Modified viral particles and uses thereof

Abstract

To provide modified viral particles and uses thereof.SOLUTION: Provided herein are compositions and methods for adapting adeno-associated virus (AAV) particles comprising capsids of non-primate animal AAV, remote AAV, or a combination thereof. AAV adapted accordingly may be a viable gene therapy platform for the treatment of a patient in need thereof, and may be particularly useful in patients excluded from current treatment modalities involving current therapeutic AAV particles due to their high titer of antibodies against the current therapeutic AAV particles.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Sequence Listing Reference Reference to sequence listing submitted as a text file via EFS Web The sequence listing set forth in file name 10364WO01_ST25.txt is 269 kilobytes, was created on May 19, 2020, and is incorporated herein by reference.

[0002] The disclosure herein relates to methods of making and using recombinant AAV particles comprising capsid proteins of non-primate AAV and / or remote AAV. [Background technology]

[0003] Gene delivery to specific target cells has become one of the most important technologies in modern medicine for the potential treatment of various chronic and genetic diseases. Currently, the lack of an ideal gene delivery vehicle has limited progress in the clinical application of gene therapy.

[0004] Ideally, a gene delivery vehicle would (1) be capable of stably transferring genetic material into desired cells, (2) avoid transferring the genetic material to non-target cells, and (3) evade neutralization by the patient's immune system, e.g., by the patient's antibodies. Although several non-pathogenic vehicles are currently available, the ability of these vehicles to transduce specific cells and remain hidden from the host immune response is still less than ideal.

[0005] For example, adeno-associated virus (AAV)-based viral particles isolated from primates, particularly humans, such as the AAV serotypes AAV2, AAV4, AAV6, AAV7, AAV8, and AAV9, have been the subject of much research due to their ability to transduce a wide range of primate species and tissues in vivo without overt evidence of toxicity or pathogenicity (Muzyczka, et al. (1992) Current Topics in Microbiology and Immunology, 158:97-129). Furthermore, AAV safely transduces postmitotic tissues. The virus can rarely integrate into host chromosomes, with very rare integration occurring at the safe harbor locus on human chromosome 19, but only when replication (Rep) proteins are supplied in trans. The AAV genome rapidly circularizes and concatenates in infected cells, remaining stable episomal within the infected cells and providing long-term, stable expression of the payload.

[0006] Moreover, in recent years, engineering and redirecting primate AAV infection to specific cells has been demonstrated. Many of the advances in targeted gene therapy using viral particles can be summarized as non-recombinant (non-genetic) or recombinant (genetic) modifications of viral vectors that result in pseudotyping, expansion, and / or retargeting of the native tropism of the viral particle. (Reviewed in Nicklin and Baker (2002) Curr. Gene Ther. 2:273-93; Verheiji and Rottier (2012) Advances Virol 2012:1-15.)

[0007] In direct recombinant targeting, a targeting ligand is directly inserted into or linked to a viral capsid. That is, the viral capsid protein gene is modified to express a capsid protein containing a heterologous targeting ligand. The targeting ligand is then redirected to, for example, bind to, a receptor or marker that is preferentially or exclusively expressed on target cells. (Stachler et al.(2006)Gene Ther.13:926-931;White et al.(2004)Circulation 109:513-519;see also Park et al.,(2007)Frontiers in Bioscience 13:2653-59;Girod et al.(1999)Nature Medicine 5:1052-56;Grifman et al. al. (2001) Molecular Therapy 3:964-75; Shi et al. (2001) Human Gene Therapy 12:1697-1711; Shi and Bartlett (2003) Molecular Therapy 7:515-525).

[0008] In the indirect recombinant approach, the viral capsid is engineered with a heterologous "scaffold" and then linked to an adapter containing a targeting ligand. The adapter binds to the scaffold and to the target cell. (Arnold et al. (2006) Mol. Ther. 5:125-132; Ponnazhagen et al. (2002) J. Virol. 76:12900-907; see also WO 97 / 05266). Scaffolds such as (1) Fc-binding molecules (e.g., Fc receptors, protein A, etc.) that bind to the Fc of an antibody adapter, (2) (strept)avidin that binds to a biotinylated adapter, (3) biotin that binds to an adapter fused to (strept)avidin, (4) detectable labels useful for detecting and / or isolating viral particles, which are attached to a bispecific adapter capable of non-covalently binding to the detectable label and to a target molecule, and recently (5) protein:protein binding pairs that form isopeptide bonds have been reported for various viral particles. (For example, Gigout et al. (2005) Molecular Therapy 11:856-865; Stachler et al. (2008) Molecular Therapy 16:1467-1473; Quetglas et al. (2010) Virus Research 153:179-196; Ohno et al. (1997) Nature Biotechnology 15:763-767; see Klimstra et al. (2005) Virology 338:9-21).

[0009] Despite advances that have provided the ability to direct AAV infection, retargeting AAV as a gene delivery vehicle is far from ideal due to the presence of neutralizing antibodies (NAb) against the AAV capsid. The presence of AAV NAb in children suggests that AAV infection occurs soon after birth (Calcedo et al. (2011) ASGCT; Huser et al. (2017) J. Virol. 91:e02137-16). Antibodies generated by AAV infection soon after birth impair the subsequent use of AAV-derived gene therapy vectors, indicating that these pre-existing antibodies recognize and neutralize the vectors. (Hurlbut et al. (2010) Mol. Ther. 18:1983-94; Jiang et al. (2006) Blood 108:3321-8; Manno et al. (2006) Nat. Med. 12:342-7; Scallan et al. (2006) Blood 107:1810-7; Wang et al. (2010) Mol. Ther. 18:126-34). Furthermore, the presence of neutralizing antibodies against AAV serotypes is clinically significant, and patients with high antibody titers are considered ineligible for any treatment including those against that serotype. (Jeune et al. (2013) Hum Gene Ther Methods 24:59-67). Thus, there remains a need for a viral system that is non-pathogenic, applicable for targeted delivery of nucleic acids of interest to a variety of target cells, and overcomes the hurdles posed by pre-existing antibodies in patients in need of treatment. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Muzyczka, et al. (1992)Current Topics in Microbiology and Immunology,158:97-129) [Non-patent document 2] Nicklin and Baker(2002)Curr.Gene Ther.2:273-93 [Non-patent document 3] Verheiji and Rottier(2012)Advances Virol2012:1-15 [Non-patent document 4] Stachler et al.(2006)Gene Ther.13:926-931 [Non-patent document 5] White et al. (2004)Circulation 109:513-519 [Non-patent document 6] Park et al.,(2007)Frontiers in Bioscience 13:2653-59 [Non-Patent Document 7] Girod et al. (1999) Nature Medicine 5:1052-56 [Non-patent document 8] Grifman et al. (2001) Molecular Therapy 3:964-75 [Non-Patent Document 9] Shi et al. (2001) Human Gene Therapy 12:1697-1711 [Non-Patent Document 10] Shi and Bartlett (2003) Molecular Therapy 7:515-525 Summary of the Invention [Means for solving the problem]

[0011] This paper describes a strategy that can simultaneously alleviate some of the problems associated with past and current adeno-associated virus (AAV) particle therapy.Without wishing to be bound by theory, it is predicted that most humans lack pre-existing NAb against AAV due to low past human exposure.However, the ability of AAV serotypes that have been successfully engineered for use as gene therapy vectors, which have the ability to target and infect specific cells and / or avoid cross-reactivity from any pre-existing antibodies in the human population, has been unknown.

[0012] Herein, we demonstrate that AAV capsid proteins from non-primate species can be modified to target nucleotides of interest into mammalian cells of different species. Furthermore, we demonstrate that such modified AAV particles from non-primate species are less likely to be recognized and / or detected by pre-existing antibodies in the human population than current AAV therapeutic modalities based on well-characterized human AAV serotypes. Thus, we describe recombinant AAV viral particles that can infect selected cells and better evade neutralization by pre-existing antibodies.

[0013] Described herein is a recombinant AAV viral particle comprising: (i) an AAV capsid comprising AAV VP1, VP2, and VP3 capsid proteins; and (ii) a nucleic acid sequence packaged within the AAV capsid comprising an AAV inverted terminal repeat (ITR), a) AAV VP1 capsid protein; b) any portion of the AAV VP1 capsid protein; c) AAV VP2 capsid protein; d) any portion of the AAV VP2 capsid protein; e) AAV VP3 capsid protein, and f) any portion of the AAV VP3 capsid protein, at least one of which comprises an amino acid sequence having significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of a capsid protein or portion thereof of a non-primate AAV or a capsid protein or portion thereof of a remote AAV; I. At least one of the AAV VP1, VP2, and VP3 capsid proteins comprises a modification selected from the group consisting of: (a) a first member of a protein:protein binding pair, wherein the protein:protein binding pair directs the tropism of AAV viral particles; (b) a detectable label; (c) a point mutation, preferably a point mutation that reduces the natural tropism of the AAV viral particle and / or generates a detectable label; (d) a chimeric amino acid sequence, and (e) any combination of (a), (b), (c), and (d); and / or II. The ITR sequence, or a portion thereof, comprises a nucleic acid sequence having significant sequence identity, e.g., at least 95% identity, to an ITR sequence, or a portion thereof, of a second AAV, wherein the second AAV is not identical to a non-primate AAV or a remote AAV; The recombinant AAV viral particles are capable of infecting a mammalian host, preferably a primate host.

[0014] In some embodiments, the recombinant AAV viral particle comprises (i) AAV VP1, VP2, and VP3 capsid proteins, and (ii) a nucleic acid sequence packaged within the AAV capsid comprising an AAV inverted terminal repeat (ITR), a) AAV VP1 capsid protein; b) AAV VP2 capsid protein, and c) at least one of the AAV VP3 capsid proteins is comprising an amino acid sequence that has significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of a capsid protein of a non-primate AAV or a remote AAV; I. At least one of the AAV VP1, VP2, and VP3 capsid proteins comprises a modification selected from the group consisting of: (a) a first member of a protein:protein binding pair, wherein the protein:protein binding pair directs the tropism of AAV viral particles; (b) a detectable label; (c) a point mutation, preferably a point mutation that reduces the natural tropism of the AAV viral particle and / or generates a detectable label; (d) a chimeric amino acid sequence, and (e) any combination of (a), (b), (c), and (d); and / or II. The ITR sequence, or a portion thereof, comprises a nucleic acid sequence having significant sequence identity, e.g., at least 95% identity, to an ITR sequence, or a portion thereof, of a second AAV, wherein the second AAV is not identical to a non-primate AAV or a remote AAV; The recombinant AAV viral particles are capable of infecting a mammalian host, preferably a primate host.

[0015] In some embodiments, the recombinant AAV viral particle comprises (i) AAV VP1, VP2, and VP3 capsid proteins, and (ii) a nucleic acid sequence packaged within the AAV capsid comprising an AAV inverted terminal repeat (ITR), any portion of the AAV VP1 capsid protein; b. any portion of the AAV VP2 capsid protein, and c. any portion of the AAV VP3 capsid protein, at least one of which comprises an amino acid sequence having significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of a capsid protein or portion thereof of a non-primate AAV or a capsid protein or portion thereof of a remote AAV; I. At least one of the AAV VP1, VP2, and VP3 capsid proteins comprises a modification selected from the group consisting of: (a) a first member of a protein:protein binding pair, wherein the protein:protein binding pair directs the tropism of AAV viral particles; (b) a detectable label; (c) a point mutation, preferably a point mutation that reduces the natural tropism of the AAV viral particle and / or generates a detectable label; (d) a chimeric amino acid sequence, and (e) any combination of (a), (b), (c), and (d); and / or II. The ITR sequence, or a portion thereof, comprises a nucleic acid sequence having significant sequence identity, e.g., at least 95% identity, to an ITR sequence, or a portion thereof, of a second AAV, wherein the second AAV is not identical to a non-primate AAV or a remote AAV; The recombinant AAV viral particles are capable of infecting a mammalian host, preferably a primate host.

[0016] In some recombinant AAV viral particle embodiments, the recombinant viral particle comprises: (i) an AAV capsid comprising AAV VP1, VP2, and VP3 capsid proteins; and (ii) a nucleic acid sequence packaged within the AAV capsid comprising an AAV inverted terminal repeat (ITR), wherein at least one of the AAV VP1 capsid protein, any portion of the AAV VP1 capsid protein, the AAV VP2 capsid protein, any portion of the AAV VP2 capsid protein, the AAV VP3 capsid protein, and any portion of the AAV VP3 capsid protein comprises an amino acid sequence having significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of a capsid protein or portion thereof of a non-primate AAV; and wherein at least one of the AAV VP1, VP2, and VP3 capsid proteins comprises a modification selected from the group consisting of: (a) a first member of a protein:protein binding pair, wherein the protein:protein binding pair directs the tropism of AAV viral particles; (b) a detectable label; (c) a point mutation, preferably a point mutation that reduces the natural tropism of the AAV viral particle and / or generates a detectable label; (d) a chimeric amino acid sequence, and (e) any combination of (a), (b), (c), and (d); In this case, the entire ITR sequence or a portion of the ITR sequence comprises a nucleic acid sequence having significant sequence identity, e.g., at least 95% identity, to the ITR of the non-primate AAV; optionally, in this case, the ITR sequence comprises a chimeric nucleic acid sequence, and in this case, the portion of the chimeric nucleic acid sequence having significant sequence identity, e.g., at least 95% identity, to the ITR or portion thereof of the non-primate AAV is operably linked to a portion of the chimeric nucleic acid sequence having significant sequence identity, e.g., at least 95% identity, to the ITR or portion thereof of a second AAV, wherein the second AAV is not identical to the non-primate AAV; and in this case, the recombinant AAV viral particle has the ability to infect a mammalian host, preferably a primate host.

[0017] In some embodiments, the recombinant AAV viral particle comprises: (i) an AAV capsid comprising AAV VP1, VP2, and VP3 capsid proteins; and (ii) a nucleic acid sequence packaged within the AAV capsid comprising an AAV inverted terminal repeat (ITR), wherein at least one of the AAV VP1 capsid protein, any portion of the AAV VP1 capsid protein, the AAV VP2 capsid protein, any portion of the AAV VP2 capsid protein, the AAV VP3 capsid protein, and any portion of the AAV VP3 capsid protein comprises an amino acid sequence having significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of a capsid protein or portion thereof of a non-primate AAV; wherein the ITR sequence, or portion thereof, comprises a nucleic acid sequence having significant sequence identity, e.g., at least 95% identity, to an ITR sequence, or portion thereof, of a second AAV, wherein the second AAV is not identical to the non-primate AAV; The recombinant AAV viral particle is capable of infecting a mammalian host, preferably a primate host; and Optionally, at least one of the AAV VP1, VP2, and VP3 capsid proteins comprises a modification selected from the group consisting of: (a) a first member of a protein:protein binding pair, wherein the protein:protein binding pair directs the tropism of AAV viral particles; (b) a detectable label; (c) a point mutation, preferably a point mutation that reduces the natural tropism of the AAV viral particle and / or generates a detectable label; and (d) Any combination of (a) to (c).

[0018] In some embodiments, the recombinant AAV viral particle comprises (i) an AAV capsid comprising AAV VP1, VP2, and VP3 capsid proteins, and (ii) a nucleic acid sequence packaged within the AAV capsid comprising an AAV inverted terminal repeat (ITR), wherein the AAV VP1 capsid protein, any portion of the AAV VP1 capsid protein, the AAV VP2 capsid protein, any portion of the AAV VP2 capsid protein, the AAV VP3 capsid protein, and the AAV At least one of any portion of the VP3 capsid protein comprises a chimeric amino acid sequence comprising: (A) an amino acid sequence having significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of a non-primate AAV capsid protein or portion thereof; (A) is operably linked to (B) an amino acid sequence having significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of a second AAV capsid protein or portion thereof, wherein the second AAV is not identical to the non-primate AAV; and wherein the recombinant AAV viral particle has the ability to infect a mammalian host, preferably a primate host; and optionally, an AAV VP1 capsid protein, any portion of the AAV VP1 capsid protein, AAV VP2 capsid protein, any portion of the AAV VP2 capsid protein, AAV VP3 capsid protein, and AAV At least one of any portion of the VP3 capsid protein further comprises a modification selected from the group consisting of: (a) protein: the first member of a protein binding pair; (b) a detectable label, and (c) A combination of (a) and (b).

[0019] In some recombinant AAV viral particle embodiments, the recombinant viral particle comprises (i) an AAV capsid comprising AAV VP1, VP2, and VP3 capsid proteins, and (ii) a nucleic acid sequence packaged within the AAV capsid comprising an AAV inverted terminal repeat (ITR), wherein at least one of the AAV VP1 capsid protein, any portion of the AAV VP1 capsid protein, the AAV VP2 capsid protein, any portion of the AAV VP2 capsid protein, the AAV VP3 capsid protein, and any portion of the AAV VP3 capsid protein comprises an amino acid sequence having significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of a capsid protein or portion thereof of a remote AAV, and wherein at least one of the AAV VP1, VP2, and VP3 capsid proteins comprises a modification selected from the group consisting of: (a) a first member of a protein:protein binding pair, wherein the protein:protein binding pair directs the tropism of AAV viral particles; (b) a detectable label; (c) a point mutation, preferably a point mutation that reduces the natural tropism of the AAV viral particle and / or generates a detectable label; (d) a chimeric amino acid sequence, and (e) any combination of (a), (b), (c), and (d); In this case, the entire ITR sequence or a portion of the ITR sequence comprises a nucleic acid sequence having significant sequence identity, e.g., at least 95% identity, to the ITR of the remote AAV; optionally, in this case, the ITR sequence comprises a chimeric nucleic acid sequence, and in this case, the portion of the chimeric nucleic acid sequence having significant sequence identity, e.g., at least 95% identity, to the ITR or portion thereof of the remote AAV is operably linked to a portion of the chimeric nucleic acid sequence having significant sequence identity, e.g., at least 95% identity, to the ITR or portion thereof of a second AAV, wherein the second AAV is not identical to the remote AAV; and in this case, the recombinant AAV viral particle is capable of infecting a mammalian host, preferably a primate host.

[0020] In some embodiments, the recombinant AAV viral particle comprises (i) an AAV capsid comprising AAV VP1, VP2, and VP3 capsid proteins, and (ii) a nucleic acid sequence packaged within the AAV capsid comprising an AAV inverted terminal repeat (ITR), wherein at least one of the AAV VP1 capsid protein, any portion of the AAV VP1 capsid protein, the AAV VP2 capsid protein, any portion of the AAV VP2 capsid protein, the AAV VP3 capsid protein, and any portion of the AAV VP3 capsid protein comprises an amino acid sequence having significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of a capsid protein or portion thereof of a remote AAV; wherein the ITR sequence, or portion thereof, comprises a nucleic acid sequence having significant sequence identity, e.g., at least 95% identity, to an ITR sequence, or portion thereof, of a second AAV, wherein the second AAV is not identical to the remote AAV; The recombinant AAV viral particle is capable of infecting a mammalian host, preferably a primate host; and Optionally, at least one of the AAV VP1, VP2, and VP3 capsid proteins comprises a modification selected from the group consisting of: (a) a first member of a protein:protein binding pair, wherein the protein:protein binding pair directs the tropism of AAV viral particles; (b) a detectable label; (c) a point mutation, preferably a point mutation that reduces the natural tropism of the AAV viral particle and / or generates a detectable label; and (d) Any combination of (a) to (c).

[0021] In some embodiments, the recombinant AAV viral particle comprises (i) an AAV capsid comprising AAV VP1, VP2, and VP3 capsid proteins, and (ii) a nucleic acid sequence packaged within the AAV capsid comprising an AAV inverted terminal repeat (ITR), wherein the AAV VP1 capsid protein, any portion of the AAV VP1 capsid protein, the AAV VP2 capsid protein, any portion of the AAV VP2 capsid protein, the AAV VP3 capsid protein, and the AAV at least one of any portion of the VP3 capsid protein comprises a chimeric amino acid sequence comprising (A) an amino acid sequence having significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of a remote AAV capsid protein or portion thereof, wherein (A) is operably linked to (B) an amino acid sequence having significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of a second AAV capsid protein or portion thereof, wherein the second AAV is not identical to the remote AAV, wherein the recombinant AAV viral particle is capable of infecting a mammalian host, preferably a primate host; and optionally, at least one of the AAV VP1 capsid protein, any portion of the AAV VP1 capsid protein, AAV VP2 capsid protein, any portion of the AAV VP2 capsid protein, AAV VP3 capsid protein, and any portion of the AAV VP3 capsid protein comprising the chimeric amino acid sequence further comprises a modification selected from the group consisting of: (a) protein: the first member of a protein binding pair; (b) a detectable label, and (c) A combination of (a) and (b).

[0022] In some embodiments of the present invention, the AAV viral particle comprises an AAV capsid, wherein the AAV capsid comprises at least one AAV capsid protein (e.g., AAV VP1 capsid protein, AAV VP2 capsid protein, and / or AAV VP3 capsid protein). VP3 capsid protein) comprises at least a portion of the amino acid sequence of a capsid protein selected from the group consisting of a capsid protein of a non-primate AAV, a capsid protein of a remote AAV, and combinations thereof, wherein at least one AAV capsid protein of the AAV capsid is modified to comprise: (a) at least a first member of a protein:protein binding pair; (b) a detectable label; (c) a point mutation; (d) a chimeric amino acid sequence comprising a portion of the amino acid sequence of another AAV capsid protein, e.g., a second, operably linked to the amino acid sequence of a capsid protein selected from the group consisting of a capsid protein of a non-primate AAV, a capsid protein of a remote AAV, or combinations thereof; and (e) any combination of (a), (b), (c), and (d).In some embodiments of the present invention, the AAV viral particle comprises an AAV capsid, wherein the AAV capsid or portion thereof comprises at least one AAV capsid protein (e.g., AAV VP1 capsid protein, AAV VP2 capsid protein, and / or AAV VP3 capsid protein). VP3 capsid protein) has significant sequence identity, e.g., at least 95% identity, to a capsid protein selected from the group consisting of a capsid protein of a non-primate AAV, a portion of a capsid protein of a non-primate AAV, a capsid protein of a remote AAV, a portion of a capsid protein of a remote AAV, and combinations thereof, wherein at least one AAV capsid protein of the AAV capsid is modified to comprise: (a) at least a first member of a protein:protein binding pair; (b) a detectable label; (c) a point mutation; (d) a chimeric amino acid sequence comprising a portion of the amino acid sequence of another AAV capsid protein, e.g., a second, operably linked to the amino acid sequence of a capsid protein selected from the group consisting of a capsid protein of a non-primate AAV, a capsid protein of a remote AAV, or combinations thereof; and (e) any combination of (a), (b), (c), and (d).In some embodiments of the present invention, the AAV viral particle comprises an AAV capsid, wherein the AAV capsid comprises at least one AAV capsid protein (e.g., AAV VP1 capsid protein, AAV VP2 capsid protein, and / or AAV VP3 capsid protein). VP3 capsid protein) comprises at least a portion of the amino acid sequence of a capsid protein of a non-primate AAV (e.g., where at least one AAV capsid protein comprises an amino acid sequence having significant sequence identity, e.g., at least 95% identity, to a capsid protein of a non-primate AAV), where at least one AAV capsid protein of the AAV capsid is modified to comprise: (a) at least a first member of a protein:protein binding pair; (b) a detectable label; (c) a point mutation; (d) a chimeric amino acid sequence comprising a portion of the amino acid sequence of another, e.g., a second, AAV capsid protein operably linked to the amino acid sequence of the capsid of the non-primate AAV; and (e) any combination of (a), (b), (c), and (d).

[0023] In some embodiments of the present invention, the AAV viral particle comprises an AAV capsid, wherein the AAV capsid comprises at least one AAV capsid protein (e.g., AAV VP1 capsid protein, AAV VP2 capsid protein, and / or AAV VP3 capsid protein). VP3 capsid protein) comprises at least a portion of the amino acid sequence of a capsid protein of a remote AAV (e.g., where at least one AAV capsid protein comprises an amino acid sequence having significant sequence identity, e.g., at least 95% identity, to a capsid protein of a remote AAV), where at least one AAV capsid protein of the AAV capsid is engineered to comprise: (a) at least a first member of a protein:protein binding pair; (b) a detectable label; (c) a point mutation; (d) a chimeric amino acid sequence comprising a portion of the amino acid sequence of another, e.g., a second, AAV capsid protein operably linked to the amino acid sequence of the capsid protein of the remote AAV; and (e) any combination of (a), (b), (c), and (d).

[0024] In some embodiments of the invention, an AAV viral particle comprises: (A) at least one AAV capsid protein, e.g., an AAV VP1 capsid protein, an AAV VP2 capsid protein, and / or an AAV VP3 capsid protein, that comprises an amino acid sequence that is identical to, or has significant identity, e.g., at least 95% sequence identity to, an amino acid sequence selected from the group consisting of: (i) the amino acid sequence of a capsid protein of a non-primate AAV, (ii) the amino acid sequence of a capsid protein of a remote primate AAV, and (iii) a combination thereof; and (B) an AAV genome that includes an AAV ITR that includes a nucleotide of interest and at least a portion of, e.g., a second, ITR sequence of another AAV, where the other AAV is not identical to the non-primate AAV or to the remote primate AAV.

[0025] In some embodiments of the invention, an AAV viral particle comprises: (A) at least one AAV capsid protein (e.g., an AAV VP1 capsid protein, an AAV VP2 capsid protein, and / or an AAV VP3 capsid protein) that comprises an amino acid sequence that is identical or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a capsid protein of a non-primate AAV; and (B) an AAV genome that includes AAV ITRs that include nucleotides of interest and at least a portion of the ITR sequence of another AAV, e.g., a second AAV, where the other AAV is not identical to the non-primate AAV.

[0026] In some embodiments of the invention, the AAV viral particle comprises: (A) at least one AAV capsid protein (e.g., an AAV VP1 capsid protein, an AAV VP2 capsid protein, and / or an AAV VP3 capsid protein) that comprises an amino acid sequence that is identical or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of the capsid protein of the remote AAV; and (B) an AAV genome that includes AAV ITRs that include nucleotides of interest and at least a portion of the ITR sequence of another AAV, e.g., a second AAV, where the other AAV is not identical to the remote primate AAV.

[0027] In some AAV viral particle embodiments of the present invention, the capsid protein comprising an amino acid sequence that is identical or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a capsid protein of a non-primate AAV, the amino acid sequence of a capsid protein of a remote AAV, or a combination thereof, is engineered to comprise (a) at least a first member of a protein:protein binding pair, (b) a detectable label, and (c) a point mutation.

[0028] In some AAV viral particle embodiments of the present invention, the capsid protein comprising an amino acid sequence that is identical or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a capsid protein of a non-primate AAV, a capsid protein of a remote AAV, or a combination thereof comprises the amino acid sequence of a VP3 capsid protein of a non-primate AAV and / or an amino acid sequence that is identical or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a VP3 capsid protein of a remote AAV. In some embodiments, the capsid protein comprising an amino acid sequence that is identical or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a capsid protein of a non-primate AAV, a capsid protein of a remote AAV, or a combination thereof comprises the amino acid sequence of a VP2 capsid protein of a non-primate AAV and / or an amino acid sequence that is identical or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a VP2 capsid protein of a remote AAV. In some embodiments, the capsid protein comprising an amino acid sequence that is identical or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a capsid protein of a non-primate AAV, a capsid protein of a remote AAV, or a combination thereof comprises the amino acid sequence of a VP1 capsid protein of a non-primate AAV and / or an amino acid sequence that is identical or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a VP1 capsid protein of a remote AAV.

[0029] In some embodiments of the AAV viral particles of the invention, the capsid of the particle comprises a VP1 capsid protein that is either (i) (a) a chimeric AAV VP1 capsid protein, optionally comprising a VP1-unique region (VP1-u) of another AAV, e.g., a second AAV, operably linked to a VP1 / VP2 common region, and a VP3 region of a non-primate AAV or a remote AAV, or (b) a VP1 capsid protein of a non-primate AAV or a remote AAV; A VP2 capsid protein, optionally the chimeric VP2 capsid protein comprising either (a) a VP2 capsid protein comprising the VP1 / VP2 common region of another AAV, e.g., a second AAV, operably linked to the VP3 region of the non-primate AAV or remote AAV, or (b) a VP2 capsid protein of a non-primate AAV or remote AAV, and (iii) a VP3 capsid protein of a non-primate AAV or remote AAV. In some embodiments, the capsid of the particle comprises: (i) a chimeric AAV VP1 capsid protein, optionally comprising a VP1-unique region (VP1-u) of another AAV, e.g., a second AAV, operably linked to a VP1 / VP2 common region, and a VP3 region of the non-primate or remote AAV; (ii) a chimeric AAV VP2 capsid protein, optionally comprising a VP1 / VP2 common region of another AAV, e.g., a second AAV, operably linked to a VP3 region of the non-primate or remote AAV; and (iii) a VP3 capsid protein of the non-primate or remote AAV.In some embodiments, the capsid of the particle comprises (i) a chimeric AAV VP1 capsid protein, optionally comprising a VP1-unique region (VP1-u) of another AAV, e.g., a second AAV, operably linked to a VP1 / VP2 common region, and a VP3 region of the non-primate AAV or remote AAV, (ii) a VP2 capsid protein of the non-primate AAV or remote AAV, and (iii) a VP3 capsid protein of the non-primate AAV or remote AAV. In some embodiments, the capsid comprises (i) a VP1 capsid protein of the non-primate or remote AAV, (ii) a VP2 capsid protein of the non-primate or remote AAV, and (iii) a VP3 capsid protein of the non-primate or remote AAV, and optionally in this case the particle comprises within the capsid an AAV genome comprising AAV ITRs that comprise at least a portion of the ITR sequences of another AAV, e.g., a second AAV. In some embodiments, the other AAV is not identical to the non-primate AAV.

[0030] In some recombinant AAV viral particle embodiments, (i) the VP1 capsid protein comprises either (a) a chimeric amino acid sequence, wherein optionally the VP1-unique region (VP1-u) of the chimeric AAV VP1 capsid protein comprises an amino acid sequence having significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of the VP1-u of a second AAV, and wherein the VP1 / VP2 common region and VP3 region of the chimeric AAV VP1 capsid comprise amino acid sequences having significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of the VP1 / VP2 common region and VP3 region of a non-primate AAV, or (b) an amino acid sequence having significant sequence identity, e.g., at least 95% identity, to the VP1 capsid protein of the non-primate AAV; and (ii) the VP2 capsid protein comprises either (a) a chimeric amino acid sequence, wherein optionally the VP1-unique region (VP1-u) of the chimeric AAV VP1 capsid protein comprises an amino acid sequence having significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of the VP1-u of a second AAV, and wherein the VP1 / VP2 common region and VP3 region of the chimeric AAV VP1 capsid comprise amino acid sequences having significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of the VP1 / VP2 common region and VP3 region of a non-primate AAV. The VP1 / VP2 common region of the VP2 capsid protein comprises an amino acid sequence that has significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of the VP1 / VP2 common region of a second AAV, in which case the VP3 region of the chimeric VP2 capsid protein comprises either (a) an amino acid sequence that has at least 95% identity to the VP3 region of a non-primate AAV, or (b) an amino acid sequence that has significant sequence identity, e.g., at least 95% identity, to the VP2 capsid protein of a non-primate AAV, and (iii) the VP3 capsid protein comprises an amino acid sequence that has significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of a non-primate AAV VP3 capsid protein.In some embodiments, (i) the VP1 capsid protein comprises a chimeric amino acid sequence, and optionally the VP1-unique region (VP1-u) of the chimeric AAV VP1 capsid protein comprises an amino acid sequence having significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of VP1-u of a second AAV, wherein the VP1 / VP2 common region and VP3 region of the chimeric AAV VP1 capsid comprise amino acid sequences having significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of the VP1 / VP2 common region and VP3 region of a non-primate AAV; and (ii) the VP2 capsid protein comprises a chimeric amino acid sequence, and optionally the VP2 capsid protein comprises a chimeric amino acid sequence. The VP1 / VP2 common region of the VP2 capsid protein comprises an amino acid sequence having significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of the VP1 / VP2 common region of a second AAV, in which case the VP3 region of the chimeric VP2 capsid protein comprises at least 95% identity to the VP3 region of a non-primate AAV, and (iii) the VP3 capsid protein comprises an amino acid sequence having significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of the VP3 capsid protein of a non-primate AAV.In some embodiments, (i) the AAV VP1 capsid protein comprises a chimeric amino acid sequence, and optionally the VP1-unique region (VP1-u) of the chimeric AAV VP1 capsid protein comprises an amino acid sequence that has significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of VP1-u of a second AAV, in which case the chimeric AAV The VP1 / VP2 common region and VP3 region of the VP1 capsid comprise amino acid sequences that have significant sequence identity, e.g., at least 95% identity, to the amino acid sequences of the VP1 / VP2 common region and VP3 region of a non-primate AAV; (ii) the VP2 capsid protein comprises an amino acid sequence that has significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of the VP2 capsid protein of a non-primate AAV; and (iii) the VP3 capsid protein comprises an amino acid sequence that has significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of the VP3 capsid protein of a non-primate AAV. In some embodiments, (i) the VP1 capsid protein comprises an amino acid sequence that has significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of the VP1 capsid protein of a non-primate AAV, (ii) the VP2 capsid protein comprises an amino acid sequence that has significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of the VP2 capsid protein of a non-primate AAV, and (iii) the VP3 capsid protein comprises an amino acid sequence that has significant sequence identity, e.g., at least 95% identity, to the amino acid sequence of the VP3 capsid protein of a non-primate AAV, and optionally in this case the particle comprises within the capsid an AAV genome that includes AAV ITRs that include at least a portion of the ITR sequence of another AAV, e.g., a second AAV. In some embodiments, the other AAV is not identical to the non-primate AAV.

[0031] In some embodiments of the AAV virus particle of the present invention, other AAV (for example, second AAV) is primate AAV or a combination of primate AAV.In some embodiments, other AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, and combinations thereof.In some embodiments, other AAV is AAV2.

[0032] In some AAV viral particle embodiments of the present invention, the non-primate AAV is a non-primate AAV listed in Table 2. In some embodiments, the non-primate AAV is an avian AAV (AAAV), a sea lion AAV, or a bearded dragon AAV. In some embodiments, the non-primate AAV is an AAAV, and optionally the amino acid sequence of the AAAV capsid protein comprises a modification at position I444 or I580 of the VP1 capsid protein of AAAV. In some embodiments, the non-primate AAV is a Sphagnum AAV, such as, for example, a Bearded Dragon AAV, and optionally the amino acid sequence of a Bearded Dragon AAV comprises a modification at position I573 or I436 of the VP1 capsid protein of a Bearded Dragon AAV. In some embodiments, the non-primate AAV is a mammalian AAV, such as a sea lion AAV, and optionally the amino acid sequence of the sea lion AAV comprises a modification at a position selected from the group consisting of I429, I430, I431, I432, I433, I434, I436, I437, and A565 of the VP1 capsid protein of the sea lion AAV.

[0033] In some AAV viral particle embodiments of the invention, the protein:protein binding pair is selected from SpyTag:SpyCatcher, SpyTag:KTag, Isopeptag:pilin C, SnoopTag:SnoopCatcher, and SpyTag002:SpyCatcher002. In some embodiments, the first member of the protein:protein binding pair comprises c-myc comprising the sequence set forth as SEQ ID NO: 44. In some embodiments, the detectable label comprises a B1 epitope comprising the amino acid sequence of IGTRYLTR (SEQ ID NO: 45).

[0034] In some embodiments, the AAV particles of the present invention comprise a VP3 capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, wherein the VP3 capsid protein is modified to include (a) at least a first member of a protein:protein binding pair, optionally the protein:protein binding pair is selected from the group consisting of SpyTag:SpyCatcher, SpyTag:KTag, Isopeptag:pilin C, SnoopTag:SnoopCatcher, and SpyTag002:SpyCatcher002; (b) a detectable label, optionally the detectable label comprises the amino acid sequence set forth as SEQ ID NO:44 or the amino acid sequence set forth as SEQ ID NO:45; (c) a point mutation; or (d) any combination of (a), (b), and (c). In some embodiments, the VP3 capsid protein of the non-primate AAV, the remote AAV, or a combination thereof is modified to include at least (a) a SpyTag comprising the amino acid sequence set forth as SEQ ID NO: 43, and / or (b) a detectable label comprising the amino acid sequence set forth as SEQ ID NO: 45.

[0035] In some embodiments, the AAV particles of the present invention comprise a first and / or second linker that operably links a first member of a protein:protein binding pair and / or a detectable label to a capsid protein of the capsid of the AAV particle. In some embodiments, the first and second linkers are not identical. In some embodiments, the first and second linkers are identical. In some embodiments, the first and second linkers are 10 amino acids in length.

[0036] In some embodiments of the viral particles of the present invention, at least one of the VP1, VP2, and VP3 capsid proteins, optionally at least the VP3 capsid, is modified to include (a) a first member of a protein:protein binding pair, (b) a detectable label, (c) a point mutation, or (d) any combination of (a), (b), and / or (c). In some embodiments, the first member of the protein:protein binding pair and / or the detectable label or the point mutation is located within a variable region of the capsid protein. In some embodiments, the first member of the protein:protein binding pair or the detectable label is flanked by a first linker and / or a second linker. In some embodiments, the first and / or second linker are 1 to 10 amino acids in length. In some embodiments, the first and second linkers are not identical. In some embodiments, the first and second linkers are identical.

[0037] In some viral particle embodiments, the AAAV VP3 capsid protein comprises a modification, optionally a first member of a protein:protein binding pair, optionally in which the modification is at position 1444 (e.g., G444) and / or 1580 (e.g., K580). In some embodiments, the AAAV VP3 capsid protein comprises a modification, optionally a first member of a protein:protein binding pair, optionally in which the modification is at position 1444 (e.g., G444) and / or 1580 (e.g., K580). In some embodiments, the bearded dragon AAV VP3 capsid protein comprises a modification, optionally a first member of a protein:protein binding pair, optionally in which the modification is at position 1573 (e.g., T573) and / or 1436 (e.g., G436). In some embodiments, the sea lion VP3 capsid protein comprises a modification, optionally a first member of a protein:protein binding pair, optionally in which the modification is at a position selected from the group consisting of I429 (e.g., N429), I430 (e.g., P430), I431 (e.g., T431), I432 (e.g., G432), I433 (e.g., S433), I434 (e.g., T434), I436 (e.g., R436), I437 (e.g., D437), and I565 (A565).

[0038] In some embodiments of viral particles of the invention, at least one capsid protein, optionally at least the VP3 capsid, is modified to comprise a first member of a protein:protein binding pair. In some embodiments, the first member of the protein:protein binding pair comprises a first member of a protein:protein binding pair. In some embodiments, the first member of the protein:protein binding pair comprises a second cognate member of the protein:protein binding pair. In some embodiments, the first and second members of the protein:protein binding pair are linked by a covalent bond, e.g., an isopeptide bond. In some embodiments, the first member of the protein:protein binding pair is SpyTag, and optionally, the second member of the protein:protein binding pair is SpyCatcher or KTag. In some embodiments, the first member of the protein:protein binding pair is KTag, and optionally, the second member of the protein:protein binding pair is SpyTag. In some embodiments, the first member of the protein:protein binding pair is SnoopTag, and the second member of the protein:protein binding pair is SnoopCatcher. In some embodiments, the first member of the protein:protein binding pair is isopeptag and the second member of the protein:protein binding pair is Pilin-C. In some embodiments, the first member of the protein:protein binding pair is SpyTag002 and the second member of the protein:protein binding pair is SpyCatcher002. In some embodiments, the second member of the protein:protein binding pair is linked to a targeting ligand, e.g., a binding moiety such as an antibody or fragment thereof. In some embodiments, the targeting ligand may be fused to the second member of the protein:protein binding pair, e.g., SpyCatcher, at the C-terminus of the second member, optionally via a linker, which is fused to SpyCatcher at the C-terminus of the linker. In some embodiments, the linker comprises the sequence GSGESG (SEQ ID NO: 49).In some embodiments, the first member of the protein:protein binding pair comprises a detectable label. In some embodiments, the first member of the protein:protein binding pair comprises a detectably labeled c-myc.

[0039] In some viral particle embodiments of the invention, at least one capsid protein, optionally at least the VP3 capsid, is modified to include a detectable label. In some embodiments, the detectable label includes an AAV B1 epitope, such as, for example, the amino acid sequence of IGTRYLTR (SEQ ID NO:45).

[0040] In some viral particle embodiments of the invention, at least one capsid protein, optionally at least the VP3 capsid, is modified to: (a) a first member of a protein:protein binding pair comprising at least one member of a protein:protein binding pair, optionally wherein the protein:protein binding pair is selected from the group consisting of SpyTag:SpyCatcher, SpyTag:KTag, Isopeptag:pilin-C, SnoopTag:SnoopCatcher, SpyTag002:SpyCatcher002, and c-myc:anti-c-myc antibody; (b) a detectable label, optionally in which case the detectable label comprises the amino acid sequence set forth as SEQ ID NO: 44 or the amino acid sequence set forth as SEQ ID NO: 45; (c) point mutation, or (d) any combination of (a), (b), and (c).

[0041] In some viral particle embodiments of the invention, at least one capsid protein, optionally at least the VP3 capsid, is modified to: (a) a protein comprising at least a SpyTag:protein binding pair comprising the amino acid sequence set forth as SEQ ID NO:43, and / or (b) a detectable label comprising the amino acid sequence set forth as SEQ ID NO:45.

[0042] In some embodiments, the AAV particles of the invention comprise: (a) the amino acid sequence set forth as SEQ ID NO:2; (b) the amino acid sequence set forth as SEQ ID NO:4; (c) the amino acid sequence set forth as SEQ ID NO:6, (d) the amino acid sequence set forth as SEQ ID NO:8, (e) the amino acid sequence set forth as SEQ ID NO:10, (f) the amino acid sequence set forth as SEQ ID NO:12, (g) the amino acid sequence set forth as SEQ ID NO:14, (h) the amino acid sequence set forth as SEQ ID NO:16, (i) the amino acid sequence set forth as SEQ ID NO:18, (j) the amino acid sequence set forth as SEQ ID NO:20, (k) the amino acid sequence set forth as SEQ ID NO:22, (l) the amino acid sequence set forth as SEQ ID NO:24, (m) the amino acid sequence set forth as SEQ ID NO:26, (n) the amino acid sequence set forth as SEQ ID NO:28, (o) the amino acid sequence set forth as SEQ ID NO:30, (p) the amino acid sequence set forth as SEQ ID NO:32, (q) the amino acid sequence set forth as SEQ ID NO:34, (r) the amino acid sequence set forth as SEQ ID NO:36, (s) the amino acid sequence set forth as SEQ ID NO:53, (t) the amino acid sequence set forth as SEQ ID NO:55, (u) the amino acid sequence set forth as SEQ ID NO:57, (v) the amino acid sequence set forth as SEQ ID NO:59 amino acid sequence, (w) the amino acid sequence set forth as SEQ ID NO: 61, (x) the amino acid sequence set forth as SEQ ID NO: 63, (y) the amino acid sequence set forth as SEQ ID NO: 65, (z) the amino acid sequence set forth as SEQ ID NO: 67, (aa) the amino acid sequence set forth as SEQ ID NO: 69, (bb) the amino acid sequence set forth as SEQ ID NO: 71, (cc) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, The capsid protein comprises an amino acid sequence selected from the group consisting of an amino acid sequence having at least 95% identity to SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71, and the amino acid sequence of any VP2 portion and / or VP3 portion of the amino acid sequence set forth in any of (dd)(a) to (cc).

[0043] In some embodiments of the viral particles of the present invention, at least one of the AAV VP1, VP2, and VP3 capsid proteins comprises a modification, e.g., a first member of a protein:protein binding pair, and the viral particle optionally further comprises a reference capsid protein that is a capsid protein corresponding to at least one of the AAV VP1, VP2, and VP3 capsid proteins, excluding the modification, thereby making the capsid a mosaic capsid. In some embodiments, the mosaic capsid comprises a VP1 capsid protein modified with a first member of a protein:protein binding pair and a reference VP1 capsid protein. In some embodiments, the mosaic capsid comprises a VP2 capsid protein modified with a first member of a protein:protein binding pair and a reference VP2 capsid protein. In some embodiments, the mosaic capsid comprises a VP3 capsid protein modified with a first member of a protein:protein binding pair and a reference VP3 capsid protein.

[0044] Also described are viral particles of the invention comprising AAV capsid proteins of the invention. In some embodiments of the invention, the AAV capsid proteins of the invention comprise an amino acid sequence that is identical, or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a capsid protein of a non-primate AAV or a remote AAV, wherein the AAV capsid protein is selected from the group consisting of: (a) a chimeric AAV VP1 capsid protein, optionally wherein the chimeric animal AAV VP1 capsid protein has been modified to include at least a first member of a protein:protein binding pair, a detectable label, and / or a point mutation; (b) a non-chimeric AAV VP1 capsid protein, optionally wherein the chimeric animal AAV VP1 capsid protein has been modified to include at least a first member of a protein:protein binding pair and / or a detectable label; and (c) a chimeric VP2 capsid protein, optionally wherein the chimeric AAV The VP2 capsid protein is selected from the group consisting of: (a) a VP2 capsid protein that has been modified to include at least a first member of a protein:protein binding pair, a detectable label, and / or a point mutation; (b) a non-chimeric AAV VP2 capsid protein that has been modified to include at least a first member of a protein:protein binding pair, a detectable label, and / or a point mutation; (c) a chimeric AAV VP3 capsid protein that has been modified to include at least a first member of a protein:protein binding pair, a detectable label, and / or a point mutation; and (d) a non-chimeric AAV VP3 capsid protein that has been modified to include at least a first member of a protein:protein binding pair, a detectable label, and / or a point mutation.

[0045] In some AAV capsid protein embodiments of the invention, the first member of the protein:protein binding pair and / or the detectable label are flanked on one or both sides by first and / or second linkers, respectively, that connect the first member of the protein:protein binding pair and / or the detectable label to the capsid protein, wherein the first and / or second linkers are each independently at least one amino acid in length. In some embodiments, the first and second linkers are not identical. In some embodiments, the first and second linkers are identical and are 10 amino acids in length.

[0046] In some embodiments, the AAV capsid proteins of the invention comprise a detectable label, optionally in which case the detectable label comprises a B1 epitope comprising the amino acid sequence set forth as SEQ ID NO: 45. In some embodiments, the detectable label comprises c-myc.

[0047] In some embodiments, the AAV capsid protein of the invention comprises both a first member and a second cognate member of a protein:protein binding pair, optionally in which the first and second members are linked by a covalent bond, optionally an isopeptide bond. In some embodiments, the first member of the protein:protein binding pair is SpyTag, and optionally the second cognate member is SpyCatcher or KTag. In some embodiments, the first member is KTag, and the second cognate member is SpyTag. In some embodiments, the first member is SnoopTag, and the second cognate member is SnoopCatcher. In some embodiments, the first member is isopeptag, and the second cognate member is Pilin-C. In some embodiments, the first member is SpyTag002, and the second cognate member is SpyCatcher002. In some embodiments, the first member comprises a detectable label, such as, but not limited to, c-myc, in which case the binding pair is an anti-c-myc antibody or portion thereof. In some embodiments, the second member is operably linked to a targeting ligand, optionally in which case the targeting ligand is a binding moiety that optionally targets a cell marker. In some embodiments, the binding moiety is an antibody or portion thereof. In some embodiments, the binding moiety is operably linked to the second member of the protein:protein binding pair, optionally via a covalent bond (such as, but not limited to, an isopeptide bond) or a linker. In some embodiments, the binding moiety is fused to the second member of the protein:protein binding pair via a linker fused at the C-terminus of the binding moiety, wherein the linker is fused to the second member at the C-terminus of the linker, optionally in which case the linker comprises the sequence set forth as SEQ ID NO:49 (GSGESG). In some embodiments, the first member of the protein:protein binding pair is fused to VR I, VR II, VR III, VR IV ... The capsid protein is optionally located at an amino acid position present in VR III, VR IV, VR V, VR VI, VR VII, VR VIII VR IX, or the HI loop, optionally in VR VIII or VR IV of the capsid protein.

[0048] In some AAV capsid protein embodiments of the present invention, the non-primate AAV is a non-primate AAV listed in Table 2. In some embodiments, the non-primate AAV is an avian AAV (AAAV), a sea lion AAV, or a bearded dragon AAV. In some embodiments, the non-primate AAV is an AAAV, and optionally the amino acid sequence of the AAAV capsid protein comprises a modification at position I444 or I580 of the VP1 capsid protein of AAAV. In some embodiments, the non-primate AAV is a Sphingidae AAV, such as, for example, a Bearded Dragon AAV, and optionally the amino acid sequence of a Bearded Dragon AAV comprises a modification at position I573 or I436 of the VP1 capsid protein of a Bearded Dragon AAV. In some embodiments, the non-primate AAV is a mammalian AAV, such as a sea lion AAV, and optionally the amino acid sequence of the sea lion AAV comprises a modification at a position selected from the group consisting of I429, I430, I431, I432, I433, I434, I436, I437, and A565 of the VP1 capsid protein of the sea lion AAV.

[0049] The non-primate VP3 capsid proteins of the present invention (a) encapsidate the genome of another AAV, e.g., a second AAV, and / or (b) comprise a mutated non-primate VP3 capsid. In some embodiments, the non-human AAV VP3 capsid proteins of the present invention encapsidate the genome of a second AAV that is not a non-primate. In some embodiments, the non-primate AAV VP3 capsid proteins of the present invention may be operably linked to a first member of a protein:protein binding pair (optionally via a first and / or second linker) and / or may comprise a point mutation, e.g., such that the capsid protein's natural targeting property is reduced or abolished and / or such that the capsid protein comprises a detectable label. In some embodiments, the first member of the protein:protein binding pair comprises a detectable label. In some embodiments, the first member of the protein:protein binding pair comprises a detectable label, including c-myc (SEQ ID NO: 44). In some embodiments, the first member of the protein:protein binding pair comprises a first member and, optionally, a second member of the protein:protein binding pair that form a covalent bond. In some embodiments, the protein:protein binding pair is selected from the group consisting of (a) SpyTag:SpyCatcher, (b) SpyTag:KTag, (c) Isopeptag:pilin C, (d) SnoopTag:SnoopCatcher, and I SpyTag002:SpyCatcher002. In some embodiments, the VP3 capsid protein of the non-primate AAV may comprise (a) the B1 epitope (SEQ ID NO: 45), (b) SpyTag, (c) SpyCatcher, or any combination of (a)-(c).

[0050] In some embodiments, the VP3 capsid protein of a non-primate AAV of the invention comprises a first member of a protein:protein binding pair operably linked to the capsid protein, optionally via a first or second linker. In some embodiments, the first member of the protein:protein binding pair is operably linked to the VP3 capsid of a non-primate AAV at an amino acid position that is present in a variable region (VR) of the VP3 capsid protein, or a portion thereof, optionally in which case the first member of the protein:protein binding pair is linked to the VP3 capsid via a first and / or second linker. In some embodiments, a first member of a protein:protein binding pair is operably linked to a non-primate AAV VP3 capsid at an amino acid position that is present in VR I, VR II, VR III, VR IV, VR V, VR VI, VR VII, VR VIII VR IX, or the HI loop of the VP3 capsid protein, optionally in which case the first member of the protein:protein binding pair is linked to the VP3 capsid via a first and / or second linker. In some embodiments, a first member of a protein:protein binding pair is operably linked to a non-primate AAV VP3 capsid at an amino acid position that is present in VR VIII or VR IV of the VP3 capsid protein, optionally in which case the first member of the protein:protein binding pair is linked to the VP3 capsid via a first and / or second linker. In some embodiments, the VP3 capsid protein of a non-primate AAV is a VP3 capsid protein of a non-primate AAV selected from the non-primate AAVs presented in Table 2. In some embodiments, the VP3 capsid protein of a non-primate AAV is a VP3 capsid protein of an avian AAV (AAAV). In some embodiments, the VP3 capsid protein of the AAAV comprises a first member of a protein:protein binding pair (e.g., a SpyTag) operably linked at position I444 or I580, optionally via a first and / or second linker.In some embodiments, the VP3 capsid protein of the non-primate AAV is the VP3 capsid protein of a bearded dragon AAV. In some embodiments, the VP3 capsid protein of a bearded dragon AAV comprises a first member of a protein:protein binding pair (e.g., a SpyTag) operably linked at position I573 or I436, optionally via a first and / or second linker. In some embodiments, the VP3 capsid protein of the non-primate AAV is the VP3 capsid protein of a sea lion AAV. In some embodiments, the VP3 capsid protein of a sea lion AAV comprises a first member of a protein:protein binding pair (e.g., a SpyTag) operably linked at a position selected from the group consisting of I429, I430, I431, I432, I433, I434, I436, I437, and I565, optionally at a position selected from the group consisting of I429, I430, I431, I432, I433, I436, and I437, optionally at position I432, optionally via a first and / or second linker.

[0051] The non-primate VP2 capsid proteins of the present invention (a) encapsidate the genome of another AAV, e.g., a second AAV, and / or (b) comprise a mutated non-primate VP2 capsid. In some embodiments, the non-primate AAV VP2 capsid proteins of the present invention encapsidate the genome of another AAV, e.g., a second AAV. In some embodiments, the non-primate AAV VP2 capsid proteins of the present invention may be operably linked to a first member of a protein:protein binding pair and / or may comprise a point mutation, e.g., such that the capsid protein's natural tropism is reduced or abolished and / or such that the capsid protein comprises a detectable label. In some embodiments, the first member of the protein:protein binding pair comprises a detectable label. In some embodiments, the first member of the protein:protein binding pair comprises a detectable label comprising c-myc (SEQ ID NO: 44). In some embodiments, the first member of the protein:protein binding pair comprises a first member and, optionally, a second member of the protein:protein binding pair that form a covalent bond. In some embodiments, the protein:protein binding pair is selected from the group consisting of (a) SpyTag:SpyCatcher, (b) SpyTag:KTag, (c) Isopeptag:pilin C, (d) SnoopTag:SnoopCatcher, and (e) SpyTag002:SpyCatcher002. In some embodiments, the VP2 capsid protein of the non-primate AAV may comprise (a) the B1 epitope (SEQ ID NO: 45), (b) SpyTag, (c) SpyCatcher, or any combination of (a)-(c).

[0052] In some embodiments, the VP2 capsid protein of a non-primate AAV of the invention comprises a first member of a protein:protein binding pair operably linked to the capsid protein, optionally via a first and second linker. In some embodiments, the first member of the protein:protein binding pair is operably linked to the VP2 capsid of a non-primate AAV at an amino acid position that is present in a variable region (VR) of the VP2 capsid protein, or a portion thereof, optionally in which case the first member of the protein:protein binding pair is linked to the VP2 capsid via a first and / or second linker. In some embodiments, a first member of a protein:protein binding pair is operably linked to a non-primate AAV VP2 capsid at an amino acid position that is present in VR I, VR II, VR III, VR IV, VR V, VR VI, VR VII, VR VIII VR IX, or the HI loop of the VP2 capsid protein, optionally in which case the first member of the protein:protein binding pair is linked to the VP2 capsid via a first and / or second linker. In some embodiments, a first member of a protein:protein binding pair is operably linked to a non-primate AAV VP2 capsid at an amino acid position that is present in VR VIII or VR IV of the VP2 capsid protein, optionally in which case the first member of the protein:protein binding pair is linked to the VP2 capsid via a first and / or second linker. In some embodiments, the VP2 capsid protein of a non-primate AAV is a VP2 capsid protein of a non-primate AAV selected from the non-primate AAVs presented in Table 2. In some embodiments, the VP2 capsid protein of a non-primate AAV is a VP2 capsid protein of an avian AAV (AAAV). In some embodiments, the VP2 capsid protein of the AAAV comprises a first member of a protein:protein binding pair (e.g., a SpyTag) operably linked at position I444 or I580, optionally via a first and / or second linker.In some embodiments, the VP2 capsid protein of the non-primate AAV is the VP2 capsid protein of a bearded dragon AAV. In some embodiments, the VP2 capsid protein of a bearded dragon AAV comprises a first member of a protein:protein binding pair (e.g., a SpyTag) operably linked at position I573 or I436, optionally via a first and / or second linker. In some embodiments, the VP2 capsid protein of the non-primate AAV is the VP2 capsid protein of a sea lion AAV. In some embodiments, the VP2 capsid protein of a sea lion AAV comprises a first member of a protein:protein binding pair (e.g., a SpyTag) operably linked at a position selected from the group consisting of I429, I430, I431, I432, I433, I434, I436, I437, and I565, optionally at a position selected from the group consisting of I429, I430, I431, I432, I433, I436, and I437, optionally at position I431, optionally via a first and / or second linker.

[0053] In some embodiments, a VP2 capsid protein of the invention can be a chimeric VP2 capsid protein comprising a portion of a VP2 capsid protein from a non-primate AAV and a portion of a VP2 capsid protein from another AAV, e.g., a second AAV, operably linked together. In some embodiments, the chimeric VP2 capsid protein comprises, from N-terminus to C-terminus, (a) a portion of a VP2 capsid protein from another AAV, e.g., a second AAV, and (b) a portion of the VP2 capsid of a non-primate AAV that includes the amino acid sequence of at least the VP3 capsid protein of the non-primate AAV, wherein (a) is operably linked to (b). In some embodiments, the chimeric VP2 capsid protein may comprise, from N- to C-terminus, (a) the amino acid sequence of the VP1 / VP2 common region of another AAV, and (b) the amino acid sequence of the VP3 capsid protein of a non-primate AAV, wherein (a) is operably linked to (b). In some embodiments, the other AAV is a non-primate AAV. In some other embodiments, the other AAV is a primate AAV.

[0054] In some embodiments, the chimeric VP2 capsid protein of the present invention comprises (a) a portion of the VP2 capsid protein of a primate AAV, and (b) a portion of the VP2 capsid of a non-primate AAV comprising the amino acid sequence of at least the VP3 capsid protein of the non-primate AAV, wherein (a) is operably linked to (b). In some embodiments, the chimeric VP2 capsid protein may comprise, from N-terminus to C-terminus, (a) the amino acid sequence of the VP1 / VP2 common region of a primate AAV, and (b) the amino acid sequence of the VP3 capsid protein of a non-primate AAV, wherein (a) is operably linked to (b). In some embodiments, the primate AAV is AAV1. In some embodiments, the primate AAV is AAV2. In some embodiments, the primate AAV is AAV3. In some embodiments, the primate AAV is AAV4. In some embodiments, the primate AAV is AAV5. In some embodiments, the primate AAV is AAV6. In some embodiments, the primate AAV is AAV7. In some embodiments, the primate AAV is AAV8. In some embodiments, the primate AAV is AAV9. In some embodiments, the non-primate AAV is selected from the group of non-primate AAVs presented in Table 2. In some embodiments, the non-primate AAV is an avian AAV, a bearded dragon AAV, or a sea lion AAV.

[0055] In some embodiments, a chimeric VP2 capsid protein of the invention comprises (a) a portion of the VP2 capsid protein of AAV2, and (b) a portion of the VP2 capsid of a non-primate AAV that comprises the amino acid sequence of at least the VP3 capsid protein of the non-primate AAV, wherein (a) is operably linked to (b). In some embodiments, a chimeric VP2 capsid protein may comprise, from N-terminus to C-terminus, (a) the amino acid sequence of the VP1 / VP2 common region of AAV2, and (b) the amino acid sequence of the VP3 capsid protein of the non-primate AAV, wherein (a) is operably linked to (b).

[0056] In some embodiments, a chimeric AAV2 / AAAV VP2 capsid protein of the invention comprises (a) a portion of the VP2 capsid protein of AAV2, and (b) a portion of the VP2 capsid protein of an avian AAV (AAAV) that comprises the amino acid sequence of at least the VP3 capsid protein of AAAV, wherein (a) is operably linked to (b). In some embodiments, a chimeric AAV2 / AAAV VP2 capsid protein may comprise, from N-terminus to C-terminus, (a) the amino acid sequence of the VP1 / VP2 common region of AAV2, and (b) the amino acid sequence of the VP3 capsid protein of AAAV, wherein (a) is operably linked to (b).

[0057] In some embodiments, a chimeric AAV2 / sea lion AAV VP2 capsid protein of the invention comprises (a) a portion of the VP2 capsid protein of AAV2, and (b) a portion of the VP2 capsid protein of a sea lion AAV that comprises the amino acid sequence of at least the VP3 capsid protein of a sea lion AAV, wherein (a) is operably linked to (b). In some embodiments, a chimeric AAV2 / sea lion AAV VP2 capsid protein may comprise, from N-terminus to C-terminus, (a) the amino acid sequence of the VP1 / VP2 common region of AAV2, and (b) the amino acid sequence of the VP3 capsid protein of a sea lion AAV, wherein (a) is operably linked to (b).

[0058] In some embodiments, a chimeric AAV2 / bearded dragon AAV VP2 capsid protein of the invention comprises (a) a portion of the VP2 capsid protein of AAV2, and (b) a portion of the VP2 capsid protein of a bearded dragon AAV that comprises the amino acid sequence of at least the VP3 capsid protein of a bearded dragon AAV, wherein (a) is operably linked to (b). In some embodiments, a chimeric AAV2 / bearded dragon AAV VP2 capsid protein may comprise, from N-terminus to C-terminus, (a) the amino acid sequence of the VP1 / VP2 common region of AAV2, and (b) the amino acid sequence of the VP3 capsid protein of a bearded dragon AAV, wherein (a) is operably linked to (b).

[0059] In some embodiments, a chimeric VP2 capsid protein of the invention may be operably linked to a first member of a protein:protein binding pair and / or may comprise a point mutation, e.g., such that the capsid protein's natural targeting property is reduced or abolished and / or such that the capsid protein comprises a detectable label. In some embodiments, the first member of the protein:protein binding pair comprises a detectable label. In some embodiments, the first member of the protein:protein binding pair comprises a detectable label comprising c-myc (SEQ ID NO: 44). In some embodiments, the first member of the protein:protein binding pair comprises a first member and, optionally, a second member of the protein:protein binding pair that form a covalent bond. In some embodiments, the protein:protein binding pair is selected from the group consisting of (a) SpyTag:SpyCatcher, (b) SpyTag:KTag, (c) Isopeptag:pilin C, (d) SnoopTag:SnoopCatcher, and (e) SpyTag002:SpyCatcher002. In some embodiments, the chimeric VP2 capsid protein may comprise (a) the B1 epitope (SEQ ID NO: 45), (b) SpyTag, (c) SpyCatcher, or any combination of (a)-(c).

[0060] In some embodiments, a chimeric primate / non-primate VP2 capsid protein of the invention (e.g., a chimeric AAV2 / AAAV VP2 capsid protein, a chimeric AAV2 / sea lion AAV VP2 capsid protein, a chimeric AAV2 / bearded dragon AAV VP2 capsid protein, etc.) comprises a first member of a protein:protein binding pair operably linked to the capsid protein, optionally via a first or second linker. In some embodiments, a first member of a protein:protein binding pair is operably linked to a chimeric primate / non-primate VP2 capsid protein (e.g., a chimeric AAV2 / AAAV VP2 capsid protein, a chimeric AAV2 / sea lion AAV VP2 capsid protein, a chimeric AAV2 / bearded dragon AAV VP2 capsid protein, etc.) at an amino acid position that is present in a variable region (VR) or portion thereof of the chimeric primate / non-primate VP2 capsid protein, optionally in which case the first member of the protein:protein binding pair is linked to the chimeric primate / non-primate VP2 capsid protein via a first and / or second linker. In some embodiments, a first member of a protein:protein binding pair is operably linked to a chimeric primate / non-primate VP2 capsid protein at an amino acid position that is present in VR I, VR II, VR III, VR IV, VR V, VR VI, VR VII, VR VIII VR IX, or the HI loop of the chimeric primate / non-primate VP2 capsid protein, optionally in which case the first member of the protein:protein binding pair is linked to the VP2 capsid via a first and / or second linker.In some embodiments, a first member of a protein:protein binding pair is operably linked to a chimeric primate / non-primate VP2 capsid protein (e.g., a chimeric AAV2 / AAAV VP2 capsid protein, a chimeric AAV2 / sea lion AAV VP2 capsid protein, a chimeric AAV2 / bearded dragon AAV VP2 capsid protein, etc.) at an amino acid position that is present in VR VIII or VR IV of the chimeric primate / non-primate VP2 capsid protein, optionally in which case the first member of the protein:protein binding pair is linked to the chimeric primate / non-primate VP2 capsid protein via a first and / or second linker. In some embodiments, the chimeric AAV2 / AAAV VP2 capsid protein comprises a first member of a protein:protein binding pair (e.g., SpyTag) operably linked, optionally via a first and / or second linker, at position 1444 or 1580. In some embodiments, the chimeric AAV2 / bearded dragon AAV VP2 capsid protein comprises a first member of a protein:protein binding pair (e.g., SpyTag) operably linked, optionally via a first and / or second linker, at position 1573 or 1436. In some embodiments, the chimeric AAV2 / sea lion AAV VP2 capsid protein comprises a first member of a protein:protein binding pair (e.g., a SpyTag) operably linked at a position selected from the group consisting of I429, I430, I431, I432, I433, I434, I436, I437, and I565, optionally at a position selected from the group consisting of I429, I430, I431, I432, I433, I436, and I437, optionally at position I431, optionally via a first and / or second linker.

[0061] Non-primate VP1 capsid proteins of the present invention (a) encapsidate the genome of another AAV, e.g., a second AAV, and / or (b) comprise a mutated non-primate VP1 capsid. In some embodiments, non-primate AAV VP1 capsid proteins of the present invention encapsidate the genome of another AAV, e.g., a second AAV. In some embodiments, non-primate VP1 capsid proteins of the present invention may be operably linked to a first member of a protein:protein binding pair and / or may comprise a point mutation, e.g., such that the capsid protein's natural tropism is reduced or abolished and / or such that the capsid protein comprises a detectable label. In some embodiments, the first member of the protein:protein binding pair comprises a detectable label. In some embodiments, the first member of the protein:protein binding pair comprises a detectable label, including c-myc (SEQ ID NO: 44). In some embodiments, the first member of the protein:protein binding pair comprises a first member and, optionally, a second member of the protein:protein binding pair that form a covalent bond. In some embodiments, the protein:protein binding pair is selected from the group consisting of (a) SpyTag:SpyCatcher, (b) SpyTag:KTag, (c) Isopeptag:pilin C, (d) SnoopTag:SnoopCatcher, and (e) SpyTag002:SpyCatcher002. In some embodiments, the VP3 capsid protein of the non-primate AAV may comprise (a) the B1 epitope (SEQ ID NO: 45), (b) SpyTag, (c) SpyCatcher, or any combination of (a)-(c).

[0062] In some embodiments, a first member of a protein:protein binding pair is operably linked to a VP1 capsid of a non-primate AAV of the invention at an amino acid position that is present in a variable region (VR) of the VP1 capsid protein, or a portion thereof, optionally in which case the first member of the protein:protein binding pair is linked to the VP1 capsid via a first and / or second linker. In some embodiments, the first member of the protein:protein binding pair is operably linked to a VP1 capsid of a non-primate AAV of the invention at an amino acid position that is present in a variable region (VR) of the VP1 capsid protein, or a portion thereof, optionally in which case the first member of the protein:protein binding pair is linked to the VP1 capsid via a first and / or second linker. In some embodiments, the first member of the protein:protein binding pair is operably linked to the non-primate AAV VP1 capsid at an amino acid position present in VR II, VR III, VR IV, VR V, VR VI, VR VII, VR VIII VR IX, or the HI loop, optionally in which case the first member of the protein:protein binding pair is linked to the VP1 capsid via a first and / or second linker. In some embodiments, the first member of the protein:protein binding pair is operably linked to the non-primate AAV VP1 capsid at an amino acid position present in VR VIII or VR IV of the VP1 capsid protein, optionally in which case the first member of the protein:protein binding pair is linked to the VP1 capsid via a first and / or second linker. In some embodiments, the non-primate AAV VP1 capsid protein is a non-primate AAV VP1 capsid protein selected from the group of non-primate AAVs presented in Table 2. In some embodiments, the VP1 capsid protein of the non-primate AAV is the VP1 capsid protein of an avian AAV (AAAV). In some embodiments, the VP1 capsid protein of the AAAV comprises a first member of a protein:protein binding pair (e.g., SpyTag) operably linked at position I444 or I580, optionally via a first and / or second linker. In some embodiments, the VP1 capsid protein of the non-primate AAV is the VP31 capsid protein of a bearded dragon AAV. In some embodiments, the VP1 capsid protein of a bearded dragon AAV comprises a first member of a protein:protein binding pair (e.g., SpyTag) operably linked at position I573 or I436, optionally via a first and / or second linker. In some embodiments, the VP1 capsid protein of non-primate AAV is the VP1 capsid protein of sea lion AAV.In some embodiments, the VP1 capsid protein of a sea lion AAV comprises a first member of a protein:protein binding pair (e.g., a SpyTag) operably linked at a position selected from the group consisting of I429, I430, I431, I432, I433, I434, I436, I437, and I565, optionally at a position selected from the group consisting of I429, I430, I431, I432, I433, I436, and I437, optionally at position I431, optionally via a first and / or second linker.

[0063] In some other embodiments, the VP1 capsid protein of the present invention may be a chimeric VP1 capsid protein, comprising a portion of a VP1 capsid protein from a non-primate AAV and a portion of a VP1 capsid protein from another AAV, operably linked together, where the other AAV is not a non-primate AAV. In some embodiments, the chimeric VP1 capsid protein comprises, from N-terminus to C-terminus, (a) a portion of a VP1 capsid protein from another AAV, the portion comprising at least the PLA2 domain of the other AAV, and (b) a portion of a VP1 capsid protein from a non-primate AAV, the portion comprising the amino acid sequence of at least the VP3 capsid protein of the non-primate AAV. In some embodiments, the chimeric VP1 capsid protein comprises, from N-terminus to C-terminus, (a) a portion of the VP1 capsid protein of another AAV, including at least the VP1-u domain of the other AAV, and (b) a portion of the VP1 capsid of a non-primate AAV, including at least the amino acid sequence of the VP3 capsid protein of the non-primate AAV. In some embodiments, the chimeric VP1 capsid protein comprises, from N-terminus to C-terminus, (a) the amino acid sequence of the VP1-u domain and VP1 / VP2 common region of another AAV, e.g., a second AAV, and (b) the amino acid sequence of the VP3 capsid protein of the non-primate AAV. In some embodiments, the other AAV is a non-primate AAV. In some other embodiments, the other AAV is a primate AAV.

[0064] In some embodiments, the chimeric VP1 capsid protein comprises, from N-terminus to C-terminus, (a) a portion of the VP1 capsid protein of a primate AAV, the portion comprising at least the PLA2 domain of the primate AAV, and (b) a portion of the VP1 capsid of a non-primate AAV, the portion comprising the amino acid sequence of at least the VP3 capsid protein of the non-primate AAV. In some embodiments, the chimeric VP1 capsid protein comprises, from N-terminus to C-terminus, (a) a portion of the VP1 capsid protein of a primate AAV, the portion comprising at least the VP1-u domain of the primate AAV, and (b) a portion of the VP1 capsid of a non-primate AAV, the portion comprising the amino acid sequence of at least the VP3 capsid protein of the non-primate AAV. In some embodiments, the chimeric VP1 capsid protein comprises, from N-terminus to C-terminus, (a) the amino acid sequence of the VP1-u domain and VP1 / VP2 common region of a primate AAV, and (b) the amino acid sequence of the VP3 capsid protein of a non-primate AAV. In some embodiments, the primate AAV is AAV1. In some embodiments, the primate AAV is AAV2. In some embodiments, the primate AAV is AAV3. In some embodiments, the primate AAV is AAV4. In some embodiments, the primate AAV is AAV5. In some embodiments, the primate AAV is AAV6. In some embodiments, the primate AAV is AAV7. In some embodiments, the primate AAV is AAV8. In some embodiments, the primate AAV is AAV9. In some embodiments, the non-primate AAV is selected from the group of non-primate AAVs presented in Table 2. In some embodiments, the non-primate AAV is an avian AAV, a bearded dragon AAV, or a sea lion AAV.

[0065] In some embodiments, the chimeric VP1 capsid protein comprises, from N-terminus to C-terminus, (a) a portion of the VP1 capsid protein of AAV2 comprising at least the PLA2 domain of AAV2, and (b) a portion of the VP1 capsid of a non-primate AAV comprising the amino acid sequence of at least the VP3 capsid protein of the non-primate AAV. In some embodiments, the chimeric VP1 capsid protein comprises, from N-terminus to C-terminus, (a) a portion of the VP1 capsid protein of AAV2 comprising at least the VP1-u domain of AAV2, and (b) a portion of the VP1 capsid of a non-primate AAV comprising the amino acid sequence of at least the VP3 capsid protein of the non-primate AAV. In some embodiments, the chimeric VP1 capsid protein comprises, from N-terminus to C-terminus, (a) the amino acid sequence of the VP1-u domain and VP1 / VP2 common region of AAV2, and (b) the amino acid sequence of a VP3 capsid protein of a non-primate.

[0066] In some embodiments, the chimeric AAV2 / AAAV VP1 capsid protein comprises, from N-terminus to C-terminus, (a) a portion of the VP1 capsid protein of AAV2 comprising at least the PLA2 domain of AAV2, and (b) a portion of the VP1 capsid of an avian AAV (AAAV) comprising the amino acid sequence of at least the VP3 capsid protein of AAAV. In some embodiments, the chimeric AAV2 / AAAV VP1 capsid protein comprises, from N-terminus to C-terminus, (a) a portion of the VP1 capsid protein of AAV2 comprising at least the VP1-u domain of AAV2, and (b) a portion of the VP1 capsid of AAAV comprising the amino acid sequence of at least the VP3 capsid protein of AAAV. In some embodiments, the chimeric AAV2 / AAAV VP1 capsid protein comprises, from N- to C-terminus, (a) the amino acid sequence of the VP1-u domain and VP1 / VP2 common region of AAV2, and (b) the amino acid sequence of the VP3 capsid protein of AAAV. In some embodiments, the chimeric AAV2 / AAAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO:2.

[0067] In some embodiments, the chimeric AAV2 / sea lion VP1 capsid protein comprises, from N-terminus to C-terminus, (a) a portion of the VP1 capsid protein of AAV2 comprising at least the PLA2 domain of AAV2, and (b) a portion of the VP1 capsid of a sea lion AAV comprising the amino acid sequence of at least the VP3 capsid protein of a sea lion. In some embodiments, the chimeric AAV2 / sea lion VP1 capsid protein comprises, from N-terminus to C-terminus, (a) a portion of the VP1 capsid protein of AAV2 comprising at least the VP1-u domain of AAV2, and (b) a portion of the VP1 capsid of a sea lion comprising the amino acid sequence of at least the VP3 capsid protein of a sea lion. In some embodiments, the chimeric AAV2 / sea lion VP1 capsid protein comprises, from N- to C-terminus, (a) the amino acid sequence of the VP1-u domain and VP1 / VP2 common region of AAV2, and (b) the amino acid sequence of the VP3 capsid protein of a sea lion. In some embodiments, the chimeric AAV2 / sea lion VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO:4.

[0068] In some embodiments, the chimeric AAV2 / bearded dragon VP1 capsid protein comprises, from N-terminus to C-terminus, (a) a portion of the VP1 capsid protein of AAV2 comprising at least the PLA2 domain of AAV2, and (b) a portion of the VP1 capsid of a bearded dragon AAV comprising the amino acid sequence of at least the VP3 capsid protein of a bearded dragon. In some embodiments, the chimeric AAV2 / bearded dragon VP1 capsid protein comprises, from N-terminus to C-terminus, (a) a portion of the VP1 capsid protein of AAV2 comprising at least the VP1-u domain of AAV2, and (b) a portion of the VP1 capsid of a bearded dragon comprising the amino acid sequence of at least the VP3 capsid protein of a bearded dragon. In some embodiments, the chimeric AAV2 / bearded dragon VP1 capsid protein comprises, from N-terminus to C-terminus, (a) the amino acid sequence of the VP1-u domain and VP1 / VP2 common region of AAV2, and (b) the amino acid sequence of the bearded dragon VP3 capsid protein. In some embodiments, the chimeric AAV2 / bearded dragon VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO:6.

[0069] In some embodiments, the chimeric VP1 capsid protein may be operably linked to a first member of a protein:protein binding pair and / or may comprise a point mutation, e.g., such that the capsid protein's natural targeting property is reduced or abolished and / or such that the capsid protein comprises a detectable label. In some embodiments, the first member of the protein:protein binding pair comprises a detectable label. In some embodiments, the first member of the protein:protein binding pair comprises a detectable label comprising c-myc (SEQ ID NO: 44). In some embodiments, the first member of the protein:protein binding pair comprises a first member and, optionally, a second member of the protein:protein binding pair that form a covalent bond. In some embodiments, the protein:protein binding pair is selected from the group consisting of (a) SpyTag:SpyCatcher, (b) SpyTag:KTag, (c) Isopeptag:pilin C, (d) SnoopTag:SnoopCatcher, and (e) SpyTag002:SpyCatcher002. In some embodiments, the chimeric VP1 capsid protein may comprise (a) the B1 epitope (SEQ ID NO: 45), (b) a SpyTag, (c) a SpyCatcher, and any combination of (a)-(c).

[0070] In some embodiments, the chimeric primate / non-primate VP1 capsid protein (e.g., a chimeric AAV2 / AAAV VP1 capsid protein, a chimeric AAV2 / sea lion AAV VP1 capsid protein, a chimeric AAV2 / bearded dragon AAV VP1 capsid protein, etc.) comprises a first member of a protein:protein binding pair operably linked to the capsid protein, optionally via a first or second linker. In some embodiments, a first member of a protein:protein binding pair is operably linked to a chimeric primate / non-primate VP1 capsid protein (e.g., a chimeric AAV2 / AAAV VP1 capsid protein, a chimeric AAV2 / sea lion AAV VP1 capsid protein, a chimeric AAV2 / bearded dragon AAV VP1 capsid protein, etc.) at an amino acid position that is present in a variable region (VR) or portion thereof of the chimeric primate / non-primate VP1 capsid protein, optionally in which case the first member of the protein:protein binding pair is linked to the chimeric primate / non-primate VP1 capsid protein via a first and / or second linker. In some embodiments, a first member of a protein:protein binding pair is operably linked to the VP1 capsid of the chimeric primate / non-primate VP1 capsid protein at an amino acid position that is present in VR I, VR II, VR III, VR IV, VR V, VR VI, VR VII, VR VIII VR IX, or the HI loop of the chimeric primate / non-primate VP1 capsid protein, optionally in which case the first member of the protein:protein binding pair is linked to the chimeric primate / non-primate VP1 capsid protein capsid via a first and / or second linker.In some embodiments, a first member of a protein:protein binding pair is operably linked to a chimeric primate / non-primate VP1 capsid protein (e.g., a chimeric AAV2 / AAAV VP1 capsid protein, a chimeric AAV2 / sea lion AAV VP1 capsid protein, a chimeric AAV2 / bearded dragon AAV VP1 capsid protein, etc.) at an amino acid position that is present in VR VIII or VR IV of the chimeric primate / non-primate VP1 capsid protein, optionally in which case the first member of the protein:protein binding pair is linked to the chimeric primate / non-primate VP1 capsid protein via a first and / or second linker.

[0071] In some embodiments, the chimeric AAV2 / AAAV VP1 capsid protein comprises a first member of a protein:protein binding pair operably linked at position 1444 or 1580, optionally via a first and / or second linker. In some embodiments, the chimeric AAV2 / AAAV VP1 capsid protein comprises a SpyTag operably linked at position 1444, optionally via a first and second linker sequence. In some embodiments, the chimeric AAV2 / AAAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 8. In some embodiments, the chimeric AAV2 / AAAV VP1 capsid protein comprises a SpyTag operably linked at position 1580, optionally via a first and second linker sequence. In some embodiments, the chimeric AAV2 / AAAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 10.

[0072] In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises a first member of a protein:protein binding pair operably linked at a position selected from the group consisting of 1429, 1430, 1431, 1432, 1433, 1434, 1436, 1437, and 1565, optionally at a position selected from the group consisting of 1429, 1430, 1431, 1432, 1433, 1436, and 1437, optionally at position 1432, optionally via a first and / or second linker. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises a SpyTag operably linked at position 1432, optionally via a first and second linker sequence. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 12. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises a SpyTag operably linked at position 1565, optionally via first and second linker sequences. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 14. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises a SpyTag operably linked at position 1429, optionally via first and second linker sequences. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 16. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises a SpyTag operably linked at position 1430, optionally via first and second linker sequences. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 18. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises a SpyTag at position 1431, optionally operably linked via first and second linker sequences. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 20.In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises a SpyTag operably linked at position 1433, optionally via first and second linker sequences. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 22. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises a SpyTag operably linked at position 1434, optionally via first and second linker sequences. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 24. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises a SpyTag operably linked at position 1435, optionally via first and second linker sequences. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 26. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises a SpyTag at position 1436, optionally operably linked via first and second linker sequences. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 28. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises a SpyTag at position 1437, optionally operably linked via first and second linker sequences. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 30. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises a SpyTag operably linked, optionally via first and second linker sequences, at position 1432. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 32. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 53.In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 55. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 57. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 59. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 61. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 63. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 65. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 67. In some embodiments, the chimeric AAV2 / sea lion AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 69. In some embodiments, the chimeric AAV2 / sea lion AAV. The VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO:71.

[0073] In some embodiments, the chimeric AAV2 / bearded dragon AAV VP1 capsid protein comprises a first member of a protein:protein binding pair operably linked at position 1436 or 1573, optionally via a first and / or second linker. In some embodiments, the chimeric AAV2 / bearded dragon AAV VP1 capsid protein comprises a SpyTag operably linked at position 1436, optionally via a first and second linker sequence. In some embodiments, the chimeric AAV2 / bearded dragon AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 34. In some embodiments, the chimeric AAV2 / bearded dragon AAV VP1 capsid protein comprises a SpyTag operably linked at position 1573, optionally via a first and second linker sequence. In some embodiments, the chimeric AAV2 / bearded dragon AAV VP1 capsid protein comprises the amino acid sequence set forth as SEQ ID NO:36.

[0074] In some embodiments, the capsid protein of the present invention further comprises a first member and a second member of a protein:protein binding pair, optionally in which the second member is operably linked to a targeting ligand, optionally in which the targeting ligand is a binding moiety. In some embodiments, the binding moiety is an antibody or a portion thereof. In some embodiments, the antibody or a portion thereof is fused to a second member, such as SpyCatcher. In some embodiments, the antibody or a portion thereof is fused to a linker at its C-terminus, optionally comprising the sequence set forth as SEQ ID NO: 49 (GSGESG), and the linker is fused to a second member, such as SpyCatcher, at the C-terminus of the linker.

[0075] In some embodiments, the capsid proteins of the present invention may comprise a detectable label, which may optionally serve as a first member of a protein:protein binding pair and / or may function for detection and / or isolation of the capsid protein. In some embodiments, the detectable label is c-myc. In some embodiments, the detectable label comprises an AAV B1 epitope, such as the amino acid sequence of IGTRYLTR (SEQ ID NO: 45).

[0076] The AAV capsid protein of the present invention is (a) the amino acid sequence set forth as SEQ ID NO:2, (b) the amino acid sequence set forth as SEQ ID NO:4, (c) the amino acid sequence set forth as SEQ ID NO:6, (d) the amino acid sequence set forth as SEQ ID NO:8, (e) the amino acid sequence set forth as SEQ ID NO:10, (f) the amino acid sequence set forth as SEQ ID NO:12, (g) the amino acid sequence set forth as SEQ ID NO:14, (h) the amino acid sequence set forth as SEQ ID NO:16, (i) the amino acid sequence set forth as SEQ ID NO:18, (j) the amino acid sequence set forth as SEQ ID NO:20. an amino acid sequence, (k) the amino acid sequence set forth as SEQ ID NO: 22, (l) the amino acid sequence set forth as SEQ ID NO: 24, (m) the amino acid sequence set forth as SEQ ID NO: 26, (n) the amino acid sequence set forth as SEQ ID NO: 28, (o) the amino acid sequence set forth as SEQ ID NO: 30, (p) the amino acid sequence set forth as SEQ ID NO: 32, (q) the amino acid sequence set forth as SEQ ID NO: 34, (r) the amino acid sequence set forth as SEQ ID NO: 36, (s) the amino acid sequence set forth as SEQ ID NO: 53, (t) the amino acid sequence set forth as SEQ ID NO: 55; (u) the amino acid sequence set forth as SEQ ID NO: 57, (v) the amino acid sequence set forth as SEQ ID NO: 59, (w) the amino acid sequence set forth as SEQ ID NO: 61, (x) the amino acid sequence set forth as SEQ ID NO: 63, (y) the amino acid sequence set forth as SEQ ID NO: 65, (z) the amino acid sequence set forth as SEQ ID NO: 67, (aa) the amino acid sequence set forth as SEQ ID NO: 69, (bb) the amino acid sequence set forth as SEQ ID NO: 71, (cc) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, or SEQ ID NO:71, and (dd) the amino acid sequence of any VP2 portion and / or VP3 portion of the amino acid sequence set forth in any of (a)-(cc);It may comprise an amino acid sequence selected from the group consisting of:

[0077] In some embodiments, the capsid proteins of the invention are encoded by nucleic acid molecules of the invention. Also provided herein are nucleic acid molecules encoding the capsid proteins of the invention.

[0078] Described herein are nucleic acid molecules comprising an AAV cap gene encoding an AAV VP1 capsid protein, an AAV VP2 capsid protein, and / or an AAV VP3 capsid protein, wherein the AAV cap gene or portion thereof comprises a nucleic acid sequence having significant sequence identity, e.g., 95% identity, to the cap gene or portion thereof of a non-primate AAV, or the nucleic acid sequence of a cap gene or portion thereof of a remote AAV, and wherein the AAV cap gene is further modified to include (a) a nucleotide sequence encoding a first member of a protein:protein binding pair, (b) a nucleotide sequence encoding a detectable label, (c) a point mutation, (d) a chimeric nucleotide sequence, or (e) any combination of (a), (b), (c), and (d). In some nucleic acid molecule embodiments, the nucleic acid comprises an AAV rep gene and an AAV cap gene, wherein the entire AAV cap gene comprises a first nucleic acid sequence that has significant sequence identity, e.g., at least 95% identity, to the nucleic acid sequence of the cap gene of a non-primate AAV or a remote AAV, and wherein the AAV rep gene or portion thereof comprises a second nucleic acid sequence that has significant sequence identity, e.g., at least 95% identity, to the nucleic acid sequence of the rep gene or portion thereof of a second AAV, wherein the non-primate AAV is not identical to the second AAV.In some embodiments, a nucleic acid molecule of the invention comprises an AAV cap gene encoding an AAV capsid protein, wherein the AAV cap gene comprises a nucleotide sequence that is identical or has significant identity, e.g., at least 95% sequence identity, to at least a portion of the nucleotide sequence of a cap gene selected from the group consisting of: (i) a cap gene of a non-primate AAV; (ii) a cap gene of a remote AAV; or (iii) a combination thereof, wherein the AAV cap gene is further modified to comprise: (a) a nucleotide sequence encoding a first member of a protein:protein binding pair; (b) a nucleotide sequence encoding a detectable label; (c) a point mutation; (d) a chimeric nucleotide sequence comprising a portion of the nucleotide sequence of a cap gene of another AAV, e.g., a second AAV, operably linked to the nucleotide sequence of an AAV cap gene selected from the group consisting of a cap gene of a non-primate AAV, a remote AAV, or a combination thereof; or (e) any combination of (a), (b), (c), and (d).

[0079] In some embodiments, a nucleic acid molecule of the invention comprises an AAV cap gene encoding an AAV capsid protein, wherein the AAV cap gene comprises a nucleotide sequence that is identical or has significant identity, e.g., at least 95% sequence identity, to at least a portion of the nucleotide sequence of a cap gene of a non-primate AAV, wherein the AAV cap gene is further modified to comprise: (a) a nucleotide sequence encoding a first member of a protein:protein binding pair; (b) a nucleotide sequence encoding a detectable label; (c) a point mutation; (d) a chimeric nucleotide sequence comprising a portion of the nucleotide sequence of a cap gene of another AAV, e.g., a second AAV, operably linked to the nucleotide sequence of the cap gene of the non-primate AAV; or (e) any combination of (a), (b), (c), and (d).

[0080] In some embodiments, a nucleic acid molecule of the present invention comprises an AAV cap gene encoding an AAV capsid protein, wherein the AAV cap gene comprises a nucleotide sequence that is identical or has significant identity, e.g., at least 95% sequence identity, to at least a portion of the nucleotide sequence of a cap gene of a remote animal AAV, wherein the AAV cap gene is further modified to comprise: (a) a nucleotide sequence encoding a first member of a protein:protein binding pair; (b) a nucleotide sequence encoding a detectable label; (c) a point mutation; (d) a chimeric nucleotide sequence comprising a portion of the nucleotide sequence of a cap gene of another AAV, operably linked to the nucleotide sequence of the cap gene of the remote animal AAV; or (e) any combination of (a), (b), (c), and (d).

[0081] In some embodiments, a nucleic acid molecule of the invention comprises an AAV rep gene and an AAV cap gene, wherein the AAV cap gene comprises a first nucleotide sequence that is identical or has significant identity, e.g., at least 95% sequence identity, to the nucleotide sequence of a cap gene selected from the group consisting of: (i) a cap gene of a non-primate AAV; (ii) a cap gene of a remote primate AAV; and (iv) a combination thereof, wherein the AAV rep gene comprises a second nucleotide sequence of an AAV rep gene of another AAV, e.g., a second AAV.

[0082] In some embodiments, a nucleic acid molecule of the invention comprises an AAV rep gene and an AAV cap gene, wherein the AAV cap gene comprises a first nucleotide sequence that is identical to or has significant identity, e.g., at least 95% sequence identity, to the nucleotide sequence of the cap gene of a non-primate AAV, and wherein the AAV rep gene comprises a second nucleotide sequence that is identical to or has significant identity, e.g., at least 95% sequence identity, to the nucleotide sequence of the AAV rep gene of another AAV, e.g., a second AAV.

[0083] In some embodiments, a nucleic acid molecule of the invention comprises an AAV rep gene and an AAV cap gene, wherein the AAV cap gene comprises a nucleotide sequence that is identical or has significant identity, e.g., at least 95% sequence identity, to a first nucleotide sequence of a cap gene of a remote animal AAV, and wherein the AAV rep gene comprises a nucleotide sequence that is identical or has significant identity, e.g., at least 95% sequence identity, to a second nucleotide sequence of an AAV rep gene of another AAV, e.g., a second AAV.

[0084] In some nucleic acid molecule embodiments of the present invention, the nucleotide sequence of a cap gene comprising a nucleotide sequence that is identical or has significant identity, e.g., at least 95% sequence identity, to the nucleotide sequence of a non-primate AAV, the nucleotide sequence of a cap gene comprising a nucleotide sequence that is identical or has significant identity, e.g., at least 95% sequence identity, to the nucleotide sequence of a remote AAV, or a combination thereof, is modified to comprise (a) a nucleotide sequence encoding at least a first member of a protein:protein binding pair, (b) a nucleotide sequence encoding a detectable label, and / or (c) a nucleotide sequence encoding a point mutation.

[0085] In some nucleic acid embodiments, the protein:protein binding pair is selected from SpyTag:SpyCatcher, SpyTag:KTag, Isopeptag:pilin-C, SnoopTag:SnoopCatcher, and SpyTag002:SpyCatcher002. In some embodiments, the first member of the protein:protein binding pair comprises a detectable label, such as, for example, c-myc comprising the sequence set forth as SEQ ID NO:44.

[0086] In some nucleic acid embodiments, the nucleotide sequence of the non-primate AAV cap gene, the remote AAV cap gene, or a combination thereof is modified to include a B1 epitope comprising the amino acid sequence of IGTRYLTR (SEQ ID NO: 45).

[0087] In some embodiments of the nucleic acid molecules of the present invention, the nucleotide sequence of the cap gene of the non-primate AAV, the cap gene of the remote AAV, or a combination thereof, comprises a nucleotide sequence encoding a VP3 capsid protein or a portion thereof, comprising an amino acid sequence that is identical to or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of the VP3 capsid protein of the non-primate AAV and / or the amino acid sequence of the VP3 capsid protein of the remote AAV. In some embodiments, the nucleotide sequence of the cap gene of the non-primate AAV, the cap gene of the remote AAV, or a combination thereof, comprises a nucleotide sequence encoding a VP2 capsid protein or a portion thereof, comprising an amino acid sequence that is identical to or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of the VP2 capsid protein of the non-primate AAV and / or the amino acid sequence of the VP2 capsid protein of the remote AAV. In some embodiments, the nucleotide sequence of the cap gene of the non-primate AAV, the cap gene of the remote AAV, or a combination thereof, comprises a nucleotide sequence encoding a VP1 capsid protein or a portion thereof, including an amino acid sequence that is identical to or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of the VP1 capsid protein of the non-primate AAV and / or the amino acid sequence of the VP1 capsid protein of the remote AAV.

[0088] In some embodiments, a nucleic acid molecule of the present invention comprises a nucleotide sequence encoding a non-primate VP3 capsid protein of the present invention. In some embodiments, a nucleic acid molecule of the present invention comprises a nucleotide sequence encoding a non-primate VP3 capsid protein of the present invention and a non-primate VP2 capsid protein of the present invention. In some embodiments, a nucleic acid molecule of the present invention comprises a nucleotide sequence encoding a non-primate VP3 capsid protein of the present invention, a VP2 capsid protein of the present invention, and a VP1 capsid protein of the present invention.

[0089] In some embodiments, the cap gene of the nucleic acid molecule of the invention is (i)(a) a chimeric AAV (ii) a VP1 capsid protein that is either (a) a VP1 / VP2 common region (VP1-u) comprising an amino acid sequence that is identical to or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a non-primate AAV or a remote AAV, operably linked to a VP1 / VP2 common region and a VP3 region that comprises an amino acid sequence that is identical to or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a non-primate AAV or a remote AAV; or (b) a VP1 capsid protein that comprises an amino acid sequence that is identical to or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a VP1 capsid protein of a non-primate AAV or a remote AAV; a VP2 capsid protein that is either (a) a VP1 / VP2 common region comprising an amino acid sequence that is identical to or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of another AAV, e.g., a second AAV, operably linked to a VP3 region comprising an amino acid sequence that is identical to or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a non-primate AAV; or (b) a VP2 capsid protein comprising an amino acid sequence that is identical to or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a VP2 capsid protein of a non-primate AAV; and / or (iii) a VP3 capsid protein comprising an amino acid sequence that is identical to or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a VP3 capsid protein of a non-primate AAV or a remote AAV.

[0090] In some embodiments, the cap gene of the nucleic acid molecule of the invention comprises: (i) a chimeric AAV VP1 capsid protein, optionally wherein the chimeric VP1 capsid protein comprises a VP1-unique region (VP1-u) comprising an amino acid sequence that is identical to, or has significant identity, e.g., at least 95% sequence identity to, the amino acid sequence of another AAV, e.g., a second AAV, operably linked to a VP1 / VP2 common region and a VP3 region comprising an amino acid sequence that is identical to, or has significant identity, e.g., at least 95% sequence identity to, the amino acid sequence of the non-primate AAV or a remote AAV; (ii) a chimeric AAV and / or (iii) a VP2 capsid protein comprising an amino acid sequence identical to or having significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of another AAV, e.g., AAV, operably linked to the VP3 region of a non-primate AAV or remote AAV, and / or (iv) a VP3 capsid protein comprising an amino acid sequence identical to or having significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a non-primate AAV or remote AAV.

[0091] In some embodiments, the cap gene of a nucleic acid molecule of the invention encodes: (i) a chimeric AAV VP1 capsid protein, optionally in which the chimeric VP1 capsid protein comprises a VP1-unique region (VP1-u) comprising an amino acid sequence that is identical to or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of another AAV, operably linked to a VP1 / VP2 common region and a VP3 region comprising an amino acid sequence that is identical to or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a non-primate AAV or a remote AAV; (ii) a VP2 capsid protein comprising an amino acid sequence that is identical to or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a non-primate AAV or a remote AAV; and (iii) a VP3 capsid protein comprising an amino acid sequence that is identical to or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a non-primate AAV or a remote AAV.

[0092] In some embodiments, the cap gene of the nucleic acid molecule of the present invention encodes (i) a VP1 capsid protein comprising an amino acid sequence that is identical or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a non-primate AAV or a remote AAV; (ii) a VP2 capsid protein comprising an amino acid sequence that is identical or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a non-primate AAV or a remote AAV; and (iii) a VP3 capsid protein comprising an amino acid sequence that is identical or has significant identity, e.g., at least 95% sequence identity, to the amino acid sequence of a non-primate AAV or a remote AAV.

[0093] In some embodiments of the nucleic acid molecule of the present invention, other AAV such as second AAV is primate AAV or a combination of primate AAV.In some embodiments, other AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, and combinations thereof.In some embodiments, other AAV is AAV2.

[0094] In some nucleic acid molecule embodiments of the present invention, the non-primate AAV is a non-primate AAV listed in Table 2. In some embodiments, the non-primate AAV is an avian AAV (AAAV), a sea lion AAV, or a bearded dragon AAV. In some embodiments, the non-primate AAV is an AAAV, and optionally, the nucleotide sequence of the AAAV capsid protein comprises a modification at position I444 or I580 of the VP1 capsid protein of AAAV. In some embodiments, the non-primate AAV is a Sphingidae AAV, such as, for example, a Bearded Dragon AAV, and optionally, the nucleotide sequence of a Bearded Dragon AAV comprises a modification at position I573 or I436 of the VP1 capsid protein of a Bearded Dragon AAV. In some embodiments, the non-primate AAV is a mammalian AAV, such as a sea lion AAV, and optionally the nucleotide sequence of the sea lion AAV comprises a modification at a position selected from the group consisting of I429, I430, I431, I432, I433, I434, I436, I437, and A565 of the VP1 capsid protein of the sea lion AAV.

[0095] Embodiments of the nucleic acid molecules of the present invention may comprise a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence set forth as SEQ ID NO:1, (b) the nucleotide sequence set forth as SEQ ID NO:3, (c) the nucleotide sequence set forth as SEQ ID NO:5, (d) the nucleotide sequence set forth as SEQ ID NO:7, (e) the nucleotide sequence set forth as SEQ ID NO:9, (f) the nucleotide sequence set forth as SEQ ID NO:11, (g) the nucleotide sequence set forth as SEQ ID NO:13, (h) the nucleotide sequence set forth as SEQ ID NO:15, (i) the nucleotide sequence set forth as SEQ ID NO:17, (j) the nucleotide sequence set forth as SEQ ID NO:19, (k) the nucleotide sequence set forth as SEQ ID NO:21, (l) the nucleotide sequence set forth as SEQ ID NO:23, (m) the nucleotide sequence set forth as SEQ ID NO:25, (n) the nucleotide sequence set forth as SEQ ID NO:27, (o) the nucleotide sequence set forth as SEQ ID NO:29, (p) the nucleotide sequence set forth as SEQ ID NO:31, (q) the nucleotide sequence set forth as SEQ ID NO:33, (r) The nucleotide sequence set forth as SEQ ID NO: 35, (s) the nucleotide sequence set forth as SEQ ID NO: 52, (t) the nucleotide sequence set forth as SEQ ID NO: 54, (u) the nucleotide sequence set forth as SEQ ID NO: 56, (v) the nucleotide sequence set forth as SEQ ID NO: 58, (w) the nucleotide sequence set forth as SEQ ID NO: 60, (x) the nucleotide sequence set forth as SEQ ID NO: 62, (y) the nucleotide sequence set forth as SEQ ID NO: 64, (z) the nucleotide sequence set forth as SEQ ID NO: 66, (aa) SEQ ID NO: 68 and (bb) the nucleotide sequence set forth as SEQ ID NO: 70; (cc) a nucleotide sequence having at least 95% identity to the nucleotide sequence set forth as SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 52, 54, 56, 58, 60, 62, 64, 66, 68, or 70; (dd) any portion of the nucleotide sequence of (a) through (cc) that encodes a VP2 capsid protein and / or a VP3 capsid protein.

[0096] In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 1, a portion thereof encoding the VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 3, a portion thereof encoding the VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 5, a portion thereof encoding the VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 7, a portion thereof encoding the VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 9, a portion thereof encoding the VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 11, a portion thereof encoding the VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 13, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 15, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 17, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 19, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 21, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof.In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO:23, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO:25, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO:27, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO:29, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO:31, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO:33, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 35, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 52, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 54, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 56, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 58, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof.In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 60, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 62, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 64, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 66, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 68, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ID NO: 70, a portion thereof encoding VP2 and / or VP3 capsid proteins, and degenerate variants thereof.

[0097] In some embodiments, the nucleotide sequences encoding the VP1 capsid protein of the present invention, and optionally the VP2 and / or VP3 capsid proteins of the present invention, are operably linked to a promoter. In some embodiments, the promoter is selected from a viral promoter, a bacterial promoter, a mammalian promoter (e.g., human or non-human), an avian promoter, a fish promoter, an insect promoter, and any combination thereof. In some embodiments, the promoter is selected from p40, SV40, EF, CMV, B19p6, and CAG. In some embodiments, the promoter is an AAV p40 promoter. In some embodiments, the promoter directs expression of the capsid proteins in a packaging cell.

[0098] In some embodiments, the cap gene of the nucleic acid molecule of the present invention is operably linked to a promoter. In some embodiments, the promoter directs expression of the capsid protein in the packaging cell. In some embodiments, the promoter is selected from p40, SV40, EF (e.g., EF1α CMV), B19p6, and CAG.

[0099] In some embodiments, the nucleic acid molecules of the invention further comprise a second nucleotide sequence encoding one or more AAV Rep proteins, optionally in this case, the second nucleotide sequence being operably linked to a promoter. In some embodiments, the one or more Rep proteins are primate AAV Rep proteins. In some embodiments, the one or more Rep proteins are non-primate AAV Rep proteins. In some embodiments, the one or more Rep proteins are selected from Rep78, Rep68, Rep52, and Rep40, optionally including Rep78. In some embodiments, the promoter operably linked to the second nucleotide sequence encoding one or more AAV Rep proteins is selected from a viral promoter, a bacterial promoter, a mammalian promoter (e.g., human or non-human), an avian promoter, a fish promoter, an insect promoter, and any combination thereof. In some embodiments, the promoter operably linked to the second nucleotide sequence encoding one or more AAV Rep proteins is selected from p19, p5, p40, SV40, EF, e.g., EF1α CMV, B19p6, and CAG. In some embodiments, the promoter directs expression of capsid proteins in a packaging cell. In some embodiments, the promoter operably linked to the second nucleotide sequence encoding one or more AAV Rep proteins is selected from p19 and / or p5.

[0100] Compositions and packaging cells comprising the nucleic acid molecules of the invention, as well as capsid proteins encoded from the nucleic acid molecules of the invention, are also part of the invention. Viral particles expressed by the packaging cells of the invention are also described.

[0101] In some embodiments, compositions and packaging cells for producing AAV viral particles of the invention comprise a nucleic acid molecule of the invention, e.g., a cap gene of the invention that encodes an AAV capsid protein of the invention. In some embodiments, compositions and / or packaging cells of the invention comprise a nucleic acid molecule of the invention.In some embodiments, the cap gene is selected from the group consisting of the nucleotide sequence set forth as SEQ ID NO:1, the nucleotide sequence set forth as SEQ ID NO:3, the nucleotide sequence set forth as SEQ ID NO:5, the nucleotide sequence set forth as SEQ ID NO:7, the nucleotide sequence set forth as SEQ ID NO:9, the nucleotide sequence set forth as SEQ ID NO:11, the nucleotide sequence set forth as SEQ ID NO:13, the nucleotide sequence set forth as SEQ ID NO:15, the nucleotide sequence set forth as SEQ ID NO:17, the nucleotide sequence set forth as SEQ ID NO:19, the nucleotide sequence set forth as SEQ ID NO:21, the nucleotide sequence set forth as SEQ ID NO:23, the nucleotide sequence set forth as SEQ ID NO:25, the nucleotide sequence set forth as SEQ ID NO:27, the nucleotide sequence set forth as SEQ ID NO:29, the nucleotide sequence set forth as SEQ ID NO:31, the nucleotide sequence set forth as SEQ ID NO:33, the nucleotide sequence set forth as SEQ ID NO:35, the nucleotide sequence set forth as SEQ ID NO:52, the nucleotide sequence set forth as SEQ ID NO:54, the nucleotide sequence set forth as SEQ ID NO:56, the nucleotide sequence set forth as SEQ ID NO:58, the nucleotide sequence set forth as SEQ ID NO:60, the nucleotide sequence set forth as SEQ ID NO:62, the nucleotide sequence set forth as SEQ ID NO:64, the nucleotide sequence set forth as SEQ ID NO:66, the nucleotide sequence set forth as SEQ ID NO:68, the nucleotide sequence set forth as SEQ ID NO:70, the nucleotide sequence set forth as SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 10 3, 25, 27, 29, 31, 33, 35, 52, 54, 56, 58, 60, 62, 64, 66, 68 or 70, any portion of the nucleotide sequence set forth as SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 52, 54, 56, 58, 60, 62, 64, 66, 68 or 70 that encodes a VP3 capsid, and any combination thereof.

[0102] In some embodiments, the compositions and packaging cells further comprise a nucleic acid molecule comprising a rep gene encoding one or more AAV Rep proteins, wherein the rep gene is operably linked to a promoter, and optionally wherein the rep gene and cap gene are two different AAV genes. In some embodiments, the promoter operably linked to the rep gene directs expression of the Rep protein in the packaging cell. For example, the promoter is selected from p5, p19 SV40, EF, CMV, B19p6, and CAG. In some embodiments, the one or more Rep proteins are selected from Rep78, Rep68, Rep52, and Rep40, and optionally, the one or more Rep proteins include Rep78. In some embodiments, the one or more Rep proteins are primate AAV Rep proteins. In some other embodiments, the one or more Rep proteins are non-primate AAV Rep proteins. In some embodiments, the one or more Rep proteins include both.

[0103] In some embodiments, the compositions and packaging cells of the invention further comprise a nucleic acid molecule comprising a nucleotide sequence of a nucleotide of interest flanked on at least one side by at least one AAV inverted terminal repeat (ITR) recognized by one or more Rep proteins. In some embodiments, the nucleotide of interest is flanked on the other side by a second ITR of the same AAV as the at least one ITR. In some embodiments, the nucleotide of interest is flanked on the other side by a second ITR, where the second ITR and the at least one ITR are from different AAVs.

[0104] In some embodiments, the compositions and / or packaging cells of the invention further comprise a nucleic acid molecule comprising nucleotides of interest (e.g., the nucleotide sequence of a transgene) flanked by 5' and 3' AAV inverted terminal repeats (ITRs), such that the viral particles of interest further comprise a genome comprising nucleotides of interest flanked by 5' and 3' AAV ITRs.

[0105] In some embodiments, the compositions and / or packaging cells of the present invention further comprise a nucleotide sequence encoding a reference capsid protein.

[0106] Thus, in some embodiments, the compositions, packaging cells, and / or viral particles of the present invention further comprise a genome including, from 5' to 3', a 5' ITR, a nucleotide of interest, and a 3' ITR. In some embodiments, the genome further comprises a promoter operably linked to the nucleotide of interest. In some embodiments, the 5' and 3' ITRs are derived from the same AAV species. In some embodiments, the 5' and 3' ITRs are derived from different AAV species.

[0107] In some embodiments, the nucleotide of interest is a reporter gene. In some embodiments, the reporter gene encodes β-galactosidase, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), MmGFP, blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof.

[0108] In some embodiments, the nucleotide of interest encodes a therapeutic protein, a suicide gene, an antibody or fragment thereof, a CRISPR / Cas system or part(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA.

[0109] Described herein are methods for producing AAV viral particles of the invention, the methods comprising culturing packaging cells under conditions sufficient to produce viral particles, the packaging cells comprising: (1) at least one nucleotide sequence encoding one or more AAV Rep proteins, e.g., a rep gene; and (2) a first nucleotide sequence encoding an AAV VP1 capsid protein, optionally a second nucleotide sequence encoding an AAV VP2 capsid protein, and a third nucleotide sequence encoding a non-primate AAV VP3 capsid protein (e.g., a first, second, and third nucleotide molecule of the invention); and (3) nucleotides of interest flanked by the first and / or second ITRs of a second AAV, wherein the one or more AAV The Rep protein recognizes a recognition site in the first and / or second ITR of a second AAV, wherein the third nucleotide sequence encodes a non-primate AAV VP3 capsid protein of the invention, and optionally wherein the first nucleotide sequence encodes an AAV VP1 capsid protein of the invention and / or the second nucleotide sequence encodes an AAV VP2 capsid protein of the invention. In some embodiments, a cap gene of the invention comprises first, second, and third nucleotide sequences. In some embodiments, a packaging plasmid comprises at least one nucleotide sequence encoding one or more AAV Rep proteins and any combination of first, second, and third nucleotide sequences encoding non-primate AAV VP1 capsid protein, AAV VP2 capsid protein, and AAV VP3 capsid protein, respectively.

[0110] In some embodiments, the methods comprise culturing a packaging cell of the invention. In some embodiments, the methods comprise culturing a packaging cell of the invention comprising a nucleic acid molecule of the invention, wherein the packaging cell optionally further comprises a helper plasmid and / or a transfer plasmid comprising a nucleotide of interest.

[0111] Some method embodiments further include isolating the self-complementing adeno-associated viral particles from the culture supernatant and / or cell lysate. Some method embodiments further include lysing the packaging cells and isolating the single-stranded adeno-associated viral particles from the culture supernatant and / or cell lysate. Some methods further include: a. removing cellular debris; b. treating the supernatant containing the viral particles with Benzonase or DNase I and MgCl2; c. concentrating the viral particles; d. purifying the viral particles; and any combination of e.a.-d.

[0112] In some embodiments, mosaic viral particles are produced by transfecting a mixture of a first cap gene encoding a VP capsid protein comprising a first member of a protein:protein binding pair and at least one reference cap gene encoding a reference VP capsid protein, in a specific ratio, into packaging cells. In some embodiments, mosaic viral particles of the present invention may be produced using a mixture of modified cap genes:reference cap genes. In some embodiments, the modified cap gene encodes at least one of AAV VP1, VP2, and VP3 capsid proteins that includes a modification, e.g., a first member of a protein:protein binding pair, and the reference cap gene encodes a reference capsid protein that corresponds to at least one of the modified AAV VP1, VP2, and VP3 capsid proteins excluding the modification. In some embodiments, the modified cap gene encodes a VP1 capsid protein modified with a first member of a protein:protein binding pair, and the reference cap gene encodes a reference VP1 capsid protein lacking the modification. In some embodiments, the modified cap gene encodes a VP2 capsid protein modified with a first member of a protein:protein binding pair, and the reference cap gene encodes a reference VP2 capsid protein lacking the modification. In some embodiments, the modified cap gene encodes a VP3 capsid protein modified with a first member of a protein:protein binding pair, and the reference cap gene encodes a reference VP3 capsid protein lacking the modification.

[0113] Generally, the viral particles described herein include a viral capsid comprising a viral capsid protein described herein, including a mosaic viral capsid, wherein the viral capsid encapsidates 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 EF, e.g., an EF1α promoter. In some embodiments, the promoter is a CAG promoter. In some embodiments, the promoter is a ubiquitin C (UbC) promoter.

[0114] Generally, the nucleotides of interest may be one or more genes, which may encode a detectable marker, e.g., a reporter, or a therapeutic polypeptide. In some embodiments, the nucleotides of interest are reporter genes. In some embodiments, the nucleotides of interest are reporter genes encoding a detectable marker selected from the group consisting of green fluorescent protein, luciferase, β-galactosidase, etc. In some embodiments, the detectable marker is green fluorescent protein. In other embodiments, the nucleotides of interest are selected from the group consisting of suicide genes, nucleotides encoding antibodies or fragments thereof, nucleotides encoding CRISPR / Cas systems or portion(s) thereof, nucleotides encoding antisense RNAs, nucleotides encoding siRNAs, secreted enzymes, genes encoding therapeutic proteins, etc. In one embodiment, the nucleotides of interest encode a multidomain therapeutic protein, e.g., a protein comprising at least two domains that provide two distinct functions.

[0115] The compositions described herein generally include viral particles comprising the viral capsid proteins described herein, e.g., capsids comprising viral capsid proteins (including mosaic capsids), where the capsid encapsidates a nucleotide of interest. In some embodiments, the compositions described herein include (1) viral particles having capsids comprising the viral capsid proteins described herein, and (2) a pharmaceutically acceptable carrier.

[0116] Also described herein are methods of using viral capsid proteins, viral particles comprising viral capsid proteins, compositions, etc. In some embodiments, the methods include contacting a target cell (which may be in vitro (e.g., ex vivo) or in vivo, e.g., in a human) with a viral particle comprising a viral capsid protein described herein, wherein the viral capsid or viral particle comprises a targeting ligand that specifically binds to a protein expressed on the surface of the target cell.

[0117] Because the viral capsid proteins described herein contain a first member of a protein:protein binding pair to its cognate second member, optionally linked to a targeting ligand, the viral particles described herein are particularly suitable for targeted introduction of a nucleotide of interest into a specific cell. In some embodiments, the targeting ligand is operably linked to the protein (second member), e.g., fused to the protein, optionally via a linker. In some embodiments, the targeting ligand may be a binding moiety, e.g., a natural ligand, an antibody, a multispecific binding molecule, etc. In some embodiments, the targeting ligand is an antibody or a portion thereof. In some embodiments, the targeting ligand is an antibody comprising a variable domain that binds to a cell surface protein on a target cell and a heavy chain constant domain. In some embodiments, the targeting ligand is an antibody comprising a variable domain that binds to a cell surface protein on a target cell and an IgG heavy chain constant domain. In some embodiments, the targeting ligand is an antibody comprising a variable domain that binds to a cell surface protein on a target cell and an IgG heavy chain constant domain, wherein the IgG heavy chain constant domain is operably linked, e.g., via a linker, to a protein (e.g., a second member of a protein:protein binding pair) that forms an isopeptide covalent bond with the first member. In some embodiments, the capsid protein described herein comprises a SpyTag operably linked to a viral capsid protein, and a second member comprising SpyCatcher linked to a targeting ligand comprising a first member covalently bound to the SpyTag, an antibody variable domain, and an IgG heavy chain domain, wherein SpyCatcher and the IgG heavy chain domain are linked via an amino acid linker, such as GSGESG (SEQ ID NO: 49). In some embodiments, the second member comprises the sequence set forth as SEQ ID NO: 47, which sequence comprises a portion of a human IgG4 heavy chain, the IgG4 portion having the sequence set forth as SEQ ID NO: 51 linked to SpyCatcher (SEQ ID NO: 43) via a linker (SEQ ID NO: 49).

[0118] Generally, the targeting ligand specifically binds to a cell surface molecule, eg, an oligosaccharide, receptor, cell surface marker, etc., expressed on the surface of a mammalian (eg, human) eukaryotic cell, eg, a target cell. [Brief explanation of the drawings]

[0119] [Figure 1] Figure 1 provides illustrative (not to scale), non-limiting, and exemplary embodiments of Rep-Cap expression plasmids of the invention that can be used to generate AAV chimeric viral particles. Primate AAV sequences are shown as open boxes, and non-primate AAV sequences are shown as black boxes. Also shown for illustrative purposes are exemplary, non-limiting locations (not to scale) for insertion of (1) a first member of a protein-protein binding pair (solid lines in the capsid sequences of non-primate AAV) that directs the tropism of the assembled capsid, including the encoded VP1, VP2, and VP3 proteins, and (2) a detectable label (dotted lines in the capsid sequences of non-primate AAV) for detection of the encoded VP1, VP2, and VP3 proteins. The Rep-Cap expression plasmids shown in Figure 1 may be used to produce AAV viral particles containing nucleotides of interest flanked by 5' and 3' inverted terminal repeats (ITRs) of primate AAV.

[0120] [Figure 2]Figure 2 shows Western blotting using the B1 antibody, which recognizes the B1 epitope engineered into the chimeric primate / non-primate AAV cap gene (see Figure 1 ). This antibody was used to analyze the resulting chimeric primate / non-primate AAV VP1, VP2, and VP3 proteins. The primate AAV was AAV2, and the non-primate AAVs were (A) avian AAV, (B) sea lion AAV, or (C) bearded dragon AAV. Western blotting was performed on protein samples collected during various steps of AAV particle purification via affinity chromatography. Samples included the input sample, the flow-through (FT) fraction, and the elution fraction from the affinity chromatography column.

[0121] [Figure 3-1] FIG. 3A provides a non-limiting predicted ribbon structure of avian AAV VP3, highlighting K580 and G444 as non-limiting insertion sites for the first member of a protein:protein binding pair. [Figure 3-2] Figure 3B presents qPCR quantification of viral titers obtained from crude viral preparations of a panel of chimeric AAV2 / avian AAV viral particles without SpyTag or containing SpyTag insertions at the indicated positions, or a panel of mosaic particles composed of a mix of AAV2 / avian AAV particles without SpyTag and AAV2 / avian AAV particles carrying SpyTag peptide insertions at the indicated positions. [Figure 3-3]Figure 3C shows Western blotting using the B1 antibody, which recognizes linear epitopes engineered into the chimeric AAV2 / avian AAV VP1, VP2, and VP3 capsid proteins, to analyze the reaction between the anti-ASGR1 antibody fused to SpyCatcher ("SpyC-anti-ASGR1 mAb") and a panel of chimeric AAV2 / avian AAV viral particles lacking or carrying SpyTag insertions at the indicated positions, or a panel of mosaic particles containing a mixture of chimeric AAV2 / avian AAV particles bearing SpyTag peptide insertions at the indicated positions and chimeric AAV2 / avian AAV particles lacking SpyTag.

[0122] [Figure 4-1] Figure 4A provides a non-limiting predicted ribbon structure of sea lion AAV VP3, highlighting A565 and G432 as non-limiting insertion sites for the first member of a protein:protein binding pair. [Figure 4-2] Figure 4B presents qPCR quantification of viral titers obtained from crude viral preparations of a panel of chimeric AAV2 / sea lion AAV viral particles without SpyTag or containing SpyTag insertions at the indicated positions, or a panel of mosaic particles composed of a mix of AAV2 / sea lion AAV particles without SpyTag and AAV2 / sea lion AAV particles carrying SpyTag peptide insertions at the indicated positions. [Figure 4-3] Figure 4C presents Western blotting using the B1 antibody, which recognizes linear epitopes engineered into the chimeric AAV2 / sea lion AAV VP1, VP2, and VP3 capsid proteins, to analyze the reaction between an anti-HER2 antibody fused to SpyCatcher (HERCEPTIN®) "SpyC-anti-HER2 mAb" and a panel of AAV2 / sea lion AAV viral particles lacking or carrying SpyTag insertions at the indicated positions, or a panel of mosaic AAV2 / sea lion AAV particles containing a mix of chimeric AAV2 / sea lion AAV particles bearing SpyTag peptide insertions at the indicated positions and chimeric AAV2 / sea lion AAV particles lacking SpyTag.

[0123] [Figure 5] Figure 5 presents (A) qPCR quantification of viral titers obtained from crude viral preparations of a panel of AAV2 / sea lion AAV viral particles lacking or carrying SpyTag insertions at the indicated positions, and (B) Western blotting using the B1 antibody, which recognizes a linear epitope engineered into the chimeric AAV2 / sea lion AAV VP1, VP2, and VP3 capsid proteins, to analyze the reaction between the anti-HER2 antibody (HERCEPTIN®) fused to SpyCatcher ("SpyC-mAb") and a panel of sea lion AAV viral particles lacking or carrying SpyTag insertions at the indicated positions.

[0124] [Figure 6-1] Figure 6A provides a non-limiting predicted ribbon structure of bearded dragon AAV VP3, highlighting T573 and G436 as non-limiting insertion sites for the first member of a protein:protein binding pair. [Figure 6-2] Figure 6B presents qPCR quantification of viral titers obtained from crude viral preparations of a panel of chimeric AAV2 / bearded dragon AAV viral particles without SpyTag or containing SpyTag insertions at the indicated positions, or a panel of mosaic particles composed of a mix of AAV2 / bearded dragon AAV particles without SpyTag and AAV2 / bearded dragon AAV particles carrying SpyTag peptide insertions at the indicated positions. [Figure 6-3]Figure 6C presents Western blotting using the B1 antibody, which recognizes linear epitopes engineered into chimeric AAV2 / bearded dragon AAV VP1, VP2, and VP3 capsid proteins, to analyze the reaction between an anti-HER2 antibody fused to SpyCatcher (HERCEPTIN®) "SpyC-anti-HER2 mAb" and a panel of chimeric AAV2 / bearded dragon AAV viral particles lacking or carrying SpyTag insertions at the indicated positions, or a panel of mosaic AAV2 / bearded dragon AAV particles containing a mix of chimeric AAV2 / bearded dragon AAV particles carrying SpyTag peptide insertions at the indicated positions and chimeric AAV2 / bearded dragon AAV particles lacking SpyTag.

[0125] [Figure 7-1] Figure 7A presents scatter plots obtained from flow cytometry evaluating green fluorescent protein (GFP) expression by HER2-positive (+) 293 hErbB2 cells infected with chimeric AAV2 / AAAV particles without SpyTag, chimeric AAV2 / AAAV G444 Linker6 SpyTag particles, or chimeric AAV2 / AAAV K580 Linker6 SpyTag particles. Chimeric AAV2 / AAAV G444 Linker6 SpyTag particles and chimeric AAV2 / AAAV K580 Linker6 SpyTag particles were bound to either an irrelevant isotype control antibody against GLP1R or an anti-HER2 antibody (HERCEPTIN®) fused to SpyCatcher (SEQ ID NO: 43) via the SpyTag (SEQ ID NO: 42). The virus expresses GFP as a marker of transduction. [Figure 7-2]Figure 7B presents a scatter plot obtained from flow cytometry evaluating green fluorescent protein (GFP) expression by parental ASGR1-negative (-) 293 cells or ASGR1-positive (+) 293 hASGR1 cells infected with chimeric AAV2 / AAAV particles without SpyTag or chimeric AAV2 / AAAV K580 Linker6 SpyTag particles. Chimeric AAV2 / AAAV K580 Linker6 SpyTag particles were bound to an irrelevant isotype control antibody against GLP1R fused to SpyCatcher via SpyTag, or to a SpyCatcher-fused antibody that specifically binds ASGR1 via SpyTag. The virus expresses GFP as a marker of transduction.

[0126] [Figure 8-1] Figure 8A presents scatter plots obtained from flow cytometry evaluating green fluorescent protein (GFP) expression by HER2-positive (+) 293 hErbB2 cells or HER2-negative (-) 293 parental cells infected with chimeric AAV2 / SeaLion particles without SpyTag or chimeric AAV2 / SeaLion G432 Linker6 SpyTag particles. Chimeric AAV2 / SeaLion G432 Linker6 SpyTag particles were coupled to an irrelevant isotype control antibody against GLP1R fused to SpyCatcher via SpyTag, or to an anti-HER2 antibody (HERCEPTIN®) fused to SpyCatcher (SEQ ID NO: 43) via SpyTag. The virus expresses GFP as a marker of transduction. [Figure 8-2]Figure 8B shows a scatter plot obtained from flow cytometry evaluating green fluorescent protein (GFP) expression by ASGR1-positive (+)293 hASGR1 or ASGR1-negative (-)293 parental cells infected with chimeric AAV2 / SeaLion particles without SpyTag, or cells infected with chimeric AAV2 / SeaLion G432 Linker6 SpyTag particles. Chimeric AAV2 / SeaLion G432 Linker6 SpyTag particles were bound to an irrelevant isotype control antibody against GLP1R fused to SpyCatcher via SpyTag, or to a SpyCatcher-fused antibody that specifically binds to ASGR1 via SpyTag. The virus expresses GFP as a marker of transduction.

[0127] [Figure 9] Figure 9 presents scatter plots obtained from flow cytometry assessing green fluorescent protein (GFP) expression by HER2-positive (+)293 infected with a panel of chimeric AAV2 / Sea Lion AAV viral particles, either without SpyTag or AAV2 / Sea Lion AAV particles containing SpyTag insertions at the indicated positions. The SpyTag inserted into the chimeric AAV2 / Sea Lion particles was conjugated to an anti-HER2 antibody (HERCEPTIN®), which was fused to SpyCatcher (SEQ ID NO: 43) via the SpyTag. The virus expresses GFP as a marker of transduction.

[0128] [Figure 10-1]10A presents scatter plots obtained from flow cytometry assessing green fluorescent protein (GFP) expression by HER2-positive (+) 293 hErbB2 cells that were "uninfected" or infected with chimeric AAV2 / bearded dragon AAV particles lacking SpyTag, chimeric AAV2 / bearded dragon T573 Linker6 SpyTag mosaic particles, or chimeric AAV2 / bearded dragon G436 Linker6 SpyTag mosaic particles. Chimeric AAV2 / bearded dragon T573 Linker6 SpyTag mosaic particles and chimeric AAV2 / bearded dragon G436 Linker6 SpyTag mosaic particles were conjugated to an anti-HER2 antibody (HERCEPTIN®), which was fused to SpyCatcher (SEQ ID NO: 43) via the SpyTag. The virus expresses GFP as a marker of transduction. [Figure 10-2] Figure 10B shows a scatter plot obtained from flow cytometry assessing green fluorescent protein (GFP) expression by ASGR1-positive (+) 293 hASGR1 cells or ASGR1-negative (-) 293 parental cells infected with chimeric AAV2 / bearded dragon AAV particles without SpyTag, chimeric AAV2 / bearded dragon T573 Linker6 SpyTag particles, or chimeric AAV2 / bearded dragon T573 Linker6 SpyTag anti-ASGR1 particles. The chimeric AAV2 / bearded dragon T573 Linker6 SpyTag anti-ASGR1 particles were conjugated to a SpyCatcher fusion antibody that specifically binds to ASGR1 via the SpyTag. The virus expresses GFP as a marker of transduction.

[0129] [Figure 11-1]Figure 11A presents the results of a Nanoluc luciferase assay evaluating Nanoluc reporter expression by hASGR1-positive (+) cells after infection with "AAV2 anti-ASGR1" particles, chimeric AAV2 / AAAV anti-ASGR1 particles, or chimeric AAV2 / Sea Lion AAV anti-ASGR1 particles in the presence of the indicated concentrations of purified human IgG. All particles were conjugated to a SpyCatcher-fused antibody, which specifically binds to ASGR1 via the SpyTag. The virus expresses Nanoluc as a marker of transduction. [Figure 11-2] Figure 11B presents a quantification of the graph in Figure 11A, but normalized to the "PBS only" condition. [Figure 11-3] FIG. 11C presents a table of IC50 values for the concentration of IgG required to neutralize the indicated viruses by 50%.

[0130] [Figure 12] Figure 12 provides luminescence images of genetically engineered mice expressing human ASGR1 on hepatocytes (ASGR1-humanized mice) 33 days after intravenous injection of (A) phosphate-buffered saline (PBS) or 5.0 x 10 viral genomes (vg) / animal with SpyTagged chimeric AAV2 / AAAV particles carrying the firefly luciferase nucleotide of interest and modified with (1) SpyCatcher-anti-human ASGR1 antibody or (2) SpyCatcher-anti-human GLP1R antibody (control mAb). The virus expresses firefly luciferase as a marker of transduction. Mice were anesthetized using isoflurane, injected with luciferin substrate, and imaged 10 minutes later using an IVIS Spectrum In Vivo Imaging System (PerkinElmer). (B) Quantification of the mean radiance of individual animals within the luminescence image depicted in panel A, and (C) quantification of the mean radiance of organs (liver and lungs) dissected from the animal depicted in panel A.

[0131] [Figure 13]Figure 13 provides luminescence images of genetically engineered mice expressing human ASGR1 on hepatocytes (ASGR1-humanized mice) 33 days after intravenous injection of (A) phosphate-buffered saline (PBS) or 5.0 x 10 viral genomes (vg) / animal with SpyTagged chimeric AAV2 / Sea Lion AAV particles carrying the firefly luciferase nucleotide of interest and modified with either (1) SpyCatcher-anti-human ASGR1 antibody or (2) SpyCatcher-anti-human GLP1R antibody (control mAb). The virus expresses firefly luciferase as a marker of transduction. Mice were anesthetized using isoflurane, injected with luciferin substrate, and imaged 10 minutes later using an IVIS Spectrum In Vivo Imaging System (PerkinElmer). (B) Quantification of the mean radiance of individual animals within the luminescence image depicted in panel A, and (C) quantification of the mean radiance of organs (liver and lungs) dissected from the animal depicted in panel A.

[0132] [Figure 14] Figure 14 shows immunofluorescence images of explant cultures of the organ of Corti from neonatal mouse inner ear 3 days after infection with chimeric AAV2 / Sea Lion AAV particles lacking SpyTag. The virus expresses GFP (green) as a marker of transduction, and hair cells are labeled with an antibody detecting Myo7a (red).

[0133] [Figure 15-1] Figure 15A presents an alignment of the C-terminal 16 amino acids of the AAV2 sequence or the AAV2 / sea lion chimeric sequence, including modifications of the B1 epitope to replace it entirely with the homologous sea lion AAV sequence, or only at residue 730. The epitope of the B1 monoclonal antibody is depicted. [Figure 15-2] Figure 15B presents qPCR quantification of viral titers obtained from purified viral preparations of chimeric AAV2 / sea lion AAV particles without SpyTag, or without SpyTag and containing modification of the B1 epitope to replace it entirely or only at residue 730 with the homologous sea lion AAV sequence. [Figure 15-3] Figure 15C presents a protein gel stain using SYPRO Ruby analyzing the expression of VP1, VP2, and VP3 capsid proteins of chimeric AAV2 / sea lion AAV particles without SpyTag, or without SpyTag and containing modification of the B1 epitope to replace it entirely or only at residue 730 with the homologous sea lion AAV sequence. [Figure 15-4] Figure 15D presents XY plots obtained from luminescence assessment of NanoLuc luciferase expression from HEK293T cell lysates infected at various multiplicities of infection (MOI) with chimeric AAV2 / sea lion AAV particles without the SpyTag, or without the SpyTag and containing modifications of the B1 epitope to replace it entirely with the homologous sea lion AAV sequence or to replace only residue 730. The virus expresses NanoLuc luciferase as a marker of transduction.

[0134] [Figure 16] Figure 16 shows quantification of the mean radiance of organs dissected from mice 34 days after intravenous injection in phosphate-buffered saline (PBS) or with 5.0 x 10 viral genomes (vg) / animal of chimeric AAV2 / sea lion AAV particles carrying the firefly luciferase nucleotide of interest and modified without a SpyTag, or without a SpyTag and modified to include a modification of the B1 epitope to replace the entire sequence or only residue 730 with the homologous sea lion AAV sequence. The virus expresses firefly luciferase as a marker of transduction. Mice were anesthetized using isoflurane, injected with luciferin substrate, and imaged 10 minutes later using an IVIS Spectrum in vivo imaging system (PerkinElmer).

[0135] [Figure 17]Figure 17 provides illustrative (not to scale), non-limiting, and exemplary embodiments of Rep-Cap expression plasmids of the present invention that can be used to generate AAV chimeric viral particles. Primate AAV sequences are shown as open boxes, and sea lion AAV sequences are shown as black boxes. Also depicted, for illustrative purposes only, are exemplary, non-limiting locations (not to scale) for (1) alternative interface placements between the primate and sea lion capsid sequences (black dotted lines in the capsid sequences of primate AAVs) and (2) detectable labels for detecting the encoded VP1, VP2, and VP3 proteins (dotted lines in the capsid sequences of non-primate AAVs). The Rep-Cap expression plasmids shown in Figure 17 may be used to produce AAV viral particles containing nucleotides of interest flanked by 5' and 3' inverted terminal repeats (ITRs) of primate AAVs.

[0136] [Figure 18-1] Figure 18A shows qPCR quantification of viral titers obtained from purified viral preparations of chimeric AAV2 / Sea Lion AAV particles lacking a SpyTag and containing alternative interface arrangements between the AAV2 and Sea Lion capsid sequences. Preparations were purified from cell lysates (v2-v4) or both cell lysates and media (v5). [Figure 18-2] Figures 18B and 18C show XY plots obtained from luminescence assessment of NanoLuc luciferase expression from HEK293T cell lysates infected at various multiplicities of infection (MOIs) with AAV2 / Sea Lion AAV particles lacking SpyTag and with alternative interface arrangements between the AAV2 and Sea Lion capsid sequences. A historical data set without AAV2 / Sea Lion AAV SpyTag is used as a reference. The virus expresses NanoLuc luciferase as a marker of transduction. [Figure 18-3] Same as above. [Figure 18-4]Figure 18D shows quantification of the mean radiance of organs dissected from mice 56 days after intravenous injection in phosphate-buffered saline (PBS) or with 5.0 x 10 viral genomes (vg) / animal of AAV2 / Sea Lion AAV particles carrying the firefly luciferase nucleotide of interest and modified without a SpyTag, or modified without a SpyTag and with the B1 epitope completely replaced with the homologous Sea Lion AAV sequence, with or without an alternative interface between the AAV2 and Sea Lion capsid sequences. The viruses express firefly luciferase as a marker of transduction. Mice were anesthetized using isoflurane, injected with luciferin substrate, and imaged 10 minutes later using an IVIS Spectrum in vivo imaging system (PerkinElmer). DETAILED DESCRIPTION OF THE INVENTION

[0137] While recombinant methods for targeting specific cells via AAV gene therapy have improved in recent years, current recombinant methods used to develop AAVs still pose challenges, likely due to their ability to evade detection and / or neutralization by preexisting antibodies developed during childhood. Described herein are methods that exploit (1) the inherent ability of non-primate AAVs or remote AAVs to infect primate cells, (2) the lack of NAb antibodies in humans against non-primate AAV capsid proteins, and, if desired or necessary, (3) the adaptability of a first member of a protein:protein binding pair engineered into an AAV capsid protein for the production of AAV viral particles useful for directed gene therapy, e.g., the introduction of a target nucleotide into specific cells of interest. Described herein are nucleotide molecules comprising at least the AAV cap gene, which can be utilized according to a desired function. The nucleotide molecules of the present invention comprise a cap gene or a portion thereof derived from a non-primate AAV and / or a remote AAV (for the purpose of producing viral capsids that are not readily recognized by preexisting NAb antibodies). The cap gene of a non-primate AAV, or a portion thereof, may be modified with a first member of a protein:protein binding pair, to which a second member comprising a targeting ligand can bind and direct the tropism of the resulting AAV viral load.

[0138] For non-primate or remote AAVs that cannot infect primate cells, the cap gene may be designed as a chimeric cap gene encoding at least the phospholipase A2 (PLA2) domain of the primate AAV VP1 capsid protein and at least a portion of the VP3 capsid protein of the non-human primate or remote AAV. The PLA2 domain, carried by the VP1 capsid protein (more specifically, the VP1-unique (VP1-u) region of the VP1 capsid), is thought to be important during AAV infection by mediating the transport of the viral genome from late endosomes / lysosomes to the nucleus to initiate replication (Zadori et al., 2001, Dev Cell, 1(2):291-302). The VP3 capsid protein is the major surface capsid protein of AAV viral particles, and therefore viral capsids containing at least a portion of the VP3 capsid protein of non-primate AAV or remote AAVs are unlikely to be recognized by NAbs generated against AAV serotypes isolated from primates during the course of primate AAV infection.

[0139] A non-limiting depiction of a nucleic acid molecule comprising a primate AAV rep gene and a chimeric cap gene is provided in Figure 1. For non-primate human AAVs, remote AAVs capable of infecting primate cells, the primate AAV rep gene may be operably linked to the chimeric cap gene described herein or the non-primate AAV cap gene. Examples herein demonstrate that when expressed in a helper plasmid and a packaging cell line containing a primate AAV genome carrying a nucleotide sequence of interest, the nucleic acid molecule encodes the appropriate replication and capsid proteins and functions to replicate and encapsidate the primate genome, respectively, into viral particles capable of infecting cells in vivo. Furthermore, these examples demonstrate that the tropism of the AAV viral particles can be easily adapted using the first and second members of a protein:protein binding pair, and further demonstrate that the insertion of the first and second members of a protein:protein binding pair does not increase the likelihood of recognition by Nabs generated against primate AAV infection. Thus, provided herein are genetically modified viral particles, compositions comprising them, and methods of making and using them.

[0140] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0141] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps of the kind described herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0142] "Percent identity" and the like can be readily determined for amino acid or nucleotide sequences spanning the entire length of a protein or a portion thereof. The portion may be at least about 5 amino acids or 24 nucleotides in length, respectively, and may be up to about 700 amino acids or 2100 nucleotides in length, respectively. Generally, when referring to "identity," "homology," or "similarity" between two different adeno-associated viruses, the "identity," "homology," or "similarity" is determined with reference to "aligned" sequences. "Aligned" sequences or "alignment" refer to multiple nucleic acid or protein (amino acid) sequences, which often include corrections for missing or added bases or amino acids compared to a reference sequence.

[0143] Alignment may be performed using any of a variety of publicly available or commercially available multiple sequence alignment programs. For amino acid sequences, sequence alignment programs are available, such as "Clustal X," "MAP," "PIMA," "MSA," "BLOCKMAKER," "MEME," and "Match-Box." Generally, one of these programs is used with default settings, but those skilled in the art can change these settings as needed. Alternatively, those skilled in the art can use a different algorithm or computer program that provides at least the same level of identity or alignment as that provided by the referenced algorithm and program. See, for example, J.D. Thomson et al., Nucl. Acids. Res., "A Comprehensive Comparison of Multiple Sequence Alignments," 27(13):2682-2690 (1999).

[0144] For nucleic acid sequences, several sequence alignment programs are also available. Examples of such programs include "Clustal W," "CAP Sequence Assembly," "MAP," and "MEME," which are accessible via web servers on the Internet. Other sources of such programs are known to those skilled in the art. Alternatively, the Vector NTI utility can be used. There are also numerous algorithms known in the art that can be used to measure the identity of nucleotide sequences, including the programs mentioned above. As another example, polynucleotide sequences can be compared using FASTA™, a program in GCG version 6.1. FASTA™ provides alignments and percentage sequence identity of the regions of best overlap between the query and search sequences. For example, the percentage sequence identity between nucleic acid sequences can be determined using FASTA™ with its default parameters (word size of 6 and NOPAM factor for the scoring matrix) provided in GCG version 6.1, which is incorporated herein by reference.

[0145] "Substantial identity" includes alignment of amino acid or nucleic acid sequences that are at least 90%, such as at least 93%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98%, for example at least 99%, or such as at least 100% identical.

[0146] The term "chimera" encompasses functional genes or polypeptides comprising nucleic acid or amino acid sequences derived from at least two different organisms, e.g., at least portions of a gene or polypeptide from a first AAV and a second AAV, where the at least first and second portions are operably linked. Unless designated as chimeric, nucleotide sequences, genes, polypeptides, and amino acids are considered non-chimeric, e.g., comprising nucleic acid or amino acid sequences from only one organism, e.g., only one AAV.

[0147] 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 contains a heavy chain variable domain (V- H ) and the heavy chain constant region (C H The heavy chain constant region comprises at least three domains: C H 1. C H 2. C H Each light chain comprises a light chain variable domain (C H ) and the light chain constant region (C L ). Heavy and light chain variable domains can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each heavy and light chain variable domain contains three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs are sometimes abbreviated as HCDR1, HCDR2, and HCDR3; light chain CDRs are sometimes abbreviated as LCDR1, LCDR2, and LCDR3). A typical tetrameric antibody structure contains two identical antigen-binding domains, each of which is a V H Domains and V L are formed by the association of domains, each of which is a separate C H Domain and C L The domains together form the antibody Fv region. Single domain antibodies contain a single antigen-binding domain, e.g., a V H or V LThe 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." The paratope is a small region (5-10 amino acids) of the Fv region, which is part of the antigen-binding fragment (Fab region) of an antibody, and may contain portions of the heavy and / or light chains of the antibody. A paratope specifically binds to a first member of a specific binding pair when it binds with high affinity to the first member of the specific binding pair. The term "high affinity" antibody refers to an antibody that binds to a target first member of a specific binding pair with an affinity of approximately 10 -9 M or less (e.g., about 1 × 10 -9 M, 1 x 10 -10 , 1×10 -11 M, or approximately 1 x 10 -12 M)'s K D In one embodiment, K D is measured by surface plasmon resonance, e.g., BIACORE™, and in another embodiment, K D is measured by ELISA.

[0148] The phrase "complementarity-determining region" or "CDR" includes an amino acid sequence encoded by a nucleic acid sequence of an organism's immunoglobulin gene, which amino acid sequence is normally (i.e., in wild-type animals) found between two framework regions in the variable region of the 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 rearranged or unrearranged sequences, e.g., by naive or mature B cells, or T cells. CDRs can be somatically mutated (e.g., different from the sequence encoded in the animal germline), humanized, and / or modified with amino acid substitutions, additions, or deletions. In some circumstances (e.g., with respect to CDR3), a CDR 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, e.g., as a result of splicing or joining of sequences (e.g., VDJ rearrangement to form a heavy chain CDR3).

[0149] The term "light chain" includes immunoglobulin light chain sequences from any organism, including human kappa and lambda light chains, as well as surrogate light chains, and VpreB, 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, from the amino terminus to the carboxyl terminus, a variable domain comprising 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 and generally includes a V segment derived from a repertoire of V and J segments present in the germline. L Segment and J L The sequences, locations, and nomenclature of V and J light chain segments of various organisms can be found in the IMGT database, www.imgt.org. Light chains include, for example, light chains that do not selectively bind either the first or second member of a specific binding pair selectively bound by the first member of the specific binding pair binding protein in which the light chain appears. Light chains also include light chains that bind to, recognize, or assist heavy chains or other light chains by binding and recognizing one or more first members of a specific binding pair selectively bound by the first member of the specific binding pair binding protein in which the light chain appears. Common or universal light chains include light chains derived from human Vκ1-39Jκ genes or human Vκ3-20Jκ genes, including somatically mutated (e.g., affinity matured) versions thereof. Exemplary human Vκ1-39Jκ genes include those derived from human Vκ3-20Jκ genes, such as those derived from human Vκ3-20Jκ genes, and those derived from human Vκ3-20Jκ genes. LThe 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 produced that include a variable domain from one organism (e.g., a human or rodent, such as a rat or mouse, or an avian, such as a chicken) and a constant region from the same or a different organism (e.g., a human or rodent, such as a rat or mouse, or an avian, such as a chicken).

[0150] The term "about" or "approximately" includes within a statistically significant range of values. Such a range may be within 10-fold, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of a given value or range. The acceptable variation encompassed by the term "about" or "approximately" will depend on the particular system under study and can be readily understood by one of ordinary skill in the art.

[0151] The term "heavy chain" or "immunoglobulin heavy chain" includes immunoglobulin heavy chain sequences (such as immunoglobulin heavy chain constant region sequences) from any organism. Unless otherwise specified, a heavy chain variable domain comprises three heavy chain CDRs and four FR regions. Fragments of heavy chains include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain comprises (from N- to C-terminus) the variable domain followed by a C H 1 domain, hinge, C H 2 domain, and C H A functional fragment of a heavy chain has three domains that specifically recognize the first member of a specific binding pair (e.g., a K in the micromolar, nanomolar, or picomolar range). DThe heavy chain variable domain is encoded by a variable region nucleotide sequence and generally corresponds to the V present in germline cells. H , D H , and J. H From the segment repertoire, V H , D H , and J. H The sequences, locations, and nomenclature of V, D, and J heavy chain segments from various organisms can be found in the IMGT database, which is accessible via the Internet on the worldwide web (www) at the URL "imgt.org".

[0152] The terms "heavy chain-only antibody," "heavy chain-only antigen-binding protein," "single domain antigen-binding protein," "single domain binding protein," and the like refer to a monomeric or homodimeric immunoglobulin molecule comprising an immunoglobulin-like chain comprising a variable region comprising a variable domain operably linked to a heavy chain constant region, where the heavy chain constant region is typically a functional C H Therefore, the terms "heavy chain-only antibody," "heavy chain-only antigen-binding protein," "single domain antigen-binding protein," "single domain binding protein," etc., refer to antibodies that lack (i) a functional C H a monomeric single-domain antigen-binding protein comprising one of the immunoglobulin-like chains comprising a variable domain operably linked to a heavy chain constant region lacking one domain; or (ii) two immunoglobulin-like chains, each of which is a functional C H In various embodiments, the homodimeric single domain antigen binding protein comprises two identical immunoglobulin-like chains, each of which is functionally linked to a heavy chain constant region lacking one domain, and a homodimeric single domain antigen binding protein, which comprises a variable domain operably linked to a heavy chain constant region lacking one domain. HIn addition, each immunoglobulin-like chain of a single domain antigen-binding protein contains a variable domain, which is derived from a heavy chain variable region gene segment (e.g., V H , D H , J H ), light chain gene segments (e.g., V L , J L ), or a combination thereof, and the sequence of the heavy chain constant region (and optionally the hinge region) gene, e.g., C encoding IgG, IgA, IgE, IgD, or a combination thereof. H heavy chain constant region (C) containing a deletion or inactivating mutation in H A single domain antigen binding protein comprising a variable domain derived from a heavy chain gene segment may be referred to as a "V" H Single domain antigen binding proteins comprising a variable domain derived from a light chain gene segment may be referred to as a "V single domain antibody" or "VH single domain antibody binding protein," see, e.g., U.S. Patent No. 8,754,287, U.S. Patent Publication Nos. 2014 / 0289876, 2015 / 0197553, 2015 / 0197554, 2015 / 0197555, 2015 / 0196015, 2015 / 0197556, and 2015 / 0197557, each of which is incorporated by reference in its entirety. L These may be referred to as "single domain antigen binding proteins," see, e.g., U.S. Publication No. 2015 / 0289489, which is incorporated by reference in its entirety.

[0153] The term "light chain" includes immunoglobulin light chain sequences from any organism, and includes human kappa (κ) and lambda (λ) light chains, as well as surrogate light chains, and VpreB, unless otherwise specified. A light chain variable domain typically includes three light chain CDRs and four framework (FR) regions, unless otherwise specified. Generally, a full-length light chain includes, from the amino terminus to the carboxyl terminus, a variable domain including FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region amino acid sequence. The light chain variable domain is encoded by a light chain variable region nucleotide sequence and generally includes a light chain V and J gene segment derived from a repertoire of light chain V and J gene segments present in germline cells. L and light chain J L The sequences, locations, and nomenclature of light chain V gene segments and light chain J gene segments of various organisms can be found in the IMGT database, which is accessible via the Internet on the worldwide web (www) at the URL "imgt.org." Light chains include, for example, light chains that do not selectively bind either the first or second member of a specific binding pair selectively bound by the first member of the specific binding pair-binding protein in which the light chain appears. Light chains also include light chains that bind to, recognize, or assist heavy chains by binding to and recognizing one or more first members of a specific binding pair selectively bound by the first member of the specific binding pair-binding protein in which the light chain appears. Light chains also include light chains that bind to, recognize, or assist heavy chains by binding to and recognizing one or more first members of a specific binding pair selectively bound by the first member of the specific binding pair-binding protein in which the light chain appears. Common or universal light chains include light chains derived from the human Vκ1-39Jκ5 gene or the human Vκ3-20Jκ1 gene, including somatically mutated (e.g., affinity matured) versions thereof.

[0154] The phrase "operably linked," as used herein, includes the physical juxtaposition (e.g., in three-dimensional space) of components or elements that interact directly or indirectly with each other or are positioned relative to each other so as to participate in a biological event, which juxtaposition achieves or enables such interaction and / or positioning. For example, a regulatory sequence (e.g., an expression control sequence) in a nucleic acid is said to be "operably linked" to a coding sequence when it is positioned relative to the coding sequence such that its presence or absence affects the expression and / or activity of the coding sequence. In many embodiments, "operably linked" includes the covalent linkage of associated components or elements. Those skilled in the art will readily appreciate that in some embodiments, covalent linkage is not required to achieve effective operable linkage. For example, in some embodiments, a nucleic acid regulatory sequence that is operably linked to a coding sequence that it controls is contiguous with the nucleotide of interest. Alternatively or additionally, in some embodiments, one or more such regulatory sequences function in trans or at a distance to regulate the coding sequence of interest. In some embodiments, the term "expression control sequences," as used herein, refers to polynucleotide sequences necessary and / or sufficient to affect the expression and processing of coding sequences to which they are linked. In some embodiments, expression control sequences may be or include appropriate transcription initiation, termination, promoter and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation signals, sequences that stabilize cytoplasmic mRNA, sequences that improve translation efficiency (e.g., Kozak consensus sequences), sequences that improve protein stability, and / or, in some embodiments, sequences that improve protein secretion. In some embodiments, one or more control sequences are preferentially or only active in a particular host cell or organism, or type thereof. By way of example, in prokaryotes, control sequences typically include a promoter, a ribosomal binding site, and a transcription termination sequence; in eukaryotes, in many embodiments, control sequences typically include a promoter, an enhancer, and / or a transcription termination sequence.Those skilled in the art will understand that in many embodiments, the term "control sequences" refers to components essential for expression and processing, and in some embodiments includes components whose presence is advantageous for expression (including, for example, leader sequences, targeting sequences, and / or fusion partner sequences).

[0155] "Retargeting" or "redirection" can include scenarios in which wild-type particles target some cells within a tissue and / or organ within an organism, and general targeting of that tissue or organ is reduced or abolished by the insertion of heterologous amino acids, and retargeting to more specific cells of a tissue or specific organ within an organism is achieved with a targeting ligand (e.g., via a targeting ligand) that binds to a marker expressed by that specific cell. Such retargeting or redirection can also include scenarios in which wild-type particles target a tissue, and targeting of that tissue is reduced or abolished by the insertion of heterologous amino acids, and retargeting to an entirely different tissue is achieved with a targeting ligand.

[0156] A "specific binding pair," "protein:protein binding pair," and the like, comprises two proteins (e.g., a first member (e.g., a first polypeptide) and a second cognate member (e.g., a second polypeptide)) that interact to form a bond (e.g., a covalent bond between an epitope of a first member and an antigen-binding portion of an antibody that recognizes the epitope) or form a covalent isopeptide bond under conditions that allow or promote bond formation. In some embodiments, the term "cognate" refers to components that function together. Epitopes and their cognate antibodies are known in the art, particularly epitopes that can also serve as detectable labels (e.g., c-myc). Specific protein:protein binding pairs capable of interacting to form covalent isopeptide bonds are reviewed in Veggiani et al. (2014) Trends Biotechnol. 32:506 and include peptide:peptide binding pairs such as SpyTag:SpyCatcher, SpyTag002:SpyCatcher002, SpyTag:KTag, Isopeptag:Pyrin C, SnoopTag:SnoopCatcher, etc. Generally, a first member of a protein:protein binding pair generally refers to a member of a protein:protein binding pair that is shorter than 30 amino acids and forms a covalent isopeptide bond with a second cognate protein, which is generally larger, but may also be shorter than 30 amino acids, such as the SpyTag:KTag system.

[0157] The term "isopeptide bond" refers to an amide bond between a carboxyl or carboxamide group and an amino group, where at least one of the amino groups is not found to originate from the protein backbone or, alternatively, is not part of the protein backbone. Isopeptide bonds can form within a single protein, or between two peptides or between a peptide and a protein. Thus, isopeptide bonds can form intramolecularly within a single protein, or intramolecularly, i.e., between two peptide / protein molecules, e.g., between two peptide linkers. Typically, an isopeptide bond can occur between a lysine residue and an asparagine, aspartic acid, glutamine, or glutamic acid residue, or the terminal carboxyl group of a protein or peptide chain, or between the alpha-amino terminus of a protein or peptide chain and an asparagine, aspartic acid, glutamine, or glutamic acid residue. Each residue in a pair involved in an isopeptide bond is referred to herein as a reactive residue. In a preferred embodiment of the present invention, an isopeptide bond can be formed between a lysine residue and an asparagine residue, or between a lysine residue and an aspartic acid residue. In particular, an isopeptide bond can occur between the side chain amine of a lysine and the carboxamide group of an asparagine or the carboxyl group of an aspartic acid.

[0158] The SpyTag:SpyCatcher system is described in U.S. Patent No. 9,547,003 and Zaveri et al. (2012) PNAS 109:E690-E697 (each of which is incorporated herein by reference in its entirety), and is derived from the CnaB2 domain of the fibronectin-binding protein FbaB of Streptococcus pyogenes. By dividing the domain, Zakeri et al. obtained the "SpyTag" peptide, which has the sequence AHIVMVDAYKPTK (SEQ ID NO: 42). This peptide forms an amide bond with its cognate protein, "SpyCatcher." SpyCatcher is a 112-amino acid polypeptide with the amino acid sequence set forth in SEQ ID NO: 43 (Zakeri (2012) supra). Another specific binding pair derived from the CnaB2 domain is SpyTag:KTag, which forms an isopeptide bond in the presence of SpyLigase. (Fierer (2014) PNAS 111:E1176-1181). SpyLigase was engineered by deleting the beta strand from SpyCatcher, which contains a reactive lysine, resulting in KTag. KTag is the 10-residue first member of a protein:protein binding pair with the amino acid sequence ATHIKFSKRD (SEQ ID NO: 72). The SpyTag002:SpyCatcher002 system was described by Keeble et al. (2017) Angew Chem Int Ed Engl 56:16521-25, which is incorporated herein by reference in its entirety. SpyTag002 has the amino acid sequence VPTIVMVDAYKRYK, set forth as SEQ ID NO: 73, and binds to SpyCatcher002.

[0159] The SnoopTag:SnoopCatcher system is described in Veggiani (2016) PNAS 113:1202-07. The D4 Ig-like domain of RrgA, an adhesin from S. pneumoniae, was split to form SnoopTag (residues 734-745) and SnoopCatcher (residues 749-860). Incubation of SnoopTag and SnoopCatcher results in the formation of specific spontaneous isopeptide bonds between the complementary proteins. See Veggiani (2016) above.

[0160] The isopeptag:pilin-C specific binding pair is The SnoopTag was derived from SpyO128, a major Pilin protein from S. pyogenes (Zakeir and Howarth (2010) J. Am. Chem. Soc. 132:4526-27). The Isopeptag has the amino acid sequence TDKDMTITFTNKKDAE, set forth as SEQ ID NO: 75, and binds to pilin-C (residues 18-299 of SpyO128). Incubation of the SnoopTag and SnoopCatcher results in the formation of a specific spontaneous isopeptide bond between the complementary proteins. See Zakeir and Howarth (2010), supra.

[0161] The term "detectable label" includes, for example, a polypeptide sequence that is a member of a specific binding pair that specifically binds with high affinity to another polypeptide sequence, such as an antibody paratope, via non-covalent bonding. Exemplary and non-limiting detectable labels include hexahistidine tag, FLAG tag, Strep II tag, streptavidin-binding peptide (SBP) tag, calcitonin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA tag, and c-myc (SEQ ID NO: 44). (Reviewed in Zhao et al. (2013) J. Analytical Meth. Chem. 1-8, incorporated herein by reference.) A common detectable label for primate AAV is the B1 epitope (SEQ ID NO: 45). The non-primate AAV capsid proteins of the present invention do not naturally contain the B1 epitope and can be modified herein to contain the B1 epitope. Generally, non-primate AAV capsid proteins may contain a sequence having substantial homology to the B1 epitope within the last 10 amino acids of the capsid protein. Thus, in some embodiments, non-primate AAV capsid proteins of the invention may be modified with one to fewer than five point mutations within the last 10 amino acids of the capsid protein, such that the AAV capsid protein contains the B1 epitope.

[0162] The term "target cell" includes any cell in which expression of a nucleotide of interest is desired. Preferably, target cells exhibit receptors on their surface that allow the cells to be targeted by targeting ligands, as described below.

[0163] Terms such as "transduction" or "infection" refer to the introduction of nucleic acid into the nucleus of a target cell by a viral particle. Efficiency related to transduction, for example, the term "transduction efficiency," refers to the fraction (e.g., percentage) of cells that express a target nucleotide after incubation with a set number of viral particles containing the target nucleotide. Known methods for determining transduction efficiency include flow cytometry of transduced cells using a fluorescent reporter gene, RT-PCR for the expression of the target nucleotide, etc.

[0164] Generally, the "reference" viral capsid protein / capsid / particle is identical to the test viral capsid protein / capsid / particle, but not with respect to the change whose effect is being tested. For example, to determine the effect of inserting a first member of a specific binding pair into a test viral particle, e.g., on transduction efficiency, the transduction efficiency of the test viral particle (in the presence or absence of an appropriate targeting ligand) can be compared to the transduction efficiency of a reference viral particle that is identical to the test viral particle in every instance (e.g., additional point mutations, target nucleotides, number of viral particles and target cells, etc.) except for the presence of the first member of the specific binding pair (in the absence or presence of an appropriate targeting ligand, as needed). In some embodiments, a reference viral capsid protein can form a capsid with a second viral capsid protein that has been modified to include at least a first member of a protein:protein binding pair, wherein the reference viral capsid protein does not include the first member of the protein:protein binding pair, and preferably the capsid formed by the reference viral capsid protein and the modified viral capsid protein is a mosaic capsid.

[0165] Adeno-associated virus (AAV)

[0166] "AAV" is an abbreviation for adeno-associated virus and can refer to the virus itself or its derivatives. AAV is a small, non-enveloped, single-stranded DNA virus. The wild-type AAV genome is typically 4.7 kb and is characterized by two inverted terminal repeats (ITRs) and two open reading frames (ORFs), rep and cap. The wild-type rep reading frame encodes four proteins with molecular weights of 78 kD (Rep78), 68 kD (Rep68), 52 kD (Rep52), and 40 kD (Rep40). Rep78 and Rep68 are transcribed from the p5 promoter, while Rep52 and Rep40 are transcribed from the p19 promoter. These proteins primarily function to regulate the transcription and replication of the AAV genome. The wild-type cap reading frame encodes three structural (capsid) viral proteins (VPs) with molecular weights of 83-85 kD (VP1), 72-73 kD (VP2), and 61-62 kD (VP3). VP3 accounts for over 80% of the total protein in the AAV virion (capsid). In mature virions, VP1, VP2, and VP3 are present in relative amounts of approximately 1:1:10, although ratios as high as 1:1:8 have also been reported. (Padron et al. (2005) J. Virology 79:5047-58)

[0167] The genome sequences of various serotypes of AAV, as well as the sequences of natural inverted terminal repeats (ITRs), Rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. For example, see GenBank accession numbers NC_002077 (AAV1), AF063497 (AAV1), NC001401 (AAV-2), AF043303 (AAV2), NC_001729 (AAV3), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF513851 (AAV7), AF513852 (AAV8), and NC_006261 (AAV8). The disclosures thereof are incorporated herein by reference for teaching the nucleic acid and amino acid sequences of AAV. For example, Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73:1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. al.(1999)J.Virology 73:3994;Muramatsu et al.(1996)Virology 221:208;Shade et al.,(1986)J.Virol.58:921;Gao et al.(2002)Proc.Nat.Acad.Sci.USA 99:11854;Moris et al. al.(2004) Virology 33:375-383; U.S. Patent Publication No. 20170130245; International Patent Application Publications WO00 / 28061, WO99 / 61601, WO 98 / 11244; and U.S. Patent No. 6,156,303, each of which is incorporated herein by reference in its entirety. Table 2 herein provides sequences of various non-primate AAVs.

[0168] "AAV" encompasses all subtypes, and both naturally occurring and modified forms, unless otherwise required. AAV includes primate AAV (e.g., AAV type 1 (AAV1), primate AAV type 2 (AAV2), primate AAV type 3 (AAV3), primate AAV type 4 (AAV4), primate AAV type 5 (AAV5), primate AAV type 6 (AAV6), primate AAV type 7 (AAV7), primate AAV type 8 (AAV8), non-primate AAV (e.g., avian AAV (AAAV)), and other non-primate AAV, such as mammalian AAV (e.g., bat AAV, sea lion AAV, bovine AAV, canine AAV, equine AAV, caprine AAV). "AAV" generally refers to AAV isolated from primates. Similarly, "non-primate AAV" refers to AAV isolated from non-primate animals. As used herein, "remote AAV" encompasses: AAV isolated from primates or non-primate animals that generally have limited contact with the general human population, and for each of the following, less than 99%, e.g., less than 95%, AAV isolated from a primate, e.g., a primate AAV, comprising a wild-type VP1 capsid protein comprising an amino acid sequence having at least, e.g., less than 90%, e.g., less than 85%, amino acid sequence identity with the AAV: AAV1 VP1 capsid protein, AAV2 VP1 capsid protein, AAV3 VP1 capsid protein, AAV4 VP1 capsid protein, AAV5 VP1 capsid protein, AAV6 VP1 capsid protein, AAV7 VP1 capsid protein, AAV8 VP1 capsid protein, an AAV isolated from a non-primate, e.g., a non-primate AAV, comprising a wild-type capsid protein comprising an amino acid sequence having less than 99%, such as less than 95%, such as less than 90%, for example less than 85%, amino acid sequence identity to a protein, an AAV9 VP1 capsid protein, an AAV10 VP1 capsid protein, an AAV11 VP1 capsid protein, an AAV12 VP1 capsid protein, and an AAV13 VP1 capsid protein, and / or each of the AAVs listed in Table 2.

[0169] Seropositivity may be assessed using known methods. For example, the absence of IgG may be determined by enzyme-linked immunosorbent assay (ELISA) or other known immune-based assays. Neutralization assays may be performed to detect neutralizing antibodies. In such assays, AAV particles are incubated with increasing amounts (serial dilution) of (i) a specific subject's serum, or a mixed serum of multiple subjects, or (ii) individual or pooled serum samples (e.g., from tens to thousands of donors representing immunoglobulins in a given population), and cell infection is then detected, for example, by monitoring reporter gene expression (e.g., luciferase gene, GFP, etc.). The infection level is then compared to the level in a control sample not exposed to serum / IVIG / IgG. See, for example, Example 8. The neutralization titer may be defined, for example, as the concentration of IVIG / IgG or the highest dilution of serum that results in a 50% or greater inhibition of reporter gene expression compared to the control. In one embodiment, the serum dilution at which a greater than 70% reduction in the number of infected cells is observed compared to the control is considered to be positive for neutralizing activity. Interaction with specific known neutralizing antibodies can be tested using, for example, an immunoblot assay.

[0170] As used herein, with respect to a gene (e.g., rep, cap, etc.), a capsid protein (e.g., VP1 capsid protein, VP2 capsid protein, VP3 capsid protein, etc.), a region of a capsid protein of a designated AAV (e.g., the PLA2 region, the VP1-u region, the VP1 / VP2 common region, the VP3 region), a nucleotide sequence (e.g., an ITR sequence), such as the cap gene or capsid protein of AAV2, "of a [designated] AAV" encompasses not only a gene or polypeptide comprising the nucleic acid sequence or amino acid sequence, respectively, described herein for that designated AAV, but also variants of that gene or polypeptide, including variants that include the minimum number of nucleotides or amino acids required to retain one or more biological functions. As used herein, a variant gene or variant polypeptide comprises a nucleic acid sequence or amino acid sequence that differs from the nucleic acid sequence or amino acid sequence described herein for the specified AAV gene or polypeptide, where the difference generally does not alter at least one biological function of the gene or polypeptide and / or does not alter the phylogenetic characteristics of the gene or polypeptide, e.g., the difference may be due to degeneracy in the genetic code, isolated variation, sequence length, etc. For example, as used herein, rep and cap genes can encompass rep and cap genes that differ from the wild-type genes in that the genes may encode one or more Rep and Cap proteins, respectively. In some embodiments, the Rep gene encodes at least Rep78 and / or Rep68. In some embodiments, the cap gene may differ from the wild type in that one or more alternative start codons, or the sequence between one or more alternative start codons, have been removed, such that the cap gene encodes only one Cap protein; for example, the VP2 and / or VP3 start codons have been removed or replaced, such that the cap gene encodes a functional VP1 capsid protein, but not a VP2 or VP3 capsid protein.Thus, as used herein, a rep gene includes any sequence that encodes a functional Rep protein, and a cap gene includes any sequence that encodes at least one functional cap gene.

[0171] Wild-type cap genes are known to express all three VP1, VP2, and VP3 capsid proteins from a single open reading frame in the cap gene under the control of the p40 promoter present in the rep ORF. The terms "capsid protein," "Cap protein," and the like include proteins that are part of the viral capsid. For adeno-associated viruses, the capsid proteins are commonly referred to as VP1, VP2, and / or VP3, and they may be encoded by a single cap gene. For AAV, the three AAV capsid proteins are naturally produced in an overlapping manner using alternative translation initiation codons in the cap ORF, but all three proteins use a common stop codon. The wild-type cap gene ORF encodes three alternative initiation codons from 5' to 3': the "VP1 initiation codon," the "VP2 initiation codon," and the "VP3 initiation codon," as well as a single "consensus stop codon." VP1, the largest viral protein, is generally encoded from the VP1 initiation codon to the "consensus stop codon." VP2 is generally encoded from the VP2 start codon to the common stop codon. VP3 is generally encoded from the VP3 start codon to the common stop codon. Therefore, VP1 contains an N-terminal sequence not shared with VP2 or VP3, which is referred to as the VP1-unique region (VP1-u). The VP1-u region is generally encoded by the sequence of the wild-type cap gene, starting from the VP1 start codon to the "VP2 start codon." VP1-u contains a phospholipase A2 domain (PLA2), which may be important for infection and a nuclear localization signal that may assist in targeting the virus to the nucleus for uncoating and genome release. VP1, VP2, and VP3 capsid proteins share the same C-terminal sequence that constitutes the entire VP3, which is sometimes referred to herein as the VP3 region. The VP3 region is encoded from the VP3 start codon to the common stop codon. VP2 shares approximately 60 additional amino acids with VP1. This region is called the VP1 / VP2 common region.

[0172] In some embodiments, one or more of the cap proteins of the present invention may be encoded by one or more cap genes having one or more ORFs. In some embodiments, the VP proteins of the present invention may be expressed from multiple ORFs containing nucleotide sequences encoding any combination of VP1, VP2, and / or VP3, each producing one or more of the VP1, VP2, and / or VP3 capsid proteins of the present invention, by using separate nucleotide sequences operably linked to at least one expression control sequence for expression in a packaging cell. In some embodiments, the VP capsid proteins of the present invention may be expressed individually from ORFs containing nucleotide sequences encoding any one of VP1, VP2, or VP3, by using separate nucleotide sequences operably linked to a single expression control sequence for expression in a viral replication cell, each producing only one of the VP1, VP2, or VP3 capsid proteins. In another embodiment, the VP proteins may be expressed from a single ORF comprising nucleotide sequences encoding VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence for expression in a viral replicating cell, each producing a VP1, VP2, and VP3 capsid protein. Thus, the amino acid positions provided herein may be provided relative to the VP1 capsid protein of the referenced AAV, and one of skill in the art would readily be able to determine the same amino acid position within the VP2 and / or VP3 capsid proteins of an AAV, and the corresponding amino acid positions among different AAVs, respectively.

[0173] The term "inverted terminal repeats" or "ITR" refers to symmetrical nucleic acid sequences in the genome of adeno-associated viruses that are required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. ITRs serve as replication origins for viral DNA synthesis and are essential cis-components for AAV particle production, including packaging into AAV particles.

[0174] The AAV ITRs contain recognition sites for the replication proteins Rep78 or Rep68. The A"D" regions of the ITRs contain DNA nick sites where DNA replication initiates, providing directionality for the nucleic acid replication process. AAV replication in mammalian cells typically involves two ITR sequences.

[0175] A single ITR may be engineered with Rep binding sites on both strands of the "A" region and the two symmetric D regions on either side of the ITR palindrome. Such engineered constructs allow Rep78- or Rep68-initiated nucleic acid replication to proceed in both directions on a double-stranded circular DNA template. A single ITR is sufficient for AAV replication in circular particles. In the methods of generating AAV viral particles of the invention, the rep coding sequence encodes a Rep protein or a Rep protein equivalent that can bind to the ITRs contained on a transfer plasmid.

[0176] The Cap proteins of the present invention, when expressed together with appropriate Rep proteins by a packaging cell, can encapsidate a transfer plasmid containing a target nucleotide and an even number of two or more ITR sequences. In some embodiments, the transfer plasmid contains one ITR sequence. In some embodiments, the transfer plasmid contains two ITR sequences.

[0177] Either Rep78 and / or Rep68 binds to a unique and known site on the ITR hairpin sequence and functions to disrupt and unravel the hairpin structure at the end of the AAV genome, thereby providing access to the replication machinery of the viral replicating cell. As is known, Rep proteins may be expressed from multiple ORFs containing nucleotide sequences encoding any combination of Rep78, Rep68, Rep52, and / or Rep40 by using separate nucleotide sequences operably linked to at least one expression control sequence for expression in viral replicating cells, each producing one or more of the Rep78, Rep68, Rep52, and / or Rep40 Rep proteins. Alternatively, the Rep proteins may be expressed individually from ORFs containing nucleotide sequences encoding any combination of Rep78, Rep68, Rep52, or Rep40, by using separate nucleotide sequences operably linked to a single expression control sequence for expression in a packaging cell, each producing only one Rep78, Rep68, Rep52, or Rep40 Rep protein. In another embodiment, the Rep proteins may be expressed from a single ORF containing nucleotide sequences encoding Rep78 and Rep52 Rep proteins operably linked to at least one expression control sequence for expression in a viral replication cell, each producing Rep78 and Rep52 Rep proteins.

[0178] In the methods of producing AAV virions, e.g., viral particles, of the invention, the rep coding sequence and cap gene of the invention may be provided in a single packaging plasmid (see, e.g., Figure 1). However, one of skill in the art will recognize that such a requirement is not necessary. Such viral particles may or may not contain a genome.

[0179] A "chimeric AAV capsid protein" includes an AAV capsid protein that contains amino acid sequences, e.g., portions, from two or more different AAVs and that has the ability to form and / or forms an AAV viral capsid / virion. A chimeric AAV capsid protein may be encoded by a chimeric AAV capsid gene, e.g., a chimeric nucleotide sequence containing multiple, e.g., at least two, nucleic acid sequences, each of which is identical to a portion of a capsid gene encoding a capsid protein of a separate AAV, and which together encode a functional chimeric AAV capsid protein. Association of a chimeric capsid protein with a particular AAV indicates that the capsid protein contains one or more portions derived from a capsid protein of that AAV and one or more portions derived from a capsid protein of a different AAV. For example, a chimeric AAV2 capsid protein includes a capsid protein that includes one or more portions of the VP1, VP2, and / or VP3 capsid proteins of AAV2 and one or more portions of the VP1, VP2, and / or VP3 capsid proteins of a different AAV.

[0180] The term "portion" refers to at least 5 amino acids or at least 15 nucleotides, but less than the full-length polypeptide or nucleic acid molecule, that has 100% identity to the sequence from which the portion is derived. See Penzes (2015) J. General Virol. 2769. A "portion" encompasses any contiguous segment of amino acids or nucleotides sufficient to determine that the polypeptide or nucleic acid molecule from which the portion is derived is "of a [designated] AAV," or has "significant identity" to a particular AAV, e.g., a non-primate AAV or a remote AAV. In some embodiments, a portion comprises at least 5 amino acids or 15 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, a portion comprises at least 10 amino acids or 30 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, a portion comprises at least 15 amino acids or 45 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 20 amino acids or 60 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 25 amino acids or 75 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 30 amino acids or 90 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 35 amino acids or 105 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 40 amino acids or 120 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 45 amino acids or 135 nucleotides that have 100% identity to a sequence associated with the designated AAV.In some embodiments, the portion comprises at least 50 amino acids or 150 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 60 amino acids or 180 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 70 amino acids or 210 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 80 amino acids or 240 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 90 amino acids or 270 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 100 amino acids or 300 nucleotides that have 100% identity to a sequence associated with the designated AAV.

[0181] Modified viral capsid proteins, viral particles, and nucleic acids

[0182] In some embodiments, a Cap protein of the invention, e.g., a VP1 capsid protein described herein, a VP2 capsid protein described herein, and / or a VP3 capsid protein described herein, is modified to include, e.g., a first member of a protein:protein binding pair, a detectable label, a point mutation, etc.

[0183] Chimeras are a type of modification described herein. Generally, modifications of a designated AAV gene or polypeptide, or variants thereof, result in a nucleic acid or amino acid sequence that differs from the nucleic acid or amino acid sequence described herein for the designated AAV, in which case the modification alters, imparts, or eliminates one or more biological functions, but does not alter the phylogenetic characteristics of the gene or polypeptide. Modifications can include, for example, the insertion of a first member of a protein:protein binding pair and point mutations, e.g., to reduce or abolish the original tropism of the capsid protein, and / or to cause the capsid protein to include a detectable label. Preferred modifications include those that do not alter, and preferably reduce low to no recognition, of the modified capsid by pre-existing antibodies present in the general population produced during infection with another AAV, such as an infection with a serotype such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, virions based on such serotypes, virions from currently used AAV gene therapy modalities, or combinations thereof. Other modifications described herein include modifications of the capsid protein, whereby the capsid protein comprises a first member of a protein:protein binding pair, a detectable label, or the like, which generally result from modifications at the genetic level, for example, via modification of the cap gene.

[0184] In some embodiments, a viral capsid comprising a modified viral capsid protein described herein is a mosaic capsid, e.g., comprising at least two pairs of VP1, VP2, and / or VP3 proteins, each pair encoded by a different cap gene. A mosaic capsid herein generally refers to a mosaic of a first viral capsid protein that has been modified to include a first member of a protein:protein binding pair and a second corresponding viral capsid protein that lacks the first member of the protein:protein binding pair. In the context of a mosaic capsid, the second viral capsid protein that lacks the first member of the protein:protein binding pair is sometimes referred to as a reference capsid protein and is encoded by a reference cap gene. In some mosaic capsid embodiments, preferably when the VP1, VP2, and / or VP3 capsid protein modified with the first member of the protein:protein pair is not a chimeric capsid protein, the VP1, VP2, and / or VP3 reference capsid protein may comprise an amino acid sequence identical to a viral VP1, VP2, and / or VP3 capsid protein modified with the first member of the protein:protein binding pair, but the reference capsid protein lacks the first member of the protein:protein binding pair. In some mosaic capsid embodiments, the VP1, VP2, and / or VP3 reference capsid protein corresponds to a viral VP1, VP2, and / or VP3 capsid protein modified with the first member of the protein:protein binding pair, but the reference capsid protein lacks the first member of the protein:protein binding pair. In some embodiments, the VP1 reference capsid protein corresponds to a viral VP1 capsid protein modified with a first member of a protein:protein binding pair, wherein the reference capsid protein lacks the first member of the protein:protein binding pair.In some embodiments, the VP2 reference capsid protein corresponds to a viral VP2 capsid protein modified with a first member of a protein:protein binding pair, where the reference capsid protein lacks the first member of the protein:protein binding pair. In some embodiments, the VP3 reference capsid protein corresponds to a viral VP3 capsid protein modified with a first member of a protein:protein binding pair, where the reference capsid protein lacks the first member of the protein:protein binding pair. In some mosaic capsid embodiments comprising chimeric VP1, VP2, and / or VP3 capsid proteins further modified to include a first member of a protein:protein binding pair, the reference protein can be a corresponding capsid protein, a portion of which forms part of the chimeric capsid protein. In some embodiments, by way of non-limiting example, a mosaic capsid comprising a chimeric AAV2 / AAAV VP1 capsid protein modified to include a first member of a protein:protein binding pair may further comprise, as a reference capsid protein, an AAV2 VP1 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, or a chimeric AAV2 / AAAV VP1 capsid protein lacking the first member. Similarly, in some embodiments, a mosaic capsid comprising a chimeric AAV2 / AAAV VP2 capsid protein modified to include a first member of a protein:protein binding pair may further comprise, as a reference capsid protein, an AAV2 VP2 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, or a chimeric AAV2 / AAAV VP2 capsid protein lacking the first member. In some embodiments, a mosaic capsid comprising a chimeric AAV2 / AAAV VP3 capsid protein modified to comprise a first member of a protein:protein binding pair may further comprise, as a reference capsid protein, an AAV2 VP2 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, or a chimeric AAV2 / AAAV VP3 capsid protein lacking the first member.In some mosaic capsid embodiments, the reference capsid protein may be any capsid protein so long as it lacks the first member of the protein:protein binding pair and so long as it is capable of forming a capsid with a first capsid protein that has been modified with the first member of the protein:protein binding pair.

[0185] Generally, mosaic particles may be generated by transfecting a mixture of modified and reference cap genes into producer cells in a specified ratio. The ratio of protein subunits in the particle, e.g., the ratio of modified VP protein:unmodified VP protein, stoichiometrically reflects, but does not necessarily reflect, the ratio of at least two of the cap gene encoding a first capsid protein modified with a first member of a protein:protein binding pair and one or more reference cap genes, e.g., modified cap gene:reference cap gene, transfected into the packaging cells. In some embodiments, the ratio of protein subunits in the particle does not stoichiometrically reflect the ratio of modified cap gene:reference cap gene transfected into the packaging cells.

[0186] In some mosaic virus particle embodiments, the ratio of protein subunits ranges from about 1:59 to about 59:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:1 (e.g., a mosaic virus particle includes about 30 modified capsid proteins and about 30 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:2 (e.g., a mosaic virus particle includes about 20 modified capsid proteins and about 40 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 3:5. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:3 (e.g., a mosaic virus particle includes about 15 modified capsid proteins and about 45 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:4 (e.g., the mosaic virus particle comprises about 12 modified capsid proteins and about 48 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:5 (e.g., the mosaic virus particle comprises about 10 modified capsid proteins and about 50 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:6. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:7. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:8. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:9 (e.g., the mosaic virus particle comprises about 6 modified capsid proteins and about 54 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:10.In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:11 (e.g., the mosaic virus particle comprises about 5 modified capsid proteins and about 55 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:12. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:13. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:14 (e.g., the mosaic virus particle comprises about 4 modified capsid proteins and about 56 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:15. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:19 (e.g., the mosaic virus particle comprises about 3 modified capsid proteins and about 57 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:29 (e.g., the mosaic virus particle comprises about 2 modified capsid proteins and about 58 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:59. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 2:1 (e.g., the mosaic virus particle comprises about 40 modified capsid proteins and about 20 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 5:3. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 3:1 (e.g., the mosaic virus particle comprises about 45 modified capsid proteins and about 15 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 4:1 (eg, the mosaic virus particle comprises about 48 modified capsid proteins and about 12 reference capsid proteins).In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 5:1 (e.g., the mosaic virus particle comprises about 50 modified capsid proteins and about 10 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 6:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 7:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 8:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 9:1 (e.g., the mosaic virus particle comprises about 54 modified capsid proteins and about 6 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 10:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 11:1 (e.g., the mosaic virus particle comprises about 55 modified capsid proteins and about 5 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 12:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 13:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 14:1 (e.g., the mosaic virus particle comprises about 56 modified capsid proteins and about 4 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 15:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 19:1 (e.g., the mosaic virus particle comprises about 57 modified capsid proteins and about 3 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 29:1 (e.g., the mosaic virus particle comprises about 58 modified capsid proteins and about 2 reference capsid proteins).In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 59:1.

[0187] In some non-mosaic virus particle embodiments, the ratio of protein subunits may be 1:0, in which case each capsid protein of the non-mosaic virus particle is modified with a first member of a protein:protein binding pair. In some non-mosaic virus particle embodiments, the ratio of protein subunits may be 0:1, in which case each capsid protein of the non-mosaic virus particle is not modified with a first member of a protein:protein binding pair.

[0188] In some embodiments, the capsid proteins of the invention are modified to include a detectable label. Many detectable labels are known in the art. (See, e.g., Nilsson et al. (1997) "Affinity fusion strategies for detection, purification, and immobilization of modified proteins," Protein Expression and Purification 11:1-16; Terpe et al. (2003) "Overview of tag protein fusions: From molecular and biochemical fundamentals to Commercial Microbiology and Biotechnology 60:523-533, and references therein.) Detectable labels include, but are not limited to, immobilized divalent cations (e.g., Ni 2+), a biotin moiety that binds to immobilized avidin (e.g., on a biotinylated polypeptide sequence in vivo), a GST (glutathione S-transferase) sequence that binds to immobilized glutathione, an S tag that binds to immobilized S protein, an antigen that binds to an immobilized antibody or domain or fragment thereof (including, for example, T7, myc, FLAG, and B tags that bind to the corresponding antibody), a FLASH® tag (a highly detectable label linked to a specific arsenic-based moiety), a receptor or receptor domain that binds to an immobilized ligand (or vice versa), protein A or a derivative thereof (e.g., Z) that binds to immobilized IgG, maltose-binding protein (MBP) that binds to immobilized amylose, an albumin-binding protein that binds to immobilized albumin, a chitin-binding domain that binds to immobilized chitin, a calmodulin-binding peptide that binds to immobilized calmodulin, and a cellulose-binding domain that binds to immobilized cellulose. Another example of a detectable label is SNAP-tag, commercially available from Covalys (www.covalys.com). In some embodiments, the detectable labels disclosed herein include detectable labels that are recognized only by antibody paratopes. In some embodiments, the detectable labels disclosed herein include detectable labels that are recognized by antibody paratopes and other specific binding pairs.

[0189] In some embodiments, the detectable label forms a binding pair with an immunoglobulin constant domain. In some embodiments, the detectable label and / or the detectable label binds to a metal ion, e.g., Ni 2+、 Co 2+ , Cu 2+ , Zn 2+ , Fe 3+ In some embodiments, the detectable label is selected from the group consisting of streptavidin, StrepII, HA, L14, 4C-RGD, LH, and protein A.

[0190] In some embodiments, the detectable label is selected from the group consisting of FLAG, HA, and c-myc (SEQ ID NO: 44). In some embodiments, the detectable label is c-myc (SEQ ID NO: 44).

[0191] In some embodiments, the detectable label is a B cell epitope. For example, the detectable label is about 1 amino acid to about 35 amino acids in length and forms a binding pair with an antibody paratope, such as an immunoglobulin variable domain. In some embodiments, the detectable label comprises the B1 epitope (SEQ ID NO: 45). In some embodiments, the capsid protein is modified to include the B1 epitope in the VP3 region.

[0192] In some embodiments, the capsid protein of the invention comprises at least a first member of a peptide:peptide binding pair.

[0193] In some embodiments, the capsid proteins of the invention comprise a first member of a protein:protein binding pair that includes a detectable label and may be used for detection and / or isolation of the Cap protein and / or may be used as the first member of a protein:protein binding pair. In some embodiments, the detectable label serves as the first member of a protein:protein binding pair for binding of a targeting ligand that includes a multispecific binding protein that can bind both the detectable label and a target expressed by a cell of interest. In some embodiments, the Cap proteins of the invention comprise a first member of a protein:protein binding pair that includes c-myc (SEQ ID NO: 44). The use of a detectable label as a first member of a protein:protein binding pair is described, for example, in WO2019006043, which is incorporated herein by reference in its entirety.

[0194] In some embodiments, the capsid protein comprises a first member of a protein:protein binding pair, wherein the protein:protein binding pair forms a covalent isopeptide bond. In some embodiments, the first member of the peptide:peptide binding pair is covalently linked to a cognate second member of the peptide:peptide binding pair via an isopeptide bond, and optionally, wherein the cognate second member of the peptide:peptide binding pair is fused to a targeting ligand, wherein the targeting ligand binds to a target expressed by a subject cell. In some embodiments, the protein:protein binding pair may be selected from the group consisting of SpyTag:SpyCatcher, SpyTag002:SpyCatcher002, SpyTag:KTag, Isopeptag:pilin-C, and SnoopTag:SnoopCatcher. In some embodiments, the first member is SpyTag (or a biologically active portion thereof) and the protein (second cognate member) is SpyCatcher (or a biologically active portion thereof). In some embodiments, the first member is SpyTag (or a biologically active portion thereof) and the protein (second cognate member) is KTag (or a biologically active portion thereof). In some embodiments, the first member is KTag (or a biologically active portion thereof) and the protein (second cognate member) is SpyTag (or a biologically active portion thereof). In some embodiments, the first member is SnoopTag (or a biologically active portion thereof) and the protein (second cognate member) is SnoopCatcher (or a biologically active portion thereof). In some embodiments, the first member is Isopeptag (or a biologically active portion thereof) and the protein (second cognate member) is Pilin-C (or a biologically active portion thereof). In some embodiments, the first member is SpyTag002 (or a biologically active portion thereof) and the protein (second cognate member) is SpyCatcher002 (or a biologically active portion thereof). In some embodiments, the Cap protein of the present invention comprises SpyTag.The use of a first member of a protein:protein binding pair is described in WO2019006046, which is incorporated herein in its entirety.

[0195] In some embodiments, the first member of the protein:protein binding pair and / or the detectable label are operably linked to the Cap protein of the invention (translated in-frame with the Cap protein of the invention, chemically attached to the Cap protein of the invention, and / or displayed by the Cap protein of the invention) via a first or second linker, e.g., an amino acid spacer that is at least one amino acid in length. In some embodiments, the first member of the protein:protein binding pair is flanked by first and / or second linkers, such as first and / or second amino acid spacers, each of which is at least one amino acid in length.

[0196] In some embodiments, the first and / or second linkers are not identical. In some embodiments, the first and / or second linkers are each independently 1 or 2 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, or 3 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, or 4 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, or 5 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, or 5 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, 5, or 6 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, 5, 6, or 7 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, 5, 6, 7, or 8 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids or more in length.

[0197] In some embodiments, the first and second linkers are identical in sequence and / or length, each being one amino acid long. In some embodiments, the first and second linkers are identical in length, each being one amino acid long. In some embodiments, the first and second linkers are identical in length, each being two amino acids long. In some embodiments, the first and second linkers are identical in length, each being three amino acids long. In some embodiments, the first and second linkers are identical in length, each being four amino acids long, e.g., the linkers are GLSG (SEQ ID NO: 37). In some embodiments, the first and second linkers are identical in length, each being five amino acids long. In some embodiments, the first and second linkers are identical in length, each being six amino acids long, e.g., the first and second linkers each comprise the sequence GLSGSG (SEQ ID NO: 38). In some embodiments, the first and second linkers are identical in length, each being seven amino acids long. In some embodiments, the first and second linkers are identical in length, each 8 amino acids in length, e.g., the first and second linkers each comprise the sequence GLSGLSGS (SEQ ID NO: 39). In some embodiments, the first and second linkers are identical in length, each 9 amino acids in length. In some embodiments, the first and second linkers are identical in length, each 10 amino acids in length, e.g., the first and second linkers each comprise GLSGLSGLSG (SEQ ID NO: 40) or GLSGGSGLSG (SEQ ID NO: 41). In some embodiments, the first and second linkers are identical in length, each more than 10 amino acids in length.

[0198] Generally, the first member amino acid sequence of a protein:protein binding pair described herein, for example, comprises a first member of a specific binding pair by itself or in combination with one or more linkers, and is from about 5 amino acids to about 50 amino acids in length. In some embodiments, the first member amino acid sequence of a protein:protein binding pair is at least 5 amino acids in length. In some embodiments, the first member amino acid sequence of a protein:protein binding pair is 6 amino acids in length. In some embodiments, the first member amino acid sequence of a protein:protein binding pair is 7 amino acids in length. In some embodiments, the first member amino acid sequence of a protein:protein binding pair is 8 amino acids in length. In some embodiments, the first member amino acid sequence of a protein:protein binding pair is 9 amino acids in length. In some embodiments, the first member amino acid sequence of a protein:protein binding pair is 10 amino acids in length. In some embodiments, the first member amino acid sequence of a protein:protein binding pair is 11 amino acids in length. In some embodiments, the first member amino acid sequence of a protein:protein binding pair is 12 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 13 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 14 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 15 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 16 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 17 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 18 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 19 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 20 amino acids in length.In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 21 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 22 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 23 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 24 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 25 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 26 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 27 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 28 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 29 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 30 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 31 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 32 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 33 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 34 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 35 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 36 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 37 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 38 amino acids in length.In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 39 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 40 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 41 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 42 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 43 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 44 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 45 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 46 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 47 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 48 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 49 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 50 amino acids in length.

[0199] Due to the high degree of conservation, at least among widespread and numerous closely related family members, corresponding insertion sites in AAVs other than those listed can be identified by amino acid alignment or comparison of capsid structure. For example, for examples of alignments of various AAV capsid proteins, see Rutledge et al. (1998) J. Virol. 72:309-19; Mietzsch et al. (2019) Viruses 11,362, 1-34, and U.S. Patent No. 9,624,274, each of which is incorporated by reference in its entirety. For example, Mietzsch et al. (2019) present a ribbon overlay of various dependoparvoviruses in Figure 7, depicting variable regions VR I through VR IX. Using structural and sequence analysis as described in the literature, one skilled in the art can determine which amino acids within the variable regions correspond to amino acid sequences of AAV that are amenable to insertion of the first member of a protein:protein binding pair and / or a detectable label.

[0200] Thus, in some embodiments, the first member of the protein:protein binding pair and / or the detectable label is inserted in the VP1 capsid protein of the non-primate AAV after an amino acid position corresponding to an amino acid position selected from the group consisting of G453 of the AAV2 capsid protein VP1, N587 of the AAV2 capsid protein VP1, G453 of the AAV9 capsid protein VP1, and A589 of the AAV9 capsid protein VP1. In some embodiments, the first member of the protein:protein binding pair and / or the detectable label is inserted in the VP1 capsid protein of the non-primate AAV between amino acids corresponding to N587 and R588 of the AAV2 VP1 capsid. Additional suitable insertion sites for non-primate VP1 capsid proteins include those corresponding to 1-1, 1-34, 1-138, 1-139, 1-161, 1-261, 1-266, 1-381, 1-447, 1-448, 1-459, 1-471, 1-520, 1-534, 1-570, 1-573, 1-584, 1-587, 1-588, 1-591, 1-657, 1-664, 1-713, and 1-716 of the VP1 capsid protein of AAV2 (Wu et al. (2000) J. Virol. 74:8635-8647). The modified viral capsid protein described herein may be a non-primate capsid protein comprising a first member of a protein:protein binding pair and / or a detectable label inserted at a position corresponding to a position in an AAV2 capsid protein selected from the group consisting of 1-1, 1-34, 1-138, 1-139, 1-161, 1-261, 1-266, 1-381, 1-447, 1-448, 1-459, 1-471, 1-520, 1-534, 1-570, 1-573, 1-584, 1-587, 1-588, 1-591, 1-657, 1-664, 1-713, 1-716, and combinations thereof. Additional suitable insertion sites in non-primate AAVs include sites equivalent to I-587 in AAV1, I-589 in AAV1, I-585 in AAV3, I-585 in AAV4, and I-585 in AAV5.In some embodiments, the modified viral capsid protein described herein may be a non-primate capsid protein comprising a first member of a protein:protein binding pair and / or a detectable label inserted at a position corresponding to a position selected from the group consisting of I-587(AAV1), I-589(AAV1), I-585(AAV3), I-585(AAV4), I-585(AAV5), and combinations thereof.

[0201] In some embodiments, the first member of the protein:protein binding pair and / or the detectable label is selected from the group consisting of: I444 of avian AAV capsid protein VP1; I580 of avian AAV capsid protein VP1; I573 of bearded dragon AAV capsid protein VP1; I436 of bearded dragon AAV capsid protein VP1; I429 of sea lion AAV capsid protein VP1; I4 ... The amino acid sequence is inserted after an amino acid position corresponding to an amino acid position selected from the group consisting of I430 of AAV capsid protein VP1, I431 of AAV capsid protein VP1, I432 of AAV capsid protein VP1, I433 of AAV capsid protein VP1, I434 of AAV capsid protein VP1, I436 of AAV capsid protein VP1, I437 of AAV capsid protein VP1, and I565 of AAV capsid protein VP1.

[0202] As used herein, the designations I-###, I#, etc. refer to an insertion site (I) where ### designates the amino acid numbering relative to the VP1 protein of the AAV capsid protein; however, such insertions may be located directly N- or C-terminal, preferably at the C-terminus of one amino acid in a sequence five amino acids N- or C-terminal to a given amino acid, and preferably at the C-terminus of one amino acid in a sequence three, more preferably two, and particularly one amino acid N- or C-terminal to a given amino acid. Furthermore, the positions referred to herein are relative to the VP1 protein encoded by the AAV capsid gene; corresponding positions (and point mutations thereof) can be readily identified in the VP2 and VP3 capsid proteins encoded by the capsid genes by performing sequence alignments of the VP1, VP2, and VP3 proteins encoded by the appropriate AAV capsid genes.

[0203] Because capsid proteins are encoded by overlapping reading frames of the same gene with shifted start codons, insertion of the coding nucleic acid of one of these sites in the cap gene at the corresponding position also results in insertion of VP1, VP2, and / or VP3. Thus, for example, for AAV2, according to this nomenclature, an insertion of amino acids 1-138 is inserted only into VP1, an insertion of 138-203 is inserted into VP1 and VP2, and an insertion of 203 to the C-terminus is inserted into VP1, VP2, and VP3, and of course, this also applies to insertion site I-587. Thus, the present invention encompasses AAV structural genes with corresponding insertions in the VP1, VP2, and / or VP3 proteins.

[0204] Also provided herein are nucleic acids encoding the VP3 capsid proteins of the present invention. AAV capsid proteins may, but do not necessarily, be encoded by overlapping reading frames of the same gene with shifted start codons. In some embodiments, nucleic acids encoding the VP3 capsid proteins of the present invention do not encode the VP2 capsid proteins or VP1 capsid proteins of the present invention. In some embodiments, nucleic acids encoding the VP3 capsid proteins of the present invention may encode the VP2 capsid proteins of the present invention, but not the VP1 capsids of the present invention. In some embodiments, nucleic acids encoding the VP3 capsid proteins of the present invention may also encode the VP2 capsid proteins of the present invention and the VP1 capsids of the present invention.

[0205] In some embodiments, a viral capsid comprising a modified viral capsid protein comprising a first and second member of a protein:protein binding pair (e.g., where the second member is operably linked to a targeting ligand and comprises a multispecific binding protein) has the ability to infect a particular cell, e.g., has an enhanced ability to target and bind to a particular cell compared to a control viral capsid. The control viral capsid is identical to the modified viral capsid protein except that it lacks either or both of the first and second members of the protein:protein binding pair, e.g., comprises a control capsid protein. In some embodiments, a viral capsid comprising a modified viral capsid protein described herein bound to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibits a detectable transduction efficiency compared to the undetectable transduction efficiency of the control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 10% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 20% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a suitable first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 30% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein bound to appropriate first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 40% greater than the transduction efficiency of a control viral capsid.In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to appropriate first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 50% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to appropriate first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 60% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to appropriate first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 70% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 75% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 80% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 85% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein bound to appropriate first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 90% greater than the transduction efficiency of a control capsid.In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 95% greater than the transduction efficiency of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 99% greater than the transduction efficiency of a control viral capsid.

[0206] In some embodiments, a viral capsid comprising a modified viral capsid protein comprising a first and second member of a protein:protein binding pair (e.g., where the second member is operably linked to a targeting ligand and comprises a multispecific binding protein) has the ability to infect a particular cell, e.g., has an enhanced ability to target and bind to a particular cell compared to a control viral capsid. The control viral capsid is identical to the modified viral capsid protein except that it lacks either or both of the first and second members of the protein:protein binding pair, e.g., comprises a control capsid protein. In some embodiments, a viral capsid comprising a modified viral capsid protein described herein bound to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibits a detectable transduction efficiency compared to the undetectable transduction efficiency of the control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 10% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 20% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a suitable first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 30% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein bound to appropriate first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 40% greater than the transduction efficiency of a control viral capsid.In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to appropriate first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 50% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to appropriate first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 60% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to appropriate first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 70% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 75% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 80% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 85% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein bound to appropriate first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 90% greater than the transduction efficiency of a control capsid.In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 95% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 99% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 1.5-fold greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency at least two-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a suitable first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency at least three-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a suitable first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency at least four-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein bound to appropriate first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 5-fold greater than the transduction efficiency of a control viral capsid.In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least six-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least seven-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least eight-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 9-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 10-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 20-fold higher than that of a control capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein bound to appropriate first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 30-fold greater than the transduction efficiency of a control viral capsid.In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 40-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 50-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 60-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 70-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 80-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 90-fold higher than that of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein described herein bound to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 100-fold greater than the transduction efficiency of a control viral capsid.In some embodiments, viral particles of the invention comprising a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, and optionally a first and second member of a protein:protein binding pair (e.g., in which the second member is operably linked to a targeting ligand and comprises a multispecific binding protein, etc.), are better able to evade neutralization by pre-existing antibodies in serum isolated from a human patient compared to a suitable control viral particle (e.g., comprising a viral capsid of an AAV serotype that includes a portion of the viral capsid protein in the viral capsid of the invention as part of the viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof). and optionally comprising a first and second member of a protein:protein binding pair (e.g., in which case the second member is operably linked to a targeting ligand and comprises a multispecific binding protein, etc.). In some embodiments, viral particles of the invention comprising a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, require at least two-fold more total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) compared to a suitable control viral particle (e.g., the viral particle of the invention has an IC50 value at least twice that of the control viral particle).

[0207] In some embodiments, viral particles of the present invention comprising a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, require at least three-fold more total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) compared to a suitable control viral particle (e.g., the viral particles of the present invention have an IC50 value at least three-fold higher than that of the control viral particle). In some embodiments, viral particles of the present invention comprising a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, require at least four-fold more total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) compared to a suitable control viral particle (e.g., the viral particles of the present invention have an IC50 value at least four-fold higher than that of the control viral particle). In some embodiments, viral particles of the present invention comprising a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, require at least five-fold more total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) compared to a suitable control viral particle (e.g., the viral particles of the present invention have an IC50 value at least five-fold greater than that of the control viral particle). In some embodiments, viral particles of the present invention comprising a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, require at least six-fold more total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) compared to a suitable control viral particle (e.g., the viral particles of the present invention have an IC50 value at least six-fold greater than that of the control viral particle). In some embodiments, viral particles of the present invention comprising a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, require at least 7-fold more total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) compared to a suitable control viral particle (e.g., the viral particles of the present invention have an IC50 value at least 7-fold higher than that of the control viral particle).In some embodiments, viral particles of the present invention comprising a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, require at least 8-fold more total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) compared to a suitable control viral particle (e.g., the viral particles of the present invention have an IC50 value at least 8-fold higher than that of the control viral particle). In some embodiments, viral particles of the present invention comprising a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, require at least 9-fold more total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) compared to a suitable control viral particle (e.g., the viral particles of the present invention have an IC50 value at least 9-fold higher than that of the control viral particle). In some embodiments, viral particles of the present invention comprising a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, require at least 10-fold more total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) compared to a suitable control viral particle (e.g., the viral particles of the present invention have an IC50 value at least 10-fold greater than that of the control viral particle). In some embodiments, viral particles of the present invention comprising a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, require at least 20-fold more total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) compared to a suitable control viral particle (e.g., the viral particles of the present invention have an IC50 value at least 20-fold greater than that of the control viral particle). In some embodiments, viral particles of the present invention comprising a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, require at least 30-fold more total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) compared to a suitable control viral particle (e.g., the viral particles of the present invention have an IC50 value at least 30-fold greater than that of the control viral particle).In some embodiments, viral particles of the present invention comprising a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, require at least 40-fold more total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) compared to a suitable control viral particle (e.g., viral particles of the present invention have an IC50 value at least 40-fold greater than that of the control viral particle). In some embodiments, viral particles of the present invention comprising a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, require at least 50-fold more total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) compared to a suitable control viral particle (e.g., viral particles of the present invention have an IC50 value at least 50-fold greater than that of the control viral particle). In some embodiments, viral particles of the present invention comprising a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, require at least 60-fold more total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) compared to a suitable control viral particle (e.g., the viral particles of the present invention have an IC50 value at least 60-fold greater than that of the control viral particle). In some embodiments, viral particles of the present invention comprising a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, require at least 70-fold more total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) compared to a suitable control viral particle (e.g., the viral particles of the present invention have an IC50 value at least 70-fold greater than that of the control viral particle). In some embodiments, viral particles of the present invention comprising a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, require at least 80-fold more total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) compared to a suitable control viral particle (e.g., the viral particles of the present invention have an IC50 value at least 80-fold greater than that of the control viral particle).In some embodiments, viral particles of the present invention comprising a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, require at least 90-fold more total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) compared to a suitable control viral particle (e.g., the viral particles of the present invention have an IC50 value at least 90-fold greater than that of the control viral particle). In some embodiments, viral particles of the present invention comprising a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, require at least 100-fold more total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) compared to a suitable control viral particle (e.g., the viral particles of the present invention have an IC50 value at least 100-fold greater than that of the control viral particle). In some embodiments, viral particles of the present invention comprising a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof have an undetectable IC50 when incubated with human serum Ig pooled from at least 100, 10,000, 20,000, 30,000, 40,000, 50,000, or more human donors.

[0208] Targeting Ligands

[0209] The viral particles described herein may further comprise a targeting ligand.

[0210] In some embodiments of the present invention comprising a detectable label, the targeting ligand comprises a multispecific binding molecule comprising (i) an antibody paratope that specifically binds to the detectable label and (ii) a second binding domain that specifically binds to a receptor that can be attached to the surface of a bead (e.g., for purification) or expressed by a target cell. Thus, a multispecific binding molecule comprising (i) an antibody paratope that specifically binds to the detectable label and (ii) a second binding domain that specifically binds to a receptor targets a viral particle. Such "targeting" or "guiding" can include a scenario in which a wild-type viral particle targets a tissue and / or some cells within some organs within an organism, and insertion of a detectable label reduces or abolishes broad targeting of the tissue or organ, and retargeting to more specific cells in a tissue or more specific organ within the organism is achieved with the multispecific binding molecule. Such retargeting or redirection may also include a scenario in which wild-type viral particles target a tissue, the targeting of that tissue is reduced or abolished by the insertion of a detectable label, and retargeting to an entirely different tissue is achieved with the multispecific binding molecule. The antibody paratopes described herein generally comprise, at a minimum, a complementarity-determining region (CDR) that specifically recognizes a detectable label, e.g., the CDR3 region of a heavy and / or light chain variable domain. In some embodiments, the multispecific binding molecule comprises an antibody (or a portion thereof) comprising an antibody paratope that specifically binds to a detectable label. For example, the multispecific binding molecule may comprise a single-domain heavy chain variable region or a single-domain light chain variable region, wherein the single-domain heavy chain variable region or the single-domain light chain variable region comprises an antibody paratope that specifically binds to a detectable label. In some embodiments, the multispecific binding molecule may comprise an Fv region, e.g., an scFv, comprising an antibody paratope that specifically binds to a detectable label. In some embodiments, the multispecific binding molecules described herein comprise an antibody paratope that specifically binds to c-myc (SEQ ID NO: 44).

[0211] One embodiment of the present invention is a multimeric structure comprising the modified viral capsid proteins of the present invention. The multimeric structure comprises at least 5, preferably at least 10, more preferably at least 30, and most preferably at least 60 modified viral capsid proteins comprising first members of specific binding pairs described herein. They can form normal viral capsids (empty viral particles) or viral particles (capsids encapsidating a target nucleotide). The formation of viral particles containing a viral genome is a highly desirable characteristic for use with the modified viral capsids described herein.

[0212] A further embodiment of the present invention is the use of at least one modified viral capsid protein and / or nucleic acid encoding same, preferably at least one multimeric structure (e.g., viral particle) for the manufacture and for use in the transfer of a nucleotide of interest to a target cell.

[0213] Use and Preparation

[0214] A further embodiment of the modified viral capsid proteins described herein is their use to deliver a target nucleotide, such as a reporter gene or a therapeutic gene, to a target cell. The target nucleotide may generally be a transfer plasmid, which generally includes 5' and 3' inverted repeat repeats (ITRs) flanking the reporter gene or therapeutic gene (which may be under the control of a viral or non-viral promoter when packaged within an AAV particle). In one embodiment, the target nucleotide is a transfer plasmid including, from 5' to 3', a 5' ITR, a promoter, a gene (e.g., a reporter gene and / or a therapeutic gene), and a 3' ITR.

[0215] Non-limiting examples of useful promoters include, for example, the cytomegalovirus (CMV) promoter, the spleen focus-forming virus (SFFV) promoter, the elongation factor 1 alpha (EF1a) promoter (1.2 kb EF1a promoter, or 0.2 kb EF1a promoter), the chimeric EF1a / IF4 promoter, and the phosphoglycerate kinase (PGK) promoter. Internal enhancers may also be present in the viral construct to increase expression of the gene of interest. For example, the CMV enhancer (Karasuyama et al. 1989. J. Exp. Med. 169:13, incorporated herein by reference in its entirety) may be used. In some embodiments, the CMV enhancer may be used in combination with the chicken β-actin promoter.

[0216] A variety of reporter genes (or detectable moieties) can be encapsidated into multimeric structures comprising the modified viral capsid proteins described herein. Exemplary reporter genes include, for example, β-galactosidase (encoded by the lacZ gene), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), MmGFP, blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or combinations thereof. The methods described herein demonstrate the construction of targeted particles employing the use of a reporter gene encoding green fluorescent protein. However, upon reading this disclosure, one of skill in the art will understand that the viral capsids described herein can be produced in the absence of a reporter gene or can be produced using any reporter gene known in the art.

[0217] A variety of therapeutic genes can also be encapsidated into multimeric structures comprising the modified viral capsid proteins described herein, e.g., as part of a transfer particle. Non-limiting examples of therapeutic genes include those encoding toxins (e.g., suicide genes), therapeutic antibodies or fragments thereof, CRISPR / Cas systems or portion(s) thereof, antisense RNA, siRNA, shRNA, etc.

[0218] A further embodiment of the present invention is a process for the preparation of a modified capsid protein, the method comprising: a) expressing a nucleic acid encoding a modified capsid protein under suitable conditions; and b) isolating the expressed capsid protein of step a).

[0219] In some embodiments, the viral particles described herein comprise a mosaic capsid, e.g., the mosaic capsid comprises a capsid protein that has been genetically modified as described herein (with or without covalent attachment to a targeting ligand) in a specific ratio to a reference capsid protein. Methods for producing such mosaic viral particles include: a) expressing under suitable conditions a nucleic acid encoding a modified capsid protein and a nucleotide encoding a reference capsid protein in a ratio (wt / wt) of at least about 60:1 to 1:60, e.g., 2:1, 1:1, 3:5, 1:2, 1:3, etc.; b) isolating the expressed capsid protein of step a).

[0220] In some embodiments, the compositions described herein comprise, or the methods described herein combine, modified cap genes:reference cap genes (or combinations of reference cap genes) in a ratio ranging from at least about 1:60 to about 60:1, e.g., 2:1, 1:1, 3:5, 1:2, 1:3, etc. In some embodiments, the ratio is at least about 1:2. In some embodiments, the ratio is at least about 1:3. In some embodiments, the ratio is at least about 1:4. In some embodiments, the ratio is at least about 1:5. In some embodiments, the ratio is at least about 1:6. In some embodiments, the ratio is at least about 1:7. In some embodiments, the ratio is at least about 1:8. In some embodiments, the ratio is at least about 1:9. In some embodiments, the ratio is at least about 1:10. In some embodiments, the ratio is at least about 1:11. In some embodiments, the ratio is at least about 1:12. In some embodiments, the ratio is at least about 1:13. In some embodiments, the ratio is at least about 1:14. In some embodiments, the ratio is at least about 1:15. In some embodiments, the ratio is at least about 1:16. In some embodiments, the ratio is at least about 1:17. In some embodiments, the ratio is at least about 1:18. In some embodiments, the ratio is at least about 1:19. In some embodiments, the ratio is at least about 1:20. In some embodiments, the ratio is at least about 1:25. In some embodiments, the ratio is at least about 1:30. In some embodiments, the ratio is at least about 1:35. In some embodiments, the ratio is at least about 1:40. In some embodiments, the ratio is at least about 1:45. In some embodiments, the ratio is at least about 1:50. In some embodiments, the ratio is at least about 1:55. In some embodiments, the ratio is at least about 1:60. In some embodiments, the ratio is at least about 2:1. In some embodiments, the ratio is at least about 3:1. In some embodiments, the ratio is at least about 4:1. In some embodiments, the ratio is at least about 5:1.In some embodiments, the ratio is at least about 6:1. In some embodiments, the ratio is at least about 7:1. In some embodiments, the ratio is at least about 8:1. In some embodiments, the ratio is at least about 9:1. In some embodiments, the ratio is at least about 10:1. In some embodiments, the ratio is at least about 11:1. In some embodiments, the ratio is at least about 12:1. In some embodiments, the ratio is at least about 13:1. In some embodiments, the ratio is at least about 14:1. In some embodiments, the ratio is at least about 15:1. In some embodiments, the ratio is at least about 16:1. In some embodiments, the ratio is at least about 17:1. In some embodiments, the ratio is at least about 18:1. In some embodiments, the ratio is at least about 19:1. In some embodiments, the ratio is at least about 20:1. In some embodiments, the ratio is at least about 25:1. In some embodiments, the ratio is at least about 30:1. In some embodiments, the ratio is at least about 35:1. In some embodiments, the ratio is at least about 40:1. In some embodiments, the ratio is at least about 45:1. In some embodiments, the ratio is at least about 50:1. In some embodiments, the ratio is at least about 55:1. In some embodiments, the ratio is at least about 60:1.

[0221] In some embodiments, the ratio of VP protein subunits in the mosaic virus particle stoichiometrically reflects, but is not necessarily, the ratio of modified cap genes to reference cap genes. As a non-limiting exemplary embodiment, a mosaic capsid formed according to the present methods may have a ratio of modified capsid protein to reference capsid protein similar to, but not necessarily, the ratio (wt:wt) of the nucleic acids encoding the same used to create the mosaic capsid. In some embodiments, the mosaic capsid comprises a protein subunit ratio of about 1:59 to about 59:1.

[0222] A further embodiment of the present invention is a method for altering the tropism of a virus, the method comprising: (a) inserting a nucleic acid encoding an amino acid sequence into a nucleic acid sequence encoding a viral capsid protein to form a nucleotide sequence encoding a genetically modified capsid protein comprising the amino acid sequence; and / or (b) culturing packaging cells under conditions sufficient to produce viral particles, wherein the packaging cells comprise the nucleic acid. A further embodiment of the present invention is a method for displaying a targeting ligand on the surface of a capsid protein, the method comprising: (a) expressing under suitable conditions a nucleic acid encoding a modified viral capsid protein described herein (optionally together with nucleotides encoding a reference capsid protein), wherein the nucleic acid encodes a capsid protein comprising a first member of a specific binding pair; (b) isolating the expressed capsid protein comprising the first member of the specific binding pair of step (a), or a capsid comprising the same; and (c) incubating the capsid protein or capsid with a second cognate member of the specific binding pair under suitable conditions to allow formation of an isopeptide bond between the first and second members, wherein the second cognate member of the specific binding pair is fused to a targeting ligand.

[0223] In some embodiments, the packaging cells further comprise a helper plasmid and / or a transfer plasmid comprising the nucleotide of interest. In some embodiments, the method further comprises isolating the self-complementary adeno-associated viral particles from the culture supernatant. In some embodiments, the method further comprises lysing the packaging cells and isolating the single-stranded adeno-associated viral particles from the cell lysate. In some embodiments, the method further comprises (a) removing cellular debris, (b) treating the supernatant containing the viral particles with a nuclease, such as DNase I and MgCl2, (c) concentrating the viral particles, (d) purifying the viral particles, and (e) any combination of (a)-(d).

[0224] Packaging cells useful for producing the viral particles described herein include, for example, animal cells that are permissive for the virus, or cells that have been modified to be permissive for the virus, or packaging cell constructs using a transforming agent such as, for example, calcium phosphate. Non-limiting examples of packaging cell lines useful for producing the viral particles described herein include, for example, human embryonic kidney 293 (HEK-293) cells (e.g., American Type Culture Collection [ATCC] No. CRL-1573), SV40 Large T-antigen-containing HEK-293 cells (HEK-293T or 293T), HEK293T / 17 cells, human sarcoma cell line HT-1080 (CCL-121), lymphoblastoid cell line Raji (CCL-86), epithelial glioblastoma-astrocytoma cell line U87-MG (HTB-14), T-lymphoma cell line HuT78 (TIB-161), NIH / 3T3 cells, Chinese hamster ovary cells (CHO) (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), HeLa cells (e.g., ATCC No. CCL-2), Vero cells, NIH Examples of such cells include 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), HLHepG2 cells, CAP cells, and CAP-T cells.

[0225] L929 cells, the FLY virus packaging cell line reviewed in Cosset et al. (1995) J Virol 69, 7430-7436, NS0 (mouse myeloma) cells, human amniotic cells (e.g., CAP, CAP-T), yeast cells (including but not limited to S. cerevisiae, Pichia pastoris), plant cells (including but not limited to tobacco NT1, BY-2), insect cells (including but not limited to SF9, S2, SF21, Tni (e.g., High5)), or bacterial cells (including but not limited to E. coli).

[0226] For additional packaging cells and systems, packaging techniques and particles for packaging nucleic acid genomes into pseudotyped viral particles, see, e.g., Polo, et al. al, Proc Natl Acad Sci USA, (1999) 96:4598-4603. Packaging methods include using packaging cells that permanently express the viral components or transiently transfecting cells with the plasmids.

[0227] Further embodiments include methods of redirecting viruses and / or delivering reporter or therapeutic genes to target cells, including methods for transducing cells in vitro (e.g., ex vivo) or in vivo, comprising contacting a target cell with a viral particle comprising a capsid described herein, wherein the capsid comprises a targeting ligand that specifically binds to a receptor expressed by the target cell. In some embodiments, the target cell is in vitro (ex vivo). In other embodiments, the target cell is in vivo in a subject, e.g., a human.

[0228] target cell

[0229] The modified viral particles disclosed herein may be used to target a wide variety of cells for delivery of a nucleotide of interest. Target cells will generally be selected based on the nucleotide of interest and the desired effect.

[0230] In some embodiments, the target nucleotide can be delivered to enable the target cells to produce proteins that compensate for the organism's deficiency, such as an enzyme deficiency or an immune deficiency, such as X-linked severe combined immunodeficiency. Thus, in some embodiments, cells that would normally produce proteins in an animal are targeted. In other embodiments, cells in the region where the protein is most beneficial are targeted.

[0231] In other embodiments, the target nucleotide, such as the gene encoding siRNA, can inhibit the expression of a specific gene in target cells.The target nucleotide can, for example, inhibit the expression of a gene involved in the pathogen life cycle.Therefore, cells that are susceptible to infection by pathogens or infected by pathogens can be targeted.In other embodiments, the target nucleotide can inhibit the expression of a gene responsible for the production of toxins in target cells.

[0232] In other embodiments, the nucleotide of interest may encode a toxic protein that kills cells that express the toxic protein, in which case tumor cells or other unwanted cells may be targeted.

[0233] In yet another embodiment, the subject nucleotides encode a therapeutic protein.

[0234] Once a specific population of target cells in which expression of the target nucleotide is desired is identified, a target receptor that is specifically expressed on that population of target cells is selected. The target receptor may be expressed only in that population of cells, or to a greater extent in that population of cells than in other populations of cells. The more specific the expression, the more specifically delivery can be directed to the target cells. Depending on the context, the desired amount of specificity of the marker (and therefore of gene delivery) may vary. For example, to introduce a toxic gene, high specificity is most preferred to avoid killing non-target cells. Lower marker specificity may be required for the expression of a protein for harvest or for the expression of a secreted product where a global effect is desired.

[0235] As discussed above, the target receptor can be any receptor for which a targeting ligand can be identified or created.Preferably, the target receptor is a peptide or polypeptide such as a receptor.However, in other embodiments, the target receptor can be a carbohydrate or other molecule that can be recognized by a binding partner.If the binding partner of the target receptor, for example, a ligand, is known, the ligand can be used as an affinity molecule.However, if the binding molecule is unknown, an antibody against the target receptor can be generated using standard procedures.The antibody can then be used as a targeting ligand.

[0236] Thus, target cells can be selected based on a variety of factors, including, for example, (1) the use (e.g., therapy, expression of harvested proteins, and conferring disease resistance), and (2) expression of a marker with a desired amount of specificity.

[0237] Target cell is not limited in any way, and includes both germline cells and cell lines, and somatic cells and cell lines.Target cell can be stem cells from any source.When target cell is germline cell, target cell is preferably selected from the group consisting of single-cell embryo and embryonic stem cell (ES).

[0238] Pharmaceutical Compositions, Dosage Forms, and Administration

[0239] A further embodiment provides a pharmaceutical product comprising at least one modified viral capsid protein and a suitable targeting ligand according to the invention, and / or a nucleic acid according to the invention. Preferably, such a pharmaceutical product is useful as a gene transfer particle.

[0240] Also disclosed herein are pharmaceutical compositions comprising the viral particles described herein and a pharmaceutically acceptable carrier and / or excipient. Additionally, disclosed herein are pharmaceutical dosage forms comprising the viral particles described herein.

[0241] As discussed herein, the viral particles described herein can be used for a variety of therapeutic applications (in vivo and in vitro) and as research tools.

[0242] Pharmaceutical compositions based on the viral particles disclosed herein can be formulated in any conventional manner using one or more physiologically acceptable carriers and / or excipients. The viral particles can be formulated for administration, for example, by injection, inhalation, or isolation (either through the mouth or nose), or by oral, buccal, parenteral, or rectal administration, or by administration directly to a tumor.

[0243] Pharmaceutical compositions can be formulated for various modes of administration, including systemic administration, topical administration, or localized administration. Techniques and formulations can be found, for example, in Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, pharmaceutical compositions can be formulated in a liquid solution, preferably in a physiologically compatible buffer such as Hank's solution or Ringer's solution. In addition, pharmaceutical compositions can be formulated in solid form and can be redissolved or suspended immediately before use. Lyophilized forms of pharmaceutical compositions are also suitable.

[0244] For oral administration, pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, or silica), disintegrants (e.g., potato starch or sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulfate). Tablets can also be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspensions, or can be presented as a dry product that is constitutional with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifying agents (e.g., lecithin or acacia), non-aqueous vehicles (e.g., oils, oily esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g., methyl- or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring agents, coloring agents, and sweetening agents, as appropriate.

[0245] The pharmaceutical composition can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion.The preparation for injection can be presented in unit dosage form, for example, in ampoules or multi-dose containers, optionally with the addition of preservatives.The pharmaceutical composition can further be formulated as a suspension, solution, or emulsion in an oily or aqueous medium, and can contain other agents, including suspending agents, stabilizing agents, and / or dispersing agents.

[0246] In addition, pharmaceutical compositions can be formulated as depot preparations. These long-acting formulations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Thus, for example, the compound can be formulated with a suitable polymeric or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin, or as a sparingly soluble derivative, e.g., as a sparingly soluble salt. Other suitable delivery systems include microspheres, which offer the possibility of localized, non-invasive delivery of drugs over long periods of time. This technology can involve microspheres with precapillary sizes that can be injected into any selected part of an organ via a coronary catheter without causing inflammation or ischemia. The administered therapeutic agent is gradually released from the microspheres and absorbed by surrounding cells present in the selected tissue.

[0247] Systemic administration can also be via transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, bile salts and fusidic acid derivatives for transmucosal administration. In addition, detergents can be used to promote penetration. Transmucosal administration can also be carried out using intranasal sprays or suppositories. For topical administration, the viral particles described herein can be formulated into ointments, salves, gels, or creams that are generally known in the art. Cleansing solutions can also be used locally to treat wounds or inflammation to accelerate healing.

[0248] Pharmaceutical forms suitable for injectable use can include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the pharmaceutical forms must be sterile and fluid. They must also be stable under the conditions of manufacture and certain storage parameters (e.g., refrigeration and freezing), and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0249] When the formulations disclosed herein are used as therapeutic agents for promoting immune responses in subjects, the therapeutic agents can be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, as well as organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0250] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof...

Claims

[Claim 1] The invention described in the specification.

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