Viral adapters and uses thereof

Abstract

The present invention relates to viral adapter proteins capable of altering the binding profile of the virus and / or modifying the cell tropism of the virus. In one aspect, the present invention relates to covalent adapter proteins for viruses capable of altering the binding profile of the virus and / or modifying the cell tropism of the virus. The present invention also relates to the use of the claimed viral adapter proteins as transduction vectors for modifying the binding profile and cell tropism of the virus and for use as medicines.

Description

[Technical field]

[0001] The present invention relates to viral adapter proteins capable of altering the binding profile of the virus and / or modifying the cell tropism of the virus. In one aspect, the present invention relates to covalent adapter proteins for viruses capable of altering the binding profile of the virus and / or modifying the cell tropism of the virus. The present invention also relates to the use of the claimed viral adapter proteins as transduction vectors for modifying the binding profile and cell tropism of the virus and for use as medicines. In one aspect, the present invention relates to adapters for adeno-associated viruses (AAV) capable of altering the binding profile of AAV and / or modifying the cell tropism of AAV. The present invention also relates to the use of the claimed AAV adapters as transduction vectors for modifying the binding profile and cell tropism of AAV and for use as medicines. [Background technology]

[0002] Adeno-associated virus The virus adeno-associated virus (AAV) belongs to the Parvoviridae family. They are distinguished by an icosahedral, non-enveloped capsid with a diameter of 18 nm to 30 nm and containing linear single-stranded DNA of about 5 kb. Efficient replication of AAV requires co-infection of the host cell with a helper virus, such as adenovirus, herpesvirus or vaccinia virus. Alternatively, replication can be promoted in vitro in the absence of helper virus by supplementing viral proteins from the helper virus required to support AAV replication in isolation as part of a helper plasmid. This production method is the most common and is referred to as helper-free AAV production. In the absence of helper virus, AAV enters a latent state and the viral genome can stably integrate into the host cell genome, although this rarely occurs. AAV is of particular interest as a transduction vector for mammalian cells because it can infect a wide range of dividing and non-dividing cells and the viral genome can either persist as an episome in infected cells or integrate into the host genome.

[0003] Generally, two inverted terminal repeats (ITRs) of about 145 bp in length are sufficient for vector function. These carry the "cis" signals required for replication, packaging, and integration into the host cell genome. For packaging into recombinant vector particles, the vector plasmid carrying the genes for the nonstructural proteins (Rep proteins) and the structural proteins (Cap proteins) is transfected into cells suitable for packaging, such as HeLa cells or HEK293 cells, and the cells are subsequently infected, for example, with adenovirus or cotransfected with a helper plasmid.

[0004] AAV capsid consists of three different proteins: VP1, VP2 and VP3, the relative ratio of which is approximately 1:1:10, which corresponds to 8.3% VP1, 8.3% VP2 and 83.3% VP3, respectively. AAV capsid genes are located at the ends of the AAV genome and are encoded by the same open reading frame (ORF) using different initiation codons. The VP1 gene contains the entire VP2 gene sequence, which contains the entire VP3 gene sequence with a specific N-terminal region. The fact that one reading frame codes for all three AAV capsid proteins is the reason why simultaneous expression of all capsid proteins is essential, albeit to different degrees.

[0005] The molecular masses of the capsid proteins are 87 kDa for VP1, 73 kDa for VP2, and 62 kDa for VP3. The sequences of the capsid genes are described, for example, in Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3, or Non-Patent Document 4 (which are incorporated herein by reference in their entirety). The physical and genetic maps of the AAV genome are described, for example, in Non-Patent Document 5 (which are incorporated herein by reference in their entirety). Exemplary AAV capsid sequences are also presented herein in Figures 7 to 19 and SEQ ID NOs: 7 to 19.

[0006] In addition, various AAV serotypes are known, among which human AAV serotype 2 (AAV2) is a viral vector with advantageous properties for somatic cell gene therapy. Its essential advantages are non-pathogenicity to humans, persistence of the viral genome as an episome in infected cells or stable integration into the cellular genome, ability to infect non-dividing cells, purification to high titers due to virion stability, low immunogenicity, and substantial absence of viral genes and gene products in recombinant AAV vectors, which are advantageous from the standpoint of safety when used in gene therapy. Currently, cloning of genes into AAV vectors is performed by methods generally known to those skilled in the art, for example as described in Patent Document 1, Non-Patent Document 6, or Non-Patent Document 5 (the entire contents of which are incorporated herein by reference).

[0007] For example, AAV2 generally has a broad spectrum of activity (tropism): it infects epithelial tissues such as human epithelial tumor cell lines, as well as primary tumor material such as cervical or ovarian cancer or melanoma, and human keratinocytes very efficiently (70%-80%), but infects hematopoietic cells such as lymphohematopoietic cells at a rate of 10-100 times lower (0.5%-5%) (Non-Patent Document 7, the entirety of which is incorporated herein by reference).

[0008] One reason for this may be that interaction between AAV and AAV receptors on the cell surface is required for AAV uptake into cells. Thus, for example, the putative primary AAV2 receptor is a 150 kDa cell membrane glycoprotein (Non-Patent Document 8, incorporated herein by reference in its entirety) or heparan sulfate proteoglycan (Non-Patent Document 9, incorporated herein by reference in its entirety). Potential secondary receptors that have been determined are α v β 5 integrin (Non-Patent Document 10, incorporated herein by reference in its entirety) and human fibroblast growth factor receptor 1 (Non-Patent Document 11, incorporated herein by reference in its entirety). Binding studies have now shown that the surface density of this receptor is reduced in cells that AAV2 inefficiently infects.

[0009] It is known that genetic modification of retroviral and adenoviral capsid proteins can introduce binding sites for receptors that are expressed only in specific cells, thereby enabling receptor-mediated targeting of the vector (see, for example, Non-Patent Document 12, Non-Patent Document 13, Non-Patent Document 14, Non-Patent Document 15, or Non-Patent Document 16, the entire contents of which are incorporated herein by reference).

[0010] Also, Patent Document 2 refers to an AAV capsid fusion protein, which is said to contain a heterologous epitope of a clinically relevant antigen, is said to induce an immune response, and is said to not interfere with encapsidation. Non-Patent Document 17 (incorporated herein in its entirety) was concerned with the in vitro assembly of AAV particles previously expressed in the baculo system. Mutations were also added to the cap gene, but this was not intended to change the tropism, but to result in a plasmid construct in which only one VP protein is expressed in each case. Non-Patent Document 18 (incorporated herein in its entirety) was intended to investigate the natural tropism of AAV2. For this purpose, the basic premise was to introduce a mutation into the C-terminus of the AAV2 VP protein, thus altering the RGD motif.

[0011] Indirect targeting of AAV has been disclosed in Non-Patent Document 19 (incorporated herein by reference in its entirety). In this case, a bispecific antibody directed against both the AAV2 capsid and the target cell was used. Non-Patent Document 20 (incorporated herein by reference in its entirety) discloses a single-chain antibody fragment against the CD34 molecule fused to the N-terminus of VP2, which is directly inserted into the N-terminus of VP1. However, this method has two obvious disadvantages. First, the infectious titer was very low, and second, for successful packaging, co-expression of the fusion protein with the unmutated capsid proteins VP1, VP2 and VP3 was required. However, this resulted in a mixture of chimeric and wild-type capsid proteins, the composition and therefore activity of which were unpredictable. Furthermore, the packaging efficiency and infectivity via the wild-type receptor in HeLa cells was also significantly reduced compared to the wild type.

[0012] Therefore, one objective of the present invention is to modify AAV to enable more specific and efficient gene transfer than known AAV vectors.

[0013] Surprisingly, it has now been found that AAV binding proteins, such as AAV receptor (AAVR, KIAA0319L) or portions thereof, can be modified to tightly bind to AAV as part of a fusion protein, and this fusion partner can be used to change the tropism of the AAV. In some embodiments, the binding protein can be covalently linked to the virus. Similarly, other viral binding proteins can be modified to consistently and covalently bind to the viral capsid as part of a fusion protein, and this fusion partner can be used to change the tropism of the virus. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] WO 95 / 23867 [Patent Document 2] International Publication No. 96 / 00587 [Non-patent literature]

[0015] [Non-Patent Document 1] Srivastava, A. et al. (1983), J. Virol., 45, 555-564 [Non-Patent Document 2] Muzyczka, N. (1992), Curr. Top. Micro. Immunol., 158, 97-129 [Non-Patent Document 3] Ruffing, N. et al. (1992), J. Virol., 66, 6922-6930 [Non-Patent Document 4] Rutledge, EA et al. (1998) J. Virol. 72, 309-319 [Non-Patent Document 5] Kotin, R.M. (1994), Human Gene Therapy, 5, 793-801 [Non-Patent Document 6] Chiorini JA et al. (1995), Human Gene Therapy, 6, 1531-1541

Non-licensed Document 7

Non-licensed literature 9

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Non-licensed Document 11

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Non-licensed Document 16

Non-licensed Document 17

[0016] This Summary introduces concepts that are described in more detail in the Detailed Description, and is not intended to identify essential features of the claimed subject matter, nor should it be used to limit the scope of the claimed subject matter.

[0017] The present invention provides an isolated polypeptide capable of binding to a viral capsid.

[0018] The present invention provides an isolated polypeptide that includes one or more cysteine ​​residues capable of covalently binding to a viral capsid.

[0019] The present invention also provides a viral adaptor molecule comprising: (i) an isolated polypeptide capable of binding to a viral capsid; and (ii) a ligand.

[0020] The present invention also provides a viral adapter molecule comprising: (i) an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid; and (ii) a ligand.

[0021] In some embodiments, the one or more cysteine ​​residues capable of covalently binding to a viral capsid are heterologous to the isolated polypeptide. In some embodiments, the viral adapter protein comprises (i) an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid, and (ii) a ligand, wherein the one or more cysteine ​​residues are heterologous to the isolated polypeptide. In some embodiments, the isolated polypeptide or the covalent viral adapter molecule is capable of binding to an unmodified wild-type viral capsid.

[0022] In some embodiments, the isolated polypeptide or viral adapter molecule is capable of binding to a viral capsid that has five or fewer (e.g., five, four, three, two, or one) amino acid point mutations relative to a wild-type viral capsid. In some embodiments, the isolated polypeptide or viral adapter molecule is capable of binding to a viral capsid that has five or fewer (i.e., one, two, three, four, or five) amino acid insertions or deletions relative to a wild-type viral capsid. In some embodiments, the isolated polypeptide or viral adapter molecule is capable of binding to a viral capsid that has no insertions or deletions relative to a wild-type viral capsid.

[0023] In some embodiments, the isolated polypeptide or viral adapter molecule comprises a portion of an adeno-associated viral receptor (AAVR, KIAA0319L) capable of binding to an adeno-associated viral capsid. In some embodiments, the isolated polypeptide or AAV adapter molecule comprises the full-length sequence of an adeno-associated viral receptor (AAVR, KIAA0319L). In some embodiments, the sequence of the adeno-associated viral receptor (AAVR, KIAA0319L) comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the isolated polypeptide or AAV adapter molecule comprises one or more point mutations in the AAVR polypeptide, optionally, the one or more point mutations are defined with reference to SEQ ID NO: 1.

[0024] In some embodiments, the isolated polypeptide or viral adapter molecule comprises a portion of an adeno-associated virus receptor (AAVR, KIAA0319L) having one or more cysteine ​​residues and capable of covalently binding to an adeno-associated virus capsid. In some embodiments, the isolated polypeptide or viral adapter molecule comprises a full-length sequence of an adeno-associated virus receptor (AAVR, KIAA0319L) having one or more cysteine ​​residues and capable of covalently binding to an adeno-associated virus capsid. In some embodiments, the sequence of the adeno-associated virus receptor (AAVR, KIAA0319L) comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the isolated polypeptide or covalent viral adapter molecule comprises one or more point mutations in the AAVR polypeptide, optionally, the one or more point mutations are defined with reference to SEQ ID NO: 1.

[0025] In some embodiments, the isolated polypeptide or covalent viral adapter molecule comprises one or more point mutations in an AAVR polypeptide to introduce one or more heterologous cysteine ​​residues, optionally, the one or more point mutations are defined with reference to SEQ ID NO:1.

[0026] In some embodiments, the isolated polypeptide or viral adapter molecule comprises a portion of a polypeptide selected from the list consisting of a DARPin, a nanobody, a suitable antibody-like protein, a protein derived from the lipocalin fold, an affibody (e.g., an affibody derived from the Z domain of protein A), a domain of fibronectin, an SH3 domain of Fyn, an affimer or scaffold derived from the protease inhibitor stefin A, a non-antibody scaffold protein (Adhiron), and an antibody or antigen-binding fragment thereof. In some embodiments, the isolated polypeptide or AAV adapter molecule comprises an antibody or antigen-binding fragment thereof.

[0027] In some embodiments, the isolated polypeptide or viral adapter molecule comprises a polypeptide selected from the list consisting of a DARPin, a nanobody, a suitable antibody-like protein, a protein derived from the lipocalin fold, an affibody (e.g., an affibody derived from the Z domain of protein A), a domain of fibronectin, an SH3 domain of Fyn, an affimer or scaffold derived from the protease inhibitor Stefin A, a non-antibody scaffold protein (Adilon), and an antibody or antigen-binding fragment thereof. In some embodiments, the isolated polypeptide or AAV adapter molecule comprises an antibody or antigen-binding fragment thereof.

[0028] In some embodiments, the isolated polypeptide or viral adapter molecule comprises an antigen-binding portion of neutralizing antibody A20. In some embodiments, the isolated polypeptide or viral adapter molecule comprises a sequence substantially identical to one or more complementarity determining regions (CDRs) of neutralizing antibody A20, e.g., one or more of the complementarity determining regions (CDRs) of neutralizing antibody A20 may comprise one, two or three amino acid point mutations, optionally, one or more of the point mutations are heterologous cysteine ​​residues. In some embodiments, the isolated polypeptide or viral adapter molecule comprises one or more sequences substantially identical to each of the complementarity determining regions (CDRs) of neutralizing antibody A20, e.g., one, two, three, four, five or six of the complementarity determining regions (CDRs) of neutralizing antibody A20 may comprise one, two or three amino acid point mutations, optionally, one or more of the point mutations are heterologous cysteine ​​residues.

[0029] In some embodiments, the isolated polypeptide or viral adapter molecule comprises the full-length sequence of one or more complementarity determining regions (CDRs) of neutralizing antibody A20, for example, the polypeptide comprises one or more of VHCDR1 of antibody A20 (SEQ ID NO:26), VHCDR2 of antibody A20 (SEQ ID NO:27), VHCDR3 of antibody A20 (SEQ ID NO:28), VLCDR1 of antibody A20 (SEQ ID NO:22), VLCDR2 of antibody A20 (SEQ ID NO:23), and VLCDR3 of antibody A20 (SEQ ID NO:24).

[0030] In some embodiments, the isolated polypeptide or viral adapter molecule comprises all of the complementarity determining regions (CDRs) of neutralizing antibody A20. In some embodiments, the isolated polypeptide or viral adapter molecule comprises one or more sequences substantially identical to the sequences of the full-length VH chain (SEQ ID NO:25) and VL chain (SEQ ID NO:21) of neutralizing antibody A20, e.g., the VH and / or VL sequences of neutralizing antibody A20 may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid point mutations, optionally, one or more of the point mutations are heterologous cysteine ​​residues.

[0031] In some embodiments, the isolated polypeptide or viral adapter molecule comprises the sequence of the full-length VH chain (SEQ ID NO:25) and VL chain (SEQ ID NO:21) of neutralizing antibody A20.

[0032] In some embodiments, the isolated polypeptide or viral adapter molecule comprises: one or more unnatural amino acids, one or more chemical moieties crosslinked to the polypeptide; Biotin tags, e.g. biotinylated AAVR, SpyTag peptides, such as AAVR-SpyTag, and / or Protein A polypeptide or Protein G polypeptide, The present invention further includes one or more of the following:

[0033] In some embodiments, the ligand is capable of binding to a cell surface molecule, hi some embodiments, the ligand is a human protein, such as an antibody or an antigen-binding fragment thereof. In some embodiments, the ligand is selected from the group consisting of interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin-12, interleukin-13, interleukin-14, interleukin-15, interleukin-16, interleukin-17, interleukin-18, interleukin-19, interleukin-20, interleukin-21, interleukin-22, interleukin-23, interleukin-24, interleukin-25, interleukin-26, interleukin-27, interleukin-28, interleukin-29, interleukin-30, interleukin-31, interleukin-32, interleukin-33, interleukin-34, interleukin-35, interleukin-36, interleukin-37, interleukin-38, interleukin-39, interleukin-40, interleukin-41, interleukin-42, interleukin-43, interleukin-44, interleukin-45, interleukin-46, interleukin-47, interleukin-48, interleukin-49, interleukin-50, interleukin-51, interleukin-52, interleukin-53, interleukin-54, interleukin-55, interleukin-56, interleukin-57, interleukin-58, interleukin-59, interleukin-60, interleukin-61, interleukin-62, interleukin-63, interleuk Interleukin-32, Interleukin-33, Interleukin-34, Interleukin-35, Insulin, Transferrin, CD2, CD58, CD59, CD2, CD40L / CD154, CD5, CD72, CD5L, CD23, CD70, CD80, CD86, S100Ap, CD178, CD155, CD106, CSF1, CD166, FasL, CD242, CD252, TRAIL, RANKL, A The ligand is one or more selected from the list consisting of PRIL, CD257, CD272, CD273, CD274, CD275, PD-L1, PD-L2, Cas13 and Cas7-11, endothelin, leptin, vasopressin, CD10, CD31, CD119, apelin, elabela, adrenomedullin, a targeting domain derived from botulinum toxin, a neuropeptide, a cytokine or a small molecule.

[0034] In some embodiments, the ligand is a small molecule, and optionally the small molecule is linked to the isolated polypeptide or viral adapter molecule via N-hydroxysuccinimide (NHS) or maleimide. In some embodiments, the ligand is a small molecule, and optionally the isolated polypeptide or viral adapter molecule includes a non-standard amino acid that links the small molecule to the isolated polypeptide or viral adapter molecule.

[0035] In some embodiments, the ligand is selected from the group consisting of Her2, interleukin-1 receptor, interleukin-2 receptor, interleukin-3 receptor, interleukin-4 receptor, interleukin-5 receptor, interleukin-6 receptor, interleukin-7 receptor, interleukin-8 receptor, interleukin-9 receptor, interleukin-10 receptor, interleukin-11 receptor, interleukin-12 receptor, interleukin-13 receptor, interleukin-15 receptor, interleukin-18 receptor, interleukin-20 receptor, interleukin-21 receptor, interleukin-22 receptor, interleukin-23 receptor, interleukin-27 receptor, interleukin-28 receptor, insulin ... and glycoRNA.

[0036] In some embodiments, the portion of the adeno-associated virus receptor (AAVR, KIAA0319L) comprises at least a portion of the PKD1 domain, at least a portion of the PKD2 domain, at least a portion of the PKD3 domain, at least a portion of the PKD4 domain, at least a portion of the PKD5 domain, or any combination thereof.

[0037] In some embodiments, the portion of the adeno-associated viral receptor (AAVR, KIAA0319L) comprises a PKD1 domain, a PKD2 domain, a PKD3 domain, a PKD4 domain, a PKD5 domain, or any combination thereof. The isolated polypeptide or covalent viral adapter molecule of any one of claims 9 to 30, wherein the portion of the adeno-associated viral receptor (AAVR, KIAA0319L) comprises a sequence selected from the list consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0038] In some embodiments, the portion of the adeno-associated virus receptor (AAVR, KIAA0319L) comprises the full-length sequence of the adeno-associated virus receptor (AAVR, KIAA0319L), optionally, the full sequence comprises SEQ ID NO:1.

[0039] The invention also provides viral particles linked to at least one isolated polypeptide or viral adaptor molecule according to the invention, in some embodiments, the viral particle is covalently linked to at least one isolated polypeptide or viral adaptor molecule according to the invention.

[0040] In some embodiments, the one or more viral capsid proteins comprise one or more cysteine ​​residues capable of covalently binding to the isolated polypeptide, hi some embodiments, the one or more viral capsid proteins comprise one or more cysteine ​​residues capable of forming disulfide bridges with one or more cysteine ​​residues in the isolated polypeptide.

[0041] In some embodiments, the virus is an adeno-associated virus, and optionally the adeno-associated virus is selected from the list consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, and AAVrh.74. In some embodiments, the adeno-associated virus is AAV1. In some embodiments, the adeno-associated virus is AAV2. In some embodiments, the adeno-associated virus is wild-type AAV.

[0042] In some embodiments, the adeno-associated virus comprises a portion of a capsid amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.

[0043] In some embodiments, the adeno-associated virus comprises a full-length capsid amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.

[0044] In some embodiments, the adeno-associated virus has one or more conservative amino acid changes relative to a wild-type amino acid sequence, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 conservative amino acid changes relative to the wild-type amino acid sequence, optionally wherein the wild-type amino acid sequence is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19. In some embodiments, the adeno-associated virus has five or fewer non-conservative amino acid changes relative to the wild-type amino acid sequence, for example five, four, three, two or one non-conservative amino acid changes relative to the wild-type amino acid sequence, optionally wherein the wild-type amino acid sequence is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19.

[0045] In some embodiments, the virus is an adenovirus, an Aichi virus, an Australian bat lyssavirus, a BK polyomavirus, a Banna virus, a Barmah Forest virus, a Bunyamwera virus, a Bunyavirus, a Lacrosse Bunyavirus, a Snowshoe Hare Bunyavirus, a Simian Herpesvirus, a Chandipura virus, a Chikungunya virus, a Cosavirus, a Cowpox virus, a Coxsackie virus, a Crimean-Congo hemorrhagic fever virus, a Dengue virus, a Dhoori virus, a Djugbe virus, a Dubenhage virus, an Eastern Equine virus, a Hepatitis B virus, a Hepatitis C ... Encephalitis virus, Ebola virus, Echovirus, Encephalomyocarditis virus, Epstein-Barr virus, European bat lyssavirus, GB virus, Hepatitis C / G virus, Hanta virus, Hendra virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Hepatitis delta virus, Horsepox virus, Human adenovirus, Human astrovirus, Human coronavirus, Human cytomegalovirus, Human enterovirus, Human herpes virus, Human immunodeficiency virus, Human papilloma virus human papillomavirus, human parainfluenza virus, human parvovirus, human respiratory syncytial virus, human rhinovirus, human SARS coronavirus, human spumaretrovirus, human T-lymphotropic virus, human torovirus, influenza A virus, influenza B virus, influenza C virus, Isfahan virus, JC polyomavirus, Japanese encephalitis virus, Junin arenavirus, KI polyomavirus, Kunjin virus, Lagos bat virus, Lake Victoria Marburg virus, Langa virus, Viruses: Lassa virus, Rosedale virus, Louping ill virus, Lymphocytic choriomeningitis virus, Machupo virus, Mayaro virus, MERS coronavirus, Measles virus, Mengo encephalomyocarditis virus, Merkel cell polyomavirus, Mokola virus, Molluscum contagiosum virus, Monkeypox virus, Mumps virus, Murray Valley encephalitis virus, New Jersey polyomavirus, New York virus, Nipah virus, Norwalk virus, O'nyong-nyong virus, Orf virus, Oropouche virus, Pichinde virus,The virus is selected from the list consisting of poliovirus, Punta Toro phlebovirus, Puumala virus, rabies virus, Rift Valley fever virus, Rosavirus A, Ross River virus, Rotavirus A, Rotavirus B, Rotavirus C, Rubella virus, Sagiyama virus, Sarivirus A, Sicilian sandfly fever virus, Sapporo virus, SARS coronavirus, Semliki forest virus, Seoul virus, Sarpholmy virus, Simian virus 40, Sindbis virus, Southampton virus, St. Louis encephalitis virus, Tick-borne Powassan virus, Torque teno virus, Toscana virus, Uukuniemi virus, Vaccinia virus, Varicella zoster virus, Smallpox virus, Venezuelan equine encephalitis virus, Vesicular stomatitis virus, Western equine encephalitis virus, WU polyomavirus, West Nile virus, Yaba monkey tumor virus, Yaba-like disease virus, Yellow fever virus, and Zika virus. In some embodiments, the virus is a wild-type virus.

[0046] In some embodiments, the virus is an adenovirus and the viral adapter molecule comprising an isolated polypeptide capable of binding to a viral capsid, or an isolated polypeptide capable of binding to a viral capsid and a ligand, comprises a portion of a Coxsackievirus-adenovirus receptor (CXADR), and optionally, the CXADR sequence is SEQ ID NO: 20.

[0047] In some embodiments, the virus is an adenovirus and the viral adapter molecule comprising an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid, or an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid and a ligand, comprises a portion of a coxsackievirus-adenovirus receptor (CXADR), and optionally, the CXADR sequence is SEQ ID NO: 20.

[0048] In some embodiments, the virus is an adenovirus and the viral adapter molecule comprising an isolated polypeptide capable of binding to a viral capsid, or an isolated polypeptide capable of binding to a viral capsid and a ligand, comprises a portion of CD46, and optionally, the CD46 sequence is SEQ ID NO:29.

[0049] In some embodiments, the virus is an adenovirus and the viral adapter molecule comprising an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid, or an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid and a ligand, comprises a portion of CD46, and optionally, the CD46 sequence is SEQ ID NO:29.

[0050] In some embodiments, the virus is an adenovirus, and the isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to the viral capsid, or the viral adapter molecule comprising an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to the viral capsid and a ligand, comprises a full-length sequence of a Coxsackievirus-adenovirus receptor (CXADR), optionally, the CXADR sequence is SEQ ID NO: 20. In some embodiments, the one or more viral capsids have one or more heterologous cysteine ​​residues introduced relative to the wild-type amino acid sequence, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 heterologous cysteine ​​residues relative to the wild-type amino acid sequence. In some embodiments, the one or more heterologous cysteine ​​residues are introduced at the interface between the isolated polypeptide or viral adapter molecule and the viral capsid.

[0051] In some embodiments, the virus is AAV1, and the isolated polypeptide or isolated fusion polypeptide comprises a portion of an adeno-associated virus receptor (AAVR, KIAA0319L) capable of covalently binding to an adeno-associated virus capsid, and the AAV1 comprises one or more mutations selected from Gly266Cys, Thr504Cys, and Asp590Cys, and the AAVR polypeptide or fusion polypeptide comprises one or more mutations selected from Thr434Cys, Asp429Cys, and Ser425Cys.

[0052] In some embodiments, the virus is AAV2, and the isolated polypeptide or isolated fusion polypeptide comprises a portion of an adeno-associated virus receptor (AAVR, KIAA0319L) capable of covalently binding to an adeno-associated virus capsid, and the AAV2 comprises one or more mutations selected from Gly265Cys, Thr503Cys, and Gln589Cys, and the AAVR polypeptide or fusion polypeptide comprises one or more mutations selected from Thr434Cys, Asp429Cys, and Ser425Cys.

[0053] In some embodiments, the virus is AAV2, and the isolated polypeptide or isolated fusion polypeptide comprises a portion of an adeno-associated virus receptor (AAVR, KIAA0319L) capable of covalently binding to an adeno-associated virus capsid, and the AAV2 comprises a Gln589Cys mutation, and the AAVR polypeptide or fusion polypeptide comprises one or more mutations selected from Ser425Cys and Val480Glu, and optionally, the AAVR polypeptide or fusion polypeptide comprises both the Ser425Cys mutation and the Val480Glu mutation.

[0054] In some embodiments, the virus is AAV2, and the isolated polypeptide or isolated fusion polypeptide comprises a PKD2 domain (e.g., SEQ ID NO: 3) of the adeno-associated virus receptor (AAVR, KIAA0319L) capable of covalently binding to an adeno-associated virus capsid, and the AAV2 comprises a Gln589Cys mutation, and the AAVR polypeptide or fusion polypeptide comprises one or more mutations selected from Ser425Cys and Val480Glu, and optionally, the AAVR polypeptide or fusion polypeptide comprises both the Ser425Cys mutation and the Val480Glu mutation.

[0055] In some embodiments, the virus is AAV1, and the isolated polypeptide or isolated fusion polypeptide comprises a portion of the adeno-associated virus receptor (AAVR, KIAA0319L) capable of covalently binding to an adeno-associated virus capsid, and the AAV2 comprises a Gln589Cys mutation, and the AAVR polypeptide or fusion polypeptide comprises one or more mutations selected from Ser425Cys and Val480Glu, and optionally, the AAVR polypeptide or fusion polypeptide comprises both the Ser425Cys mutation and the Val480Glu mutation.

[0056] In some embodiments, the virus is AAV1, and the isolated polypeptide or isolated fusion polypeptide comprises a PKD2 domain (e.g., SEQ ID NO: 3) of the adeno-associated virus receptor (AAVR, KIAA0319L) capable of covalently binding to an adeno-associated virus capsid, and the AAV2 comprises a Gln589Cys mutation, and the AAVR polypeptide or fusion polypeptide comprises one or more mutations selected from Ser425Cys and Val480Glu, and optionally, the AAVR polypeptide or fusion polypeptide comprises both the Ser425Cys mutation and the Val480Glu mutation.

[0057] In some embodiments, the virus is AAV5, and the isolated polypeptide or isolated fusion polypeptide comprises a portion of the adeno-associated virus receptor (AAVR, KIAA0319L) capable of covalently binding to an adeno-associated virus capsid, and the AAV5 comprises a Gln697Cys mutation, and the AAVR polypeptide or fusion polypeptide comprises a Ser356Cys mutation.

[0058] In some embodiments, the virus is AAV5, and the isolated polypeptide or isolated fusion polypeptide comprises a PKD1 domain (e.g., SEQ ID NO: 2) of the adeno-associated virus receptor (AAVR, KIAA0319L) capable of covalently binding to an adeno-associated virus capsid, and the AAV5 comprises a Gln697Cys mutation, and the AAVR polypeptide or fusion polypeptide comprises a Ser356Cys mutation.

[0059] In some embodiments, the virus is AAV2, and the isolated polypeptide or isolated fusion polypeptide comprises an antigen-binding portion of neutralizing antibody A20, wherein the AAV2 comprises one or more mutations selected from Ser264Cys, Val708Cys, and Asn717Cys, and the neutralizing antibody A20 polypeptide or fusion polypeptide comprises one or more mutations selected from VH Tyr102Cys, VH Ser56Cys, and VL Ile93Cys.

[0060] In some embodiments, the virus is AAV2, and the isolated polypeptide or isolated fusion polypeptide comprises an antigen-binding portion of neutralizing antibody A20, wherein the AAV2 comprises one or more mutations selected from Gly266Cys, Thr504Cys, and Asp590Cys, and the neutralizing antibody A20 polypeptide or fusion polypeptide comprises one or more mutations selected from VH Tyr102Cys, VH Ser56Cys, and VL Ile93Cys.

[0061] In some embodiments, the adeno-associated virus particle comprises one or more amino acid changes in the capsid protein that mediate interactions with non-protein binding agents, such as heparan sulfate proteoglycans (HSPGs), O-linked sialic acid, N-linked sialic acid, or N-linked galactose.

[0062] In some embodiments, the adeno-associated viral particle comprises mutations at arginine residues 585 and 588 of AAV2.

[0063] The present invention also provides a pharmaceutical composition comprising a viral particle according to the invention.

[0064] The present invention also provides a method for covalently modifying a viral particle, comprising the steps of: Providing viral particles; Providing an isolated polypeptide capable of covalently binding to a viral capsid of a viral particle; combining the viral particle with the isolated polypeptide such that the isolated polypeptide is covalently attached to the viral particle; The present invention provides a method comprising:

[0065] The present invention also provides a method for covalently modifying a viral particle, comprising the steps of: Providing viral particles; providing a viral adapter molecule comprising an isolated polypeptide and a ligand capable of covalently binding to a viral capsid of a viral particle; combining the viral particle with a viral adapter molecule such that the isolated fusion polypeptide is covalently attached to the viral particle; The present invention provides a method comprising:

[0066] In some embodiments, the method further comprises introducing one or more heterologous cysteine ​​residues, e.g., one, two or three heterologous cysteine ​​residues, into the isolated polypeptide or viral adapter molecule. In some embodiments, the method further comprises introducing one or more heterologous cysteine ​​residues, e.g., one, two or three heterologous cysteine ​​residues, into the viral capsid.

[0067] In some embodiments, the method further comprises introducing one or more heterologous cysteine ​​residues into both the isolated polypeptide or viral adapter molecule and the viral capsid, such as one, two or three heterologous cysteine ​​residues into both the isolated polypeptide or viral adapter molecule and the viral capsid.

[0068] In some embodiments, conjugation of the isolated polypeptide or viral adapter molecule to the viral particle reduces or eliminates the natural tropism of one or more viral capsid proteins, hi some embodiments, conjugation of the isolated polypeptide or viral adapter molecule to the viral particle increases the tropism of the viral particle for one or more cell types.

[0069] In some embodiments, the covalent attachment of the isolated polypeptide or viral adapter molecule to the viral particle increases and / or decreases the tropism of the viral particle for one or more cell types selected from the list consisting of neurons, macrophages, microglia, T cells, B cells, dendritic cells, antigen presenting cells, NK cells, cancer cells, muscle cells, hepatocytes, photoreceptors, pancreatic beta cells, kidney cells, and lung cells.

[0070] In some embodiments, the viral particle has a tropism that is at least 10%, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% decreased or increased compared to the native tropism of the viral particle without the isolated polypeptide or viral adapter molecule. In some embodiments, association of the isolated polypeptide or viral adapter molecule with the viral particle enables the viral particle to present protein antigens in a multimeric manner using the virus as a scaffold.

[0071] In some embodiments, the covalent attachment of the isolated polypeptide or viral adapter molecule to the viral particle selectively induces cytolysis, and optionally, cytolysis is induced in one or more cell types selected from the list consisting of neurons, macrophages, microglia, T cells, B cells, dendritic cells, antigen presenting cells, NK cells, cancer cells, muscle cells, hepatocytes, photoreceptor cells, pancreatic beta cells, and lung cells.

[0072] The present invention also provides a method of targeting a viral particle to a target cell, comprising the steps of: Providing viral particles; providing a viral adapter molecule comprising an isolated polypeptide capable of binding to a viral capsid of a viral particle and a ligand specific for a target cell; combining the viral particle with a viral adapter molecule such that the viral adapter molecule binds to the viral particle, thereby producing a modified viral particle; contacting a mixture of cells including target cells with the modified viral particles; The present invention provides a method comprising:

[0073] In some embodiments of the methods of the invention, the isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid comprises a portion of the adeno-associated virus receptor (AAVR, KIAA0319L). In some embodiments of the methods of the invention, the isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid comprises at least a portion of the PKD1 domain, at least a portion of the PKD2 domain, at least a portion of the PKD3 domain, at least a portion of the PKD4 domain, at least a portion of the PKD5 domain, or any combination thereof, of the adeno-associated virus receptor (AAVR, KIAA0319L). The isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid of the methods of the invention comprises the complete PKD1 domain, the complete PKD2 domain, the complete PKD3 domain, the complete PKD4 domain, the complete PKD5 domain, or any combination thereof, of the adeno-associated virus receptor (AAVR, KIAA0319L). In some embodiments, the isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid comprises one or more mutations in an AAVR polypeptide, optionally, the one or more mutations are defined with reference to SEQ ID NO: 1. The isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid comprises at least a portion of the PKD1 domain, at least a portion of the PKD2 domain, at least a portion of the PKD3 domain, at least a portion of the PKD4 domain, at least a portion of the PKD5 domain, or any combination thereof, of the adeno-associated virus receptor (AAVR, KIAA0319L), the isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid comprises one or more mutations in an AAVR polypeptide, optionally, the one or more mutations are defined with reference to SEQ ID NO: 1.In some embodiments, the isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid comprises the complete PKD1 domain, the complete PKD2 domain, the complete PKD3 domain, the complete PKD4 domain, the complete PKD5 domain of the adeno-associated virus receptor (AAVR, KIAA0319L), or any combination thereof, and the isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid comprises one or more mutations in the AAVR polypeptide, optionally, the one or more mutations are defined with reference to SEQ ID NO:1.

[0074] In some embodiments of the methods of the invention, the isolated polypeptide capable of covalently binding to a viral capsid comprises a portion of the adeno-associated viral receptor (AAVR, KIAA0319L). In some embodiments of the methods of the invention, the isolated polypeptide capable of covalently binding to a viral capsid comprises at least a portion of the PKD1 domain, at least a portion of the PKD2 domain, at least a portion of the PKD3 domain, at least a portion of the PKD4 domain, at least a portion of the PKD5 domain, or any combination thereof, of the adeno-associated viral receptor (AAVR, KIAA0319L). In some embodiments of the methods of the invention, the isolated polypeptide capable of covalently binding to a viral capsid comprises the PKD1 domain, the PKD2 domain, the PKD3 domain, the PKD4 domain, the PKD5 domain, or any combination thereof, of the adeno-associated viral receptor (AAVR, KIAA0319L).

[0075] In some embodiments of the methods of the invention, the isolated polypeptide capable of covalently binding to an adeno-associated virus (AAV) capsid comprises a sequence selected from the list consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. In some embodiments of the methods of the invention, the isolated polypeptide capable of binding to a viral capsid comprises the full-length sequence of the adeno-associated virus receptor (AAVR, KIAA0319L), optionally, the full sequence comprises SEQ ID NO: 1.

[0076] The present invention also provides a method of delivering a nucleic acid sequence of interest to a target cell comprising contacting the target cell with a modified viral particle according to the invention or a pharmaceutical composition according to the invention, wherein the viral particle comprises the nucleic acid sequence of interest.

[0077] In some embodiments of the methods of the invention, the nucleic acid sequence of interest encodes a protein selected from the list consisting of RAB escort protein 1, RPE65, Factor VIII, Factor IX, Cochlin, CLN7, acid alpha-glucosidase (GAA), aquaporin 1, glial cell line-derived neurotrophic factor, aspartoacylase, aromatic L-amino acid decarboxylase, retinitis pigmentosa GTPase regulator deficiency, sarcoplasmic reticulum calcium ATPase, cyclic nucleotide-gated channel beta 3, neurturin, galactosidase beta 1, glucose-6-phosphatase, phenylalanine hydroxylase, ornithine transcarbamylase, dystrophin and carnitine palmitoyltransferase II, phenylalanine hydroxylase (PAH), cystic fibrosis transmembrane conductance regulator (CFTR).

[0078] In some embodiments, the target cell is in vitro. In some embodiments, the target cell is in vivo in a subject. In some embodiments, the target cell is selected from the list consisting of a neuron, a macrophage, a microglia cell, a T cell, a B cell, a dendritic cell, an antigen presenting cell, a NK cell, a cancer cell, a muscle cell, a liver cell, a photoreceptor cell, a pancreatic beta cell, a kidney cell, and a lung cell. In some embodiments, the subject is a human. In some embodiments, the target cell is a human target cell.

[0079] The present invention also provides a viral particle according to the invention or a pharmaceutical composition according to the invention for use as a medicament.The present invention also provides a method for treating a patient in need of treatment comprising administering a therapeutically effective amount of a viral particle according to the invention or a pharmaceutical composition according to the invention.

[0080] The present invention also provides a viral particle according to the invention or a pharmaceutical composition according to the invention for use in a method for treating a disease in a subject in need of treatment comprising delivering a nucleic acid sequence of interest to a target cell by contacting the target cell with the viral particle.The present invention also provides a method for treating a disease in a subject in need of treatment comprising administering a therapeutically effective amount of a viral particle according to the invention or a pharmaceutical composition according to the invention, thereby delivering a nucleic acid sequence of interest to a target cell by contacting the target cell with the viral particle.

[0081] In some embodiments, the disease is a neurodegenerative disorder, cancer, Duchenne muscular dystrophy, hemophilia, congenital blindness, diabetes, cystic fibrosis, choroideremia, hemophilia A, hemophilia B, CLN7 disease, Pompe disease, Parkinson's disease, Canavan disease, demyelinating diseases, inherited retinal dystrophies due to RPE65 mutations, aromatic L-amino acid decarboxylase (AADC) deficiency, X-linked retinitis pigmentosa, Leber's congenital amaurosis, Churg-Strauss syndrome (CSS), critical limb ischemia, color blindness, Alzheimer's disease, macular degeneration, ornithine transcatheterization syndrome, orthotopic retinal dystrophy ...

[0033] The present invention relates to a method for treating rheumatoid arthritis, including the treatment of ...

[0082] In some embodiments of the invention, the viral particles or compositions of the invention are used in a method of treating a disease in a subject in need of treatment, comprising delivering a nucleic acid sequence of interest to a target cell by contacting the target cell with the viral particle, comprising any one of the following listed combinations: Neuronal cells for the treatment of neurodegenerative disorders, Immune cells (e.g. macrophages, microglia, T cells, B cells, dendritic cells, antigen-presenting cells, NK cells) for the treatment of cancer; Immune cells (e.g., macrophages, microglia, T cells, B cells, dendritic cells, antigen-presenting cells, NK cells) to elicit an immune response; Cancer or tumor cells for the treatment of cancer; Muscle cells for the treatment of Duchenne muscular dystrophy, Hepatocytes for the treatment of hemophilia, Photoreceptors for congenital blindness, Pancreatic beta cells for the treatment of diabetes, or Lung cells for the treatment of cystic fibrosis, Targeting of the designated target cells by the therapeutic agent treats or ameliorates the designated disease.

[0083] In some embodiments, the viral particles or pharmaceutical compositions are administered to the subject by aerosol (e.g., into lung tissue), intramuscularly, intra-arterially (e.g., via the hepatic artery), intra-articularly, subretinal, intracranial, intravenous, intrathecal, intracoronary, or subcutaneously.

[0084] In some embodiments of the invention, the ligand may consist of a sequence-specific RNA-binding molecule, such as a Cas13 molecule, which then confers tropism to the virus for cells expressing the target RNA on their surface. Specifically, in this case, the adapter molecule consists of a fusion of Cas13 with a partial or full-length PKD2 domain. Cas13 can then complex with a suitable single guide RNA (sgRNA) for Cas13, endowing it with the ability to bind RNA complementary to the protospacer region of the sgRNA.

[0085] In some embodiments of the present invention, a method for producing a viral adaptor protein includes providing a viral capsid protein and reacting the viral capsid protein with a targeting molecule that has high specificity for a target cell. The reaction may be performed using NHS or maleimide chemistry, or by incorporating a non-standard amino acid with a reactive group (such as azide or alkyne or tetrazine or trans-cyclooctene) at a specific position of the viral capsid molecule. In some embodiments of the present invention, the viral adaptor protein includes a viral capsid protein and a targeting molecule that has high specificity for a target cell.

[0086] In some embodiments, the viral capsid molecule is combined with a specific peptide sequence capable of binding to a targeting molecule, such as a split intein, a SpyTag, a tetracysteine ​​FCM motif (FLNCCPGCCMEP), or a ybbR motif (TVLDSLEFIASKLA).

[0087] In this case, the targeting molecule can be chosen to bind to a specific receptor, hi some embodiments, the targeting molecule can be a protein, such as an antibody, directed against a receptor expressed in the target cell type.

[0088] In some embodiments, the targeting molecule can be a small molecule, for example, neurotransmitters such as serotonin or dopamine can be attached to viral capsid proteins to create adapter molecules that, when combined with the viral particle, generate viral particles specific for cells expressing serotonin or dopamine receptors, respectively.

[0089] In some embodiments, the AAV particles may be conjugated to two or more isolated polypeptides or AAV adapter molecules according to the invention. In some embodiments, the AAV particles may be conjugated to two, three, four, five, six, seven, eight, nine, ten or more different isolated polypeptides or different AAV adapter molecules according to the invention. In some embodiments, the AAV particles may be conjugated to two different isolated polypeptides or different AAV adapter molecules according to the invention. In some embodiments, the AAV particles may be conjugated to three different isolated polypeptides or different AAV adapter molecules according to the invention. In some embodiments, the AAV particles may be conjugated to four different isolated polypeptides or different AAV adapter molecules according to the invention. In some embodiments, the AAV particles may be conjugated to five different isolated polypeptides or different AAV adapter molecules according to the invention. Such embodiments provide specificity for multiple different cell types. For example, by combining a single AAV particle with a mixture of adapter molecules, some specific for CD4 and some specific for CD8, a single AAV-based gene therapy can be formulated that targets both CD4+ and CD8+ T cell lineages.

[0090] In some embodiments, a viral particle may be covalently linked to two or more isolated polypeptides or viral adapter molecules according to the invention. In some embodiments, a viral particle may be covalently linked to two, three, four, five, six, seven, eight, nine, ten or more different isolated polypeptides or different viral adapter molecules according to the invention. In some embodiments, a viral particle may be covalently linked to two different isolated polypeptides or different viral adapter molecules according to the invention. In some embodiments, a viral particle may be covalently linked to three different isolated polypeptides or different viral adapter molecules according to the invention. In some embodiments, a viral particle may be covalently linked to four different isolated polypeptides or different viral adapter molecules according to the invention. In some embodiments, a viral particle may be covalently linked to five different isolated polypeptides or different viral adapter molecules according to the invention. Such embodiments provide specificity for multiple different cell types. For example, by combining a single viral particle with a mixture of adapter molecules, some specific for CD4 and some specific for CD8, a single viral gene therapy can be formulated that targets both CD4+ and CD8+ T cell lineages.

[0091] In some embodiments, the AAV particles may be conjugated to two or more isolated polypeptides or AAV adapter molecules according to the invention. In some embodiments, the AAV particles may be conjugated to two, three, four, five, six, seven, eight, nine, ten or more different isolated polypeptides or different AAV adapter molecules according to the invention. In some embodiments, the AAV particles may be conjugated to two different isolated polypeptides or different AAV adapter molecules according to the invention. In some embodiments, the AAV particles may be conjugated to three different isolated polypeptides or different AAV adapter molecules according to the invention. In some embodiments, the AAV particles may be conjugated to four different isolated polypeptides or different AAV adapter molecules according to the invention. In some embodiments, the AAV particles may be conjugated to five different isolated polypeptides or different AAV adapter molecules according to the invention. Such embodiments provide specificity for multiple different cell types. For example, by combining a single AAV particle with a mixture of adapter molecules, some specific for CD4 and some specific for CD8, a single AAV gene therapy can be formulated that targets both CD4+ and CD8+ T cell lineages.

[0092] In some embodiments, the isolated polypeptide or viral adapter molecule of the invention comprises at least a portion of a CD4-specific designed ankyrin repeat protein (DARPin) as disclosed in Schweizer A, Rusert P, Berlinger L, Ruprecht CR, Mann A, Corthesy S, et al. (2008) CD4-Specific Designed Ankyrin Repeat Proteins Are Novel Potent HIV Entry Inhibitors with Unique Characteristics. PLoS Pathog 4(7): e1000109. In some embodiments, the isolated polypeptide or viral adapter molecule of the invention comprises a full-length CD4-specific designed ankyrin repeat protein (DARPin) as disclosed in Schweizer A, Rusert P, Berlinger L, Ruprecht CR, Mann A, Corthesy S, et al. (2008) CD4-Specific Designed Ankyrin Repeat Proteins Are Novel Potent HIV Entry Inhibitors with Unique Characteristics. PLoS Pathog 4(7): e1000109.

[0093] In some embodiments of the invention, the isolated polypeptide or viral adapter molecule of the invention comprises at least a portion of any suitable antibody-like protein capable of binding to a viral capsid (Kondo T et al., Antibody-like proteins that capture and neutralize SARS-CoV-2. Sci Adv. 2020 Oct 14;6(42):eabd3916). In some embodiments of the invention, the isolated polypeptide or viral adapter molecule of the invention comprises a full-length protein of any suitable antibody-like protein capable of binding to a viral capsid.

[0094] In some embodiments of the invention, an isolated polypeptide or viral adapter molecule of the invention comprises at least a portion of a protein derived from the lipocalin fold capable of binding to a viral capsid (Beste et al., Small antibody-like proteins with prescribed ligand specificities derived from the lipocalin fold, PNAS March 2, 1999 96 (5) 1898-1903). In some embodiments of the invention, an isolated polypeptide or viral adapter molecule of the invention comprises a full-length protein derived from the lipocalin fold capable of binding to a viral capsid.

[0095] In some embodiments of the invention, an isolated polypeptide or viral adapter molecule of the invention comprises at least a portion of an affibody derived from the Z domain of protein A capable of binding to a viral capsid (Nord et al., Binding proteins selected from combinatorial libraries of an α-helical bacterial receptor domain, Nature Biotechnology, volume 15, pages 772-777 (1997)). In some embodiments of the invention, an isolated polypeptide or viral adapter molecule of the invention comprises a full-length affibody derived from the Z domain of protein A capable of binding to a viral capsid.

[0096] In some embodiments of the invention, an isolated polypeptide or viral adapter molecule of the invention comprises at least a portion of a domain of fibronectin capable of binding to a viral capsid (Koide, et al., The fibronectin type III domain as a scaffold for novel binding proteins, Journal of Molecular Biology, Volume 284, Issue 4, 1998, Pages 1141-1151). In some embodiments of the invention, an isolated polypeptide or viral adapter molecule of the invention comprises the full-length sequence of a domain of fibronectin capable of binding to a viral capsid.

[0097] In some embodiments of the invention, an isolated polypeptide or viral adapter molecule of the invention comprises at least a portion of an SH3 domain of Fyn capable of binding to a viral capsid (Grabulovski, et al., A Novel, Non-immunogenic Fyn SH3-derived Binding Protein with Tumor Vascular Targeting Properties, Journal of Biological Chemistry, Volume 282, Issue 5, 2007, Pages 3196-3204). In some embodiments of the invention, an isolated polypeptide or viral adapter molecule of the invention comprises the full-length sequence of an SH3 domain of Fyn capable of binding to a viral capsid.

[0098] In some embodiments of the invention, an isolated polypeptide or viral adapter molecule of the invention comprises at least a portion of an affimer or scaffold derivative derived from the protease inhibitor Stefin A capable of binding to a viral capsid (Woodman R, Yeh JT, Laurenson S, Ko Ferrigno P. Design and validation of a neutral protein scaffold for the presentation of peptide aptamers. J Mol Biol. 2005 Oct 7;352(5):1118-33). In some embodiments of the invention, an isolated polypeptide or viral adapter molecule of the invention comprises the full length sequence of an affimer or scaffold derivative derived from the protease inhibitor Stefin A capable of binding to a viral capsid.

[0099] In some embodiments of the invention, an isolated polypeptide or viral adapter molecule of the invention comprises at least a portion of a non-antibody scaffold protein (Adhiron) capable of binding to a viral capsid (Tiede et al., Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications, Protein Eng Des Sel. 2014 May; 27(5): 145-155). In some embodiments of the invention, an isolated polypeptide or viral adapter molecule of the invention comprises the full-length sequence of a non-antibody scaffold protein (Adhiron) capable of binding to a viral capsid.

[0100] In some embodiments, the isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid, or an AAV adapter molecule comprising an isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid and a ligand, comprises one or more point mutations that increase the solubility of the isolated polypeptide or adapter molecule, such as one, two, three, four, five or more point mutations that increase the solubility of the isolated polypeptide or adapter molecule.

[0101] In some embodiments, an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid, or a viral adapter molecule comprising an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid and a ligand, comprises one or more point mutations that increase the solubility of the isolated polypeptide or adapter molecule, for example, one, two, three, four, five or more point mutations that increase the solubility of the isolated polypeptide or adapter molecule.

[0102] In some embodiments, the isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid, or an AAV adapter molecule comprising an isolated polypeptide and a ligand capable of binding to an adeno-associated virus (AAV) capsid, comprises one or more point mutations that increase the yield of protein expression and / or purification of the isolated polypeptide or adapter molecule in bacterial and / or mammalian systems, such as one, two, three, four, five or more point mutations that increase the yield of protein expression and / or purification of the isolated polypeptide or adapter molecule in bacterial and / or mammalian systems.

[0103] In some embodiments, an isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid, or an AAV adapter molecule comprising an isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid and a ligand, comprises a PKD2 domain of AAVR comprising one or more point mutations that increase the solubility of the isolated polypeptide or adapter molecule, such as one, two, three, four, five or more point mutations that increase the solubility of the isolated polypeptide or adapter molecule.

[0104] In some embodiments, an isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid, or an AAV adapter molecule comprising an isolated polypeptide and a ligand capable of binding to an adeno-associated virus (AAV) capsid, comprises a PKD2 domain of AAVR comprising one or more point mutations that increase the yield of protein expression and / or purification of the isolated polypeptide or adapter molecule in bacterial and / or mammalian systems, such as one, two, three, four, five or more point mutations that increase the yield of protein expression and / or purification of the isolated polypeptide or adapter molecule in bacterial and / or mammalian systems.

[0105] A particular advantage of the present invention is that the use of AAV binding proteins as part of the adapter molecule allows the tropism of AAV to be essentially altered, in particular to increase infectivity towards less sensitive cells several fold or to decrease infectivity towards more sensitive cells several fold, without compromising the efficiency of packaging of recombinant AAV vectors into the viral capsid. Thus, the present invention is particularly suitable for improving the in vitro and in vivo transduction of specific cells, e.g. for somatic cell gene therapy.

[0106] In some embodiments, the isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid, or the viral adapter molecule comprising an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid and a ligand, comprises one or more point mutations that increase the yield of protein expression and / or purification of the isolated polypeptide or adapter molecule in bacterial and / or mammalian systems, such as one, two, three, four, five or more point mutations that increase the yield of protein expression and / or purification of the isolated polypeptide or adapter molecule in bacterial and / or mammalian systems.

[0107] In some embodiments, an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid, or a viral adapter molecule comprising an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid and a ligand, comprises a PKD2 domain of AAVR comprising one or more point mutations that increase the solubility of the isolated polypeptide or adapter molecule, for example, one, two, three, four, five or more point mutations that increase the solubility of the isolated polypeptide or adapter molecule.

[0108] In some embodiments, an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid, or a viral adapter molecule comprising an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid and a ligand, comprises a PKD2 domain of AAVR comprising one or more point mutations that increase the yield of protein expression and / or purification of the isolated polypeptide or adapter molecule in bacterial and / or mammalian systems, such as one, two, three, four, five or more point mutations that increase the yield of protein expression and / or purification of the isolated polypeptide or adapter molecule in bacterial and / or mammalian systems.

[0109] In some embodiments of the invention, the isolated polypeptide, AAV adapter molecule or covalent viral adapter molecule of the invention can be coupled to the AAV particle, AAV capsid, viral particle or viral capsid in the presence of a reducing agent. In some embodiments of the invention, the reducing agent is tris(2-carboxyethyl)phosphine hydrochloride (TCEP). In some embodiments of the invention, the isolated polypeptide, AAV adapter molecule or covalent viral adapter molecule of the invention can be partially oxidized prior to the coupling reaction. In some embodiments of the invention, the AAV particle, AAV capsid, viral particle or viral capsid of the invention can be partially oxidized prior to the coupling reaction. In some embodiments of the invention, the isolated polypeptide, AAV adapter molecule or covalent viral adapter molecule of the invention and the AAV particle, AAV capsid, viral particle or viral capsid of the invention can be partially oxidized prior to the coupling reaction.

[0110] In some embodiments of the invention, the partially oxidized isolated polypeptide, AAV adapter molecule, or covalent viral adapter molecule of the invention can bind to an AAV particle, AAV capsid, viral particle, or viral capsid in the presence of a reducing agent. In some embodiments of the invention, the isolated polypeptide, AAV adapter molecule, or covalent viral adapter molecule of the invention can bind to an AAV particle, AAV capsid, viral particle, or viral capsid in the presence of a reducing agent. In some embodiments of the invention, the partially oxidized isolated polypeptide, AAV adapter molecule, or covalent viral adapter molecule of the invention can bind to an AAV particle, AAV capsid, viral particle, or viral capsid in the presence of a reducing agent. In some embodiments of the invention, the reducing agent is tris(2-carboxyethyl)phosphine hydrochloride (TCEP).

[0111] In some embodiments of the invention, a "virus particle" or "AAV particle" may be a virus-like particle. In some embodiments of the invention, a "virus particle" or "AAV particle" may be a virus-like particle assembled in vitro after recombinant expression of capsid proteins. In some embodiments of the invention, a "virus particle" or "AAV particle" may be a virus-like particle assembled in vivo in a cellular expression system. In some embodiments of the invention, the cellular expression system may be a bacterial (e.g., E. coli), yeast (e.g., S. cerevisiae), plant, insect (e.g., buttercup) or mammalian (e.g., human) cellular expression system.

[0112] A particular advantage of the present invention is that by using a binding protein capable of being covalently bound to the viral capsid as part of an adapter molecule, the tropism of the virus can be essentially altered, in particular increasing the infectivity towards less sensitive cells several fold or decreasing the infectivity towards more sensitive cells several fold, without compromising the efficiency of packaging of the recombinant vector into the viral capsid. The present invention is therefore particularly suitable for improving the in vitro and in vivo transduction of specific cells, e.g. for somatic cell gene therapy.

[0113] In any of the embodiments of the invention described herein, the isolated polypeptide capable of binding to a viral capsid, or a viral adapter molecule comprising an isolated polypeptide capable of binding to a viral capsid and a ligand ("viral adapter molecule"), may be capable of covalently binding to a viral capsid. Any statements made above regarding non-covalent binding of an isolated polypeptide to a viral capsid apply mutatis mutandis to an isolated polypeptide capable of covalently binding to a viral capsid. Particular embodiments regarding covalent binding of an isolated polypeptide or viral adapter molecule to a viral capsid are described herein.

[0114] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0115] [Figure 1] 1 is an image showing the expression of the mScarlet gene in cells exposed to AAV. HEK293FT expressing synthetic membrane-anchored anti-sfGFP nanobody was treated as described. A - HEK293FT cells were infected with wild-type AAV2 carrying the mScarlet gene. B - HEK293FT cells were infected with AAV2 virus Arg585Ala Arg588Ala carrying the mScarlet gene. C - HEK293FT cells were exposed to sfGFP-PKD2 adaptor protein and then infected with AAV2 virus Arg585Ala Arg588Ala carrying the mScarlet gene. The data show that sfGFP-PKD2 protein fusion significantly restores the infectivity of AAV2 Arg585Ala Arg588Ala in cells expressing the partner receptor (see Example 6). [Diagram 2] Figure 1 shows SKBR3 cell line experiments showing infection with wild type AAV2 with and without the DAPRin-PKD2 adaptor protein. A - SKBR3+AAV2 wt(-)DAPRin-PKD2, B - SKBR3+AAV2 wt(+)DAPRin-PKD2. The images show that the Her2-specific DARPin-PKD2 fusion protein strongly enhances the infectivity of AAV2 wild type in SKBR3 cells. [Diagram 3] Figure 1 shows an experiment with HEK293FT cells showing infection with mScarlet-PKD2+AAV2 mix. The data show that the presence of mScarlet-PKD2 fusion protein in solution inhibits AAV2 wild-type infectivity in a concentration-dependent manner, suggesting that the PKD2 domain of the fusion protein can effectively interact with the virus and outcompete the cellular AAVR for binding, preventing infection. [Figure 4]Figure 1 shows surface plasmon resonance (SPR) measurements of the binding kinetics of PKD2 fusion protein with AAV2. The SPR signal confirms the interaction of the fusion protein with immobilized AAV2, with a measured affinity of around 6 μM. [Diagram 5] Figure 1 shows surface plasmon resonance (SPR) measurements of the binding kinetics of PKD2 fusion protein with AAV1. The SPR signal confirms the interaction of the fusion protein with immobilized AAV2, with a measured affinity of around 2 μM. [Figure 6] FIG. 1 shows exemplary AAV receptor amino acid sequences, taken from Uniprot entry Q8IZA0 and disclosed as SEQ ID NO: 1. PKD1 is residues 312-401, PKD2 is residues 409-498, PKD3 is residues 504-594, PKD4 is residues 600-688, and PKD5 is residues 694-785, all highlighted in bold and represented as SEQ ID NO: 2-SEQ ID NO: 6. [Figure 7] 1 shows an exemplary AAV1 cap gene amino acid sequence, obtained from NCBI entry NP_049542 and disclosed as SEQ ID NO:7. [Figure 8] 1 shows an exemplary AAV2 cap gene amino acid sequence, obtained from NCBI entry YP_680426 and disclosed as SEQ ID NO:8. [Figure 9] 1 shows an exemplary AAV3 cap gene amino acid sequence, obtained from NCBI entry NP_043941.1 and disclosed as SEQ ID NO:9. [Figure 10] 1 shows an exemplary AAV4 cap gene amino acid sequence, taken from NCBI entry NP_044927 and disclosed as SEQ ID NO:10. [Figure 11] 1 shows an exemplary AAV5 cap gene amino acid sequence, obtained from NCBI entry YP_068409 and disclosed as SEQ ID NO:11. [Figure 12]1 shows an exemplary AAV6 cap gene amino acid sequence, obtained from Uniprot entry O56137_9VIRU and disclosed as SEQ ID NO:12. [Figure 13] 1 shows an exemplary AAV7 cap gene amino acid sequence, taken from NCBI entry YP_077178 and disclosed as SEQ ID NO: 13. [Figure 14] 1 shows an exemplary AAV8 cap gene amino acid sequence, taken from NCBI entry YP_077180 and disclosed as SEQ ID NO: 14. [Figure 15] FIG. 1 shows an exemplary AAV9 cap gene amino acid sequence, taken from Uniprot entry Q6JC40_9VIRU and disclosed as SEQ ID NO: 15. [Figure 16] 1 shows an exemplary AAV10 cap gene amino acid sequence, obtained from NCBI entry AAT46337 and disclosed as SEQ ID NO: 16. [Figure 17] 1 shows an exemplary AAV11 cap gene amino acid sequence, taken from Uniprot entry Q5Y9B2_9VIRU and disclosed as SEQ ID NO:17. [Figure 18] FIG. 1 shows an exemplary AAV12 cap gene amino acid sequence, taken from Uniprot entry A9RAI0 and disclosed as SEQ ID NO: 18. [Figure 19] 1 shows an exemplary AAV13 cap gene amino acid sequence, taken from Uniprot entry B5SUY7 and disclosed as SEQ ID NO:19. [Figure 20]Figure 1 shows the formation of disulfide bonds between residues Cys590 of the AAV1 (Asp590Cys) mutant and Cys425 of the sfGFP-PKD2 (Ser425Cys) adaptor fusion protein. The figure shows the formation of three bands migrating between the 150 kDa and 100 kDa markers, corresponding to disulfide-linked sfGFP-PKD2+VP1, sfGFP-PKD2+VP2, and sfGFP-PKD2+VP3, respectively. These three bands can be observed in a non-reducing gel (top) only when AAV1 capsid with the Asp590Cys mutation is mixed with sfGFP-PKD2 with the Ser425Cys mutation, but not otherwise. Furthermore, these bands are unstable in the presence of reducing agents such as β-mercaptoethanol (βME) (bottom gel). See Example 6. [Figure 21] Figure 1 shows the isolation of covalently bound AAV1 complexed with adaptor proteins. The figure shows the composition of AAV1-sfGFP CAR-V complexes purified by iodixanol step gradient ultracentrifugation. Stably bound heterodimers of PKD2 mutant fusion proteins with viral capsid proteins VP1, VP2 and VP3 could be recovered from the 40% iodixanol fraction (bands above 100 kDa). This demonstrates the successful formation of covalently bound AAV1Cys<>sfGFP particles (see Example 2). [Figure 22] Figure 1 shows the tropism of AAV1 covalently linked to an adaptor. The data show that AAV1(Asp590Cys)<>sfGFP CAR-V has reduced tropism for HEK293T cells that do not express the chimeric sfGFP receptor, but infectivity is restored when the target cells express the chimeric receptor. The data further show that HEK293T cells are susceptible to infection by AAV1(Asp590Cys) without the adaptor protein (see Example 3). [Figure 23]FIG. 1 shows an exemplary Coxsackievirus and Adenovirus Receptor (CxAdR) cap gene amino acid sequence, taken from Uniprot entry P78310 and disclosed as SEQ ID NO:20. [Figure 24] FIG. 1 shows exemplary A20 antibody sequences, taken from McCraw et al., Structure of adeno-associated virus-2 in complex with neutralizing monoclonal antibody, Volume 431, Issues 1-2, 15-30 September 2012, Pages 40-49 and disclosed as SEQ ID NO:21 to SEQ ID NO:28. [Diagram 25] FIG. 1 shows an exemplary CD46 sequence, taken from Uniprot entry P15529 and disclosed as SEQ ID NO:29. [Figure 26] Figure 1 shows the affinity of PKD2(V480E) mutant to AAV1 capsid. Response-concentration plot (Y-axis: arbitrary units, X-axis: molar concentration) for the affinity between either wild-type (shown as squares, fitted with solid line) or Val480Glu mutant (shown as circles, fitted with dashed line) AAV1 capsid and the PKD2 domain of AAVR. The plot shows that the measured Kd for PKD2 wild-type is 12.18 μM and for the PKD2(V480E) mutant is 6.13 μM, indicating that the mutant has a higher affinity for AAV1 capsid. Thus, we found that the PKD2(V480E) mutant has improved affinity for AAV capsid and is a superior adaptor protein. [Figure 27]Figure 3 shows AdV-5 fiber knob coupling with CAR-derived adapter protein. Reaction of fiber knob protein from adenovirus-5 with mutation V441C with adapter protein from human coxsackievirus-adenovirus receptor (CAR) with mutation V70C. Reactions were performed in 20 mM Tris-HCl (pH 8), 150 mM NaCl and 0.1 mM CuSO4. The final concentration of knob protein (22 kDa) was 2 μM and that of adapter protein (42 kDa) was 4 μM. Protein bands were separated on a non-reducing 4%-12% Bis-Tris gel. Lane 3 shows the formation of a band with a molecular weight of around 65 kDa, corresponding to a covalent adduct of knob protein with CAR-derived adapter protein. Thus, it was demonstrated that the use of adapter proteins to retarget viruses can be applied to adenoviruses using CAR fusion proteins as adapters. [Figure 28]Negative stain electron microscopy of AAV particles covalently bound to AAVR-derived adapter proteins. Negative stain electron microscopy of AAV particles at 52000x magnification. A) AAV1(Asp590Cys) particles were purified by affinity chromatography (AVB Sepharose resin from Cytiva) followed by iodixanol gradient ultracentrifugation, stained with phosphotungstic acid, and imaged by electron microscopy. The figure reveals that the particle outlines are smooth. B) AAV1(Asp590Cys) particles were purified by affinity chromatography (AVB Sepharose resin from Cytiva) and then incubated with sfGFP-PKD2(Ser425Cys) adapter protein (3 μM) (20 h at room temperature). The particles were then purified by iodixanol gradient ultracentrifugation, stained with phosphotungstic acid, and imaged by electron microscopy. The figure reveals that the particle outlines are rough and larger than the native particles. C) Quantification of particle diameter. The mean diameter of the native particles in A) is 26.0 nm, while the mean diameter of the adaptor protein-coated particles in B) is 29.0 nm. This figure shows the complex of AAV1(Asp590Cys) with the adaptor protein. [Figure 29]Figure 1. AAV1Asp590Cys coupling with PKD2-derived adaptor protein. Coomassie stained SDS PAGE showing that PKD2-derived fusion proteins containing cysteines and encompassing various fusion partners can form covalent bonds with AAV1Asp590Cys. The fusion partners used were a single domain antibody against eGFP (a:eGFP sdAb), a DARPin evolved to bind Her2 (a:Her2 DARPin), a monomeric variant of superfolder GFP (msfGFP), and maltose binding protein (MBP) from E. coli. All adaptor proteins were coupled with AAV1Asp590Cys at a final concentration of 3 μM for 2 h at room temperature. Proteins were run on a non-reducing 4%-12% Bis-Tris gradient gel. This figure shows that the identity of the fusion partner that constitutes the adaptor protein together with PKD2 (Ser425Cys) can be customized in a modular manner while maintaining the ability to form covalent adducts. This suggests that our technology may be used to retarget AAV to many different receptors by changing the identity of the fusion partner. Figure legend: * represents capsid protein, † represents adapter protein, †† represents adapter protein dimer, *† represents conjugated species of adapter protein and viral protein. [Diagram 30]AAV1Asp590Cys coupling to PKD2 derived adaptor protein in 1 mM TCEP. Blot for AAV1Asp590Cys capsid protein when virus was incubated separately (first lane after marker) or with PKD2 (Ser425Cys-Val480Glu) with 3 μM of adaptor protein composed of fusion partners as indicated. Incubation was performed at room temperature for 20 h in the presence of 1 mM TCEP. After incubation, reactions were run on an 8% Bis-Tris non-reducing gel in MOPS buffer at 200 V for 37 min. After electrophoresis, the gel was transferred to a PVDF membrane and immunoblotted with a primary mouse monoclonal B1 antibody against AAV VP1 / 2 / 3 (Progen, Cat. No. 690058) and an infrared IRDye™ 800CW goat anti-mouse IgG secondary antibody (Li-COR, Cat. No. 926-32210). The figure shows that covalent adducts between the adapter protein and the viral capsid protein can form even in the presence of 1 mM of the reducing agent TCEP, indicating that disulfide bonds between the proteins form at interfaces that are not fully exposed to solvent. Of note, adducts can form between viral proteins and adapters both when the adapters are expressed in bacterial cells (e.g., sfGFP and DARPin) and when the adapters are expressed in mammalian cells (e.g., a:ICAM scFv). [Diagram 31]Figure 1 shows infection of SKOV3 cells with AAV1 wild type or anti-EGFR-AAV1. Infection of SKOV3 cells with AAV coated with (anti-EGFR)DARPin-PKD2(Ser425Cys-Val480Glu) fusion protein shows that this cell line is less sensitive to AAV1 normally administered at high doses (1.5x107vg per μL of medium) and that this cell line is 3-fold more sensitive to infection with the coated virus. Infectivity was measured by the percentage of cells expressing GFP (whose gene was delivered by the virus) after 72 hours of infection. Data are plotted as the mean of three replicates and analyzed by unpaired two-tailed t-test. Error bars indicate standard deviation. Significance is expressed as ns: p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001. This figure shows that retargeting AAV1(Asp590Cys) with an adapter protein can substantially increase its infectivity. [Diagram 32] ADK1a neutralization - Effect of anti-AAV1 mAb on rAAV1 transgene expression. 0.5x109vg of recombinant AAV1 or 0.5x109vg of AAV1(Asp590Cys)<>(anti-EGFR)DARPin-PKD2(Ser425Cys-Val480Glu)CAR-V were incubated with serial dilutions of ADK1a neutralizing antibody (Progen, Cat. No. 610150) for 1 hour at room temperature, and the mixture was then used to infect HEK293T cells seeded in 96-well plates. The percentage of infected cells, as measured by expression of the GFP reporter, was measured after 24 hours and plotted as a function of the concentration of antibody used. The plot shows that recombinant AAV1 is neutralized by ADK1a antibody, whereas coated CAR-V particles maintain the same level of infectivity and are therefore resistant to neutralization, regardless of the concentration of neutralizing antibody. [Diagram 33]Infection of 293T, CHO and SKBR3 cell lines with coated AAV(Asp590Cys). Percentage of transduced cells 48 hours after infection with coated and uncoated AAV1(Asp590Cys) viral particles at a final concentration of 2.0×106 vg per μL of cell culture medium. Virus coating was performed for 2 hours at room temperature in the presence of 1 μM PKD2(Ser425Cys-Val480Glu) adaptor protein. Quantification was based on the number of cells expressing the reporter delivered by the virus as assessed by laser scanning confocal microscopy. The x-axis indicates the receptor targeted by the adaptor protein used to coat the virus. Data are plotted as the average of two replicates. Infection of the SKBR3 cell line (C), which expresses high levels of HER2 and EGFR, shows higher infectivity with AAV1(Asp590Cys) viral particles coated with the corresponding PKD2(Ser425Cys-Val480Glu) adaptor protein. Coating of AAV1(Asp590Cys) reduces infectivity in CHO cells (B), which do not have the corresponding receptor. This data indicates that coating of AAV1(Asp590Cys) with adaptor proteins can enhance its specificity for cell types expressing receptors that interact with the adaptor proteins. [Diagram 34]Figure 1: Liver AAV transduction in a TZM-bl xenograft model. Liver infectivity by sfGFP-PKD2 (Ser425Cys-Val480Glu) and (anti-CD4)DARPin-PKD2 (Ser425Cys-Val480Glu) coated AAV1 (Asp590Cys) compared to uncoated AAV1 (Asp590Cys). Livers were harvested 14 days after intravenous injection of coated and uncoated AAV1 (Asp590Cys) particles at 3.2x1010vg / mouse into C57BL / 6Jax mice. Mice harbored tumors composed of TZM-bl cells. Data show that coating of virus with adaptor proteins abolishes infection of the liver, the main site of off-target infection of AAV. Quantification was based on three 50 μm sections of liver from each mouse. Images were acquired with a laser scanning confocal microscope. Data are plotted as the average of three replicates. [Diagram 35]Figure 1 shows AAV5Q697C coupling with PKD1S356C-derived AP. Blot against AAV5 capsid protein on AAV5Gln697Cys of virus either in isolation (first lane after marker) or incubated with 10 μM adapter protein (38.2 kDa) composed of superfolder GFP fused to PKD1 (Ser356Cys). Incubation was performed at room temperature for 16 hours. After incubation, reactions were run on an 8% Bis-Tris non-reducing gel in MOPS buffer at 200V for 37 minutes. The gel was then transferred to a PVDF membrane and immunoblotted with a primary mouse monoclonal B1 antibody against AAV VP1 / 2 / 3 (Progen, Cat. No. 690058) and an infrared IRDye™ 800CW goat anti-mouse IgG secondary antibody (Li-COR, Cat. No. 926-32210). The figure shows the covalent adduct of adaptor protein with viral capsid protein, as indicated by the shift in the molecular weight of the capsid protein. Importantly, no measurable band corresponding to free VP protein could be observed after incubation with adaptor protein, indicating that coupling went to completion. This indicates that PKD1-derived adaptor protein can be used for retargeting AAV5Q697C. [Diagram 36]Figure 1. AdV B3 knob coupling with DSG2-derived AP. Coomassie stained gel showing covalent coupling of adenovirus B3 fiber knob (residues 128-319, Uniprot ID P04501, strep-tagged, Asn192Cys mutant, 23.4 kDa) with an adaptor protein (AP) obtained by fusing superfolder GFP to desmoglein 2 (residues 150-385, Ala174Cys mutant). The molecular weight of the AP is 54.4 kDa. 3 μg of purified knob was incubated with 4 μg of purified adaptor protein in the absence (left) or presence (right) of 1 mM CaCl2 in PBS at room temperature for 18 h in a total reaction volume of 15 μL. The reaction was then run on a 4%-12% bis-tris acrylamide gel in MES running buffer at 200 V for 40 min under non-reducing conditions. The gel was then stained with Coomassie dye. The gels show the formation of covalent adducts between the fiber knob and the adaptor protein that are only observed in the presence of calcium ions, indicating that bond formation is dependent on a Ca2+-dependent interaction between the knob and desmoglein 2. Thus, DSG2-derived adaptor proteins can be used to covalently bind adenovirus B3 and redirect its tropism. [Figure 37] 1 shows an exemplary human adenovirus B3 fiber protein sequence, taken from Uniprot entry P04501 and disclosed as SEQ ID NO:30. [Figure 38] FIG. 1 shows an exemplary Desmoglein 2 (DSG2) sequence, taken from Uniprot entry Q14126 and disclosed as SEQ ID NO:31. [Figure 39]Infection of 293T cells with coated AAV2(Gln589Cys). Percentage of transduced cells 48 hours after infection with coated and uncoated AAV2(Gln589Cys) viral particles at a final concentration of 1.6×106 vg per μL of cell medium. Virus coating was performed at room temperature for 24 hours in the presence of 3 μM PKD2(Ser425Cys-Val480Glu) adaptor protein. Quantification was based on the number of cells expressing the reporter delivered by the virus as assessed by laser scanning microscopy. The x-axis indicates the receptor targeted by the adaptor protein used to coat the virus. Data are plotted as the average of two replicates. Viruses are more infectious to 293T cells when coated with adaptor proteins against HER2 and EGFR expressed on the cell surface. Coating with GFP, for which no cell surface receptor is present, reduces infectivity. The data indicate that coating AAV2(Gln589Cys) with an adaptor protein can enhance its specificity for cells expressing a receptor that interacts with the adaptor protein. [Diagram 40]Figure 1. AAV2(Gln589Cys) coupling to PKD2 derived adaptor protein. Blot for AAV1Asp590Cys and AAV2Gln589Cys capsid proteins with viruses either separated (first and third lanes after marker, respectively) or incubated with 5 μM superfolder GFP fused to PKD2(Ser425Cys-Val480Glu). Incubation was for 22 h at room temperature. After incubation, reactions were run on an 8% Bis-Tris non-reducing gel in MOPS buffer at 200V for 40 min. The gel was then transferred to a PVDF membrane and immunoblotted with a primary mouse monoclonal B1 antibody against AAV VP1 / 2 / 3 (Progen, Cat. No. 690058) and an infrared IRDye™ 800CW goat anti-mouse IgG secondary antibody (Li-COR, Cat. No. 926-32210). The figure shows covalent adducts of adapter proteins with viral capsid proteins, as indicated by a shift in the molecular weight of the capsid protein in lanes 2 and 5 after the marker. Importantly, adduct bands are formed for both AAV1 and AAV2 mutant viruses. This confirms that PKD2-derived adapter proteins can be used to redirect the tropism of AAV1 and AAV2. [Diagram 41]Infection of SKBR3 cells with coated AAV5(Gln697Cys). Percentage of transduced cells 6 days after infection with coated and uncoated AAV5(Gln697Cys) viral particles at a final concentration of 1.0×107vg per μL of cell medium. Virus coating was performed at room temperature for 24 hours in the presence of 0.5 μM PKD1(Ser356Cys) adaptor protein. Quantification was based on the number of cells expressing the reporter delivered by the virus as assessed by laser scanning microscopy. The x-axis indicates the receptor targeted by the adaptor protein used to coat the virus. Data are plotted as the average of two replicates. Viruses are more infectious to SKBR3 cells when coated with an adaptor protein against HER2, which is highly expressed on the cell surface. Coating with GFP, for which no cell surface receptor is present, reduces infectivity. The data indicate that coating of AAV5(Gln697Cys) with an adapter protein may enable retargeting by enhancing its specificity for cells expressing the corresponding receptor. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0116] In order that the present invention may be more readily understood, certain terms are first defined below. Further definitions for these terms and other terms are set forth throughout the specification.

[0117] As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that is included within (greater or less than) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%), 6%), 5%, 4%, 3%, 2%, 1%) in either direction of the stated reference value, unless otherwise specified or clear from the context (except where such number exceeds 100% of the possible values).

[0118] As used herein, the term "amelioration" refers to the prevention, reduction or alleviation of a condition, or the betterment of a subject's condition. Amelioration includes, but does not require, complete recovery or complete prevention of a disease condition.

[0119] As used herein, the term "comparable" refers to a system, set of conditions, effect or result that is sufficiently similar to a test system, set of conditions, effect or result to allow a scientifically reasonable comparison. One of ordinary skill in the art will recognize and understand which system, set of conditions, effect or result is sufficiently similar to be "comparable" to any particular test system, set of conditions, effect or result described herein.

[0120] As used herein, the term "correlates" has its ordinary meaning of "correlating with." One of skill in the art will recognize that two features, items, or values ​​correlate with each other when they show a tendency to appear and / or vary together. In some embodiments, a correlation is statistically significant when its p-value is less than 0.05, and in some embodiments, a correlation is statistically significant when its p-value is less than 0.01. In some embodiments, the correlation is assessed by regression analysis. In some embodiments, the correlation is a correlation coefficient.

[0121] As used herein, the terms "improve," "increase," or "reduce," or grammatical equivalents, refer to a value relative to a reference (e.g., baseline) measurement, e.g., a measurement taken under comparable conditions (e.g., in the same individual prior to the initiation of a treatment described herein, or in a control individual (or control individuals) in the absence of treatment).

[0122] As used herein, a "polypeptide" generally refers to a string of at least two amino acids linked together by peptide bonds. In some embodiments, a polypeptide can include at least 3-5 amino acids, each of which is linked to another amino acid by at least one peptide bond. Those of skill in the art will recognize that polypeptides can optionally include "unnatural" amino acids or other entities, which can optionally be incorporated into the polypeptide chain.

[0123] As used herein, the term "protein" refers to a polypeptide (i.e., a string of at least two amino acids linked together by peptide bonds). A protein may contain moieties other than amino acids (e.g., may be a glycoprotein, proteoglycan, etc.) and / or may be otherwise processed or modified. Those of skill in the art will recognize that a "protein" may be an entire polypeptide chain (with or without a signal sequence) produced by a cell, or a characteristic portion thereof. Those of skill in the art will recognize that a protein may also include two or more polypeptide chains, for example, linked by one or more disulfide bonds or linked by other means. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, a protein may include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids.

[0124] As used herein, the term "ligand" refers to a molecule, such as a cell surface receptor, that can mediate an interaction with a biomolecule. A ligand can include a portion of a protein or polypeptide, or the full length sequence of a protein or polypeptide. A ligand can also include any other molecule, such as a cell surface receptor, that can mediate an interaction with a biomolecule, such as a suitable small molecule. In some embodiments of the invention, the ligand mediates a biological interaction with a target cell. In some embodiments of the invention, the ligand mediates a biological interaction that is different from the interaction of an isolated polypeptide or adapter molecule with a viral particle or viral capsid.

[0125] As used herein, the term "full length" of a protein or polypeptide refers to the entire canonical protein, including substantially all of the amino acids shown in the sequence. As used herein, the term "portion" of a protein or polypeptide refers to a characteristic portion of the full length protein that can substantially perform the same function as the full length protein (e.g., binding a target). The term "portion" of a protein or polypeptide may refer to a protein or polypeptide portion that includes at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the amino acids of the full length protein or polypeptide. As used herein, a "portion" of a protein or polypeptide may include at least 100, 200, 300, 400, 500 or more amino acids.

[0126] As used herein, the term "conservative" or "conservative amino acid change" refers to cases where the substituted amino acid has similar structural or chemical properties. In the context of a protein or polypeptide, a conservative change is an amino acid change that does not affect at least one function of the protein or polypeptide (e.g., an amino acid change that does not affect the function of a viral capsid). One type of conservative amino acid substitution refers to the interchangeability of residues with similar side chains. For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine, the group of amino acids with aliphatic hydroxyl side chains is serine and threonine, the group of amino acids with amide-containing side chains is asparagine and glutamine, the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan, the group of amino acids with basic side chains is lysine, arginine, and histidine, and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0127] As used herein, the term "non-conservative" change or "non-conservative amino acid change" refers to the replacement of an amino acid with another amino acid having different structural or chemical properties, for example, replacing glycine with tryptophan. With respect to a protein or polypeptide, a non-conservative change is an amino acid change that affects at least one function of the protein or polypeptide (e.g., an amino acid change that affects the function of a viral capsid). Guidance for determining which and how many amino acid residues can be replaced, inserted or deleted without affecting biological activity can be found using computer programs known in the art, such as DNAStar software. Mutants can be tested in functional assays. Certain mutants have less than 10%, preferably less than 5%, even more preferably less than 2% or less than 1% of amino acids replaced. As used herein, the term "point mutation" refers to a single amino acid substitution.

[0128] As used herein, the term "insertion" refers to the introduction of one or more consecutive heterologous amino acid residues (i.e., amino acids that do not appear at the corresponding position in the wild-type amino acid sequence). An insertion may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in length. When referring to a viral capsid that does not have an insertion, preferably the viral capsid does not contain an insertion of more than 5 amino acids in length. Means of introducing an insertion will be known to those skilled in the art, for example, via genetic or chemical means.

[0129] As used herein, the term "deletion" refers to the removal of one or more consecutive amino acid residues from an amino acid sequence. The deletion may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in length. When referring to a viral capsid that does not have a deletion, preferably the viral capsid does not contain a deletion of more than 5 amino acids in length. Means for introducing deletions will be known to those skilled in the art, for example, via genetic or chemical means.

[0130] As used herein, the terms "subject," "individual," or "patient" refer to any organism to which an embodiment of the invention can be used or administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals, e.g., mice, rats, rabbits, non-human primates, and humans; insects; worms, etc.). In a preferred embodiment of the invention, the subject is a human.

[0131] As used herein, the term "target cell" or "target tissue" refers to any cell, tissue, or organism. In some embodiments, the target cell or tissue is a cell or tissue involved in a pathological condition amenable to treatment by viral vector-mediated gene therapy.

[0132] As used herein, the term "therapeutic regimen" refers to any method that partially or completely relieves, improves, eliminates, inhibits, prevents, delays the onset, reduces the severity, and / or reduces the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. This can include the administration of one or more doses, optionally spaced by regular or varying time intervals. In some embodiments, a therapeutic regimen is one whose implementation is designed to achieve and / or correlate with a particular effect (e.g., across a relevant population of cells, tissues, or organisms), such as reduction or elimination of a harmful condition or disease. In some embodiments, treatment includes the administration of one or more therapeutic agents, either simultaneously, sequentially, or at different times, for the same or different amounts of time. In some embodiments, a "therapeutic regimen" includes genetic methods, e.g., gene therapy, gene disruption, or other methods known to induce or reduce expression (e.g., transcription, processing, and / or translation of a particular gene product, e.g., the primary transcript or mRNA).

[0133] As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic agent that confers a therapeutic effect on the treated subject at a reasonable benefit / risk ratio applicable to any medical treatment. Such therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject shows an indication of or feels an effect). In some embodiments, "therapeutically effective amount" refers to an amount of a therapeutic agent or composition that is effective to treat, ameliorate or prevent (e.g., delay the onset of an associated disease or condition) and / or exhibit a detectable therapeutic or prophylactic effect, for example, by improving symptoms associated with the disease, by preventing or delaying the onset of the disease, and / or by reducing the severity or frequency of symptoms of the disease. Therapeutically effective amounts are generally administered in a dosing regimen that may include multiple unit doses. For any particular therapeutic agent, the therapeutically effective amount (and / or the appropriate unit dose within an effective dosing regimen) may vary, for example, depending on the route of administration or combination with other therapeutic agents. Alternatively or additionally, the particular therapeutically effective amount (and / or unit dose) for any particular patient may depend on a variety of factors, including the activity of the particular therapeutic agent used; the particular composition used; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and / or rate of excretion or metabolism of the particular therapeutic agent used; the duration of treatment; and factors such as are known in the medical arts.

[0134] In some embodiments of the invention, the method of treatment comprises administering a therapeutically effective amount of a composition or viral particle described herein. In some embodiments of the invention, the compositions described for use herein are administered in a therapeutically effective amount.

[0135] As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of a therapeutic agent according to a treatment regimen that achieves a desired effect in terms of partially or completely relieving, ameliorating, eliminating, inhibiting, delaying the onset of, reducing the severity of, and / or reducing the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. In some embodiments, the administration of a therapeutic agent according to a treatment regimen is correlated with achieving a desired effect. Such treatment may be of subjects who do not show signs of the associated disease, disorder, and / or condition, and / or of subjects who show only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be of subjects who show one or more established signs of the associated disease, disorder, and / or condition. In some embodiments, treatment may be of subjects who have been diagnosed as suffering from the associated disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the relevant disease, disorder and / or condition.

[0136] The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable domain (VH) and a heavy chain constant region (CH). The heavy chain constant region comprises at least three domains, CH1, CH2, CH3, and optionally CH4. Each light chain comprises a light chain variable domain (CH) and a light chain constant region (CL). The heavy and light chain variable domains can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each heavy and light chain variable domain comprises three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 and HCDR3, and light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3). A typical tetrameric antibody structure comprises two identical antigen-binding domains, each of which is formed by the association of a VH domain and a VL domain, each of which, together with its respective CH and CL domains, forms the Fv region of the antibody. A single domain antibody comprises a single antigen-binding domain, e.g., VH or VL. The antigen-binding domain of an antibody, e.g., the portion of an antibody that recognizes and binds to the first member of a specific binding pair of an antigen, is also referred to as the "paratope". It is a portion of a small region (of 5-10 amino acids) of the Fv region of an antibody, i.e., the antigen-binding fragment (Fab region), and may contain a portion of the heavy and / or light chain of the antibody. A paratope specifically binds to a first member of a specific binding pair if the paratope binds to the first member of the specific binding pair with high affinity. The term "high affinity" antibody refers to an antibody that binds to a first member of the specific binding pair with a high affinity of about 10 -9 M or less (for example, about 1×10 -9 M, 1×10M -10 M, 1×10 -11 M, or approximately 1 x 10 -12M) In one embodiment, the KD is measured by surface plasmon resonance, e.g., BIACORE™, and in another embodiment, the KD is measured by ELISA.

[0137] The phrase "complementarity determining region" or "CDR" includes amino acid sequences encoded by nucleic acid sequences of an organism's immunoglobulin genes that normally (i.e., in a wild-type animal) appear between two framework regions in the variable region of a light or heavy chain of an immunoglobulin molecule (e.g., an antibody or T cell receptor). CDRs can be encoded, for example, by germline sequences, or by rearranged or unrearranged sequences, and by, for example, naive or mature B or T cells. CDRs can be somatically mutated (e.g., different from the sequence encoded in the germline of the animal), humanized, and / or modified by amino acid substitution, insertion, or deletion. In some situations (e.g., in the case of CDR3), CDRs can be encoded by two or more sequences (e.g., germline sequences) that are not contiguous (e.g., in an unrearranged nucleic acid sequence) but are contiguous in the B cell nucleic acid sequence, for example, as a result of splicing or joining of sequences (e.g., VDJ recombination to form a heavy chain CDR3).

[0138] The phrase "heavy chain" or "immunoglobulin heavy chain" includes immunoglobulin heavy chain sequences, including immunoglobulin heavy chain constant region sequences, from any organism. The heavy chain variable domain includes three heavy chain CDRs and four FR regions, unless otherwise specified. Fragments of heavy chains include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has a variable domain (from N-terminus to C-terminus) followed by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain. Functional fragments of heavy chains include fragments that can specifically recognize a first member of a specific binding pair (e.g., recognize a first member of a specific binding pair with a KD in the micromolar, nanomolar, or picomolar range), can be expressed and secreted from a cell, and contain at least one CDR. The heavy chain variable domain is encoded by a variable region nucleotide sequence that collectively includes VH, DH, and JH segments derived from a repertoire of VH, DH, and JH segments present in the germline.

[0139] The term "light chain" includes immunoglobulin light chain sequences from any organism, including human kappa and lambda light chains, and VpreB, as well as surrogate light chains, unless otherwise specified. A light chain variable domain typically includes three light chain CDRs and four framework (FR) regions, unless otherwise specified. In general, a full-length light chain includes a variable domain including, from the amino terminus to the carboxyl terminus, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region. The light chain variable domain is encoded by a light chain variable region gene sequence that includes, in total, VL and JL segments derived from the repertoire of V and J segments present in the germline. The sequences, locations and nomenclature of the V and J light chain segments of various organisms can be found in the IMGT database (www.imgt.org). Light chains include, for example, those that do not selectively bind to either the first or second first members of the specific binding pair in which they appear, selectively bound by the first member of the protein that binds the specific binding pair. Light chains also include those that bind, recognize, or assist a heavy chain or another light chain by binding to and recognizing one or more first members of the specific binding pair in which they appear, selectively bound by the first member of the protein that binds the specific binding pair. Common or universal light chains include those derived from the human Vκ1-39Jκ gene or the human Vκ3-20Kκ gene, including somatically mutated (e.g., affinity matured) forms thereof. Exemplary human VL segments include the human Vκ1-39 gene segment, the human Vκ3-20 gene segment, the human Vλ1-40 gene segment, the human Vλ1-44 gene segment, the human Vλ2-8 gene segment, the human Vλ2-14 gene segment, and the human Vλ3-21 gene segment, including somatically mutated (e.g., affinity matured) forms thereof. Light chains can be generated that contain a variable domain from one organism (e.g., human, or a rodent, such as rat or mouse, or an avian, such as chicken) and a constant region from the same or a different organism (e.g., human, or a rodent, such as rat or mouse, or an avian, such as chicken).

[0140] The term "capsid protein" includes proteins that are part of the viral capsid. In the case of adeno-associated viruses, capsid proteins generally refer to VP1, VP2 and / or VP3 and are encoded by a single cap gene. In the case of AAV, the three AAV capsid proteins are produced redundantly from the cap open reading frame (ORF) through alternative splicing of mRNA and / or alternative use of translation initiation codons, although all three proteins share a common stop codon.

[0141] The term "wild type" as used herein includes entities having structure and / or activity as found in nature in a "normal" state or situation (as opposed to mutant, diseased, denatured, etc.). Those skilled in the art will understand that a wild type viral vector, e.g., a wild type capsid protein, may be used as a reference viral vector in comparative studies with modified capsids. Generally, the reference viral capsid protein / capsid / vector is identical to the test viral capsid protein / capsid / vector, except for the changes to be tested for effect. When used in reference to a polynucleotide or polypeptide, "wild type" refers to the natural (unmodified) form of the polynucleotide or polypeptide found in or expressed by a wild type organism.

[0142] As used herein, the terms "covalent", "covalent bond" or "covalently bonded" refer to a chemical bond involving the sharing of electron pairs between atoms. These electron pairs are known as shared or bond electron pairs, and a stable balance of attractive and repulsive forces between atoms results in the formation of a covalent bond. In many molecules, the sharing of electrons allows each atom to reach the equivalent of a full valence shell, which corresponds to a stable electronic configuration. In a preferred embodiment of the present invention, the covalent bond is a disulfide (SS bond), which can be induced by the coupling of two thiol groups. The covalent bond between the thiol groups of two cysteine ​​residues is an important element of the secondary and tertiary structure of proteins. A covalent bond can provide a stronger link between one or more biomolecules than other forms of bonds, such as ionic bonds or electromagnetic intermolecular forces.

[0143] As used herein, the term "viral adapter molecule" refers to a protein, fusion protein, or conjugated compound that includes a polypeptide and a ligand that can bind to a viral capsid. The term "viral adapter" may also be used to refer to such a compound. In some embodiments, the term "viral adapter molecule" refers to a protein, fusion protein, or conjugated compound that includes a polypeptide and a ligand that includes one or more cysteine ​​residues that can be covalently bound to a viral capsid. The terms "viral adapter" or "covalent viral adapter" may also be used to refer to such a compound. As used herein, the term "AAV adapter molecule" refers to a protein, fusion protein, or conjugated compound that includes a polypeptide and a ligand that can bind to an adeno-associated virus (AAV) capsid. The term "AAV adapter" may also be used to refer to such a compound.

[0144] Gene therapy In some embodiments, the recombinant viral vector described herein comprises a viral particle bound to an isolated polypeptide capable of binding to a viral capsid, or a viral adapter molecule comprising an isolated polypeptide capable of binding to a viral capsid and a ligand, wherein the viral particle encapsulates a nucleotide of interest. In some embodiments, the nucleotide of interest is under the control of a promoter selected from the group consisting of a viral promoter, a bacterial promoter, a mammalian promoter, an avian promoter, a fish promoter, an insect promoter, and any combination thereof. In some embodiments, the nucleotide of interest is under the control of a non-human promoter. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. In some embodiments, the promoter is an EF1α promoter. In some embodiments, the promoter is a CAGG promoter. In some embodiments, the promoter is a Ubiquitin C (UbC) promoter.

[0145] In some embodiments, the recombinant viral vector described herein comprises a viral particle covalently linked to an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of covalently linking to a viral capsid, or a viral adapter molecule comprising an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of covalently linking to a viral capsid and a ligand, wherein the viral particle encapsulates a nucleotide of interest. In some embodiments, the nucleotide of interest is under the control of a promoter selected from the group consisting of a viral promoter, a bacterial promoter, a mammalian promoter, an avian promoter, a fish promoter, an insect promoter, and any combination thereof. In some embodiments, the nucleotide of interest is under the control of a non-human promoter. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. In some embodiments, the promoter is an EF1α promoter. In some embodiments, the promoter is a CAGG promoter. In some embodiments, the promoter is a Ubiquitin C (UbC) promoter.

[0146] Generally, the nucleotide or gene of interest can be one or more genes that can code for a detectable marker, such as a reporter, or a therapeutic polypeptide. In some embodiments, the nucleotide of interest is a reporter gene. In some embodiments, the nucleotide or gene of interest is a reporter gene that codes for a detectable marker selected from the group consisting of green fluorescent protein, luciferase, β-galactosidase, and the like. In some embodiments, the detectable marker is green fluorescent protein. In other embodiments, the nucleotide of interest is selected from the group consisting of a suicide gene, a nucleotide that codes for an antibody or a fragment thereof, a nucleotide that codes for a CRISPR / Cas system or a portion(s) thereof, a nucleotide that codes for an antisense RNA, a nucleotide that codes for an siRNA, a secreted enzyme, a gene that codes for a therapeutic protein, and the like. In one embodiment, the nucleotide of interest codes for a multi-domain therapeutic, for example, a protein that includes at least two domains that provide two different functions.

[0147] The compositions described herein include recombinant viral particles as described herein, and may also include viral vectors, e.g., viral particles bound to an isolated polypeptide capable of binding to a viral capsid, or a viral adapter molecule comprising an isolated polypeptide capable of binding to a viral capsid and a ligand, wherein the viral particle encapsulates a nucleotide or gene of interest.

[0148] The compositions described herein include recombinant viral particles as described herein, and may also include viral vectors, e.g., viral particles covalently linked to an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of covalently linking to a viral capsid, or viral adapter molecules comprising an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of covalently linking to a viral capsid and a ligand, wherein the viral particle encapsulates a nucleotide or gene of interest.

[0149] Also described herein are methods of making and using recombinant viral capsid proteins, viral vectors, compositions, etc. comprising the same. In some embodiments, a method of redirecting a virus, e.g., adenovirus, adeno-associated virus, etc., to deliver a diagnostic / therapeutic cargo to a target cell, etc., comprises contacting a target cell (which may be in vitro or in vivo, e.g., in a human) with a recombinant viral vector comprising a recombinant viral capsid protein as described herein. Such a method may include, as a first step, producing a viral vector, e.g., culturing a packaging cell under conditions sufficient for the production of the viral vector, where the packaging cell comprises a plasmid encoding an isolated polypeptide capable of binding to a viral capsid, or a viral adapter molecule comprising an isolated polypeptide capable of binding to a viral capsid and a ligand. Such a method may include, as a first step, producing a viral vector, e.g., culturing a packaging cell under conditions sufficient for producing the viral vector, wherein the packaging cell comprises a plasmid encoding an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of being covalently bound to a viral capsid, or a viral adapter molecule comprising an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of being covalently bound to a viral capsid and a ligand.

[0150] In some embodiments, the target cell is a (human) hepatocyte and the (mosaic) recombinant viral vector comprises a targeting ligand that specifically binds to an asialoglycoprotein receptor, e.g., (h)ASGR1. In some embodiments, the target cell is a (human) neuronal cell and the (mosaic) recombinant viral vector comprises a targeting ligand that specifically binds to GABA, transferrin receptor, etc. In some embodiments, the target cell is a (human) T cell and the (mosaic) recombinant viral vector comprises a targeting ligand that specifically binds to CD3, e.g., CD3ε. In some embodiments, the target cell is a (human) hematopoietic stem cell and the (mosaic) recombinant viral vector comprises a targeting ligand that specifically binds to CD34. In some embodiments, the target cell is a (human) kidney cell. In some embodiments, the target cell is a (human) muscle cell and the (mosaic) recombinant viral vector comprises a targeting ligand that specifically binds to an integrin. In some embodiments, the target cell is a (human) cancer cell and the (mosaic) recombinant viral vector comprises a targeting ligand that specifically binds to a tumor associated antigen, e.g., E6 and E7, Her2, etc. In some embodiments, the targeting ligand binds to the human glucagon receptor (hGCGR).

[0151] The genetic cargo capacity (e.g., of a nucleotide of interest) of the viral particles provided herein can vary. The genetic cargo capacity of the viral particles can be 5.0kb, 5.1kb, 5.2kb, 5.3kb, 5.4kb, 5.5kb, 5.6kb, 5.7kb, 5.8kb, 5.9kb, 6.0kb, 6.1kb, 6.2kb, 6.3kb, 6.4kb, 6.5kb, 6.6kb, 6.7kb, 6.8kb, 6.9kb, 7.0kb, 7.1kb, 7.2kb, 7.3kb, 7.4kb, 7.5kb, 7.6kb, 7.7kb, 7.8kb, 7.9kb, 8kb, 9kb, 10kb, 11kb, 12kb, 13kb, 14kb, 15kb, 16kb, 17kb, 18kb, 19kb, 20kb, 21kb, 22kb, 23kb, 24kb, 25kb, 26kb, 27kb, 28kb, 29kb, 30kb, 31kb, 32kb, 33kb, 34kb, 35kb, 36kb, 37kb, 38kb, 39kb, 40kb, 41kb, 42kb, 43kb, 44kb, 45kb, 46kb, 47kb, 48kb, 49kb, 50kb, 51kb, 52 .8kb, 7.9kb, 8.0kb, 8.1kb, 8.2kb, 8.3kb, 8.4kb, 8.5kb, 8.6kb, 8.7kb, 8.8kb, 8.9kb, 9.0kb, 9.1kb, 9.2kb, 9.3kb, 9.4kb, 9.5kb, 9.6kb, 9.7kb, 9.8kb, 9.9kb, 10.0kb, or a number or range between any two of these values, or it may be approximately these. The genetic cargo capacity of the viral particles is at least 5.0kb, 5.1kb, 5.2kb, 5.3kb, 5.4kb, 5.5kb, 5.6kb, 5.7kb, 5.8kb, 5.9kb, 6.0kb, 6.1kb, 6.2kb, 6.3kb, 6.4kb, 6.5kb, 6.6kb, 6.7kb, 6.8kb, 6.9kb, 7.0kb, 7.1kb, 7.2kb, 7.3kb, 7.4kb, 7.5kb, 7.6kb, 7.7kb, 7.8kb, 7.9kb, 8 ... , 7.6kb, 7.7kb, 7.8kb, 7.9kb, 8.0kb, 8.1kb, 8.2kb, 8.3kb, 8.4kb, 8.5kb, 8.6kb, 8.7kb, 8.8kb, 8.9kb, 9.0kb, 9.1kb, 9.2kb, 9.3kb, 9.4kb, 9.5kb, 9.6kb, 9.7kb, 9.8kb, 9.9kb, or 10.0kb. The genetic cargo capacity may be (i) the maximum length of a single-stranded DNA molecule that the viral particle can protect from DNAse I digestion, and / or (ii) the maximum length of a double-stranded DNA molecule that the viral particle can protect from DNAse I digestion. The single-stranded DNA molecule can self-hybridize to form a double-stranded region. The single stranded DNA molecule may comprise a self-complementary AAV (scAAV) vector.

[0152] Adeno-associated virus (AAV) Adeno-associated viruses (AAV) are small viruses that infect humans and several other primate species. They belong to the genus Dependoparvovirus and further to the family Parvoviridae. They are small (20 nm), replication-defective, non-enveloped viruses with a linear, single-stranded DNA (ssDNA) genome of approximately 4.8 kilobases (kb).

[0153] AAV is not currently known to cause disease. The virus induces a very mild immune response. Several additional features make AAV an attractive candidate for the generation of viral vectors for gene therapy and for the generation of isogenic human disease models. Gene therapy vectors using AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integration into the host cell genome, whereas native viruses allow integration of virally delivered genes into the host genome.

[0154] The AAV genome is constructed of either positive-sense or negative-sense single-stranded deoxyribonucleic acid (ssDNA) and is approximately 4.7 kilobases in length. The genome contains inverted terminal repeat (ITR) sequences at both ends of the DNA strand and two open reading frames (ORFs): rep and cap. The former consists of four overlapping genes encoding the Rep proteins required for the AAV life cycle, while the latter contains overlapping nucleotide sequences of the capsid proteins VP1, VP2, and VP3, which interact to form the capsid with icosahedral symmetry.

[0155] The inverted terminal repeat (ITR) sequences contain 145 bases each. They are so named due to a symmetry that has been shown to be necessary for efficient propagation of the AAV genome. A feature of these sequences that confers this property is their ability to form hairpins, which contribute to the so-called self-priming that allows primase-independent synthesis of the second DNA strand. It has also been shown that the ITRs are required for both integration of AAV DNA into and rescue from the host cell genome (chromosome 19 in humans), as well as for efficient encapsidation of AAV DNA combined with the generation of fully assembled deoxyribonuclease-resistant AAV particles.

[0156] On the "left" side of the genome are two promoters, called p5 and p19, from which two overlapping messenger ribonucleic acids (mRNAs) of different lengths can be generated. Each of these contains an intron that may or may not be spliced ​​out. Taking these possibilities into account, four mRNAs, and therefore four Rep proteins, with overlapping sequences, can be synthesized. Their names, Rep78, Rep68, Rep52, and Rep40, refer to their size in kilodaltons (kDa). Rep78 and Rep68 can specifically bind to the hairpin formed by the ITR in a self-priming action and cleave at a specific region within the hairpin that is a designated terminal release site. They have also been shown to be required for AAVS1-specific integration of the AAV genome. All four Rep proteins have been shown to bind ATP and have helicase activity. They have also been shown to upregulate transcription from the p40 promoter (see below) and downregulate both the p5 and p19 promoters.

[0157] The right side of the positive-sense AAV genome encodes overlapping sequences of three capsid proteins, VP1, VP2, and VP3, starting from a single promoter designated p40. The molecular weights of these proteins are 87 kDa, 72 kDa, and 62 kDa, respectively. The AAV capsid is composed of a mixture of VP1, VP2, and VP3, a total of 60 monomers, arranged in an icosahedral symmetry in a 1:1:10 ratio, with an estimated size of 3.9 megadaltons.

[0158] The cap gene produces an additional nonstructural protein called the assembly activating protein (AAP), which is produced from ORF2 and is essential for the capsid assembly process.

[0159] All three VPs are translated from one mRNA. After synthesis, this mRNA can be spliced ​​in two different ways. Either the long or short intron can be excised, resulting in two mRNA pools: a 2.3 kb-long and a 2.6 kb-long mRNA pool. Usually, especially in the presence of adenovirus, the long intron is preferred, so the 2.3 kb-long mRNA corresponds to the so-called "major splice". In this form, the first AUG codon, from which synthesis of the VP1 protein is initiated, is spliced ​​out, reducing the overall level of VP1 protein synthesis. The first AUG codon remaining in the major splice is the start codon of the VP3 protein. However, upstream of that codon in the same open reading frame is an ACG sequence surrounded by an optimal Kodak context.

[0160] The larger intron is preferentially spliced ​​out, and in the major splice, the ACG codon is a much weaker translation initiation signal, so that AAV structural proteins are synthesized in vivo at a ratio of approximately 1:1:10, the same as in mature virus particles. A unique fragment at the N-terminus of the VP1 protein was shown to have phospholipase A2 (PLA2) activity, probably required for the release of AAV particles from late endosomes.

[0161] AAV is highly prevalent in humans and other primates, and several serotypes have been isolated from various tissue samples. Serotypes 2, 3, 5, and 6 have been found in human cells, and AAV serotypes 1, 4, and 7-11 have been found in non-human primate samples. Although the AAV capsid protein contains 12 hypervariable surface regions, with most variation occurring in threefold proximal peaks, parvovirus genomes generally display highly conserved replication and structural genes across serotypes. All known serotypes can infect cells of multiple diverse tissue types. It is believed that tissue specificity is determined by the capsid serotype, and pseudotyping to alter the tropism range of AAV vectors may be important for their use in therapy.

[0162] Serotype 2 (AAV2) has been the most extensively examined to date. AAV2 shows natural tropism for skeletal muscle, neurons, vascular smooth muscle cells and hepatocytes. Three cellular receptors have been described for AAV2: heparan sulfate proteoglycans (HSPGs), aVβ5 integrin and fibroblast growth factor receptor 1 (FGFR-1). The former functions as a primary receptor, whereas the latter two have co-receptor activity, allowing AAV to enter cells by receptor-mediated endocytosis. Although HSPGs function as primary receptors, their abundance in the extracellular matrix may trap AAV particles, compromising infection efficiency.

[0163] Studies have shown that the virus serotype 2 (AAV2) cancer cells do not harm healthy cells. "Our results suggest that the adeno-associated virus type 2, which infects a large proportion of the population but has no known adverse effects, kills multiple types of cancer cells but has no effect on healthy cells," said Craig Meyers, professor of immunology and microbiology at the Pennsylvania State University College of Medicine in Pennsylvania, in 2005.

[69]

[70] This could lead to new anticancer drugs.

[0164] Other serotypes. Although AAV2 is the most common serotype in various AAV-based studies, other serotypes have been shown to be more effective as gene delivery vectors. For example, AAV6 appears to be much better at infecting airway epithelial cells, AAV7 shows very high transduction rates of murine skeletal muscle cells (as do AAV1 and AAV5), AAV8 excels at transducing hepatocytes, and AAV1 and AAV5 have been shown to be very efficient at gene delivery to vascular endothelial cells. In the brain, most AAV serotypes show neurotropism, while AAV5 also transduces astrocytes. Also, AAV6, a hybrid of AAV1 and AAV2, shows lower immunogenicity than AAV2.

[0165] Serotypes can differ with respect to the receptors they bind, for example, AAV4 and AAV5 transduction can be inhibited by soluble sialic acid (in different forms for each of these serotypes), and AAV5 has been shown to enter cells via the platelet-derived growth factor receptor.

[0166] Much effort has been made to engineer and improve new AAV variants for both clinical and research purposes. Such modifications include new tropisms to target specific tissues and modified surface residues to avoid detection by the immune system. In addition to choosing specific recombinant AAV (rAAV) strains to target specific cells, researchers have also explored AAV pseudotyping, the practice of creating hybrids of certain AAV strains to approach more precise targets. Hybrids are created by taking the capsid from one strain and the genome from another. For example, studies involving AAV2 / 5, a hybrid of the genome of AAV2 and the capsid of AAV5, were able to achieve greater accuracy and range in brain cells than unhybridized AAV2 could achieve. Researchers have continued to experiment with pseudotyping by creating strains with hybrid capsids. AAV-DJ has hybrid capsids from eight different AAV strains, allowing it to infect a variety of cells in many regions of the body, a property not present in single-strain AAVs with limited tropism. Other efforts to engineer and improve new AAV variants have involved reconstructing the ancestors of viral variants to generate new vectors with enhanced properties for clinical use and for the study of AAV biology.

[0167] Chimeric antigen receptor virus (CAR-V) As used herein, chimeric antigen receptor viruses, or CAR-Vs, are viral particles whose tropism has been altered by the surface presentation of a synthetic protein (chimeric antigen receptor) capable of binding to a cell surface ligand or antigen. In the example presented, CAR-Vs can be obtained from AAV1 Asp590Cys virions by coupling 0.1 μg to 1 μg of capsids overnight in a 500 μL reaction at room temperature with a synthetic receptor in the form of a 15 μM concentration of sfGFP-PKD2 adaptor molecule capable of binding to a synthetic surface antigen in the form of a modified surface-expressed anti-sfGFP nanobody.

[0168] Virus-like particles (VLPs) Virus-like particles (VLPs) are molecules that resemble viruses, but are non-infectious because they do not contain viral genetic material. They can occur naturally or can be synthesized by the individual expression of viral structural proteins that then self-assemble into virus-like structures (Zeltins, Molecular Biotechnology volume 53, pages 92-107 (2013)). Combinations of structural capsid proteins from different viruses can be used to create recombinant VLPs. Both in vivo assembly (i.e., assembly within cells such as E. coli via recombinant co-expression of multiple proteins) and in vitro assembly (i.e., self-assembly of proteins in a reaction vessel using stoichiometric amounts of purified proteins) can be used to form virus-like particles.

[0169] VLPs have been produced from components of a wide variety of viral families, including Parvoviridae (e.g., adeno-associated virus), Retroviridae (e.g., HIV), Flaviviridae (e.g., Hepatitis C virus), Paramyxoviridae (e.g., Nipah), and bacteriophages (e.g., Qβ, AP205). VLPs can be produced in multiple cell culture systems, including bacteria, mammalian cell lines, insect cell lines, yeast, and plant cells.

[0170] VLPs can also refer to structures generated by some naturally occurring LTR retrotransposons. These are defective, immature virions that may contain genetic material but are generally non-infectious because they lack a functional viral envelope.

[0171] Pharmaceutical Compositions of Agents Another aspect of the invention also relates to a pharmaceutical or diagnostic aid comprising an isolated polypeptide according to the invention, an AAV adapter molecule, a viral adapter molecule, a virus particle, a virus like particle, a nucleic acid or a cell, and optionally suitable excipients and additives, such as, for example, saline, stabilizers or proteinase inhibitors.

[0172] kit In some embodiments of the invention, an agent described herein (e.g., an isolated polypeptide, an AAV adapter molecule, a viral adapter molecule, a viral particle, a virus-like particle, a nucleic acid, a cell, or a pharmaceutical composition) can be provided in a kit. In some examples, the kit includes (a) a container that includes an agent described herein, and optionally (b) informational material. The informational material can be explanatory, instructional, marketing, or other material relating to the methods described herein and / or the use of the agent, e.g., for therapeutic benefit.

[0173] The informational material of the kit is not limited in format. In some examples, the informational material can include information regarding the manufacture of the therapeutic agent, the molecular weight of the therapeutic agent, concentration, expiration date, batch or manufacturing site information, etc. In other aspects, the informational material relates to, for example, how to administer the therapeutic agent in an appropriate amount, mode or mode of administration (e.g., a dosage, dosage form or mode of administration described herein).

[0174] In some cases, the informational material, e.g., instructions, is provided in printed matter, e.g., printed text, drawings, and / or photographs, e.g., labels or printed sheets. The informational material can also be provided in other formats, e.g., Braille, computer readable material, video recordings, or audio recordings. In other examples, the informational material of the kit is a contact address, e.g., a physical address, email address, website, or phone number, where a user of the kit can obtain essential information regarding the therapeutic agent therein and / or its use in the methods described herein. The informational material can also be provided in any combination of formats.

[0175] In addition to the therapeutic agent, the kit can include other components, such as a solvent or buffer, a stabilizer or a preservative. The kit can also include additional agents, such as a second or third agent, such as other therapeutic agents. The components can be provided in any form, such as liquid, dried or lyophilized form. The components can be substantially pure (but they can be combined together or delivered separately from each other) and / or sterile. If the components are provided in a liquid solution, the liquid solution can be an aqueous solution, such as a sterile aqueous solution. If the components are provided as a dry form, reconstitution is generally by the addition of a suitable solvent. A solvent, such as sterile water or a buffer, can optionally be provided in the kit.

[0176] The kit may include one or more containers for the therapeutic agent or other agent. In some cases, the kit includes separate containers, dividers, or compartments for the therapeutic agent and the informational material. For example, the therapeutic agent may be in a bottle, vial, or syringe, and the informational material may be in a plastic sleeve or packet. In other aspects, the separate elements of the kit are included in a single undivided container. For example, the therapeutic agent may be in a bottle, vial, or syringe with the informational material attached thereto in the form of a label. In some cases, the kit may include multiple (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., dosage forms described herein) of the therapeutic agent. The containers may include unit doses, e.g., units containing the therapeutic agent. For example, the kit may include multiple syringes, ampoules, foil packets, blister packs, or medical devices, each containing, e.g., unit doses. The containers of the kit may be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation), and / or light-tight.

[0177] The kit can optionally include a device suitable for administration of the therapeutic agent, e.g., a syringe or other suitable delivery device. The device can be provided pre-filled, e.g., with a unit dose of the therapeutic agent, or it can be empty but suitable for loading.

[0178] Combination therapy In some embodiments, the invention features a composition (e.g., one or more compositions, formulations, or dosage forms) or pharmaceutical combination comprising an isolated polypeptide capable of binding to a viral capsid, or a viral adapter molecule comprising an isolated polypeptide capable of binding to a viral capsid and a ligand, in accordance with the invention, and a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from one or more agents selected from the list consisting of analgesics, anesthetics, antibacterials, anticonvulsants, anti-dementia drugs, antidepressants, antidotes, antiemetics, antifungals, anti-gout drugs, anti-inflammatory drugs, antimigraine drugs, antimyasthenic drugs, antimycobacterial drugs, antineoplastic drugs, antiparasitic drugs, antiparkinsonian drugs, antipsychotic drugs, antispasmodics, antiviral drugs, anxiolytic drugs, bipolar drugs, blood glucose regulating drugs, cardiovascular drugs, central nervous system acting drugs, dental and oral drugs, dermatological drugs, enzyme supplements / modifiers, gastrointestinal drugs, genitourinary drugs, hormonal drugs, inflammatory bowel disease drugs, bone metabolic disease drugs, ophthalmic drugs, otic drugs, airway drugs, sedatives / hypnotics, skeletal muscle relaxants, therapeutic nutrients / minerals, and electrolytes.

[0179] In some embodiments, the composition comprises a pharma- ceutically acceptable carrier. In some embodiments, the isolated polypeptide capable of binding to a viral capsid, or the viral adapter molecule comprising an isolated polypeptide capable of binding to a viral capsid and a ligand, and the second agent can be present in a single composition or as two or more different compositions. The isolated polypeptide capable of binding to a viral capsid, or the viral adapter molecule comprising an isolated polypeptide capable of binding to a viral capsid and a ligand, and the second agent can be administered via the same or different administration routes. The isolated polypeptide capable of binding to a viral capsid, or the viral adapter molecule comprising an isolated polypeptide capable of binding to a viral capsid and a ligand, and the second agent can be administered simultaneously or sequentially. In some embodiments, the pharmaceutical combination comprises the isolated polypeptide capable of binding to a viral capsid, or the viral adapter molecule comprising an isolated polypeptide capable of binding to a viral capsid and a ligand, and the second agent, separately or together.

[0180] Route of administration The agent or pharmaceutical composition can be administered by a variety of routes, including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, by inhalation, buccal administration, intrapleurally, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intrathecally, and / or intraarticularly, or combinations thereof. In some embodiments, the agent or pharmaceutical composition is administered orally.

[0181] Transduction efficiency In some embodiments, viral particles bound to isolated polypeptides capable of binding to viral capsids of the invention exhibit at least a 10% reduction in transduction efficiency compared to appropriate control wild-type viral particles. In some embodiments, viral particles bound to isolated polypeptides capable of binding to viral capsids of the invention exhibit at least a 20% reduction in transduction efficiency compared to appropriate control wild-type viral particles. In some embodiments, viral particles bound to isolated polypeptides capable of binding to viral capsids of the invention exhibit at least a 30% reduction in transduction efficiency compared to appropriate control wild-type viral particles. In some embodiments, viral particles bound to isolated polypeptides capable of binding to viral capsids of the invention exhibit at least a 40% reduction in transduction efficiency compared to appropriate control wild-type viral particles. In some embodiments, viral particles bound to isolated polypeptides capable of binding to viral capsids of the invention exhibit at least a 50% reduction in transduction efficiency compared to appropriate control wild-type viral particles. In some embodiments, viral particles bound to isolated polypeptides capable of binding to viral capsids of the invention exhibit at least a 60% reduction in transduction efficiency compared to appropriate control wild-type viral particles. In some embodiments, viral particles bound to isolated polypeptides capable of binding to viral capsids of the present invention exhibit at least a 70% reduction in transduction efficiency compared to appropriate control wild-type viral particles. In some embodiments, viral particles bound to isolated polypeptides capable of binding to viral capsids of the present invention exhibit at least an 80% reduction in transduction efficiency compared to appropriate control wild-type viral particles. In some embodiments, viral particles bound to isolated polypeptides capable of binding to viral capsids of the present invention exhibit at least a 90% reduction in transduction efficiency compared to appropriate control wild-type viral particles. In some embodiments, viral particles bound to isolated polypeptides capable of binding to viral capsids of the present invention exhibit at least a 95% reduction in transduction efficiency compared to appropriate control wild-type viral particles.In some embodiments, viral particles bound to an isolated polypeptide capable of binding to a viral capsid of the invention exhibit at least a 99% reduction in transduction efficiency compared to a suitable control wild-type viral particle. In some embodiments, transduction of control cells by viral particles comprising a recombinant viral particle molecule described herein is ineffective, e.g., undetectable, e.g., by a method that measures expression of a nucleotide of interest, such as a reporter assay.

[0182] Conversely, viral particles bound to viral adapter molecules comprising a ligand are capable of infecting target cells, e.g., partially or completely restored in their ability to target and bind reference cells that are otherwise permissive for transduction, as compared to the ability of a reference viral capsid, e.g., a capsid comprising a reference viral capsid protein, e.g., a wild-type control viral capsid protein. In some embodiments, viral particles bound to viral adapter molecules comprising an isolated polypeptide capable of binding to a viral capsid of the invention and a ligand exhibit a transduction efficiency that is at least 10% higher than that of a suitable control wild-type viral capsid. In some embodiments, viral particles bound to viral adapter molecules comprising an isolated polypeptide capable of binding to a viral capsid of the invention and a ligand exhibit a transduction efficiency that is at least 20% higher than that of a suitable control wild-type viral capsid. In some embodiments, viral particles bound to viral adapter molecules comprising an isolated polypeptide capable of binding to a viral capsid of the invention and a ligand exhibit a transduction efficiency that is at least 30% higher than that of a suitable control wild-type viral capsid. In some embodiments, viral particles bound to viral adapter molecules comprising an isolated polypeptide capable of binding to a viral capsid of the invention and a ligand exhibit a transduction efficiency that is at least 40% higher than that of a suitable control wild-type viral capsid. In some embodiments, viral particles bound to viral adapter molecules comprising an isolated polypeptide capable of binding to a viral capsid of the invention and a ligand exhibit a transduction efficiency that is at least 50% higher than that of a suitable control wild-type viral capsid. In some embodiments, viral particles bound to viral adapter molecules comprising an isolated polypeptide capable of binding to a viral capsid of the invention and a ligand exhibit a transduction efficiency that is at least 60% higher than that of a suitable control wild-type viral capsid.In some embodiments, viral particles bound to viral adapter molecules comprising an isolated polypeptide capable of binding to a viral capsid of the present invention and a ligand exhibit a transduction efficiency that is at least 70% higher than that of a suitable control wild-type viral capsid. In some embodiments, viral particles bound to viral adapter molecules comprising an isolated polypeptide capable of binding to a viral capsid of the present invention and a ligand exhibit a transduction efficiency that is at least 80% higher than that of a suitable control wild-type viral capsid. In some embodiments, viral particles bound to viral adapter molecules comprising an isolated polypeptide capable of binding to a viral capsid of the present invention and a ligand exhibit a transduction efficiency that is at least 90% higher than that of a suitable control wild-type viral capsid. In some embodiments, viral particles bound to viral adapter molecules comprising an isolated polypeptide capable of binding to a viral capsid of the present invention and a ligand exhibit a transduction efficiency that is at least 100% higher than that of a suitable control wild-type viral capsid.

[0183] In some embodiments, viral particles covalently linked to an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of covalently linking to a viral capsid of the invention exhibit at least a 10% reduction in transduction efficiency compared to a suitable control wild-type viral particle. In some embodiments, viral particles covalently linked to an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of covalently linking to a viral capsid of the invention exhibit at least a 20% reduction in transduction efficiency compared to a suitable control wild-type viral particle. In some embodiments, viral particles covalently linked to an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of covalently linking to a viral capsid of the invention exhibit at least a 30% reduction in transduction efficiency compared to a suitable control wild-type viral particle. In some embodiments, viral particles covalently linked to an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of covalently linking to a viral capsid of the invention exhibit at least a 40% reduction in transduction efficiency compared to a suitable control wild-type viral particle. In some embodiments, viral particles covalently linked to an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of covalently linking to a viral capsid of the invention exhibit at least a 50% reduction in transduction efficiency compared to a suitable control wild-type viral particle. In some embodiments, viral particles covalently linked to an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of covalently linking to a viral capsid of the invention exhibit at least a 60% reduction in transduction efficiency compared to a suitable control wild-type viral particle. In some embodiments, viral particles covalently linked to an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of covalently linking to a viral capsid of the invention exhibit at least a 70% reduction in transduction efficiency compared to a suitable control wild-type viral particle. In some embodiments, viral particles covalently linked to an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of covalently linking to a viral capsid of the invention exhibit at least an 80% reduction in transduction efficiency compared to a suitable control wild-type viral particle.In some embodiments, viral particles covalently linked to an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of covalently linking to a viral capsid of the invention exhibit at least a 90% reduction in transduction efficiency compared to a suitable control wild-type viral particle. In some embodiments, viral particles covalently linked to an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of covalently linking to a viral capsid of the invention exhibit at least a 95% reduction in transduction efficiency compared to a suitable control wild-type viral particle. In some embodiments, viral particles covalently linked to an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of covalently linking to a viral capsid of the invention exhibit at least a 99% reduction in transduction efficiency compared to a suitable control wild-type viral particle. In some embodiments, transduction of a control cell by a viral particle comprising a recombinant viral particle molecule described herein is ineffective, e.g., undetectable, e.g., by a method that measures expression of a nucleotide of interest, e.g., a reporter assay.

[0184] Conversely, viral particles covalently linked to a viral adapter molecule comprising a ligand are capable of infecting target cells, e.g., partially or completely regaining the ability to target and bind reference cells that are otherwise permissive for transduction, as compared to the ability of a reference viral capsid, e.g., a capsid comprising a reference viral capsid protein, e.g., a wild-type control viral capsid protein. In some embodiments, viral particles covalently linked to a viral adapter molecule comprising an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of covalently binding to a viral capsid of the invention and a ligand exhibit a transduction efficiency that is at least 10% higher than that of a suitable control wild-type viral capsid. In some embodiments, viral particles covalently linked to a viral adapter molecule comprising an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of covalently binding to a viral capsid of the invention and a ligand exhibit a transduction efficiency that is at least 20% higher than that of a suitable control wild-type viral capsid. In some embodiments, viral particles covalently linked to viral adapter molecules comprising an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of being covalently linked to a viral capsid of the invention and a ligand exhibit a transduction efficiency that is at least 30% higher than that of a suitable control wild-type viral capsid. In some embodiments, viral particles covalently linked to viral adapter molecules comprising an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of being covalently linked to a viral capsid of the invention and a ligand exhibit a transduction efficiency that is at least 40% higher than that of a suitable control wild-type viral capsid. In some embodiments, viral particles covalently linked to viral adapter molecules comprising an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of being covalently linked to a viral capsid of the invention and a ligand exhibit a transduction efficiency that is at least 50% higher than that of a suitable control wild-type viral capsid.In some embodiments, viral particles covalently linked to viral adapter molecules comprising an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of being covalently linked to a viral capsid of the invention and a ligand exhibit a transduction efficiency that is at least 60% higher than that of a suitable control wild-type viral capsid. In some embodiments, viral particles covalently linked to viral adapter molecules comprising an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of being covalently linked to a viral capsid of the invention and a ligand exhibit a transduction efficiency that is at least 70% higher than that of a suitable control wild-type viral capsid. In some embodiments, viral particles covalently linked to viral adapter molecules comprising an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of being covalently linked to a viral capsid of the invention and a ligand exhibit a transduction efficiency that is at least 80% higher than that of a suitable control wild-type viral capsid. In some embodiments, viral particles covalently linked to a viral adapter molecule comprising an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of being covalently linked to a viral capsid of the invention and a ligand exhibit a transduction efficiency that is at least 90% greater than the transduction efficiency of a suitable control wild-type viral capsid. In some embodiments, viral particles covalently linked to a viral adapter molecule comprising an isolated polypeptide comprising one or more heterologous cysteine ​​residues capable of being covalently linked to a viral capsid of the invention and a ligand exhibit a transduction efficiency that is at least 100% greater than the transduction efficiency of a suitable control wild-type viral capsid.

[0185] Identification of suitable residues for mutation in capsid and binder proteins To identify potential residue pairs to be mutated to cysteines capable of forming disulfide bonds, structures of viruses bound to receptors or ligands were searched from public databases such as the PDB (e.g., AAV1 structure complexed with the PKD2 domain of AAVR, PBD entry 6jcq). By inspecting residues that line the interface between the two partners, pairs of residues (i) on the virus particle and (ii) on the receptor or ligand were identified as follows: When mutated in silico to a cysteine ​​residue using software such as Pymol or Chimera, the side chain does not induce a major steric clash that would disrupt the interface (i.e., the side chain of the cysteine ​​residue does not occupy a region of space already occupied by another residue); The distance between the beta carbons of the cysteines substituting residues (i) and (ii) is less than 5.5 Å; The side chains of cysteine ​​residues (i) and (ii) have an orientation obtained by rotation of the side chains about C(α)-C(β) such that the distance between the gamma sulfur atoms of the side chains is less than 2.5 Å; The angles formed by the atoms C(β)1-S(γ)1-S(γ)2, S(γ)1-S(γ)2-C(β) do not differ significantly (more than 40%) from the optimal angles observed in naturally occurring disulfide bonds, as shown in the literature (e.g., Dombkowski et al., Protein disulphide engineering, FEBS Letters Volume 588, Issue 2, 21 January 2014, Pages 206-212).

[0186] To take into account that protein flexibility is not accurately represented by static structures, arbitrary deviations from the reference parameters were allowed for surface residues that exhibit minimal structural constraints.

[0187] The adaptor protein (A) may be derived from a viral binding protein (B) that is capable of interacting with a viral particle (V) as follows: 1) the binding protein (B) is capable of binding to the virus (V) with an affinity of Kd<100 μM, as measured by standard methods in the art; 2) there is at least one pair of residues X and Y, where X is part of binding protein B and Y is part of virus particle V, and their respective β-carbons are spaced less than 5.5 Å apart when binding protein B is complexed with virus particle V; 3) Residues X and Y can be mutated to cysteines such that the cysteine ​​side chains do not induce major steric clashes that would disrupt the interface (i.e., the side chains of the cysteine ​​residues do not occupy regions of space where another residue already resides); 4) the side chains of residues X and Y, after mutation to cysteine, have an orientation obtained by rotation of the side chains about C(α)-C(β) such that the distance between the sulfur atoms at the gamma positions of the side chains is less than 2.5 Å; and / or 5) The angles formed by the atoms C(β)1-S(γ)1-S(γ)2 and S(γ)1-S(γ)2-C(β) do not differ significantly (e.g., by 40% or less) from the optimal angles observed in naturally occurring disulfide bonds as shown in the literature (e.g., Dombkowski et al., Protein disulphide engineering, FEBS Letters Volume 588, Issue 2, 21 January 2014, Pages 206-212).

[0188] If one or more of these criteria are met, an adapter protein (A) derived from a binding protein (B) by mutation of X to cysteine, and a modified virus (V') derived from V by mutation of Y to cysteine, are candidate adapter protein-virus pairs that, according to the present invention, may be expected to covalently bind to each other with a reasonable probability.

[0189] In some embodiments of the invention, a candidate adaptor protein-virus pair may be selected based on one or more of the above criteria. In some embodiments of the invention, a candidate adaptor protein-virus pair meets at least one requirement in the above list. In some embodiments of the invention, a candidate adaptor protein-virus pair meets at least two requirements in the above list. In some embodiments of the invention, a candidate adaptor protein-virus pair meets at least three requirements in the above list. In some embodiments of the invention, a candidate adaptor protein-virus pair meets at least four requirements in the above list. In some embodiments of the invention, a candidate adaptor protein-virus pair meets all five requirements in the above list.

[0190] In some embodiments of the invention, residues in a viral capsid and / or in an isolated polypeptide suitable for binding to a viral capsid may be selected for mutation to a cysteine ​​residue by using the methods described herein. Means for mutating amino acid residues to cysteine ​​residues are known to those of skill in the art, for example, by genetic or chemical means.

[0191] The present invention will be further described in the following examples. It should be understood that these examples, although showing embodiments of the present invention, are provided for illustrative purposes only. From the above discussion and these examples, those skilled in the art can ascertain the essential features of the present invention, and can make various changes and modifications to adapt the present invention to various applications and conditions without departing from the spirit and scope of the present invention. Thus, in addition to those shown and described herein, various modifications of the present invention will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to be included within the scope of the appended claims. EXAMPLES

[0192] Example 1 - Disulfide bond formation between residue Cys590 of the AAV1 (Asp590Cys) mutant and Cys425 of the sfGFP-PKD2 (Ser425Cys) adapter fusion protein See top gel of Figure 20. 2 μL of either wild type (AAV1WT in legend) or Asp590Cys (AAV1Cys in legend) virus was mixed with 1.5 μL of 100 μM stock of either wt (PKD2WT in legend) or Ser425Cys (PKD2Cys in legend) sfGFP-PKD2 fusion protein in the combinations indicated in the figure in a final reaction volume of 10 μL (20 mM Tris HCl pH 8, 100 mM NaCl). Reactions were incubated at room temperature for 1 hour and then run on a 4%-12% NuPAGE at 200V for 1 hour. Appropriate individual components were also run as controls. No reducing agent was included in the gel loading dye.

[0193] See bottom gel in Figure 20. 4 μL of either wild type (AAV1WT in legend) or Asp590Cys (AAV1Cys in legend) virus stock was mixed with 3 μL of 100 μM stock of either wt (PKD2WT in legend) or Ser425Cys (PKD2Cys in legend) sfGFP-PKD2 fusion protein in the combinations shown in the figure in a final reaction volume of 20 μL (20 mM Tris HCl pH 8, 100 mM NaCl). Reactions were incubated at room temperature for 1 h, then samples were split in two. Half of the reaction was mixed with non-reducing loading dye (left lane) and half of the reaction was mixed with reducing loading dye containing βME. Samples were run on a 4%-12% NuPAGE at 200V for 1 h. Appropriate individual components were also run as controls.

[0194] The gel showed the formation of bands corresponding to sfGFP-PKD2+AAV1, sfGFP-PKD2+VP2, and sfGFP-PKD2+VP3 migrating between 150 and 100 kDa, which could only be observed if the AAV1 capsid and sfGFP-PKD2 adapter protein both contain cysteine ​​residues, which are destabilized in the presence of reducing agents, consistent with a disulfide-linked heterodimer (Figure 20).

[0195] Example 2 - Purification of covalently linked AAV1 CAR-V complexes AAV1(Asp590Cys) virus was incubated overnight in PBS in the presence of 50 μM sfGFP-PKD2(Ser425Cys) fusion protein, where PKD2(Ser425Cys) refers to the PKD2 domain of the AAVR(Ser425Cys) mutant (fusion protein 50 μM).

[0196] After incubation, the reaction was subjected to ultracentrifugation using an iodixanol step gradient to separate the viral particles from the excess unreacted sfGFP-PKD2(Ser425Cys) fusion polypeptide. After ultracentrifugation, the particles that partitioned into the 40% iodixanol fraction were collected, buffer exchanged, and run on a 4%-12% SDS PAGE.

[0197] The gel shows that the gradient was effective in purifying and removing most of the unbound sfGFP-PKD2(Ser425Cys) adaptor protein (compare the bands between 37 kDa and 50 kDa and the bands between 75 kDa and 100 kDa). Furthermore, the gel shows that we were able to recover stably associated heterodimers of the PKD2 mutant fusion protein with the viral capsid proteins VP1, VP2, and VP3 (bands above 100 kDa). This demonstrated the successful formation of covalently linked AAV1Cys<>sfGFP particles (Figure 21).

[0198] Example 3 -Targeting of AAV1 covalently linked to an adaptor HEK293T cells were seeded in 24-well plates. When the cells reached approximately 60% cell occupancy, two wells were transfected with a plasmid expressing the chimeric sfGFP receptor in the form of a membrane-bound anti-sfGFP nanobody (right column), while the other wells were not transfected (left column). The cells were incubated at 37°C for 24 h to allow expression of the chimeric receptor in the transfected cells. Subsequently, the cells were infected with either AAV1(Asp590Cys)<>sfGFP (CAR-V), or AAV1(Asp590Cys). Both viruses carried a gene encoding the red fluorescent protein mScarlet. Images show phase contrast (grey) or mScarlet (red).

[0199] The data show that AAV1(Asp590Cys)<>sfGFP CAR-V (described in Example 1) has reduced tropism for HEK293T cells that do not express the chimeric sfGFP receptor, but infectivity is restored when the target cells express the chimeric receptor. The data further show that HEK293T cells are highly susceptible to infection by AAV1(Asp590Cys) without the adapter protein. These data demonstrate that the adapter protein reduces the infectivity of AAV1 for cells that do not express the corresponding chimeric receptor, but restores infectivity for cells that express the corresponding chimeric receptor (Figure 22).

[0200] Example 4 - Use of small molecule ligands Adapter proteins can be generated by obtaining an isolated polypeptide that contains one or more cysteine ​​residues capable of covalently binding to a viral capsid and reacting it with a targeting molecule that has high specificity for a target cell. This reaction can be performed using NHS or maleimide chemistry, or by incorporating a non-standard amino acid with a reactive group (such as azide or alkyne or tetrazine or transcyclooctene) at a specific position within the viral capsid binding molecule.

[0201] It can also be achieved by fusing an isolated polypeptide containing one or more cysteine ​​residues capable of covalently binding to a viral capsid with a specific peptide sequence capable of binding to a targeting molecule, such as a split intein, SpyTag, tetracysteine ​​FCM motif (FLNCCPGCCMEP) or ybbR motif (TVLDSLEFIASKLA). In such cases, the targeting molecule is designed to contain opposite reactive elements, such as opposite a split intein; SpyCatcher; a fluorescein arsenic hairpin, or coenzyme A, respectively.

[0202] The targeting molecule can then be selected to bind to a specific receptor.In some cases, the targeting molecule can be a protein, for example, an antibody against a receptor expressed on a target cell type.In the case of an antibody, the antibody can be modified with an alkyne group and then reacted with the azide group on the viral capsid molecule via click chemistry.

[0203] In some cases, the targeting molecule may be a small molecule. For example, by connecting a neurotransmitter such as serotonin or dopamine to the viral capsid, it is possible to create an adapter molecule that, when combined with the viral particle, produces a viral particle specific to cells that express serotonin receptors or dopamine receptors, respectively. This approach (i.e., creating an adapter protein that includes an isolated polypeptide that includes one or more cysteine ​​residues that can be covalently bound to the viral capsid and a reactive group, and then reacting it with the targeting molecule) is particularly useful when the targeting molecule cannot be expressed as a fusion with the isolated polypeptide that includes one or more cysteine ​​residues that can be covalently bound to the viral capsid (e.g., when the targeting molecule is not a single-chain protein or is a small molecule), or when the targeting molecule cannot be efficiently purified by fusing to the isolated polypeptide that includes one or more cysteine ​​residues that can be covalently bound to the viral capsid.

[0204] Example 5 - Use of various viruses Adenovirus The present invention can be practiced using adenoviruses for delivery of payloads that are too large for AAV. For example, adenoviruses (AdVs) are used in the COVID-19 vaccine from AstraZeneca to deliver the full-length spike protein, and adenoviruses are also being tested in some other gene therapies. As with AAVs, cell type-specific AdVs allow for more specific, more efficient, and less toxic gene therapy. Some adenoviruses are specific for the Coxsackievirus and Adenovirus Receptor (CXADR). It is therefore believed that an adapter molecule comprising a fusion of CXADR with a ligand can be used to retarget adenoviruses to specific cell types. In some embodiments of the present invention, the adapter molecule comprises a fusion of at least a portion of CXADR with a ligand. Some adenoviruses are specific for CD46. It is therefore believed that an adapter molecule comprising a fusion of a portion or the full length of CD46 with a ligand can be used to retarget adenoviruses to specific cell types. In some embodiments of the present invention, the adapter molecule comprises a fusion of at least a portion of CD46 with a ligand. It is contemplated that the present invention can be practiced using adenovirus-CXADR CAR-V or adenovirus-CD46 CAR-V, or AAV-A20 antibody CAR-V. In some embodiments of the present invention, the CXADR protein may comprise a portion of SEQ ID NO:20. In some embodiments of the present invention, the CXADR protein may comprise the full length sequence of SEQ ID NO:20.

[0205] Lentivirus The present invention can be implemented using lentiviruses to deliver payloads for genome integration. For example, lentiviruses are often used for in vitro cell modification, as they can integrate their payloads into target cells, allowing the creation of stable cell lines with specific gene insertions. It is believed that cell type-specific lentiviral vectors can be used to increase the specificity and efficiency of gene editing steps in cell therapy. Since lentiviruses are highly specific for the CD4 receptor, an adapter molecule consisting of a portion of the CD4 receptor fused to a ligand may facilitate retargeting of lentiviral vectors to specific cell types.

[0206] Combination of residues on the virus with binders For the combination of human adenovirus D37 fiber protein with human (h)CxAdR (crystal structure accession number PDB 2j12, see Seiradake et al., Structural and Mutational Analysis of Human (h) Ad37 and Canine Adenovirus 2 Fiber Heads in Complex with the D1 Domain of Coxsackie and Adenovirus Receptor, JBC, Volume 281, Issue 44, 3 November 2006, Pages 33704-33716), a covalently linked adenovirus (hAdV) CAR-V can be obtained by using hAdV-D37 containing the following mutations in combination with an adapter fusion protein containing at least a portion of hCxAdR, where the hCxAdR contains the following corresponding point mutations [numbering shown in the PDB file]: Val226Cys on hAdV-D37 and Val70Cys in hCxAdR Ser193Cys in hAdV-D37 and Glu56Cys in hCxAdR Ser274Cys in hAdV-D37 and Val128Cys in hCxAdR

[0207] In some embodiments of the invention, an isolated polypeptide or adapter protein of the invention may comprise at least a portion of hCxAdR in combination with hAdV-D37, which comprises one or more of the above point mutations. In some embodiments of the invention, an isolated polypeptide or adapter protein of the invention may comprise at least a portion of hCxAdR in combination with human adenovirus 5 (e.g., comprising hAdV-D37), where hCxAdR comprises a Val70Cys mutation and AdV-5 comprises a Val441Cys mutation.

[0208] Other adenoviruses may not bind CxAdR, but may bind other cellular receptors, including CD46 (see Seiradake et al., Structural and Mutational Analysis of Human Ad37 and Canine Adenovirus 2 Fiber Heads in Complex with the D1 Domain of Coxsackie and Adenovirus Receptor, JBC, Volume 281, Issue 44, 3 November 2006, Pages 33704-33716). Thus, the invention can be practiced using a combination of an adenovirus capsid or virus particle with another suitable adenovirus binding protein, such as CD46.

[0209] The structure of the fiber protein of hAdV11p complexed with hCD46 (see PDB entry 2O39, Persson et al., Adenovirus type 11 binding alters the conformation of its receptor CD46, Nature Structural & Molecular Biology volume 14, pages164-166 (2007)) shows that covalently linked hAdV CAR-Vs can be obtained by using hAdV-11p containing the following point mutations, paired with an adapter fusion protein containing a fragment of hCD46, where hCD46 contains the corresponding point mutations: Asn283Cys on hAdV-11p and Thr42Cys in hCD46

[0210] In some embodiments of the invention, an isolated polypeptide or adapter protein of the invention may comprise at least a portion of CD46 in combination with hAdV-11p comprising a point mutation as described above.

[0211] AAV2 can bind to the A20 neutralizing antibody (see PDB entry 3j1s (McCraw et al., Structure of adeno-associated virus-2 in complex with neutralizing monoclonal antibody, Volume 431, Issues 1-2, 15-30 September 2012, Pages 40-49).

[0212] An AAV2 CAR-V covalently linked to the A20 antibody can be obtained by using an AAV2 containing the following mutations paired with an adapter fusion protein containing a portion of the A20 neutralizing antibody (nAb), where the A20 nAb contains the following corresponding mutations: AAV2 (Ser264Cys) bound to the A20 antibody (heavy chain Tyr102Cys, which is located in CDR3 of A20) AAV2 (Val708Cys) bound to the A20 antibody (heavy chain Ser56Cys, which is located in CDR2 of A20) AAV2 (Asn717Cys) bound to the A20 antibody (light chain Ile93Cys, which is located in CDR3 of A20)

[0213] In some embodiments of the invention, an isolated polypeptide or adaptor protein of the invention may comprise at least a portion of an A20 antibody in combination with an AAV2 comprising one or more of the point mutations described above. In some embodiments of the invention, the AAV2 comprises a sequence having at least 85%, 90%, 95%, 99% or 100% identity to SEQ ID NO:8, and the portion of the A20 antibody comprises one or more sequences having at least 85%, 90%, 95%, 99% or 100% identity to any one of SEQ ID NOs:21-28.

[0214] AAV5 can bind to the PKD1 domain of AAVR. Covalent binding between AAV5 and the PKD1 domain of AAVR can be achieved by using AAV5 containing one or more of the following mutations in a pair with an adapter fusion protein containing at least a portion of the PKD1 domain of AAVR, where the PKD1 domain of AAVR contains the following corresponding mutations: mutations Gln697Cys on AAV5 and Ser356Cys on PKD1; Mutation Leu543Cys on AAV5 and Leu376Cys on PKD1

[0215] In some embodiments of the invention, an isolated polypeptide or adaptor protein of the invention may comprise at least a portion of the PKD1 domain of AAVR in combination with an AAV5 comprising one or more of the point mutations described above. In some embodiments of the invention, the AAV5 comprises a sequence having at least 85%, 90%, 95%, 99% or 100% identity to SEQ ID NO:11, and the PKD1 domain of AAVR comprises a sequence having at least 85%, 90%, 95%, 99% or 100% identity to SEQ ID NO:2.

[0216] Human adenovirus B3 (through its fiber protein, Uniprot sequence P04501) can bind to Desmoglein 2 (DSG2, Uniprot sequence ID: Q14126). A covalent bond between human adenovirus B3 and Desmoglein 2 can be obtained by using human adenovirus B3 containing one or more of the following mutations paired with an adapter fusion protein containing at least a portion of Desmoglein 2, where Desmoglein 2 contains the corresponding mutations: mutation Tyr147Cys on fiber protein and mutation His175Cys on DSG2; mutation Asn188Cys on fiber protein and mutation Thr226Cys on DSG2; mutation Asn192Cys on fiber protein and mutation Ala174Cys on DSG2; mutation Asp261Cys on fiber protein and mutation Lys362Cys on DSG2; Mutation Phe265Cys on fiber protein and mutation Ser366Cys on DSG2

[0217] In some embodiments of the invention, an isolated polypeptide or adapter protein of the invention may comprise at least a portion of Desmoglein 2 in combination with human adenovirus B3 comprising one or more of the above point mutations. In some embodiments of the invention, human adenovirus B3 comprises a sequence having at least 85%, 90%, 95%, 99% or 100% identity to SEQ ID NO: 30, and Desmoglein 2 comprises a sequence having at least 85%, 90%, 95%, 99% or 100% identity to SEQ ID NO: 31. In some embodiments, an isolated polypeptide or adapter protein of the invention may comprise at least a portion of Desmoglein 2 in combination with human adenovirus B3, wherein human adenovirus B3 comprises an Asn192Cys mutation on the fiber protein, and wherein the portion of Desmoglein 2 comprises an Ala174Cys mutation.

[0218] Example 6 - sfGFP-PKD2 fusion protein In a 24-well plate, HEK293FT cells were seeded in well A, well B, and well C and waited until they reached 70%-80% cell occupancy. Cells were then transfected with a plasmid in all three wells to allow expression of membrane-anchored anti-sfGFP nanobodies. 24 h after transfection, 5 μL of sfGFP-PKD2 fusion protein (~2 mg / mL) was added to well A3 and incubated for 30 min to allow capture of the fusion protein by the nanobody, after which unbound fusion protein was washed away and cell medium was replaced.

[0219] Well A (Figure 1A) was infected with AAV2 wild type carrying the mScarlet gene. Well B and well C (Figure 1B and Figure 1C) were infected with AAV2 virus Arg585Ala Arg588Ala carrying the mScarlet gene. 24 hours after infection, cells were imaged to visualize mScarlet. The images show that sfGFP-PKD2 fusion protein enhances the infectivity of AAV2 Arg585Ala Arg588Ala in cells expressing synthetic membrane-anchored anti-sfGFP nanobody (Figure 1).

[0220] Example 7 - SKBR3 cells with DARPin-PKD2 fusion protein Two 35 mm dishes were seeded with SKBR3 cells. When the cells reached a cell coverage of approximately 60%, the medium was replaced with 1 mL of DMEM + 10% FBS and 100 μL of DARPin-PKD2 (2 μM) (the DARPin ligand was a high affinity binder for Her2) was added to one of the dishes, the other dish was kept as a control. The wells were incubated for 30 min to allow the DARPin domain of the DARPin-PKD2 fusion protein to bind to the Her2 receptor endogenously expressed by the SKBR3 cell line, after which the medium was removed, the cells were washed with PBS and fresh medium was added.

[0221] Both dishes were then infected with AAV2 wild type carrying the mScarlet gene. After 24 hours, cells were fixed for immunostaining (DNA = blue, Her2 = green, mScarlet = red, phase contrast = grey). The images show that the DARPin-PKD2 fusion protein significantly enhances the infectivity of AAV2 wild type in SKBR3 cells (Figure 2).

[0222] Example 8 - mScarlet-PKD2 fusion protein blocks AAV2 infectivity HEK293FT cells were seeded on Matrigel-coated 24-well plates and cultured until the cells reached approximately 90% cell occupancy. mScarlet-PKD2 fusion protein (at concentrations shown in Figure 3) was mixed with AAV2 wild type carrying the GFP gene under a strong promoter (at concentrations shown in Figure 3), and the amount of virus was fixed with varying concentrations of fusion protein in each sample. After 10 min of incubation, cells were infected with the mScarlet-PKD2+AAV2 mixture. After 24 h, cells were imaged for GFP expression as a marker of infection.

[0223] The data showed that the presence of mScarlet-PKD2 fusion protein in solution inhibited AAV2 wild-type infectivity in a concentration-dependent manner, suggesting that the PKD2 domain of the fusion protein effectively interacts with the virus to compete with cellular AAVR for binding and prevent infection (Fig. 3 ).

[0224] Example 9 - Binding of PKD2 fusion proteins to AAV2 10 μL of commercially available AAV2 virus (10 13 We immobilized AAV2 at 1000 ng / mL (vg / mL) on a CM3 chip and performed surface plasmon resonance (SPR) by amine coupling. The SPR signal of the AAV2 channel was measured relative to a reference channel obtained by adding increasing concentrations of sfGFP-PKD2 fusion protein.

[0225] The SPR signal confirmed the interaction between immobilized AAV2 and the fusion protein, with a measured affinity of approximately 6 μM (Figure 4).

[0226] Example 10 - Binding of PKD2 fusion proteins to AAV1 10 μL of commercially available AAV1 virus (10 13 We immobilized AAV2 at 1000 ng / mL (vg / mL) on a CM3 chip and performed surface plasmon resonance (SPR) by amine coupling. The SPR signal of the AAV2 channel was measured relative to a reference channel obtained by adding increasing concentrations of sfGFP-PKD2 fusion protein.

[0227] The SPR signal confirmed the interaction between immobilized AAV1 and the fusion protein, with a measured affinity of approximately 2 μM (Figure 5).

[0228] Example 11 - Use of small molecule ligands It is possible to generate an adapter protein by taking an isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid (e.g., a PKD2 domain-containing protein) and reacting it with a targeting molecule that has high specificity for a target cell. This reaction can be performed using NHS or maleimide chemistry, or by incorporating a non-standard amino acid with a reactive group (such as azide or alkyne or tetrazine or transcyclooctene) at a specific position within the isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid.

[0229] It can also be performed by fusing an isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid molecule with a specific peptide sequence capable of binding to a targeting molecule, such as a split intein, SpyTag, a tetracysteine ​​FCM motif (FLNCCPGCCMEP) or a ybbR motif (TVLDSLEFIASKLA) (see Fernandes, DD, et al. Characterization of Fluorescein Arsenical Hairpin (FlAsH) as a Probe for Single-Molecule Fluorescence Spectroscopy. Sci Rep 7, 13063 (2017)). In such cases, the targeting molecule is designed to contain opposite reactive elements, such as opposite a split intein; SpyCatcher; a fluorescein arsenical hairpin, or coenzyme A, respectively.

[0230] The targeting molecule can then be selected to bind to a specific receptor.In some cases, the targeting molecule can be a protein, for example, an antibody against a receptor expressed on a target cell type.In the case of an antibody, the antibody can be modified with an alkyne group and then reacted via click chemistry with the azide group on an isolated polypeptide that can bind to adeno-associated virus (AAV) capsid.

[0231] In some cases, the targeting molecule can be a small molecule.In some cases, the isolated polypeptide capable of binding to adeno-associated virus (AAV) capsid is a PKD2 domain-containing protein.For example, by connecting a neurotransmitter such as serotonin or dopamine to the isolated polypeptide capable of binding to adeno-associated virus (AAV) capsid, it is possible to create an adapter molecule that, when combined with AAV, produces AAV specific to cells that express serotonin receptors or dopamine receptors, respectively. This approach (i.e., generating an adapter molecule that comprises an isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid and a reactive group, and then reacting it with a targeting molecule) is particularly useful when the targeting molecule cannot be expressed as a fusion with an isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid (e.g., when the targeting molecule is not a single-chain protein or is a small molecule) or when it cannot be efficiently purified by fusion of the targeting molecule to an isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid.

[0232] Example 12 - Further examples of the present invention The inventors have identified a number of aspects of the present invention which result in particularly advantageous features of the present invention.

[0233] The PKD2(V480E) mutant of AAVR has improved affinity for the AAV capsid, making it a superior adaptor protein (see Figure 26).

[0234] The use of viral adapter proteins to retarget viruses can be applied to adenoviruses using CAR-fusion proteins as adapters (see Figure 27).

[0235] A complex can be generated between AAV1(Asp590Cys) and an adapter protein (see Figure 28).

[0236] The identity of the fusion partner that constitutes the adaptor protein with PKD2(Ser425Cys) can be customized in a modular manner while maintaining the ability to form covalent adducts. This means that this technology can be used to retarget AAV to many different receptors by altering the identity of the fusion partner (see Figure 29).

[0237] Covalent adducts between adapter proteins and viral capsid proteins can also be formed in the presence of 1 mM reducing agent tris(2-carboxyethyl)phosphine hydrochloride (TCEP), indicating that disulfide bonds between the proteins form at interfaces that are not fully exposed to solvent (see FIG. 30). Coupling in the presence of a reducing agent such as TCEP is also favorable for coupling of adapter proteins to viruses, and the proteins and viruses may be partially oxidized prior to the coupling reaction.

[0238] Retargeting AAV1(Asp590Cys) with an adaptor protein can substantially increase its infectivity (see Figure 31).

[0239] Although recombinant AAV1 is neutralized by ADK1a antibody, the coated viral particles (CAR-V particles) maintain the same level of infectivity regardless of the concentration of neutralizing antibody and are therefore resistant to neutralization (see Figure 32).

[0240] Coating AAV1(Asp590Cys) with an adaptor protein can increase its specificity for cell types that express receptors that interact with the adaptor protein (see Figure 33).

[0241] Coating the virus with an adaptor protein prevents it from infecting the liver, a major site of off-target infection by AAV (see Figure 34).

[0242] After incubation with the adaptor protein, no visible bands corresponding to free VP protein could be observed, indicating that coupling was complete. This demonstrates that the PKD1-derived adaptor protein can be used to transfect AAV5 Q697C It has been shown that the antibody can be retargeted to the IL-1 domain (see FIG. 35).

[0243] The formation of covalent adducts between the fiber knob and adaptor proteins was observed exclusively in the presence of calcium ions, thus indicating that bond formation is mediated by the Ca ion pathway between the knob and desmoglein 2. 2+ These results indicate that the DSG2-derived adaptor protein is dependent on a ubiquitin-dependent interaction, and thus can be used to covalently bind to adenovirus B3 and redirect its tropism (see FIG. 36).

[0244] FIG. 39 shows that coating AAV2(Gln589Cys) with an adaptor protein can increase its specificity for cells expressing a receptor that interacts with the adaptor protein.

[0245] Figure 40 shows covalent adducts between the adapter protein and the viral capsid protein, as indicated by a shift in the molecular weight of the capsid protein in lanes 2 and 5 after the marker. Importantly, the adduct bands form in both AAV1 and AAV2 mutant viruses, confirming that the tropism of AAV1 and AAV2 can be altered using the adapter protein from PKD2.

[0246] FIG. 41 shows that coating AAV5(Gln697Cys) with an adapter protein enables retargeting by increasing its specificity to cells expressing the corresponding receptor.

[0247] These additional data demonstrate that the principles described herein are applicable globally to multiple different viral adaptor proteins and multiple different virus types. In particular, the present application provides evidence supporting a global effect across five distinct viral-adaptor protein pairs: PKD2(Ser425Cys-Val480Glu)+AAV1(Asp590Cys) PKD2(Ser425Cys-Val480Glu)+AAV2(Gln589Cys) PKD1(Ser356Cys)+AAV5(Gln697Cys) DSG2(Ala174Cys)+AdV-B3(Asn192Cys) CAR(Val70Cys)+AdV-5(Val441Cys)

[0248] It will be apparent to those skilled in the art that various modifications and variations can be made to the compositions and methods of the present invention. Thus, it is intended that the present invention cover such modifications and variations provided they come within the scope of the appended claims and their equivalents.

[0249] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent section headings are used, they should not be construed as necessarily limiting.

[0250] Further embodiments of the present invention are disclosed below: 1. An isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid. 2. An adeno-associated virus (AAV) adapter molecule comprising: (i) an isolated polypeptide capable of binding to an AAV capsid; and (ii) a ligand. 3. An isolated polypeptide or AAV adapter molecule according to any one of embodiments 1 or 2, which is capable of binding to an unmodified wild-type viral capsid. 4. An isolated polypeptide or AAV adapter molecule according to any one of embodiments 1-3, which is capable of binding to a viral capsid having five or fewer (e.g., five, four, three, two or one) amino acid point mutations compared to a wild-type viral capsid. 5. The isolated polypeptide or AAV adapter molecule according to any one of embodiments 1 to 4, which is capable of binding to a viral capsid having an amino acid insertion or deletion of 5 amino acids or less (i.e. 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids or 5 amino acids) relative to a wild-type viral capsid. 6. An isolated polypeptide or AAV adapter molecule according to any one of embodiments 1 to 5, which is capable of binding to a viral capsid that has no insertions or deletions relative to a wild-type viral capsid. 7. An isolated polypeptide or AAV adapter molecule according to any one of embodiments 1 to 6, comprising a portion of the adeno-associated virus receptor (AAVR, KIAA0319L) capable of binding to an adeno-associated virus capsid. 8. An isolated polypeptide or AAV adapter molecule according to any one of embodiments 1 to 7, comprising the full-length sequence of the adeno-associated virus receptor (AAVR, KIAA0319L). 9. The isolated polypeptide or AAV adapter molecule according to any one of embodiments 7 or 8, wherein the sequence of the adeno-associated virus receptor (AAVR, KIAA0319L) comprises the amino acid sequence of SEQ ID NO:1. 10. The isolated polypeptide or AAV adapter molecule according to any one of embodiments 7-9, comprising one or more point mutations in the AAVR polypeptide, optionally wherein the one or more point mutations are defined with reference to SEQ ID NO:1. 11. The isolated polypeptide or AAV adapter molecule according to any one of embodiments 7-10, comprising one or more point mutations in the AAVR polypeptide, optionally wherein the one or more point mutations are defined with reference to a sequence selected from the list consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. 12. The isolated polypeptide or AAV adapter molecule according to any one of embodiments 7 to 10, comprising a V480E mutation in the PKD2 domain, and optionally one or more point mutations defined with reference to SEQ ID NO:1. 13. The isolated polypeptide or AAV adapter molecule according to any one of embodiments 7 to 12, comprising the sequence of SEQ ID NO:1 with the V480E mutation. 14. The isolated polypeptide or AAV adapter molecule according to any one of embodiments 7 to 11, comprising the sequence of SEQ ID NO:3 with the V480E mutation. 15. An isolated polypeptide or AAV adapter molecule according to any one of embodiments 7 to 14, comprising an S425C mutation in the PKD2 domain, and optionally one or more point mutations defined with reference to SEQ ID NO:1. 16. An isolated polypeptide or AAV adapter molecule according to any one of embodiments 7 to 14, comprising the sequence of SEQ ID NO:1 with the S425C mutation. 17. An isolated polypeptide or AAV adapter molecule according to any one of embodiments 7 to 14, comprising the sequence of SEQ ID NO: 3, having an S435C mutation. 18. An isolated polypeptide or AAV adapter molecule according to any one of embodiments 7 to 17, comprising an S425C mutation and a V480E mutation in the PKD2 domain, and optionally one or more point mutations defined with reference to SEQ ID NO:1 or SEQ ID NO:3. 19. The isolated polypeptide or AAV adapter molecule according to any one of embodiments 1-6, comprising a portion of a polypeptide selected from the list consisting of a DARPin, a nanobody, a suitable antibody-like protein, a protein derived from the lipocalin fold, an affibody (e.g. an affibody derived from the Z domain of Protein A), a domain of fibronectin, an SH3 domain of Fyn, an affimer or scaffold derived from the protease inhibitor Stefin A, a non-antibody scaffold protein (Adilon), and an antibody or antigen-binding fragment thereof. 20. The isolated polypeptide or AAV adapter molecule according to any one of embodiments 1-6, comprising a polypeptide selected from the list consisting of a DARPin, a nanobody, a suitable antibody-like protein, a protein derived from the lipocalin fold, an affibody (e.g. an affibody derived from the Z domain of Protein A), a domain of fibronectin, an SH3 domain of Fyn, an affimer or scaffold derived from the protease inhibitor Stefin A, a non-antibody scaffold protein (Adilon), and an antibody or antigen-binding fragment thereof. twenty one. a) one or more unnatural amino acids, b) one or more chemical moieties crosslinked to the polypeptide; c) a biotin tag, e.g., biotinylated AAVR; d) SpyTag peptides, such as AAVR-SpyTag, and / or e) a Protein A polypeptide or a Protein G polypeptide, 21. The isolated polypeptide or AAV adapter molecule according to any one of embodiments 1 to 20, further comprising one or more of: 22. The isolated polypeptide or AAV adapter molecule according to any one of embodiments 1 to 21, wherein the ligand is capable of binding to a cell surface molecule. 23. The isolated polypeptide or AAV adapter molecule according to any one of embodiments 1-22, wherein the ligand is a human protein, such as an antibody or an antigen-binding fragment thereof. 24. The ligand is interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin-12, interleukin-13, interleukin-14, interleukin-15, interleukin-16, interleukin-17, interleukin-18, interleukin-19, interleukin-20, interleukin-21, interleukin-22, interleukin-23, interleukin-24, interleukin-25, interleukin-26, interleukin-27, interleukin-28, interleukin-29, interleukin-30, interleukin-31, interleukin-32, interleukin-3 3, interleukin-34, interleukin-35, insulin, transferrin, CD2, CD58, CD59, CD2, CD40L / CD154, CD5, CD72, CD5L, CD23, CD70, CD80, CD86, S100Ap, CD178, CD155, CD106, CSF1, CD166, FasL, CD242, CD252, TRAIL, RANKL, APRIL, CD257, CD272, CD273, CD 24. The isolated polypeptide or AAV adaptor molecule according to any one of embodiments 1-23, which is one or more ligands selected from the list consisting of 274, CD275, PD-L1, PD-L2, Cas13 and Cas7-11, endothelin, leptin, vasopressin, CD10, CD31, CD119, apelin, elabella, adrenomedullin, a targeting domain derived from botulinum toxin, a neuropeptide, a cytokine or a small molecule. 25. The isolated polypeptide or AAV adapter molecule according to any one of embodiments 1 to 24, wherein the ligand is a small molecule, and optionally the small molecule is linked to the isolated polypeptide or AAV adapter molecule via N-hydroxysuccinimide (NHS) or maleimide. 26. The isolated polypeptide or AAV adapter molecule according to any one of embodiments 1-24, wherein the ligand is a small molecule and optionally the isolated polypeptide or AAV adapter molecule comprises a non-standard amino acid that links the small molecule to the isolated polypeptide or AAV adapter molecule. 27. The ligand is Her2, interleukin-1 receptor, interleukin-2 receptor, interleukin-3 receptor, interleukin-4 receptor, interleukin-5 receptor, interleukin-6 receptor, interleukin-7 receptor, interleukin-8 receptor, interleukin-9 receptor, interleukin-10 receptor, interleukin-11 receptor, interleukin-12 receptor, interleukin-13 receptor, interleukin-15 receptor, interleukin-18 receptor, interleukin-20 receptor, interleukin-21 receptor, interleukin-22 receptor, interleukin-23 receptor, interleukin-27 receptor, interleukin-28 receptor, insulin receptor, transferrin receptor, CD58, CD2 27. The isolated polypeptide or AAV adaptor molecule according to any one of embodiments 1-26, which binds to one or more cell surface molecules selected from the list consisting of: CD2, CD59, CD40, CD72, CD5, CD36, CD19, CD21, CD81, CD27, CD28, CTLA-4, CD85j, CD95, CD96, alpha4beta1 integrin, CD115, CD6, CD178, LFA-1, TNFRSF4, DR4, DR5, RANK / CD265, TACI / CD267, CD267, CD268, CD269, HVEM, PD1 / CD279, B7-1 / CD80, CD278, CD4, CD8, CD19, NMDAR, AMPAR, mGluR5, DRD1, DRD2, Bmp4, GLP1R, leptin receptor, alpha5beta5 integrin and glycoRNA. 28. The isolated polypeptide or AAV adapter molecule according to any one of embodiments 7-27, wherein the portion of the adeno-associated virus receptor (AAVR, KIAA0319L) comprises at least a portion of the PKD1 domain, at least a portion of the PKD2 domain, at least a portion of the PKD3 domain, at least a portion of the PKD4 domain, at least a portion of the PKD5 domain, or any combination thereof. 29. The isolated polypeptide or AAV adapter molecule according to any one of embodiments 7 to 28, wherein the portion of the adeno-associated virus receptor (AAVR, KIAA0319L) comprises a complete PKD1 domain, a complete PKD2 domain, a complete PKD3 domain, a complete PKD4 domain, a complete PKD5 domain, or any combination thereof. 30. The isolated polypeptide or AAV adapter molecule according to any one of embodiments 7 to 29, wherein the portion of the adeno-associated virus receptor (AAVR, KIAA0319L) comprises a sequence selected from the list consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. 31. The isolated polypeptide or AAV adapter molecule according to any one of embodiments 7 to 30, wherein the portion of the adeno-associated virus receptor (AAVR, KIAA0319L) comprises the full-length sequence of the adeno-associated virus receptor (AAVR, KIAA0319L), optionally, the full sequence comprises SEQ ID NO:1. 32. An adeno-associated virus particle linked to at least one isolated polypeptide or AAV adapter molecule according to any one of embodiments 1 to 31. 33. An adeno-associated virus particle linked to at least two, three, four or five isolated polypeptides or AAV adapter molecules according to any one of embodiments 1 to 32. 34. The adeno-associated viral particle according to embodiment 33, having specificity for two or more different target cells. 35. An adeno-associated viral particle according to embodiment 33, having increased tropism for two or more different target cells. 36. The adeno-associated viral particle according to embodiment 34 or 35, wherein two or more target cells express different cell surface molecules. 37. The adeno-associated virus particle according to any one of embodiments 34 to 36, wherein at least two, three, four or five isolated polypeptides or AAV adapter molecules specifically bind to different cell surface molecules on two or more target cells. 38. An adeno-associated viral particle according to embodiment 33, having specificity for target cells expressing two or more different cell surface molecules. 39. An adeno-associated viral particle according to embodiment 33, having increased tropism for target cells expressing two or more distinct cell surface molecules. 40. The adeno-associated virus particle according to embodiment 38 or 39, wherein at least two, three, four or five isolated polypeptides or AAV adapter molecules specifically bind to two or more distinct cell surface molecules on a target cell. 41. The adeno-associated virus particle according to embodiment 32, wherein the adeno-associated virus is selected from the list consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.74. 42. The adeno-associated virus particle according to any one of embodiments 32 or 41, wherein the adeno-associated virus is AAV2. 43. The adeno-associated virus particle according to any one of embodiments 32 to 42, wherein the adeno-associated virus is a wild-type AAV. 44. The adeno-associated virus particle according to any one of embodiments 32 to 43, wherein the adeno-associated virus comprises a portion of a capsid amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19. 45. The adeno-associated virus particle according to any one of embodiments 32 to 44, wherein the adeno-associated virus comprises a full-length capsid amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19. 46. ​​The adeno-associated virus particle according to any one of embodiments 32 to 45, wherein the adeno-associated virus has one or more conservative amino acid changes relative to the wild-type amino acid sequence, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 conservative amino acid changes relative to the wild-type amino acid sequence, and optionally, the wild-type amino acid sequence is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19. 47. The adeno-associated virus particle according to any one of embodiments 32 to 46, wherein the adeno-associated virus has five or fewer non-conservative amino acid changes relative to the wild-type amino acid sequence, such as five, four, three, two or one non-conservative amino acid changes relative to the wild-type amino acid sequence, and optionally the wild-type amino acid sequence is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19. 48. An adeno-associated virus particle according to any one of embodiments 32 to 47, comprising one or more amino acid changes in the capsid protein that mediate interactions with non-protein binders, such as heparan sulfate proteoglycans (HSPGs), O-linked sialic acid, N-linked sialic acid, N-linked galactose, etc. 49. The adeno-associated virus particle according to embodiment 48, wherein the one or more mutations include arginine residues 585 and 588 of AAV2. 50. A pharmaceutical composition comprising an adeno-associated virus particle according to any one of embodiments 32 to 49. 51. A method for modifying AAV particles, comprising: Preparing AAV particles; Providing an isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid; combining the AAV particles with an isolated polypeptide such that the isolated polypeptide is bound to the AAV particles; A method comprising: 52. A method for modifying AAV particles, comprising: Preparing AAV particles; providing an adeno-associated virus (AAV) adaptor molecule comprising an isolated polypeptide capable of binding to an AAV capsid and a ligand; combining the AAV particle with an AAV adapter molecule such that the isolated fusion polypeptide is attached to the AAV particle; A method comprising: 53. A method for modifying AAV particles, comprising: Preparing AAV particles; Providing two or more (e.g., two, three, four, or five) isolated polypeptides capable of binding to an adeno-associated virus (AAV) capsid; combining the AAV particles with an isolated polypeptide such that the isolated polypeptide is bound to the AAV particles; A method comprising: 54. A method for modifying AAV particles, comprising: Preparing AAV particles; Providing two or more (e.g., two, three, four, or five) adeno-associated virus (AAV) adaptor molecules comprising an isolated polypeptide capable of binding to an AAV capsid and a ligand; combining an AAV particle with an AAV adapter molecule such that the AAV adapter molecule is attached to the AAV particle; A method comprising: 55. A method for modifying an AAV particle according to any one of embodiments 51 to 54, wherein binding of an isolated polypeptide or an AAV adapter molecule to the AAV particle reduces or eliminates the native tropism of one or more AAV capsid proteins. 56. A method for modifying an AAV particle according to any one of embodiments 51 to 55, wherein binding of the isolated polypeptide or AAV adapter molecule to the AAV particle increases the tropism of the AAV particle for one or more cell types. 57. A method for modifying an AAV particle according to any one of embodiments 51 to 56, wherein binding of the isolated polypeptide or AAV adapter molecule to the AAV particle increases the tropism of the AAV particle for one or more cell types selected from the list consisting of neurons, macrophages, microglia, T cells, B cells, dendritic cells, antigen-presenting cells, NK cells, cancer cells, muscle cells, hepatocytes, photoreceptor cells, pancreatic beta cells, renal cells and lung cells. 58. A method for modifying an AAV particle according to any one of embodiments 51 to 57, wherein the AAV particle has a tropism that is reduced or increased by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% compared to the native tropism of the AAV particle not comprising the isolated polypeptide or AAV adapter molecule. 59. A method for modifying AAV particles according to any one of embodiments 51 to 58, wherein the AAV particles have specificity for two or more different target cells. 60. A method for modifying AAV particles according to any one of embodiments 51 to 58, wherein the AAV particles have increased tropism for two or more different target cells. 61. A method for modifying AAV particles according to embodiment 59 or 60, wherein two or more target cells express different cell surface molecules. 62. A method for modifying an AAV particle according to any one of embodiments 59 to 61, wherein at least two, three, four or five isolated polypeptides or AAV adapter molecules specifically bind to different cell surface molecules on two or more target cells. 63. A method for modifying AAV particles according to any one of embodiments 51 to 58, wherein the adeno-associated viral particles have specificity for target cells expressing two or more different cell surface molecules. 64. A method for modifying AAV particles according to any one of embodiments 51 to 58, wherein the adeno-associated viral particles have increased tropism for target cells expressing two or more distinct cell surface molecules. 65. A method for modifying an AAV particle according to embodiment 63 or 64, wherein at least two, three, four or five isolated polypeptides or AAV adapter molecules specifically bind to two or more different cell surface molecules on a target cell. 66. A method for targeting AAV particles to a target cell, comprising: Preparing AAV particles; providing an adeno-associated virus (AAV) adaptor molecule comprising an isolated polypeptide capable of binding to an AAV capsid and a ligand specific for a target cell; combining the AAV particle with an AAV adapter molecule such that the AAV adapter molecule is attached to the AAV particle, thereby generating a modified AAV particle; contacting a mixture of cells including target cells with the modified AAV particles; A method comprising: 67. A method for targeting AAV particles to a target cell, comprising: Preparing AAV particles; Providing two or more (e.g., two, three, four, or five) adeno-associated virus (AAV) adaptor molecules comprising an isolated polypeptide capable of binding to an AAV capsid and a ligand specific for a target cell; combining the AAV particle with two or more AAV adapter molecules such that the AAV adapter molecules are attached to the AAV particle, thereby generating the modified AAV particle; contacting a mixture of cells including target cells with the modified AAV particles; wherein the target cell expresses two or more distinct cell surface molecules, and optionally the AAV adapter molecule specifically binds to two or more distinct cell surface molecules on the target cell. 68. A method for targeting AAV particles to two or more target cells, comprising: Preparing AAV particles; providing two or more (e.g., two, three, four, or five) adeno-associated virus (AAV) adaptor molecules comprising an isolated polypeptide capable of binding to an AAV capsid and a ligand specific for at least one of two or more target cells; combining the AAV particle with two or more AAV adapter molecules such that the AAV adapter molecules are attached to the AAV particle, thereby generating the modified AAV particle; contacting a mixture of cells comprising two or more target cells with the modified AAV particles; wherein the two or more target cells express two or more different cell surface molecules, and optionally the AAV adapter molecule specifically binds to different cell surface molecules on the two or more target cells. 69. A method for modifying an adeno-associated virus (AAV) particle or for targeting an AAV particle to a target cell according to any one of embodiments 51 to 68, wherein the isolated polypeptide capable of binding to an AAV capsid comprises a portion of the adeno-associated virus receptor (AAVR, KIAA0319L). 70. A method for modifying an adeno-associated virus (AAV) particle or a method for targeting an AAV particle to a target cell according to any one of embodiments 51 to 69, wherein the isolated polypeptide capable of binding to an AAV capsid comprises at least a portion of the PKD1 domain, at least a portion of the PKD2 domain, at least a portion of the PKD3 domain, at least a portion of the PKD4 domain, at least a portion of the PKD5 domain, or any combination thereof, of the adeno-associated virus receptor (AAVR, KIAA0319L). 71. A method for modifying an adeno-associated virus (AAV) particle or a method for targeting an AAV particle to a target cell according to any one of embodiments 51 to 70, wherein the isolated polypeptide capable of binding to an AAV capsid comprises a complete PKD1 domain, a complete PKD2 domain, a complete PKD3 domain, a complete PKD4 domain, a complete PKD5 domain of the adeno-associated virus receptor (AAVR, KIAA0319L), or any combination thereof. 72. A method for modifying an adeno-associated virus (AAV) particle or a method for targeting an AAV particle to a target cell according to any one of embodiments 51 to 71, wherein the isolated polypeptide capable of binding to an AAV capsid comprises one or more mutations in an AAVR polypeptide, optionally wherein the one or more mutations are defined with reference to SEQ ID NO:1. 73. A method for modifying an AAV particle or a method for targeting an AAV particle to a target cell according to any one of embodiments 51 to 72, wherein the isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid comprises at least a portion of the PKD1 domain, at least a portion of the PKD2 domain, at least a portion of the PKD3 domain, at least a portion of the PKD4 domain, at least a portion of the PKD5 domain, or any combination thereof, of the adeno-associated virus receptor (AAVR, KIAA0319L), and the isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid comprises one or more mutations in the AAVR polypeptide, optionally wherein the one or more mutations are defined with reference to SEQ ID NO:1. 74. A method for modifying an AAV particle or a method for targeting an AAV particle to a target cell according to any one of embodiments 51 to 73, wherein the isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid comprises a complete PKD1 domain, a complete PKD2 domain, a complete PKD3 domain, a complete PKD4 domain, a complete PKD5 domain of the adeno-associated virus receptor (AAVR, KIAA0319L), or any combination thereof, and wherein the isolated polypeptide capable of binding to an adeno-associated virus (AAV) capsid comprises one or more mutations in the AAVR polypeptide, optionally wherein the one or more mutations are defined with reference to SEQ ID NO:1. 75. A method for modifying an adeno-associated virus (AAV) particle or a method for targeting an AAV particle to a target cell according to any one of embodiments 51 to 74, wherein the isolated polypeptide capable of binding to an AAV capsid comprises a sequence selected from the list consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. 76. A method for modifying an adeno-associated virus (AAV) particle or a method for targeting an AAV particle to a target cell according to any one of embodiments 51 to 75, wherein the isolated polypeptide capable of binding to an AAV capsid comprises the full-length sequence of the adeno-associated virus receptor (AAVR, KIAA0319L), optionally wherein the full sequence comprises SEQ ID NO:1. 77. A method for modifying an adeno-associated virus (AAV) particle or a method for targeting an AAV particle to a target cell according to any one of embodiments 51 to 76, wherein the isolated polypeptide capable of binding to an AAV capsid comprises a portion of an adeno-associated virus receptor having one or more point mutations, optionally wherein the one or more point mutations are defined with reference to SEQ ID NO:1. 78. A method for modifying an adeno-associated virus (AAV) particle or a method for targeting an AAV particle to a target cell according to any one of embodiments 51 to 76, wherein the isolated polypeptide capable of binding to an AAV capsid comprises a portion of an adeno-associated virus receptor having one or more point mutations, and optionally the one or more point mutations are defined with reference to a sequence selected from the list consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. 79. A method for modifying an adeno-associated virus (AAV) particle or a method for targeting an AAV particle to a target cell according to any one of embodiments 51 to 77, wherein the isolated polypeptide capable of binding to an AAV capsid comprises a V480E mutation in the PKD2 domain, and optionally one or more point mutations are defined with reference to SEQ ID NO:1. 80. A method for modifying an adeno-associated virus (AAV) particle or a method for targeting an AAV particle to a target cell according to any one of embodiments 51 to 79, wherein the isolated polypeptide capable of binding to an AAV capsid comprises the sequence of SEQ ID NO: 1 having a V480E mutation. 81. A method for modifying an adeno-associated virus (AAV) particle or a method for targeting an AAV particle to a target cell according to any one of embodiments 51 to 78, wherein the isolated polypeptide capable of binding to an AAV capsid comprises the sequence of SEQ ID NO: 3 having a V480E mutation. 82. A method for modifying an adeno-associated virus (AAV) particle or a method for targeting an AAV particle to a target cell according to any one of embodiments 51 to 81, wherein the isolated polypeptide capable of binding to an AAV capsid comprises an S425C mutation in the PKD2 domain, and optionally one or more point mutations are defined with reference to SEQ ID NO:1. 83. A method for modifying an adeno-associated virus (AAV) particle or a method for targeting an AAV particle to a target cell according to any one of embodiments 51 to 82, wherein the isolated polypeptide capable of binding to an AAV capsid comprises the sequence of SEQ ID NO: 1 having an S425C mutation. 84. A method for modifying an adeno-associated virus (AAV) particle or a method for targeting an AAV particle to a target cell according to any one of embodiments 51 to 83, wherein the isolated polypeptide capable of binding to an AAV capsid comprises the sequence of SEQ ID NO: 3 having an S425C mutation. 85. A method for modifying an adeno-associated virus (AAV) particle or a method for targeting an AAV particle to a target cell according to any one of embodiments 51 to 84, wherein the isolated polypeptide capable of binding to an AAV capsid comprises an S425C mutation and a V480E mutation in the PKD2 domain, and optionally one or more point mutations are defined with reference to SEQ ID NO: 1 or SEQ ID NO: 3. 86. A method for delivering a nucleic acid sequence of interest to a target cell, comprising contacting the target cell with a modified AAV particle according to any one of embodiments 32 to 49 or a pharmaceutical composition according to embodiment 50, wherein the AAV particle comprises the nucleic acid sequence of interest. 87. The method of embodiment 86, wherein the nucleic acid sequence of interest encodes a protein selected from the list consisting of RAB escort protein 1, RPE65, factor VIII, factor IX, cochlin, CLN7, acid alpha-glucosidase (GAA), aquaporin 1, glial cell line-derived neurotrophic factor, aspartoacylase, aromatic L-amino acid decarboxylase, retinitis pigmentosa GTPase regulator deficiency, sarcoplasmic reticulum calcium ATPase, cyclic nucleotide-gated channel beta 3, neurturin, galactosidase beta 1, glucose-6-phosphatase, phenylalanine hydroxylase, ornithine transcarbamylase, dystrophin and carnitine palmitoyltransferase II, phenylalanine hydroxylase (PAH), cystic fibrosis transmembrane conductance regulator (CFTR). 88. The method according to any one of embodiments 66 to 87, wherein the target cell is in vitro. 89. The method according to any one of embodiments 66 to 87, wherein the target cell is in vivo in a subject. 90. The method according to any one of embodiments 66 to 89, wherein the target cells are selected from the list consisting of neurons, macrophages, microglia, T cells, B cells, dendritic cells, antigen-presenting cells, NK cells, cancer cells, muscle cells, liver cells, photoreceptor cells, pancreatic beta cells, kidney cells and lung cells. 91. The method according to embodiment 89 or 90, wherein the subject is a human. 92. The method according to any one of embodiments 66 to 93, wherein the target cell is a human target cell. 93. An AAV particle according to any one of embodiments 32 to 49 or a pharmaceutical composition according to embodiment 50 for use as a medicament. 94. An AAV particle according to any one of embodiments 32 to 49 or a pharmaceutical composition according to embodiment 50 for use in a method for treating a disease in a subject in need of treatment, comprising contacting the target cell with the AAV particle, thereby delivering a nucleic acid sequence of interest to the target cell. 95. Diseases include neurodegenerative disorders, cancer, Duchenne muscular dystrophy, hemophilia, congenital blindness, diabetes, cystic fibrosis, choroideremia, hemophilia A, hemophilia B, CLN7 disease, Pompe disease, Parkinson's disease, Canavan disease, demyelinating diseases, inherited retinal dystrophies due to RPE65 mutations, aromatic L-amino acid decarboxylase (AADC) deficiency, X-linked retinitis pigmentosa, Leber congenital amaurosis, Churg-Strauss syndrome (CSS), critical limb ischemia, color vision disorders, Alzheimer's disease, macular degeneration, ornithine transcarbamylase deficiency, Wilson's disease, glycogen storage disease type IA, Crigler-Nager syndrome, Tay-Sachs disease, Sandhoff disease, multiple myeloma, multiple system atrophy, gangliosidosis, Danon disease, Fabry disease, Batten disease, phenylketonuria, rheumatoid arthritis, mucopolysaccharidosis type IIIa, Sanfilippo syndrome B, mucopolysaccharidosis type VI, alpha 1-antitrypsin deficiency, spinal muscular atrophy type 1, Krabbe disease, Becker muscular dystrophy, Charcot-Marie-Tooth neuropathy type 1a, carnitine palmitoyltransferase II (CPT II) deficiency and trimethylaminuria, cystic fibrosis, phenylketonuria. 96. A method for treating a disease in a subject in need of treatment, comprising delivering a nucleic acid sequence of interest to a target cell by contacting the target cell with an AAV particle, comprising: a) Neuronal cells for the treatment of neurodegenerative disorders; b) immune cells (e.g. macrophages, microglia, T cells, B cells, dendritic cells, antigen presenting cells, NK cells) for the treatment of cancer; c) immune cells (e.g., macrophages, microglia, T cells, B cells, dendritic cells, antigen-presenting cells, NK cells) to elicit an immune response; d) cancer cells or tumor cells for the treatment of cancer; e) muscle cells for the treatment of Duchenne muscular dystrophy; f) Hepatocytes for the treatment of hemophilia; g) Photoreceptor cells for congenital blindness; h) pancreatic beta cells for the treatment of diabetes; or i) Lung cells for the treatment of cystic fibrosis; The AAV particle according to any one of embodiments 32 to 49 or the pharmaceutical composition according to embodiment 50, wherein targeting of the designated target cell by the AAV particle treats or ameliorates the designated disease. 97. AAV particles or pharmaceutical compositions for use according to any one of embodiments 93 to 97, wherein the AAV particles or pharmaceutical compositions are administered to a subject by aerosol (e.g. to lung tissue), intramuscularly, intraarterially (e.g. via the hepatic artery), intraarticularly, subretinal, intracranially, intravenously, intrathecally, intracoronarily or subcutaneously. 98. An isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid. 99. A viral adaptor molecule comprising: (i) an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid; and (ii) a ligand. 100. The isolated polypeptide according to embodiment 98, wherein one or more cysteine ​​residues capable of covalently binding to a viral capsid are heterologous to the isolated polypeptide. 101. A viral adapter molecule according to embodiment 99, comprising (i) an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid, and (ii) a ligand, wherein the one or more cysteine ​​residues are heterologous to the isolated polypeptide. 102. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 98 to 101, capable of binding to an unmodified wild-type viral capsid. 103. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 98 to 102, capable of binding to a viral capsid having five or fewer (e.g., five, four, three, two or one) amino acid point mutations compared to a wild-type viral capsid. 104. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 98 to 103, capable of binding to a viral capsid having an amino acid insertion or deletion of 5 amino acids or less (i.e., 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids or 5 amino acids) relative to a wild-type viral capsid. 105. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 98 to 103, which is capable of binding to a viral capsid that has no insertions or deletions relative to a wild-type viral capsid. 106. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 98 to 105, comprising a portion of an adeno-associated virus receptor (AAVR, KIAA0319L) having one or more cysteine ​​residues and capable of covalently binding to an adeno-associated virus capsid. 107. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 98 to 106, comprising a full-length sequence of an adeno-associated virus receptor (AAVR, KIAA0319L) having one or more cysteine ​​residues and capable of covalently binding to an adeno-associated virus capsid. 108. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 106 or 107, wherein the sequence of the adeno-associated viral receptor (AAVR, KIAA0319L) comprises the amino acid sequence of SEQ ID NO:1. 109. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 106 to 108, comprising one or more point mutations in the AAVR polypeptide, optionally wherein the one or more point mutations are defined with reference to SEQ ID NO:1. 110. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 106 to 109, comprising one or more point mutations in an AAVR polypeptide, optionally wherein the one or more point mutations are defined with reference to a sequence selected from the list consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. 111. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 106 to 110, comprising a V480E mutation in the PKD2 domain, and optionally one or more point mutations defined with reference to SEQ ID NO:1. 112. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 106 to 110, comprising the sequence of SEQ ID NO:1 with the V480E mutation. 113. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 106 to 110, comprising the sequence of SEQ ID NO: 3, having the V480E mutation. 114. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 106 to 113, comprising an S425C mutation in the PKD2 domain, and optionally one or more point mutations defined with reference to SEQ ID NO:1. 115. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 106 to 114, comprising the sequence of SEQ ID NO:1 with the S425C mutation. 116. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 106 to 115, comprising the sequence of SEQ ID NO: 3 with the S425C mutation. 117. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 106 to 116, comprising an S425C mutation and a V480E mutation in the PKD2 domain, and optionally one or more point mutations defined with reference to SEQ ID NO: 1 or SEQ ID NO: 3. 118. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 106 to 117, comprising one or more point mutations in the AAVR polypeptide for introducing one or more heterologous cysteine ​​residues, optionally wherein the one or more point mutations are defined with reference to SEQ ID NO:1. 119. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 106 to 118, comprising one or more point mutations in the AAVR polypeptide for introducing one or more heterologous cysteine ​​residues, optionally wherein the one or more point mutations are defined with reference to a sequence selected from the list consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. 120. The isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 98-105, wherein the isolated polypeptide or covalent viral adapter molecule comprises a portion of a polypeptide selected from the list consisting of a DARPin, a nanobody, a suitable antibody-like protein, a protein derived from the lipocalin fold, an affibody (e.g. an affibody derived from the Z domain of protein A), a domain of fibronectin, an SH3 domain of Fyn, an affimer or scaffold derived from the protease inhibitor Stefin A, a non-antibody scaffold protein (Adilon), and an antibody or an antigen-binding fragment thereof. In some embodiments, the isolated polypeptide or AAV adapter molecule comprises an antibody or an antigen-binding fragment thereof. 121. The isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 98-105, wherein the isolated polypeptide or viral adapter molecule comprises a polypeptide selected from the list consisting of a DARPin, a nanobody, a suitable antibody-like protein, a protein derived from the lipocalin fold, an affibody (e.g. an affibody derived from the Z domain of protein A), a domain of fibronectin, an SH3 domain of Fyn, an affimer or scaffold derived from the protease inhibitor Stefin A, a non-antibody scaffold protein (Adilon), and an antibody or an antigen-binding fragment thereof. In some embodiments, the isolated polypeptide or AAV adapter molecule comprises an antibody or an antigen-binding fragment thereof. 122. The isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 98 to 105, 120 or 121, wherein the isolated polypeptide or covalent viral adapter molecule comprises a portion of the Coxsackievirus-Adenovirus Receptor (CXADR), and optionally, the CXADR sequence is SEQ ID NO: 20. 123. The isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 98 to 105, 120 or 121, wherein the isolated polypeptide or covalent viral adapter molecule comprises the full-length sequence of the Coxsackievirus-adenovirus receptor (CXADR), and optionally, the CXADR sequence is SEQ ID NO: 20. 124. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 98-105, 120 or 121, comprising an antigen-binding portion of the neutralizing antibody A20. 125. An isolated polypeptide or covalent viral adapter molecule according to embodiment 124, wherein the isolated polypeptide or covalent viral adapter molecule comprises a sequence substantially identical to one or more complementarity determining regions (CDRs) of neutralizing antibody A20, e.g., one or more complementarity determining regions (CDRs) of neutralizing antibody A20 may comprise one, two or three amino acid point mutations, and optionally, one or more of the point mutations are heterologous cysteine ​​residues. 126. An isolated polypeptide or covalent viral adapter molecule according to embodiment 125, wherein the isolated polypeptide or covalent viral adapter molecule comprises one or more sequences substantially identical to each of the complementarity determining regions (CDRs) of neutralizing antibody A20, e.g., one, two, three, four, five or six of the complementarity determining regions (CDRs) of neutralizing antibody A20 may contain one, two or three amino acid point mutations, and optionally, one or more of the point mutations are heterologous cysteine ​​residues. 127. An isolated polypeptide or covalent viral adapter molecule according to embodiment 124, wherein the isolated polypeptide or covalent viral adapter molecule comprises the full-length sequence of one or more complementarity determining regions (CDRs) of neutralizing antibody A20, e.g., the polypeptide comprises one or more of VHCDR1 of antibody A20 (sequence number 26), VHCDR2 of antibody A20 (sequence number 27), VHCDR3 of antibody A20 (sequence number 28), VLCDR1 of antibody A20 (sequence number 22), VLCDR2 of antibody A20 (sequence number 23) and VLCDR3 of antibody A20 (sequence number 24). 128. An isolated polypeptide or covalent viral adapter molecule according to embodiment 127, comprising all of the complementarity determining regions (CDRs) of the neutralizing antibody A20. 129. An isolated polypeptide comprising one or more cysteine ​​residues according to any one of embodiments 98-105 or 120-128, wherein the polypeptide or covalent viral adapter molecule comprises one or more sequences substantially identical to the sequences of the full-length VL chain (SEQ ID NO: 21) and VH chain (SEQ ID NO: 25) of neutralizing antibody A20, e.g., the VL sequence and / or the VH sequence of neutralizing antibody A20 may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid point mutations, optionally wherein one or more of the point mutations are heterologous cysteine ​​residues. 130. An isolated polypeptide comprising one or more cysteine ​​residues according to any one of embodiments 98-105 or 120-128, wherein the polypeptide or covalent viral adapter molecule comprises the sequence of the full-length VL chain (sequence number 21) and VH chain (sequence number 25) of neutralizing antibody A20. 131. a) one or more unnatural amino acids, b) one or more chemical moieties crosslinked to the polypeptide; c) biotin tag, d) SpyTag peptide, and / or e) a Protein A polypeptide or a Protein G polypeptide, 131. The isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 98 to 130, further comprising one or more of: 132. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 98 to 131, wherein the ligand is capable of binding to a cell surface molecule. 133. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 98 to 132, wherein the ligand is a human protein, such as an antibody or an antigen-binding fragment thereof. 134. The ligand is interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin-12, interleukin-13, interleukin-14, interleukin-15, interleukin-16, interleukin-17, interleukin-18, interleukin-19, interleukin-20, interleukin-21, interleukin-22, interleukin-23, interleukin-24, interleukin-25, interleukin-26, interleukin-27, interleukin-28, interleukin-29, interleukin-30, interleukin-31, interleukin-32, interleukin-33, interleukin-34, interleukin-35, interleukin-36, interleukin-37, interleukin-38, interleukin-39, interleukin-40, interleukin-41, interleukin-42, interleukin-43, interleukin-44, interleukin-45, interleukin-46, interleukin-47, interleukin-48, interleukin-49, interleukin-50, interleukin-51, interleukin-52, interleukin-53, interleukin-54, interleukin-55, interleukin-56, interleukin-57, interleukin-58, interleukin-59, interleukin-60, interleukin-61, interleukin-62, interleukin-63, interleukin-64, Interleukin-34, Interleukin-35, Insulin, Transferrin, CD2, CD58, CD59, CD2, CD40L / CD154, CD5, CD72, CD5L, CD23, CD70, CD80, CD86, S100Ap, CD178, CD155, CD106, CSF1, CD166, FasL, CD242, CD252, TRAIL, RANKL, APRIL, CD257, CD272, CD273, CD274, C 134. The isolated polypeptide or covalent viral adaptor molecule according to any one of embodiments 98-133, which is one or more ligands selected from the list consisting of D275, PD-L1, PD-L2, Cas13 and Cas7-11, endothelin, leptin, vasopressin, CD10, CD31, CD119, apelin, elabella, adrenomedullin, a targeting domain derived from botulinum toxin, a neuropeptide, a cytokine or a small molecule. 135. The isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 98 to 134, wherein the ligand is a small molecule, and optionally the small molecule is linked to the isolated polypeptide or viral adapter molecule via N-hydroxysuccinimide (NHS) or maleimide. 136. The isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 98 to 134, wherein the ligand is a small molecule and optionally the isolated polypeptide or viral adapter molecule comprises a non-standard amino acid that links the small molecule to the isolated polypeptide or viral adapter molecule. 137. Ligands include Her2, interleukin-1 receptor, interleukin-2 receptor, interleukin-3 receptor, interleukin-4 receptor, interleukin-5 receptor, interleukin-6 receptor, interleukin-7 receptor, interleukin-8 receptor, interleukin-9 receptor, interleukin-10 receptor, interleukin-11 receptor, interleukin-12 receptor, interleukin-13 receptor, interleukin-15 receptor, interleukin-18 receptor, interleukin-20 receptor, interleukin-21 receptor, interleukin-22 receptor, interleukin-23 receptor, interleukin-27 receptor, interleukin-28 receptor, insulin receptor, transferrin receptor, CD58, CD2, CD 2. The isolated polypeptide or covalent viral adaptor molecule according to any one of embodiments 98-136, which binds to one or more cell surface molecules selected from the list consisting of CD59, CD40, CD72, CD5, CD36, CD19, CD21, CD81, CD27, CD28, CTLA-4, CD85j, CD95, CD96, alpha4beta1 integrin, CD115, CD6, CD178, LFA-1, TNFRSF4, DR4, DR5, RANK / CD265, TACI / CD267, CD267, CD268, CD269, HVEM, PD1 / CD279, B7-1 / CD80, CD278, CD4, CD8, CD19, NMDAR, AMPAR, mGluR5, DRD1, DRD2, Bmp4, GLP1R, leptin receptor, alpha5beta5 integrin and glycoRNA. 138. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 106 to 137, wherein the portion of the adeno-associated viral receptor (AAVR, KIAA0319L) comprises at least a portion of the PKD1 domain, at least a portion of the PKD2 domain, at least a portion of the PKD3 domain, at least a portion of the PKD4 domain, at least a portion of the PKD5 domain, or any combination thereof. 139. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 106 to 137, wherein the portion of the adeno-associated viral receptor (AAVR, KIAA0319L) comprises a PKD1 domain, a PKD2 domain, a PKD3 domain, a PKD4 domain, a PKD5 domain, or any combination thereof. 140. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 106 to 138, wherein the portion of the adeno-associated viral receptor (AAVR, KIAA0319L) comprises a sequence selected from the list consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. 141. An isolated polypeptide or covalent viral adapter molecule according to any one of embodiments 106 to 140, wherein the portion of the adeno-associated viral receptor (AAVR, KIAA0319L) comprises the full-length sequence of the adeno-associated viral receptor (AAVR, KIAA0319L), optionally wherein the full sequence comprises SEQ ID NO:1. 142. A viral particle covalently linked to at least one isolated polypeptide or viral adapter molecule according to any one of embodiments 98 to 141. 143. A viral particle covalently linked to at least two, three, four or five isolated polypeptides or viral adapter molecules according to any one of embodiments 98 to 141. 144. A viral particle according to embodiment 143, having specificity for two or more different target cells. 145. A viral particle according to embodiment 143, having increased tropism for two or more different target cells. 146. A viral particle according to embodiment 144 or 145, wherein two or more target cells express different cell surface molecules. 147. A viral particle according to any one of embodiments 144 to 146, wherein at least two, three, four or five isolated polypeptides or viral adapter molecules specifically bind to different cell surface molecules on two or more target cells. 148. A viral particle according to embodiment 143, having specificity for target cells expressing two or more different cell surface molecules. 149. A viral particle according to embodiment 143, having increased tropism for target cells expressing two or more different cell surface molecules. 150. A viral particle according to embodiment 148 or 149, wherein at least two, three, four or five isolated polypeptides or viral adapter molecules specifically bind to two or more different cell surface molecules on a target cell. 151. A viral particle according to any one of embodiments 142 to 150, wherein one or more viral capsid proteins comprise one or more cysteine ​​residues capable of covalently binding to the isolated polypeptide. 152. A viral particle according to any one of embodiments 142 to 151, wherein one or more viral capsid proteins comprise one or more cysteine ​​residues capable of forming disulfide bridges with one or more cysteine ​​residues in the isolated polypeptide. 153. A viral particle according to any one of embodiments 142 to 152, wherein the virus is an adeno-associated virus, optionally wherein the adeno-associated virus is selected from the list consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh.74. 154. A viral particle according to any one of embodiments 142 to 153, wherein the adeno-associated virus is AAV2. 155. A viral particle according to any one of embodiments 142 to 154, wherein the adeno-associated virus is a wild-type AAV. 156. A viral particle according to any one of embodiments 142 to 155, wherein the adeno-associated virus comprises a portion of a capsid amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19. 157. A viral particle according to any one of embodiments 142 to 156, wherein the adeno-associated virus comprises a full-length capsid amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19. 158. A viral particle according to any one of embodiments 142 to 155, wherein the adeno-associated virus has one or more conservative amino acid changes relative to the wild-type amino acid sequence, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 conservative amino acid changes relative to the wild-type amino acid sequence, and optionally, the wild-type amino acid sequence is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19. 159. A viral particle according to any one of embodiments 142 to 158, wherein the adeno-associated virus has five or fewer non-conservative amino acid changes relative to the wild-type amino acid sequence, such as five, four, three, two or one non-conservative amino acid changes relative to the wild-type amino acid sequence, and optionally, the wild-type amino acid sequence is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19. 160. Viruses include Adenovirus, Aichi virus, Australian bat lyssavirus, BK polyomavirus, Banna virus, Barmah forest virus, Bunyamwera virus, Bunyavirus, Lacrosse bunyavirus, Snowshoe hare bunyavirus, Simian herpesvirus, Chandipura virus, Chikungunya virus, Cosavirus, Cowpox virus, Coxsackievirus, Crimean-Congo hemorrhagic fever virus, Dengue virus, Dori virus, Djugbe virus, Dubenhage virus, Eastern equine encephalitis virus, Ebola virus, Viruses, echoviruses, encephalomyocarditis viruses, Epstein-Barr virus, European bat lyssavirus, GB virus, hepatitis C / G virus, Hantaan virus, Hendra virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, hepatitis delta virus, horsepox virus, human adenovirus, human astrovirus, human coronavirus, human cytomegalovirus, human enterovirus, human herpes virus, human immunodeficiency virus, human papillomavirus, human papilloma virus Viruses, human parainfluenza virus, human parvovirus, human respiratory syncytial virus, human rhinovirus, human SARS coronavirus, human spumaretrovirus, human T-lymphotropic virus, human torovirus, influenza A virus, influenza B virus, influenza C virus, Isfahan virus, JC polyomavirus, Japanese encephalitis virus, Junin arenavirus, KI polyomavirus, Kunjin virus, Lagos bat virus, Lake Victoria Marburg virus, Langat virus, Rat Savirus, Rosedale virus, Louping ill virus, Lymphocytic choriomeningitis virus, Machupo virus, Mayaro virus, MERS coronavirus, Measles virus, Mengo encephalomyocarditis virus, Merkel cell polyomavirus, Mokola virus, Molluscum contagiosum virus, Monkeypox virus, Mumps virus, Murray Valley encephalitis virus, New Jersey polyomavirus, New York virus, Nipah virus, Norwalk virus, O'nyong-nyong virus, Orf virus, Oropouche virus, Pichinde virus, Polio virus,The virus particle according to any one of embodiments 142 to 159, selected from the list consisting of Punta Toro phlebovirus, Puumala virus, Rabies virus, Rift Valley fever virus, Rosavirus A, Ross River virus, Rotavirus A, Rotavirus B, Rotavirus C, Rubella virus, Sagiyama virus, Sarivirus A, Sicilian sandfly fever virus, Sapporo virus, SARS coronavirus, Semliki forest virus, Seoul virus, Sarpholmy virus, Simian virus 40, Sindbis virus, Southampton virus, St. Louis encephalitis virus, Tick-borne Powassan virus, Torque teno virus, Toscana virus, Uukuniemi virus, Vaccinia virus, Varicella zoster virus, Smallpox virus, Venezuelan equine encephalitis virus, Vesicular stomatitis virus, Western equine encephalitis virus, WU polyomavirus, West Nile virus, Yaba monkey tumor virus, Yaba-like disease virus, Yellow fever virus and Zika virus. 161. The virus particle according to embodiment 160, wherein the virus is a wild-type virus. 162. A viral particle according to embodiment 160, wherein the virus is an adenovirus and the viral adapter molecule comprising an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid, or an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid and a ligand, comprises a portion of CD46, and optionally the CD46 sequence is SEQ ID NO: 29. 163. A viral particle according to embodiment 162, wherein the virus is an adenovirus and the isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to the viral capsid, or the viral adapter molecule comprising an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to the viral capsid and a ligand, comprises the full-length sequence of CD46, and optionally the CD46 sequence is SEQ ID NO: 29. 164. A viral particle according to embodiment 160, wherein the virus is an adenovirus and the viral adapter molecule comprising an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid, or an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid and a ligand, comprises a portion of a coxsackievirus-adenovirus receptor (CXADR), and optionally the CXADR sequence is SEQ ID NO: 20. 165. A viral particle according to embodiment 164, wherein the virus is an adenovirus and the viral adapter molecule comprising an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid, or an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid and a ligand, comprises the full-length sequence of a coxsackievirus-adenovirus receptor (CXADR), and optionally, the CXADR sequence is SEQ ID NO: 20. 166. A viral particle according to any one of embodiments 142 to 165, wherein one or more viral capsids have one or more heterologous cysteine ​​residues introduced relative to the wild-type amino acid sequence, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 heterologous cysteine ​​residues introduced relative to the wild-type amino acid sequence. 167. A viral particle according to embodiment 166, in which one or more heterologous cysteine ​​residues are introduced into the interface between the isolated polypeptide or viral adapter molecule and the viral capsid. 168. The virus is AAV1, and the isolated polypeptide or isolated fusion polypeptide comprises a portion of the adeno-associated virus receptor (AAVR, KIAA0319L) capable of covalently binding to an adeno-associated virus capsid; AAV1 comprises one or more mutations selected from Gly266Cys, Thr504Cys, and Asp590Cys; 168. The viral particle according to embodiment 166 or 167, wherein the AAVR polypeptide or fusion polypeptide comprises one or more mutations selected from Thr434Cys, Asp429Cys, and Ser425Cys. 169. The virus is AAV2, and the isolated polypeptide or isolated fusion polypeptide comprises a portion of the adeno-associated virus receptor (AAVR, KIAA0319L) capable of covalently binding to an adeno-associated virus capsid; AAV2 comprises one or more mutations selected from Gly265Cys, Thr503Cys, and Gln589Cys; 168. The viral particle according to embodiment 166 or 167, wherein the AAVR polypeptide or fusion polypeptide comprises one or more mutations selected from Thr434Cys, Asp429Cys, and Ser425Cys. 170. The virus is AAV2, and the isolated polypeptide or isolated fusion polypeptide comprises an antigen-binding portion of the neutralizing antibody A20; AAV2 comprises one or more mutations selected from Ser264Cys, Val708Cys, and Asn717Cys; 168. The viral particle according to embodiment 166 or 167, wherein the neutralizing antibody A20 polypeptide or fusion polypeptide comprises one or more mutations selected from VH Tyr102Cys, VH Ser56Cys, and VL Ile93Cys. 171. The virus is hAdV-11p, and the isolated polypeptide or isolated fusion polypeptide comprises a portion of hCD46; hAdV-11p contains the mutation Asn283Cys 168. The viral particle according to embodiment 166 or 167, wherein hCD46 comprises the mutation Thr42Cys. 172. A viral particle according to any one of embodiments 142 to 171, wherein the adeno-associated viral particle comprises one or more amino acid changes in the capsid protein that mediate interactions with non-protein binders, such as heparan sulfate proteoglycans (HSPGs), O-linked sialic acid, N-linked sialic acid, N-linked galactose, and the like. 173. A pharmaceutical composition comprising a viral particle according to any one of embodiments 142 to 172. 174. A method for covalently modifying a viral particle, comprising: Providing viral particles; Providing an isolated polypeptide capable of covalently binding to a viral capsid of a viral particle; combining the viral particle with the isolated polypeptide such that the isolated polypeptide is covalently attached to the viral particle; A method comprising: 175. A method for covalently modifying a viral particle, comprising the steps of: Providing viral particles; providing a viral adapter molecule comprising an isolated polypeptide and a ligand capable of covalently binding to a viral capsid of a viral particle; combining a viral particle with a viral adapter molecule such that the viral adapter molecule is covalently attached to the viral particle; A method comprising: 176. A method for covalently modifying a viral particle, comprising: Providing viral particles; Providing two or more (e.g., two, three, four, or five) isolated polypeptides capable of covalently binding to a viral capsid of a viral particle; combining a viral particle with two or more isolated polypeptides such that the isolated polypeptides are covalently attached to the viral particle; A method comprising: 177. A method for covalently modifying a viral particle, comprising: Providing viral particles; Providing two or more (e.g., two, three, four, or five) viral adapter molecules comprising an isolated polypeptide and a ligand capable of covalently binding to a viral capsid of a viral particle; combining a viral particle with two or more viral adapter molecules such that the viral adapter molecules are covalently attached to the viral particle; A method comprising: 178. A method for covalently modifying a viral particle according to any one of embodiments 174 to 177, further comprising introducing one or more heterologous cysteine ​​residues, such as one, two or three heterologous cysteine ​​residues, into the isolated polypeptide or viral adapter molecule. 179. A method for covalently modifying a viral particle according to any one of embodiments 174 to 178, further comprising introducing one or more heterologous cysteine ​​residues, such as one, two or three heterologous cysteine ​​residues, into the viral capsid. 180. A method for covalently modifying a viral particle according to any one of embodiments 174 to 179, further comprising introducing one or more heterologous cysteine ​​residues into both the isolated polypeptide or viral adapter molecule and the viral capsid, such as one, two or three heterologous cysteine ​​residues into both the isolated polypeptide or viral adapter molecule and the viral capsid. 181. A method for covalently modifying a viral particle according to any one of embodiments 174 to 180, wherein covalent binding of an isolated polypeptide or a viral adapter molecule to the viral particle reduces or eliminates the native tropism of one or more viral capsid proteins. 182. A method for covalently modifying a viral particle according to any one of embodiments 174 to 180, wherein covalent attachment of an isolated polypeptide or a viral adapter molecule to the viral particle increases the tropism of the viral particle for one or more cell types. 183. A method for covalently modifying a viral particle according to any one of embodiments 174 to 182, wherein the AAV particle has specificity for two or more different target cells. 184. A method for covalently modifying a viral particle according to any one of embodiments 174 to 182, wherein the covalent attachment of an isolated polypeptide or a viral adapter molecule to the viral particle increases the tropism of the viral particle towards two or more different target cells. 185. A method for covalently modifying a viral particle according to embodiment 183 or 184, wherein two or more target cells express different cell surface molecules. 186. A method for covalently modifying a viral particle according to any one of embodiments 183 to 185, wherein at least two, three, four or five isolated polypeptides or viral adapter molecules specifically bind to different cell surface molecules on two or more target cells. 187. A method for covalently modifying a viral particle according to any one of embodiments 174 to 184, wherein the viral particle has specificity for target cells expressing two or more different cell surface molecules. 188. A method for covalently modifying a viral particle according to any one of embodiments 174 to 184, wherein the viral particle has increased tropism for target cells expressing two or more distinct cell surface molecules. 189. A method for covalently modifying a viral particle according to embodiment 187 or 188, wherein at least two, three, four or five isolated polypeptides or viral adapter molecules specifically bind to two or more distinct cell surface molecules on a target cell. 190. A method for covalently modifying a viral particle according to any one of embodiments 174 to 189, wherein the covalent attachment of the isolated polypeptide or viral adapter molecule to the viral particle increases and / or decreases the tropism of the viral particle for one or more cell types selected from the list consisting of neurons, macrophages, microglia, T cells, B cells, dendritic cells, antigen-presenting cells, NK cells, cancer cells, muscle cells, hepatocytes, photoreceptor cells, pancreatic beta cells, renal cells and lung cells. 191. A method for covalently modifying a viral particle according to any one of embodiments 174 to 190, wherein the viral particle has a tropism that is reduced or increased by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% compared to the native tropism of the viral particle not comprising the isolated polypeptide or viral adapter molecule. 192. A method for covalently modifying a viral particle according to any one of embodiments 174 to 191, wherein the covalent attachment of an isolated polypeptide or a viral adapter molecule to the viral particle enables the viral particle to present protein antigens in a multimeric manner using the virus as a scaffold. 193. A method for covalently modifying a viral particle according to any one of embodiments 174 to 192, wherein the covalent attachment of the isolated polypeptide or viral adapter molecule to the viral particle selectively induces cell lysis, and optionally, cell lysis is induced in one or more cell types selected from the list consisting of neurons, macrophages, microglia, T cells, B cells, dendritic cells, antigen-presenting cells, NK cells, cancer cells, muscle cells, hepatocytes, photoreceptor cells, pancreatic beta cells, renal cells and lung cells. 194. A method for targeting a viral particle to a target cell, comprising: Providing viral particles; providing a viral adapter molecule comprising an isolated polypeptide capable of covalently binding to a viral capsid of a viral particle and a ligand specific for a target cell; combining the viral particle with a viral adapter molecule such that the viral adapter molecule is covalently attached to the viral particle, thereby producing a modified viral particle; contacting a mixture of cells including target cells with the modified viral particles; A method comprising: 195. A method for targeting a viral particle to a target cell, comprising: Providing viral particles; Providing two or more (e.g., two, three, four, or five) viral adapter molecules comprising an isolated polypeptide capable of covalently binding to a viral capsid of a viral particle and a ligand specific for a target cell; combining a viral particle with two or more viral adapter molecules such that the viral adapter molecules bind to the viral particle, thereby producing a modified viral particle; contacting a mixture of cells including target cells with the modified viral particles; wherein the target cell expresses two or more distinct cell surface molecules, and optionally the viral adapter molecule specifically binds to two or more distinct cell surface molecules on the target cell. 196. A method for targeting a viral particle to two or more target cells, comprising: Providing viral particles; Providing two or more (e.g., two, three, four, or five) viral adaptor molecules comprising an isolated polypeptide capable of covalently binding to a viral capsid of a viral particle and a ligand specific for at least one of two or more target cells; combining a viral particle with two or more viral adapter molecules such that the viral adapter molecules bind to the viral particle, thereby producing a modified viral particle; contacting a mixture of cells comprising two or more target cells with the modified viral particles; wherein the two or more target cells express two or more different cell surface molecules, and optionally the viral adapter molecule specifically binds to different cell surface molecules on the two or more target cells. 197. A method for covalently modifying a viral particle or targeting a viral particle to a target cell according to any one of embodiments 174 to 196, wherein the isolated polypeptide capable of covalently binding to a viral capsid comprises a portion of the adeno-associated viral receptor (AAVR, KIAA0319L). 198. A method for covalently modifying a viral particle or targeting a viral particle to a target cell according to any one of embodiments 174 to 197, wherein the isolated polypeptide capable of covalently binding to a viral capsid comprises a portion of an adeno-associated virus receptor having one or more point mutations, optionally wherein the one or more point mutations are defined with reference to SEQ ID NO:1. 199. A method for covalently modifying a viral particle or for targeting a viral particle to a target cell according to any one of embodiments 174 to 198, wherein the isolated polypeptide capable of covalently binding to a viral capsid comprises a portion of an adeno-associated virus receptor having one or more point mutations, optionally wherein the one or more point mutations are defined with reference to a sequence selected from the list consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. 200. A method for covalently modifying a viral particle or targeting a viral particle to a target cell according to any one of embodiments 174 to 198, wherein the isolated polypeptide capable of covalently binding to a viral capsid comprises a V480E mutation in the PKD2 domain, and optionally one or more point mutations are defined with reference to SEQ ID NO:1. 201. A method for covalently modifying a viral particle or targeting a viral particle to a target cell according to any one of embodiments 174 to 199, wherein the isolated polypeptide capable of covalently binding to a viral capsid comprises the sequence of SEQ ID NO: 1 having a V480E mutation. 202. A method for covalently modifying a viral particle or targeting a viral particle to a target cell according to any one of embodiments 174 to 200, wherein the isolated polypeptide capable of covalently binding to a viral capsid comprises the sequence of SEQ ID NO: 3 having a V480E mutation. 203. A method for covalently modifying a viral particle or targeting a viral particle to a target cell according to any one of embodiments 174 to 202, wherein the isolated polypeptide capable of covalently binding to a viral capsid comprises an S425C mutation in the PKD2 domain, and optionally one or more point mutations are defined with reference to SEQ ID NO:1. 204. A method for covalently modifying a viral particle or targeting a viral particle to a target cell according to any one of embodiments 174 to 203, wherein the isolated polypeptide capable of covalently binding to a viral capsid comprises the sequence of SEQ ID NO: 1 having the S425C mutation. 205. A method for covalently modifying a viral particle or targeting a viral particle to a target cell according to any one of embodiments 174 to 204, wherein the isolated polypeptide capable of covalently binding to a viral capsid comprises the sequence of SEQ ID NO: 3 having an S425C mutation. 206. A method for covalently modifying a viral particle or targeting a viral particle to a target cell according to any one of embodiments 174 to 205, wherein the isolated polypeptide capable of covalently binding to a viral capsid comprises an S425C mutation and a V480E mutation in the PKD2 domain, and optionally one or more point mutations are defined with reference to SEQ ID NO: 1 or SEQ ID NO: 3. 207. A method for covalently modifying a viral particle or targeting a viral particle to a target cell according to any one of embodiments 174 to 206, wherein the isolated polypeptide capable of covalently binding to the viral capsid comprises at least a portion of the PKD1 domain, at least a portion of the PKD2 domain, at least a portion of the PKD3 domain, at least a portion of the PKD4 domain, at least a portion of the PKD5 domain, or any combination thereof, of the adeno-associated viral receptor (AAVR, KIAA0319L). 208. A method for covalently modifying a viral particle or targeting a viral particle to a target cell according to any one of embodiments 174 to 207, wherein the isolated polypeptide capable of covalently binding to the viral capsid comprises the PKD1 domain, the PKD2 domain, the PKD3 domain, the PKD4 domain, the PKD5 domain of the adeno-associated viral receptor (AAVR, KIAA0319L), or any combination thereof. 209. A method for covalently modifying a viral particle or a method for targeting a viral particle to a target cell according to any one of embodiments 174 to 208, wherein the isolated polypeptide capable of covalently binding to an adeno-associated virus (AAV) capsid comprises a sequence selected from the list consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. 210. A method for covalently modifying a viral particle or targeting a viral particle to a target cell according to any one of embodiments 174 to 209, wherein the isolated polypeptide capable of covalently binding to the viral capsid comprises the full-length sequence of the adeno-associated viral receptor (AAVR, KIAA0319L), optionally wherein the full sequence comprises SEQ ID NO:1. 211. A method for delivering a nucleic acid sequence of interest to a target cell, comprising contacting the target cell with a modified viral particle according to any one of embodiments 142 to 172 or a pharmaceutical composition according to embodiment 173, wherein the viral particle comprises the nucleic acid sequence of interest. 212. The method of embodiment 211, wherein the nucleic acid sequence of interest encodes a protein selected from the list consisting of RAB escort protein 1, RPE65, factor VIII, factor IX, cochlin, CLN7, acid alpha-glucosidase (GAA), aquaporin 1, glial cell line-derived neurotrophic factor, aspartoacylase, aromatic L-amino acid decarboxylase, retinitis pigmentosa GTPase regulator deficiency, sarcoplasmic reticulum calcium ATPase, cyclic nucleotide-gated channel beta 3, neurturin, galactosidase beta 1, glucose-6-phosphatase, phenylalanine hydroxylase, ornithine transcarbamylase, dystrophin and carnitine palmitoyltransferase II, phenylalanine hydroxylase (PAH), cystic fibrosis transmembrane conductance regulator (CFTR). 213. The method according to any one of embodiments 194 to 212, wherein the target cell is in vitro. 214. The method according to any one of embodiments 194 to 212, wherein the target cell is in vivo in a subject. 215. The method according to any one of embodiments 194 to 214, wherein the target cells are selected from the list consisting of neurons, macrophages, microglia, T cells, B cells, dendritic cells, antigen-presenting cells, NK cells, cancer cells, muscle cells, liver cells, photoreceptor cells, pancreatic beta cells, kidney cells and lung cells. 216. The method according to embodiment 214 or 215, wherein the subject is a human. 217. The method according to any one of embodiments 194 to 216, wherein the target cell is a human target cell. 218. A viral particle according to any one of embodiments 142 to 172 or a pharmaceutical composition according to embodiment 173 for use as a medicament. 219. A viral particle according to any one of embodiments 142 to 172 or a pharmaceutical composition according to embodiment 173 for use in a method for treating a disease in a subject in need of treatment, comprising contacting the target cell with the viral particle, thereby delivering a nucleic acid sequence of interest to the target cell. 220. Diseases include neurodegenerative disorders, cancer, Duchenne muscular dystrophy, hemophilia, congenital blindness, diabetes, cystic fibrosis, choroideremia, hemophilia A, hemophilia B, CLN7 disease, Pompe disease, Parkinson's disease, Canavan disease, demyelinating diseases, inherited retinal dystrophies due to RPE65 mutations, aromatic L-amino acid decarboxylase (AADC) deficiency, X-linked retinitis pigmentosa, Leber congenital amaurosis, Churg-Strauss syndrome (CSS), critical limb ischemia, color vision disorders, Alzheimer's disease, macular degeneration, ornithine transcarbamylase deficiency, Wilson's disease, glycogen storage disease type IA, Crigler-Nager syndrome, Tay-Sachs disease, Sandhoff disease, multiple myeloma, multiple system atrophy, gangliosidosis, Danon disease, Fabry disease, Batten disease, phenylketonuria, rheumatoid arthritis, mucopolysaccharidosis type IIIa, Sanfilippo syndrome B, mucopolysaccharidosis type VI, alpha 1-antitrypsin deficiency, spinal muscular atrophy type 1, Krabbe disease, Becker muscular dystrophy, Charcot-Marie-Tooth neuropathy type 1a, carnitine palmitoyltransferase II (CPT II) deficiency and trimethylaminuria, cystic fibrosis, phenylketonuria. 221. A method for treating a disease in a subject in need of treatment, comprising delivering a nucleic acid sequence of interest to a target cell by contacting the target cell with a viral particle, comprising: a) Neuronal cells for the treatment of neurodegenerative disorders; b) immune cells (e.g. macrophages, microglia, T cells, B cells, dendritic cells, antigen presenting cells, NK cells) for the treatment of cancer; c) immune cells (e.g., macrophages, microglia, T cells, B cells, dendritic cells, antigen-presenting cells, NK cells) to elicit an immune response; d) cancer cells or tumor cells for the treatment of cancer; e) muscle cells for the treatment of Duchenne muscular dystrophy; f) Hepatocytes for the treatment of hemophilia; g) Photoreceptor cells for congenital blindness; h) pancreatic beta cells for the treatment of diabetes; or i) Lung cells for the treatment of cystic fibrosis; The viral particle according to any one of embodiments 142 to 172 or the pharmaceutical composition according to embodiment 173, wherein targeting of the designated target cells by the viral particle treats or ameliorates the designated disease. 222. A viral particle or pharmaceutical composition for use according to any one of embodiments 218 to 221, wherein the viral particle or pharmaceutical composition is administered to a subject by aerosol (e.g. to lung tissue), intramuscularly, intraarterially (e.g. via the hepatic artery), intraarticularly, subretinally, intracranially, intravenously, intrathecally, intracoronarily or subcutaneously. 223. A viral particle covalently linked to at least one isolated polypeptide or viral adapter molecule, wherein the isolated polypeptide or viral adapter molecule has an affinity (Kd) between the virus and the virus of less than 100 μM. 224. The viral particle of embodiment 223, wherein there is at least one pair of residues X and Y, where X is part of the isolated polypeptide or viral adapter molecule and Y is part of the viral particle, and the respective beta carbons are less than 5.5 Å apart when the isolated polypeptide or viral adapter molecule is complexed with the viral particle. 225. The virus particle of embodiment 224, wherein residues X and Y can be mutated to cysteine ​​such that the cysteine ​​side chains do not induce major steric clashes that disrupt the interface. 226. The virus particle of embodiment 224 or 225, wherein for the side chains of residues X and Y, after mutation to cysteine, there is an orientation obtained by rotation of the side chains around C(α)-C(β) such that the sulfur atoms at the γ positions of the side chains are less than 2.5 Å apart. 227. A viral particle covalently linked to at least one isolated polypeptide or viral adapter molecule, wherein there is at least one pair of residues X and Y, where X is part of the isolated polypeptide or viral adapter molecule and Y is part of the viral particle, and where the respective beta carbons are less than 5.5 Å apart when the isolated polypeptide or viral adapter molecule is complexed with the viral particle. 228. The virus particle of embodiment 227, wherein residues X and Y can be mutated to cysteine ​​such that the cysteine ​​side chains do not induce major steric clashes that disrupt the interface. 229. The virus particle of embodiment 227 or 228, wherein for the side chains of residues X and Y, after mutation to cysteine, there is an orientation obtained by rotation of the side chains around C(α)-C(β) such that the sulfur atoms at the γ positions of the side chains are less than 2.5 Å apart. 230. A virus particle of any one of embodiments 223 to 229, wherein the angles formed by the atoms C(β)1-S(γ)1-S(γ)2, S(γ)1-S(γ)2-C(β) are not significantly different (e.g., not more than 40% greater) than the optimal angles observed in naturally occurring disulfide bonds. 231. A viral particle covalently linked to at least one isolated polypeptide or viral adaptor molecule, wherein the angles formed by the atoms C(β)1-S(γ)1-S(γ)2, S(γ)1-S(γ)2-C(β) do not differ significantly (e.g., not more than 40% greater) than the optimal angles observed in naturally occurring disulfide bonds. 232. A virus-like particle (VLP) linked to at least one isolated polypeptide or AAV adapter molecule according to any one of embodiments 1 to 31 or at least one isolated polypeptide or viral adapter molecule according to any one of embodiments 98 to 141. 233. A virus-like particle (VLP) linked to at least two, three, four or five isolated polypeptides or AAV adapter molecules according to any one of embodiments 1 to 31 or at least one isolated polypeptide or viral adapter molecule according to any one of embodiments 98 to 141. 234. A virus-like particle (VLP) according to embodiment 233, having specificity for two or more different target cells. 235. A virus-like particle (VLP) according to embodiment 233, having increased tropism for two or more different target cells. 236. A virus-like particle (VLP) according to embodiment 234 or 235, wherein two or more target cells express different cell surface molecules. 237. A virus-like particle (VLP) according to any one of embodiments 233 to 235, wherein at least two, three, four or five isolated polypeptides or AAV adapter molecules specifically bind to different cell surface molecules on two or more target cells. 238. A virus-like particle (VLP) according to embodiment 233, having specificity for target cells expressing two or more different cell surface molecules. 239. A virus-like particle (VLP) according to embodiment 233, having increased targeting for target cells expressing two or more different cell surface molecules. 240. A virus-like particle (VLP) according to embodiment 238 or 239, wherein at least two, three, four or five isolated polypeptides or AAV adapter molecules specifically bind to two or more distinct cell surface molecules on a target cell. 241. A virus-like particle (VLP) according to any one of embodiments 232 to 240, comprising a portion of a capsid amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19. 242. A virus-like particle (VLP) according to any one of embodiments 232 to 241, comprising a full-length capsid amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19. 243. The capsid protein of virus-like particles is expressed in Adenovirus, Aichi virus, Australian bat lyssavirus, BK polyomavirus, Banna virus, Barmah forest virus, Bunyamwera virus, Bunyavirus, Lacrosse bunyavirus, Snowshoe hare bunyavirus, Simian herpesvirus, Chandipura virus, Chikungunya virus, Cosavirus, Cowpox virus, Coxsackievirus, Crimean-Congo hemorrhagic fever virus, Dengue virus, Dhoori virus, Djugbe virus, Dubenhage virus, Eastern U virus, and others. Encephalitis virus, Ebola virus, Echovirus, Encephalomyocarditis virus, Epstein-Barr virus, European bat lyssavirus, GB virus, Hepatitis C / G virus, Hantavirus, Hendra virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Hepatitis Delta virus, Horsepox virus, Human adenovirus, Human astrovirus, Human coronavirus, Human cytomegalovirus, Human enterovirus, Human herpesvirus, Human immunodeficiency virus, Human papillomavirus , human papillomavirus, human parainfluenza virus, human parvovirus, human respiratory syncytial virus, human rhinovirus, human SARS coronavirus, human spumaretrovirus, human T-lymphotropic virus, human torovirus, influenza A virus, influenza B virus, influenza C virus, Isfahan virus, JC polyomavirus, Japanese encephalitis virus, Junin arenavirus, KI polyomavirus, Kunjin virus, Lagos bat virus, Lake Victoria Marburg virus, Langa virus, Viruses: Lassa virus, Rosedale virus, Louping ill virus, Lymphocytic choriomeningitis virus, Machupo virus, Mayaro virus, MERS coronavirus, Measles virus, Mengo encephalomyocarditis virus, Merkel cell polyomavirus, Mokola virus, Molluscum contagiosum virus, Monkeypox virus, Mumps virus, Murray Valley encephalitis virus, New Jersey polyomavirus, New York virus, Nipah virus, Norwalk virus, O'nyong-nyong virus, Orf virus, Oropouche virus, Pichinde virus,Poliovirus, Punta Toro phlebovirus, Puumala virus, Rabies virus, Rift Valley fever virus, Rosavirus A, Ross River virus, Rotavirus A, Rotavirus B, Rotavirus C, Rubella virus, Sagiyama virus, Sarivirus A, Sicilian sandfly fever virus, Sapporo virus, SARS coronavirus, Semliki Forest virus, Seoul virus, Sarfomy virus, Simian virus 40, Sindbis virus, Southampton virus, St. Louis encephalitis virus Virus-like particles (VLPs) according to any one of embodiments 232 to 242, selected from one or more viruses in the list consisting of: varicella-zoster virus, Torque Teno virus, Toscana virus, Ukuniemi virus, Vaccinia virus, Varicella-zoster virus, Smallpox virus, Venezuelan equine encephalitis virus, Vesicular stomatitis virus, Western equine encephalitis virus, WU polyomavirus, West Nile virus, Yaba monkey tumor virus, Yaba-like disease virus, Yellow fever virus and Zika virus. 244. A virus-like particle (VLP) according to any one of embodiments 232 to 243, wherein the virus-like particle has one or more conservative amino acid changes relative to the wild-type viral capsid amino acid sequence, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 conservative amino acid changes relative to the wild-type viral capsid amino acid sequence, optionally wherein the wild-type viral capsid amino acid sequence is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19. 245. A virus-like particle (VLP) according to any one of embodiments 232 to 243, wherein the virus-like particle has no more than 5 non-conservative amino acid changes relative to the wild-type viral capsid amino acid sequence, such as 5, 4, 3, 2 or 1 non-conservative amino acid changes relative to the wild-type viral capsid amino acid sequence, optionally wherein the wild-type viral capsid amino acid sequence is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19. 246. A virus-like particle (VLP) according to any one of embodiments 232 to 245, comprising one or more amino acid changes in the capsid protein that mediate interactions with non-protein binders, such as heparan sulfate proteoglycans (HSPGs), O-linked sialic acid, N-linked sialic acid, N-linked galactose, etc. 247. A virus-like particle (VLP) according to embodiment 246, wherein the one or more mutations include arginine residues 585 and 588 of AAV2. 248. A pharmaceutical composition comprising a virus-like particle (VLP) according to any one of embodiments 232 to 247. 249. A method for modifying a virus-like particle, comprising: Providing a virus-like particle; Providing an isolated polypeptide capable of binding to a virus-like particle capsid; combining the virus-like particle with the isolated polypeptide such that the isolated polypeptide is bound to the virus-like particle; A method comprising: 250. A method for modifying a virus-like particle, comprising: Providing a virus-like particle; providing an AAV adapter molecule comprising an isolated polypeptide and a ligand capable of binding to a virus-like particle capsid; combining a virus-like particle with an AAV adapter molecule such that the isolated fusion polypeptide is attached to the virus-like particle; A method comprising: 251. A method for modifying a virus-like particle, comprising: Providing a virus-like particle; Providing two or more (e.g., two, three, four, or five) isolated polypeptides capable of binding to a virus-like particle capsid; combining the virus-like particle with the isolated polypeptide such that the isolated polypeptide is bound to the virus-like particle; A method comprising: 252. A method for modifying a virus-like particle, comprising: Providing a virus-like particle; Providing two or more (e.g., two, three, four, or five) AAV adapter molecules comprising an isolated polypeptide capable of binding to a virus-like particle capsid and a ligand; combining a virus-like particle with an AAV adapter molecule such that the AAV adapter molecule is attached to the virus-like particle; A method comprising: 253. A method for modifying a virus-like particle according to any one of embodiments 249 to 252, wherein binding of the isolated polypeptide or AAV adapter molecule to the AAV particle reduces or eliminates the native tropism of one or more AAV capsid proteins. 254. A method for modifying a virus-like particle according to any one of embodiments 249 to 253, wherein binding of the isolated polypeptide or AAV adapter molecule to the AAV particle increases the tropism of the AAV particle for one or more cell types. 255. A method for targeting a virus-like particle to a target cell, comprising: Providing a virus-like particle; providing an AAV adapter molecule comprising an isolated polypeptide capable of binding to a virus-like particle capsid and a ligand specific for a target cell; combining a virus-like particle with an AAV adapter molecule such that the AAV adapter molecule binds to the virus-like particle, thereby generating a virus-like particle; contacting a mixture of cells including target cells with the modified virus-like particles; A method comprising: 256. A method for targeting a virus-like particle to a target cell, comprising: Providing a virus-like particle; Providing two or more (e.g., two, three, four, or five) AAV adapter molecules comprising an isolated polypeptide capable of binding to a virus-like particle capsid and a ligand specific for a target cell; combining a virus-like particle with two or more AAV adapter molecules such that the AAV adapter molecules are attached to the virus-like particle, thereby generating a modified virus-like particle; contacting a mixture of cells including target cells with the modified virus-like particles; wherein the target cell expresses two or more distinct cell surface molecules, and optionally the AAV adapter molecule specifically binds to two or more distinct cell surface molecules on the target cell. 257. A method for targeting a virus-like particle to two or more target cells, comprising: Providing a virus-like particle; providing two or more (e.g., two, three, four, or five) AAV adapter molecules comprising an isolated polypeptide capable of binding to a virus-like particle capsid and a ligand specific for at least one of two or more target cells; combining a virus-like particle with two or more AAV adapter molecules such that the AAV adapter molecules are attached to the virus-like particle, thereby generating a modified virus-like particle; contacting a mixture of cells comprising two or more target cells with a modified virus-like particle; wherein the two or more target cells express two or more different cell surface molecules, and optionally the AAV adapter molecule specifically binds to different cell surface molecules on the two or more target cells. 258. A method for covalently modifying a virus-like particle, comprising: Providing a virus-like particle; Providing an isolated polypeptide capable of covalently binding to a viral capsid of a virus-like particle; combining the virus-like particle with the isolated polypeptide such that the isolated polypeptide is covalently attached to the virus-like particle; A method comprising: 259. A method for covalently modifying a virus-like particle, comprising: Providing a virus-like particle; providing a viral adapter molecule comprising an isolated polypeptide and a ligand capable of covalently binding to a viral capsid of a virus-like particle; combining a virus-like particle with a viral adapter molecule such that the viral adapter molecule is covalently attached to the virus-like particle; A method comprising: 260. A method for covalently modifying a virus particle, comprising: Providing a virus-like particle; Providing two or more (e.g., two, three, four, or five) isolated polypeptides capable of covalently binding to a viral capsid of a virus-like particle; combining a virus-like particle with two or more isolated polypeptides such that the isolated polypeptides are covalently attached to the virus-like particle; A method comprising: 261. A method for covalently modifying a virus-like particle, comprising: Providing a virus-like particle; Providing two or more (e.g., two, three, four, or five) viral adaptor molecules comprising an isolated polypeptide and a ligand capable of covalently binding to a viral capsid of a virus-like particle; combining a virus-like particle with two or more viral adapter molecules such that the viral adapter molecules are covalently attached to the virus-like particle; A method comprising: 262. A method for covalently modifying a virus-like particle according to any one of embodiments 258 to 262, further comprising introducing one or more heterologous cysteine ​​residues, such as one, two or three heterologous cysteine ​​residues, into the isolated polypeptide or the viral adapter molecule. 263. A method for covalently modifying a virus-like particle according to any one of embodiments 258 to 263, further comprising introducing one or more heterologous cysteine ​​residues, such as one, two or three heterologous cysteine ​​residues, into the viral capsid of the virus-like particle.

Claims

1. An isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to one or more cysteine ​​residues of a viral capsid having five or fewer amino acid point mutations compared to a wild-type viral capsid at an interface between the isolated polypeptide and the viral capsid, has an affinity (Kd) for the viral capsid of less than 100 μM; When the isolated polypeptide is complexed with the viral capsid, the respective beta carbons of one or more cysteine ​​residues of the isolated polypeptide and one or more cysteine ​​residues of the viral capsid are separated by less than 5.5 Å. Isolated polypeptide.

2. 2. The isolated polypeptide of claim 1, wherein one or more cysteine ​​residues capable of covalently binding to the viral capsid are heterologous to the isolated polypeptide.

3. A viral adapter molecule comprising a fusion protein comprising (i) the isolated polypeptide of claim 1 and (ii) a ligand capable of binding to a cell surface molecule.

4. 4. The viral adapter molecule of claim 3, comprising: (i) an isolated polypeptide comprising one or more cysteine ​​residues capable of covalently binding to a viral capsid; and (ii) a ligand capable of binding to a cell surface molecule, wherein the one or more cysteine ​​residues are heterologous to the isolated polypeptide.

5. 10. The isolated polypeptide of claim 1 or the viral adapter molecule of claim 3, comprising a portion of an adeno-associated virus receptor (AAVR, KIAA0319L) having one or more heterologous cysteine ​​residues capable of forming a covalent disulfide bond with an adeno-associated virus capsid.

6. An isolated polypeptide described in claim 1 or a viral adapter molecule described in claim 3, comprising a portion of a coxsackievirus-adenovirus receptor (CXADR) having one or more heterologous cysteine ​​residues capable of forming a covalent disulfide bond with a viral capsid, and optionally the CXADR sequence is SEQ ID NO:

20.

7. 10. The isolated polypeptide of claim 1 or the viral adapter molecule of claim 3, comprising one or more complementarity determining regions (CDRs) of the neutralizing antibody A20.

8. The ligand is selected from the group consisting of Her2, interleukin-1 receptor, interleukin-2 receptor, interleukin-3 receptor, interleukin-4 receptor, interleukin-5 receptor, interleukin-6 receptor, interleukin-7 receptor, interleukin-8 receptor, interleukin-9 receptor, interleukin-10 receptor, interleukin-11 receptor, interleukin-12 receptor, interleukin-13 receptor, interleukin-15 receptor, interleukin-18 receptor, interleukin-20 receptor, interleukin-21 receptor, interleukin-22 receptor, interleukin-23 receptor, interleukin-27 receptor, interleukin-28 receptor, insulin receptor, and transferrin receptor. , CD58, CD2, CD2, CD59, CD40, CD72, CD5, CD36, CD19, CD21, CD81, CD27, CD28, CTLA-4, CD85j, CD95, CD9 6, α4β1 integrin, CD115, CD6, CD178, LFA-1, TNFRSF4, DR4, DR5, RANK / CD265, TACI / CD267, CD267, CD268, 4. The viral adapter molecule of claim 3, which binds to one or more cell surface molecules selected from the list consisting of CD269, HVEM, PD1 / CD279, B7-1 / CD80, CD278, CD4, CD8, CD19, NMDAR, AMPAR, mGluR5, DRD1, DRD2, Bmp4, GLP1R, leptin receptor, α5β5 integrin, and glycoRNA.

9. 6. The isolated polypeptide or viral adapter molecule of claim 5, wherein the portion of the adeno-associated viral receptor (AAVR, KIAA0319L) comprises at least 80% of the entire length of the PKD1 domain, at least 80% of the entire length of the PKD2 domain, at least 80% of the entire length of the PKD3 domain, at least 80% of the entire length of the PKD4 domain, at least 80% of the entire length of the PKD5 domain, or any combination thereof.

10. 10. A viral particle covalently linked to at least one isolated polypeptide of claim 1 or at least one viral adapter molecule of claim 3.

11. 11. The viral particle of claim 10, wherein the virus is an adeno-associated virus, and optionally the adeno-associated virus is selected from the list consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, and AAVrh.

74.

12. A viral particle as described in claim 10, comprising an unmodified wild-type viral capsid.

13. A viral particle as described in claim 10, comprising a viral capsid having five or fewer (e.g., five, four, three, two or one) amino acid point mutations compared to a wild-type viral capsid.

14. A viral particle as described in claim 10, comprising a viral capsid having an amino acid insertion or deletion of 5 amino acids or less (i.e., 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids or 5 amino acids) relative to a wild-type viral capsid.

15. A pharmaceutical composition comprising the virus particle of claim 10.

16. 1. A method for covalently modifying a viral particle, comprising: Providing viral particles; Providing an isolated polypeptide capable of covalently binding to the viral capsid of the viral particle of claim 1; combining the viral particle with the isolated polypeptide such that the isolated polypeptide is covalently attached to the viral particle; A method comprising:

17. 1. A method for covalently modifying a viral particle, comprising: Providing viral particles; providing a viral adapter molecule comprising an isolated fusion polypeptide capable of covalently binding to a viral capsid of said viral particle and a ligand capable of binding to the cell surface molecule of claim 3; combining the viral particle with the viral adapter molecule such that the isolated fusion polypeptide is covalently attached to the viral particle; A method comprising:

18. 18. A method for covalently modifying a viral particle according to claim 16 or 17, further comprising introducing one or more heterologous cysteine ​​residues, such as one, two or three heterologous cysteine ​​residues, into the isolated polypeptide or viral adapter molecule.

19. 18. A method for covalently modifying a viral particle as described in claim 16 or 17, wherein the covalent attachment of the isolated polypeptide or viral adapter molecule to the viral particle reduces or eliminates the native tropism of one or more viral capsid proteins by at least 50%.

20. 18. A method for covalently modifying a viral particle as described in claim 16 or 17, wherein covalent attachment of the isolated polypeptide or viral adapter molecule to the viral particle increases the tropism of the viral particle for one or more cell types by at least 50%.

21. 16. The pharmaceutical composition of claim 15 for use as a medicament.

22. 16. The pharmaceutical composition of claim 15, for use in a method for treating a disease in a subject in need thereof, comprising contacting the target cell with a viral particle, thereby delivering a nucleic acid sequence of interest to the target cell.

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