsids, which are then crosslinked with a functionalized ligand. The surface functionalized capsid and functionalized ligand each comprise a member of a crosslinker reactive pair. The crosslinker reactive pair members react to form a moiety, Q, that covalently cross-links the viral capsid to the ligand.

[0097] In some embodiments, the first and second members of the crosslinker reactive pair participate in a reaction selected from: a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction, a strain- promoted alkyne-nitrone cycloaddition (SPANC) reaction, an inverse electron demand Diels- Alder (IEDDA) reaction, and a Staudinger ligation and a [4+1] cycloaddition reaction. In some embodiments, the first and second members of the crosslinker reactive pair participate in a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. In some embodiments,the first and second members of the crosslinker reactive pair participate in a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. In some embodiments, the first and second members of the crosslinker reactive pair participate in a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction. In some embodiments, the first and second members of the crosslinker reactive pair participate in an inverse electron demand Diels-Alder (IEDDA) reaction. In some embodiments, the first and second members of the crosslinker reactive pair participate in a Staudinger ligation and a [4+1] cycloaddition reaction.

[0098] In some embodiments, the first and second members of the crosslinker reactive pair participate in a reaction selected from: a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction, a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction, an inverse electron demand Diels-Alder (IEDDA) reaction, and a Staudinger ligation and a [4+1] cycloaddition reaction.

[0099] In typical embodiments, the crosslinker reactive pair members are bioorthogonal. As used herein, the term bioorthogonal chemistry refers to any chemical process that can occur inside of living systems without interfering with native biochemical processes or can occur in vitro without interfering with biochemical / biological activity of the reaction products. A number of chemical conjugation strategies have been developed that fulfill the requirements of bioorthogonality, including the 1,3 -dipolar cycloaddition between azides and cyclooctynes (also termed copper-free click chemistry), between nitrones and cyclooctynes, oxime / hydrazone formation from aldehydes and ketones, the tetrazine ligation, e.g., the cycloaddition of s-tetrazine and trans-cyclooctene derivatives or isocyanide-based click reaction, and most recently, the quadricyclane ligation.5.3.1.1.1 CuAAC

[0100] In certain embodiments, the crosslinker reactive pair is selected from chemical moieties that participate in a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). In certain embodiments, the crosslinker reactive pair comprises an azide and an alkyne. Derivatives of these moieties that retain the desired chemical reactivity are also contemplated herein. In certain embodiments, the crosslinked moiety Q comprises a 5-membered heteroatom ring. In certain embodiments, the crosslinked moiety Q comprises a 1,4 triazole.5.3.1.1.2 SPAAC and SPANC

[0101] Unlike CuAAC, Cu-free click chemistry has been modified to be bioorthogonal by eliminating a cytotoxic copper catalyst, allowing reaction to proceed quickly and without live cell toxicity. Instead of copper, the reaction is a strain-promoted alkyne-azide cycloaddition (SPAAC). Copper-free click chemistry has been adapted to use nitrones as the 1,3 -dipole rather than azides and has been used in the modification of peptides.

[0102] In certain embodiments, the crosslinker reactive pair is selected from chemical moieties that participate in a strain-promoted alkyne-nitrone cycloaddition (SPANC). In certain embodiments, the crosslinker reactive pair comprises an azide and a nitrone. Derivatives of these moieties that retain the desired chemical reactivity are also contemplated herein. In certain embodiments, the crosslinked moiety Q comprises an isoxazoline.

[0103] In some embodiments, the crosslinker reactive pair is an azide and a nitrone, as illustrated below, where the R group represents the point of attachment to the capsid-reactive linker or functionalized ligand. Derivatives of these moieties that retain the desired chemical reactivity are also contemplated herein. For example, substitution on both the carbon and nitrogen atoms of the nitrone dipole, and acyclic and endocyclic nitrones are all tolerated.RN — N+=N'azide nitrone

[0104] In some embodiments, the crosslinker reactive pair comprises a cyclooctyne analogue. In certain embodiments, the crosslinker reactive pair comprises a cyclooctyne analogue, e.g., those illustrated below where the R group represents the point of attachment to the capsid- reactive linker or functionalized ligand. Derivatives of these moieties that retain the desired chemical reactivity are also contemplated herein.BCN TMTH

[0105] In certain embodiments, the crosslinker reactive pair comprises a dibenzylcyclooctyne analog selected from the group dibenzylcyclooctyne (DIBO), dibenzoazacyclooctyne (DIBAC or DBCO), and biarylazacyclooctynone (BARAC). Derivatives of these moieties that retain the desired chemical reactivity are also contemplated herein.

[0106] In certain embodiments, the crosslinker reactive pair comprises a nitrone according to the structure below, where the R1group represents the point of attachment to the capsid- reactive linker or functionalized ligand. R2and R3are not particularly limited. In some embodiments, R2and R3are independently selected from hydrogen and C1-C4 alkyl groups such as methyl, ethyl, propyl and butyl groups. Derivatives of these moieties that retain the desired chemical reactivity are also contemplated herein.

[0107] In certain embodiments, the crosslinker reactive pair comprises a dibenzylcyclooctyne analog as identified above and either a 1,3 -nitrone or an azide. In certain embodiments, the crosslinker reactive pair comprises a dibenzylcyclooctyne (or analog thereof) and either a 1,3-nitrone or an azide, as shown below, where the R1group represents the point of attachment to a viral capsid or a capsid-reactive linker, and wherein R2 group on either the azide or the nitrone represents the point of attachment to a functionalized ligand. Inalternative embodiments, the crosslinker reactive pair comprises a dibenzylcyclooctyne (or analog thereof) and either a 1,3 -nitrone or an azide, as shown below, where the R1group represents the point of attachment to a ligand and wherein R2group on either the azide or the nitrone represents the point of attachment to a surface functionalized viral capsid or a capsid- reactive linker.

[0108] In certain embodiments, the crosslinked moiety Q comprises a cyclic moiety according to any one of those illustrated below, where R1and R2represent the point of attachment to the viral capsid. R3and R4may be H or any substituent described herein, provided the substituted derivatives retain the desired chemical reactivity are also contemplated herein.IEDDA

[0109] In certain embodiments, the crosslinker reactive pair comprises chemical moieties that participate in an inverse electron demand Diels-Alder (IEDDA) reaction. In certain embodiments, the crosslinker reactive pair comprises an electron poor diene and an electron rich dienophile. Examples of such groups are known in the art and described elsewhere, for example, F. Thalhammer, et al., Tetrahedron Lett., 1990, 31, 6851-6854; and B. L. Oliveira, Chem. Soc. Rev., 2017, 46, 4895-4950. In some embodiments, the electron poor diene has an electron withdrawing group substituted on the diene as exemplified below. In some embodiments, the electron rich dienophile has an electron donating group substituted on the dienophile, as exemplified below.Inverse electron demandElectron poor dieneElectron rich dienophile

[0110] In certain embodiments, the crosslinker reactive pair comprises chemical moieties that participate in a Diels-Alder [4+2]-cycloaddition, the reaction between a diene and a dienophile to form a six-membered ring in a 7i4s + 7i2s fashion via suprafacial / suprafacial interaction of 47i-electrons of the diene with the 27i-electrons of the dienophile. In contrast to a normal electron demand Diels-Alder reaction, where an electron-rich diene reacts with an electron-poor dienophile, in an inverse-electron-demand Diels- Alder reaction (IEDDA), an electron-rich dienophile reacts with an electron-poor diene. Alkyne dienophiles directly yield the respective pyridazine upon reaction.

[0111] In certain embodiments, the crosslinker reactive pair comprises a triazine (e.g., 1, 2, 4 triazine), a tetrazine (Tz) (e.g., 1,2,4,5-tetrazines, also referred to as an s-tetrazine) or a strained dienophile such as noroborene, transcyclooctene (TCO), cyclopropene, or N- acylazetine. In certain embodiments, the crosslinker reactive pair comprises a crosslinker reactive moiety exemplified below, where the R group represents the point of attachment to a capsid-reactive linker, surface functionalized viral capsid, or functionalized ligand of the present disclosure. Derivatives of these moieties that retain the desired chemical reactivity are also contemplated herein. In certain embodiments, the crosslinker reactive pair comprises TCO and tetrazine.norbornene transcyclooctene cyclopropene N-acylazetine5.3.1.1.1 Staudinger ligation

[0112] In certain embodiments, the crosslinker reactive pair is selected from a crosslinker reactive moiety that participates in a Staudinger reaction such as an azide, a phosphine (PPI12) or phosphite that are able to react to produce an iminophosphorane.

[0113] In certain embodiments, the crosslinker reactive moiety is a triphenylphosphine, such as the triphenylphosphine shown below where the R group represents the point of attachment to the capsid-reactive linker of the present disclosure. Derivatives of this crosslinker reactive moiety that retains the desired chemical reactivity is also contemplated herein.crosslinker reactive moiety5.3.1.1.2 [4+1] Cycloaddition

[0114] In certain embodiments, the crosslinker reactive pair is selected from a crosslinker reactive moiety that participates in an a [4+1] cycloaddition followed by a retro-Diels Alder elimination of N2, e.g., an isocyanide or a 1,2, 4, 5, tetrazine.

[0115] In some embodiments, the crosslinker reactive moiety is an isocyanide as shown below, where the R1group represents the point of attachment, e.g., to the capsid-reactive linker. In some embodiments, the crosslinker reactive moiety is a 1,2, 4, 5, tetrazine as shown below, where R2group represents the point of attachment, e.g., to the ligand. Derivatives of these moieties that retain the desired chemical reactivity are also contemplated herein.R1— N+=C5.3.1.1.1 Tag reactions

[0116] In certain embodiments, the crosslinker reactive moiety is a bioorthogonal tag known in the art, such as a SNAP -tag, a CLIP tag, a Halo-tag, or LUMIO-tag or a chemical group that reacts with these tags, e.g., benzylguanine group, a benzylcytosine group, or a chloroalkane group. In certain embodiments, one member of the crosslinker reactive moiety comprises a SNAP -tag and the other member of the crosslinker reactive moiety comprises a benzylguanine group.5.3.1.2 Crosslinked Moiety - Q

[0117] In an aspect of the present disclosure, the surface modified viral capsid comprises a moiety, Q, that is a moiety formed by the reaction between a crosslinker reactive pair as described herein. In some embodiments, the first and second members of the crosslinker reactive pair participate in a reaction selected from: a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction, a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction, an inverse electron demand Diels-Alder (IEDDA) reaction, and a Staudinger ligation and a [4+1] cycloaddition reaction. In some embodiments, the first and second members of the crosslinker reactive pair participate in a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. In some embodiments, the first and second members of the crosslinker reactive pair participate in a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. In some embodiments, the first and second members of the crosslinker reactive pair participate in a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction. In some embodiments, the first and second members of the crosslinker reactive pair participate in an inverse electron demand Diels-Alder (IEDDA) reaction. In some embodiments, the first and second members of the crosslinker reactive pair participate in a Staudinger ligation and a [4+1] cycloaddition reaction.

[0118] In certain embodiments, Q comprises the product of a CuAAC reaction. In certain embodiments, Q comprises the product of a SPAAC reaction. In certain embodiments, Q is the product of a SPANC reaction. In certain embodiments, Q comprises the product of an IEDDA reaction. In certain embodiments, Q comprises the product of a Staudinger ligation. In certain embodiments, Q comprises the product of a [4+1] cycloaddition reaction. In some embodiments, Q comprises the product of a strain promoted reaction, e.g., SPAAC, SPANC, and IEDDA.

[0119] In some embodiments, the crosslinked moiety comprises at least one of an eight membered ring and a triazole ring.

[0120] In certain embodiments, Q comprises a cyclic group. In certain embodiments, Q comprises a bicyclic group. In certain embodiments, Q comprises a tricyclic group. In some embodiments, Q is a fused polycyclic ring. In certain embodiments, Q comprises a 5-8 membered carbocyclic ring comprising from 0 to 3 heteroatoms selected from O, S or N. In certain embodiments, Q comprises an eight membered ring comprising 0 to 1 heteroatomselected from O and N. In certain embodiments, Q comprises a five membered ring comprising 0 to 3 heteroatoms selected from O and N. In certain embodiments, Q is a triazole ring. In certain embodiments, Q comprises a six membered ring comprising 0-3 heteroatoms selected from O and N. In certain embodiments, Q comprises a six membered ring comprising 2 N heteroatoms.

[0121] In some embodiments, the crosslinker reactive pair comprises a cyclooctyne and an azide.

[0122] In some embodiments, the crosslinker reactive pair comprises an optionally substituted cyclooctyne and an azide.

[0123] In some embodiments, the crosslinker reactive pair comprises an optionally substituted cyclooctyne and a nitrone.

[0124] In some embodiments, the cyclooctyne is selected from dibenzylcyclooctyne (DIBO), dibenzoazacyclooctyne (DBCO), and biarylazacyclooctynone (BARAC), or a derivative thereof. In some embodiments, the cyclooctyne is dibenzylcyclooctyne (DIBO). In some embodiments, the cyclooctyne is dibenzoazacyclooctyne (DBCO). In some embodiments, the cyclooctyne is biarylazacyclooctynone (BARAC).

[0125] In some embodiments, where Q comprises a cyclic group, Q is according to a structure below, where Z is a 7 or 8 membered carbocycle comprising from 0-3 heteroatoms selected from O or N. In some embodiments, Z is optionally substituted with one or more arylene. In some embodiments, Z is unsubstituted. In some embodiments, Z is substituted with one arylene. In some embodiments, Z is substituted with two arylenes. In some embodiments, the arylenes are independently selected. In some embodiments, the arylenes are identical.

[0126] In some embodiments, Q comprises the reaction product of a cyclooctyne reactive moiety and an azide reactive moiety.

[0127] In some embodiments, the crosslinked moiety comprises the following structure:

[0128] wherein R1and R2indicate the points of attachment to the linker.

[0129] In some embodiments, Q comprises one of the following structures:

[0130] In some embodiments, the crosslinker reactive pair comprises a transcyclooctene and a tetrazine.

[0131] In some embodiments, the crosslinked moiety (Q) comprises the following structure:wherein R1and R2indicate the points of attachment to the linker.

[0132] In some embodiments, the crosslinked moiety (Q) comprises a phosphine oxide derivative having the following structure:wherein R1and R2indicate the points of attachment to the linker.

[0133] In some embodiments, the crosslinked moiety (Q) comprises a 4H-pyrazol-4-imine derivative having the following structure:wherein R1and R2indicate the points of attachment to the linker.

[0134] In some embodiments, Q comprises a structure below:5.3.1.3 Surface Functionalized Capsid

[0135] In an aspect of the present disclosure, a surface functionalized capsid is provided wherein the surface of the capsid is functionalized to comprise a member of a crosslinker reactive pair, i.e., a crosslinker reactive moiety.

[0136] In certain embodiments, an amino acid in a capsid protein primary sequence is functionalized by reaction with a capsid-reactive linker, as provided herein. In certain embodiments, a genetically introduced in-frame enzyme recognition sequence (e.g., abioorthogonal tag) in a capsid protein primary sequence is functionalized by enzymatic reaction with an enzyme-reactive linker (a recognition sequence-reactive linker), as provided herein. In certain embodiments, a capsid protein primary sequence comprises a non-natural amino acid, wherein the non-natural amino acid comprises a crosslinker reactive moiety.5.3.1.3.1 Capsid-Reactive Linker

[0137] Exemplary capsid-reactive linkers useful according to certain embodiments, include, e.g., those as disclosed in patent publication WO 2022 / 101363, the disclosure of which is herein expressly incorporated by reference in its entirety.

[0138] The capsid-reactive linker, in accordance with the present disclosure, comprises i) a capsid surface reactive moiety available to form a covalent attachment with the capsid surface as described herein, and ii) a member of a crosslinker reactive pair or a ligand. In some embodiments, a first member of a crosslinker reactive pair is selected to be mutually reactive with a ligand functionalized with a second member of the crosslinker reactive pair as described in the present disclosure.5.3.1.3.2 Spacer

[0139] The capsid-reactive linker optionally further comprises one or more spacer moiety. The spacer moiety is not particularly limited and may be any spacer known in the art. In accordance with the present disclosure, the spacer is not particularly limited and includes any divalent moiety that is able to covalently attach to the linker. In some embodiments, the divalent moiety comprises alkylene, alkenylene, alkynylene, arylene, heteroarylene, fused arylene, fused heteroarylene, cycloalkylene, heterocyclylene, fused cycloalkylene, or fused heterocyclylene.

[0140] In some embodiments the spacer comprises one or more monomers of ethylene glycol, i.e., polyethylene glycol, -(O-CH2-CH2)n- or [PEG]n, also known as “dPEGn” for “discrete polyethylene glycol”, where “n” is the number of ethylene oxide (or “ethylene glycol”) units. In certain embodiments, n is 0. In certain 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. In certain embodiments, the spacer is [PEG]n where n is 4, 8 or 12. In certain embodiments the spacer is [PEG]nwhere n is 4 and is understood to comprise four -(O-CH2-CH2)- groups without limitation to the relative position of the ether oxygen.

[0141] In some embodiments, the spacer comprises one or more divalent groups such as - CH2- (methylene), -O- (ether), -C(=O)- (carbonyl), and -N(R)- (amine, where R is H or C1-3 alkyl).

[0142] In some embodiments, the spacer comprises one or more -Y-C(O)-, -Y-C(O)O-, -Y- NHC(O)-, -Y-NHC(S)-, or -Y-C(O), wherein Y is a bond or one or more monomers of ethylene glycol.

[0143] In some embodiments, the spacer comprises one or -([PEG]n)-C(O)-, wherein n is from 1 to 100, such as, for example, from 1 to 25, from 1 to 20, from 1 to 15, from 1 to 10, from 2 to 25, from 2 to 20, from 2 to 15, from 2 to 10, from 4 to 15, or from 4 to 10.

[0144] In some embodiments, the spacer comprises one or more -(CH2)n-, wherein n is from 1 to 100, such as, for example, from 1 to 25, from 1 to 20, from 1 to 15, from 1 to 10, from 2 to 25, from 2 to 20, from 2 to 15, from 2 to 10, from 4 to 15, or from 4 to 10. In certain 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, 100.5.3.1.3.3 Capsid Surface Reactive Moiety

[0145] In accordance with the present disclosure, the capsid surface reactive moiety is not particularly limited and includes any moiety that is able to covalently attach to the desired capsid surface.

[0146] In some embodiments, the capsid surface reactive moiety covalently attaches to a surface exposed amino acid residue in the capsid protein primary sequence using known techniques in residue specific protein labeling.

[0147] In some embodiments, the amino acid residue is present in the wild-type capsid protein. In some embodiments the capsid protein is a known variant of a wild-type capsid protein.

[0148] In some embodiments, the amino acid residue is a naturally occurring residue. In particular of these embodiments, the primary sequence of the capsid protein is wild-type or includes regions have been genetically engineered.

[0149] In other embodiments, the amino acid residue is engineered into the primary amino acid sequence of the capsid.i. Capsid Surface Primary Amine

[0150] In some embodiments, the capsid surface reactive moiety comprises a chemical group that reacts with primary amines (-NH2). Primary amines exist at the N-terminus of each capsid protein and in the side chain (epsilon) of lysine (Lys, K) amino acid residues in the capsid protein sequence.

[0151] Exemplary chemical groups that react with primary amines include isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters. Most of these conjugate to amines by either acylation or alkylation. In some embodiments, the capsid surface reactive moiety comprises an NHS ester or an imidoester, e.g., such as those illustrated below where the R group represents the point of attachment to remainder of the capsid-reactive linker (further comprising a member of a crosslinker reactive pair as defined herein or a reaction product thereof).R— N=C=SIsothiocyanateR— N=C=OIsocyanate Sulfonyl Chloride Aldehyde CarbodiimideNHS Ester Imidoester Epoxide Fluorophenyl Ester

[0152] In some embodiments, the capsid surface reactive moiety covalently attaches to a surface exposed lysine residue of the capsid protein primary sequence. In certain of these embodiments, the capsid surface reactive moiety comprises an NHS-ester, a fluorophenyl ester (e.g. TFP), an isocyanate, an isothiocyanate, or a benzyl fluoride as shown below, wherethe R group represents the point of attachment to the remainder of the capsid-reactive linker (further comprising a member of a crosslinker reactive pair as defined herein or a reaction product thereof) and thesymbols denote the points of attachment of the lysine residue within the capsid protein sequence.Isothiocyanates

[0153] In some embodiments, the capsid-reactive linker comprises an N-hydroxysuccinimide ester (NHS ester). NHS esters are reactive groups formed by carbodiimide-activation of carboxylate molecules. The NHS ester-activated capsid-reactive linker reacts with primary amines in physiologic to slightly alkaline conditions (pH 7.2 to 9) to yield stable amide bonds. The reaction releases N-hydroxysuccinimide (NHS).

[0154] In some embodiments, the capsid-reactive linker comprises a fluorophenyl ester, e.g., tetrafluorophenyl (TFP) or pentaflurophenyl ester (PFP). Fluorophenylesters are reactive groups formed by carbodiimide-activation of carboxylate molecules. Fluorophenylester ofcarboxylic acids react with primary amines forming a covalent amide bond that is identical to one formed by the reaction between primary amines and NHS esters. ii. Capsid Surface Sulfhydryl Group

[0155] In some embodiments, the capsid surface reactive moiety covalently attaches to a surface exposed sulfhydryl group. In some embodiments, the capsid surface reactive moiety covalently attaches to a surface exposed cysteine residue of the capsid protein primary sequence.

[0156] In certain of these embodiments, the capsid surface reactive moiety comprises a maleimide, an iodoacetamide, a 2-thiopyridne, or a 3-arylpropiolonitrile as exemplified below, where the R group represents the point of attachment to the capsid-reactive linker and the ''*1'' symbols denote the points of attachment of the lysine residue in the capsid protein sequence.

[0157] In some embodiments, the capsid-reactive linker comprises a maleimide. Maleimide and its derivatives are prepared from maleic anhydride by treatment with amines followed by dehydration. The maleimide group reacts specifically with sulfhydryl groups when the pH of the reaction mixture is between 6.5 and 7.5; the result is formation of a stable thioether linkage that is not reversible. iii. Non-Natural Amino Acids

[0158] In some embodiments, the surface of the viral capsid comprises one or more proteins that have a non-natural amino acid comprising a crosslinker reactive moiety.

[0159] In certain embodiments, the non-natural amino acid selected from: 1 : 3-(6- acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), 2: (S)-l-carboxy-3-(7-hydroxy-2-oxo-2H-chromen-4-yl)propan-l-aminium (CouAA), 3: 3-(5-(dimethylamino)naphthalene- 1 -sulfonamide) propanoic acid (Dansylalanine), 4: Y- -azidobenzyloxy carbonyl lysine (PABK), 5: Propargyl-L-lysine (PrK), 6: 7VE-(l-methylcycloprop-2-enecarboxamido) lysine (CpK), 7: TY-acryllysine (AcrK), 8: 7VE-(cyclooct-2-yn-l-yloxy)carbonyl)L-lysine (CoK), 9: bicyclo[6.1.0]non-4-yn-9-ylmethanol lysine (BCNK), 10: trans-cyclooct-2-ene lysine (2'- TCOK), 11 : trans-cyclooct-4-ene lysine (4'-TCOK), 12: dioxo-TCO lysine (DOTCOK), 13:3-(2-cyclobutene-l-yl)propanoic acid (CbK), 14: 7VE-5-norbomene-2-yloxycarbonyl-L-lysine (NBOK), 15: cyclooctyne lysine (SCOK), 16: 5 -norbornen-2-ol tyrosine (NOR), 17: cyclooct-2-ynol tyrosine (COY), 18: (E)-2-(cyclooct-4-en-l-yloxyl)ethanol tyrosine (DS 1 / 2), 19: azidohomoalanine (AHA), 20: homopropargylglycine (HPG), 21 : azidonorleucine (ANL), and 22: 7VE-2-azideoethyloxycarbonyl-L-lysine (NEAK), as illustrated below.5.3.2. Surface modification by recognition sequence-reactive linker (chemogenetic capsid modification)

[0160] The recognition sequence-reactive linker in accordance with this aspect of the present disclosure comprises i) an enzyme substrate moiety, and ii) a member of a crosslinker reactive pair, i.e., a crosslinker reactive moiety. The member of a crosslinker reactive moietyis preferably selected to be mutually reactive with the crosslinker reactive moiety on a functionalized ligand of the present disclosure.5.3.2.1.1 Linker

[0161] In some embodiments, a “linker” or linking moiety is derived from a molecule with a reactive terminus, e.g., suitable for conjugation to a protein of interest. In some instances, the reactive terminus of the linker precursor includes a chemoselective ligation group capable of conjugating to amino acid residue(s) of a polypeptide. In certain instances, the chemoselective ligation group conjugates to a cysteine thiol group, or a lysine sidechain amine group of the polypeptide that is accessible. A variety of conjugation chemistries can be utilized in the conjugates of this disclosure (e.g., as described herein). In some embodiments, the chemoselective ligation group is a thiol -reactive group such as mal eimide or dibromomaleimide. In some embodiments, the chemoselective ligation group is an aminereactive group such as an active ester, e.g., perfluorophenyl ester or tetrafluorophenyl ester, or N-hydroxysuccinimidyl ester (NHS) or sulfo-NHS, or as defined herein.

[0162] It is understood that a variety of substituents and chemistries can be utilized to connect a particular X ligand moiety (e.g., as described herein) to an adjacent linker. In some embodiments, a linking moiety of the linker comprises a triazole that derives from a Click chemistry conjugation. i. Chemoselective Ligation Group

[0163] A chemoselective ligation group is a group having a reactive functionality or functional group capable of conjugation to a compatible group of a second moiety (e.g., a polypeptide or chemically modified polypeptide). For example, chemoselective ligation groups (or a precursor thereof) may be one of a pair of groups associated with a conjugation chemistry such as azido-alkyne click chemistry, copper free click chemistry, Staudinger ligation, tetrazine ligation, hydrazine-iso-Pictet-Spengler (HIPS) ligation, cysteine-reactive ligation chemistry (e.g., thiol-maleimide, thiol-haloacetamide or alkyne hydrothiolation), lysine-reactive ligation chemistry (e.g., amine-active ester coupling, e.g., PFP ester or NHS ester), tyrosine specific conjugation chemistry (e.g., e-Y-CLICK), methionine specific conjugation chemistry (e.g., oxaziridine-based or ReACT chemistry), reductive amination, dialkyl squarate chemistry, etc.

[0164] Chemoselective ligation groups that may be utilized in linking two moieties include, but are not limited to, amino (e.g., a N-terminal amino or a lysine sidechain group of a polypeptide), azido, aryl azide, alkynyl (e.g., ethynyl or cyclooctyne or derivative), active ester (e.g., N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, pentafluorophenyl (PFP) ester, tetrafluorophenyl (TFP) ester, or thioester), haloacetamide (e.g., iodoacetamide or bromoacetamide), chloroacetyl, bromoacetyl, hydrazide, maleimide, vinyl sulfone, 2-sulfonyl pyridine, cyano-alkyne, thiol (e.g., a cysteine residue), disulfide or protected thiol, isocyanate, isothiocyanate, aldehyde, ketone, alkoxyamine, hydrazide, aminooxy, phosphine, HIPS hydrazinyl-indolyl group, or aza-HIPS hydrazinyl-pyrrolo-pyridinyl group, tetrazine, cyclooctene, squarate, and the like.

[0165] In some embodiments, the chemoselective ligation group is capable of spontaneous conjugation to a compatible chemical group when the two groups come into contact under suitable conditions (e.g., copper free Click chemistry conditions). In some instances, the chemoselective ligation group is capable of conjugation to a compatible chemical group when the two groups come into contact in the presence of a catalyst or other reagent under suitable conditions (e.g., copper catalyzed Click chemistry conditions).

[0166] In some embodiments, the chemoselective ligation group is a photoactive ligation group. For example, upon irradiation with ultraviolet light, a diazirine group can form reactive carbenes, which can insert into C-H, N-H, and O-H bonds of a second moiety.

[0167] In some embodiments, the terminal of a linker precursor includes a precursor of the reactive functionality or functional group capable of conjugation (e.g., forming a covalent bond) to a compatible group of a polypeptide (e.g., with a compatible amino acid sidechain of the polypeptide). The reactive moiety can be referred to as a chemoselective ligation group. For example, a carboxylic acid is a precursor of an active ester chemoselective ligation group.

[0168] In certain embodiments, the terminal of a linker precursor is a thiol -reactive chemoselective ligation group (e.g., as described herein). A residual moiety Z (e.g., of formula (la) can result from the covalent linkage of the thiol -reactive chemoselective ligation group to one or more cysteine residue(s) (Cys) of a protein, e.g., carrier protein and / or MuSK polypeptide. In certain embodiments, the Cys-reactive chemoselective ligation group is a maleimide derivative, e.g., as described herein. In some cases, the Cys-reactive chemoselective ligation group is a maleimide.

[0169] In certain embodiments, the terminal of a linker precursor is an amino-reactive chemoselective ligation group (e.g., as described herein). A residual moiety Z (e.g., of formula (la)) can result from the covalent linkage of the amine -re active chemoselective ligation group to one or more lysine residue(s) (Lys) of a protein, e.g., carrier protein and / or MuSK polypeptide. For amineactive ester couplings, the residual moiety Z is an amide bond. In some embodiments the Lys-reactivechemoselective ligation group is a PFP ester. In some embodiments the Lys-reactive chemoselective ligation group is a TFP ester. In some embodiments the Lys-reactive chemoselective ligation group is a NHS or sulfo-NHS ester.5.3.2.1.2 Spacer

[0170] The recognition sequence-reactive linker optionally further comprises one or more spacer moiety. The spacer moiety is not particularly limited and may be any spacer known in the art. In accordance with the present disclosure, the spacer is not particularly limited and includes any divalent moiety. In some embodiments, the divalent moiety comprises alkylene, alkenylene, alkynylene, arylene, heteroarylene, fused arylene, fused heteroarylene, cycloalkylene, heterocyclylene, fused cycloalkylene, or fused heterocyclylene.

[0171] In some embodiments the spacer comprises one or more monomers of ethylene glycol, i.e., polyethylene glycol, -(O-CH2-CH2)n- or [PEG]n, also known as “dPEGn” for “discrete polyethylene glycol”, where “n” is the number of ethylene oxide (or “ethylene glycol”) units. In certain embodiments, n is 0. In certain 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. In certain embodiments, the spacer is [PEG]n where n is 4, 8 or 12. In certain embodiments the spacer is [PEG]nwhere n is 4 and is understood to comprise four -(O-CH2-CH2)- groups.

[0172] In some embodiments, the surface modified viral capsid comprises one or more spacers. In some embodiments, the one or more spacers comprise from 1 to 20 monomers of ethylene glycol. In some embodiments, the one or more spacers comprise from 2 to 8 monomers of ethylene glycol. In some embodiments, at least one of the one or more spacers comprise 4 monomers of ethylene glycol. In some embodiments, the surface modified viral capsid comprises two spacers that comprise 4 monomers of ethylene glycol.

[0173] In some embodiments, the spacer comprises one or more divalent groups such as - CH2- (methylene), -O- (ether), -C(=O)- (carbonyl), and -N(R)- (amine, where R is H or C1-3 alkyl).

[0174] In some embodiments, the spacer comprises one or more -Y-C(O)-, -Y-C(O)O-, -Y- NHC(O)-, -Y-NHC(S)-, or -Y-C(O), wherein Y is a bond or one or more monomers of ethylene glycol.

[0175] In some embodiments, the spacer comprises one or -([PEG]n)-C(O)-, wherein n is from 1 to 100, such as, for example, from 1 to 25, from 1 to 20, from 1 to 15, from 1 to 10, from 2 to 25, from 2 to 20, from 2 to 15, from 2 to 10, from 4 to 15, or from 4 to 10.

[0176] In some embodiments, the spacer comprises one or more -(CH2)n-, wherein n is from 1 to 100, such as, for example, from 1 to 25, from 1 to 20, from 1 to 15, from 1 to 10, from 2 to 25, from 2 to 20, from 2 to 15, from 2 to 10, from 4 to 15, or from 4 to 10. In certain 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, 100.5.3.2.1.3 Enzyme Substrate Moiety

[0177] The enzyme substrate moiety as referred to herein is a chemical or biological functional group that is recognized as a substrate by the relevant enzyme and participates in the enzymatic ligation (formation of a covalent attachment) between the recognition sequence and the enzyme substrate moiety.

[0178] In some embodiments, the enzyme substrate moiety is selected from an oligoglycine, (i.e., an oligopeptide comprising at least two glycine residues, e.g., GG, GGG, GGGG etc.), or a primary amine.5.3.2.1.4 Recognition Sequence

[0179] The recognition sequence in accordance with the present disclosure is (i) engineered into a capsid protein, (ii) recognized by a relevant enzyme, and (iii) participates in enzyme catalyzed enzymatic ligation, i.e., formation of a covalent attachment between the recognition sequence and the enzyme substrate moiety.

[0180] In certain embodiments, the enzyme cleaves between amino acids in the recognition sequence and enables the formation of a covalent bond between an enzyme substrate and an amino acid in the remaining, i.e., residual recognition sequence, as described in more detail herein.

[0181] In embodiments of the present disclosure, the recognition sequence is more than three amino acids, e.g., an oligopeptide. In certain embodiments, the recognition sequence is less than fifteen amino acids in length. In some embodiments the recognition sequence is 3 amino acids. In some embodiments the recognition sequence is 4 amino acids. In some embodiments the recognition sequence is 5 amino acids. In some embodiments therecognition sequence is 6 amino acids. In some embodiments the recognition sequence is 7 amino acids. In some embodiments the recognition sequence is 8 amino acids. In some embodiments the recognition sequence is 9 amino acids. In some embodiments the recognition sequence is 10 amino acids. In some embodiments the recognition sequence is 11 amino acids. In some embodiments the recognition sequence is 12 amino acids. In some embodiments the recognition sequence is 13 amino acids. In some embodiments the recognition sequence is 14 amino acids. In some embodiments the recognition sequence is 15 amino acids.

[0182] In certain embodiments, the recognition sequence is selected from LPXTA (SEQ ID NO: 15), LPXTG (SEQ ID NO: 45), NXXTN (SEQ ID NO: 67), PGF, VPXXXP (SEQ ID NO: 68), PEF, or PEP, where X can be any amino acid. In a particular embodiment the recognition sequence is LPXTG (SEQ ID NO: 45). In certain embodiments, the recognition sequence is LPETG (SEQ ID NO: 12).5.3.2.1.5 Residual Recognition Sequence

[0183] The residual recognition sequence refers to the amino acids of the recognition sequence that remain after an enzyme cleaves the recognition sequence during enzymatic ligation.

[0184] In certain embodiments, the residual recognition sequence is the recognition sequence without the final amino acid in the sequence. In embodiments where recognition sequence is selected from LPXTA (SEQ ID NO: 15), LPXTG (SEQ ID NO: 45), NXXTN (SEQ ID NO: 67), PGF, VPXXXP (SEQ ID NO: 68), PEF, or PEP, in certain embodiments the corresponding residual recognition sequence is LPXT (residues 1 to 4 of SEQ ID NO: 15), LPXT (residues 1 to 4 of SEQ ID NO: 45), NXXT (residues 1 to 4 of SEQ ID NO: 67), PG, VPXXX (residues 1 to 5 of SEQ ID NO: 68), PE, or PE, respectively, where X is any amino acid. In a particular embodiment, the recognition sequence is LPXTG (SEQ ID NO: 45) and the residual recognition sequence is LPXT (residues 1 to 4 of SEQ ID NO: 15). For example, when the recognition sequence is LPETG (SEQ ID NO: 12), the residual recognition sequence is LPET (residues 1 to 4 of SEQ ID NO: 12).

[0185] 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 someembodiments 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.5.3.2.1.6 Enzymes

[0186] An enzyme suitable for use with the present disclosure is able to catalyze site-specific enzymatic ligation of an enzyme substrate to a corresponding recognition sequence engineered into a capsid protein, and thereby form a product of enzymatic ligation.Exemplary enzymes for use with the present disclosure are not particularly limited and include transpeptidases such as sortase A, sortase B, archaeosortase A, exosrotase A, rhombosortase, and PorU, and ligases such as Spyligase, and Snoopligase and engineered variants of any of these. i. Transpeptidases

[0187] Transpeptidases are enzymes that catalyze nucleophilic carbonyl substitution reactions. In some embodiments, the transpeptidase first associates with and then cleaves a recognition sequence and then catalyzes a nucleophilic ligation with a linker comprising an enzyme substrate moiety.

[0188] Examples of transpeptidases for use with the present disclosure include: sortase A, sortase B, archaeosortase A, archaeosortase B, archaeosortase C, exosortase A, rhombosortase, and PorU, and functional mutants thereof. ii. Sortases

[0189] In certain embodiments, an enzyme that catalyzes the enzymatic ligation of an enzyme-reactive linker to a recognition sequence is a sortase enzyme. In certain embodiments, the sortase enzyme, the recognition sequence (or “motif’) and the enzyme substrate moiety are as described in Bradshaw, W.J., et al. (2015), Molecular features of the sortase enzyme family. FEBS J, 282: 2097-2114, which is incorporated in its entirety.

[0190] SrtA is a thiol transpeptidase that cleaves protein substrates between the threonine and glycine residues of the LPXTG (SEQ ID NO: 45) and subsequently links the protein to the enzyme substrate moiety. SrtA catalyzes the formation of an amide bond (i.e., thebioconjugated moiety) between the carboxyl group of the threonine of LPXT (residues 1 to 4 of SEQ ID NO: 45) and the amino group of the enzyme substrate moiety.

[0191] In certain embodiments, the enzyme that bioconjugates the enzyme-reactive linker to the recognition sequence is Sortase A (SrtA) from S. aureus (Sa-SrtA), the recognition sequence is LPXTG (SEQ ID NO: 45), the residual recognition sequence is LPXT (residues 1 to 4 of SEQ ID NO: 45) and the enzyme substrate moiety is a primary amine, including glycine or an oligoglycine.

[0192] The transpeptidase can also be mutated from the natural sequence to include mutations. In some embodiments the enzyme is a transpeptidase harboring a mutation that increases the efficiency of the cleavage and hence the overall reaction. Mutated sortases identified in J. Biol. Chem. (2020) 295:9, 2664-2675 and Scientific reports 2016 (6:31899) are incorporated herein by refence. In certain embodiments, the sortase for use with the present disclosure harbors one or more of the following mutations: P94R, E105K, El 08 A, D124G, D160N, D165A, D186G, Y187L, E189R, K190E, and K196T. In certain embodiments, the transpeptidase is Hepta-Mutant Staphylococcus aureus Sortase A (SrtA7m).

[0193] In certain embodiments, the recognition sequence, corresponding enzyme and enzyme substrate moiety for functionalizing a ligand are selected from those described in the Peptide Ligation Table below.Table A - Peptide ligationRecognition sequence Ezyme(s) Enzyme substrate moietyHeptamutantLPXTG (SEQ ID NO: 45) Sortase A oligo glycine, primary amineNPQTN (SEQ ID NO: 22) Sortase B oligo glycine any AA except Pro, Asp, and Glu, at the second position He, Leu, Vai,N-HV or D-HV Butelase or CysRH-X (X = peptide, tag)Y -RH Trypsiligase nucleophilic acyl acceptor peptideKDPGA (SEQ ID NO: 69) Connectase PGAAHI VMVDA YKPTK (SEQ ID NO : 70) Spy ligase ATHIKF SKRD (SEQ ID NO : 71 )DIPATYEFTDGKHYITNEPIPPKKLGSIEFIKVNK (SEQ ID NO: 72) Snoophgase (SEQ ID NO: 73)5.3.2.1.7 Functionalized Ligand

[0194] In an aspect of the present disclosure, a functionalized ligand is provided wherein the ligand comprises a member of a crosslinker reactive pair or an enzyme substrate. In some embodiments, the ligand is functionalized by reaction with a ligand-reactive linker. In some embodiments, the functionalized ligand is a polypeptide mutated to include a non-natural amino acid comprising a crosslinker reactive moiety; enzyme substrate moiety or a bioorthogonal tag. In some of these embodiments, the enzyme substrate moiety is an oligoglycine enzyme substrate moiety or amine moiety.5.3.2.1 Ligand-Reactive Linker

[0195] Exemplary ligand-reactive pairs useful according to certain embodiments, include, e.g., those as disclosed in WO 2022 / 101363, the disclosure of which is herein expressly incorporated by reference in its entirety.

[0196] The ligand-reactive linker, in accordance with the present disclosure, comprises (i) a ligand-reactive moiety available to form a covalent attachment with the ligand, and (ii) a member of a crosslinker reactive pair available for crosslinking with the surface functionalized viral capsid of the present disclosure.5.3.2.2.1 Linker

[0197] In some embodiments, a “linker” or linking moiety is derived from a molecule with a reactive terminus, e.g., suitable for conjugation to a protein of interest. In some instances, the reactive terminus of the linker precursor includes a chemoselective ligation group capable of conjugating to amino acid residue(s) of a polypeptide. In certain instances, the chemoselective ligation group conjugates to a cysteine thiol group, or a lysine sidechain amine group of the polypeptide that is accessible. A variety of conjugation chemistries can be utilized in the conjugates of this disclosure (e.g., as described herein). In some embodiments, the chemoselective ligation group is a thiol -reactive group such as mal eimide or dibromomaleimide. In some embodiments, the chemoselective ligation group is an aminereactive group such as an active ester, e.g., perfluorophenyl ester or tetrafluorophenyl ester, or N-hydroxysuccinimidyl ester (NHS) or sulfo-NHS, or as defined herein.

[0198] It is understood that a variety of substituents and chemistries can be utilized to connect a particular X ligand moiety (e.g., as described herein) to an adjacent linker. In some embodiments, a linking moiety of the linker comprises a triazole that derives from a Click chemistry conjugation.5.3.2.2.2 Spacer

[0199] The ligand-reactive linker optionally further comprises at least one spacer moiety. The spacer moiety is not particularly limited and may be any spacer known in the art. In some embodiments the spacer comprises monomers of ethylene glycol, i.e., polyethylene glycol, - (O- CH2- CH2)n~ or [PEG]n, also known as “dPEGn” for “discrete polyethylene glycol”, where “n” is the number of ethylene oxide (or “ethylene glycol”) units. In certain embodiments, n is 0. In certain 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. In certain embodiments, n is 4, 8, 12 or16. In certain embodiments, the spacer is [PEG]n where n is 4 and is understood to comprise four -(O-CH2-CH2)- groups.

[0200] In some embodiments, the surface modified viral capsid comprises one or more spacers. In some embodiments, the one or more spacers comprise from 1 to 20 monomers of ethylene glycol. In some embodiments, the one or more spacers comprise from 2 to 8 monomers of ethylene glycol. In some embodiments, at least one of the one or more spacers comprise 4 monomers of ethylene glycol. In some embodiments, the surface modified viral capsid comprises two spacers that comprise 4 monomers of ethylene glycol.

[0201] In some embodiments, the spacer comprises one or more divalent groups such as - CH2- (methylene), -O- (ether), -C(=O)- (carbonyl), and -N(R)- (amine, where R is H or C1-3 alkyl).

[0202] In some embodiments, the spacer comprises one or more -Y-C(O)-, -Y-C(O)O-, -Y- NHC(O)-, -Y-NHC(S)-, or -Y-C(O), wherein Y is a bond or one or more monomers of ethylene glycol.

[0203] In some embodiments, the spacer comprises one or -([PEG]n)-C(O)-, wherein n is from 1 to 100, such as, for example, from 1 to 25, from 1 to 20, from 1 to 15, from 1 to 10, from 2 to 25, from 2 to 20, from 2 to 15, from 2 to 10, from 4 to 15, or from 4 to 10.

[0204] In some embodiments, the spacer comprises one or more -(CH2)n-, wherein n is from 1 to 100, such as, for example, from 1 to 25, from 1 to 20, from 1 to 15, from 1 to 10, from 2 to 25, from 2 to 20, from 2 to 15, from 2 to 10, from 4 to 15, or from 4 to 10. In certain 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, 100.5.3.2.2.3 Ligand-Reactive Moiety

[0205] In accordance with the present disclosure, the ligand-reactive moiety is not particularly limited and includes any moiety that is able to covalently attach to the desired ligand.

[0206] In some embodiments the ligand is a peptide, an oligopeptide, or a polypeptide, and the ligand-reactive moiety attaches (via covalent bond) to an amino acid in the ligand protein primary sequence using, e.g., known techniques in residue specific protein labeling. In further embodiments, 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. i. Ligand Primary Amine

[0207] In some embodiments, the ligand-reactive moiety comprises a chemical group that reacts with primary amines (-NH2). In embodiments in which the ligand is a polypeptide, primary amines exist at the N-terminus of each ligand protein and in the side chain of lysine (Lys, K) amino acid residues in the ligand protein sequence. Exemplary chemical groups that react with primary amines include isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters. Most of these chemical groups conjugate to amines by either acylation or alkylation. In some embodiments, the ligand surface reactive moiety comprises and NHS ester or an imidoester, e.g., such as those illustrated below where the R group represents the point of attachment to the ligand reactive linker.R— N=C=SIsothiocyanateR— N=C=OIsocyanate Sulfonyl Chloride Aldehyde CarbodiimideNHS Ester Imidoester Epoxide Fluorophenyl Ester

[0208] In some embodiments in which the ligand is a polypeptide, the ligand-reactive moiety covalently attaches to a surface exposed lysine residue of the ligand protein primary sequence. In certain of these embodiments, the ligand surface reactive moiety comprises an NHS-ester, a TFP ester, an isocyanate, an isothiocyanate, or a benzyl fluoride as shown below, where the R group represents the point of attachment to the ligand reactive linker and the symbols denote the points of attachment of the lysine residue in the ligand protein sequence.Isothiocyanates

[0209] In some embodiments, the ligand-reactive linker comprises an N-hydroxysuccinimide ester (NHS ester). NHS esters are reactive groups formed by carbodiimide-activation of carboxylate molecules. The NHS ester-activated ligand reactive linker reacts with primary amines in physiologic to slightly alkaline conditions (pH 7.2 to 9) to yield stable amide bonds. The reaction releases N-hydroxysuccinimide (NHS).

[0210] In some embodiments, the capsid-reactive linker comprises a fluorophenyl ester, e.g., tetrafluorophenyl (TFP) or pentaflurophenyl ester (PFP). Fluorophenylesters are reactive groups formed by carbodiimide-activation of carboxylate molecules. Fluorophenyl ester of carboxylic acids react with primary amines forming a covalent amide bond that is identical to one formed by the reaction between primary amines and NHS esters. ii. Ligand Sulfhydryl Group

[0211] In some embodiments, the ligand-reactive moiety covalently attaches to a surface exposed sulfhydryl group. In some embodiments in which the ligand is a polypeptide, the ligand-reactive moiety covalently attaches to a cysteine residue of the ligand protein primary sequence.

[0212] In certain of these embodiments, the ligand reactive moiety comprises an maleimide, an iodoacetamide, a 2-thiopyridne, or a 3-arylpropiolonitrile as exemplified below, where theR group represents the point of attachment to the ligand reactive linker and thesymbols denote the points of attachment of the lysine residue in the ligand protein sequence.

[0213] In some embodiments, the ligand-reactive linker comprises a maleimide. Maleimide and its derivatives are prepared from maleic anhydride by treatment with amines followed by dehydration. The maleimide group reacts specifically with sulfhydryl groups when the pH of the reaction mixture is between 6.5 and 7.5; the result is formation of a stable thioether linkage that is not reversible. iii. Non-Natural Amino Acids

[0214] In some embodiments, the ligand is a polypeptide that has been mutated to include a non-natural amino acid that comprises a crosslinker-reactive moiety.

[0215] In certain embodiments, a ligand polypeptide is mutated to comprise one or more of a non-natural amino acid selected from: 1 : 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), 2: (S)-l-carboxy-3-(7-hydroxy-2-oxo-2H-chromen-4- yl)propan-l-aminium (CouAA), 3: 3-(5-(dimethylamino)naphthalene-l-sulfonamide) propanoic acid (Dansylalanine), 4: AE- / ?-azidobenzyloxycarbonyl lysine (PABK), 5: Propargyl-L-lysine (PrK), 6: AE-(l-methylcycloprop-2-enecarboxamido) lysine (CpK), 7: TY- acryllysine (AcrK), 8: AE-(cyclooct-2-yn-l-yloxy)carbonyl)L-lysine (CoK), 9: bicyclo[6.1.0]non-4-yn-9-ylmethanol lysine (BCNK), 10: trans-cyclooct-2-ene lysine (2'- TCOK), 11: trans-cyclooct-4-ene lysine (4'-TCOK), 12: dioxo-TCO lysine (DOTCOK), 13: 3-(2-cyclobutene-l-yl)propanoic acid (CbK), 14: AE-5-norbomene-2-yloxycarbonyl-L-lysine (NBOK), 15: cyclooctyne lysine (SCOK), 16: 5-norbornen-2-ol tyrosine (NOR), 17: cyclooct-2-ynol tyrosine (COY), 18: (E)-2-(cyclooct-4-en-l-yloxyl)ethanol tyrosine (DS 1 / 2), 19: azidohomoalanine (AHA), 20: homopropargylglycine (HPG), 21: azidonorleucine (ANL), 22: TY^-azideoethyloxycarbonyl-L-lysine (NEAK).iv. Fusion Proteins with Tag-Reactive Molecules

[0216] In embodiments of the present disclosure, the ligand is a fusion protein comprising a tag that is able to bind to their corresponding counterpart with high affinity, such as SNAP- tag, CLIP -tag, Halo Tag, Lumio Tag, and others known to those in the art.

[0217] Benzylguanine, benzylcytosine and chloroalkane are recognized by a “suicide” enzyme, such as SNAP. In the context of this disclosure, benzylguanine, or benzylcytosine may be optionally substituted to form derivatives of benzylguanine, or benzylcytosine. Benzylguanine derivatives or benzylcytosine derivatives are understood to mean a benzylguanine or benzylcytosine group, which is modified but which is nevertheless recognized by the suicide enzyme.

[0218] The tag molecule may be any molecule or biomolecule, which is capable of specifically binding to a further molecule. The examples may include SNAP-tag, CLIP -tag, Lumio-Tag, or Halo-Tag. For example, the affinity tag may be a SNAP-tag, a mutant of an alkylguanine-DNA alkyltransferase. Importantly, one of the substrates for SNAP-tag is benzylguanine. Commercially available products useful for the present disclosure include, e.g., HaloTag from Promega, Lumio Tag from Life Technologies, and SNAP / CLIP Tags from NEB.

[0219] Self-labeling protein tags are commercially available in various expression vectors. SNAP-tag is a 182 residues polypeptide (19.4 kDa) that can be fused to any protein of interest and further specifically and covalently tagged with a suitable ligand, such as a fluorescent dye. 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 using a directed evolution strategy, leading to a hAGT variant that accepts O6-benzylguanine derivatives instead of repairing alkylated guanine derivatives in damaged DNA.

[0220] CLIP -tag, was further engineered from SNAP -tag to accept O2-benzylcytosine derivatives as substrates, instead of O6-benzylguanine. A split-SNAP-tag version suitable for protein complementation assay and protein-protein interaction studies was later developed.

[0221] HaloTag is a self-labeling protein tag. It is a 297-residue peptide (33 kDa) derived from a bacterial enzyme, designed to covalently bind to a synthetic ligand. The HaloTag is a hydrolase, which has a genetically modified active site, which specifically binds the reactive chloroalkane linker and has an increased rate of ligand binding. The reaction that forms the bond between the protein tag and chloroalkane linker is fast and essentially irreversible under physiological conditions due to the terminal chlorine of the linker portion. In the aforementioned reaction, nucleophilic attack of the chloroalkane reactive linker causes displacement of the halogen with an amino acid residue, which results in the formation of a covalent alkyl-enzyme intermediate. This intermediate would then be hydrolyzed by an amino acid residue within the wild-type hydrolase. This would lead to regeneration of the enzyme following the reaction. However, in the modified haloalkane dehalogenase (HaloTag), the reaction intermediate cannot proceed through a subsequent reaction because it cannot be hydrolyzed due to the mutation in the enzyme. This causes the intermediate to persist as a stable covalent adduct with which there is no associated back reaction.

[0222] There are two steps to using this system: cloning and expression of the protein of interest as a SNAP -tag® fusion and labeling of the fusion with the SNAP -tag substrate of choice. The SNAP-tag is a small protein based on human 06-alkylguanine-DNA- alkyltransferase (hAGT), a DNA repair protein. The SNAP-tag substrate in this case is the guanine leaving group connected to a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag.

[0223] The SNAP-tag protein labeling system enables the specific, covalent attachment of virtually any molecule to a protein of interest.5.3.2.3 Examples of Reactive Linkers

[0224] The following reactive linkers are examples of those suitable for use either as a capsid-reactive linker or as a ligand-reactive linker in accordance with various embodiments of the present disclosure. A person of ordinary skill in the art would understand that the number of PEG monomers (i.e., -(O-CH2-CH2)- groups in each of the examples below is easily adapted to reflect the number of PEG monomers described herein. In additionalembodiments of the below exemplified linkers, the number of PEG monomers is 4, 8, 12 or 16.5.3.2.3.1 TCO-PEG4-NHS

[0225] Synonym(s): trans-Cyclooctene-PEG4-NHS; Empirical Formula (Hill Notation): C24H38N2O10; Molecular Weights 14.57.5.3.2.3.1 Tetrazine-PEG5-NHS

[0226] Tetrazine-PEG5-NHS Ester is an amine-reactive linker often used for modification of proteins, peptides, or amine-modified oligonucleotides with a tetrazine moiety.5.3.2.3.3 Azido-PEG4-NHS

[0227] Also referred to as “Azide-PEG4-NHS” herein.5.3.2.3.4 Phosphine-NHS

[0228] Molecular Weight: 461.40.5.3.2 .5 DBCO-PEGn-NHSwherein y is an integer from 1 to 4; and n is an integer from 1 to 10, e.g., 4, 5, 6, 7 or 8. i. DBCO-PEG4-NHS

[0229] Molecular Weight: 649.26.5.3.2.3.6 DBCO-PEGn-TFP ester9 DBCO-amine5.3.2.3.10 Maleimide-PEGn-succinimidyl ester

[0230] Maleimide PEG8 succinimidyl ester, 31-(2,5-Dihydro-2,5-dioxo-lH-pyrrol-l-yl)-29- oxo-4, 7, 10,13,16,19,22,25-octaoxa-28-azahentriacontanoic acid 2, 5-dioxo- 1 -pyrrolidinyl ester, Maleimide-PEG8-NHS ester, 31-(2,5-Dihydro-2,5-dioxo-lH-pyrrol-l-yl)-29-oxo- 4,7,10,13,16,19,22,25-octaoxa-28-azahentriacontanoic acid 2,5-dioxo-l-pyrrolidinyl ester, Maleimide-PEG8-NHS ester.5.3.2.3.12 N3-PEGn-Maleimide5.3.2.3.13 Dibenzocyclooctyne- TFP

[0231] In some embodiments, the capsid-reactive linker is of the following structure:wherein SP1is a bond or spacer; and“a” is an integer from 1 to 5, from 2 to 4, e.g., 1, 2, 3, 4, or 5.In certain embodiments, a is 4.5.3.3. Surface modification by insertion of a peptide ligand

[0232] In various embodiments, the surface modified viral capsid comprises a peptide inserted in-frame into a capsid protein. In some embodiments, the inserted peptide is a ligand that facilitates passage of the rAAV from CSF into the brain parenchyma. In particular embodiments, a region flanking 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-conjugated ligands that facilitate transduction of CNS cells, optionally transduction of one or more desired CNS cell types.

[0233] In some embodiments, the peptide ligand is inserted into the capsid protein of a naturally occurring AAV serotype or an engineered or synthesized AAV capsid protein. For example, the peptide ligand can be inserted into a capsid protein of a naturally occurring human AAV serotype, e.g., AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, AAV9 or hu68, or into naturally occurring rhesus serotype, such as rh34. In other embodiments, the peptide ligand is inserted into an artificial AAV capsid sequence, such as the bioinformatically predicted ancestral capsids described in U.S. Pat. Nos. 9,695,220 and 10,738,087, the disclosures of which are incorporated herein by reference in their entireties.

[0234] In some embodiments, the peptide ligand is inserted into the variable region 4 (VR4) of the AAV capsid protein, corresponding to an exposed loop on the capsid surface. In otherembodiments, the peptide ligand is inserted into the 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 that reduce or abrogate binding to HSPG.

[0235] The specific insertion site in VR4 or VR8 can be different depending on the capsid protein into which the modification is engineered.

[0236] 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.

[0237] 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.

[0238] Exemplary capsid insertion sites of naturally occurring AAV serotypes AAV1 to AAV9 are provided in Table 1. For other (e.g. modified) capsid, the insertion site can be determined by identification of the amino acid position corresponding to the insertion site listed in Table 1 by sequence alignment. For example, the insertion site can be an amino acid position corresponding to a site after Serine (S) 443 when the sequence is aligned with the AAV1 capsid protein. In another example, the insertion site is an amino acid position corresponding to a site after Serine (S) 442 when the sequence is aligned with the AAV2 capsid protein.

[0239] In some embodiments, the surface modified viral capsid protein comprises a capsid protein of AAV1 with a peptide insertion after Serine (S) 443. In some embodiments, thesurface modified viral capsid protein comprises a capsid protein of AAV2 with a peptide insertion after Serine (S) 442. In some embodiments, the surface modified viral capsid protein comprises a capsid protein of AAV6 with a peptide insertion after Serine (S) 443. In some embodiments, the surface modified viral capsid protein comprises a capsid protein of AAV7 with a peptide insertion after Proline (P) 454. In some embodiments, the surface modified viral capsid protein comprises a capsid protein of AAV8 with a peptide insertion after Threonine (T) 454. In some embodiments, the surface modified viral capsid protein comprises a capsid protein of AAV9 with a peptide insertion after Serine (S) 454.

[0240] In some embodiments, the surface modified viral capsid protein comprises a capsid protein of AAV2 with a peptide insertion after Arginine (R) 589. In some embodiments, the surface modified viral capsid protein comprises a capsid protein of AAV5 with a peptide insertion after Serine (S) 576. In some embodiments, the surface modified viral capsid protein comprises a capsid protein of AAV6 with a peptide insertion after Serine (S) 591. In some embodiments, the surface modified viral capsid protein comprises a capsid protein of AAV7 with a peptide insertion after Alanine (A) 591. In some embodiments, the surface modified viral capsid protein comprises a capsid protein of AAV8 with a peptide insertion after Glutamine (Q) 593.

[0241] In some embodiments, the peptide ligand is a peptide agonist of the PARI receptor. In some embodiments, the peptide ligand is the PARI A peptide. In some embodiments, the peptide ligand has a sequence of SFLLRN (residues 5 to 10 of SEQ ID NO: 61). In some embodiments, the peptide ligand has a sequence of LDPRSFLLRN (SEQ ID NO: 61).

[0242] In some embodiments, the PARI A peptide is inserted into VR-IV (VR-4) of the AAV capsid protein sequence.

[0243] In some embodiments, the PARI A peptide may be inserted into VR-VIII (VR-8) of the AAV capsid protein sequence.5.3.4. Surface Modified Viral Capsid of Formula I

[0244] In certain embodiments, the surface modified viral capsid comprises a ligand and linker according to Formula IY is an attachment moiety;Y’ is an attachment moiety;Q is a crosslinked moiety;PEG is a monomer of ethylene glycol; n and n’ are independently an integer from 0 to 100, Sp and Sp’ are independently an optional spacer; L is a ligand, and x is not particularly limited. In some embodiments, x is an integer from 1 to 300, from 100 to 200, from 120 to 180 or around 150.

[0245] In some embodiments, x ranges from 100-200. In some embodiments, x ranges from 130-170.

[0246] In certain embodiments, the attachment moiety Y is formed by reaction between a capsid-reactive moiety and a capsid protein. In certain embodiments, the attachment moietyY is formed by reaction between an NHS or TFP ester and a primary amino group of an amino acid of a capsid protein. In some embodiments, the amino group is the sidechain of a lysine present in the primary sequence of a capsid protein. In some embodiments, the amino group is a lysine present in the primary sequence of an AAV capsid protein.

[0247] In certain embodiments, the attachment moiety Y’ is formed by any known chemistry for coupling reaction between a ligand-reactive moiety and a ligand. In certain embodiments, the attachment moiety Y’ is formed by reaction between an ester, e.g., NHS or TFP, and an amino group of the ligand.

[0248] In certain embodiments, Q is a product formed by the reaction between members of a crosslinker reactive pair. In certain embodiments, Q is a crosslinked moiety formed by the reaction of DBCO and an azido group.

[0249] In certain embodiments, Q is selected from:wherein, Z is a 7 or 8 membered cyclic or heterocyclic structure. In some embodiments, Z is optionally substituted with one or more arylene. In some embodiments, Z is unsubstituted. In some embodiments, Z is substituted with one arylene. In some embodiments, Z is substituted with two arylenes. In some embodiments, the arylenes are independently selected. In some embodiments, the arylenes are identical.

[0250] In certain embodiments, the surface modified viral capsid is according to Formula 1-1 :n and n’ are independently an integer selected from 0 to 30;L is a ligand; and x is an integer from 50 to 250.

[0251] In some embodiments, x ranges from 100-200. In some embodiments, the ligand per capsid ratio (x) is in the range from 130 to 170.

[0252] In certain embodiments, n is an integer selected from 0 to 100. In certain embodiments, 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.

[0253] In certain embodiments, n’ is an integer selected from 0 to 100. In certain embodiments, 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 100.5.3.5. Engineered viral capsid

[0254] 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 second targeting ligand are distinct.

[0255] In some embodiments, the first targeting ligand promotes crossing of a 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) translocating peptide or a lectin ligand.

[0256] In some embodiments, one of the first and second targeting ligands is covalently attached (e.g., conjugated) to a lysine or cysteine residue of a viral capsid protein via a linker. In some embodiments, the other of the first and second targeting ligands is covalently attached (e.g., conjugated) to a recognition sequence motif residue via a linker.

[0257] In some embodiments, the viral capsid is an adeno-associated virus (AAV) capsid. In some embodiments, the first targeting ligand or second targeting ligand is a peptide, such as a PAR-1 agonist.

[0258] In some embodiments, the first targeting ligand or second targeting ligand is a peptide as set forth in SEQ ID NO: 3 or portion thereof. In some embodiments, the first targeting ligand or second targeting ligand is nerve growth factor. In some embodiments, the first targeting ligand or second targeting ligand binds to brain microvascular endothelial cells (BMECs). In some embodiments, the first targeting ligand or second targeting ligand binds to neuronal membranes, such as wheat germ agglutinin (WGA).

[0259] In some embodiments, the engineered viral capsid conjugated to a first targeting ligand and a second targeting ligand further comprises: (a) a modified viral capsid comprising a plurality of recognition sequence motif residues; (b) a first targeting ligand conjugated to the modified viral capsid via a first linker covalently attached to the recognition sequence motif residue comprising: bl. the residue covalently attached to an amide, - C(O)NH-; b2. a first spacer (Sp)n; b3. a bivalent fused polycyclic 11- to 20-membered heterocyclic ring, a phosphine oxide derivative, or a 4H-pyrazol-4-imine derivative; b4. a second spacer (Sp')n'; b5. a first targeting ligand; and, (c) a second targeting ligand conjugated to the modified viral capsid via a second linker covalently attached to a primary amino group or a thiol of the modified viral capsid primary sequence comprising: cl. an amide, -NHC(O)-; c2. a first spacer (Sp)n; c3. a bivalent fused polycyclic 11- to 20- membered heterocyclic ring, a phosphine oxide derivative, or a 4H-pyrazol-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.

[0260] In some embodiments, the recognition sequence motif residue is selected from any one of the peptide sequences as set forth in SEQ ID NOs: 1-58 or 61-75 or portion thereof. In some embodiments, the recognition sequence motif residue comprises at least four consecutive amino acids selected from the peptide sequences as set forth in SEQ ID NOs: 8- 33, 35-44, 62-64, and 65. In some embodiments, the recognition sequence motif residue comprises at least six consecutive amino acids as set forth in SEQ ID NO: 61 or SEQ ID NO: 66.

[0261] In some embodiments, the engineered viral capsid conjugated to a first targeting ligand and a second targeting ligand further comprises: (a) a modified viral capsid; (b) a first targeting ligand conjugated to the modified viral capsid via a first linker covalently attached to a primary amino group or a thiol of the modified viral capsid primary sequence comprising: bl. the residue covalently attached to an amide, -C(O)NH-; b2. a first spacer (Sp)n; b3. a bivalent fused polycyclic 11- to 20-membered heterocyclic ring, a phosphine oxide derivative, or a 4H-pyrazol-4-imine derivative; b4. a second spacer (Sp')n'; b5. a first targeting ligand; and, (c) a second targeting ligand conjugated to the modified viral capsid via a second linker covalently attached to a primary amino group or a thiol of the modified viral capsid primary sequence comprising: cl. an amide, -NHC(O)-; c2. a first spacer (Sp)n; c3. a bivalent fused polycyclic 11- to 20-membered heterocyclic ring, a phosphine oxidederivative, or a 4H-pyrazol-4-imine derivative; c4. a second spacer (Sp')n'; and, c5. a second targeting ligand; wherein n and if are each independently an integer from 0 to 20.

[0262] In some embodiments, the primary amino group is selected from an N-terminal amino group, a lysine epsilon amino group and an arginine amino acid group.

[0263] In some embodiments, the bivalent fused polycyclic 11- to 20-membered heterocyclic ring is:wherein Z is an optionally substituted 7- to 9-membered carbocycle or heterocyclic ring.

[0264] In some embodiments, Z is substituted with one or more arylenes.

[0265] In some embodiments, the phosphine oxide derivative is:[ diments, the 4H-pyrazol-4-imine derivative is:5.3.6. Ligands

[0267] In accordance with the present disclosure, a surface-modified viral capsid, typically an AAV viral capsid, is provided. The surface modification comprises a first ligand that facilitates passage of the rAAV from CSF into the brain parenchyma.

[0268] Optionally, the surface modified viral capsid comprises one or more additional surface-conjugated ligands. In particular embodiments, at least one additional surface- conjugated ligand facilitate transduction of CNS cells, optionally transduction of one or more desired CNS cell types. In various embodiments, the optional ligands are not particularly limited, as long as the ligand is amenable to conjugation to the viral capsid surface as described herein. In some embodiments, the ligand is selected from a protein ligand having a cognate that is located on the surface of mammalian cells, such as receptors. In some of these embodiments, the cognate protein is involved in transduction of the surface modified viral capsid. In some embodiments, the ligand is a protein known to promote permeabilization of tissue barriers in the body such as the meninges, including the pia mater.

[0269] In some embodiments, the surface modified viral capsid comprises a ligand. In some embodiments, the ligand is a cell-type specific ligand.

[0270] In some embodiments, the ligand is a cell-type or receptor specific ligand. In certain embodiments, the ligand is selected from polypeptides, proteins, monosaccharides or polysaccharides, from steroid hormones, from RGD motif peptide, from vitamins, from small molecules or from targeting peptides. Also contemplated are antibodies (e.g., single chain) and nanobodies; enzymes such as proteases, glycosidases, lipases, peptidases; immunoglobulins such as CD47 (don't eat me signal); IgG proteases such as IdeZ and IdeS; protein based and small molecule adjuvants for vaccination.

[0271] According to one embodiment, a cell-type specific ligand is derived from proteins such as transferrin, Epidermal Growth Factor EGF, basic Fibroblast Growth Factor bFGF.

[0272] According to one embodiment, a cell-type specific ligand is derived from mono- or polysaccharides such as galactose, N-acetylgalactosamine and mannose.

[0273] According to one embodiment, a cell-type specific ligand is derived from vitamins such as folates.

[0274] According to one embodiment, a cell-type specific ligand is derived from small molecules including naproxen, ibuprofen or other known protein-binding molecules.

[0275] In certain embodiments, the ligand is selected from a protein ligand, such as a growth factor or a cytokine; a toxin subunit, such as a cholera toxin B subunit; a lectin, such asisolectin B4 or wheat germ agglutinin; an adhesion factor, such as lactadherin; an antibody or a single chain variable fragment thereof, such as an anti-CD-34 antibody; more specifically, an E. coli recombinant scFv CD-34 antibody fragment, a peptide, such as deltorphin opioid receptor ligand; and a gene editing nuclease, 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 multiple chain antibody. In some embodiments, the ligand is an antigen binding antibody fragment. In some embodiments, the ligand is a peptide.

[0276] In certain embodiments, the ligand is selected from agents that are known to promote permeabilization of tissue barriers, such as Tissue Plasminogen Activator (TP A), Zonulin, nanobodies, single chain antibodies, metalloproteases, kallikreins (subgroup of serine proteases), bacterial proteases (e.g., from bacteria that cross the BBB), Zonula Occludens Toxin / deltaG, EphA2 / EPhAl agonist / ligand (known to modulate astrocytes / pericytes), and bacteriophage derived peptides. In certain embodiments, the ligand is Tissue Plasminogen Activator (TP A).

[0277] In certain embodiments, the ligand is selected from agents that are known to target receptors that are highly expressed on the endothelial cells forming the BBB. In some embodiments, the receptor is a receptor-mediated transporter. These include the insulin receptor, transferrin receptor, LDL receptor and its related protein, and others known in the art.

[0278] In some embodiments, the ligand is a protein known to promote permeabilization of tissue barriers in the body such as the meninges, including the pia mater.

[0279] In some embodiments, the ligand is known to bind to a receptor endogenous to the brain microvascular endothelial cells (BMECs). In some embodiments, the receptor is protease activated receptors (PARs). In some embodiments, the receptor is an Endothelial Protease Activated Receptor 1 (PARI). In some embodiments, the ligand binds to PARI. In some embodiments, the ligand is a PAR-1 activating peptide, e.g., thrombin or derivatives thereof. In some embodiments, the ligand binds to thrombin or derivatives thereof. In some embodiments, the ligand is a peptide comprising the sequence SFLLR (residues 1 to 5 ofSEQ ID NO: 3) . In some embodiments, the ligand is a peptide having the sequence SFLLRNPNDKC (SEQ ID NO: 3).

[0280] In certain embodiments, the ligand is selected from agents that are known to promote permeabilization of tissue barriers, such as Tissue Plasminogen Activator (TP A), Zonulin, nanobodies, single chain antibodies, metalloproteases, kallikreins (subgroup of serine proteases), bacterial proteases (e.g., from bacteria that cross the BBB), Zonula Occludens Toxin / deltaG, EphA2 / EPhAl agonist / ligand (known to modulate astrocytes / pericytes), and bacteriophage derived peptides. In certain embodiments, the ligand is Tissue Plasminogen Activator (TP A).

[0281] In some embodiments, the ligand is a lecithin. In some embodiments, the ligand is a wheat germ agglutinin (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 PARI or the ligand is a WGA. In some embodiments, the ligand binds to PARI. In some embodiments, the ligand is a WGA. In some embodiments, the capsid comprises a first ligand that binds to binds to PARI and a second ligand comprises a WGA.5.3.7. Viral Capsids

[0282] In embodiments of the present disclosure, the viral capsid (referred to simply as capsid, herein) is not particularly limited. In some embodiments, the capsid is selected from non-enveloped viruses, such as adenovirus or adeno-associated virus. In some embodiments, the capsid is selected from an enveloped virus, such as retroviruses, lentiviruses, herpes simplex virus, and baculoviruses. Embodiments include non-naturally occurring capsids and includes a biologic or chemical alteration or variation of a naturally occurring capsid protein other than or in addition to a change in the primary amino acid sequence.

[0283] In embodiments of the present disclosure, the capsid comprises an engineered capsid protein, e.g., as prepared by site directed mutagenesis, wherein the engineered capsid protein comprises at least one recognition sequence, as described elsewhere herein.

[0284] In some embodiments, the surface modified viral capsid comprises a first ligand that binds to PARI and a second ligand that comprises a WGA.

[0285] In some embodiments, the surface modified viral capsid comprises a linker. In some embodiments, the linker is covalently attached to a primary amino group of the capsid protein primary sequence.

[0286] In some embodiments, the primary amino group is selected from an N-terminal amino group, a lysine epsilon amino group and an arginine amino acid group. In some embodiments, the primary amino group is the N-terminal amino group. In some embodiments, the primary amino group is the epsilon amino group of a lysine amino acid residue. In some embodiments, the primary amino group is the arginine amino acid group.

[0287] In some embodiments, the surface modified viral capsid comprises a linker, wherein the linker is covalently attached to the ligand via a primary amino group of the ligand.

[0288] In some embodiments, the linker is covalently attached to the targeting ligand via a non-natural amino acid residue of the primary sequence of the targeting ligand.

[0289] In some embodiments, the non-natural amino acid residue comprises a member of the crosslinker reactive pair that participates in a reaction selected from: a Cu(I)-catalyzed azidealkyne cycloaddition (CuAAC) reaction, a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction, a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction, an inverse electron demand Diels-Alder (IEDDA) reaction, a Staudinger ligation and a [4+1] cycloaddition reaction. In some embodiments, the non-natural amino acid residue comprises a member of the crosslinker reactive pair that participates in a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. In some embodiments, the non-natural amino acid residue comprises a member of the crosslinker reactive pair that participates in a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. In some embodiments, the non-natural amino acid residue comprises a member of the crosslinker reactive pair that participates in a strain- promoted alkyne-nitrone cycloaddition (SPANC) reaction. In some embodiments, the non- natural amino acid residue comprises a member of the crosslinker reactive pair that participates in an inverse electron demand Diels-Alder (IEDDA) reaction. In some embodiments, the non-natural amino acid residue comprises a member of the crosslinker reactive pair that participates in a Staudinger ligation and a [4+1] cycloaddition reaction.

[0290] In some embodiments, the non-natural amino acid residue comprises a member of the crosslinker reactive pair that participates in a reaction selected from: a Cu(I)-catalyzed azidealkyne cycloaddition (CuAAC) reaction, a strain-promoted alkyne-azide cycloaddition(SPAAC) reaction, a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction, an inverse electron demand Diels-Alder (IEDDA) reaction, a Staudinger ligation and a [4+1] cycloaddition reaction.

[0291] In some embodiments, the surface modified viral capsid comprises a crosslinker reactive pair. In some embodiments, the crosslinker reactive pair comprises an azide, cyclooctyne, cyclooctene or 1,2,4,5-tetrazine moiety.

[0292] In some embodiments, the surface modified viral capsid comprises a protein sequence. In some embodiments, the protein sequence of the viral capsid has been mutated to attenuate or abrogate binding of the capsid to mammalian cell polysaccharides or proteoglycans.

[0293] In some embodiments, the surface modified viral capsid is characterized by increased infectivity compared to an unmodified viral capsid having the same capsid protein sequence.

[0294] In some embodiments, the viral capsid is selected from an adenovirus capsid, adeno- associated virus capsid, retro virus capsid, lentivirus capsid, herpes simplex virus capsid, and a baculovirus capsid. 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 retro virus capsid. In some embodiments, the viral capsid is a herpes simplex virus capsid. In some embodiments, the viral capsid is a baculovirus capsid.

[0295] In some embodiments, surface modified viral capsid comprises at least one of the arginine residues at 585 and 588 of VP1, or analogous positions in VP2 or VP3, that have been mutated.

[0296] In some embodiments, surface modified viral capsid comprises the arginine residues at 585 and 588 of VP1, that have been mutated to alanine residues.

[0297] In some embodiments, the surface modified viral capsid is characterized by altered tropism compared to an unmodified viral capsid.

[0298] In some embodiments, the surface modified viral capsid demonstrates evasion of preexisting neutralizing antibodies.5.3.7.1 AAV

[0299] All recombinant adeno-associated viruses (rAAV, or AAV used interchangeably herein) may be implemented in the framework of the present disclosure. Such AAV particles are capable of transducing a wide range of post-mitotic cells in vivo in the mammal, e.g, (including but not limited to) muscle cells, hepatocytes and neurons.

[0300] In some embodiments, the AAV capsid comprises a VP1, VP2, and / or VP3 capsid protein of a naturally occurring AAV serotype. In some embodiments, the AAV comprises one or more of a non-naturally occurring VP1, VP2, and / or VP3 capsid protein. In certain of these embodiments, the non-naturally occurring VP1, VP2, or VP3 capsid protein differs in primary amino acid sequence from naturally occurring capsids. In certain embodiments, the non-naturally occurring capsid includes a biologic or chemical alteration or variation of a naturally occurring AAV capsid protein other than or in addition to a change in the primary amino acid sequence.

[0301] In various embodiments, the capsid proteins are those of an AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, or AAV9 naturally occurring AAV serotype. In various embodiments, the capsid protein is selected from capsid proteins 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 entireties.

[0302] The adeno-associated virus capsid may be chosen among all identified natural serotypes and in particular AAV2, AAV3b, AAV5, AAV8, AAV9 and AAV 10 and may be even more particularly AAV2.

[0303] Also, the adeno-associated virus may be chosen among synthetic serotypes generated by non-natural methods, such as, but not limited to: capsid mutagenesis, peptide insertions into, or deletions from, the capsid sequence, capsid shuffling from various serotypes or ancestral reconstruction.

[0304] The AAV capsids for use with the present disclosure are produced by any method known in the art, without limitation. For example, the AAV capsids 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 andtransgene) or insect cells infected with baculovirus vectors (expressing rep-cap and transgene). AAV capsids produced by any of these methods can be used to produce the surface functionalized and surface modified viral capsid described herein. In certain embodiments, the vectors are produced by transient transfection of HEK293 cells with calcium phosphate-HeBS method with two plasmids: pHelper, PDP2-KANA encoding AAV Rep2-Cap2 and adenovirus helper genes (E2A, VA RNA, and E4) and pVector ss-CAG- eGFP as illustrated in the provided Examples.

[0305] In some embodiments, the AAV capsid of the present disclosure comprises one or more sequences from extraviral origin, as desired.

[0306] In some embodiments, the capsid of AAV is composed of three overlapping capsid proteins (VP1, VP2, VP3) containing a unique VP1 N-terminus, a VP1 / VP2 common portion and a portion which is common to VP1, VP2 and VP3.

[0307] In certain embodiments one or more capsid proteins comprise amino groups that are naturally occurring, that is the primary sequence corresponds to a wild-type capsid protein. In alternative embodiments, the primary sequence of one or more capsid proteins comprises amino acids that are engineered into a wild-type capsid protein sequence. In certain of these embodiments, the engineered amino acids include one or more amino groups present at the surface of the capsid and are involved in the surface functionalization of one or more capsid protein. In certain embodiments, the naturally occurring or engineered amino groups that are involved in surface functionalization of the capsid are selected from lysine, arginine and cysteine. In certain embodiments, the amino acid is lysine.

[0308] According to certain embodiments, the AAV capsid comprises one or more wild-type capsid proteins from naturally occurring serotypes.

[0309] According to certain embodiments, AAV capsid comprises a genetically modified capsid protein. In certain embodiments, the genetically modified capsid protein is a naturally occurring serotype engineered to comprise one or more genetic modifications (mutation, insertions or deletions). In an alternative embodiment, the rAAV capsid is composed of one or more of a synthetic capsid protein. In some embodiments, the AAV capsid is engineered to modify the natural tropism, e.g., to reduce heparin binding.

[0310] In the framework of the present disclosure, a synthetic capsid includes any combination of capsid proteins from natural, genetically modified and artificially created (random mutations, sequence shuffling, in silico design, etc.) serotypes that are able to assemble and produce a new AAV virus capsid that is not known to exist in nature.

[0311] Currently, there are more than 100 AAV serotypes identified that differ in the binding capacity of capsid proteins to specific cell surface receptors that can transduce different cell types. 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 tested thoroughly for their ability to transduce specific cell types and differentiated between capsid protein motifs that bind specific cell surface receptors for cell attachment. In the context of this disclosure, an AAV capsid is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 is preferred. However, it should be understood that any other AAV capsid can be used in the context of the present disclosure.

[0312] In one embodiment, the adeno associated virus (AAV) particle of the present disclosure is 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 derivatives of viruses, e.g., adeno-associated virus type 2 (AAV2), AAV9, and AAV8. In some embodiments, the rAAV capsid AAV-2 and AAV-9, where the capsid proteins are optionally further engineered to reduce or modify native tropism, e.g., to reduce heparin binding.5.3.7.1 Removal of Natural Binding Moiety

[0313] In some embodiments, of the adeno associated virus (rAAV) capsid of the present disclosure, the rAAV is selected from a naturally occurring serotype having a natural cell binding site that enables binding to heparan sulfate proteoglycans that has been removed.

[0314] In some embodiments, removal of the heparin binding has been engineered by replacing at least one of arginine 585 or arginine 588 of VP1 and / or an analogous arginine in VP2 or VP3 with a different amino acid, such as alanine. In some embodiments, at least one of arginine 448 and arginine 451 in VP2 or 383 and 386 in VP3 is altered.

[0315] In some embodiments, the adeno associated virus (AAV) capsid of the present disclosure is comprised of at least one protein that is mutated from wild-type, e.g., wherein the engineered / mutated protein is selected from wild-type protein is VP1, VP2, and / or VP3. Alternatively, two of the proteins VP1, VP2 and / or VP3 in said capsid are mutated, or all three of the proteins VP1, VP2 and VP3 in said capsid are modified. In some embodiments, at least one part, e.g., one amino acid, of the at least one of the proteins to be modified in said capsid is mutated (replaced, inserted or deleted). However, it is also possible to mutate multiple parts of the proteins VP1, VP2 and VP3 in said capsid, e.g. multiple amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or any other number of parts or amino acids. In some embodiments, at least one of arginines 484, 487, 585 and 588 and lysine 532 of VP1, and / or an analogous arginine in VP2 or VP3, are removed by replacing them with a different amino acid, such as alanine.5.3.7.3 PEG immune cloaking

[0316] According to certain embodiments, the viral capsid surface may be modified according to methods known in the art to comprise a steric shielding agent for avoiding interaction with neutralizing antibodies. In some embodiments, the steric shielding agent is derived from synthetic polymers such as polyethylene glycol (PEG) or pHPMA. Polymers of PEG are prepared by polymerization processes and comprise a heterogeneous mixture of sizes and molecular weights that may be characterized by a Poisson distribution of chain lengths and molecular weights, also known as the poly dispersity index (PDI), dispersity index or simply dispersity (indicated by the symbol “D”). The reported molecular weight is an average molecular weight, and D (or PDI) gives an indication of the range of molecular weights in the sample.5.4. Engineered Capsid Proteins

[0317] In certain aspects of the present disclosure, an engineered adeno-associated virus (AAV) capsid protein is provided wherein the capsid protein comprises: a functional sequence motif, wherein the functional sequence motif has the sequence: X1X2X3X4X5X6 (SEQ ID NO: 5); wherein Xi, X2, X3, X4, X5, and Xe are each independently selected from any amino acid residue and wherein X4, X5, and Xe are optional; wherein the functional sequence motif is a substrate for a transamidase enzyme.

[0318] In some embodiments, the enzyme is selected from a sortase A, sortase B, archaeosortase A, exosortase A, rhombosortase, and PorU. In some embodiments, the enzyme is sortase A. In some embodiments, the enzyme is sortase B. In some embodiments, the enzyme is archaeosortase A. In some embodiments, the enzyme is exosortase A. In some embodiments, the enzyme is rhombosortase. In some embodiments, the enzyme is PorU.

[0319] In some embodiments, the engineered rAAV capsid protein is heptamutant (SrtA7M) or a pentamutant (SrtA5M). In some embodiments, the engineered rAAV capsid protein is heptamutant (SrtA7M). In some embodiments, the engineered rAAV capsid protein is a pentamutant (SrtA5M).

[0320] In some embodiments, the enzyme is Hepta-Mutant Staphylococcus aureus Sortase A (SrtA7m).

[0321] In certain embodiments, the engineered AAV capsid protein of the present disclosure comprises one or more sequence changes. In certain embodiments, the sequence changes are selected from insertions, deletions, or substitutions, as compared to a known wild-type protein. A single insertion, deletion, or mutation comprises one or more sequential amino acid residue. These engineered sequence changes serve to introduce the functional sequence motif into the capsid protein. In certain of these embodiments, the sequence change comprises at least one insertion of an insertion sequence that forms the functional sequence motif.5.4.1. Transamidation Enzymes

[0322] In certain embodiments, the functional sequence motif of the present disclosure comprises any known transamidase enzyme recognition sequence. Recognition sequences are specific amino acid sequences found in proteins that are recognized and targeted by transamidase enzymes, such as transpeptidases. Transpeptidases, such as sortase enzymes, are a group of enzymes that play a crucial role in the process of protein cross-linking, particularly in the formation of the bacterial cell wall. The transamidase 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 the amino acids vary depending on the bacterial species.

[0323] The transamidase for use with the present disclosure can also be selected from those mutated from the natural sequence to include mutations. In some embodiments the enzyme is a transamidase harboring a mutation that increases the efficiency of the cleavage and hence the overall reaction. Example mutated sortases for use with the present disclosure are those identified in J. Biol. Chem. (2020) 295:9, 2664-2675 and Scientific reports 2016 (6:31899) are incorporated herein by refence. In certain embodiments, the sortase for use with the present disclosure harbors one or more of the following mutations: P94R, E105K, E108A, D124G, D160N, D165A, D186G, Y187L, E189R, K190E, and K196T. In a particular embodiment the transamidase is Hepta-Mutant Staphylococcus aureus Sortase A (SrtA7m).

[0324] In certain embodiments, the functional sequence motif, corresponding enzyme and enzyme substrate moiety for functionalizing a ligand are selected from those described in the Peptide Ligation Table below.

[0325] Peptide LigationFunctional sequence motif Enzyme(s) Enzyme substrate moietyLPXTG (SEQ ID NO: 45) Heptamutant Sortase A oligo glycine, primary amineNPQTN (SEQ ID NO: 22) Sortase B oligo glycine any AA except Pro, Asp, and Glu, at theN-HV or D-HV Butelase second position He, Leu, Vai, or CysRH-X (X = peptide, tag) nucleophilicY-RH Trypsiligase acyl acceptor peptideKDPGA (SEQ ID NO: 69) Connectase PGAAHI VMVD A YKPTK (SEQ ID NO : 70) Spy ligase ATHIKF SKRD (SEQ ID NO : 71 )DIPATYEFTDGKHYITNEPIPPK (SEQKLGSIEFIKVNK (SEQ ID NO: 72) Snoopligase ID NO: 735.4.1.1 Sortase Transamidase Enzyme

[0326] The term “sortase,” as used herein, refers to an enzyme able to carry out a transpeptidation reaction, e.g., conjugating the C-terminus of a protein to the N-terminus of a protein, via transamidation. Sortases are also referred to as transamidases, and typically exhibit both a protease and a transpeptidation activity. Various sortases from prokaryotic organisms have been identified. For example, some sortases from Gram-positive bacteria cleave and translocate proteins to proteoglycan moieties in intact cell walls. Among the sortases that have been isolated from Staphylococcus aureus, are sortase A (Srt A) and sortase B (Srt B). Thus, in certain embodiments, a transamidase used in accordance with the present disclosure is sortase A, e.g., from S. aureus, also referred to herein as SrtAaureus. Incertain embodiments, a transamidase is a sortase B, e.g., from S. aureus, also referred to herein as SrtBaureus.

[0327] Sortases have been classified into 4 classes, designated A, B, C, and D, designated sortase A, sortase B, sortase C, and sortase D, respectively, based on sequence alignment and phylogenetic analysis of 61 sortases from Gram-positive bacterial genomes (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 classes correspond to the following subfamilies, into which sortases have also been classified by Comfort and Clubb (Comfort D., Clubb R. T. 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), Class D (Subfamilies 4 and 5). The aforementioned references disclose numerous sortases and recognition motifs. See also Pallen, M. J.; Lam, A. C.; 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 be able to assign a sortase to the correct class based on its sequence and / or other characteristics such as those described in Drami, et al., supra. The term “sortase A” is used herein to refer to a class A sortase, usually named SrtA in any particular bacterial species, e.g., SrtA from S. aureus. Likewise, “sortase B” is used herein to refer to a class B sortase, usually named SrtB in any particular bacterial species, e.g., SrtB from S. aureus. The disclosure encompasses embodiments relating to a sortase A from any bacterial species or strain. The disclosure encompasses embodiments relating to a sortase B from any bacterial species or strain. The disclosure encompasses embodiments relating to a class C sortase from any bacterial species or strain. The disclosure encompasses embodiments relating to a class D sortase from any bacterial species or strain.

[0328] Amino acid sequences of Srt A and Srt B and the nucleotide sequences that encode them are known to those of skill in the art and are disclosed in a number of references cited herein, the entire contents of all of which are incorporated herein by reference. The amino acid sequences of S. aureus SrtA and SrtB are homologous, sharing, for example, 22% sequence identity and 37% sequence similarity. The amino acid sequence of a sortase- transamidase from Staphylococcus aureus also has substantial homology with sequences of enzymes from other Gram-positive bacteria, and such transamidases can be utilized in theligation processes described herein. For example, for SrtA there is about a 31% sequence identity (and about 44% sequence similarity) with best alignment over the entire sequenced region of the S. pyogenes open reading frame. There is about a 28% sequence identity with best alignment over the entire sequenced region of the A. naeslundii open reading frame. It will be appreciated that different bacterial strains may exhibit differences in sequence of a particular polypeptide, and the sequences herein are exemplary.5.4.2. Functional Sequence motifs

[0329] In some embodiments, the engineered rAAV capsid protein comprises the functional sequence motif that is located within a surface accessible variable region (VR) of the capsid primary sequence.

[0330] In some embodiments, the functional sequence motif has a sequence of X1X2X3 (SEQ ID NO: 6); wherein Xi, X2, and X3, are each independently selected from any amino acid residue.

[0331] In some embodiments, the functional sequence motif has a sequence of X1X2X3 (SEQ ID NO: 7), and wherein two or more of Xi, X2, and X3, are selected from: Xi is valine (V); X2 is proline (P); and X3 is proline (P).

[0332] In some embodiments, Xi, X2, and X3, are selected from: Xi is independently proline (P); X2 is independently selected from glycine (G) and glutamic acid (E); and X3 is independently selected from proline (P), and phenylalanine (F).

[0333] 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 a sequence PEF (SEQ ID NO: 50). In some embodiments, the functional sequence motif has a sequence PGF (SEQ ID NO: 51). In some embodiments, the functional sequence motif has a sequence PEP (SEQ ID NO: 52).

[0334] In some embodiments, the functional sequence motif has a sequence of X1X2X3X4 (SEQ ID NO: 53); wherein Xi, X2, X3, and X4, are each independently selected from any amino acid residue.

[0335] In some embodiments, wherein the functional sequence motif has a sequence of X1X2X3X4X5 (SEQ ID NO: 54); wherein Xi, X2, X3, X4, and X5, are each independently selected from any amino acid residue.

[0336] In some embodiments, the functional sequence motif has a sequence of X1X2X3X4X5 (SEQ ID NO: 54), wherein: Xi is independently selected from lysine (L), valine (V), (I) and asparagine (N); X2 is independently proline (P); 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).

[0337] In some embodiments, two or more of Xi, X2, X3, X4, and X5, are selected from: Xi is independently selected from lysine (L); X2 is independently proline (P); X3 is independently selected from any amino acid residue; X4 is independently threonine (T); and X5 is independently selected from alanine (A), a glycine (G), and asparagine (N).

[0338] In some embodiments, three or more of Xi, X2, X3, X4, and X5 are selected from: Xi is independently selected from lysine (L), valine (V), (I) and an asparagine (N); X2 is independently proline (P); 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).

[0339] In some embodiments, Xi 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).

[0340] 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 that is LPX3TG (SEQ ID NO: 48). In some embodiments, the functional sequence motif comprises a sequence that is LPX3TA (SEQ ID NO: 55). In some embodiments, the functional sequence motif comprises a sequence that is LPETG (SEQ ID NO: 12). In some embodiments, the functional sequence motif comprises a sequence that is LPETA (SEQ ID NO: 56).

[0341] In some embodiments, the functional sequence motif has a sequence of X1X2X3X4X5X6 (SEQ ID NO: 5); wherein Xi, X2, X3, X4, X5, and Xe are each independently selected from any amino acid residue.

[0342] In some embodiments, the functional sequence motif has a X1X2X3X4X5X6 (SEQ ID NO: 5); wherein two or more of Xi, X2, X3, X4, X5, and Xe are: Xi is independently asparagine (N); X4 is independently threonine (T); X5 is independently asparagine (N); and Xe is independently proline (P).

[0343] In some embodiments, the functional sequence motif has a X1X2X3X4X5X6 (SEQ ID NO: 5); wherein at least three of Xi, X2, X3, X4, X5, and Xe are: Xi is independently asparagine (N); X4 is independently threonine (T); X5 is independently asparagine (N); and Xe is independently proline (P).

[0344] 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 a sequence that is NX2X3TNX6 (SEQ ID NO: 57). In some embodiments, the functional sequence motif comprises a sequence that is VPX3X4X5P (SEQ ID NO: 58).

[0345] In some embodiments, the engineered rAAV capsid protein comprises one or more sequence changes selected from insertions, deletions, or substitutions, compared to a wild type protein that serve to introduce the functional sequence motif. In some embodiments, a sequence change is an insertion. In some embodiments, a sequence change is a deletion. In some embodiments, a sequence change is a substitution.

[0346] In some embodiments, the engineered rAAV capsid protein comprises at least one insertion mutation wherein the insertion forms the functional sequence motif.

[0347] In some embodiments, the insertion mutation is the insertion of an exogenous 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).

[0348] 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.

[0349] 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 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.

[0350] In embodiments of the present disclosure, an engineered adeno-associated virus (AAV) capsid protein comprising one or more functional sequence motif(s), wherein the functional sequence motif comprises a sortase recognition motif.

[0351] 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.

[0352] In some embodiments, the engineered rAAV capsid protein comprises a functional sequence motif within variable region IV or VIII.

[0353] In some embodiments, the engineered rAAV capsid protein comprises a functional sequence motif within variable region VIII.

[0354] In some embodiments, the engineered rAAV capsid protein comprises a functional sequence motif within variable region IV and VIII, wherein each functional sequence motif is independently selected.

[0355] In some embodiments, each functional sequence motif is the same.

[0356] In some embodiments, the rAAV capsid protein is selected from an AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9 serotype. 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.

[0357] In some embodiments, the functional sequence motif begins after S453 in VR4 of an AAV1 capsid serotype; S452 in VR4 or R585 VR8 of an AAV2 capsid serotype; S576 in VR8 of an AAV5 capsid serotype; S453 in VR4 or S587 VR8 of an AAV6 capsid serotype; P454 in VR4 or A587 VR8 of an AAV7 capsid serotype; T454 in VR4 or Q589 VR8 of an AAV8 capsid serotype; and S454 in VR8 of an AAV9 capsid serotype.

[0358] In some embodiments, the functional sequence motif begins after S452 in VR4 or R585 in VR8 of an AAV2 capsid serotype. In some embodiments, the functional sequence motif begins after S452 in VR4. In some embodiments, the functional sequence motif begins after R585 in VR8.

[0359] In some embodiments, LPET (SEQ ID NO: 49) is inserted after S452 to form the functional sequence motif.

[0360] In some embodiments, the engineered rAAV capsid protein does not comprise additional mutations when compared to the wildtype capsid protein.

[0361] In one aspect, a polynucleotide encoding the engineered rAAV capsid protein of the present disclosure is provided.

[0362] In one aspect, a vector comprising the polynucleotide of the present disclosure is provided.

[0363] In some embodiments, the vector comprises a promoter operably linked to the polynucleotide.

[0364] In one aspect, a host cell comprising the engineered rAAV capsid protein, the polynucleotide, or the vector of the present disclosure is provided.

[0365] In one aspect, a recombinant rAAV virion (rAAV) comprising the engineered rAAV capsid protein of the present disclosure is provided.

[0366] In some embodiments, the rAAV virion further comprises an exogenous cargo polynucleotide.

[0367] In some embodiments, the exogenous cargo polynucleotide comprises a template for homology directed repair.

[0368] 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 miRNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding gene editing guide RNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding RNA-editing guide RNA.

[0369] In some embodiments, the exogenous polynucleotide comprises an expressible polynucleotide encoding a therapeutic protein.

[0370] In some embodiments, the rAAV virion comprises a functional sequence motif, wherein the functional sequence motif attenuates or abrogates binding of the virion to mammalian cell polysaccharides or proteoglycans compared to a reference virion having the same capsid protein sequence but without the functional sequence motif. In some embodiments, the functional sequence motif attenuates or abrogates binding of the virion to mammalian cell polysaccharides compared to a reference virion having the same capsid protein sequence but without the functional sequence motif. In some embodiments, the functional sequence motif attenuates or abrogates binding of the virion to mammalian cellproteoglycans compared to a reference virion having the same capsid protein sequence but without the functional sequence motif.

[0371] In some embodiments, the rAAV virion comprises at least one cross linker reactive moiety covalently connected 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.

[0372] In some embodiments, the amino acid residue is a 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.

[0373] In one aspect, 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) a N-terminal cleavage fragment and (ii) a C- terminal cleavage fragment; and at least one cross linker reactive moiety covalently connected to a C-terminal amino acid of the N-terminal cleavage fragment.

[0374] In some embodiments, the covalent linkage comprises an amide bond. In some embodiments, the C-terminal amino acid of the N-terminal cleavage fragment is a threonine.

[0375] In one aspect, 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) a N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and wherein at least one cross linker reactive moiety is covalently connected to a C-terminal amino acid of the N-terminal cleavage fragment; and optionally at least one cross linker reactive moiety is covalently connected to an amino acid residue in the engineered rAAV capsid protein, wherein the amino acid residue is not within the functional sequence motif.

[0376] In some embodiments, the surface functionalized rAAV virion comprises at least one crosslinker moiety, wherein each crosslinker reactive moiety is independently selected from a crosslinker reactive moiety that participates in a reaction selected from: a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strain-promoted alkyne-azide cycloaddition(SPAAC) reaction, a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction, an inverse electron demand Diels-Alder (IEDDA) reaction, and a Staudinger ligation and a [4+1] cycloaddition reaction. In some embodiments, a crosslinker reactive moiety that participates in a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. In some embodiments, a crosslinker reactive moiety that participates in a strain-promoted alkyneazide cycloaddition (SPAAC) reaction. In some embodiments, a crosslinker reactive moiety that participates in a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction. In some embodiments, a crosslinker reactive moiety that participates in an inverse electron demand Diels-Alder (IEDDA) reaction. In some embodiments, a crosslinker reactive moiety that participates in a Staudinger ligation and a [4+1] cycloaddition reaction.

[0377] In some embodiments, the crosslinker reactive moiety comprises at least one of an eight membered ring and a triazole ring. In some embodiments, the crosslinker reactive moiety is selected from a cyclooctyne and an azide.

[0378] In some embodiments, the cyclooctyne is selected from dibenzylcyclooctyne (DIBO), dibenzoazacyclooctyne (DBCO), and biarylazacyclooctynone (BARAC), or a derivative thereof. In some embodiments, the cyclooctyne is dibenzylcyclooctyne (DIBO). In some embodiments, the cyclooctyne is dibenzoazacyclooctyne (DBCO). In some embodiments, the cyclooctyne is biarylazacyclooctynone (BARAC).

[0379] In some embodiments of the present disclosure, the sortase is a sortase A (SrtA). SrtA recognizes the motif LPX1TX2 (SEQ ID NO: 8); wherein each occurrence of Xi and X2 represents independently any amino acid residue), with common recognition motifs being, e.g., LPKTG (SEQ ID NO: 9), LPATG (SEQ ID NO: 10), LPNTG (SEQ ID NO: 11). In some embodiments LPETG (SEQ ID NO: 12) is used as the sortase recognition motif. However, motifs falling outside this consensus may also be recognized. For example, in some embodiments the motif comprises an ‘A’ rather than a ‘T’ at position 4, e.g., LPXAG (SEQ ID NO: 13), e.g., LPNAG (SEQ ID NO: 14). In some embodiments the motif comprises an ‘A’ rather than a ‘G’ at position 5, e.g., LPXTA (SEQ ID NO: 15), e.g., LPNTA (SEQ ID NO: 16). In some embodiments the motif comprises a ‘G’ rather than ‘P’ at position 2, e.g., LGXTG (SEQ ID NO: 17), e.g., LGATG (SEQ ID NO: 18). In some embodiments the motif comprises an ‘I’ rather than ‘L’ at position 1, e.g., IPXTG (SEQ ID NO: 19), e.g., IPNTG (SEQ ID NO: 20) or IPETG (SEQ ID NO: 21). Additional suitable sortase recognition motifs will be apparent to those of skill in the art, and the disclosure is not limited in this respect. Itwill be appreciated that the terms “recognition motif’ and “recognition sequence”, with respect to sequences recognized by a transamidase, transpeptidase or sortase, are used interchangeably.

[0380] In some embodiments of the present disclosure, “recognition sequence motif residue” will be apparent to those of skill in the art. In embodiments, a recognition sequence motif residue refers to the portion of the enzymatic substrate that has been cleaved and then functionalized. Functionalization may refer to transforming the free carboxylic acid into an activated ester, such as a thioester, and reacting 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 the sortase A enzyme. Sortase A recognizes substrates such as SEQ ID NO: 12 and cleaves the peptide bond between threonine and glycine to generate an intermediate (e.g., thioester-linked acyl enzyme intermediate), which is then ligated (or functionalized). Accordingly, the recognition sequence motif residue in this example refers to LPET (residues 1 to 4 of SEQ ID NO: 12) or (SEQ ID NO: 49).

[0381] In some embodiments of the disclosure the sortase is a sortase B (SrtB), e.g., a sortase B of S. aureus, B. anthracis, or L. monocytogenes. Motifs recognized by sortases of the B class (SrtB) often fall within the consensus sequences NPXTX (SEQ ID NO: 74), e.g., NP[Q / K]-T[sH / N / G / sG], such as NPQTN (SEQ ID NO: 22) or NPKTG (SEQ ID NO: 23). For example, sortase B of S. aureus or B. anthracis cleaves the NPQTN (SEQ ID NO: 24) or NPKTG (SEQ ID NO: 25) motif of IsdC in the respective bacteria (see, e.g., Marraffini, L. and Schneewind, O., Journal of Bacteriology, 189(17), p. 6425-6436, 2007). Other recognition motifs found in putative substrates of class B sortases are 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 L. monocytogenes recognizes certain motifs lacking P at position 2 and / or lacking Q or K at position 3, such as NAKTN (SEQ ID NO: 30) and NPQSS (SEQ ID NO: 31) (Mariscotti J. F., Garcia-Del Portillo F., Pucciarelli M. G. The listeria monocytogenes sortase-B recognizes varied amino acids at position two of the sorting motif. J. Biol. Chem., 2009)

[0382] In certain embodiments, the sortase enzyme, the functional sequence motif (or “motif’) and the enzyme substrate moiety are as described in Bradshaw W.J., et al. (2015), Molecular Features of the Sortase Enzyme Family. FEBS J, 282: 2097-2114, which is incorporated herein in its entirety.

[0383] In some embodiments, the sortase is a sortase C (Srt C). Sortase C may utilize LPXTX (SEQ ID NO: 8) as a recognition motif, with each occurrence of X independently representing any amino acid residue.

[0384] In some embodiments, the sortase is a sortase D (Srt D). Sortases in this class are predicted to recognize motifs with a consensus sequence NA-[E / A / S / H]-TG (SEQ ID NO: 75) (Comfort D, supra). Sortase D has been found, e.g., in Streptomyces spp., Corynebacterium spp., Tropheryma whipplei, Thermobifida fusca, and Bifidobacterium longhum.

[0385] LPXTA (SEQ ID NO: 15) or LAXTG (SEQ ID NO: 32) may serve as a recognition sequence for sortase D, e.g., of subfamilies 4 and 5, respectively subfamily-4 and subfamily-5 enzymes process the motifs LPXTA (SEQ ID NO: 15) and LAXTG (SEQ ID NO: 32), respectively). For example, B. anthracis Sortase C has been shown to specifically cleave the LPNTA (SEQ ID NO: 16) motif in B. anthracis BasI and BasH (see Marrafini, supra).

[0386] See Barnett and Scott for description of a sortase that recognizes QVPTGV (SEQ ID NO: 33) motif (Barnett, T. C. and Scott, J. R., 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).Additional sortases, including, but not limited to, sortases recognizing additional sortase recognition motifs are also suitable for use in some embodiments of this disclosure. For example, sortases described in Chen I., Dorr B. M., and Liu D. R., 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.

[0387] The use of sortases found in any gram-positive organism, such as those mentioned herein and / or in the references (including databases) cited herein is contemplated in the context of some embodiments of this disclosure. Also contemplated is the use of sortases found in gram negative bacteria, e.g., Colwellia psychrerythraea, Microbulbifer degradans, Bradyrhizobium japonicum, Shewanella oneidensis, and Shewanella putrefaciens. Such sortases recognize sequence motifs outside the LPXTX (SEQ ID NO: 8) consensus, for example, 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) . In keeping with the variation tolerated at position 3 in sortases from gram-positive organisms, asequence motif LPXT[A / S] (SEQ ID NO: 8), e g., LPXTA (SEQ ID NO: 15) or LPSTS (SEQ ID NO: 35) may be used.

[0388] Those of skill in the art will appreciate that any sortase recognition motif known in the art can be used in some embodiments of this disclosure, and that the disclosure is not limited in this respect. For example, in some embodiments the sortase 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), LAHTG (SEQ ID NO: 41), LASTG (SEQ ID NO: 42), LPLTG (SEQ ID NO: 43), LSRTG (SEQ ID NO: 44), LPETG (SEQ ID NO: 12), The disclosure encompasses embodiments in which ‘X’ in any sortase recognition motif disclosed herein or known in the art is amino acid, for example, any naturally-occurring or any non-naturally occurring amino acid. In some embodiments, X is selected from the 20 standard amino acids found most commonly in proteins found in living organisms. In some embodiments, e.g., where the recognition motif is LPXTG (SEQ ID NO: 45) or LPXT, wherein X is D, E, A, N, Q, K, or R. In some embodiments, X in a particular recognition motif is selected from those amino acids that occur naturally at position 3 in a naturally occurring sortase substrate. For example, in some embodiments X is selected from K, E, N, Q, A in an LPXTG (SEQ ID NO: 45) or LPXT motif (residues 1 to 4 of SEQ ID NO: 45) where the sortase is a sortase A. In some embodiments X is selected from K, S, E, L, A, N in an LPXTG (SEQ ID NO: 45) or LPXT (residues 1 to 4 of SEQ ID NO: 45) motif and a class C sortase is used.

[0389] In certain of these embodiments, the functional sequence motif is LPETG (SEQ ID NO 12). In certain embodiments, the functional sequence motif has a sequence selected from: PEF, PGF, or PEP. In certain embodiments, the functional sequence motif has a sequence of X1X2X3X4 (SEQ ID NO: 53); wherein Xi, X2, X3, and X4, are each independently selected from any amino acid residue. In certain embodiments, the functional sequence motif has a sequence of X1X2X3X4X5 (SEQ ID NO: 54), wherein Xi, X2, X3, X4, X5, are each independently selected from any amino acid residue. In certain 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 certain embodiments, the functional sequence motif comprises a sequence selected from: NX2X3TNX6 (SEQ ID NO: 57), and VPX3X4X5P (SEQ ID NO: 58).

[0390] In one aspect, a surface modified rAAV virion is provided, wherein the virion comprises an exogenous cargo polynucleotide and at least one 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 region 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 crosslinked moiety that covalently connects the T of the N-terminal cleavage fragment and a targeting ligand wherein the covalent linkage comprises amide bond; and optionally (b) wherein the virion comprises a crosslinked moiety that covalently connects a lysine residue or a N terminal amine of a capsid protein and a targeting ligand.

[0391] In certain embodiments, the functional sequence motif is selected from one of: SEQ ID NOs 1-58.

[0392] 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 without the functional sequence motif. In some embodiments, the increased infectivity is with respect to at least one cell or tissue type.

[0393] In some embodiments, the cell or tissue type is selected from cardiac, nerve, central and peripheral nervous system, hematopoietic system, liver, muscle, connective tissue, lung, kidney and retinal. In some embodiments, the cell or tissue type is cardiac. In some embodiments, the cell or tissue type is nerve. In some embodiments, the cell or tissue type is central and peripheral nervous system. In some embodiments, the cell or tissue type is hematopoietic system. In some embodiments, the cell or tissue type is liver. In some embodiments, the cell or tissue type is connective tissue. In some embodiments, the cell or tissue type is lung. In some embodiments, the cell or tissue type is kidney and retinal.

[0394] The term “sortase substrate,” as used herein refers to any molecule that is recognized by a sortase, for example, any molecule that can partake in a sortase-mediated transpeptidation reaction. A typical sortase-mediated transpeptidation reaction involves a substrate comprising a C-terminal sortase recognition motif, e.g., an LPXTX motif (SEQ ID NO: 8), and a second substrate comprising an N-terminal sortase recognition motif, e.g., an N-terminal polyglycine or polyalanine. A sortase substrate may be a peptide or a protein, forexample, a target protein on the surface of a virus, or a peptide comprising a sortase recognition motif such as an LPXTX motif (SEQ ID NO: 8) or a polyglycine or polyalanine, wherein the peptide is conjugated to an agent, e.g., a small molecule, a binding agent, or a fluorophore. Accordingly, both proteins and non-protein molecules can be sortase substrates as long as they comprise a sortase recognition motif. Some examples of sortase substrates are described in more detail elsewhere herein and additional suitable sortase substrates will be apparent to the skilled artisan. The disclosure is not limited in this respect.

[0395] The term “sortagging,” as used herein, refers to the process of functionalizing the surface of the AAV virion with a reactive moiety, tag or ligand, for example, a protein, polypeptide, detectable label, binding agent, or click chemistry handle, via a sortase-mediated transpeptidation reaction. Examples of additional 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 of skill in the art and the disclosure is not limited in this aspect. In some embodiments, a tag comprises a sequence useful for purifying, expressing, solubilizing, and / or detecting a polypeptide. In some embodiments, a tag can serve multiple functions. In some embodiments, the tag is relatively small, e.g., ranging from a few amino acids up to about 100 amino acids long. In some embodiments, a tag is more than 100 amino acids long, e.g., up to about 500 amino acids long, or more.

[0396] The term “target protein,” as used herein in the context of sortase-mediated modification of viral particles, refers to a capsid protein that comprises the surface of a virus that is the target of a sortase-mediated conjugation. For example, in an embodiment where AAV2 is modified by sortagging, e.g., by adding a targeting ligand to VR4 on the surface of AAV2 particle, VP1 of AAV2 is the target protein. The term “target protein” may refer to a wild type or naturally occurring form of the respective protein, or to an engineered form, for example, to a recombinant protein variant comprising a sortase recognition motif not contained in a wild-type form of the protein. The term “modifying a target protein,” as used herein in the context of sortase-mediated protein modification, refers to a process of altering a target protein comprising a sortase recognition motif via a sortase-mediated transpeptidation reaction. Typically, the modifying results in the target protein being conjugated to ligand, for example, a peptide, protein, binding agent, detectable label, or small molecule.5.4.3. AAV Sequence Changes

[0397] In certain embodiments, the engineered rAAV capsid protein comprises one or more sequence changes as described above. In certain embodiments, the sequence change comprises the insertion of an exogenous peptide (insertion peptide) selected from LPET, LPETX (SEQ ID NO: 46) and LPETG (SEQ ID NO: 12).

[0398] The sequence change is present in one or more of VP1, VP2, and VP3 that comprise the virion capsid of the present disclosure.

[0399] In embodiments of the present disclosure, the functional sequence motif is located within a surface loop region selected from: the AB, BC, CD, DE, EF, FG, GH, and HI loops. For example, in certain embodiments, the functional sequence motif is located within a GH surface loop region.

[0400] In embodiments of the present disclosure, the functional sequence motif is located within one or more variable regions (VR) selected from VR1, VR2, VR3, VR4, VR5, VR6, VR7, VR8 and VR9. In certain of these embodiments, the functional sequence motif is located within a surface accessible variable region the capsid primary sequence. In certain of these embodiments, the engineered rAAV capsid protein comprises a functional sequence motif within variable region IV or VIII. For example, in certain embodiments, the engineered rAAV capsid protein comprises a functional sequence motif within variable region VIII. In certain embodiments, the engineered rAAV capsid protein comprises a functional sequence motif within variable region IV. In other embodiments, the engineered rAAV capsid protein comprises a functional sequence motif within variable region IV and VIII. In certain of these embodiments having more than one functional sequence, each functional sequence motif is independently selected.

[0401] In certain embodiments, the engineered rAAV capsid protein comprises more than one functional sequence motif. In certain embodiments, the rAAV virion of the present disclosure comprises more than one engineered rAAV capsid protein. In certain embodiments, each functional sequence motif (in the same or in different capsid proteins) is the same. In alternative embodiments, each functional sequence motif is different.5.4.4. AAV Serotypes and Insertion sites

[0402] In the framework of the present disclosure, an AAV capsid includes any combination of capsid proteins from natural, genetically modified and artificially created (random mutations, sequence shuffling, in silico design, etc.) serotypes that are able to assemble and produce a new AAV virus capsid that is not known to exist in nature.

[0403] Currently, there are more than 100 AAV serotypes identified that differ in the binding capacity of capsid proteins to specific cell surface receptors that can transduce different cell types. 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 tested thoroughly for their ability to transduce specific cell types and differentiated between capsid protein motifs that bind specific cell surface receptors for cell attachment. In the context of this disclosure, an rAAV capsid protein is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 is preferred. However, any other AAV capsid proteins can be used in the context of the present disclosure.

[0404] In one embodiment, the AAV capsid protein of the present disclosure comprises capsid proteins selected from those of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In particular, the capsid of the present disclosure comprises capsid proteins comprising those of AAV2, AAV9, and AAV8.

[0405] In certain embodiments, the engineered AAV capsid protein is selected from an AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9 serotype.

[0406] In particular embodiments of the present disclosure, the AAV serotype, variable region, insertion location and insertion sequence that forms the functional sequence motif are according to the following table:Table 2. The AAV serotype, variable region, insertion location and insertion sequence that forms the functional sequence motif.AAV VR4 Insertion VR8 Insertion Insertion sequence serotype Insertion sequence Location_ Location _AAV1 S(453) LPXTAAV2 S(452) LPXT R(585) LPXT (residues 1 to 4 ofSEQ ID NO: 45)AAV5 S(576) LPXTG (SEQ ID NO: 45)

[0407] In certain embodiments, the capsid is an AAV2 subtype and the functional sequence motif begins after S452 or R585 or both S452 and R585. In certain of these embodiments, LPET (SEQ ID NO. 49) is inserted to form the functional sequence motif. In certain of these embodiments, LPETG (SEQ ID NO. 12) is inserted to form the functional sequence motif.

[0408] In certain embodiments, a “VR4 mutant” comprises a functional sequence motif formed by the insertion of LPET, i.e., CTGCCCGAGACC (SEQ ID NO: 47) between S452 and G 453 (in variable region 4) of VP1 of AAV2. In this case, this section of the sequence of the engineered capsid protein becomes S(452)-L(453)-P(454)-E(455)-T(456)-G(457) and so on with the remaining AA residues incremented by the number of AAs inserted, in this case, four.

[0409] In certain embodiments, a “VR8 mutant” comprises a functional sequence motif formed by the insertion of LPET, i.e., CTGCCCGAGACC (SEQ ID NO: 47) between R 585 and G 586 (in variable region 8) of VP1 of AAV2. In this case, this section of the sequence of the engineered capsid protein becomes R(585)-L(586)-P(587)-E(589)-T(590)-G(591) etc.

[0410] In certain embodiments, a “VR4 / VR8 mutant” comprises a functional sequence motif formed by the insertion of LPET, i.e., CTGCCCGAGACC (SEQ ID NO: 47) between S452 and G 453 (in variable region 4) of VP1 of AAV2 and a functional sequence motif formed by the insertion of LPET, i.e., CTGCCCGAGACC (SEQ ID NO: 47) between R585 and G586 (in variable region 8) of VP1 of AAV2. In this case, this section of the sequence of the engineered capsid protein becomes R589-L590-P591-E592-T593-G594.

[0411] In certain embodiments, the engineered rAAV capsid protein does not comprise additional mutations beyond the sequence changes that form the functional sequence motifs when compared to the wildtype capsid protein.5.5. rAAV Cargo

[0412] The nucleic acid cargo packaged inside the surface modified rAAV capsid of the present disclosure can be any kind of nucleic acid molecule usefully transduced into cells by rAAV.

[0413] In some embodiments, the payload or cargo of the rAAV capsid is an expressible polynucleotide. In certain embodiments, the expressible polynucleotide encodes a protein (e.g., encoding a therapeutic protein). In certain embodiments, the expressible polynucleotide encodes a transgene. In certain embodiments, the expressible polynucleotide can be transcribed to provide a guide RNA, a trans-activating CRISPR RNA (tracrRNA), a messenger RNA (mRNA), a microRNA (miRNA), or a shRNA.

[0414] In some embodiments, the payload provides a DNA homology construct for homology directed repair.

[0415] In some embodiments, said nucleic acid molecule is encoding intracellular antibodies (for example to neutralize certain proteins inside cells), nucleic acid molecules encoding peptide toxins (for example to block ion channels in the pain pathway), nucleic acid molecules encoding optogenetic actuators (for example to turn on or turn off neuronal activity using light), nucleic acid molecules encoding pharmacogenetic tools (for example to turn on or off neuronal signaling using chemical ligands that have no interfering pharmacological effect), nucleic acid molecules encoding CRISPR based-editors for precision gene editing, nucleic acid molecules encoding CRISPR-epigenetic tools to regulate gene expression, and / or nucleic acid molecules encoding suicide genes to induce cell death.

[0416] Preferably, when the cargo comprises a gene editing nuclease, such as Cas9, the cargo further comprises a nucleic acid molecule, such as a gRNA and / or a specific DNA to be inserted into a host genome. In certain of these embodiments, the cargo comprises a transgene known to be associated with a genetic disorder.

[0417] The person of skill is aware of other gene editing nucleases, apart from Cas9, such as Cpfl, TALEN, ZFN, or a homing endonuclease. Further, it may be convenient to engineer using DNA-guided Argonaute interference systems (DAIS). Basically, said Argonaute (Ago) protein is heterologously expressed from a polynucleotide introduced into said cell in the presence of at least one exogenous oligonucleotide (DNA guide) providing specificity of cleavage to said Ago protein to a preselected locus. The TALEN and Cas9 systems are respectively described in WO 2013 / 176915 and WO 2014 / 191128. The Zinc-finger nucleases (ZFNs) are initially described in Kim, YG; Cha, J.; Chandrasegaran, S. (“Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain” (1996), Proc. Natl. Acad. Sci. USA 93 (3): 1156-60). Cpfl 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 initially described in Guo S, Kemphues KJ. (Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser / Thr kinase that is asymmetrically distributed. (1995), Cell; 81 (4): 611-20).

[0418] 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 miRNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding gene editing guide RNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding RNA-editing guide RNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic protein.5.6. Methods of Making Surface Modified rAAV Virions

[0419] Another aspect of this disclosure relates to a method of producing a surface modified recombinant AAV virion.

[0420] The method comprises the step of crosslinking, i.e., covalently conjugating, a ligand to an engineered viral capsid protein via a linker comprising a crosslinked moiety, Q. In some embodiments, the ligand introduces at least one mammalian cell surface target binding site into said capsid, optionally wherein a natural cell surface target binding site in said capsid is removed, such as is previously removed.

[0421] In one embodiment, a method of a making a surface modified viral capsid described herein comprises the steps: obtaining a surface functionalized viral capsid by conjugating an engineered viral capsid protein as described herein with a bifunctional linker as described herein;conjugating the surface functionalized viral capsid with a functionalized ligand to form a crosslinked moiety, Q; and obtaining the surface modified viral capsid.

[0422] In one embodiment, a method of a making a surface modified viral capsid described herein comprises the steps: obtaining a surface functionalized viral capsid by enzymatic conjugation between: a functional motif in a viral capsid protein; and an enzyme-reactive linker; wherein the linker comprises an enzyme substrate moiety and a crosslinker reactive moiety; and reacting the obtained surface functionalized viral capsid with a functionalized ligand comprising a second member of the crosslinker reactive moiety.

[0423] In certain embodiments, the method further comprises the step of producing an AAV capsid comprising at least one viral capsid protein comprising 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 8.5.7. Methods of Making Surface Modified Viral Capsids

[0424] In one aspect, a method of a making a surface modified viral capsid is provided, the method comprising the steps: 1) obtaining a surface functionalized viral capsid by reacting a viral capsid protein with a capsid-reactive linker, the linker comprising a first member of a crosslinker reactive pair and optionally one or more of a spacer; 2) conjugating the surface functionalized viral capsid with a functionalized ligand comprising a second member of the crosslinker reactive pair and optionally one or more of a spacer; wherein the first and second members of the crosslinker reactive pair react to form a crosslinked moiety, Q.

[0425] In some embodiments, the method of a making a surface modified viral capsid of the present disclosure is provided.

[0426] Another aspect of this disclosure relates to a method of producing a surface-modified recombinant viral capsid. In certain embodiments, the provided capsid is for use in transducing nucleic acids into eukaryotic, typically mammalian, particularly human, cells. Insome embodiments, the surface-modified viral capsid is a recombinant adenoviral virion. In some embodiments, the surface-modified viral capsid is a recombinant AAV virion.

[0427] The method comprises the step of crosslinking, i.e., covalently conjugating, a ligand to a viral capsid protein via a linker comprising a crosslinked moiety, Q. Preferably, the ligand introduces at least one mammalian cell surface target binding site into said capsid, optionally wherein a natural cell surface target binding site in said capsid is removed, such as is previously removed.

[0428] In one embodiment, a method of a making a surface modified viral capsid described herein comprises the steps: obtaining a surface functionalized viral capsid by reacting a viral capsid protein with a capsid-reactive linker comprising a first member of a crosslinker reactive pair and optionally one or more of a spacer; conjugating the surface functionalized viral capsid with a functionalized ligand comprising a second member of the crosslinker reactive pair and optionally one or more of a spacer, wherein the first and second members of the crosslinker reactive pair react to form a crosslinked moiety, Q; and obtaining the surface modified viral capsid.

[0429] In another embodiment, a method of a making a surface modified viral capsid described herein comprises the steps: obtaining a surface functionalized viral capsid by enzymatic conjugation between: a recognition sequence located in a surface loop region in a viral capsid protein; and an enzyme-reactive linker; wherein the linker comprises an enzyme substrate and a first member of a crosslinker reactive pair; and reacting the obtained surface functionalized viral capsid with a functionalized ligand comprising a second member of the crosslinker reactive pair.

[0430] In certain embodiments, the method further comprises the step of producing an AAV capsid comprising at least one viral capsid protein comprising at least one recognition sequence. Optionally, the recognition sequence is located in a surface loop region, such as variable region 4 or 8.

[0431] As mentioned above, if said natural mammalian cell surface target binding site in said capsid is present and not removed, and the capsid is surface modified to comprise at least one ligand according to the present disclosure, the provided surface modified viral capsid has a higher infectivity rate (i.e., improved transduction - greater efficiency or similar efficiency at lower titer), compared to the capsid that has not been surface modified as described herein. In alternative embodiments, if said natural mammalian cell target binding site in said capsid is removed, (e.g., genetic modification of the known heparin binding site) prior to surface modification of the capsid to comprise a ligand, the provided surface modified capsid has one or more of i) modified tropism and ii) improved transduction compared to the capsid that has not been surface modified as described herein.

[0432] The adeno associated virus (AAV) particle produced by the above method is preferably selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, hu68, rh.10, and mixtures thereof.

[0433] Any of the proteins of the adeno associated virus (AAV) particle to be produced by the above method can be modified. Preferably, at least one of the proteins VP1, VP2 or VP3 in said capsid is modified in the above method. Alternatively, two of the proteins VP1, VP2 and / or VP3 in said capsid are modified, or all three of the proteins VP1, VP2 and VP3 in said capsid are modified. Preferably, at least one part, e.g. at least one amino acid, of the at least one of the proteins to be modified in said capsid is modified. However, it is also possible to modify multiple parts of the proteins VP1, VP2 and VP3 in said capsid, e.g. multiple amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or any other number of parts or amino acids.Preferably at least one of arginines 484, 487, 585 and 588 and lysine 532 of VP1 and / or an analogous arginine in VP2 or VP3 are removed by replacing them with a different amino acid, such as alanine.

[0434] A capsid protein from a natural AAV serotype (further genetically modified if desired), such as VP1, VP2 or VP3, are modified chemically at specific amino acids. Examples for such modifications are well known in the art and are summarized e.g., in R.Lundblad, Chemical Reagents for Protein Modification, 3rd ed. CRC Press, 2005, which is incorporated herein by reference. Chemical modification of amino acids includes but is not limited to, modification by acylation, amidination, pyridoxylation of lysine, reductive alkylation, trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amide modification of carboxyl groups and sulphydryl modification by performic acid oxidation of cysteine to cysteic acid, formation of mercurial derivatives, formation of mixed disulphides with other thiol compounds, reaction with maleimide, carboxymethylation with iodoacetic acid or iodoacetamide and carbamoylation with cyanate at alkaline pH, although without limitation thereto. In this regard, the skilled person is referred to Chapter 15 of Current Protocols in Protein Science, Eds. Coligan et al. (John Wiley & Sons NY 1995- 2000, the entire contents of which are expressly incorporated herein) for more extensive methodology relating to chemical modification of proteins.

[0435] In some embodiments of the above method for producing an improved adeno associated virus (AAV), the capsid modification comprises both removing of natural binding sites and introducing of ligand binding sites, e.g., via functionalizing of the surface of the capsid with a capsid surface reactive moiety. In certain other embodiments, the natural binding site of the AAV capsid is unchanged, i.e. not be removed, and at least one ligand binding site or ligand is introduced in accordance with the present disclosure.

[0436] In some embodiments the natural binding site is removed by the above method for producing an improved adeno associated virus (AAV) particle, wherein the natural binding site enables binding to heparan sulfate proteoglycans. In certain of these embodiments, the natural binding site is removed by replacing at least one of arginines 585 and 588 of VP1 and / or an analogous arginine in VP2 or VP3 with a different amino acid, such as alanine.

[0437] In some embodiments the ligand binding site as introduced in accordance with the present disclosure is one that enables the covalent attachment of ligands. In certain of these embodiments the ligand binding site is selected from a benzylguanine group that is attached to available lysine residues, more preferably by reacting said capsid with benzylguanine N- hydroxysuccinimide (BG-NHS), and / or benzylcytosine N-hydroxysuccinimide (BC-NHS).

[0438] The present disclosure preferably utilizes tags that are able to bind to their specific ligands with high affinity, such as SNAP -tag, CLIP -tag, Halo-Tag, Lumio-Tag, and others. The tag molecule as introduced in the above method may be any molecule or biomolecule,which is capable of specifically binding to a further molecule. The examples may include SNAP-tag, CLIP-tag, Lumio-Tag, or Halo-Tag. For example, the affinity tag may be a SNAP-tag, a mutant of an alkylguanine-DNA alkyltransferase. Importantly, one of the substrates for SNAP-tag is benzylguanine. Commercially available products useful for the present disclosure include, e.g., HaloTag from Promega, Lumio Tag from Life Technologies, and SNAP / CLIP Tags from NEB. Said ligand binding site as introduced is preferably attached to the epsilon-amino group or the primary amine of said available lysine residue.

[0439] Accordingly, the above method for producing an improved adeno associated virus (AAV) particle is further preferred, wherein said method further comprises the step of attaching a ligand to said benzylguanine and / or said benzylcytosine group, in particular a HaloTagTM, a SNAP-tagTM or a CLIP-tagTM.

[0440] Said ligand to be attached can be any kind of ligand, but is preferably selected from a protein ligand, such as a growth factor or a cytokine; a toxin subunit, such as a cholera toxin B subunit; a lectin, such as isolectin B4 or wheat germ agglutinin; an adhesion factor, such as lactadherin; an antibody, such as an anti CD-34 antibody; a peptide, such as deltorphin opioid receptor ligand; and a gene editing nuclease, such as Cas9.5.8. Formulations

[0441] Yet another embodiment of the disclosure pertains to the afore-described surface modified viral capsid for use in the treatment of a disease, wherein said AAV is administered to a subject in a liquid, dry or semi-solid form, such as, for example, in the form of a tablet, coated tablet, effervescent tablet, capsule, powder, granulate, sugar-coated tablet, lozenge, pill, ampoule, drop, suppository, emulsion, ointment, gel, tincture, paste, cream, moist compress, gargling solution, plant juice, nasal agent, inhalation mixture, aerosol, mouthwash, mouth spray, nose spray, or room spray.

[0442] In certain embodiments, a pharmaceutical composition is provided comprising a recombinant virion, the recombinant virion comprising a surface modified viral capsid as provided herein with a recombinant nucleic acid cargo contained therein, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier, diluents, solubilizer, filler, preservative and / or excipient. Such pharmaceutically acceptable carrier, diluents, solubilizer, filler, preservative and / or excipient may for instance be found in Remington: TheScience and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins, 2000.

[0443] A further aspect of the present disclosure then relates to a pharmaceutical composition, comprising the surface modified viral capsid according to the present disclosure, together with at least one pharmaceutically acceptable carrier and / or diluent, i.e. in combination with pharmaceutically acceptable additives, carriers, diluents, solvents, filters, lubricants, excipients, binders or stabilizers. Preferably, said composition is administered to said subject in form of sprays, coatings, foams, lotions, gels, mouthwash, oral formulations or injections. Said composition can be administered to said subject systemically, orally or by any other clinically / medically accepted method.

[0444] In one aspect, a composition for use in treating a disease treatable by intracellular delivery of a recombinant polynucleotide cargo is provided, the composition comprising a therapeutically effective amount of the pharmaceutical composition according to the present disclosure, wherein the recombinant polynucleotide cargo is capable of treating the disease.

[0445] A further aspect of the present disclosure then relates to a kit comprising: a) the surface modified viral capsid as disclosed and / or for use according to the present disclosure, or a pharmaceutical composition comprising the surface modified viral capsid as disclosed according to the present disclosure, b) written instructions to apply said surface modified viral capsid or said pharmaceutical composition to a target said; and optionally, a container holding the surface modified viral capsid for use or the composition and the written instructions.

[0446] Another aspect of the present disclosure relates to the use of the above-described kit for preventing, treating, and / or inhibiting a viral infection in a subject in need of said treatment.5.9. Methods of Treating Disease

[0447] The present disclosure also includes a method for treating a subject at risk for development and / or progression of a disease, including a monogenic or polygenic genetic disease, wherein a therapeutically effective amount of the AAV particle as provided by the present disclosure is administered to the patient. In this context, therapeutically effective describes an amount of AAV particles sufficient to treat the disease, such as a geneticdisease, by resolution of symptoms. Therapeutically effective can also be an amount sufficient to prevent symptoms of a disease, such as a genetic disease, from occurring. Being at risk for the disease can result from, e.g., genetic and / or phenotypic symptoms, which predispose to the disease. In some embodiments, a patient at risk for a genetic disease has been determined to carry or be deficient in a gene associated with a genetic disease.

[0448] In one aspect, a method of treating a patient having a disease treatable by intracellular delivery of a recombinant polynucleotide cargo is provided, 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.

[0449] In one aspect, a method of treating a disease or disorder is provided, the method comprising administering a therapeutically effective amount of an engineered viral capsid according to the present disclosure. In some embodiments, the disease or disorder is a central nervous system disease or disorder, 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. In some embodiments, the disease or disorder is aromatic amino acid decarboxylase (AADC) deficiency. In some embodiments, the disease or disorder is Canavan disease. In some embodiments, the disease or disorder is Parkinson’s disease. In some embodiments, the disease or disorder is Alzheimer’s disease. In some embodiments, the disease or disorder is giant axonal neuropathy.

[0450] A further aspect of this disclosure then relates to a method for treating a disease that can be treated by gene therapy, the method comprising administering the surface modified viral capsid according to the present disclosure to a subject in need thereof.

[0451] Cells and / or subjects to be treated with the surface modified viral capsids of this disclosure are preferably of mammalian origin, such as of human origin. Nevertheless, the present disclosure can advantageously be used also in veterinary medicine, cell culture procedures, or even in plant cell diseases, depending on the similarities of the mechanisms of entry into the cells. In some embodiments, said cell to be treated is a mammalian cell, a prokaryotic cell, or a plant cell. In particular embodiments, said cell to be treated is a human cell.

[0452] Yet another embodiment of the disclosure pertains to the afore-described method for treating a disease, comprising administering the surface modified viral capsid according to the present disclosure to a subject in need thereof, wherein said surface modified viral capsid is administered to a subject in a liquid, dry or semi-solid form, such as, for example, in the form of a tablet, coated tablet, effervescent tablet, capsule, powder, granulate, sugar-coated tablet, lozenge, pill, ampoule, drop, suppository, emulsion, ointment, gel, tincture, paste, cream, moist compress, gargling solution, plant juice, nasal agent, inhalation mixture, aerosol, mouthwash, mouth spray, nose spray, or room spray.

[0453] The disease to be treated by the above method for treating a disease that comprises administering the surface modified viral capsid to a subject. In certain embodiments, the disease selected from cancer, an inherited monogenic disease, such as inherited retinal disease, a genetic skin disease, such as Olmsted Syndrome or Familiar Primary Localized Cutaneous Amyloidosis, an infectious disease, adrenoleukodystrophy, alpha- 1 antitrypsin deficiency, aromatic L-amino acid deficiency, Batten disease, Becker muscular dystrophy, beta 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, gyrate atrophy, hemophilia A, hemophilia B, Hurler syndrome (mucopolysaccharidosis type I), Hunter syndrome (mucopolysaccharidosis type II), Huntington's chorea, junctional epidermolysis bullosa, late infantile neuronal ceroid lipofuscinosis, leukocyte adherence deficiency, limb girdle muscular dystrophy, lipoprotein lipase deficiency, metachromatic leukodystrophy, Sly syndrome (mucopolysaccharidosis type VII), Netherton syndrome, ornithine transcarbamylase deficiency, Pompe disease, purine nucleoside phosphorylase deficiency, recessive dystrophic epidermolysis bullosa, Sanfilippo A (mucopolysaccharidosis type IIIA), Sanfilippo B (mucopolysaccharidosis type IILB), sickle cell disease, severe combined immunodeficiency, spinal muscular atrophy, Tay Sachs disease, Wiskott-Aldrich syndrome, von Gierke disease (glycogen storage disease type la), X-linked myotubular myopathy, anemia of end stage renal disease, angina pectoris (stable, unstable, refractory), coronary artery stenosis, critical limb ischemia, heart failure, intermittent claudication, myocardial ischemia, peripheral vascular disease, pulmonary hypertension, venous ulcers, adenovirus infection, cytomegalovirus infection, Epstein-Barr virus infection, hepatitis B infection, hepatitis C infection, HIV / AIDS, influenza, Japanese encephalitis, malaria, pediatric respiratory disease,respiratory syncytial virus, tetanus, tuberculosis, gynecological cancer, breast cancer, ovary cancer, cervix cancer, vulva cancer, nervous system cancer, glioblastoma, leptomeningeal carcinomatosis, glioma, astrocytoma, neuroblastoma, retinoblastoma, gastrointestinal cancer, colon, colorectal, liver metastases, post-hepatitis liver cancer, pancreas, gall bladder, hepatocellular carcinoma, genitourinary cancer, prostate, renal, bladder, ano-genital neoplasia, skin cancer, melanoma (malignant / metastatic), head and neck cancer, nasopharyngeal carcinoma, squamous cell carcinoma, esophageal cancer, lung cancer, adenocarcinoma, small cell / non-small cell, mesothelioma, hematological cancer, leukemia, lymphoma, multiple myeloma, sarcoma, germ cell cancer, Li-Fraumeni syndrome, thyroid cancer, Alzheimer's disease, amyotrophic lateral sclerosis, carpal tunnel syndrome, chronic traumatic brain injury, cubital tunnel syndrome, diabetic neuropathy, epilepsy, giant axonal neuropathy, late infantile neuronal ceroid lipofuscinosis, multiple sclerosis, myasthenia gravis, pain, Parkinson disease, peripheral neuropathy, spinal muscular atrophy type 2, achromatopsia, age-related macular degeneration, choroideraemia, diabetic macular edema, glaucoma, Leber congenital amaurosis, macular telangiectasia type 2, retinitis pigmentosa, superficial corneal opacity, X-linked retinoschisis, arthritis (rheumatoid, inflammatory, degenerative), degenerative joint disease, severe inflammatory disease of the rectum, ulcerative colitis, chronic renal disease, diabetic ulcer, foot ulcer, detrusor overactivity, erectile dysfunction, fractures, hearing loss, hereditary inclusion body myopathy, graft versus host disease / transplant patients, oral mucositis, parotid salivary hypofunction, systemic scleoderma, type I diabetes, and wound healing, or combinations thereof.

[0454] Also provided is a method for treating a disease, comprising administering the surface modified viral capsid according to the present disclosure to a subject in need thereof, wherein said surface modified viral capsid is administered to said subject or to a cell, in the form of a pharmaceutical composition, e.g., in combination with pharmaceutically acceptable additives, carriers, diluents, solvents, filters, lubricants, excipients, binders or stabilizers. In certain embodiments, said composition is administered to said subject in form of sprays, coatings, foams, lotions, gels, mouthwash, oral formulations or injections. Said composition can be administered to said subject systemically, orally or by any other clinically / medically accepted method.

[0455] Yet another aspect of this disclosure relates to the surface modified viral capsid according to the present disclosure for use in the transfection of a cell, for example as a geneI l ldelivery tool in research. Said use can also be for cosmetic purposes, and the present disclosure includes a method for cosmetic treatment in analogy to the medical treatment as disclosed herein. For this, administering the surface modified viral capsid according to the present disclosure to a subject or to a cell can be also achieved in form of a cosmetic composition, e.g. in combination with cosmetically safe and acceptable additives, carriers, diluents, solvents, filters, lubricants, excipients, binders or stabilizers. In certain embodiments, said composition is administered to said subject in form of sprays, coatings, foams, lotions, gels, mouthwash, oral formulations or injections. Said composition can be administered to said subject systemically, orally or by any other clinically / cosmetically accepted method.

[0456] The person of skill is aware of methods of using vectors derived from AAV for transferring genes in vitro and in vivo, such as those that have been described in WO 93 / 09239, US4797368, US 5139941 and EP 488 528.

[0457] In one aspect, a composition for use in treating a disease treatable by intracellular delivery of a recombinant polynucleotide cargo is provided, 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.

[0458] In one aspect, a use of the surface modified viral capsid of the present disclosure for the manufacture of a medicament for treating a disease treatable by intracellular delivery of a recombinant polynucleotide cargo is provided.

[0459] In one aspect, a surface functionalized viral capsid is provided, wherein the surface functionalized viral capsid comprises a first member of a crosslinker reactive pair and optionally one or more spacers. In some embodiments, the surface functionalized viral capsid is suitable for reaction with a functionalized ligand comprising a second member of the crosslinker reactive pair and optionally one or more spacers. In some embodiments, the members of the crosslinker reactive pair participate in a reaction selected from: a Cu(I)- catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction, a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction, an inverse electron demand Diels-Alder (IEEDD) reaction, and a Staudinger ligation and a [4+1] cycloaddition reaction. In some embodiments, the members of the crosslinker reactive pair participate in a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC)reaction. In some embodiments, the members of the crosslinker reactive pair participate in a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. In some embodiments, the members of the crosslinker reactive pair participate in a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction. In some embodiments, the members of the crosslinker reactive pair participate in an inverse electron demand Diels-Alder (IEDDA) reaction. In some embodiments, the members of the crosslinker reactive pair participate in a Staudinger ligation and a [4+1] cycloaddition reaction.

[0460] An additional aspect of the present disclosure relates to a kit comprising: a) the surface modified viral capsid for the transfection of cells, b) written instructions to use the surface modified viral capsid for the transfection of cells; and optionally, a container holding the surface modified viral capsid and the written instructions.5.9.1. Indications

[0461] Another aspect of this disclosure relates to recombinant virions comprising the surface modified viral capsid according to the present disclosure for use in the treatment of a disease, and methods of treating disease by administering an effective amount of recombinant virions comprising surface modified capsid as described herein. In certain embodiments, the compositions provided herein are for use in a treatment of a patient having a disease treatable by intracellular delivery of a recombinant polynucleotide cargo. In certain embodiments, the compositions provided herein are for use in a treatment comprising gene therapy. Furthermore, the disclosure provides for the use of the surface modified viral capsid composition for the preparation of a medicament for gene therapy. Also, the disclosure provides for a method of treatment comprising gene therapy, wherein the method comprises the administration of a rAAV composition comprising the surface modified capsid.

[0462] In preferred embodiments, the disease is a disease with central nervous system involvement. See, e.g., Hocquemiller et al., Hum. Gene Ther. 27(7):478-496 (2016) and Daci and Flotte, hit. J. Mol. Sci. 25(2): 1050 (2024), the disclosures of which are incorporated herein by reference in their entireties. In some of these embodiments, the disease is frontotemporal dementia (FTD), ALS, Huntington’s disease, Krabbe disease, Alzheimer’s disease, metachromatic leukodystrophy, or GM1 gangliosidosis.

[0463] The kind of disease that can be treated or prevented by administration of the rAAV according to the present disclosure is not particularly limited. Diseases include those diseasesthat can be treated by gene therapy, such as cancer, an inherited monogenic disease, such as inherited retinal disease, a genetic skin disease, such as Olmsted Syndrome or Familiar Primary Localized Cutaneous Amyloidosis, an infectious disease, ataxia, adrenoleukodystrophy, alpha- 1 antitrypsin deficiency, aromatic L-amino acid deficiency, Batten disease, Becker muscular dystrophy, beta thalassemia, Canavan disease, chronic granulomatous disease, Crigler-Najjar syndrome, cystic fibrosis, Duchenne muscular dystrophy, Fabry disease, familial adenomatous polyposis, familial hypercholesterolaemia, familial lecithin-cholesterol acyltransferase deficiency, Fanconi anaemia, galactosialidosis, Gaucher's disease, gyrate atrophy, hemophilia A and B, Hurler syndrome (mucopolysaccharidosis type I), Hunter syndrome (mucopolysaccharidosis type II), Huntington's chorea, junctional epidermolysis bullosa, late infantile neuronal ceroid lipofuscinosis, leukocyte adherence deficiency, limb girdle muscular dystrophy, lipoprotein lipase deficiency, metachromatic leukodystrophy, Sly syndrome (mucopolysaccharidosis type VII), Netherton syndrome, ornithine transcarbamylase deficiency, Pompe disease, purine nucleoside phosphorylase deficiency, recessive dystrophic epidermolysis bullosa, Sanfilippo A (mucopolysaccharidosis type IIIA), Sanfilippo B (mucopolysaccharidosis type IIIB), sickle cell disease, severe combined immunodeficiency, spinal muscular atrophy, Tay Sachs disease, Wiskott-Aldrich syndrome, von Gierke disease (glycogen storage disease type la), X-linked myotubular myopathy, anemia of end stage renal disease, angina pectoris (stable, unstable, refractory), coronary artery stenosis, critical limb ischemia, heart failure, intermittent claudication, myocardial ischemia, peripheral vascular disease, pulmonary hypertension, venous ulcers, adenovirus infection, cytomegalovirus infection, Epstein-Barr virus infection, hepatitis B infection, hepatitis C infection, HIV / AIDS, influenza, Japanese encephalitis, malaria, pediatric respiratory disease, respiratory syncytial virus, tetanus, tuberculosis, gynaecological cancer, breast, ovary, cervix, vulva, nervous system cancer, glioblastoma, leptomeningeal carcinomatosis, glioma, astrocytoma, neuroblastoma, retinoblastoma, gastrointestinal cancer, colon, colorectal, liver metastases, post-hepatitis liver cancer, pancreas, gall bladder, hepatocellular carcinoma, genitourinary cancer, prostate, renal, bladder, ano-genital neoplasia, skin cancer, melanoma (malignant / metastatic), head and neck cancer, nasopharyngeal carcinoma, squamous cell carcinoma, esophageal cancer, lung cancer, adenocarcinoma, small cell / non-small cell, mesothelioma, hematological cancer, leukemia, lymphoma, multiple myeloma, sarcoma, germ cell cancer, Li-Fraumeni syndrome, thyroid cancer, Alzheimer's disease, amyotrophic lateral sclerosis, carpal tunnel syndrome, chronic traumatic brain injury, cubital tunnel syndrome, diabetic neuropathy, epilepsy, giant axonalneuropathy, late infantile neuronal ceroid lipofuscinosis, multiple sclerosis, myasthenia gravis, pain, Parkinson disease, peripheral neuropathy, spinal muscular atrophy type 2, achromatopsia, age-related macular degeneration, choroideraemia, diabetic macular oedema, glaucoma, Leber congenital amaurosis, macular telangiectasia type 2, retinitis pigmentosa, superficial corneal opacity, X-linked retinoschisis, arthritis (rheumatoid, inflammatory, degenerative), degenerative joint disease, severe inflammatory disease of the rectum, ulcerative colitis, chronic renal disease, diabetic ulcer / foot ulcer, detrusor overactivity, erectile dysfunction, fractures, hearing loss, hereditary inclusion body myopathy, graft versus host disease / transplant patients, oral mucositis, parotid salivary hypofunction, systemic scleroderma, type I diabetes, and / or wound healing.

[0464] In some embodiments, the condition to be treated in accordance with the present disclosure is a CNS disorder or disease. In some embodiments, the CNS disease or disorder is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is selected from Aromatic amino acid decarboxylase (AADC) deficiency, Canavan disease, Parkinson’s disease, Alzheimer’s disease, and giant axonal neuropathy.

[0465] In certain embodiments, the ataxia to be treated in accordance with the present disclosure is ataxia associated with a hereditary disorder consisting of degeneration of the cerebellum or of the spine and may present with overlapping cerebellar and sensory ataxia, even. Hereditary disorders causing ataxia include autosomal dominant ones such as spinocerebellar ataxia, episodic ataxia, and dentatorubropallidoluysian atrophy, as well as autosomal recessive disorders such as Friedreich's ataxia (sensory and cerebellar, with the former predominating) and Niemann Pick disease, ataxia-telangiectasia (sensory and cerebellar, with the latter predominating), and abetalipoproteinaemia. An example of X- linked ataxic condition is the rare fragile X-associated tremor / ataxia syndrome or FXTAS.

[0466] In certain embodiments, the indication to be treated is lipoprotein lipase deficiency, large B-cell lymphoma, beta thalassemia, mantle cell lymphoma, vascular endothelial growth factor peripheral artery disease, head and neck squamous cell carcinoma, spinal muscular atrophy, adenosine deaminase deficiency (ADA-SCID), melanoma in patients who have recurring skin lesions, B cell lymphoblastic leukemia, or Leber congenital amaurosis.

[0467] In some embodiments, the condition to be treated in accordance with the present disclosure is a neuromuscular disease. In some embodiments, the neuromuscular disease is spinal muscular atrophy or amyotrophic lateral sclerosis.

[0468] In some embodiments, the condition to be treated in accordance with the present 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 gangliosidosis.

[0469] In certain embodiments, the indication to be treated include Charcot-Marie-Tooth (all types), Gangliosidosis (all types), Genetic epilepsy (i.e. Dravet), tuberous sclerosis complex, Spinal cord injury, all demyelinating hereditary motor and sensory neuropathies (HMSN), Krabbe disease, fibrodysplasia ossificans progressive, Neurofibromatosis 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, myelopathies such as tabes dorsalis (syphilitic myelopathy), leukoencephalopathies such as progressive multifocal leukoencephalopathy, leukodystrophies, and Guillain-Barre syndrome and its chronic counterpart, chronic inflammatory demyelinating polyneuropathy.

[0470] In certain embodiments, the indication to be treated is anti-MAG peripheral neuropathy, or copper deficiency-associated conditions (peripheral neuropathy, myelopathy, and rarely optic neuropathy), or progressive inflammatory neuropathy.5.9.2. Modes of Administration

[0471] Another aspect of this disclosure relates to modes of administration of the surface- modified rAAV according to the present disclosure, for use in the treatment of a disease.

[0472] In some embodiments, the rAAV is administered directly to the cerebral spinal fluid (CSF) by intrathecal administration, such as by injection into the spinal canal, intracerebroventricular (ICV) administration, or intra cisterna magna (ICM) administration.

[0473] In some embodiments, the surface modified viral capsid according to the present disclosure may be directly or indirectly administered using suitable means known in the art. Methods and uses of the disclosure include delivery and administration of the surface modified viral capsid according to the present disclosure composition systemically,regionally or locally, or by any route, for example, by injection, infusion. Exemplary administration and delivery routes include intravenous (i.v.), intra-articular, intraperitoneal (i.p.), intra-arterial, intramuscular, parenteral, subcutaneous, intra-pleural, topical, dermal, intradermal, transdermal, parenterally, e.g., transmucosal, intra-cranial, intra-spinal, oral (alimentary), mucosal, respiration, intranasal, intubation, intrapulmonary, intrapulmonary instillation, buccal, sublingual, intravascular, intrathecal, intracavity, iontophoretic, intraocular, ophthalmic, optical, intraglandular, intraorgan, intralymphatic, intrathecal, intra ci sterna magna.

[0474] In certain embodiments, the mode of administration is systemic. Systemic administration includes systemic routes of injection, such as intramyocardialy, intramuscularly, intravenously, and intravascularly. In some embodiments, the mode of administration is intramyocardialy. In some embodiments, the mode of administration is intramuscularly. In some embodiments, the mode of administration is intravenously. In some embodiments, the mode of administration is intravascularly. In certain embodiments, the surface modified viral capsid comprises an enzyme ligand (e.g., Tissue Plasminogen Activator) that permeabilizes the blood brain barrier, e.g., the pia matter, allow transduction of spinal cord and brain parenchyma following, e.g., intrathecal injection. In embodiments, the surface modified viral capsid further comprises a cell -targeting ligand.

[0475] Improvements in means for providing an individual or a cell, tissue, organ of said individual with the surface modified viral capsid according to the present disclosure composition are anticipated considering the progress that has already thus far been achieved. Such future improvements may of course be incorporated to achieve the mentioned effect of the disclosure.

[0476] In certain embodiments, the step of administering the surface modified viral capsid according to the present disclosure, the capsid composition is dissolved in a solution that is compatible with the delivery method. In certain embodiments formulation for intravenous, subcutaneous, intramuscular, intrathecal, intraarticular and / or intraventricular administration, is the capsid composition is formulated as a physiological salt solution.

[0477] In some embodiments, the surface-modified rAAV is administered intrathecally and IVIG is administered intravenously. Exemplary intrathecal surface-modified rAAV administration and intravenous IVIG administration useful according to certain embodiments,include, e.g., those as disclosed in WO 2022 / 173847, the disclosure of which is herein expressly incorporated by reference in its entirety. In typical embodiments, IVIG is administered at a time and in an amount effective to reduce transduction of cells outside the CNS by rAAV that migrate to peripheral locations. In certain of these embodiments, the surface-modified rAAV is administered by lumbar puncture. In certain of these embodiments, the surface-modified rAAV is administered intracerebroventricularly (ICV). In certain of these embodiments, the surface-modified rAAV is administered to the cisterna magna by intra-ci sterna magna (ICM) injection.

[0478] In some embodiments, the present disclosure provides a modified version of Pari agonist SFLLRN (residues 1 to 6 of SEQ ID NO: 66), in which an extra cysteine (C) residue is included at its amidated C-termini. In some embodiments, the resulting peptide, SFLLRNC (SEQ ID NO: 66), is subsequently modified with linkers (e.g., mal eimide linkers) in order to accept an azide motif directly linked to its extra C residue. In some embodiments, AHSPG- SFLLRNC (SEQ ID NO: 66) is effectively transported by the bloodstream to different regions of the CNS. In some embodiments, modified vectors (e.g., AHSPG-SFLLRNC vectors (SEQ ID NO: 66)) are delivered systemically with intravenous injection (IV). In some embodiments, AHSPG-SFLLRNC vectors (SEQ ID NO: 66)are injected IV at the dose of 9xlO10VG / animal. In some embodiments, the virus is delivered through the bloodstream and its distribution is not localized in a specific region, but will be uniform throughout the nervous system. In some embodiments, the presence of positive neurons in different regions of the brain of a subject (e.g., mice) injected with AHSPG-SFLLRNC (SEQ ID NO: 66) may indicate the effective ability of the Pari agonist SFLLRNC (SEQ ID NO: 66) to confer enhanced BBB permeability to the AHSPG vectors.5.10. Additional Embodiments1. A surface modified viral capsid, comprising one or more of: a ligand covalently conjugated to a viral capsid protein via a linker, the linker comprising: a crosslinked moiety, wherein the crosslinked moiety is formed by a reaction between first and second members of a crosslinker reactive pair; and optionally one or more spacers.The surface modified viral capsid according to embodiment 1, wherein the first and second members of the crosslinker reactive pair participate in a reaction selected from: a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strain- promoted alkyne-azide cycloaddition (SPAAC) reaction, a strain-promoted alkyne- nitrone cycloaddition (SPANC) reaction, an inverse electron demand Diels-Alder (IEDDA) reaction, and a Staudinger ligation and a [4+1] cycloaddition reaction. In some embodiments, the first and second members of the crosslinker reactive pair participate in a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. In some embodiments, the first and second members of the crosslinker reactive pair participate in a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. In some embodiments, the first and second members of the crosslinker reactive pair participate in a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction. In some embodiments, the first and second members of the crosslinker reactive pair participate in an inverse electron demand Diels- Alder (IEDDA) reaction. In some embodiments, the first and second members of the crosslinker reactive pair participate in a Staudinger ligation and a [4+1] cycloaddition reaction. The surface modified viral capsid according to any preceding embodiment, wherein the crosslinked moiety comprises at least one of: an eight membered ring and a triazole ring. The surface modified viral capsid according to embodiment 2, wherein the reaction is a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. The surface modified viral capsid according to any preceding embodiment, wherein the crosslinker reactive pair comprises a cyclooctyne and an azide. The surface modified viral capsid according to embodiment 1, wherein the cyclooctyne is selected from dibenzylcyclooctyne (DIBO), dibenzoazacyclooctyne (DBCO), and biarylazacyclooctynone (BARAC), or a derivative thereof. In some embodiments, the cyclooctyne is dibenzoazacyclooctyne (DBCO). In some embodiments, the cyclooctyne is biarylazacyclooctynone (BARAC).The surface modified viral capsid according to embodiment 1, wherein the cyclooctyne is a DBCO. The surface modified viral capsid according to any preceding embodiment, wherein the crosslinked moiety comprises the following structure:wherein R1and R2indicate the points of attachment to the linker. The surface modified viral capsid according to embodiment 2, wherein the reaction is an inverse electron demand Diels-Alder (IEDDA) reaction. The surface modified viral capsid according to any one of embodiments 1, 2, and 9, wherein the crosslinker reactive pair comprise a transcyclooctene and a tetrazine. The surface modified viral capsid according to any one of embodiments 1, 2, 9, and10, wherein the crosslinked moiety comprises the following structurewherein R1and R2indicate the points of attachment to the linker. The surface modified viral capsid according to any preceding embodiment, wherein the linker comprises one or more spacers.The surface modified viral capsid according to embodiment 12, wherein the one or more spacers comprise from 1 to 20 monomers of polyethylene glycol. In some embodiments, the one or more spacers comprise from 1 to 20 monomers of ethylene glycol. The surface modified viral capsid according to embodiment 13, wherein the one or more spacers comprise from 2 to 8 monomers of polyethylene glycol. In some embodiments, the one or more spacers comprise from 2 to 8 monomers of ethylene glycol. The surface modified viral capsid according to embodiment 14, wherein at one of the one or more spacers comprise 4 monomers of polyethylene glycol. In some embodiments, at least one of the one or more spacers comprise 4 monomers of ethylene glycol. The surface modified viral capsid according to embodiment 15, comprising two spacers that comprise 4 monomers of polyethylene glycol. In some embodiments, the surface modified viral capsid comprises two spacers that comprise 4 monomers of ethylene glycol. The surface modified viral capsid according to any preceding embodiment, wherein the ligand is a cell-type specific ligand. The surface modified viral capsid according to any preceding embodiment, wherein the ligand is selected from cytokines, growth factors, lectins, toxins, single chain antibodies, multiple chain antibodies or antigen binding antibody fragments, peptides and combinations thereof. In some embodiments, the ligand is known to bind to a receptor endogenous to the brain microvascular endothelial cells (BMECs). In some embodiments, the surface modified viral capsid comprises a receptor. In some embodiments, the receptor is a protease activated receptor (PARs).In some embodiments, the receptor is an Endothelial Protease Activated Receptor 1 (PARI). In some embodiments, the ligand binds to PARI . In some embodiments, the ligand is a PAR-1 activating peptide. In some embodiments, the ligand is thrombin or derivatives thereof. In some embodiments, the ligand is a peptide comprising the sequence SFLLR (SEQ ID NO.: 2). In some embodiments, the ligand comprises a peptide having the sequence SFLLRNPNDKC (SEQ ID NO: 3). In some embodiments, the ligand comprises a wheat germ agglutinin (WGA). In some embodiments, the ligand is selected from WGA1, WGA2, and WGA3. In some embodiments, the ligand binds to PARI or the ligand is a WGA. In some embodiments, the ligand binds to PARI. In some embodiments, the ligand is a WGA. In some embodiments, the surface modified viral capsid comprises a first ligand that binds to binds to PARI and a second ligand that comprises a WGA. The surface modified viral capsid according to any preceding embodiment, wherein the linker is covalently attached to a primary amino group of the capsid protein primary sequence. In some embodiments, the primary amino group is selected from an N-terminal amino group, a lysine epsilon amino group and an arginine amino acid group. In some embodiments, the primary amino group is the epsilon amino group of a lysine amino acid residue.The surface modified viral capsid according to any preceding embodiment, wherein the linker is covalently attached to the ligand via a primary amino group of the ligand. The surface modified viral capsid according to any preceding embodiment, wherein the linker is covalently attached to the targeting ligand via a non-natural amino acid residue of the primary sequence of the targeting ligand. The surface modified viral capsid according to embodiment 23, wherein the nonnatural amino acid residue comprises a member of the crosslinker reactive pair that participates in a reaction selected from: a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction, a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction, an inverse electron demand Diels- Alder (IEEDD) reaction, a Staudinger ligation and a [4+1] cycloaddition reaction. The surface modified viral capsid according to embodiment 24, wherein the crosslinker reactive pair comprises an azide, cyclooctyne, cyclooctene or 1, 2,4,5- tetrazine moiety. The surface modified viral capsid according to any one of embodiments 1-25, wherein the protein sequence of the viral capsid has been mutated to attenuate or abrogate binding of the capsid to mammalian cell polysaccharides or proteoglycans. The surface modified viral capsid according to any one of embodiments 1-25, wherein the protein sequence of the viral capsid has not been mutated to attenuate or abrogate binding of the capsid protein to mammalian cell polysaccharides or proteoglycans. The surface modified viral capsid according to embodiment 1, characterized by increased infectivity compared to an unmodified viral capsid having the same capsid protein sequence. The surface modified viral capsid according to any preceding embodiment, wherein the viral capsid is selected from an adenovirus capsid, adeno-associated virus capsid, retro virus capsid, lentivirus capsid, herpes simplex virus capsid, and a baculoviruscapsid. 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 retro virus capsid. In some embodiments, the viral capsid is a herpes simplex virus capsid. In some embodiments, the viral capsid is a baculovirus capsid. The surface modified viral capsid according to embodiment 1, wherein the viral capsid is an adeno-associated virus (AAV) capsid. The surface modified viral capsid according to embodiment 1, wherein at least one of the arginine residues at 585 and 588 of VP1, or analogous positions in VP2 or VP3, have been mutated. The surface modified viral capsid according to embodiment 31, wherein the arginine residues at 585 and 588 of VP1, have been mutated to alanine residues. The surface modified viral capsid according to embodiment 31 or 32, characterized by altered tropism compared to an unmodified viral capsid. The surface modified viral capsid according to any preceding embodiment, wherein the surface modified viral capsid demonstrates evasion of pre-existing neutralizing antibodies. A surface modified viral capsid according to Formula I:Y and Y’ are independently an attachment moiety; n and n’ are independently 0 or an integer from 1 to 50;Sp and Sp’ are independently an optional spacer;L is a ligand; x is the ligand per capsid ratio that is in a range from 1 to 500; andQ is selected from:wherein, Z is a 7 or 8 membered cyclic or heterocyclic structure. The surface modified viral capsid according to embodiment 35, wherein x ranges from 100-200. The surface modified viral capsid according to embodiment 35, wherein x ranges from 130-170. A surface modified viral capsid according to Formula 1-1 :n and n’ are independently an integer selected from 0 to 30;T is a ligand; and x is an integer from 1 to 500. The surface modified viral capsid according to embodiment 38, wherein x ranges from 100-200.The surface modified viral capsid according to any one of embodiments 1 to 38, wherein the ligand per capsid ratio (x) is in the range from 130 to 170. In some embodiments, the ligand is known to bind to a receptor endogenous to the brain microvascular endothelial cells (BMECs). In some embodiments, the receptor is protease activated receptors (PARs). In some embodiments, the receptor is an Endothelial Protease Activated Receptor 1 (PARI). In some embodiments, the ligand binds to PARI. In some embodiments, the ligand is a PAR-1 activating peptide. In some embodiments, the ligand is thrombin or derivatives thereof. In some embodiments, the ligand is a peptide comprising the sequence SFLLR (SEQ ID NO.: 2). In some embodiments, the ligand comprises a peptide having the sequence SFLLRNPNDKC (SEQ ID NO: 3). In some embodiments, the ligand comprises a wheat germ agglutinin (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 PARI or wherein the ligand comprises a WGA. In some embodiments, the capsid comprises a first ligand that binds to binds to PARI and a second ligand comprises a WGA.A pharmaceutical composition comprising at least one recombinant virion, the recombinant virion comprising the surface modified viral capsid according to the present disclosure and a recombinant polynucleotide cargo, further comprising a pharmaceutically acceptable carrier, diluent, solubilizer, filler, preservative, excipient or combination thereof. A method of treating a patient having a disease treatable by intracellular delivery of a recombinant polynucleotide cargo, the method comprising: administering a therapeutically effective amount of the pharmaceutical composition according to embodiment 41, wherein the recombinant polynucleotide cargo is capable of treating the disease. A composition for use in treating a disease treatable by intracellular delivery of a recombinant polynucleotide cargo, the composition comprising: a therapeutically effective amount of the pharmaceutical composition according to embodiment 41, wherein the recombinant polynucleotide cargo is capable of treating the disease. Use of the surface modified viral capsid according to any one of embodiments 1-39 for the manufacture of a medicament for treating a disease treatable by intracellular delivery of a recombinant polynucleotide cargo. A surface functionalized viral capsid, comprising a first member of a crosslinker reactive pair and optionally one or more spacers, wherein the surface functionalized viral capsid is suitable for reaction with a functionalized ligand comprising a second member of the crosslinker reactive pair and optionally one or more spacers, wherein the members of the crosslinker reactive pair participate in a reaction selected from: a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction, a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction, an inverse electron demand Diels- Alder (IEEDD) reaction, and a Staudinger ligation and a [4+1] cycloaddition reaction.68. A method of a making a surface modified viral capsid, the method comprising the steps: obtaining a surface functionalized viral capsid by reacting a viral capsid protein with a capsid-reactive linker, the linker comprising a first member of a crosslinker reactive pair and optionally one or more of a spacer; conjugating the surface functionalized viral capsid with a functionalized ligand comprising a second member of the crosslinker reactive pair and optionally one or more of a spacer; wherein the first and second members of the crosslinker reactive pair react to form a crosslinked moiety, Q.69. The method according to embodiment 46, wherein the surface modified viral capsid is according to any one of embodiments 1 to 39.70. A recombinant virion comprising a surface modified capsid of any one of embodiments 1 to 39.71. The recombinant virion of embodiment 48, wherein the recombinant virion is rAAV.5.11. Further additional embodiments72. In one aspect, an engineered adeno-associated virus (AAV) capsid protein is provided, wherein the capsid protein comprises a functional sequence motif, wherein the functional sequence motif has the sequence: X1X2X3X4X5X6 (SEQ ID NO: 5); wherein Xi, X2, X3, X4, X5, and Xe are each independently selected from any amino acid residue and wherein X4, X5, and X6 are optional; wherein the functional sequence motif is a substrate for a sortase transamidase enzyme.73. In some embodiments, the enzyme is selected from a sortase A, sortase B, archaeosortase A, exosortase A, rhombosortase, and PorU. In some embodiments, the enzyme is sortase A. In some embodiments, the enzyme is sortase B. In some embodiments, the enzyme is archaeosortase A. In some embodiments, the enzyme is exosortase A. In some embodiments, the enzyme is rhombosortase. In some embodiments, the enzyme is PorU.74. In some embodiments, the enzyme is a sortase A.75. In some embodiments, the engineered rAAV capsid protein is heptamutant (SrtA7M) or a pentamutant (SrtA5M). In some embodiments, the engineered rAAV capsid protein is heptamutant (SrtA7M). In some embodiments, the engineered rAAV capsid protein is a pentamutant (SrtA5M).76. In some embodiments, the enzyme is Hepta-Mutant Staphylococcus aureus Sortase A (SrtA7m).77. In some embodiments, the engineered rAAV capsid protein comprises the functional sequence motif that is located within a surface accessible variable region (VR) of the capsid primary sequence.78. In some embodiments, the functional sequence motif has a sequence of X1X2X3 (SEQ ID NO: 6); wherein Xi, X2, and X3, are each independently selected from any amino acid residue.79. In some embodiments, the functional sequence motif has a sequence of X1X2X3 (SEQ ID NO: 7), and wherein two or more of XI, X2, and X3, are selected from: XI is valine (V); X2 is proline (P); and X3 is proline (P).80. In some embodiments, XI, X2, and X3, are selected from: XI is independently proline (P); X2 is independently selected from glycine (G) and glutamic acid (E); and X3 is independently selected from proline (P), and phenylalanine (F).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 a sequence PEF (SEQ ID NO: 50). In some embodiments, the functional sequence motif has a sequence PGF (SEQ ID NO: 51). In some embodiments, the functional sequence motif has a sequence PEP (SEQ ID NO: 52).82. In some embodiments, the functional sequence motif has a sequence of X1X2X3X4 (SEQ ID NO: 53); wherein XI, X2, X3, and X4, are each independently selected from any amino acid residue.83. In some embodiments, wherein the functional sequence motif has a sequence of X1X2X3X4X5 (SEQ ID NO: 54); wherein XI, X2, X3, X4, X5, are each independently selected from any amino acid residue.84. In some embodiments, the functional sequence motif has a sequence of X1X2X3X4X5 (SEQ ID NO: 54), wherein: XI is independently selected from lysine (L), valine (V), (I) and asparagine (N); X2 is independently proline (P); 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).85. In some embodiments, two or more of XI, X2, X3, X4, and X5, are selected from: XI is independently selected from lysine (L); X2 is independently proline (P); X3 is independently selected from any amino acid residue; X4 is independently threonine (T); and X5 is independently selected from alanine (A), a glycine (G), and asparagine (N).86. In some embodiments, three or more of XI, X2, X3, X4, and X5 are selected from: XI is independently selected from lysine (L), valine (V), (I) and an asparagine (N); X2 is independently proline (P); 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).87. In some embodiments, XI 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).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 that is LPX3TG (SEQ ID NO: 48). In some embodiments, the functional sequence motif comprises a sequence that is LPX3TA (SEQ ID NO: 55). In some embodiments, the functional sequence motif comprises a sequence that is LPETG (SEQ ID NO: 12). In some embodiments, the functional sequence motif comprises a sequence that is LPETA (SEQ ID NO: 56).89. In some embodiments, the functional sequence motif has a sequence of X1X2X3X4X5X6 (SEQ ID NO: 5); wherein XI, X2, X3, X4, X5, and X6 are each independently selected from any amino acid residue.90. In some embodiments, the functional sequence motif has a X1X2X3X4X5X6 (SEQ ID NO: 5); wherein two or more of XI, X2, X3, X4, X5, and X6 are: XI is independentlyasparagine (N); X4 is independently threonine (T); X5 is independently asparagine (N); and X6 is independently proline (P).91. In some embodiments, the functional sequence motif has a X1X2X3X4X5X6 (SEQ ID NO: 5); wherein at least three of XI, X2, X3, X4, X5, and X6 are: XI is independently asparagine (N); X4 is independently threonine (T); X5 is independently asparagine (N); and X6 is independently proline (P).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 a sequence that is NX2X3TNX6 (SEQ ID NO: 57). In some embodiments, the functional sequence motif comprises a sequence that is VPX3X4X5P (SEQ ID NO: 58).93. In some embodiments, the engineered rAAV capsid protein comprises one or more sequence changes selected from insertions, deletions, or substitutions, compared to a wild type protein that serve to introduce the functional sequence motif. In some embodiments, a sequence change is an insertion. In some embodiments, a sequence change is a deletion. In some embodiments, a sequence change is a substitution.94. In some embodiments, the engineered rAAV capsid protein comprises at least one insertion mutation wherein the insertion forms the functional sequence motif.95. In some embodiments, the insertion mutation is the insertion of an exogenous 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).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 VP3.97. 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 AB, BC, CD, DE, EF, FG, GH, and HI loops. In some embodiments, thefunctional 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.98. In some embodiments, the functional sequence motif is located within a GH surface loop region.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 VR9.100. In some embodiments, the engineered rAAV capsid protein comprises a functional sequence motif within variable region IV or VIII.101. In some embodiments, the engineered rAAV capsid protein comprises a functional sequence motif within variable region VIII.102. In some embodiments, the engineered rAAV capsid protein comprises a functional sequence motif within variable region IV and VIII, wherein each functional sequence motif is independently selected.103. In some embodiments, each functional sequence motif is the same.104. In some embodiments, the rAAV capsid protein is selected from an AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9 serotype. 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 AAV9.105. In some embodiments, the functional sequence motif begins after S453 in VR4 of an AAV1 capsid serotype; S452 in VR4 or R585 VR8 of an AAV2 capsid serotype; S576 in VR8 of an AAV5 capsid serotype; S453 in VR4 or S587 VR8 of an AAV6 capsid serotype; P454 in VR4 or A587 VR8 of an AAV7 capsid serotype; T454 in VR4 or Q589 VR8 of an AAV8 capsid serotype; and S454 in VR8 of an AAV9 capsid serotype.106. In some embodiments, the functional sequence motif begins after S452 in VR4 or R585 in VR8 of an AAV2 capsid serotype. In some embodiments, the functional sequence motif begins after S452 in VR4. In some embodiments, the functional sequence motif begins after R585 in VR8.107. In some embodiments, LPET (SEQ ID NO: 49) is inserted after S452 to form the functional sequence motif.108. In some embodiments, the engineered rAAV capsid protein does not comprise additional mutations when compared to the wildtype capsid protein.109. In one aspect, a polynucleotide encoding the engineered rAAV capsid protein of the present disclosure is provided.110. In one aspect, a vector comprising the polynucleotide of the present disclosure is provided.111. In some embodiments, the vector comprises a promoter operably linked to the polynucleotide.112. In one aspect, a host cell comprising the engineered rAAV capsid protein, the polynucleotide, or the vector of the present disclosure is provided.113. In one aspect, a recombinant rAAV virion (rAAV) comprising the engineered rAAV capsid protein of the present disclosure is provided.114. In some embodiments, the rAAV virion further comprises an exogenous cargo polynucleotide.115. In some embodiments, the exogenous cargo polynucleotide comprises a template for homology directed repair.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 miRNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding gene editing guide RNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding RNA-editing guide RNA.117. In some embodiments, the exogenous polynucleotide comprises an expressible polynucleotide encoding a therapeutic protein.118. In some embodiments, the rAAV virion comprises a functional sequence motif, wherein the functional sequence motif attenuates or abrogates binding of the virion to mammalian cell polysaccharides or proteoglycans compared to a reference virion having the same capsid protein sequence but without the functional sequence motif. In some embodiments, the functional sequence motif attenuates or abrogates binding of the virion to mammalian cell polysaccharides compared to a reference virion having the same capsid protein sequence but without the functional sequence motif. In some embodiments, the functional sequence motif attenuates or abrogates binding of the virion to mammalian cell proteoglycans compared to a reference virion having the same capsid protein sequence but without the functional sequence motif.119. In some embodiments, the rAAV virion comprises at least one cross linker reactive moiety covalently connected 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.120. In some embodiments, the amino acid residue is a lysine.121. In some embodiments, the amino acid residue in the viral capsid protein is an N terminal amine.122. In some embodiments, the amino acid residue in the viral capsid protein is a nonnatural amino acid residue.123. In one aspect, 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) a N-terminal cleavage fragment and (ii) a C- terminal cleavage fragment; and at least one cross linker reactive moiety covalently connected to a C-terminal amino acid of the N-terminal cleavage fragment.124. In some embodiments, the covalent linkage comprises an amide bond.125. In some embodiments, the C-terminal amino acid of the N-terminal cleavage fragment is a threonine.126. In one aspect, 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) a N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and wherein at least one cross linker reactive moiety is covalently connected to a C-terminal amino acid of the N-terminal cleavage fragment; and optionally at least one cross linker reactive moiety is covalently connected to an amino acid residue in the engineered rAAV capsid protein, wherein the amino acid residue is not within the functional sequence motif.127. In some embodiments, the surface functionalized rAAV virion comprises at least one crosslinker moiety, wherein each crosslinker reactive moiety is independently selected from a crosslinker reactive moiety that participates in a reaction selected from: a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strain-promoted alkyne-azide cycloaddition(SPAAC) reaction, a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction, an inverse electron demand Diels-Alder (IEDDA) reaction, and a Staudinger ligation and a [4+1] cycloaddition reaction. In some embodiments, a crosslinker reactive moiety that participates in a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. In some embodiments, a crosslinker reactive moiety that participates in a strain-promoted alkyneazide cycloaddition (SPAAC) reaction. In some embodiments, a crosslinker reactive moiety that participates in a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction. In some embodiments, a crosslinker reactive moiety that participates in an inverse electron demand Diels-Alder (IEDDA) reaction. In some embodiments, a crosslinker reactive moiety that participates in a Staudinger ligation and a [4+1] cycloaddition reaction.128. In some embodiments, the crosslinker reactive moiety comprises at least one of an eight membered ring and a triazole ring.129. In some embodiments, the reaction is a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction.130. In some embodiments, the crosslinker reactive moiety is selected from a cyclooctyne and an azide.131. In some embodiments, the cyclooctyne is selected from dibenzylcyclooctyne (DIBO), dibenzoazacyclooctyne (DBCO), and biarylazacyclooctynone (BARAC), or a derivative thereof. In some embodiments, the cyclooctyne is dibenzylcyclooctyne (DIBO). In some embodiments, the cyclooctyne is dibenzoazacyclooctyne (DBCO). In some embodiments, the cyclooctyne is biarylazacyclooctynone (BARAC).132. In some embodiments, the cyclooctyne is a DBCO.133. In some embodiments, the reaction is an inverse electron demand Diels-Alder (IEDDA) reaction.134. In some embodiments, the crosslinker reactive moiety is selected from a transcyclooctene and a tetrazine.135. In one aspect, a surface modified 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 iscleaved within the functional sequence motif to form (i) a N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and wherein the virion comprises a crosslinked moiety that covalently connects a C-terminal amino acid of the N-terminal cleavage fragment and a targeting ligand.136. In some embodiments, the covalent linkage comprises an amide bond.137. In some embodiments, the C-terminal amino acid of the N-terminal cleavage fragment is a threonine.138. In some embodiments, the crosslinked moiety comprises a product of a reaction selected from: a CuAAC reaction, a SPAAC reaction, a SPANC reaction, an IEDDA reaction, a Staudinger ligation, a [4+1] cycloaddition reaction. In some embodiments, the crosslinked moiety comprises a product of a CuAAC reaction. In some embodiments, the crosslinked moiety comprises a product of a SPAAC reaction. In some embodiments, the crosslinked moiety comprises a product of a SPANC reaction. In some embodiments, the crosslinked moiety comprises a product of an IEDDA reaction. In some embodiments, the crosslinked moiety comprises a product of a Staudinger ligation. In some embodiments, the crosslinked moiety comprises a product of a [4+1] cycloaddition reaction.139. In some embodiments, the reaction is selected from: a SPAAC, a SPANC, and a IEDDA reaction.140. In some embodiments, the crosslinked moiety comprises a 7 or 8 membered carbocycle comprising from 0-3 heteroatoms selected from O or N.141. In some embodiments, the crosslinked moiety comprises142. In some embodiments, the functional sequence motif is selected from one of: SEQ ID Nos: 1-58.143. In some embodiments, the functional sequence motif is LPETG (SEQ ID NO: 12).144. In one aspect, a surface modified rAAV virion is provided, wherein the virion comprises an exogenous cargo polynucleotide and at least one 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 region 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 crosslinked moiety that covalently connects the T of the N-terminal cleavage fragment and a targeting ligand wherein the covalent linkage comprises amide bond; and optionally (b) wherein the virion comprises a crosslinked moiety that covalently connects a lysine residue or a N terminal amine of a capsid protein and a targeting ligand.145. In some embodiments, the crosslinked moiety comprises146. In some embodiments, the surface modified rAAV virion comprises a targeting ligand, wherein the targeting ligand is a cell-type specific ligand.147. In some embodiments, the targeting ligand is selected from enzymes, peptide mimetic, ligands of endogenous receptors, lipid binding proteins, toxins, bacteriophage peptidases, nucleotides 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 mimetic. 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 bacteriophage 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 ananoparticle. In some embodiments, the targeting ligand is a lipid. In some embodiments, the targeting ligand is a small molecule.148. In some embodiments, the targeting ligand binds a receptor-mediated transporter expressed on the endothelial cells forming the blood brain barrier (BBB).149. In some embodiments, the receptor is selected from an insulin receptor, transferrin receptor, a choline transporter, an amino acid transporter, e.g., a large neutral amino acid carrier, and an LDL receptor. In some embodiments, the receptor is an insulin receptor. In some embodiments, the receptor is 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.150. In some embodiments, the ligand is known to bind to a receptor endogenous to the brain microvascular endothelial cells (BMECs).151. In some embodiments, the receptor is protease activated receptors (PARs).152. In some embodiments, the receptor is an Endothelial Protease Activated Receptor 1 (PARI).153. In some embodiments, the ligand binds to PARI .154. In some embodiments, the ligand is a PAR-1 activating peptide. In some embodiments, the ligand is thrombin or derivatives thereof.155. In some embodiments, the ligand is a peptide comprising the sequence SFLLR (SEQ ID NO: 2).156. In some embodiments, the ligand comprises a peptide having the sequence SFLLRNPNDKC (SEQ ID NO: 3).157. In some embodiments, the ligand comprises a wheat germ agglutinin (WGA).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 WGA3.159. In some embodiments, the ligand binds to PARI or the ligand comprises WGA. In some embodiments, the ligand binds to PARI. In some embodiments, the ligand comprises WGA.160. In some embodiments, the capsid comprises a first ligand that binds to binds to PARI and a second ligand that comprises WGA.161. In some embodiments, the surface modified rAAV virion comprises a functional sequence motif, wherein the functional sequence motif attenuates or abrogates binding of the virion to mammalian cell polysaccharides or proteoglycans compared to a reference virion having the same capsid protein sequence but without the functional sequence motif. In some embodiments, the functional sequence motif attenuates or abrogates binding of the virion to mammalian cell polysaccharides compared to a reference virion having the same capsid protein sequence but without the functional sequence motif. In some embodiments, the functional sequence motif attenuates or abrogates binding of the virion to mammalian cell proteoglycans compared to a reference virion having the same capsid protein sequence but without the functional sequence motif.162. 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 without the functional sequence motif.163. In some embodiments, the increased infectivity is with respect to at least one cell or tissue type.164. In some embodiments, the cell or tissue type is selected from cardiac, nerve, central and peripheral nervous system, hematopoietic system, liver, muscle, connective tissue, lung, kidney and retinal. In some embodiments, the cell or tissue type is cardiac. In some embodiments, the cell or tissue type is nerve. In some embodiments, the cell or tissue type is central and peripheral nervous system. In some embodiments, the cell or tissue type is hematopoietic system. In some embodiments, the cell or tissue type is liver. In some embodiments, the cell or tissue type is connective tissue. In some embodiments, the cell or tissue type is lung. In some embodiments, the cell or tissue type is kidney and retinal.165. In some embodiments, wherein the exogenous cargo polynucleotide comprises a template for homology directed repair.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 miRNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding gene editing guide RNA. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding RNA-editing guide RNA.167. In some embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic protein.168. In one aspect, a pharmaceutical composition comprising a plurality of surface modified rAAV virions according to the present disclosure is provided, the composition further comprising a pharmaceutically acceptable carrier, diluent, solubilizer, filler, preservative, excipient or combination thereof.169. In one aspect, a method of treating a patient having a disease treatable by intracellular delivery of a recombinant polynucleotide cargo is provided, the method comprising administering a therapeutically effective amount of the pharmaceutical composition according to the present disclosure, wherein the recombinant polynucleotide cargo is capable of treating the disease.170. In one aspect, a composition for use in treating a disease treatable by intracellular delivery of a recombinant polynucleotide cargo is provided, the composition comprising a therapeutically effective amount of the pharmaceutical composition according to the present disclosure, wherein the recombinant polynucleotide cargo is capable of treating the disease.171. In one aspect, use of the rAAV virion according to the present disclosure, for the manufacture of a medicament for treating a disease treatable by intracellular delivery of a recombinant polynucleotide cargo, is provided.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 ligandconjugated to a second site of the surface modified viral capsid, wherein the first targeting ligand and second targeting ligand are distinct.173. In some embodiments, the first targeting ligand promotes crossing of a blood-brain barrier (BBB), and the second targeting ligand is a cell targeting ligand.174. In some embodiments, the first targeting ligand is a blood-brain barrier (BBB) translocating peptide or a lectin ligand.175. In some embodiments, one of the first and second targeting ligands is covalently attached (e.g., conjugated) to a lysine or cysteine residue of a viral capsid protein via a linker.176. In some embodiments, the other of the first and second targeting ligands is covalently attached (e.g., conjugated) to a recognition sequence motif residue via a linker.177. In some embodiments, the viral capsid is an adeno-associated virus (AAV) capsid.178. In some embodiments, the first targeting ligand or second targeting ligand is a peptide, such as a PAR-1 agonist.179. In some embodiments, the first targeting ligand or second targeting ligand is a peptide as set forth in SEQ ID NO: 3 or portion thereof.180. In some embodiments, the first targeting ligand or second targeting ligand is nerve growth factor.181. In some embodiments, the first targeting ligand or second targeting ligand binds to brain microvascular endothelial cells (BMECs).182. In some embodiments, the first targeting ligand or second targeting ligand binds to neuronal membranes, such as wheat germ agglutinin (WGA).183. In some embodiments, the engineered viral capsid conjugated to a first targeting ligand and a second targeting ligand further comprises: (a) a modified viral capsid comprising a plurality of recognition sequence motif residues; (b) a first targeting ligand conjugated to the modified viral capsid via a first linker covalently attached to the recognition sequence motif residue comprising: bl. the residue covalently attached to an amide, -C(O)NH-; b2. a first spacer (Sp)n; b3. a bivalent fused polycyclic 11- to 20-membered heterocyclic ring, aphosphine oxide derivative, or a 4H-pyrazol-4-imine derivative; b4. a second spacer (Sp')n'; b5. a first targeting ligand ; and, (c) a second targeting ligand conjugated to the modified viral capsid via a second linker covalently attached to a primary amino group or a thiol of the modified viral capsid primary sequence comprising: cl. an amide, -NHC(O)-; c2. a first spacer (Sp)n; c3. a bivalent fused polycyclic 11- to 20-membered heterocyclic ring, a phosphine oxide derivative, or a 4H-pyrazol-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 20.184. In some embodiments, the recognition sequence motif residue is selected from any one of the peptide sequences as set forth in SEQ ID NOs: 1-58 or 61-75 or portion thereof.185. In some embodiments, the recognition sequence motif residue comprises at least four consecutive amino acids selected from the peptide sequences as set forth in SEQ ID NOs: 8- 33, 35-44, 62-64, and 65.186. In some embodiments, the recognition sequence motif residue comprises at least six consecutive amino acids as set forth in SEQ ID NO: 61 or SEQ ID NO: 66.187. In some embodiments, the engineered viral capsid conjugated to a first targeting ligand and a second targeting ligand further comprises: (a) a modified viral capsid; (b) a first targeting ligand conjugated to the modified viral capsid via a first linker covalently attached to a primary amino group or a thiol of the modified viral capsid primary sequence comprising: bl. the residue covalently attached to an amide, -C(O)NH-; b2. a first spacer (Sp)n; b3. a bivalent fused polycyclic 11- to 20-membered heterocyclic ring, a phosphine oxide derivative, or a 4H-pyrazol-4-imine derivative; b4. a second spacer (Sp')n'; b5. a first targeting ligand; and, (c) a second targeting ligand conjugated to the modified viral capsid via a second linker covalently attached to a primary amino group or a thiol of the modified viral capsid primary sequence comprising: cl. an amide, -NHC(O)-; c2. a first spacer (Sp)n; c3. a bivalent fused polycyclic 11- to 20-membered heterocyclic ring, a phosphine oxide derivative, or a 4H-pyrazol-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 20.188. In some embodiments, the primary amino group is selected from an N-terminal amino group, a lysine epsilon amino group and an arginine amino acid group.189. In some embodiments, the thiol is a cysteine amino acid group.190. In some embodiments, each first spacer is selected from -(CH2)n-, -(CH2CH2O)n-, - C(O)-, -C(O)NH-, 5-membered heterocyclic ring, and combinations thereof, wherein n is 0 to 20, such as n is 1 to 20, such as n is 1 to 8, such as n is 2 to 8, such as n is 1 to 4, such as n is 2 to 4, such as n is 4.191. In some embodiments, each second spacer is selected from -(CH2)n’-, -(CH2CH2O)n’- , -C(O)-, -C(O)NH-, 5 -membered heterocyclic ring, and combinations thereof, wherein n' is 0 to 20, such as n' is 1 to 20, such as n' is 1 to 8, such as n' is 2 to 8, such as n' is 1 to 4, such as n' is 2 to 4, such as n' is 4.192. In some embodiments, the bivalent fused polycyclic 11- to 20-membered heterocyclicwherein Z is an optionally substituted 7- to 9-membered carbocycle or heterocyclic ring.193. In some embodiments, Z is substituted with one or more arylenes.194. In some embodiments, the phosphine oxide derivative is: ents, the 4H-pyrazol-4-imine derivative is:196. In one aspect, a method of treating a disease or disorder is provided, the method comprising administering a therapeutically effective amount of an engineered viral capsid according to the present disclosure.197. In some embodiments, the disease or disorder is a central nervous system disease or disorder, 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. In some embodiments, the disease or disorder is Aromatic amino acid decarboxylase (AADC) deficiency. In some embodiments, the disease or disorder is Canavan disease. In some embodiments, the disease or disorder is Parkinson’s disease. In some embodiments, the disease or disorder is Alzheimer’s disease. In some embodiments, the disease or disorder is giant axonal neuropathy.5.12. ExamplesSurface modification of viral capsids

[0479] Recombinant adeno-associated virus (rAAV) has emerged as the in vivo gene delivery vector of choice, both in basic research and for clinical use. Recombinant AAV vectors do not undergo site-specific integration in the host genome, and this, coupled with modest immunogenicity, renders them one of the safest strategies for gene therapy (Naso M.F., Tomkowicz B., Perry W.L., 3rd, Strohl WR. Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. BioDrugs 2017; 31 :317-34). Despite their clear advantages over other viral vectors for in vivo use, rAAVs still have some limitations. For example, they are ineffective at transducing some cell types; as a result, high titers are often required for efficient gene transfer. This in turn leads to off-target effects through transduction of inappropriate cell types, raises production costs substantially, and leads to toxicity.

[0480] Efforts to improve AAV-mediated gene delivery initially focused on exploiting wildtype serotypes that display distinct tropism for different cell types. By generating pseudotyped AAV containing transgenes flanked by ITRs (inverted terminal repeats) from serotype 2 and capsids from other wildtype serotypes, the transduction specificity of the recombinant vector can be modified. More recently, synthetic AAV capsids have been engineered, which contain capsid proteins derived from directed evolution or rational design (Colella P., Ronzitti G., Mingozzi F. Emerging Issues in AAV-Mediated In vivo GeneTherapy. Mol. Ther. Methods Clin. Dev. 2018; 8:87-104). This approach is exemplified by the development of the engineered AAV-PHP.eB and AAV-PHP.S capsids that transduce the central and peripheral nervous systems (Chan K.Y., Jang M.J., Yoo B.B., Greenbaum A., Ravi N., Wu WL, et al. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat. Neurosci. 2017; 20: 1172-9). These variants can be injected systemically in mice to target the entire brain or peripheral ganglia. However, despite the success of such approaches, AAV vectors in which the primary amino acid sequence of the capsid proteins, VP1, VP2, and / or VP3, has been engineered still suffer from some drawbacks such as the high titers needed for systemic transduction, and questions about their translational potential beyond rodent models.

[0481] An aspect of the solution to these problems has been to provide a protein chemistrybased method to functionalize the capsid surface in ways that facilitate the targeted delivery of a viral capsid (as part of a recombinant AAV virion), with its encapsidated cargo, into cells of choice. Chemical modification of AAV capsids using NHS esters produces a heterogeneous mixture of modified virus depending upon the extent to which the available lysine residues are reacted in each capsid. To standardize the modification process for certain applications, a hybrid approach was developed, where a peptide is genetically incorporated into the virus allowing for site specific modification with DBCO. In early attempts efforts were focused on protein or peptide-based tags such as the Clip tag or Spytag, but were hindered by substantial reductions in AAV yield upon incorporation of the tag. Similar problems had been reported for encoding of unnatural amino acids into the AAV capsid. Thus, an alternative method, termed sortagging, which is based upon a transpeptidation reaction catalyzed by a bacterial sortase enzyme was evaluated. This involved the incorporation of an LPXTG (SEQ ID NO: 45)peptide sequence into the protein of interest (i.e., the AAV capsid), and subsequent incubation with the Sortase A enzyme from Staphylococcus aureus and a labelled oligoglycine peptide. The LPXTG (SEQ ID NO: 45) sequence is cleaved between the T and G, generating a thioester-linked acyl enzyme intermediate, which can then be attacked by the aminoglycine nucleophile, resulting in sitespecific ligation of the labelled oligoglycine to the LPETG sequence. Recently it has been demonstrated that the reaction can also proceed with diverse amines instead of the oligoglyine peptide (Polavarapu R., et al. Tissue-type plasminogen activator-mediated shedding of astrocytic low-density lipoprotein receptor-related protein increases the permeability of the neurovascular unit. Blood 2007; 109:3270-8). It was determined ifAmine-DBCO could be incorporated directly into exposed loops of the AAV2 capsid via sortase mediated ligation.

[0482] Provided herein are the crosslinking of ligands to the surface functionalized AAV capsid (whether functionalized using a capsid reactive linker, e.g., comprising an ester such as NHS or TFP, a recognition sequence-reactive linker, or engineering an un-natural amino acid comprising a crosslinker reactive moiety into the primary sequence) through bioorthogonal chemistry to penetrate biological tissue barriers, improve tropism, and / or to enhance transduction efficiency.

[0483] In certain embodiments, a ligand of the surface-modified viral capsid binds to its cognate receptors on the surface of mammalian cells to mediate gene delivery selectively into cell types which display the appropriate cognate receptor thus enabling targeted viral gene delivery. In certain embodiments, a ligand of the surface modified viral capsid enhances penetration of certain barrier tissues. In certain embodiments, the surface modified viral capsid comprises at least two different kinds of ligands, e.g., a cell-targeting ligand and a tissue permeating ligand. In some embodiments, the targeting ligand binds a receptor- mediated transporter expressed on the endothelial cells forming the blood brain barrier (BBB). In some embodiments, the receptor is selected from an insulin receptor, transferrin receptor, a choline transporter, an amino acid transporter, e.g., a large neutral amino acid carrier, and an LDL receptor. In some embodiments, the receptor is an insulin receptor. In some embodiments, the receptor is transferrin receptor. In some embodiments, the receptor is a...

Claims

WHAT IS CLAIMED IS:

1. An engineered viral capsid 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 second targeting ligand are distinct.

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

3. The engineered viral capsid of claim 1 or 2, wherein the first targeting ligand is a bloodbrain barrier (BBB) translocating peptide or a lectin ligand.

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

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

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

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

8. The engineered viral capsid of any one of claims 1 to 7, wherein the first targeting ligand or second targeting ligand is a peptide as set forth in SEQ ID NO: 3 or portion thereof.

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

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

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

12. The engineered viral capsid conjugated to a first targeting ligand and a second targeting ligand of any one of claims 1 to 11 further comprising:(a) a modified viral capsid comprising a plurality of recognition sequence motif residues;(b) a first targeting ligand conjugated to the modified viral capsid via a first linker covalently attached to the recognition sequence motif residue comprising: bl. the residue covalently attached to an amide, -C(O)NH-; b2. a first spacer (Sp)n; b3. a bivalent fused polycyclic 11- to 20-membered heterocyclic ring, a phosphine oxide derivative, or a 4H-pyrazol-4-imine derivative; b4. a second spacer (Sp')n'; b5. a first targeting ligand ; and,(c) a second targeting ligand conjugated to the modified viral capsid via a second linker covalently attached to a primary amino group or a thiol of the modified viral capsid primary sequence comprising: cl. an amide, -NHC(O)-; c2. a first spacer (Sp)n; c3. a bivalent fused polycyclic 11- to 20-membered heterocyclic ring, a phosphine oxide derivative, or a 4H-pyrazol-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.

13. The engineered viral capsid of any one of claims 1 to 12, wherein the recognition sequence motif residue is selected from any one of the peptide sequences as set forth in SEQ ID NOs: 1-58 or 61-75 or portion thereof.

14. The engineered viral capsid of 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 as set forth in SEQ ID NOs: 8-33, 35-44, 62-64, and 65.

15. The engineered viral capsid of any one of claims 1 to 14, wherein the recognition sequence motif residue comprises at least six consecutive amino acids as set forth in SEQ ID NO: 61 or SEQ ID NO: 66.

16. The engineered viral capsid conjugated to a first targeting ligand and a second targeting ligand of any one of claims 1 to 11 further comprising:(a) a modified viral capsid;(b) a first targeting ligand conjugated to the modified viral capsid via a first linker covalently attached to a primary amino group or a thiol of the modified viral capsid primary sequence comprising: bl. the residue covalently attached to an amide, -C(O)NH-; b2. a first spacer (Sp)n; b3. a bivalent fused polycyclic 11- to 20-membered heterocyclic ring, a phosphine oxide derivative, or a 4H-pyrazol-4-imine derivative; b4. a second spacer (Sp')n'; b5. a first targeting ligand; and,(c) a second targeting ligand conjugated to the modified viral capsid via a second linker covalently attached to a primary amino group or a thiol of the modified viral capsid primary sequence comprising: cl. an amide, -NHC(O)-; c2. a first spacer (Sp)n; c3. a bivalent fused polycyclic 11- to 20-membered heterocyclic ring, a phosphine oxide derivative, or a 4H-pyrazol-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.

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

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

19. The engineered viral capsid 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 heterocyclic ring, and combinations thereof, wherein n is 0 to 20, such as n is 1 to 20, such as n is 1 to 8, such as n is 2 to 8, such as n is 1 to 4, such as n is 2 to 4, such as n is 4.

20. The engineered viral capsid 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 heterocyclic ring, and combinations thereof, wherein n' is 0 to 20, such as n' is 1 to 20, such as n' is 1 to 8, such as n' is 2 to 8, such as n' is 1 to 4, such as n' is 2 to 4, such as n' is 4.

21. The engineered viral capsid any one of claims 1 to 20, wherein the bivalent fused polycyclic 11- to 20-membered heterocyclic ring is:wherein Z is an optionally substituted 7- to 9-membered carbocycle or heterocyclic ring.

22. The engineered viral capsid of claim 21, wherein Z is substituted with one or more arylenes.

23. The engineered viral capsid any one of claims 1 to 20, wherein the phosphine oxide derivative is:

24. The engineered viral capsid any one of claims 1 to 20, wherein the 4H-pyrazol-4-imine derivative is:

25. A method of treating a disease or disorder 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 disorder of claim 25, wherein the disease or disorder is a central nervous system disease or disorder, 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 surface modified viral capsid according to Formula I:Y and Y’ are independently an attachment moiety; n and n’ are independently 0 or an integer from 1 to 50;Sp and Sp’ are independently an optional spacer;L is a ligand; x is the ligand per capsid ratio that is in a range from 1 to 500; andQ is selected from:wherein, Z is a 7 or 8 membered cyclic or heterocyclic structure.

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

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

30. The surface modified viral capsid according to claim 27, having a structure according toFormula (1-1):(1-1) wherein:viral capsid; n and n’ are independently an integer 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 100-200.

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

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

34. The surface modified viral capsid according to claim 33, wherein the receptor is protease activated receptors (PARs).

35. The surface modified viral capsid according to claim 34, wherein the receptor is an Endothelial Protease Activated Receptor 1 (PARI).

36. The surface modified viral capsid according to claim 31, wherein the ligand binds to PARI.

37. The surface modified viral capsid according to claim 36, wherein the ligand is a PAR-1 activating peptide, e.g., thrombin or derivatives thereof.

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

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

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

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

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

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

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

45. A method of treating a patient having a disease treatable by intracellular delivery of a recombinant polynucleotide cargo, the method comprising: administering a therapeutically effective amount of the pharmaceutical composition according to claim 44, wherein the recombinant polynucleotide cargo is capable of treating the disease.

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

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

48. A surface functionalized viral capsid, comprising a first member of a crosslinker reactive pair and optionally one or more spacers, wherein the surface functionalized viral capsid is suitable for reaction with a functionalized ligand comprising a second member of the crosslinker reactive pair and optionally one or more spacers, wherein the members of the crosslinker reactive pair participate in a reaction selected from: a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction, a strain- promoted alkyne-nitrone cycloaddition (SPANC) reaction, an inverse electron demand Diels- Alder (IEEDD) reaction, and a Staudinger ligation and a [4+1] cycloaddition reaction.

49. A method of a making a surface modified viral capsid, the method comprising the steps: obtaining a surface functionalized viral capsid by reacting a viral capsid protein with a capsid-reactive linker, the linker comprising a first member of a crosslinker reactive pair and optionally one or more of a spacer; conjugating the surface functionalized viral capsid with a functionalized ligand comprising a second member of the crosslinker reactive pair and optionally one or more of a spacer; wherein the first and second members of the crosslinker reactive pair react to form a crosslinked moiety, Q.

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

51. A recombinant virion comprising a surface modified capsid according to any one of claims 27-43.

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

53. An engineered adeno-associated virus (AAV) capsid protein, wherein: the capsid protein comprises: a functional sequence motif, wherein the functional sequence motif has the sequence: X1X2X3X4X5X6 (SEQ ID NO: 5); wherein Xi, X2, X3, X4, X5, and X6are each independently selected from any amino acid residue and wherein X4, X5, and Xe are optional;wherein the functional sequence motif is a substrate for a sortase transamidase enzyme.

54. The engineered rAAV capsid protein of claim 53, wherein the enzyme is selected from a sortase A, sortase B, archaeosortase A, exosortase A, rhombosortase, and PorU.

55. The engineered rAAV capsid protein of claim 54, wherein the enzyme is a sortase A.

56. The engineered rAAV capsid protein of claim 55, heptamutant (SrtA7M)or a pentamutant (SrtA5M).

57. The engineered rAAV capsid protein of claim 55, wherein the enzyme is Hepta-Mutant Staphylococcus aureus Sortase A (SrtA7m).

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

59. The engineered rAAV capsid protein of claim any one of claims 27-58, wherein the functional sequence motif has a sequence of X1X2X3 (SEQ ID NO: 6); wherein Xi, 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 a sequence of X1X2X3 (SEQ ID NO: 7), and wherein two or more of Xi, X2, and X3, are selected from:Xi is valine (V);X2 is proline (P); andX3 is proline (P).

61. The engineered rAAV capsid protein of claim 59, wherein Xi, X2, and X3, are selected from:Xi is independently proline (P);X2 is independently selected from glycine (G) and glutamic acid (E); andX3 is independently selected from proline (P), and phenylalanine (F).

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

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

65. The engineered rAAV capsid protein of claim 64, wherein the functional sequence motif has a sequence of X1X2X3X4X5 (SEQ ID NO: 54), and wherein:Xi is independently selected from lysine (L), valine (V), (I) and asparagine (N);X2 is independently proline (P);X3 is independently selected from any amino acid residue;X4 is independently threonine (T); andX5 is independently selected from alanine (A), glycine (G), and asparagine (N).

66. The engineered rAAV capsid protein of claim 65, wherein two or more of Xi, X2, X3, X4, and X5, are selected from:Xi is independently selected from lysine (L);X2 is independently proline (P);X3 is independently selected from any amino acid residue;X4 is independently threonine (T); andX5 is independently selected from alanine (A), a glycine (G), and asparagine (N).

67. The engineered rAAV capsid protein of claim 66, wherein three or more of Xi, X2, X3, X4, and X5 are selected from:Xi is independently selected from lysine (L), valine (V), (I) and an asparagine (N);X2 is independently proline (P);X3 is independently selected from any amino acid residue;X4 is independently threonine (T); andX5 is independently selected from alanine (A) and glycine (G).

68. The engineered rAAV capsid protein of claim 67, whereinXi is lysine (L);X2 is proline (P);X3 is glutamic acid (E), serine (S), or alanine (A);X4 is threonine (T); andX5 is alanine (A) or glycine (G).

69. The engineered rAAV capsid protein of any one of claims 64-68, wherein 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).

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

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

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

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

74. The engineered rAAV capsid protein according to any one of claims 27-73, wherein the engineered rAAV capsid protein comprises one or more sequence changes selected from insertions, deletions, or substitutions, compared to a wild type protein that serve to introduce the functional sequence motif.

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

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

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

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

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

83. The engineered rAAV capsid protein according to claim 80, wherein the engineered rAAV capsid protein comprises a functional sequence motif within variable region IV and VIII; and wherein each functional sequence motif is independently selected.

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

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

86. The engineered rAAV capsid protein according to claim 85, wherein the functional sequence motif begins after:S453 in VR4 of an AAV1 capsid serotype;5452 in VR4 or R585 VR8 of an AAV2 capsid serotype;S576 in VR8 of an AAV5 capsid serotype;5453 in VR4 or S587 VR8 of an AAV6 capsid serotype;P454 in VR4 or A587 VR8 of an AAV7 capsid serotype;T454 in VR4 or Q589 VR8 of an AAV8 capsid serotype; and5454 in VR8 of an AAV9 capsid serotype.

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

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 53-88, wherein the engineered rAAV capsid protein does not comprise additional mutations when compared to the wildtype capsid protein.

90. A polynucleotide encoding the engineered rAAV capsid protein of any one of claims 27- 89.

91. A vector comprising the polynucleotide of claim 90.

92. The vector of claim 91, further comprising a promoter operably linked to the polynucleotide.

93. A host cell comprising the engineered rAAV capsid protein of any one of claims 1-37, the polynucleotide specified in claim 90, or the vector of claim 91 or 92.

94. A recombinant rAAV virion (rAAV) comprising the engineered rAAV capsid protein of any one of claims 53-89.

95. The rAAV virion of claim 94, further comprising an exogenous cargo polynucleotide.

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

97. The rAAV virion 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 virion of claim 94, wherein the exogenous polynucleotide comprises an expressible polynucleotide encoding a therapeutic protein.

99. The rAAV virion according to any one of claims 94-98, wherein the functional sequence motif attenuates or abrogates binding of the virion to mammalian cell polysaccharides or proteoglycans compared to a reference virion having the same capsid protein sequence but without the functional sequence motif.

100. A surface functionalized rAAV virion according to any one of claims 94-99, wherein the rAAV virion comprises: at least one cross linker reactive moiety covalently connected 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.

101. The surface functionalized rAAV virion according to claim 100, wherein the amino acid residue is a lysine.

102. The surface functionalized rAAV virion according to claim 100, wherein the amino acid residue in the viral capsid protein is an N terminal amine.

103. The surface functionalized rAAV virion according to claim 100, wherein the amino acid residue in the viral capsid protein is a non-natural amino acid residue.

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

105. The surface functionalized rAAV virion according to claim 104, wherein the covalent linkage comprises an amide bond.

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

107. A surface functionalized rAAV virion, wherein the virion comprises at least one engineered rAAV capsid protein according to any one of claims 53-89, and an exogenous cargo polynucleotide, and wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form (i) a N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and wherein at least one cross linker reactive moiety is covalently connected to a C-terminal amino acid of the N-terminal cleavage fragment; and optionally at least one cross linker reactive moiety is covalently connected 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 virion according to any one of claims 100-107, wherein each crosslinker reactive moiety is independently selected from a crosslinker reactive moiety that participates in a reaction selected from: a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction, a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction, an inverse electron demand Diels-Alder (IEEDD) reaction, and a Staudinger ligation and a [4+1] cycloaddition reaction.

109. The surface functionalized rAAV virion according to claim 108, wherein the crosslinker reactive moiety comprises at least one of an eight membered ring and a triazole ring.

110. The surface functionalized rAAV virion according to claim 108, wherein the reaction is a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction.

111. The surface functionalized rAAV virion according to claim 108, wherein the crosslinker reactive moiety is selected from a cyclooctyne and an azide.

112. The surface functionalized rAAV virion according to claim 108, wherein the cyclooctyne is selected from dibenzylcyclooctyne (DIBO), dibenzoazacyclooctyne (DBCO), and biarylazacyclooctynone (BARAC), or a derivative thereof.

113. The surface functionalized rAAV virion according to claim 112, wherein the cyclooctyne is a DBCO.

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

115. The surface functionalized rAAV virion according to claim 108, wherein the crosslinker reactive moiety is selected from a transcyclooctene and a tetrazine.

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

117. The surface modified rAAV virion according to claim 116, wherein the covalent linkage comprises an amide bond.

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

119. The surface modified rAAV virion according to any one of claims 116-118, wherein the crosslinked moiety comprises a product of a reaction selected from: a CuAAC reaction, a SPAAC reaction, a SPANC reaction, an IEEDD reaction, a Staudinger ligation, a [4+1] cycloaddition reaction.

120. The surface modified rAAV virion according to claim 119, wherein the reaction is selected from: a SPAAC, a SPANC, and a IEEDD reaction.

121. The surface modified rAAV virion according to any one of claims 116-120, wherein the crosslinked moiety comprises a 7 or 8 membered carbocycle comprising from 0-3 heteroatoms selected from O or N.

122. The surface modified rAAV virion according to claim 121, wherein the crosslinked moiety comprises123. The surface modified rAAV virion according to any one of claims 116-122, wherein the functional sequence motif is selected from one of: SEQ ID Nos: 1-58.

124. The surface modified rAAV virion according to claim 123, wherein the functional sequence motif is LPETG (SEQ ID NO: 12).

125. A surface modified rAAV virion, wherein the virion comprises an exogenous cargo polynucleotide and at least one 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 region 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 crosslinked moiety that covalently connects the T of the N- terminal cleavage fragment and a targeting ligand wherein the covalent linkage comprises amide bond; and optionally (b) wherein the virion comprises a crosslinked moiety that covalently connects a lysine residue or a N terminal amine of a capsid protein and a targeting ligand.

126. The surface modified rAAV virion according to claim 125, wherein the crosslinked moiety comprises127. The surface modified rAAV virion according to any one of claims 116-126, wherein the targeting ligand is a cell-type specific ligand.

128. The surface modified rAAV virion according to according to claim 127, wherein the targeting ligand is selected from enzymes, peptide mimetic, ligands of endogenous receptors, lipid binding proteins, toxins, bacteriophage peptidases, nucleotides 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 virion according to any one of claims 116-128, wherein the targeting ligand binds a receptor-mediated transporter expressed on the endothelial cells forming the blood brain barrier (BBB).

130. The surface modified rAAV virion according to claim 129, wherein the receptor is selected from an insulin receptor, transferrin receptor, a choline transporter, an amino acid transporter, e.g., a large neutral amino acid carrier, and an LDL receptor.

131. The surface modified viral capsid according to any preceding claim, wherein the ligand is known to bind to a receptor endogenous to the brain microvascular endothelial cells (BMECs).

132. The surface modified viral capsid according to claim 131, wherein the receptor is protease activated receptors (PARs).

133. The surface modified viral capsid according to claim 132, wherein the receptor is an Endothelial Protease Activated Receptor 1 (PARI).

134. The surface modified viral capsid according to claim 133, wherein the ligand binds to PARI.

135. The surface modified viral capsid according to claim 134, wherein the ligand is a PAR-1 activating peptide, e.g., thrombin or derivatives thereof.

136. The surface modified viral capsid according to claim 131, wherein the ligand is a peptide comprising the sequence SFLLR (SEQ ID NO: 2).

137. The surface modified viral capsid according to claim 136, wherein the ligand comprises a peptide having the sequence SFLLRNPNDKC (SEQ ID NO: 3).

138. The surface modified viral capsid according to claim 131, wherein the ligand comprises a wheat germ agglutinin (WGA).

139. The surface modified viral capsid according to claim 138, wherein the ligand is selected from WGA1, WGA2, and WGA3.

140. The surface modified viral capsid according to any one of claims 116-128, wherein the ligand binds to PARI or wherein the ligand comprises WGA.

141. The surface modified viral capsid according to any one of claims 116-128, wherein the capsid comprises a first ligand that binds to binds to PARI and a second ligand that comprises WGA.

142. The surface modified rAAV virion according to any one of claims 116-141, wherein the functional sequence motif attenuates or abrogates binding of the virion to mammalian cell polysaccharides or proteoglycans compared to a reference virion having the same capsid protein sequence but without the functional sequence motif.

143. The surface modified rAAV virion according to any one of claims 116-141, characterized by increased infectivity compared to a reference virion having the same capsid protein sequence but without the functional sequence motif.

144. The surface modified rAAV virion according to claim 143, wherein the increased infectivity is with respect to at least one cell or tissue type.

145. The surface modified rAAV virion according to claim 143, wherein the cell or tissue type is selected from cardiac, nerve, central and peripheral nervous system, hematopoietic system, liver, muscle, connective tissue, lung, kidney and retinal.

146. The surface modified rAAV virion of any one of claims 116-145, wherein the exogenous cargo polynucleotide comprises a template for homology directed repair.

147. The surface modified rAAV virion 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 virion 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 virions according to any one of claims 116-148, the composition further comprising a pharmaceutically acceptable carrier, diluent, solubilizer, filler, preservative, excipient or combination thereof.

150. A method of treating a patient having a disease treatable by intracellular delivery of a recombinant polynucleotide cargo, the method comprising: administering a therapeutically effective amount of the pharmaceutical composition according to claim 149, wherein the recombinant polynucleotide cargo is capable of treating the disease.

151. A composition for use in treating a disease treatable by intracellular delivery of a recombinant polynucleotide cargo, the composition comprising: a therapeutically effective amount of the pharmaceutical composition according to claim 149, wherein the recombinant polynucleotide cargo is capable of treating the disease.

152. Use of the rAAV virion according to any one of claims 116-148, for the manufacture of a medicament for treating a disease treatable by intracellular delivery of a recombinant polynucleotide cargo.

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