Adeno-associated virus variant capsid with improved pulmonary tropism and uses thereof

Abstract

The present disclosure provides variant AAV capsid proteins that confer tropism to lung cells, and recombinant adeno-associated viruses comprising the variant AAV capsid proteins, and pharmaceutical compositions comprising the same, and their use in delivering heterologous nucleic acids to lung cells for the treatment of lung conditions.

Description

[0001] Cross-reference to related applications This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 317,450, filed on March 7, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] Submitting a sequence listing via EFS-WEB A computer-readable XML file entitled "090400-5019-WO-Sequence-Listing" created on or about March 3, 2023, with a file size of approximately 144,000 bytes, contains the sequence listing of the present application and is hereby incorporated by reference in its entirety. Background of the Invention Gene delivery vectors based on adeno-associated virus (AAV) have shown promise in both preclinical disease models and recent human clinical trials for several disease targets. AAV-based vectors are extremely safe because wild-type AAV is not pathogenic and has no etiological association with any known disease. In addition, AAV provides the ability to deliver efficient genes and sustained transgene expression in a variety of tissues including liver, muscle, lung, retina, and brain.

[0004] AAV is a single-stranded DNA virus containing two open reading frames, rep and cap. The first gene encodes four proteins (Rep78, Rep68, Rep52 and Rep40) necessary for genome replication, and the second gene expresses three structural proteins (VP1-3) that can be assembled to form the viral capsid. As its name implies, AAV relies on the presence of a helper virus such as adenovirus or herpes virus for active replication. In the absence of a helper virus, it establishes a latent state in which its genome is maintained episomally or integrated into the host chromosome. A variety of homologous primate AAV serotypes and a variety of non-human primate serotypes have been identified. AAV2 is the best characterized as a gene delivery vehicle.

[0005] AAV has produced promising results in a growing number of clinical trials (Bainbridge et al., 2008; Carpentier et al., 2012; Gaudet et al., 2010; MacLaren et al., 2014; AM Maguire et al., 2009; A. Maguire & Simonelli, 2008; Nathwani et al., 2011, 2014; Stroes et al., 2008). However, there are gene delivery barriers that may limit the utility of AAV, such as anti-capsid immune responses, limited transduction of certain tissues, inability to target delivery to specific cell types, and relatively low packaging capacity. In many cases, insufficient knowledge of mechanisms that enable efficient rational design of AAVs is available. As an alternative, directed evolution has become a strategy for generating new AAV variants that meet specific biomedical needs. Directed evolution strategies utilize genetic diversification and selection processes to enable the accumulation of beneficial mutations that gradually improve the function of biomolecules. In the process, wild-type AAV cap genes are diversified using several different methods to generate large gene libraries, which are packaged to produce viral particle libraries, and then selection pressure is applied to isolate new variants that can overcome barriers to gene delivery.

[0006] Previously, proof-of-concept applications have shown that AAV directed evolution can be used to generate optimized AAV variants for gene delivery to human lung epithelium in vitro or to porcine lungs in vivo. These earlier studies demonstrated that AAV variants optimized for lung epithelial transduction can lead to significantly improved gene therapy treatments for CF when evaluated in the context of the model for which they were selected. However, evaluation of these vectors has shown species differences between porcine and human lung epithelium, which has resulted in limited transduction of human lung epithelium by vectors evolved for porcine lung epithelium transduction.

[0007] There is a need in the art to develop new AAV variants with improved delivery to human lung cells, such as human lung epithelium, for the treatment of lung disorders. Summary of the invention

[0008] Provided herein are variant adeno-associated virus (AAV) capsid proteins having one or more modifications in the amino acid sequence relative to the parent AAV capsid protein, which, when present in an AAV virion, confer increased infectivity to one or more types of lung cells compared to the infectivity of the AAV virion comprising the unmodified parent AAV capsid protein to lung cells. Also provided are recombinant AAV virions and pharmaceutical compositions thereof comprising the variant AAV capsid proteins as described herein, methods for preparing variant rAAV capsid proteins and virions, and methods for using these rAAV capsid proteins and virions in research and in clinical practice, for example, in delivering nucleic acid sequences to one or more cells of the lung for the treatment of lung disorders and diseases.

[0009] In some aspects of the present disclosure, variant adeno-associated virus (AAV) capsid proteins are provided that have one or more modifications in the amino acid sequence relative to the parent AAV capsid, and when present in AAV virions, the variant AAV capsid proteins confer increased infectivity to one or more types of lung cells (e.g., respiratory epithelial cells, including but not limited to alveolar epithelial cells, bronchial (primary, secondary or tertiary) epithelial cells or tracheal epithelial cells, ciliated respiratory epithelial cells, alveolar epithelial type 1 (AECI) or type 2 (AECII) cells, smooth muscle or endothelial cells) compared to the infectivity of AAV virions comprising the parent AAV capsid protein that does not comprise the amino acid sequence modification to lung cells.

[0010] In some aspects of the present disclosure, recombinant AAV (rAAV) virions are provided, which contain variant capsid proteins as described herein, wherein the rAAV virions exhibit increased infectivity to one or more types of lung cells relative to the infectivity of AAV virions containing the corresponding unmodified parent AAV capsid proteins to lung cells. In some embodiments, the rAAV virions exhibit increased infectivity to all lung cells relative to AAV virions containing parent AAV capsid proteins. In other embodiments, the rAAV virions exhibit increased infectivity to certain cell types of the lung rather than other cell types relative to AAV virions containing parent AAV capsid proteins. In other words, the rAAV virions exhibit increased infectivity to certain cell types of the lung rather than to other cell types, for example, rAAV exhibits increased infectivity to one or more upper respiratory tract cells of the lung preferentially, but does not exhibit increased infectivity to all cell types. In some aspects, variant capsid proteins as described herein confer increased resistance to human AAV neutralizing antibodies to these rAAV virions.

[0011] In some embodiments, the rAAV virion comprises a heterologous nucleic acid. In some such embodiments, the heterologous nucleic acid encodes an RNA encoding a polypeptide. In other such embodiments, the heterologous nucleic acid sequence encodes an RNA that does not encode a polypeptide, for example, a heterologous nucleic acid sequence RNA interfering agent, a guide RNA for a nuclease, etc. In some embodiments, the heterologous nucleic acid comprises a nucleotide sequence encoding a polypeptide and a nucleotide sequence encoding an interfering RNA.

[0012] Also provided herein is a pharmaceutical composition comprising a subject infectious rAAV virion and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated as a liquid / suspension suitable for aerosolized delivery. In a related embodiment, the pharmaceutical composition is administered as an aerosol suspension of respirable particles comprising rAAV virions inhaled by the subject. The respirable particles may be liquid or solid. An aerosol of liquid particles comprising rAAV virions can be produced by any suitable method, such as using a pressure-driven aerosol nebulizer or an ultrasonic nebulizer. An aerosol of solid particles comprising rAAV virions can be produced with any solid particle aerosol generator.

[0013] Also provided is the use of rAAV virions comprising variant capsid proteins as described herein in methods for delivering heterologous nucleic acids to target cells (e.g., lung cells) by contacting the target cells with rAAV virions. In some embodiments, the target cells are in vivo, such as in the lungs of an individual in need of treatment for a lung disease. In other embodiments, the target cells are in vitro.

[0014] Also provided are methods of treating a lung disease by administering to a subject in need of such treatment an effective amount of rAAV virions, or a pharmaceutical composition comprising an effective amount of rAAV virions, wherein the rAAV virions comprise a variant capsid protein as described herein.

[0015] Also provided are isolated nucleic acids and host cells comprising the isolated nucleic acids, the isolated nucleic acids comprising sequences encoding variant AAV capsid proteins as described herein. In yet other embodiments, the isolated nucleic acids and / or isolated host cells comprise rAAV.

[0016] In some aspects, the variant AAV capsid protein comprises an insertion of about 5 amino acids to about 20 amino acids in the GH-loop of the capsid protein, preferably in a surface-exposed region of the GH-loop (a "heterologous peptide" or "peptide insertion") relative to the corresponding parent AAV capsid protein, wherein when present in an AAV virion, the variant capsid protein confers increased infectivity to retinal cells compared to the infectivity of AAV virions comprising the corresponding parent AAV capsid protein to retinal cells. In some embodiments, the peptide comprises, consists essentially of, or consists of a sequence selected from the group consisting of HDITKNI (SEQ ID NO: 12), NQDYTKT (SEQ ID NO: 13), DNTVTRS (SEQ ID NO: 14), SNSVQSI (SEQ ID NO: 15), NSTRHTD (SEQ ID NO: 16), TNRTSPD (SEQ ID NO: 17), ISDQTKH (SEQ ID NO: 18), QADTTKN (SEQ ID NO: 19), NAVKTDF (SEQ ID NO: 20), TNQTLSA (SEQ ID NO: 21), ENRTTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TNNSKPD (SEQ ID NO: 25), SKLTLNN (SEQ ID NO: 26), VTAGMGA (SEQ ID NO: 27), PNSTTNN (SEQ ID NO: 28), NSTSRID (SEQ ID NO: 29), NO:29), VASHTNN (SEQ ID NO:30), RSHQEIP (SEQ ID NO:31), LNTTKDI (SEQ ID NO:32), IIDATKN (SEQ ID NO:33), NHISQTN (SEQ ID NO:34), SNSAHIT (SEQ ID NO:35), STHQSNN (SEQ ID NO:36), KTPNLTS (SEQ ID NO:37), SNTPALS (SEQ ID NO:38), SPGATTN (SEQ ID NO:39), LAHDITKNIA (SEQ ID NO:40), LANQDYTKTA (SEQ ID NO:41), LADNTVTRSA (SEQ ID NO:42), LASNSVQSIA (SEQ ID NO:43), LANSTRHTDA (SEQ ID NO:44), LATNRTSPDA (SEQ ID NO:45), LAISDQTKHA (SEQ IDNO: 46), LAQADTTKNA (SEQ ID NO: 47), LANAVKTDFA (SEQ ID NO: 48), LATNQTLSAA (SEQ ID NO: 49), LAENRTTSNA (SEQ ID NO: 50), LAPQQDTTHA (SEQ ID NO: 51), LANATNHVIA (SEQ ID NO: 52), LATNNSKPDA (SEQ ID NO: 53), LASKLTLNNA (SEQ ID NO: 54), LAVTAGMGAA (SEQ ID NO: 55), LAPNSTTNNA (SEQ ID NO: 56), LANSTSRIDA (SEQ ID NO: 57), LAVASHTNNA (SEQ ID NO: 58), LARSHQEIPA (SEQ ID NO: 59), LALNTTKDIA (SEQ ID NO: 60), LAIIDATKNA (SEQ ID NO: 61), LANHISQTNA (SEQ ID NO: 62), LASNSAHITA (SEQ ID NO: 63), LASTHQSNNA (SEQ ID NO: 64), LAKTPNLTSA (SEQ ID NO: 65), LASNTPALSA (SEQ ID NO: 66) and LASPGATTNA (SEQ ID NO: 67).

[0017] In some aspects, the variant AAV capsid protein comprises a peptide insertion in the GH-loop of the capsid protein relative to the corresponding parent AAV capsid protein, and further comprises one or more amino acid substitutions relative to the corresponding parent AAV capsid protein, wherein when present in an AAV virion, the variant capsid protein confers increased infectivity to pneumocytes compared to the infectivity of AAV virions comprising the corresponding parent AAV capsid protein to pneumocytes. In some preferred aspects, the variant AAV capsid protein comprises, consists essentially of, or consists of a sequence selected from the group consisting of HDITKNI (SEQ ID NO: 12), NQDYTKT (SEQ ID NO: 13), DNTVTRS (SEQ ID NO: 14), SNSVQSI (SEQ ID NO: 15), NSTRHTD (SEQ ID NO: 16), TNRTSPD (SEQ ID NO: 17), ISDQTKH (SEQ ID NO: 18), QADTTKN (SEQ ID NO: 19), NAVKTDF (SEQ ID NO: 20), TNQTLSA (SEQ ID NO: 21), ENRTTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TNNSKPD (SEQ ID NO: 25), SKLTLNN (SEQ ID NO: 26), VTAGMGA (SEQ ID NO: 27), PNSTTNN (SEQ ID NO: 28), NSTSRID (SEQ ID NO: 29), NO:29), VASHTNN (SEQ ID NO:30), RSHQEIP (SEQ ID NO:31), LNTTKDI (SEQ ID NO:32), IIDATKN (SEQ ID NO:33), NHISQTN (SEQ ID NO:34), SNSAHIT (SEQ ID NO:35), STHQSNN (SEQ ID NO:36), KTPNLTS (SEQID NO:37), SNTPALS (SEQ ID NO:38), SPGATTN (SEQ ID NO:39), LAHDITKNIA (SEQ ID NO:40), LANQDYTKTA (SEQ ID NO:41), LADNTVTRSA (SEQ ID NO:42), LASNSVQSIA (SEQ ID NO:43), LANSTRHTDA (SEQ ID NO:44), LATNRTSPDA (SEQ ID NO:45), LAISDQTKHA (SEQ ID NO:46), LAQADTTKNA (SEQ IDNO:47), LANAVKTDFA (SEQ ID NO:48), LATNQTLSAA (SEQ ID NO:49), LAENRTTSNA (SEQ ID NO:50), LAPQQDTTHA (SEQ ID NO:51), LANATNHVIA (SEQ ID NO:52), LATNNSKPDA (SEQ ID NO:53), LASKLTLNNA (SEQ ID NO:54), LAVTAGMGAA (SEQ ID NO:55), LAPNSTTNNA (SEQ ID NO:56), LANSTSRIDA (SEQ ID NO:57), LAVASHTNNA (SEQ ID NO:58), LARSHQEIPA (SEQ ID NO:59), LALNTTKDIA (SEQ ID NO:60), LAIIDATKNA (SEQ ID NO:61), LANHISQTNA (SEQ ID NO:62), LASNSAHITA (SEQ ID NO:63), LASTHQSNNA (SEQ ID NO:64), LAKTPNLTSA (SEQ ID NO:65), LASNTPALSA (SEQ ID NO:66) and LASPGATTNA (SEQ ID NO:67), and further comprises a V708I substitution relative to VP1 of AAV2.

[0018] Also disclosed herein are methods for making and / or delivering rAAV comprising a variant AAV capsid as disclosed herein. In addition, provided herein are kits comprising rAAV comprising a variant AAV capsid as disclosed herein and for use in the methods described herein.

[0019] In other embodiments, the AAV virus particles comprising variant capsid proteins in the preceding paragraphs may comprise any previously or later disclosed embodiments. In fact, it should be understood that certain features of the invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. On the contrary, the various features of the invention described in the context of a single embodiment for brevity may also be provided individually or in any suitable sub-combination. All combinations of embodiments related to the present invention are specifically included by the present invention and disclosed herein, as if each combination were individually and explicitly disclosed. In addition, all sub-combinations of various embodiments and their elements are specifically included by the present invention and disclosed herein, as if each such sub-combination were individually and explicitly disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Illustrated is a directed evolution process used to identify variant AAV capsids that confer improved respiratory tract transduction.

[0021] Figure 2 Figure 1 is a schematic diagram of the model system decision tree. Decisions were based on sequencing analysis performed after the second and third rounds of NHP delivery.

[0022] Figure 3A -B Figure 3a illustrates the estimated genetic diversity of the capsid library, where the total diversity is >1 billion variants. Figure 3b illustrates the productivity of the capsid library, all of which were manufactured at a level sufficient to produce material for in vivo therapeutic vector evolution processes. The administered viral genome (vg) represents the target dose, excluding losses associated with the delivery device and route of administration.

[0023] Figure 4 Illustrated is ddPCR quantification of viral genomes present in the trachea and primary, secondary, and tertiary bronchi of NHPs administered the capsid library for selection rounds 1-3.

[0024] Figure 5 The frequency of "hits" in the third round of sequencing analysis is illustrated. The sequencing analysis is based on the total frequency within the population sequenced in each region. Left panel (trachea and primary bronchi) The frequencies are as follows: (i) point mutant #1 = 13%; (ii) peptide insertion #1 = 11%; (iii) peptide insertion #2 = 4%; (iv) peptide insertion #3 = 6%; (v) AAV2 point mutant = 11%; (vi) AAV2 peptide insertion = 40%; (vii) chimera = 7%; (viii) other = 8%. Right panel (secondary and tertiary bronchi) The frequencies are as follows: (i) point mutant #1 = 9%; (ii) peptide insertion #1 = 3%; (iii) peptide insertion #2 = 10%; (iv) peptide insertion #3 = 3%; (v) peptide insertion #4 = 7%; (vi) AAV2 point mutant = 9%; (vii) AAV2 peptide insertion = 54%; (viii) other = 5%.

[0025] Figure 6 AB illustrates the 4th round selection ( Figure 6 a) and the 5th round selection ( Figure 6 b) ddPCR quantification of viral genomes present in human upper respiratory tract ALI cultures from the capsid library administered in the absence and presence of human IVIG.

[0026] Figure 7The frequency of "hits" in round 5 sequencing analysis is illustrated. Sequencing analysis was performed based on the overall frequency within the sequencing population selected with and without IVIG. Left panel (in vitro round 5A - IVIG) Frequencies are as follows: (i) point mutant #1 = 10%: (ii) peptide insertion #1 = 17%; (iii) peptide insertion #2 = 8%; (iv) peptide insertion #3 = 1%; (v) peptide insertion #4 = 4%; (vi) peptide insertion #5 = 9%; (vi) peptide insertion #6 = 15%; (vii) AAV2 peptide insertion = 35%; (viii) others = 1%. Right panel (in vitro round 5B + IVIG) frequencies are as follows: (i) point mutant #1 = 6.7%: (ii) peptide insertion #1 = 11.5%; (iii) peptide insertion #2 = 9.6%; (iv) peptide insertion #3 = 2.9%; (v) peptide insertion #4 = 1.9%; (vi) peptide insertion #5 = 16.3%; (vi) peptide insertion #6 = 10.6%; (vii) AAV2 peptide insertion = 40.4%.

[0027] Fig. 8A -C AAV capsid transduction efficiency in lung upper airway epithelial ALI cultures measured by reporter gene EGFP expression using fluorescence microscopy. Figures 8a-b show human (Figure 8a) and NHP (Figure 8b) apical transduction of six novel variants compared to AAV2, AAV5, and AAV101. Figure 8c shows human basal transduction of six novel variants compared to AAV2, AAV5, and AAV101. Adeno-associated virus (AAV), air-liquid interface (ALI), non-transduced (NT), multiplicity of infection (MOI).

[0028] Fig.9A -C Secondary analysis of the highest new capsids compared to AAV2 and AAV101 by immunocytochemistry (Fig. 9a, 9b) and ddPCR (Fig. 9c). AAV capsid transduction efficiency in lung upper airway epithelial ALI cultures was measured using fluorescence microscopy by reporter gene EGFP expression (Fig. 9a). Quantification of micrograph pixel intensity (Fig. 9b). Quantification of transcripts after transduction (Fig. 9c). Adeno-associated virus (AAV), air-liquid interface (ALI), non-transduced (NT), multiplicity of infection (MOI), mean fluorescence intensity (MFI), housekeeper (HK). Error bars = standard deviation. n = 3 per condition.

[0029] Fig. 10A-B illustrates EGFP expression in human airway epithelial cell cultures following transduction with the indicated AAV capsids carrying nucleic acid comprising an EGFP reporter gene operably linked to a CAG promoter in the presence or absence of mucus. Cultures grown in an air-liquid interface for ≥30 days; time post infection 7 days; -mucus, no mucus; +mucus, with mucus; apical transduction; multiplicity of infection (MOI) 25,000. DETAILED DESCRIPTION OF THE INVENTION definition As used in the specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0031] As used herein, the term "comprising" is intended to mean that compositions and methods include the elements described, but do not exclude other elements. "Consisting essentially of" when used to define compositions and methods should mean excluding other elements that are of any importance to the combination for the intended use. For example, a composition consisting essentially of the elements as defined herein will not exclude trace contaminants from separation and purification methods and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, etc. "Consisting of" should mean excluding more than trace elements of other ingredients and substantial method steps for applying the compositions disclosed herein. Each of the defined aspects of these transitional terms is within the scope of the present disclosure.

[0032] Adeno-associated virus is a non-pathogenic parvovirus consisting of a 4.7 kb single-stranded DNA genome within a non-enveloped icosahedral capsid. The genome contains three open reading frames (ORFs) flanked by inverted terminal repeats (ITRs) that function as viral replication origins and packaging signals. The rep ORF encodes four nonstructural proteins that play a role in viral replication, transcriptional regulation, site-specific integration, and virion assembly. The cap ORF encodes three structural proteins (VP1-3) that assemble to form a 60-mer viral capsid. Finally, the ORF, which exists as a separate reading frame within the cap gene, produces the assembly activation protein (AAP), a viral protein that localizes the AAV capsid proteins to the nucleolus and plays a role in the capsid assembly process.

[0033] There are several naturally occurring ("wild-type") serotypes of AAV and over 100 known variants, each of which differs in amino acid sequence, particularly within the hypervariable regions of the capsid protein, and therefore in its gene delivery properties. No AAV has been associated with any human disease, making recombinant AAV attractive for clinical applications.

[0034] For the purposes of this disclosure, the term "AAV" is an abbreviation for adeno-associated virus, including but not limited to the virus itself and its derivatives. Unless otherwise indicated, the term refers to all subtypes or serotypes and both replication-competent and recombinant forms. The term "AAV" includes, but is not limited to, AAV1 (AAV-1 or AAV1), AAV2 (AAV-2 or AAV2), AAV3A (AAV-3A or AAV3A), AAV3B (AAV-3B or AAV3B), AAV4 (AAV-4 or AAV4), AAV5 (AAV-5 or AAV5), AAV6 (AAV-6 or AAV6), AAV7 (AAV-7 or AAV7), AAV8 (AAV-8 or AAV8), AAV9 (AAV-9 or AAV9), AAV10 (AAV-10 or AAV10 or AAVrh10), avian AAV, bovine AAV, canine AAV, caprine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, "bovine AAV" refers to AAV that infects bovine mammals, etc.

[0035] The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), 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. See, for example, GenBank Accession Nos. NC_002077.1 (AAV1), AF063497.1 (AAV1), NC_001401.2 (AAV2), AF043303.1 (AAV2), J01901.1 (AAV2), U48704.1 (AAV3A), NC_001729.1 (AAV3A), AF028705.1 (AAV3B), NC_001829.1 (AAV4), U89790.1 (AAV4), NC_0061.1 (AAV4). 52.1 (AAV5), AF085716.1 (AAV-5), AF028704.1 (AAV6), NC_006260.1 (AAV7), AF513851.1 (AAV7), AF513852.1 (AAV8) NC_006261.1 (AAV-8), AY530579.1 (AAV9), AAT46337 (AAV10) and AA088208 (AAVrhlO); for teaching AAV nucleic acid and amino acid sequences, their disclosures are incorporated herein by reference. See also, e.g., 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. (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. (2004) Virology 33:375-383; International Patent Publication Nos. WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; and U.S. Patent No. 6,156,303.

[0036] The sequences of naturally occurring cap (capsid) proteins associated with AAV serotypes are known in the art and include those disclosed herein as AAV1 (SEQ ID NO: 1), AAV2 (SEQ ID NO: 2), AAV3A (SEQ ID NO: 3), AAV3B (SEQ ID NO: 4), AAV4 (SEQ ID NO: 5), AAV5 (SEQ ID NO: 6), AAV6 (SEQ ID NO: 7), AAV7 (SEQ ID NO: 8), AAV8 (SEQ ID NO: 9), AAV9 (SEQ ID NO: 10), and AAV10 (SEQ ID NO: 11). The term "variant AAV capsid protein" or "AAV variant" refers to an AAV capsid protein comprising an amino acid sequence that comprises at least one modification or substitution (including deletions, insertions, point mutations, etc.) relative to a naturally occurring or "wild-type" AAV capsid protein sequence (e.g., as described in SEQ ID NOs: 1-11 herein). The variant AAV capsid protein may have about 80% identity or more to the amino acid sequence of the wild-type capsid protein, such as 85% identity or more, 90% identity or more, or 95% identity or more, such as 98% or 99% identity to the amino acid sequence of the wild-type capsid protein. The variant AAV capsid protein may not be a wild-type capsid protein.

[0037] For purposes of this disclosure, an "AAV virion" or "AAV viral particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated AAV polynucleotide.

[0038] For the purposes of this disclosure, the term "rAAV" refers to the abbreviation for recombinant adeno-associated virus. "Recombinant" when applied to polynucleotides means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is different from the polynucleotide found in nature. Recombinant viruses are viral particles that contain recombinant polynucleotides. The terms include copies of the original polynucleotide construct and progeny of the original viral construct, respectively.

[0039] The term "rAAV vector" includes rAAV virions (rAAV viral particles) (e.g., infectious rAAV virions), which by definition include rAAV polynucleotides; and also include polynucleotides encoding rAAV (e.g., single-stranded polynucleotides encoding rAAV (ss-rAAV); double-stranded polynucleotides encoding rAAV (ds-rAAV), such as plasmids encoding rAAV; etc.).

[0040] If an AAV virion contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, e.g., a transgene to be delivered to a target cell, an RNAi agent or a CRISPR agent to be delivered to a target cell, etc.), it is generally referred to as a "recombinant AAV (rAAV) virion" or "rAAV viral particle." Typically, the heterologous polynucleotide is flanked by at least one, and typically two, AAV inverted terminal repeats (ITRs).

[0041] The term "packaging" refers to the series of intracellular events that lead to the assembly and encapsidation of AAV particles. The AAV "rep" and "cap" genes refer to polynucleotide sequences encoding the replication and encapsidation proteins of the adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

[0042] The term "helper virus" of AAV refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells. Various such helper viruses for AAV are known in the art, including adenovirus, herpes virus, and pox virus such as vaccinia. Adenovirus includes many different subgroups, although adenovirus type 5 of subgroup C is the most commonly used. Many adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as ATCC. Viruses of the herpes family include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV); these can also be obtained from depositories such as ATCC.

[0043] The term "one or more helper virus functions" refers to one or more functions encoded in the helper virus genome that allow AAV replication and packaging (together with other requirements for replication and packaging as described herein). As described herein, "helper virus functions" can be provided in a variety of ways, including by providing a helper virus or providing a polynucleotide sequence encoding one or more necessary functions to the producer cell in trans. For example, a plasmid or other expression vector containing a nucleotide sequence encoding one or more adenoviral proteins is transfected into the producer cell together with the rAAV vector.

[0044] The term "infectious" virus or virus particle is a virus or virus particle that contains a competently assembled viral capsid and is capable of delivering a polynucleotide component to a cell tropistic for the viral species. The term does not necessarily imply any replication capability of the virus. Assays for counting infectious virus particles are described elsewhere in this disclosure and in the art. Viral infectivity can be expressed as a ratio of infectious virus particles to total virus particles. Methods for determining the ratio of infectious virus particles to total virus particles are known in the art. See, e.g., Grainger et al., (2005) Mol, Ther. 11: S337 (describing TCID50 infectivity titer assay); and Zolotukhin et al. (1999) Gene Ther. 6: 973. See also the Examples.

[0045] As used herein, the term "tropism" refers to a virus (e.g., AAV) preferentially targeting cells of a particular host species or a specific cell type within a host species. For example, relative to viruses that may only infect lung and muscle cells, viruses that can infect heart, lung, liver and muscle cells have a wider (i.e., increased) tropism. Tropism can also include the dependence of viruses on cell surface molecules of a particular type of host. For example, some viruses can only infect cells with surface glycosaminoglycans, while other viruses can only infect cells with sialic acid (this dependence can be tested as potential host cells infected by viruses using various cell lines lacking specific class molecules). In some cases, the tropism of a virus describes the relative preference of the virus. For example, a first virus may be able to infect all cell types, but is much more successful in infecting those cells with surface glycosaminoglycans. If a second virus also prefers the same features (e.g., a second virus is also more successful in infecting those cells with surface glycosaminoglycans), it can be considered that the second virus has a tropism similar to (or identical to) the first virus, even if the absolute transduction efficiency is not similar. For example, a second virus may be more effective than a first virus at infecting each given cell type tested, but if the relative preferences are similar (or identical), the second virus can still be considered to have a tropism similar (or identical) to the first virus. In some embodiments, the tropism of the virions comprising the subject variant AAV capsid protein is not altered relative to naturally occurring virions. In some embodiments, the tropism of the virions comprising the subject variant AAV capsid protein is expanded (i.e., widened) relative to naturally occurring virions. In some embodiments, the tropism of the virions comprising the subject variant AAV capsid protein is reduced relative to naturally occurring virions.

[0046] The term "replication-competent" virus (e.g., replication-competent AAV) refers to a phenotypically wild-type virus that is infectious and also capable of replicating in infected cells (i.e., in the presence of a helper virus or helper virus function). In the case of AAV, replication competence generally requires the presence of functional AAV packaging genes. Typically, rAAV vectors as described herein are not replicable in mammalian cells (especially in human cells) due to the lack of one or more AAV packaging genes. Generally, such rAAV vectors lack any AAV packaging gene sequences to minimize the possibility of producing replication-competent AAV by recombination between AAV packaging genes and entry into the rAAV vector. In many embodiments, rAAV vector preparations as described herein are those containing very few, if any, replication-competent AAV (rcAAV, also referred to as RCA) rAAV vector preparations (e.g., 10 cells per 10 cells). 2 Each rAAV particle is smaller than about 1 rcAAV particle. 4 Each rAAV particle is smaller than about 1 rcAAV, each 10 rAAV particles are smaller than about 1 rcAAV, each 10 12 less than about 1 rcAAV particle or no rcAAV).

[0047] The term "polynucleotide" refers to nucleotides in a polymeric form of any length, including deoxyribonucleotides or ribonucleotides or their analogs. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, the modification of the nucleotide structure may be given before or after polymer assembly. As used herein, the term polynucleotide refers to double-stranded and single-stranded molecules interchangeably. Unless otherwise specifically stated or required, any embodiment of polynucleotides herein includes each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

[0048] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, thereby meaning that when compared, the bases or amino acids of the percentage are identical when comparing two sequences. Sequence similarity can be determined in a variety of different ways. In order to determine sequence identity, the method and computer program including BLAST available on the World Wide Web at nebi.nlm.nih.gov / BLAST / can be used to compare sequences. Another comparison algorithm is PASTA, which can be obtained from the Genetics Computing Group (GCG) software package of Madison, Wis., the U.S., a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for comparison are described in Methods in Enzymology, Vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), compiled by Doolittle, Academic Press, Inc., Harcourt Brace & Co., San Diego, Calif., USA. Of particular interest is the comparison program that allows the gap in the sequence. Smith-Waterman is an algorithm that allows gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Similarly, the GAP program using the Needleman and Wunsch alignment method can be used to align sequences, see J. Mol. Biol. 48: 443-453 (1970).

[0049] The term "gene" refers to a polynucleotide that performs a certain type of function in a cell. For example, a gene can contain an open reading frame that can encode a gene product. An example of a gene product is a protein transcribed and translated from a gene. Another example of a gene product is an RNA that is transcribed but not translated, for example, a functional RNA product, for example, an aptamer, an interfering RNA, a ribosomal RNA (rRNA), a transfer RNA (tRNA), a non-coding RNA (ncRNA), a guide RNA for a nuclease, etc.

[0050] The term "gene expression product" or "gene product" is a molecule produced by the expression of a specific gene as defined above. Gene expression products include, for example, polypeptides, aptamers, interfering RNAs, messenger RNAs (mRNAs), rRNAs, tRNAs, non-coding RNAs (ncRNAs), etc.

[0051] The term "siRNA agent" ("small interfering" or "short interfering RNA" (or siRNA)) is an RNA duplex of nucleotides that targets a gene of interest ("target gene"). "RNA duplex" refers to a structure formed by complementary pairing between two regions of an RNA molecule, thereby forming a double-stranded RNA (dsRNA) region. siRNA "targets" a gene because the nucleotide sequence of the duplex portion of the siRNA is complementary to the nucleotide sequence of the targeted gene. In some embodiments, the length of the duplex of the siRNA is less than 30 nucleotides. In some embodiments, the length of the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotides. In some embodiments, the length of the duplex is 19-25 nucleotides. In some embodiments, siRNA-mediated gene targeting is achieved by using DNA-guided RNA interference (ddRNAI), which is a gene silencing technology that utilizes DNA constructs to activate endogenous RNA interference (RNAi) pathways of animal cells. This DNA construct is designed to express self-complementary double-stranded RNAs that cause one or more target genes to be silenced once processed, generally short hairpin RNAs (shRNA). Any RNA including endogenous mRNAs or viral RNAs can be silenced by designing the construct to express double-stranded RNA complementary to the desired mRNA target. Therefore, the RNA duplex portion of the siRNA agent can be a part of a short hairpin structure referred to as shRNA. In addition to the duplex portion, the hairpin structure can contain a loop portion between two sequences forming a duplex. The length of the loop can vary. In some embodiments, the length of the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides. The hairpin structure can also contain a 3' or 5' overhang portion. In some embodiments, the overhang is a 3' or 5' overhang of 0, 1, 2, 3, 4 or 5 nucleotides in length. Typically, the level of the expression product (e.g., mRNA, polypeptide, etc.) of the target gene is reduced by an siRNA agent (e.g., siRNA, shRNA, etc.) containing a specific double-stranded nucleotide sequence, which is complementary to a segment of at least 19-25 nucleotides (e.g., 2021 nucleotide sequence) of the target gene transcript, including a 5' untranslated (UT) region, an ORF or a 3'UT region. In some embodiments, the length of the short interfering RNA is about 19-25 nt.See, e.g., PCT applications WO0144895, WO99 / 32619, WO01 / 75164, WO01 / 92513, WO01 / 29058, WO01 / 89304, WO02 / 16620, and WO02 / 29858; and U.S. Patent Publication No. 20040023390 describing siRNA technology. siRNA and / or shRNA can be encoded by a nucleic acid sequence, and the nucleic acid sequence can also include a promoter. The nucleic acid sequence can also include a polyadenylation signal. In some embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal.

[0052] The term "antisense RNA" includes RNA that is complementary to a gene expression product. For example, an antisense RNA targeting a specific mRNA is an RNA-based agent (or modified RNA) that is complementary to the mRNA, wherein hybridization of the antisense RNA with the mRNA changes the expression of the mRNA (e.g., via changing the stability of the RNA, changing the translation of the RNA, etc.). "Antisense RNA" also includes nucleic acids encoding antisense RNA.

[0053] About "CRISPR / Cas9 agent", the term "CRISPR" includes regularly spaced clustered short palindromic repeats / CRISPR-related (Cas) systems, which have evolved to provide adaptive immunity to viruses and plasmids to bacteria and archaea by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. Cas9 protein (or its functional equivalent and / or its variant, i.e., Cas9-like protein) naturally contains DNA endonuclease activity, which depends on the association of protein with two naturally occurring or synthetic RNA molecules referred to as crRNA and tracrRNA (also referred to as guide RNAs). In some cases, two molecules are covalently linked to form a single molecule (also referred to as a single guide RNA ("sgRNA")). Therefore, Cas9 or Cas9-like protein associates with DNA-targeting RNA (the term includes both two-molecule guide RNA configuration and single-molecule guide RNA configuration), and the DNA-targeting RNA activates Cas9 or Cas9-like protein and guides the protein to the target nucleic acid sequence.

[0054] If Cas9 or Cas9-like protein retains its native enzymatic function, it will cut the target DNA to produce double-strand breaks, which can lead to genomic changes (i.e., editing: deletion, insertion (when a donor polynucleotide is present), substitution, etc.), thereby changing gene expression. Some variants of Cas9 (variants of which are included by the term Cas9-like) have been altered so that they have reduced DNA cleavage activity (in some cases, they cut a single strand of the target DNA instead of two strands, and in other cases, they are severely reduced to no DNA cleavage activity). Cas9-like proteins with reduced DNA cleavage activity (or even no DNA cleavage activity) can still be guided to the target DNA to block RNA polymerase activity. On the other hand, Cas9 or Cas9-like proteins can be modified by fusing the VP64 transcriptional activation domain to the Cas9 protein and co-delivering the fusion protein with the MS2-P65-HSF1 auxiliary protein and a single guide RNA containing an MS2 RNA aptamer on the tetranucleotide loop and the stem loop to form a synergistic activation mediator (Cas9-SAM) complex in cells that activate transcription. Therefore, the enzymatically inactivated Cas9-like protein can be targeted to a specific location in the target DNA by the DNA-targeting RNA to block or activate the transcription of the target DNA. As used herein, the term "CRISPR'Cas9 agent" includes all forms of CRISPR / Cas9 as described above or known in the art.

[0055] Detailed information about CRISPR agents can be found, for example, in the following references: (a) Jinek et al., Science. 2012 Aug 17; 337(6096):816-21: "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity"; (b) Qi et al., Cell. 2013 Feb 228; 152(5):1173-83: "Repurposing CRISPR. as an RNA-guided platform for sequence-specific control of gene expression", and (c) U.S. patent application serial number 13 / 842,859 and PCT application number PCT / US13 / 32589; all of which are hereby incorporated by reference in their entirety. Thus, the term "CRISPR agent" as used herein includes any agent (or nucleic acid encoding such an agent) that can be used in a Cas9-based system (e.g., Cas9 or Cas9-like protein; any component of a DNA-targeting RNA, such as crRNA-like RNA, tracrRNA-like RNA, a single guide RNA, etc.; a donor polynucleotide; etc.), including naturally occurring and / or synthetic sequences.

[0056] So-called "zinc finger nucleases" (ZFNs) refer to artificial DNA endonucleases produced by fusing a zinc finger DNA binding domain with a DNA cleavage domain. ZFNs can be artificially engineered to target a desired DNA sequence, and this enables the zinc finger nuclease to cut a unique target sequence. When introduced into a cell, ZFNs can be used to edit target DNA in a cell (e.g., the genome of a cell) by inducing double-strand breaks. For more information on the use of ZFNs, see, e.g., Asuri et al. Mol Ther. 2012 Feb;20(2):329-38; Bibikova et al. Science. 2003 May 2;300(5620):764; Wood et al. Science. 2011 Jul 15;333(6040):307; Ochiai et al. Genes Cells. 2010 Aug;15(8):875-85; Takasu et al. Insect Biochem Mol 2010 Oct;40(10):759-65; Ekker et al. Zebrafish 2008 Summer;5(2):121-3; Young et al. Proc Natl Acad Sci US A. 2011 Apr 26;108(17):7052-7; Goldberg et al. Cell. 2010 Mar 5;140(5):678-91; Geurts et al. Science. 2009 Jul 24;325(5939):433; Flisikowska et al. PLoS One. 2011;6(6):e21045.doi:10.1371 / journal.pone.0021045.Epub 2011 Jun 13; Flauschild et al. Proc Nati Acad Sci US A. 2011 Jul 19;108(29):12013-7; and Yu et al. Cell. 2009 Jul 24;325(5939):433; Flisikowska et al. PLoS One. 2011;6(6):e21045.doi:10.1371 / journal.pone.0021045.Epub 2011 Jun 13; Flauschild et al. Proc Nati Acad Sci US A. 2011 Jul 19;108(29):12013-7; and Yu et al. Cell. Res. 2011 Nov;21(11):1638-40; which is incorporated herein by reference in its entirety for its teachings related to ZFNs. The term "ZFN agent" includes zinc finger nucleases and / or polynucleotides comprising a nucleotide sequence encoding a zinc finger nuclease.

[0057] The term "Transcription activator-like effector nuclease" or "TALEN" agent refers to transcription activator-like effector nucleases (TALENs) are artificial DNA endonucleases produced by fusing TAL (transcription activator-like) effector DNA binding domains to DNA cleavage domains. TALENS can be rapidly artificially engineered to bind virtually any desired DNA sequence, and when introduced into a cell, TALENs can edit target DNA in the cell (e.g., the genome of the cell) by inducing double-strand breaks. For more information on the use of TALENs, see, e.g., Hockemeyer et al. Nat Biotechnol. 2011 Jul 7;29(8):731-4; Wood et al. Science. 2011 Jul 15;333(6040):307; Tesson et al. Nat Biotechnol, 2011 Aug 5;29(8):695-6; and Huang et al., Nat Biotechnol. 2011 Aug 5;29(8):699-700, which are incorporated herein by reference in their entirety for their teachings related to TALENs. The term "TALEN agent" includes TALEN and / or a polynucleotide comprising a nucleotide sequence encoding a TALEN.

[0058] The term "control element" or "control sequence" refers to a nucleotide sequence involved in molecular interactions that contributes to the functional regulation of a polynucleotide, including replication, repetition, transcription, splicing, translation or degradation of the polynucleotide. The regulation can affect the frequency, speed or specificity of the process and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences, such as promoters and enhancers. A promoter is a DNA region that is capable of binding to RNA polymerase under certain conditions and initiating transcription of a coding region that is usually located downstream (3' direction) of the promoter. Promoters can be ubiquitous, i.e., active in many cell types, such as CAG or CMV promoters; or tissue or cell specific.

[0059] The term "operatively linked" or "operably linked" refers to a juxtaposition of genetic elements wherein the elements are in a relationship that permits them to function in an intended manner. For example, a promoter is operably linked to a coding region if it helps initiate transcription of a coding sequence. Intervening residues may be present between the promoter and the coding region as long as the functional relationship is maintained.

[0060] The term "expression vector" includes a vector comprising a polynucleotide region encoding a polypeptide of interest, and is used to achieve expression of the protein in a desired target cell. The expression vector may also include a control element operably linked to the coding region to promote expression of the protein in the target. Control elements and combinations of one or more genes operably linked thereto for expression are sometimes referred to as "expression cassettes," many of which are known and available in the art, or can be easily constructed from components available in the art.

[0061] The term "heterologous" means an entity that is derived from a genotypically different entity from the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide. A promoter removed from its native coding sequence and operably linked to a coding sequence not found linked in nature is a heterologous promoter. Thus, for example, an rAAV comprising a heterologous nucleic acid sequence encoding a heterologous gene product is an rAAV comprising a polynucleotide that is not normally included in a naturally occurring wild-type AAV, and the encoded heterologous gene product is a gene product that is not normally encoded by a naturally occurring wild-type AAV.

[0062] The terms "genetic alteration" and "genetic modification" (and grammatical variations thereof) are used interchangeably herein to refer to a process in which a genetic element (e.g., a polynucleotide) is introduced into a cell other than by mitosis or meiosis. The element may be heterologous to the cell, or it may be an additional copy or improved version of an element already present in the cell. Genetic alteration can be achieved, for example, by transfecting the cell with a recombinant plasmid or other polynucleotide by any method known in the art, such as electroporation, calcium phosphate precipitation, or contact with a polynucleotide-liposome complex. Genetic alteration can also be achieved, for example, by transduction or infection with a DNA or RNA virus or viral vector. Typically, genetic elements are introduced into a chromosome or mini-chromosome in a cell; however, any alteration that changes the phenotype and / or genotype of the cell and its progeny is included in the term.

[0063] With respect to cell modification, the term "genetically modified" or "transformed" or "transfected" or "transduced" by exogenous DNA (e.g., via a recombinant virus) refers to when such DNA has been introduced into a cell. The presence of exogenous DNA results in permanent or transient genetic changes. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. A "clone" is a population of cells derived from a single cell or a common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.

[0064] As used herein, a cell is said to be "stably" altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence can be used to perform its function during prolonged cell culture in vitro and / or for extended periods of time in vivo. Typically, such cells are "heritably" altered (genetically modified) because a genetic alteration has been introduced that is also heritable by progeny of the altered cell.

[0065] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The term also includes modified amino acid polymers; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation or conjugation with a labeling component. When discussed in the context of delivering gene products to a mammalian subject, polypeptides such as anti-angiogenic polypeptides, neuroprotective polypeptides, etc. and compositions thereof refer to respective intact polypeptides or any fragments or genetically engineered derivatives thereof that retain the desired biochemical function of the intact protein. Similarly, reference to nucleic acids encoding anti-angiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for delivering gene products to a mammalian subject (which may be referred to as "transgenes" to be delivered to recipient cells) includes polynucleotides encoding intact polypeptides or any fragments or genetically engineered derivatives having the desired biochemical function.

[0066] As used herein, an "isolated" plasmid, nucleic acid, vector, virus, virion, host cell, protein or other substance refers to a preparation of the substance that lacks at least some other components, which may also be present where the substance or a similar substance is naturally present or was originally prepared therefrom. Thus, for example, an isolated substance can be prepared by using purification techniques to enrich the isolated substance from a source mixture. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or can be measured relative to a second potentially interfering substance present in the source mixture. The gradually increasing enrichment of embodiments of the present disclosure is increasingly isolated. In some embodiments, the isolated plasmid, nucleic acid, vector, virus, host cell or other substance is purified, for example, about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99% pure or higher.

[0067] As used herein, the terms "treatment", "treating", and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or a symptom thereof, and / or may be therapeutic in terms of partially or completely curing a disease and / or a side effect attributable to a disease. As used herein, "treatment" includes any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the occurrence of a disease (and / or symptoms caused by a disease) in a subject who may be susceptible to the disease or at risk of the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease (and / or symptoms caused by the disease), i.e., inhibiting its development; and (c) alleviating the disease (and / or symptoms caused by the disease), i.e., causing regression of the disease (and / or symptoms caused by the disease), i.e., ameliorating the disease and / or one or more symptoms of the disease. For example, the subject compositions and methods may relate to the treatment of a lung disease.

[0068] The terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including but not limited to humans; non-human primates, including monkeys; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0069] In some embodiments, the individual is a human who has previously been naturally exposed to AAV and therefore carries anti-AAV antibodies (i.e., AAV neutralizing antibodies). In some embodiments, the individual is a human who has previously been administered an AAV vector (and therefore may carry anti-AAV antibodies) and requires re-administration of the vector for treatment of a different condition or further treatment of the same condition. There are many such therapeutic applications / disease targets based on positive results in clinical trials involving AAV gene delivery to, for example, liver, muscle, and retina - all tissues affected by neutralizing antibodies to the vector.

[0070] As used herein, the term "effective amount" is an amount sufficient to achieve a beneficial or desired clinical outcome. The effective amount can be administered in one or more administrations. For the purposes of this disclosure, an effective amount of a compound (e.g., an infectious rAAV virion) is an amount sufficient to alleviate, improve, stabilize, reverse, prevent, slow down or delay the progress of a specific disease state (e.g., lung disease) (and / or symptoms associated therewith). Therefore, an effective amount of infectious rAAV virions is the amount of infectious rAAV virions that can effectively deliver heterologous nucleic acids to individual target cells (or multiple target cells). The effective amount can be determined preclinically by, for example, detecting a gene product (RNA, protein) encoded by a heterologous nucleic acid sequence in a cell or tissue using techniques well known in the art such as RT-PCR, protein blotting, ELISA, fluorescence or other reporter molecule indication readings. The effective amount can be determined clinically by, for example, detecting changes in the onset or progression of a disease using methods known in the art.

[0071] The term "directed evolution" refers to an in vitro and / or in vivo method of artificially modifying the shell that mimics natural evolution through iterative rounds of genetic diversification and selection processes, thereby accumulating beneficial mutations that gradually improve the function of the biomolecule. Directed evolution often involves an in vivo method known as "biopanning," which is used to select AAV variants from a library that have more effective levels of infectivity in the cell or tissue type of interest.

[0072] The term "interfering RNA" includes both small interfering RNAs and microRNAs (miRNAs), including artificial miRNAs.

[0073] "2A peptide" refers to a "self-cleaving" peptide of about 20 amino acids that produces equimolar levels of multiple genes from the same mRNA and can be used in place of the IRES element in a polycistronic vector. Non-limiting examples include T2A, P2A, E2A, and F2A peptide sequences. In embodiments where the heterologous nucleic acid comprises a nucleotide sequence encoding multiple gene products, expression of the multiple (e.g., 2) gene products can be mediated by multiple (e.g., 2) independent promoters, or can be mediated by a single promoter, with the multiple transgenes separated by an internal ribosome entry site (IRES) or a 2A peptide sequence.

[0074] The so-called "increased resistance" refers to the increased infectivity of the subject infectious rAAV virus particles in the presence of human anti-AAV antibodies. Viral infectivity can be expressed as the ratio of infectious virus particles to total virus particles. Therefore, increased infectivity means the ratio of increased infectious virus particles to total virus particles. In order to determine the resistance of AAV to human anti-AAV antibodies, the infectivity of AAV is measured in the presence of various concentrations of human anti-AAV antibodies to obtain the antibody concentration (e.g., serum concentration, IVIG concentration, etc.) (mg / mL) required to reduce the gene delivery efficiency (i.e., infectivity) to 50% in the absence of human anti-AAV antibodies. It is said that a higher antibody concentration is required to reduce the gene delivery efficiency to 50% of the virus in the absence of human anti-AAV antibodies with increased resistance to antibody neutralization. Therefore, a two-fold increase in resistance means a two-fold increase in the antibody concentration required to reduce the gene delivery efficiency to 50% in the absence of human anti-AAV antibodies. In some embodiments, a subject infectious rAAV virion exhibits at least about 1.5-fold (e.g., at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold, at least about 17-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, etc.) resistance to human AAV neutralizing antibodies that is exhibited by wild-type AAV (e.g., AAV2 (wild-type AAV serotype 2)) or AAV comprising a wild-type capsid protein.

[0075] It can be said that in the presence of human AAV neutralizing antibodies, the subject infectious rAAV virions exhibit increased transduction of mammalian cells. In some embodiments, in the presence of human AAV neutralizing antibodies, a subject infectious rAAV virion exhibits at least about 1.5-fold (e.g., at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold, at least about 17-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, etc.) transduction of mammalian cells exhibited by wild-type AAV (e.g., AAV2 (wild-type AAV serotype 2)) or AAV comprising a wild-type capsid protein.

[0076] In some embodiments, a subject infectious rAAV virion exhibits reduced binding to neutralizing antibodies that bind to wild-type AAV capsid proteins. For example, a subject infectious rAAV virion can exhibit reduced binding (e.g., reduced affinity) to a neutralizing antibody that binds to a wild-type AAV capsid protein as compared to the antibody's binding affinity for the wild-type AAV capsid protein.

[0077] In some embodiments, the anti-AAV neutralizing antibody binds to the subject infectious rAAV virion at a rate of less than about 10 -7 M, less than about 5×10 -6 M, less than about 10 -6 M, less than about 5×10 -5 M, less than about 10 -5 M, less than about 10 -4 M or lower affinity binding.

[0078] Adeno-associated viruses (AAVs) are a family of parvoviruses with a 4.7 kb single-stranded DNA genome contained within a non-enveloped capsid. The viral genome of naturally occurring AAV has two inverted terminal repeats (ITRs) flanking two major open reading frames (ORFs): rep (encoding proteins that play a role in viral replication, transcriptional regulation, site-specific integration, and virion assembly) and cap - which function as an origin of viral replication and packaging signal. The cap ORF encodes three structural proteins that assemble to form a 60-mer viral capsid. Many naturally occurring AAV variants and serotypes have been isolated, and none have been associated with human disease.

[0079] Recombinant versions of AAV can be used as gene delivery vectors, in which a marker or therapeutic gene of interest is inserted between the ITRs in place of rep and cap. These vectors have been shown to transduce both dividing and non-dividing cells in vitro and in vivo, and can result in stable transgene expression in post-mitotic tissues for many years. See, eg, Knipe DM, Howley PM, Fields' Virology. Lippincott Williams & Wilkins, Philadelphia, Pa., USA, 2007; Gao GP, Alvira MR, Wang L, Calcedo R, Johnston J, Wilson S M. Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Nati AcadSci USA 2002;99:11854-9; Atchison RW, Casty BC, Hammon W M. Adenovirus-Associated Defective Virus Particles, Science 1965;149:754-6; Hoggan MD, Blacklow NR, Rowe W P. Studies of small DNA viruses found in various adenovirus preparations: physical, biological, and immunological characteristics, Proc NatlAcad USA 1966; 55:1467-74; Blacklow NR, Hoggan MD, Rowe WP. Isolation of adenovirus-associated viruses from man. Proc Natl Acad Sci USA 1967; 58:1410-5; Bantel-Schaal U, zur Hauser" H. Characterization of the DNA of a defective human parvovirus isolated from a genital site. Virology 1984;134:52-63;Mayor HD,Melnick J L.Small deoxyribonucleic acid-containingviruses(picodnavirus group).Nature 1966;210:331-2;Mori S,Wang L,Takeuchi T,Kanda T.Two novel adeno-associated viruses from cynomolgus monkey:pseudotyping characterization ofcapsidprotein.Virology 2004;330:375-83;FlotteT R.Gene therapy progress and prospects:recombinant adeno-associated virus(rAAV)vectors. Gene Ther 2004;11:805-10. .

[0080] Recombinant AAV (interchangeably referred to herein as "AAV" or "rAAV") has produced promising results in an increasing number of clinical trials. However, there are gene delivery barriers that may limit the effectiveness of AAV, such as anti-capsid immune responses, low transduction of certain tissues, inability to target delivery to specific cell types, and relatively low carrying capacity. In many cases, insufficient knowledge of mechanisms that enable the rational design of AAV to be effectively improved is required. As an alternative, directed evolution has become a strategy for generating new AAV variants that meet specific biomedical needs. The directed evolution strategy utilizes genetic diversification and selection processes to enable the accumulation of beneficial mutations that gradually improve the function of biomolecules. In the process, the wild-type AAV cap gene is diversified by several methods to produce a large gene library, which is packaged to generate a viral particle library, and then selection pressure is applied to isolate new variants that can overcome gene delivery barriers. Importantly, the directed evolution of function does not require knowledge of the mechanistic basis that underlies the gene delivery problem, which can therefore accelerate the development of enhanced vectors.

[0081] In general, the variants disclosed herein are generated by using an AAV library and / or multiple AAV libraries. Such an AAV library or multiple AAV libraries are generated by mutating the gene cop encoding the structural protein of the AAV capsid using a series of directed evolution techniques known and readily available to those skilled in the art of viral genome engineering. See, for example, Bartel et al. Am. Soc. Gene Cell Then. 15 thAnnu.Meet.20,5140(2012); Bowles, D. et al. J.Virol.77,423-432(2003); Gray et al. Mol.Ther.18,570-578(2010); Grimm, D. et al. J.Virol.82,5887-5911; Koerber, JT et al. Mol.Then.16,1703-1709(2008); Li W. et al. Mol.Ther.16,1252-1260(2008); Koerber, JT et al. Methods Mol.Biol,434,161-170(2008); Koerber, JT et al. Hum.Gene Then. 18, 367-378 (2007); and Koerber, JT et al. Mol. Ther. 17, 2088-2095 (2009). Such techniques are as follows, without limitation: i) error-prone PCR to introduce random point mutations into the AAV cap open reading frame (ORF) at a predetermined, modifiable rate; ii) in vitro or in vivo viral recombination or "DNA shuffling" to generate random chimeras of the AAV cap gene to generate a gene library with multiple AAV serotypes; iii) random peptide insertion at defined sites of the capsid by ligating degenerate oligonucleotides in the cap ORF; iv) defined insertion of peptide coding sequences into random positions of the AAV cap ORF using transposase mutagenesis; v) replacement of surface loops of the AAV capsid with a library of peptide sequences bioinformatically designed based on the conservation level of each amino acid position in natural AAV serotypes and variants to generate a "loop-swap" library; vi) random amino acid substitutions at degenerate positions between AAV serotypes to generate a library of ancestral variants (Santiago-Ortiz et al., 2015); and combinations of such techniques.

[0082] DNA shuffling produces chimeras that combine the properties of their parents in a unique and often beneficial way; however, some may not be packaged, which actually reduces the diversity of the library. Diversity enrichment of the library is achieved by peptide insertion techniques, such as, but not limited to, iii-iv above. The diversity of the library is also concentrated in techniques such as v) above, and this concentration is directed to multiple hypervariable regions located on the surface-exposed loops of the AAV capsid. Although many techniques produce variant capsids in which only small regions of the capsid are mutated, these techniques can be paired with additional mutagenesis strategies to modify the complete capsid.

[0083] Once an AAV library or multiple AAV libraries are generated, the virus is then packaged so that each AAV particle consists of a mutant capsid surrounding a cap gene encoding a mutant capsid, and purified. The variants of the library are then subjected to in vitro and / or in vivo selective pressure techniques known and readily available to those skilled in the AAV field. See, e.g., Maheshri, N. et al. Nature Biotech, 24, 198-204 (2006); Dalkara, D. et al. Sci. Tran. sl. Med. 5, 189ra76 (2013); Lisowski, L. et al. Nature. 506, 382-286 (2013); Yang, L. et al. 1PNAS, 106, 3946-3951 (2009); Gao, G. et al. Mol. Them. 13, 77-87 (2006); and Bell, P. et al. Hum. Gene. Ther. 22, 985-997 (2011). For example, but not by way of limitation, AAV variants may be selected using: i) affinity columns, where elution of different fractions produces variants with altered binding properties; ii) primary cells - isolated from tissue samples or immortalized cell lines that mimic the behavior of human cells - which produce AAV variants with improved efficiency and / or tissue specificity; iii) animal models - which mimic the clinical gene therapy environment - which produce AAV variants that have successfully infected target tissues; iv) human xenograft models, which produce AAV variants that have infected transplanted human cells; and / or combinations of selection techniques thereof.

[0084] Once the viruses are selected, they can be recovered by known techniques, such as, but not limited to, adenovirus-mediated replication, PCR amplification, next generation sequencing and cloning, etc. Viral clones are then enriched by repeated rounds of selection techniques, and AAV DNA is isolated to recover the selected variant cap gene of interest. Such selected variants can undergo further modification or mutation, and thus serve as a new starting point for further selection steps to iteratively increase AAV virus fitness, however, in some cases, successful capsids have been generated without additional mutations.

[0085] The AAV variants disclosed herein are generated at least in part by using an in vivo directed evolution method, such as the above-mentioned technique, which involves the use of primate lung screens after aerosol administration. Thus, the AAV variant capsids disclosed herein comprise one or more modifications in the amino acid sequence that confer more efficient transduction of primate lung cells than the corresponding parent AAV capsid protein. As used herein, "corresponding parent AAV capsid protein" refers to an AAV capsid protein of the same wild-type or variant AAV serotype as the subject variant AAV capsid protein, but does not comprise one or more amino acid sequence modifications of the subject variant AAV capsid protein.

[0086] In some embodiments, the subject variant AAV capsid protein comprises a heterologous peptide of about 5 amino acids to about 20 amino acids inserted into the GH loop or loop IV of the AAV capsid protein by covalent attachment relative to the corresponding parent AAV capsid protein. The so-called "GH loop" or loop IV of the AAV capsid protein refers to the solvent-accessible portion of the OH loop or loop IV of the AAV capsid protein in the art. For the OH loop / loop IV of the AAV capsid, see, e.g., van Viet et al., (2006) Mol. Ther. 14: 809; Padron et al. (2005) J. Virol, 79: 5047; and Shen et al., (2007) Mol. Ther. 15: 1955, so, for example, the insertion site can be within about amino acids 411-650 of the AAV VP1 capsid protein. For example, the insertion site can be within amino acids 571-612 of AAV1 VP1, within amino acids 570-611 of AAV2 VP1, within amino acids 571-612 of AAV3A VP1, within amino acids 571-612 of AAV3B VP1, within amino acids 569-610 of AAV4 VP1, within amino acids 560-601 of AAV5 VP1, within amino acids 571-612 of AAV6 VP1, within amino acids 572-613 of AAV7 VP1, within amino acids 573-614 of AAV8 VP1, within amino acids 571-612 of AAV9 VP1, or within amino acids 573-614 of AAV10 VP1, or the corresponding amino acids of any variants thereof. Based on a comparison of the amino acid sequences of the capsid proteins of various AAV serotypes, one skilled in the art will know where the insertion site of the "amino acid corresponding to AAV2" will be in the capsid protein of any given AAV serotype. For an alignment of wild-type AAV SEQ ID NOS: 1-11, see also U.S. Application Publication No. 2019 / 0255192. Figure 6, which provides amino acid positions between and across wild-type (naturally occurring) serotypes AAV1, AAV2, AAV3A, AAV3B, and AAV4-10, the entire contents of which are incorporated herein by reference.

[0087] In certain embodiments, the insertion site is a single insertion site between two adjacent amino acids between amino acids 570-614 of VP1 of any wild-type AAV serotype or AAV variant, e.g., the insertion site is between two adjacent amino acids located in amino acids 570-610, amino acids 580-600, amino acids 570-575, amino acids 575-580, amino acids 5809585, amino acids 585-590, amino acids 590-600, or amino acids 600-614 of VP1 of any AAV serotype or variant. For example, the insertion site can be between amino acids 580 and 581, between amino acids 581 and 582, between amino acids 583 and 584, between amino acids 584 and 585, between amino acids 585 and 586, between amino acids 586 and 587, between amino acids 587 and 588, between amino acids 588 and 589, or between amino acids 589 and 590. The insertion site can be between amino acids 575 and 576, between amino acids 576 and 577, between amino acids 577 and 578, between amino acids 578 and 579, or between amino acids 579 and 580. The insertion site can be between amino acids 590 and 591, between amino acids 591 and 592, between amino acids 592 and 593, between amino acids 593 and 594, between amino acids 594 and 595, between amino acids 595 and 596, between amino acids 596 and 597, between amino acids 597 and 598, between amino acids 598 and 599, or between amino acids 599 and 600. For example, the insertion site can be between amino acids 587 and 588 of AAV2, between amino acids 590 and 591 of AAV1, between amino acids 588 and 589 of AAV3A, between amino acids 588 and 589 of AAV3B, between amino acids 584 and 585 of AAV4, between amino acids 575 and 576 of AAV5, between amino acids 590 and 591 of AAV6, between amino acids 589 and 590 of AAV7, between amino acids 590 and 591 of AAV8, between amino acids 588 and 589 of AAV9, or between amino acids 588 and 589 of AAV10. In certain embodiments, the insertion site is between amino acids 587 and 588 of AAV2 or between amino acids 588 and 589 of AAV2.

[0088] In some embodiments, the peptide insertion disclosed herein has a length of 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids or 20 amino acids. In another embodiment, the peptide insertion disclosed herein comprises 1 to 4 spacer amino acids at the amino terminal (N-terminus) and / or carboxyl terminal (C-terminus) of any one of the peptide insertions disclosed herein. Exemplary spacer amino acids include, but are not limited to, leucine (L), alanine (A), glycine (G), serine (S), threonine (T) and proline (P). In certain embodiments, the peptide insertion comprises 2 spacer amino acids at the N-terminus and 2 spacer amino acids at the C-terminus. In other embodiments, the peptide insertion comprises 2 spacer amino acids at the N-terminus and 1 spacer amino acid at the C-terminus.

[0089] In some embodiments, the insertion peptide comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of: HDITKNI (SEQ ID NO: 12), NQDYTKT (SEQ ID NO: 13), DNTVTRS (SEQ ID NO: 14), SNSVQSI (SEQ ID NO: 15), NSTRHTD (SEQ ID NO: 16), TNRTSPD (SEQ ID NO: 17), ISDQTKH (SEQ ID NO: 18), QADTTKN (SEQ ID NO: 19), NAVKTDF (SEQ ID NO: 20), TNQTLSA (SEQ ID NO: 21), ENRTTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TNNSKPD (SEQ ID NO: 25), SKLTLNN (SEQ ID NO: 26), VTAGMGA (SEQ ID NO: 27), PNSTTNN (SEQ ID NO: 28), NSTSRID (SEQ ID NO: 29), NO:29), VASHTNN (SEQ ID NO:30), RSHQEIP (SEQ ID NO:31), LNTTKDI (SEQ ID NO:32), IIDATKN (SEQ ID NO:33), NHISQTN (SEQ ID NO:34), SNSAHIT (SEQ ID NO:35), STHQSNN (SEQ ID NO:36), KTPNLTS (SEQ ID NO:37), SNTPALS (SEQ ID NO:38), SPGATTN (SEQ ID NO:39).

[0090] In other preferred embodiments, the insertion peptide comprises, consists essentially of, or consists of 1 to 3 spacer amino acids (Y1-Y3) at the amino and / or carboxyl terminus of an amino acid sequence selected from the group consisting of: HDITKNI (SEQ ID NO: 12), NQDYTKT (SEQ ID NO: 13), DNTVTRS (SEQ ID NO: 14), SNSVQSI (SEQ ID NO: 15), NSTRHTD (SEQ ID NO: 16), TNRTSPD (SEQ ID NO: 17), ISDQTKH (SEQ ID NO: 18), QADTTKN (SEQ ID NO: 19), NAVKTDF (SEQ ID NO: 20), TNQTLSA (SEQ ID NO: 21), ENRTTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TNNSKPD (SEQ ID NO: 25), SKLTLNN (SEQ ID NO: 26), VTAGMGA (SEQ ID NO: 27), NO:27), PNSTTNN (SEQ ID NO:28), NTSSRID (SEQ ID NO:29), VASHTNN (SEQ ID NO:30), RSHQEIP (SEQ ID NO:31), LNTTKDI (SEQ ID NO:32), IIDATKN (SEQ ID NO:33), NHISQTN (SEQ ID NO:34), SNSAHIT (SEQ ID NO:35), STHQSNN (SEQ ID NO:36), KTPNLTS (SEQ ID NO:37), SNTPALS (SEQ ID NO:38), SPGATTN (SEQ ID NO:39).The manufacturers of LAHDITKNIA(SEQ ID NO:40), LANQDYTKTA(SEQ ID NO:41), LADNTVTRSA(SEQ IDNO:42), and LASNSVQSIA(SEQ ID). NO:43、LANSTRHTDA(SEQ ID NO:44)、LATNRTSPDA(SEQ IDNO:45)、LAISDQTKHA(SEQ ID NO:46)、LAQADTTKNA(SEQ ID NO:47)、LANAVKTDFA(SEQ IDNO:48)、LATNQTLSAA(SEQ ID NO:48). NO:49)、LAENRTTSNA(SEQ ID NO:50)、LAPQQDTTHA(SEQ IDNO:51)、LANATNHVIA(SEQ ID NO:52)、LATNNSKPDA(SEQ ID NO:53)、LASKLTLNNA(SEQ IDNO:54)、LAVTAGMGAA(SEQ ID NO:54). NO:55)、LAPNSTTNNA(SEQ ID NO:56)、LANSTSRIDA(SEQ ID NO:57)、LAVASHTNNA(SEQ ID NO:58)、LARSHQEIPA(SEQ ID NO:59)、LALNTTKDIA(SEQ ID NO:60)、LAIIDATKNA(SEQ ID NO:60). NO:61, LANHISQTNA (SEQ ID NO:62), LASNSAHITA (SEQ ID NO:63), LASTHQSNNA (SEQ ID NO:64), LAKTPNLTSA (SEQ ID NO:65), LASNTPALSA (SEQ ID NO:66), and LASPGATTNA (SEQ ID NO:67).

[0091] In some embodiments, a subject variant AAV capsid protein does not comprise any other amino acid sequence modifications other than a peptide insertion of about 5 amino acids to about 20 amino acids in the GH loop or loop IV. For example, in some embodiments, the subject variant AAV capsid protein comprises, consists essentially of, or consists of a peptide insertion comprising an amino acid sequence selected from the group consisting of: HDITKNI (SEQ ID NO: 12), NQDYTKT (SEQ ID NO: 13), DNTVTRS (SEQ ID NO: 14), SNSVQSI (SEQ ID NO: 15), NSTRHTD (SEQ ID NO: 16), TNRTSPD (SEQ ID NO: 17), ISDQTKH (SEQ ID NO: 18), QADTTKN (SEQ ID NO: 19), NAVKTDF (SEQ ID NO: 20), TNQTLSA (SEQ ID NO: 21), ENRTTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TNNSKPD (SEQ ID NO: 25), SKLTLNN (SEQ ID NO: 26), VTAGMGA (SEQ ID NO: 27), PNSTTNN (SEQ ID NO: 28), NO:28), NTSSRID (SEQ ID NO:29), VASHTNN (SEQ ID NO:30), RSHQEIP (SEQ ID NO:31), LNTTKDI (SEQ ID NO:32), IIDATKN (SEQ ID NO:33), NHISQTN (SEQ ID NO:34), SNSAHIT (SEQ ID NO:35), STHQSNN (SEQID NO:36), KTPNLTS (SEQ ID NO:37), SNTPALS (SEQ ID NO:38), SPGATTN (SEQ ID NO:39), LAHDITKNIA (SEQ ID NO:40), LANQDYTKTA (SEQ ID NO:41), LADNTWTRSA (SEQ ID NO:42), LASNSVQSIA (SEQ ID NO:43), LANSTRHTDA (SEQ ID NO:44), LATNRTSPDA (SEQ ID NO:45), LAISDQTKHA (SEQ ID NO:46), LAQADTTKNA (SEQ ID NO:47), LANAVKTDFA (SEQ ID NO:48), LATNQTLSAA (SEQ ID NO:49), LAENRTTSNA (SEQ IDNO:50), LAPQQDTTHA (SEQ ID NO:51), LANATNHVIA (SEQ ID NO:52), LATNNSKPDA (SEQ ID NO:53), LASKLTLNNA (SEQ ID NO:54), LAVTAGMGAA (SEQ ID NO:55), LAPNSTTNNA (SEQ ID NO:56), LANSTSRIDA (SEQ ID NO:57), LAVASHTNNA (SEQ ID NO:58), LARSHQEIPA (SEQ ID NO:59), LALNTTKDIA (SEQ ID NO:60), LAIIDATKNA (SEQ ID NO:61), LANHISQTNA (SEQ ID NO:62), LASNSAHITA (SEQ ID NO:63), LASTHQSNNA (SEQ ID NO:64), LAKTPNLTSA (SEQ ID NO:65), LASNTPALSA (SEQ ID NO:66) and LASPGATTNA (SEQ ID NO:67), and the variant AAV capsid does not contain any other amino acid substitutions, insertions or deletions (i.e., the variant AAV capsid protein contains the insertion and is otherwise identical to the corresponding AAV capsid protein). In other words, the variant AAV capsid protein containing the insertion is otherwise identical to the parent AAV capsid protein into which the peptide has been inserted. As another example, a subject variant AAV capsid protein comprises, consists essentially of, or consists of a peptide insertion having an amino acid sequence selected from the group consisting of: HDITKNI (SEQ ID NO: 12), NQDYTKT (SEQ ID NO: 13), DNTVTRS (SEQ ID NO: 14), SNSVQSI (SEQ ID NO: 15), NSTRHTD (SEQ ID NO: 16), TNRTSPD (SEQ ID NO: 17), ISDQTKH (SEQ ID NO: 18), QADTTKN (SEQ ID NO: 19), NAVKTDF (SEQ ID NO: 20), TNQTLSA (SEQ ID NO: 21), ENRTTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TNNSKPD (SEQ ID NO: 25), SKLTLNN (SEQ ID NO: 26), VTAGMGA (SEQ ID NO: 27), PNSTTNN (SEQ ID NO: 28), NO:28)、NSTSSRID(SEQ IDNO:29), VASHTNN (SEQ ID NO:30), RSHQEIP (SEQ ID NO:31), LNTTKDI (SEQ ID NO:32), IIDATKN (SEQ ID NO:33), NHISQTN (SEQ ID NO:34), SNSAHIT (SEQ ID NO:35), STHQSNN (SEQ ID NO:36), KTPNLTS (SEQ ID NO:37), SNTPALS (SEQ ID NO:38), SPGATTN (SEQ ID NO:39), LAHDITKNIA (SEQ ID NO:40), LANQDYTKTA (SEQ ID NO:41), LADNTVTRSA (SEQ ID NO:42), LASNSVQSIA (SEQ ID NO:43), LANSTRHTDA (SEQ ID NO:44), LATNRTSPDA (SEQ ID NO:45), LAISDQTKHA (SEQ ID NO:46), LAQADTTKNA (SEQ ID NO:47), LANAVKTDFA (SEQ ID NO:48), LATNQTLSAA (SEQ ID NO:49), LAENRTTSNA (SEQ ID NO:50), LAPQQDTTHA (SEQ ID NO:51), LANATNHVIA (SEQ ID NO:52), LATNNSKPDA (SEQ ID NO:53), LASKLTLNNA (SEQ ID NO:54), LAVTAGMGAA (SEQ ID NO:55), LAPNSTTNNA (SEQ ID NO:56), LANSTSRIDA (SEQ ID NO:57), LAVASHTNNA (SEQ ID NO:58), LARSHQEIPA (SEQ ID NO:59), LALNTTKDIA (SEQ ID NO:60), LAIIDATKNA (SEQ ID NO:61), LANHISQTNA (SEQ ID NO:62), LASNSAHITA (SEQ ID NO:63), LASTHQSNNA (SEQ ID NO:64), LAKTPNLTSA (SEQ ID NO:65), LASNTPALSA (SEQ ID NO:66) and LASPGATTNA (SEQ IDNO:67), wherein the peptide insertion is located between amino acids 587 and 588 of VP1 of AAV2 capsid or the corresponding amino acids of VP1 of another parent AAV, such as between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6 or AAV9, between amino acids 586 and 587 of VP1 of AAV4, between amino acids 577 and 578 of VP1 of AAV5, between amino acids 589 and 590 of VP1 of AAV7, between amino acids 590 to 591 of VP1 of AAV8 or AAV10, etc., wherein the variant AAV capsid protein sequence is otherwise identical to the corresponding parent AAV capsid protein sequence, such as any one of SEQ ID NOs:1-12.

[0092] In other embodiments, in addition to comprising a peptide insertion in the GH loop, e.g., as disclosed herein or as known in the art, the subject variant AAV capsid protein comprises from about 1 to about 100 amino acid substitutions or deletions, e.g., 1 to about 5, from about 2 to about 4, from about 2 to about 5, from about 5 to about 10, from about 10 to about 15, from about 15 to about 20, from about 20 to about 25, from about 25 to about 50, from about 50 to about 100 amino acid substitutions or deletions compared to the parent AAV capsid protein. Thus, in some embodiments, the subject variant capsid protein comprises an amino acid sequence having a sequence identity of 85% or more, 90% or more, 95% or more, or 98% or more, e.g., or 99% or more, to the corresponding parent AAV capsid, e.g., the wild-type capsid protein set forth in SEQ ID NOs: 1-12. In some embodiments, a subject variant capsid protein comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence of an AAV2 capsid protein (SEQ ID NO: 2).

[0093] In a further embodiment, the one or more amino acid substitutions are at amino acid residues 1, 6, 15, 16, 18, 30, 34, 37, 38, 57, 65, 66, 81, 91, 99, 101, 103, 109, 118, 120, 133, 134, 135, 136, 137, 138, 144, 164, 176, 188, 196, 200, 213, 220, 226, 236, 247, 250, 261, 269, 370, 381, 391, 402, 413, 424, 430, 449, 451, 462, 471, 480, 491, 503, 514, 525, 536, 540, 551 40, 250, 283, 312, 344, 347, 363, 368, 371, 376, 399, 428, 436, 449, 451, 456, 463, 469, 472, 484, 491, 524, 532, 535, 551, 585, 591, 593, 594, 608, 641, 688, 698, 705, 708, 719, 721, and / or 735, or one or more corresponding amino acid residues of another AAV capsid protein. In some such embodiments, the one or more amino acid substitutions are selected from AAV2 as numbered prior to peptide insertion. VP1 capsid protein Y6F, S16Y, G18E, P30L, R37L, H38Q, V65A, L91I, E99D, R103L, R103C, S109T, V118A, Q120H, E133D, E134Q, P135A, V136G, K137E, T138R, T200I, D213Y, G220R, P250S, D283E, N312K, T344 S, E347D, G376A, P399H, G406E, Q428H, P436H, N449D, P451Q, N469D, D472N, T491I, K532E, K544E, R585K, A591D, A593E, D594N, D608N, H641N, K688R, N705S, and V708I, or one or more corresponding amino acid residues of another AAV capsid protein. In other such embodiments, the one or more amino acid substitutions are selected from M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, 1456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V708I, V719M, S721L, and L735Q of the AAV2 VP1 capsid protein as numbered prior to peptide insertion, or one or more corresponding amino acid residues of another AAV capsid protein.In some preferred embodiments, the subject variant capsid protein comprises an amino acid substitution at amino acid residue 708 of the AAV2 VP1 capsid protein (SEQ ID NO: 2). In particularly preferred embodiments, the subject variant capsid protein comprises a V708I amino acid substitution relative to the AAV2 VP1 capsid protein (SEQ ID NO: 2), and optionally further comprises one or more amino acid substitutions described herein.

[0094] In some embodiments, the subject variant AAV capsid protein confers rAAV increased infectivity to one or more lung cell types, and also confers rAAV increased resistance to human AAV neutralizing antibodies compared to the resistance exhibited by AAV2 (wild-type AAV serotype 2). In some cases, rAAV exhibits increased resistance to human AAV neutralizing antibodies compared to the resistance exhibited by AAV2 (wild-type AAV serotype 2). In some cases, the resistance to human AAV neutralizing antibodies exhibited by rAAV is at least about 1.5-fold (e.g., at least about 3-fold, at least about 5-fold, at least about -10-fold, at least about -30-fold, etc.) of the resistance exhibited by AAV2. In some cases, rAAV exhibits increased transduction of one or more mammalian lung cell types in the presence of human AAV neutralizing antibodies compared to mammalian cell transduction exhibited by wild-type AAV serotype 2 (AAV2).

[0095] In a preferred embodiment, a variant AAV capsid protein is provided, which comprises a) a peptide insertion in the GH-loop of the capsid protein, wherein the peptide insertion comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of: HDITKNI (SEQ ID NO: 12), NHDITKNI (SEQ ID NO: 12), NQDYTKT (SEQ ID NO: 13), DNTVTRS (SEQ ID NO: 14), SNSVQSI (SEQ ID NO: 15), NSTRHTD (SEQ ID NO: 16), TNRTSPD (SEQ ID NO: 17), ISDQTKH (SEQ ID NO: 18), QADTTKN (SEQ ID NO: 19), NAVKTDF (SEQ ID NO: 20), TNQTLSA (SEQ ID NO: 21), ENRTTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TNNSKPD (SEQ ID NO: 25), SKLTLNN (SEQ ID NO: 26), TNQTLSA (SEQ ID NO: 27), ENRTTSN (SEQ ID NO: 28), PQQDTTH (SEQ ID NO: 29), NATNHVI (SEQ ID NO: 30), TNNSKPD (SEQ ID NO: 31), SKLTLNN (SEQ ID NO: 32), NO:26), VTAGMGA (SEQ ID NO:27), PNSTTNN (SEQ ID NO:28), NTSSRID (SEQ ID NO:29), VASHTNN (SEQ ID NO:30), RSHQEIP (SEQ ID NO:31), LNTTKDI (SEQ ID NO:32), IIDATKN (SEQ ID NO:33), NHISQTN (SEQ ID NO:34), SNSAHIT (SEQ ID NO:35), STHQSNN (SEQ ID NO:36), KTPNLTS (SEQ ID NO:37), SNTPALS (SEQ ID NO:38), SPGATTN (SEQ ID NO:39), LAHDITKNIA (SEQ ID NO:40), LANQDYTKTA (SEQ ID NO:41), LADNTVTRSA (SEQ ID NO:42), LASNSVQSIA (SEQ ID NO:43), LANSTRHTDA (SEQ ID NO:44), LATNRTSPDA (SEQ ID NO:45), LAISDQTKHA (SEQ ID NO:46), LAQADTTKNA (SEQ ID NO:47), LANAVKTDFA (SEQ ID NO:48), LATNQTLSAA (SEQ ID NO:49), LAENRTTSNA (SEQ ID NO:50), LAPQQDTTHA (SEQNO:54), LAVTAGMGAA (SEQ ID NO:55), LAPNSTTNNA (SEQ ID NO:56), LANSTSRIDA (SEQ ID NO:57), LAVASHTNNA (SEQ ID NO:58), LARSHQEIPA (SEQ ID NO:59), LALNTTKDIA (SEQ ID NO:60), LAIIDATKNA (SEQ ID NO:61), LANHISQTNA (SEQ ID NO:62), LASNSAHITA (SEQ ID NO:63), LASTHQSNNA (SEQ ID NO:64), LAKTPNLTSA (SEQ ID NO:65), LASNTPALSA (SEQ ID NO:66), and LASPGATTNA (SEQ ID NO:67), and b) the amino acid sequence of AAV2 (SEQ ID NO:68). NO:2) compared to one or more of the following amino acid substitutions or a corresponding substitution in another AAV parent serotype (i.e., other than AAV2), wherein one or more of the substituted amino acids does not naturally occur at the corresponding position: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L 88I, S196Y, G226E, G236V, I240I, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524I, P535S, N551S, A593E, I698V, V708I, V719M, S721L, L735Q, and combinations thereof. In a preferred embodiment, the one or more amino acid substitutions include a V708I substitution. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or a corresponding position in the capsid protein of another AAV serotype.

[0096] In particularly preferred embodiments, the variant AAV capsid comprises, consists essentially of, or consists of a peptide insertion between amino acids 587 and 588 of VP1 of AAV2, or the corresponding amino acids of another AAV capsid, the peptide insertion comprising, consisting essentially of, or consisting of the amino acid sequence HDITKNI (SEQ ID NO: 12), or comprising, consisting essentially of, or consisting of the amino acid sequence LAHDITKNIA (SEQ ID NO: 40), and further comprises a V708I amino acid substitution at residue 708 relative to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2), or the corresponding residue of another AAV capsid. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or more amino acid sequence identity to the entire length of the amino acid sequence set forth in SEQ ID NO: 2, or the corresponding parent AAV capsid. In particularly preferred embodiments, the variant AAV capsid has an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 98% identical, at least about 99% identical, or 100% identical to the following amino acid sequence (AAV102): In another embodiment, a variant AAV capsid protein is provided that comprises, consists essentially of, or consists of: a) a peptide insertion between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6, or AAV9, amino acids 586 and 587 of AAV4, amino acids 577 and 578 of AAV5, amino acids 589 and 590 of AAV7, or amino acids 590 to 591 of AAV8 or AAV10, the peptide insertion comprising LAHDITKNIA (SEQ ID NO: 40) and HDITKNI (SEQ ID NO: 51). ID NO: 12), and b) a valine to isoleucine substitution at amino acid 709 of AAV3A or AAV3B, an alanine to isoleucine substitution at position 709 of AAV1 or AAV6, an asparagine to isoleucine substitution at amino acid 707 of AAV4 or amino acid 709 of AAV9, or a threonine to isoleucine substitution at amino acid 710 of AAV7 or amino acid 711 of AAV8 or AAV10, or a glutamine to isoleucine substitution at amino acid 697 of AAV5, and optionally otherwise identical to any one of SEQ ID NOs: 1 and 3-12. In preferred embodiments, the variant capsid protein comprises a) a peptide insertion comprising an amino acid sequence comprising, consisting of, or consisting essentially of HDITKNI (SEQ ID NO: 12) between amino acids 587 and 588 of the AAV2 capsid, comprising, consisting of, or consisting essentially of the amino acid sequence LAHDITKNIA (SEQ ID NO: 40), and b) an amino acid substitution of valine to isoleucine at amino acid 708 compared to the amino acid sequence of AAV2, wherein the variant capsid protein comprises 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions.

[0097] In yet another embodiment, the variant capsid protein comprises a) a peptide insertion comprising, consisting of, or consisting essentially of the amino acid sequence HDITKNI (SEQ ID NO: 12) or comprising, consisting essentially of, or consisting of the amino acid sequence LAHDITKNIA (SEQ ID NO: 40) between amino acids 587 and 588 of the AAV2 capsid and is otherwise identical to the amino acid sequence of SEQ ID NO: 2.

[0098] In particularly preferred embodiments, the variant AAV capsid comprises a peptide insertion between amino acids 587 and 588 of VP1 of AAV2, or the corresponding amino acids of another AAV capsid, comprising, consisting of, or consisting essentially of the amino acid sequence NQDYTKT (SEQ ID NO: 13), or comprising, consisting essentially of, or consisting of the amino acid sequence LANQDYTKTA (SEQ ID NO: 41), and further comprising a V708I amino acid substitution at residue 708 relative to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2), or the corresponding residue of another AAV capsid. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or more amino acid sequence identity to the entire length of the amino acid sequence set forth in SEQ ID NO: 2, or the corresponding parent AAV capsid. In particularly preferred embodiments, the variant AAV capsid has an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 98% identical, at least about 99% identical, or 100% identical to the following amino acid sequence (AAV103): In another embodiment, a variant AAV capsid protein is provided, comprising: a) a peptide insertion between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6, or AAV9, amino acids 586 and 587 of AAV4, amino acids 577 and 578 of AAV5, amino acids 589 and 590 of AAV7, or amino acids 590 to 591 of AAV8 or AAV10, the peptide insertion comprising LANQDYTKTA (SEQ ID NO: 41) and NQDYTKT (SEQ ID NO: 42). NO:13), and b) a valine to isoleucine substitution at amino acid 709 of AAV3A or AAV3B, an alanine to isoleucine substitution at position 709 of AAV1 or AAV6, an asparagine to isoleucine substitution at amino acid 707 of AAV4 or amino acid 709 of AAV9, or a threonine to isoleucine substitution at amino acid 710 of AAV7 or amino acid 711 of AAV8 or AAV10, or a glutamine to isoleucine substitution at amino acid 697 of AAV5, and optionally otherwise identical to any one of SEQ ID NOs: 1 and 3-12. In preferred embodiments, the variant capsid protein comprises a) a peptide insertion between amino acids 587 and 588 of the AAV2 capsid, the peptide insertion comprising the amino acid sequence, or comprising, consisting of, or consisting essentially of NQDYTKT (SEQ ID NO: 13), or comprising, consisting of, or consisting essentially of the amino acid sequence LANQDYTKTA (SEQ ID NO: 41), and b) an amino acid substitution of valine to isoleucine at amino acid 708 compared to the amino acid sequence of AAV2, wherein the variant capsid protein comprises 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions.

[0099] In yet another embodiment, the variant capsid protein comprises a) a peptide insertion comprising, consisting essentially of, or consisting of the amino acid sequence NQDYTKT (SEQ ID NO: 13) or comprising, consisting of, or consisting of the amino acid sequence LANQDYTKTA (SEQ ID NO: 41) between amino acids 587 and 588 of the AAV2 capsid and is otherwise identical to the amino acid sequence of SEQ ID NO: 2.

[0100] In a particularly preferred embodiment, the variant AAV capsid comprises a peptide insertion between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid, the peptide insertion comprising, consisting of, or consisting essentially of the amino acid sequence DNTVTRS (SEQ ID NO: 14), or comprising, consisting essentially of, or consisting of the amino acid sequence LADNTVTRSA (SEQ ID NO: 42), and further comprising a V708I amino acid substitution at residue 708 relative to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2) or the corresponding residue of another AAV capsid. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or more amino acid sequence identity to the entire length of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding parent AAV capsid. In particularly preferred embodiments, the variant AAV capsid has an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 98% identical, at least about 99% identical, or 100% identical to the following amino acid sequence (AAV104): In another embodiment, a variant AAV capsid protein is provided, comprising: a) a peptide insertion between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6, or AAV9, amino acids 586 and 587 of AAV4, amino acids 577 and 578 of AAV5, amino acids 589 and 590 of AAV7, or amino acids 590 to 591 of AAV8 or AAV10, the peptide insertion comprising LADNTVTRSA (SEQ ID NO: 42) and DNTVTRS (SEQ ID NO: 43). NO:14), and b) a valine to isoleucine substitution at amino acid 709 of AAV3A or AAV3B, an alanine to isoleucine substitution at position 709 of AAV1 or AAV6, an asparagine to isoleucine substitution at amino acid 707 of AAV4 or amino acid 709 of AAV9, or a threonine to isoleucine substitution at amino acid 710 of AAV7 or amino acid 711 of AAV8 or AAV10, or a glutamine to isoleucine substitution at amino acid 697 of AAV5, and optionally otherwise identical to any one of SEQ ID NOs: 1 and 3-12. In preferred embodiments, the variant capsid protein comprises a) a peptide insertion between amino acids 587 and 588 of the AAV2 capsid, the peptide insertion comprising, or comprising, consisting of, or consisting essentially of the amino acid sequence DNTVTRS (SEQ ID NO: 14), comprising, consisting of, or consisting essentially of the amino acid sequence LADNTVTRSA (SEQ ID NO: 42), and b) an amino acid substitution of valine to isoleucine at amino acid 708 compared to the amino acid sequence of AAV2, wherein the variant capsid protein comprises 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions.

[0101] In yet another embodiment, the variant capsid protein comprises a) a peptide insertion comprising, consisting of, or consisting essentially of the amino acid sequence DNTVTRS (SEQ ID NO: 14) or comprising, consisting essentially of, or consisting of the amino acid sequence LADNTVTRSA (SEQ ID NO:42) between amino acids 587 and 588 of the AAV2 capsid and is otherwise identical to the amino acid sequence of SEQ ID NO:2.

[0102] In particularly preferred embodiments, the variant AAV capsid comprises a peptide insertion between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid, the peptide insertion comprising, consisting of, or consisting essentially of the amino acid sequence SNSVQSI (SEQ ID NO: 15), or comprising, consisting essentially of, or consisting of the amino acid sequence LASNSVQSIA (SEQ ID NO: 43), and further comprising a V708I amino acid substitution at residue 708 relative to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2) or the corresponding residue of another AAV capsid. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or more amino acid sequence identity to the entire length of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding parent AAV capsid. In particularly preferred embodiments, the variant AAV capsid has an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 98% identical, at least about 99% identical, or 100% identical to the following amino acid sequence (AAV105): In another embodiment, a variant AAV capsid protein is provided, comprising: a) a peptide insertion between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6, or AAV9, amino acids 586 and 587 of AAV4, amino acids 577 and 578 of AAV5, amino acids 589 and 590 of AAV7, or amino acids 590 to 591 of AAV8 or AAV10, the peptide insertion comprising LASNSVQSIA (SEQ ID NO: 43) and SNSVQSI (SEQ ID NO: 44). NO:15), and b) a valine to isoleucine substitution at amino acid 709 of AAV3A or AAV3B, an alanine to isoleucine substitution at position 709 of AAV1 or AAV6, an asparagine to isoleucine substitution at amino acid 707 of AAV4 or amino acid 709 of AAV9, or a threonine to isoleucine substitution at amino acid 710 of AAV7 or amino acid 711 of AAV8 or AAV10, or a glutamine to isoleucine substitution at amino acid 697 of AAV5, and optionally otherwise identical to any one of SEQ ID NOs: 1 and 3-12. In preferred embodiments, the variant capsid protein comprises a) a peptide insertion between amino acids 587 and 588 of the AAV2 capsid, the peptide insertion comprising the amino acid sequence, or comprising, consisting of, or consisting essentially of SNSVQSI (SEQ ID NO: 15), or comprising, consisting of, or consisting essentially of the amino acid sequence LASNSVQSIA (SEQ ID NO: 43), and b) an amino acid substitution of valine to isoleucine at amino acid 708 compared to the amino acid sequence of AAV2, wherein the variant capsid protein comprises 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions.

[0103] In yet another embodiment, the variant capsid protein comprises a) a peptide insertion comprising, consisting of, or consisting essentially of the amino acid sequence SNSVQSI (SEQ ID NO: 15) or comprising, consisting essentially of, or consisting of the amino acid sequence LASNSVQSIA (SEQ ID NO: 43) between amino acids 587 and 588 of the AAV2 capsid, and is otherwise identical to the amino acid sequence of SEQ ID NO: 2.

[0104] In particularly preferred embodiments, the variant AAV capsid comprises a peptide insertion between amino acids 587 and 588 of VP1 of AAV2, or the corresponding amino acids of another AAV capsid, comprising, consisting of, or consisting essentially of the amino acid sequence NSTRHTD (SEQ ID NO: 16), or comprising, consisting essentially of, or consisting of the amino acid sequence LANSTRHTDA (SEQ ID NO: 44), and further comprising a V708I amino acid substitution at residue 708 relative to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2), or the corresponding residue of another AAV capsid. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or more amino acid sequence identity to the entire length of the amino acid sequence set forth in SEQ ID NO: 2, or the corresponding parent AAV capsid. In particularly preferred embodiments, the variant AAV capsid has an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 98% identical, at least about 99% identical, or 100% identical to the following amino acid sequence (AAV106): In another embodiment, a variant AAV capsid protein is provided, comprising: a) a peptide insertion between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6, or AAV9, amino acids 586 and 587 of AAV4, amino acids 577 and 578 of AAV5, amino acids 589 and 590 of AAV7, or amino acids 590 to 591 of AAV8 or AAV10, the peptide insertion comprising LANSTRHTDA (SEQ ID NO: 44) and NSTRHTD (SEQ ID NO: 45). NO:16), and b) a valine to isoleucine substitution at amino acid 709 of AAV3A or AAV3B, an alanine to isoleucine substitution at position 709 of AAV1 or AAV6, an asparagine to isoleucine substitution at amino acid 707 of AAV4 or amino acid 709 of AAV9, or a threonine to isoleucine substitution at amino acid 710 of AAV7 or amino acid 711 of AAV8 or AAV10, or a glutamine to isoleucine substitution at amino acid 697 of AAV5, and optionally otherwise identical to any one of SEQ ID NOs: 1 and 3-12. In preferred embodiments, the variant capsid protein comprises a) a peptide insertion between amino acids 587 and 588 of the AAV2 capsid, the peptide insertion comprising the amino acid sequence, or comprising, consisting of, or consisting essentially of NSTRHTD (SEQ ID NO: 16), or comprising, consisting of, or consisting essentially of the amino acid sequence LANSTRHTDA (SEQ ID NO: 44), and b) an amino acid substitution of valine to isoleucine at amino acid 708 compared to the amino acid sequence of AAV2, wherein the variant capsid protein comprises 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions.

[0105] In yet another embodiment, the variant capsid protein comprises a) a peptide insertion comprising, consisting of, or consisting essentially of the amino acid sequence NSTRHTD (SEQ ID NO: 16) or comprising, consisting essentially of, or consisting of the amino acid sequence LANSTRHTDA (SEQ ID NO:44) between amino acids 587 and 588 of the AAV2 capsid, and is otherwise identical to the amino acid sequence of SEQ ID NO:2.

[0106] In particularly preferred embodiments, the variant AAV capsid comprises a peptide insertion between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid, the peptide insertion comprising, consisting of, or consisting essentially of the amino acid sequence TNRTSPD (SEQ ID NO: 17), or comprising, consisting essentially of, or consisting of the amino acid sequence LATNRTSPDA (SEQ ID NO: 45), and further comprising a V708I amino acid substitution at residue 708 relative to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2) or the corresponding residue of another AAV capsid. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or more amino acid sequence identity to the entire length of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding parent AAV capsid. In particularly preferred embodiments, the variant AAV capsid has an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, at least about 99% sequence identity, or 100% identical to the following amino acid sequence (AAV107): In another embodiment, a variant AAV capsid protein is provided, comprising: a) a peptide insertion between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6, or AAV9, amino acids 586 and 587 of AAV4, amino acids 577 and 578 of AAV5, amino acids 589 and 590 of AAV7, or amino acids 590 to 591 of AAV8 or AAV10, the peptide insertion comprising LATNRTSPDA (SEQ ID NO: 45) and TNRTSPD (SEQ ID NO: 59). NO:17), and b) a valine to isoleucine substitution at amino acid 709 of AAV3A or AAV3B, an alanine to isoleucine substitution at position 709 of AAV1 or AAV6, an asparagine to isoleucine substitution at amino acid 707 of AAV4 or amino acid 709 of AAV9, or a threonine to isoleucine substitution at amino acid 710 of AAV7 or amino acid 711 of AAV8 or AAV10, or a glutamine to isoleucine substitution at amino acid 697 of AAV5, and optionally otherwise identical to any one of SEQ ID NOs: 1 and 3-12. In preferred embodiments, the variant capsid protein comprises a) a peptide insertion between amino acids 587 and 588 of the AAV2 capsid, the peptide insertion comprising the amino acid sequence, or comprising, consisting of, or consisting essentially of TNRTSPD (SEQ ID NO: 17), or comprising, consisting of, or consisting essentially of the amino acid sequence LATNRTSPDA (SEQ ID NO: 45), and b) an amino acid substitution of valine to isoleucine at amino acid 708 compared to the amino acid sequence of AAV2, wherein the variant capsid protein comprises 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions.

[0107] In yet another embodiment, the variant capsid protein comprises a) a peptide insertion comprising, consisting of, or consisting essentially of the amino acid sequence TNRTSPD (SEQ ID NO: 17) or comprising, consisting essentially of, or consisting of the amino acid sequence LATNRTSPDA (SEQ ID NO:45) between amino acids 587 and 588 of the AAV2 capsid and is otherwise identical to the amino acid sequence of SEQ ID NO:2.

[0108] In other embodiments, the variant AAV capsid comprises, consists of, or consists essentially of a peptide insertion between amino acids 587 and 588 of VP1 of AAV2, or the corresponding amino acids of another AAV capsid, the peptide insertion comprising an amino acid sequence selected from the group consisting of ISDQTKH (SEQ ID NO: 18), QADTTKN (SEQ ID NO: 19), NAVKTDF (SEQ ID NO: 20), TNQTLSA (SEQ ID NO: 21), ENRTTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TNNSKPD (SEQ ID NO: 25), SKLTLNN (SEQ ID NO: 26), VTAGMGA (SEQ ID NO: 27), PNSTTNN (SEQ ID NO: 28), NSTSRID (SEQ ID NO: 29), VASHTNN (SEQ ID NO: 30), RSHQEIP (SEQ ID NO: 31), LNTTKDI (SEQ ID NO: 32), NO:32), IIDATKN (SEQ ID NO:33), NHISQTN (SEQ ID NO:34), SNSAHIT (SEQ ID NO:35), STHQSNN (SEQ ID NO:36), KTPNLTS (SEQ ID NO:37), SNTPALS (SEQ ID NO:38) and SPGATTN (SEQ ID NO:39),:54), LAVTAGMGAA (SEQ ID NO:55), LAPNSTTNNA (SEQ ID NO:56), LANSTSRIDA (SEQ ID NO:57), LAVASHTNNA (SEQ ID NO:58), LARSHQEIPA (SEQ ID NO:59), LALNTTKDIA (SEQ ID NO:60), LAIIDATKNA (SEQ ID NO:61), LANHISQTNA (SEQ ID NO:62), LAENRTTSNA (SEQ ID NO:63), LAPQQDTTHA (SEQ ID NO:64), LANATNHVIA (SEQ ID NO:65), LATNNSKPDA (SEQ ID NO:66), LASKLTLNNA (SEQ ID NO:67), LAVTAGMGAA (SEQ ID NO:68), LAPNSTTNNA (SEQ ID NO:69), LANSTSRIDA (SEQ ID NO:70), LAVASHTNNA (SEQ ID NO:71), LARSHQEIPA (SEQ ID NO:72), LALNTTKDIA (SEQ ID NO:73), LAIIDATKNA (SEQ ID NO:74), LANHISQTNA (SEQ ID NO:75), LATNRTTSNA (SEQ ID NO:76), LAPQQDTTHA (SEQ ID NO:77), LANATNHVIA (SEQ ID NO:78), LATNNSKPDA (SEQ ID NO:79), LASKLTLNNA (SEQ ID NO:80), NO:62), LASNSAHITA (SEQ ID NO:63), LASTHQSNNA (SEQ ID NO:64), LAKTPNLTSA (SEQ ID NO:65), LASNTPALSA (SEQ ID NO:66) and LASPGATTNA (SEQ ID NO:67), and further comprises one or more amino acid substitutions relative to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2). In some aspects, the one or more amino acid substitutions include a V708I amino acid substitution at residue 708 relative to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2) or the corresponding residue of another AAV capsid. The variant AAV capsid can have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more amino acid sequence identity to the entire length of the amino acid sequence set forth in SEQ ID NO:2 or the corresponding parent AAV capsid. ,

[0109] In related embodiments, a variant AAV capsid protein is provided that comprises: a) a peptide insertion located between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6, or AAV9, amino acids 586 and 587 of AAV4, amino acids 577 and 578 of AAV5, amino acids 589 and 590 of AAV7, or amino acids 590 to 591 of AAV8 or AAV10, the peptide insertion comprising, consisting of, or consisting essentially of an amino acid sequence selected from the group consisting of HDITKNI (SEQ ID NO: 12), NQDYTKT (SEQ ID NO: 13), DNTVTRS (SEQ ID NO: 14), SNSVQSI (SEQ ID NO: 15), NSTRHTD (SEQ ID NO: 16), TNRTSPD (SEQ ID NO: 17), ISDQTKH (SEQ ID NO: 18), QADTTKN (SEQ ID NO: 19), NAVKTDF (SEQ ID NO: 20), NO:20), TNQTLSA (SEQ ID NO:21), ENRTTSN (SEQ ID NO:22), PQQDTTH (SEQ ID NO:23), NATNHVI (SEQ ID NO:24), TNNSKPD (SEQ ID NO:25), SKLTLNN (SEQ ID NO:26), VTAGMGA (SEQ ID NO:27), PNSTTNN (SEQ ID NO:28), NTSSRID (SEQ ID NO:29), VASHTNN (SEQ ID NO:30), RSHQEIP (SEQ ID NO:31), LNTTKDI (SEQ ID NO:32), IIDATKN (SEQ ID NO:33), NHISQTN (SEQ ID NO:34), SNSAHIT (SEQ ID NO:35), STHQSNN (SEQ ID NO:36), KTPNLTS (SEQ ID NO:37), SNTPALS (SEQ ID NO:38), SPGATTN (SEQ ID NO:39), LAHDITKNIA (SEQ ID NO:40), LANQDYTKTA (SEQ ID NO:41), LADNTWTRSA (SEQ ID NO:42), LASNSVQSIA (SEQ ID NO:43), LANSTRHTDA (SEQ ID NO:44), LATNRTSPDA (SEQ ID NO:45), LAISDQTKHA (SEQ ID NO:46), LAQADTTKNA (SEQ IDNO:47), LANAVKTDFA (SEQ ID NO:48), LATNQTLSAA (SEQ ID NO:49), LAENRTTSNA (SEQ ID NO:50), LAPQQDTTHA (SEQ ID NO:51), LANATNHVIA (SEQ ID NO:52), LATNNSKPDA (SEQ ID NO:53), LASKLTLNNA (SEQ ID NO:54), LAVTAGMGAA (SEQ ID NO:55), LAPNSTTNNA (SEQ ID NO:56), LANSTSRIDA (SEQ ID NO:57), LAVASHTNNA (SEQ ID NO:58), LARSHQEIPA (SEQ ID NO:59), LALNTTKDIA (SEQ ID NO:60), LAIIDATKNA (SEQ ID NO:61), LANHISQTNA (SEQ ID NO:62), LASNSAHITA (SEQ ID NO:63), LASTHQSNNA (SEQ ID NO:64), LAKTPNLTSA (SEQ ID NO:65), LASNTPALSA (SEQ ID NO:66) and LASPGATTNA (SEQ ID NO:67), and b) a valine to isoleucine substitution at amino acid 709 of AAV3A or AAV3B, an alanine to isoleucine substitution at position 709 of AAV1 or AAV6, an asparagine to isoleucine substitution at amino acid 707 of AAV4 or amino acid 709 of AAV9, or a threonine to isoleucine substitution at amino acid 710 of AAV7 or amino acid 711 of AAV8 or AAV10, or a glutamine to isoleucine substitution at amino acid 697 of AAV5, and optionally otherwise identical to any one of SEQ ID NOs: 1 and 3-12. In preferred embodiments, the variant capsid protein comprises a) a peptide insertion between amino acids 587 and 588 of the AAV2 capsid, the peptide insertion comprising, consisting of, or consisting essentially of an amino acid sequence selected from the group consisting of ISDQTKH (SEQ ID NO: 18), QADTTKN (SEQ ID NO: 19), NAVKTDF (SEQ ID NO: 20), TNQTLSA (SEQ ID NO: 21), ENRTTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TNNSKPD (SEQ ID NO: 25), SKLTLNN (SEQ ID NO: 26), VTAGMGA (SEQ ID NO: 27),NO:39), or consists of an amino acid sequence selected from the group consisting of LAISDQTKHA (SEQ ID NO:46), LAQADTTKNA (SEQ ID NO:47), LANAVKTDFA (SEQ ID NO:48), LATNQTLSAA (SEQ ID NO:49), LAENRTTSNA (SEQ ID NO:50), LAPQQDTTHA (SEQ ID NO:51), LAQADTTKNA (SEQ ID NO:52), LANAVKTDFA (SEQ ID NO:53), LATNQTLSAA (SEQ ID NO:54), LAENRTTSNA (SEQ ID NO:55), LAQDTTHA (SEQ ID NO:56), LAQDTTKNA (SEQ ID NO:57), LANAVKTDFA (SEQ ID NO:58), LATNQTLSAA (SEQ ID NO:59), LAENRTTSNA (SEQ ID NO:50), LAQDTTHA (SEQ ID NO:51), LAQDTTKNA (SEQ ID NO:52), LANAVKTDFA (SEQ ID NO:53), LATNQTLSAA (SEQ ID NO:54), LAENRTTSNA (SEQ ID NO:55), LAQDTTHA (SEQ ID NO:56), LAQD NO:51), LANATNHVIA (SEQ ID NO:52), LATNNSKPDA (SEQ ID NO:53), LASKLTLNNA (SEQ ID NO:54), LAVTAGMGAA (SEQ ID NO:55), LAPNSTTNNA (SEQ ID NO:56), LANSTSRIDA (SEQ ID NO:57), LAVASHTNNA (SEQ IDNO: 58), LARSHQEIPA (SEQ ID NO: 59), LALNTTKDIA (SEQ ID NO: 60), LAIIDATKNA (SEQ ID NO: 61), LANHISQTNA (SEQ ID NO: 62), LASNSAHITA (SEQ ID NO: 63), LASTHQSNNA (SEQ ID NO: 64), LAKTPNLTSA (SEQ ID NO:65), LASNTPALSA (SEQ ID NO:66) and LASPGATTNA (SEQ IDNO:67), and b) an amino acid substitution of valine to isoleucine at amino acid 708 compared to the amino acid sequence of AAV2, wherein the variant capsid protein comprises 2 to 5, 5 to 10 or 10 to 15 amino acid substitutions.

[0110] In yet another embodiment, the variant capsid protein comprises a) a peptide insertion between amino acids 587 and 588 of the AAV2 capsid, the peptide insertion comprising, consisting of, or consisting essentially of an amino acid sequence selected from the group consisting of ISDQTKH (SEQ ID NO: 18), QADTTKN (SEQ ID NO: 19), NAVKTDF (SEQ ID NO: 20), TNQTLSA (SEQ ID NO: 21), ENRTTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TNNSKPD (SEQ ID NO: 25), SKLTLNN (SEQ ID NO: 26), VTAGMGA (SEQ ID NO: 27), PNSTTNN (SEQ ID NO: 28), NSTSRID (SEQ ID NO: 29), VASHTNN (SEQ ID NO: 30), RSHQEIP (SEQ ID NO: 31), LNTTKDI (SEQ ID NO: 32), IIDATKN (SEQ ID NO: 33), NO:33), NHISQTN (SEQ ID NO:34), SNSAHIT (SEQ ID NO:35), STHQSNN (SEQ ID NO:36), KTPNLTS (SEQ ID NO:37), SNTPALS (SEQ ID NO:38) and SPGATTN (SEQ ID NO:39),or consisting of an amino acid sequence selected from the group consisting of LAISDQTKHA (SEQ ID NO:46), LAQADTTKNA (SEQ ID NO:47), LANAVKTDFA (SEQ ID NO:48), LATNQTLSAA (SEQ ID NO:49), LAENRTTSNA (SEQ ID NO:50), LAPQQDTTHA (SEQ ID NO:51), LANATNHVIA (SEQ ID NO:52), LATNNSKPDA (SEQ ID NO:53), LASKLTLNNA (SEQ ID NO:54), LAVTAGMGAA (SEQ ID NO:55), LAPNSTTNNA (SEQ ID NO:56), LANSTSRIDA (SEQ ID NO:57), LAVASHTNNA (SEQ ID NO:58), LARSHQEIPA (SEQ ID NO:59), LALNTTKDIA (SEQ ID NO:60), LAIIDATKNA (SEQ ID NO:61), LANHISQTNA (SEQ ID NO:62), NO:62), LASNSAHITA (SEQ ID NO:63), LASTHQSNNA (SEQ ID NO:64), LAKTPNLTSA (SEQ ID NO:65), LASNTPALSA (SEQ ID NO:66) and LASPGATTNA (SEQ ID NO:67), and is otherwise identical to the amino acid sequence of SEQ ID NO:2.

[0111] In another embodiment, a variant AAV capsid protein is provided, comprising a substitution of amino acids 586-597 of wt AAV2 of SEQ ID NO:2, comprising, consisting of, or consisting essentially of the following amino acid sequence: VPTGaEtLNvnG (SEQ ID NO:74; lowercase letters correspond to amino acids in the wild-type AAV2 sequence). In other words, the variant AAV capsid protein comprises the following amino acid substitutions relative to AAV2: G586V, N587P, R588T, Q589, A591E, A593L, D594N, and T597G. The variant AAV capsid protein may comprise the above amino acid substitutions and be otherwise identical to SEQ ID NO:2, or may be at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:2.

[0112] In another embodiment, a variant AAV capsid protein with improved tropism for one or more lung cells is provided, comprising substitutions of amino acids 588-597 of wt AAV2 of SEQ ID NO: 2, comprising, consisting of, or consisting essentially of the following amino acid sequence: LAPDFTTLDA (SEQ ID NO: 75). In other words, the variant AAV capsid protein comprises the following amino acid substitutions relative to AAV2: R588L, Q589A, A590P, A591D, T592F, A593T, D594T, V595L, N596D, and T597A. The variant AAV capsid protein may comprise the above amino acid substitutions and be otherwise identical to SEQ ID NO: 2, or may be at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 2.

[0113] In another embodiment, a variant AAV capsid protein with improved tropism for one or more lung cells is provided, which comprises, consists of, or consists essentially of a substitution of amino acids 575-586 (SSTTAPATGTYN; SEQ ID NO:76) of wt AAV5 of SEQ ID NO:6 with an amino acid sequence selected from the group consisting of TGRQNPDMSGLS (SEQ ID NO:77) TGQRALDLRGLS (SEQ ID NO:78), TGWMSNQWLGLS (SEQ ID NO:79), TGVSQEPWAGLS (SEQ ID NO:80), TGVSLLVPSGLS (SEQ ID NO:81), TGGMGSWHSGLS (SEQ ID NO:82), TGSPLVFQAGLS (SEQ ID NO:83), TGLYDNSHVGLS (SEQ ID NO:84), TGDGDVGgGGLS (SEQ ID NO:85), TGPSpNPYtGLS (SEQ ID NO:86), TGNSGLAEAGLS (SEQ ID NO:87). The variant AAV capsid protein may comprise the aforementioned amino acid substitutions and be otherwise identical to SEQ ID NO:6, or may be at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:6.

[0114] In another embodiment, a variant AAV capsid protein with improved tropism for one or more lung cells is provided, which comprises, consists of, or consists essentially of a substitution of amino acids 533-545 (PANPGTTATYLEG; SEQ ID NO:89) of wt AAV5 of SEQ ID NO:6 having an amino acid sequence selected from the group consisting of FSPTYPSVWWFQR (SEQ ID NO:90), VMPWgLVFVCFDF (SEQ ID NO:91), CMTAWPVDASFLN (SEQ ID NO:92), IYLRLGIYWCAGV (SEQ ID NO:93), GLGGSStGSRTSA (SEQ ID NO:94), LFICFCCFYA(l)FF (SEQ ID NO:95), IDDDCSVaGyRSW (SEQ ID NO:96), SNGITFKDRRCLL (SEQ ID NO:97), FMIGNKVPIA(l)Pg (SEQ ID NO:98), and IYLRLGIYWCAGN (SEQ ID NO:99). In a related embodiment, a variant AAV capsid protein with improved tropism for one or more lung cells is provided, comprising replacing amino acids 533-544 (PANPGTTATYLE; SEQ ID NO: 100) of wt AAV5 of SEQ ID NO: 6 with the following amino acid sequence: LSTpFIVaGSGI (SEQ ID NO: 101). The lowercase letters in each sequence correspond to the amino acids in the wild-type AAV5 sequence. The variant AAV capsid protein may comprise the aforementioned amino acid substitutions and be otherwise identical to SEQ ID NO: 6, or may be at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 6.

[0115] The AAV variants disclosed herein are generated by using in vivo directed evolution, including screening using primate lungs after aerosol administration. In some embodiments, the variant capsid proteins disclosed herein, when present in AAV virions, confer increased transduction of lung cells compared to transduction of lung cells by AAV virions containing the corresponding parent AAV capsid protein or wild-type AAV. For example, in some embodiments, when present in AAV virions, the variant capsid proteins disclosed herein confer more efficient transduction of primate lung cells than AAV virions containing the corresponding parent AAV capsid protein or wild-type AAV capsid protein, for example, lung cells receive more AAV virions containing the subject variant AAV capsid protein than AAV virions containing the parent AAV capsid protein or wild-type AAV. In some such embodiments, AAV variant virions or variant rAAVs exhibit at least 2-fold, at least 5-fold, at least 10-fold, at least -15-fold, at least 20-fold, at least 25-fold, at least 50-fold or more than 50-fold increased transduction of lung cells compared to transduction of lung cells by wild-type AAV virions or rAAVs containing corresponding parental AAV capsid proteins. In certain such embodiments, when present in AAV virions, the variant capsid proteins disclosed herein confer broader transduction to primate lung cells than AAV virions containing corresponding parental AAV capsid proteins or wild-type AAV capsid proteins. In other words, variant AAV virions transduce cell types that are not transduced by virions containing corresponding parental AAV capsid proteins, and therefore transduce more types of cells in the lung than corresponding parental AAV virions. In some embodiments, the AAV variant virions preferentially transduce lung cells, for example, the subject rAAV virions infect one lung cell with a specificity of 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold or more than 50-fold for another lung cell or non-lung cell, such as a cell outside the lung. In some embodiments, the transduced lung cells are upper respiratory tract cells. In some embodiments, the lung cells are upper respiratory tract epithelial cells. In some embodiments, the lung cells are alveolar epithelial cells. In some embodiments, the lung cells are primary, secondary or tertiary bronchial epithelial cells. In some embodiments, the lung cells are tracheal epithelial cells. In some embodiments, the lung cells are ciliated respiratory epithelial cells. In some embodiments, the lung cells are alveolar epithelial type 1 (AECI) cells or type 2 (AECII) cells. In some embodiments, the lung cells are smooth muscle cells. In some embodiments, the lung cells are endothelial cells. Increased transduction of lung cells, e.g., increased transduction efficiency, more extensive transduction, more preferential transduction, etc., can be readily assessed in vitro or in vivo by any number of methods known in the art for measuring gene expression.For example, an AAV can be packaged with a genome comprising an expression cassette comprising a reporter gene, such as a fluorescent protein, under the control of a ubiquitous or tissue-specific promoter, and the extent of transduction is assessed by detecting the fluorescent protein by, for example, fluorescence microscopy. As another example, an AAV can be packaged with a genome comprising a barcoded nucleic acid sequence, and the extent of transduction is assessed by detecting the nucleic acid sequence by, for example, PCR. As another example, an AAV can be packaged with a genome comprising an expression cassette comprising a therapeutic gene for treating a lung disease, and the extent of transduction is assessed by detecting treatment of the lung disease in afflicted patients administered the AAV.

[0116] Provided herein are methods for delivering a heterologous nucleic acid to a lung cell, comprising contacting the lung cell with an rAAV virion comprising (i) a capsid protein comprising, consisting of, or consisting essentially of a peptide insertion selected from the group consisting of HDITKNI (SEQ ID NO: 12), NQDYTKT (SEQ ID NO: 13), DNTVTRS (SEQ ID NO: 14), SNSVQSI (SEQ ID NO: 15), NSTRHTD (SEQ ID NO: 16), TNRTSPD (SEQ ID NO: 17), ISDQTKH (SEQ ID NO: 18), QADTTKN (SEQ ID NO: 19), NAVKTDF (SEQ ID NO: 20), TNQTLSA (SEQ ID NO: 21), ENRTTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TNNSKPD (SEQ ID NO: 25), SKLTLNN (SEQ ID NO: 26), NO:26), VTAGMGA (SEQ ID NO:27), PNSTTNN (SEQ ID NO:28), NTSSRID (SEQ ID NO:29), VASHTNN (SEQ ID NO:30), RSHQEIP (SEQ ID NO:31), LNTTKDI (SEQ ID NO:32), IIDATKN (SEQ ID NO:33), NHISQTN (SEQ ID NO:34), SNSAHIT (SEQ ID NO:35), STHQSNN (SEQ ID NO:36), KTPNLTS (SEQ ID NO:37), SNTPALS (SEQ ID NO:38), SPGATTN (SEQ ID NO:39), LAHDITKNIA (SEQ ID NO:40), LANQDYTKTA (SEQ ID NO:41), LADNTVTRSA (SEQ ID NO:42), LASNSVQSIA (SEQ ID NO:43), LANSTRHTDA (SEQ ID NO:44), LATNRTSPDA (SEQ ID NO:45), LAISDQTKHA (SEQ ID NO:46), LAQADTTKNA (SEQ ID NO:47), LANAVKTDFA (SEQ ID NO:48), LATNQTLSAA (SEQ ID NO:49), LAENRTTSNA (SEQ ID NO:50), LAPQQDTTHA (SEQ IDNO:54), LAVTAGMGAA (SEQ ID NO:55), LAPNSTTNNA (SEQ ID NO:56), LANSTSRIDA (SEQ ID NO:57), LAVASHTNNA (SEQ ID NO:58), LARSHQEIPA (SEQ ID NO:59), LALNTTKDIA (SEQ ID NO:60), LAIIDATKNA (SEQ ID NO:61), LANHISQTNA (SEQ ID NO:62), LASNSAHITA (SEQ ID NO:63), LASTHQSNNA (SEQ ID NO:64), LAKTPNLTSA (SEQ ID NO:65), LASNTPALSA (SEQ ID NO:66), and LASPGATTNA (SEQ ID NO:67), and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products. In some embodiments, the method is an in vitro or ex vivo method. In some embodiments, heterologous nucleic acids encode proteins and / or short interfering RNAs. In some preferred embodiments, lung cells are any cells of the lungs or trachea. In other preferred embodiments, lung cells are respiratory epithelial cells, including but not limited to alveolar epithelial cells, bronchial (primary, secondary or tertiary) epithelial cells or tracheal epithelial cells. In some preferred aspects, lung cells are ciliated respiratory epithelial cells. In some preferred aspects, lung cells are alveolar epithelial type 1 (AECI) or type 2 (AECII) cells. In other embodiments, lung cells are smooth muscle or endothelial cells. In other embodiments, lung cells are basal cells, goblet cells or oocytes.

[0117] Also provided herein are methods of delivering a heterologous nucleic acid to the lungs of a subject (e.g., a human subject), comprising administering to the subject an rAAV virion comprising (i) a capsid protein comprising, consisting of, or consisting essentially of a peptide insertion selected from the group consisting of HDITKNI (SEQ ID NO: 12), NQDYTKT (SEQ ID NO: 13), DNTVTRS (SEQ ID NO: 14), SNSVQSI (SEQ ID NO: 15), NSTRHTD (SEQ ID NO: 16), TNRTSPD (SEQ ID NO: 17), ISDQTKH (SEQ ID NO: 18), QADTTKN (SEQ ID NO: 19), NAVKTDF (SEQ ID NO: 20), TNQTLSA (SEQ ID NO: 21), ENRTTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TNNSKPD (SEQ ID NO: 25), SKLTLNN (SEQ ID NO: 26), TNQTLSA (SEQ ID NO: 27), ENRTTSN (SEQ ID NO: 28), PQQDTTH (SEQ ID NO: 29), NATNHVI (SEQ ID NO: 30), TNNSKPD (SEQ ID NO: 31), SKLTLNN (SEQ ID NO: 32), NO:26), VTAGMGA (SEQ ID NO:27), PNSTTNN (SEQ ID NO:28), NTSSRID (SEQ ID NO:29), VASHTNN (SEQ ID NO:30), RSHQEIP (SEQ ID NO:31), LNTTKDI (SEQ ID NO:32), IIDATKN (SEQ ID NO:33), NHISQTN (SEQ ID NO:34), SNSAHIT (SEQ ID NO:35), STHQSNN (SEQ ID NO:36), KTPNLTS (SEQ ID NO:37), SNTPALS (SEQ ID NO:38), SPGATTN (SEQ ID NO:39), LAHDITKNIA (SEQ ID NO:40), LANQDYTKTA (SEQ ID NO:41), LADNTVTRSA (SEQ ID NO:42), LASNSVQSIA (SEQ ID NO:43), LANSTRHTDA (SEQ ID NO:44), LATNRTSPDA (SEQ ID NO:45), LAISDQTKHA (SEQ ID NO:46), LAQADTTKNA (SEQ ID NO:47), LANAVKTDFA (SEQ ID NO:48), LATNQTLSAA (SEQ ID NO:49), LAENRTTSNA (SEQ ID NO:50), LAPQQDTTHA (SEQ IDNO:54), LAVTAGMGAA (SEQ ID NO:55), LAPNSTTNNA (SEQ ID NO:56), LANSTSRIDA (SEQ ID NO:57), LAVASHTNNA (SEQ ID NO:58), LARSHQEIPA (SEQ ID NO:59), LALNTTKDIA (SEQ ID NO:60), LAIIDATKNA (SEQ ID NO:61), LANHISQTNA (SEQ ID NO:62), LASNSAHITA (SEQ ID NO:63), LASTHQSNNA (SEQ ID NO:64), LAKTPNLTSA (SEQ ID NO:65), LASNTPALSA (SEQ ID NO:66), and LASPGATTNA (SEQ ID NO:67), and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products. In some embodiments, the heterologous nucleic acid encodes a protein and / or a short interfering RNA. In related embodiments, a method for delivering a heterologous nucleic acid to the upper respiratory tract, nasopharynx, sinuses, oral / buccal area, and / or salivary glands of a subject (e.g., a human subject) comprises administering to the subject an rAAV virion comprising (i) a capsid protein comprising, consisting of, or consisting essentially of a peptide insertion selected from the group consisting of HDITKNI (SEQ ID NO: 12), NQDYTKT (SEQ ID NO: 13), DNTVTRS (SEQ ID NO: 14), SNSVQSI (SEQ ID NO: 15), NSTRHTD (SEQ ID NO: 16), TNRTSPD (SEQ ID NO: 17), ISDQTKH (SEQ ID NO: 18), QADTTKN (SEQ ID NO: 19), NAVKTDF (SEQ ID NO: 20), TNQTLSA (SEQ ID NO: 21), ENRTTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), NO:24), TNNSKPD (SEQ ID NO:25), SKLTLNN (SEQ ID NO:26), VTAGMGA (SEQ ID NO:27), PNSTTNN (SEQ ID NO:28), NTSSRID (SEQ ID NO:29), VASHTNN (SEQ IDNO: 30), RSHQEIP (SEQ ID NO: 31), LNTTKDI (SEQ ID NO: 32), IIDATKN (SEQ ID NO: 33), NHISQTN (SEQ ID NO: 34), SNSAHIT (SEQ ID NO: 35), STHQSNN (SEQ ID NO: 36), KTPNLTS (SEQ ID NO: 37), SNTPALS (SEQ ID NO: 38), SPGATTN (SEQ ID NO: 39), LAHDITKNIA (SEQ ID NO: 40), LANQDYTKTA (SEQ ID NO: 41), LADNTVTRSA (SEQ ID NO: 42), LASNSVQSIA (SEQ ID NO: 43), LANSTRHTDA (SEQ ID NO: 44), LATNRTSPDA (SEQ ID NO: 45), LAISDQTKHA (SEQ ID NO: 46), LAQADTTKNA (SEQ ID NO: 47), LANAVKTDFA (SEQ ID NO: 48), LATNQTLSAA (SEQ ID NO: 49), LAENRTTSNA (SEQ ID NO: 50), LAPQQDTTHA (SEQ ID NO: 51), LANATNHVIA (SEQ ID NO: 52), LATNNSKPDA (SEQ ID NO: 53), LASKLTLNNA (SEQ ID NO: 54), LAVTAGMGAA (SEQ ID NO: 55), LAPNSTTNNA (SEQ ID NO: 56), LANSTSRIDA (SEQ ID NO: 57), LAVASHTNNA (SEQ ID NO: 58), LARSHQEIPA (SEQ ID NO: 59), LALNTTKDIA (SEQ ID NO: 60), LAIIDATKNA (SEQ ID NO: 61), LANHISQTNA (SEQ ID NO: 62), LASNSAHITA (SEQ ID NO: 63), LASTHQSNNA (SEQ ID NO: 64), LAKTPNLTSA (SEQ ID NO: 65), LASNTPALSA (SEQ ID NO: 66) and LASPGATTNA (SEQ IDNO:67), and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products. In related aspects, rAAV or a pharmaceutical composition comprising the same is administered to the subject via the lungs, intrabronchial, intranasal, intratracheal and / or intrabronchial administration. In preferred aspects, the heterologous nucleic acid is delivered to the lungs of the subject to deliver one or more encoded gene products to the lungs of the subject. The uses of the gene products include, but are not limited to, enhancing the level of the factor in the cell, enhancing the level of the factor in the neighboring cell by the secretion of the factor, reducing the level of the factor in the cell, or reducing the level of the factor in the neighboring cell by the secretion of the factor. The gene product can be designed to supplement the level of the defective or missing gene product (for example, the gene product can be a therapeutic replacement gene), reduce the level of the defective gene product (for example, the gene product can be an interfering RNA, such as siRNA or miRNA, which reduces the expression of the defective gene product), introduce a new supporting gene product, supplement the level of the supporting gene product, reduce the level of the obstructive gene product, or reduce the level of the obstructive gene product and introduce or supplement the level of both the supporting gene product.

[0118] Also provided herein are methods for treating a pulmonary disease, comprising administering to a subject in need thereof a therapeutically effective amount of a recombinant AAV (rAAV), the recombinant AAV comprising (i) a capsid protein comprising, consisting of, or consisting essentially of a peptide insertion selected from the group consisting of HDITKNI (SEQ ID NO: 12), NQDYTKT (SEQ ID NO: 13), DNTVTRS (SEQ ID NO: 14), SNSVQSI (SEQ ID NO: 15), NSTRHTD (SEQ ID NO: 16), TNRTSPD (SEQ ID NO: 17), ISDQTKH (SEQ ID NO: 18), QADTTKN (SEQ ID NO: 19), NAVKTDF (SEQ ID NO: 20), TNQTLSA (SEQ ID NO: 21), ENRTTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TNNSKPD (SEQ ID NO: 25), SKLTLNN (SEQ ID NO: 26), TNQTLSA (SEQ ID NO: 27), ENRTTSN (SEQ ID NO: 28), PQQDTTH (SEQ ID NO: 29), NATNHVI (SEQ ID NO: 30), TNNSKPD (SEQ ID NO: 31), SKLTLNN (SEQ ID NO: 32), NO:26), VTAGMGA (SEQ ID NO:27), PNSTTNN (SEQ ID NO:28), NTSSRID (SEQ ID NO:29), VASHTNN (SEQ ID NO:30), RSHQEIP (SEQ ID NO:31), LNTTKDI (SEQ ID NO:32), IIDATKN (SEQ ID NO:33), NHISQTN (SEQ ID NO:34), SNSAHIT (SEQ ID NO:35), STHQSNN (SEQ ID NO:36), KTPNLTS (SEQ ID NO:37), SNTPALS (SEQ ID NO:38), SPGATTN (SEQ ID NO:39), LAHDITKNIA (SEQ ID NO:40), LANQDYTKTA (SEQ ID NO:41), LADNTVTRSA (SEQ ID NO:42), LASNSVQSIA (SEQ ID NO:43), LANSTRHTDA (SEQ ID NO:44), LATNRTSPDA (SEQ ID NO:45), LAISDQTKHA (SEQ ID NO:46), LAQADTTKNA (SEQ ID NO:47), LANAVKTDFA (SEQ ID NO:48), LATNQTLSAA (SEQ ID NO:49), LAENRTTSNA (SEQ ID NO:50), LAPQQDTTHA (SEQ IDNO:51), LANATNHVIA (SEQ ID NO:52), LATNNSKPDA (SEQ ID NO:53), LASKLTLNNA (SEQ ID NO:54), LAVTAGMGAA (SEQ ID NO:55), LAPNSTTNNA (SEQ ID NO:56), LANSTSRIDA (SEQ ID NO:57), LAVASHTNNA (SEQ ID NO:58), LARSHQEIPA (SEQ ID NO:59), LALNTTKDIA (SEQ ID NO:60), LAIIDATKNA (SEQ ID NO:61), LANHISQTNA (SEQ ID NO:62), LASNSAHITA (SEQ ID NO:63), LASTHQSNNA (SEQ ID NO:64), LAKTPNLTSA (SEQ ID NO:65), LASNTPALSA (SEQ ID NO:66) and LASPGATTNA (SEQ ID NO:67), and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products, wherein the one or more gene products are operably linked to a promoter. Also provided herein is a use of a recombinant AAV (rAAV) for treating a lung disease, wherein the recombinant AAV comprises (i) a capsid protein, wherein the capsid protein consists of, or consists essentially of a peptide insertion selected from the group consisting of HDITKNI (SEQ ID NO: 12), NQDYTKT (SEQ ID NO: 13), DNTVTRS (SEQ ID NO: 14), SNSVQSI (SEQ ID NO: 15), NSTRHTD (SEQ ID NO: 16), TNRTSPD (SEQ ID NO: 17), ISDQTKH (SEQ ID NO: 18), QADTTKN (SEQ ID NO: 19), NAVKTDF (SEQ ID NO: 20), TNQTLSA (SEQ ID NO: 21), ENRTTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TNNSKPD (SEQ ID NO: 25), SKLTLNN (SEQ ID NO: 26), VTAGMGA (SEQ ID NO: 27), VGAQTS (SEQ ID NO: 28), VGAQTS (SEQ ID NO: 29), VGAQTS (SEQ ID NO: 30), VGAQTS (SEQ ID NO: 31), VGAQTS (SEQ ID NO: 32), VGAQTS (SEQ ID NO: 33), VGAQTS (SEQ ID NO: 34), VGAQTS (SEQ ID NO: 35), VGAQTS (SEQ ID NO: 36), VGAQTS (SEQ ID NO: 37), VGAQTS (SEQ ID NO: 38), VGAQTS (SEQ ID NO: 39), VGAQTS (SEQ ID NO: 40), VGAQTS (SEQ ID NO: 41), VGAQTS (SEQ ID NO: NO:27), PNSTTNN (SEQ ID NO:28), NTSSRID (SEQ ID NO:29), VASHTNN (SEQ ID NO:30), RSHQEIP (SEQ ID NO:31), LNTTKDI (SEQ IDNO:32), IIDATKN (SEQ ID NO:33), NHISQTN (SEQ ID NO:34), SNSAHIT (SEQ ID NO:35), STHQSNN (SEQ ID NO:36), KTPNLTS (SEQ ID NO:37), SNTPALS (SEQ ID NO:38), SPGATTN (SEQ ID NO:39), LAHDITKNIA (SEQ ID NO:40), LANQDYTKTA (SEQ ID NO:41), LADNTVTRSA (SEQ ID NO:42), LASNSVQSIA (SEQ ID NO:43), LANSTRHTDA (SEQ ID NO:44), LATNRTSPDA (SEQ ID NO:45), LAISDQTKHA (SEQ ID NO:46), LAQADTTKNA (SEQ ID NO:47), LANAVKTDFA (SEQ ID NO:48), LATNQTLSAA (SEQ ID NO:49), LAENRTTSNA (SEQ ID NO:50), LAPQQDTTHA (SEQ ID NO:51), LANATNHVIA (SEQ ID NO:52), LATNNSKPDA (SEQ ID NO:53), LASKLTLNNA (SEQ ID NO:54), LAVTAGMGAA (SEQ ID NO:55), LAPNSTTNNA (SEQ ID NO:56), LANSTSRIDA (SEQ ID NO:57), LAVASHTNNA (SEQ ID NO:58), LARSHQEIPA (SEQ ID NO:59), LALNTTKDIA (SEQ ID NO:60), LAIIDATKNA (SEQ ID NO:61), LANHISQTNA (SEQ ID NO:62), LASNSAHITA (SEQ ID NO:63), LASTHQSNNA (SEQ ID NO:64), LAKTPNLTSA (SEQ ID NO:65), LASNTPALSA (SEQ ID NO:66) and LASPGATTNA (SEQ IDNO:67), and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products, wherein the one or more gene products are operably linked to a promoter. Also provided herein is a use of a recombinant AAV (rAAV) in the preparation of a medicament for treating a lung disease, wherein the recombinant AAV comprises (i) a capsid protein comprising, consisting of, or consisting essentially of a peptide insertion selected from the group consisting of HDITKNI (SEQ ID NO: 12), NQDYTKT (SEQ ID NO: 13), DNTVTRS (SEQ ID NO: 14), SNSVQSI (SEQ ID NO: 15), NSTRHTD (SEQ ID NO: 16), TNRTSPD (SEQ ID NO: 17), ISDQTKH (SEQ ID NO: 18), QADTTKN (SEQ ID NO: 19), NAVKTDF (SEQ ID NO: 20), TNQTLSA (SEQ ID NO: 21), ENRTTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TNNSKPD (SEQ ID NO: 25), SKLTLNN (SEQ ID NO: 26), VTAGMGA (SEQ ID NO: 27), ID NO:27), PNSTTNN (SEQ ID NO:28), NTSSRID (SEQ ID NO:29), VASHTNN (SEQ ID NO:30), RSHQEIP (SEQ ID NO:31), LNTTKDI (SEQ ID NO:32), IIDATKN (SEQ ID NO:33), NHISQTN (SEQ ID NO:34), SNSAHIT (SEQ ID NO:35), STHQSNN (SEQ ID NO:36), KTPNLTS (SEQ ID NO:37), SNTPALS (SEQ ID NO:38), SPGATTN (SEQ ID NO:39), LAHDITKNIA (SEQ ID NO:40), LANQDYTKTA (SEQ ID NO:41), LADNTWTRSA (SEQ ID NO:42), LASNSVQSIA (SEQ ID NO:43), LANSTRHTDA (SEQ ID NO:44), LATNRTSPDA (SEQ ID NO:45), LAISDQTKHA (SEQ ID NO:46), LAQADTTKNA (SEQ ID NO:47), LANAVKTDFA (SEQ ID NO:48), LATNQTLSAA (SEQ IDNO:49), LAENRTTSNA (SEQ ID NO:50), LAPQQDTTHA (SEQ ID NO:51), LANATNHVIA (SEQ ID NO:52), LATNNSKPDA (SEQ ID NO:53), LASKLTLNNA (SEQ ID NO:54), LAVTAGMGAA (SEQ ID NO:55), LAPNSTTNNA (SEQ ID NO:56), LANSTSRIDA (SEQ ID NO:57), LAVASHTNNA (SEQ ID NO:58), LARSHQEIPA (SEQ ID NO:59), LALNTTKDIA (SEQ ID NO:60), LAIIDATKNA (SEQ ID NO:61), LANHISQTNA (SEQ ID NO:62), LASNSAHITA (SEQ IDNO:63), LASTHQSNNA (SEQ ID NO:64), LAKTPNLTSA (SEQ ID NO:65), LASNTPALSA (SEQ IDNO:66) and LASPGATTNA (SEQ ID NO:67), and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products, the one or more gene products being operably linked to a promoter. In some aspects, the heterologous nucleic acid comprises a nucleotide sequence encoding multiple gene products, in which case the expression of the multiple (e.g., 2) gene products can be mediated by multiple (e.g., 2) independent promoters, or can be mediated by a single promoter, wherein multiple transgenes are separated by an internal ribosome entry site (IRES) or a 2A peptide sequence. In a preferred embodiment, the heterologous nucleic acid encodes a therapeutic protein and / or a therapeutic short interfering RNA. In related aspects, the one or more gene products delivered by rAAV reduce the level of an obstructive gene product and / or introduce or supplement the level of a supportive gene product.

[0119] Pulmonary diseases that can be treated using the variant rAAV vectors or viral particles and / or methods disclosed herein include, but are not limited to, monogenic diseases, complex genetic diseases, acquired diseases, and traumatic injuries. In some aspects, the pulmonary disease is selected from chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), cystic fibrosis, pulmonary hypertension, pulmonary hypertension, lung cancer (primary, secondary, and metastatic), surfactant deficiency, viral and / or bacterial infection, acute bronchitis, pneumonia (including viral, bacterial, and fungal pneumonia), respiratory tract infections (including pharyngitis, croup, Aspergillus, coocidiomycosis, Hantavirus pulmonary syndrome, and histoplasmosis), chemical and hypersensitivity pneumonitis, tuberculosis and other mycobacterial infections (including but not limited to Mycobacterium avium), sarcoidosis, respiratory syncytial virus, pulmonary edema, acute respiratory distress syndrome Acute respiratory syndrome (ARDS), pneumoconiosis (including black lung disease, asbestosis, and silicosis), interstitial lung disease (including sarcoidosis and autoimmune diseases), pulmonary embolism, pleural effusion, pleurisy, mesothelioma, pneumothorax, acute bronchitis, bronchiolitis (including bronchiolitis obliterans), sudden infant death syndrome, sleep apnea, bronchiectasis, bronchopulmonary dysplasia, cryptogenic organizing pneumonia, electronic cigarette use-associated lung injury (EVALI), Middle East respiratory syndrome (MERS), primary ciliary dyskinesia, severe acute respiratory syndrome (SARS), alpha-1-antitrypsin deficiency, asthma, interstitial lung disease, and COVID-19 (coronavirus disease 2019).

[0120] In certain aspects, genes that can be targeted for treatment of IPF include, but are not limited to, SFTPA1 (Surfactant A1) and Caveolin-1. Genes that can be targeted for treatment of COPD include, but are not limited to, genes encoding alpha-1-antitrypsin, alpha-1-antichymotrypsin, alpha-1-macroglobulin, matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 12 (MMP12), microsomal epoxidase, CYP1A1, glutathione S-transferase, heme oxygenase-1, TGF-β-1, TNF-α, IL-1 complex, IL-8, IL-13, human leukocyte antigens (HLA-B7 and Bw16), vitamin D binding protein, and β-2-adrenergic receptor or biologically active portions thereof.

[0121] In some aspects, methods of treating COVID-19 are provided, comprising administering to a subject in need thereof a therapeutically effective amount of a recombinant AAV (rAAV), wherein the recombinant AAV comprises (i) a subject variant AAV capsid protein and (ii) a heterologous nucleic acid, wherein the heterologous nucleic acid comprises a nucleotide sequence encoding one or more gene products operably linked to one or more promoters, or a pharmaceutical composition comprising the rAAV, wherein the one or more gene products knock down, modify and / or overexpress viral gene products or host cell genes to reduce or eliminate viral pathogenicity or replication in the lungs or nasopharynx and / or express neutralizing antibodies against epitopes on the virus.

[0122] In some aspects, methods for treating IPF are provided, comprising administering to a subject in need thereof a therapeutically effective amount of a recombinant AAV (rAAV), the recombinant AAV comprising (i) a subject variant AAV capsid protein and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products operably linked to one or more promoters, or a pharmaceutical composition comprising the rAAV. In some preferred embodiments, rAAV for treating IPF is provided, the rAAV comprising a subject variant AAV capsid protein and a nucleic acid comprising a nucleotide sequence encoding SFTPA1 and / or caveolin-1 or a biologically active portion thereof.

[0123] In some aspects, a method for treating COPD is provided, comprising administering to a subject in need thereof a therapeutically effective amount of a recombinant AAV (rAAV), the recombinant AAV comprising (i) a subject variant AAV capsid protein and (ii) a heterologous nucleic acid, the heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products operably linked to one or more promoters, or a pharmaceutical composition comprising the rAAV. In some preferred embodiments, rAAV for treating COPD is provided, the rAAV comprising a subject variant AAV capsid protein and a nucleic acid comprising a nucleotide sequence encoding alpha-1-antitrypsin or a biologically active portion thereof.

[0124] In some preferred aspects, rAAV comprising a subject variant AAV capsid and a nucleic acid encoding CFTR or a biologically active portion thereof is provided for use in treating cystic fibrosis or a lung disease associated therewith as described herein, or for use in the preparation of a medicament for treating cystic fibrosis or a lung disease associated therewith. Preferably, the nucleotide sequence encoding CFTR or a biologically active portion thereof is operably linked to an expression control sequence. In some embodiments, the nucleotide sequence encoding human CFTR or a biologically active portion thereof encodes a native human CFTR protein and has the following sequence or a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical thereto: In preferred embodiments, the nucleotide sequence encoding human CFTR or a biologically activated truncated CFTR protein is codon optimized for expression in humans. In some embodiments, the nucleotide sequence encodes a biologically active truncated human CFTR protein lacking amino acids 708-759 and comprises the following nucleotide sequence or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical thereto: Preferably, the nucleotide sequence encoding CFTR or its biologically active part is operably connected to an expression control sequence. In some aspects, the promoter is a constitutive promoter, optionally a truncated cytomegalovirus immediate / early (CMVie) enhancer / promoter, and is operably connected to the nucleotide sequence encoding human CFTR or its biologically active part. In other aspects, the promoter is a tissue-specific promoter, preferably, wherein the promoter guides the preferential expression of nucleic acid in pneumocytes, and is operably connected to the nucleotide sequence encoding human CFTR or its biologically active part. In a preferred embodiment, the promoter is a truncated CMVie promoter and is operably connected to the nucleotide sequence encoding human CFTR or its biologically active part. In a particularly preferred embodiment, the CMVie promoter is CMV173 with the following sequence or a sequence identical to it at least 90%, at least 95%, at least 98% or at least 99%: In a particularly preferred embodiment, the rAAV vector comprises (i) a capsid protein comprising, consisting of, or consisting essentially of a peptide insertion selected from the group consisting of HDITKNI (SEQ ID NO: 12), NQDYTKT (SEQ ID NO: 13), DNTVTRS (SEQ ID NO: 14), SNSVQSI (SEQ ID NO: 15), NSTRHTD (SEQ ID NO: 16), TNRTSPD (SEQ ID NO: 17), ISDQTKH (SEQ ID NO: 18), QADTTKN (SEQ ID NO: 19), NAVKTDF (SEQ ID NO: 20), TNQTLSA (SEQ ID NO: 21), ENRTTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TNNSKPD (SEQ ID NO: 25), SKLTLNN (SEQ ID NO: 26), VTAGMGA (SEQ ID NO: 27), PNSTTNN (SEQ ID NO: 28), NO:28), NTSSRID (SEQ ID NO:29), VASHTNN (SEQ ID NO:30), RSHQEIP (SEQ ID NO:31), LNTTKDI (SEQ ID NO:32), IIDATKN (SEQ ID NO:33), NHISQTN (SEQ ID NO:34), SNSAHIT (SEQ ID NO:35), STHQSNN (SEQ ID NO:36), KTPNLTS (SEQ ID NO:37), SNTPALS (SEQ ID NO:38), SPGATTN (SEQ ID NO:39), LAHDITKNIA (SEQ ID NO:40), LANQDYTKTA (SEQ ID NO:41), LADNTWTRSA (SEQ ID NO:42), LASNSVQSIA (SEQ ID NO:43), LANSTRHTDA (SEQ ID NO:44), LATNRTSPDA (SEQ ID NO:45), LAISDQTKHA (SEQ ID NO:46), LAQADTTKNA (SEQ IDNO:47), LANAVKTDFA (SEQ ID NO:48), LATNQTLSAA (SEQ ID NO:49), LAENRTTSNA (SEQ ID NO:50), LAPQQDTTHA (SEQ ID NO:51), LANATNHVIA (SEQ IDNO:52), LATNNSKPDA (SEQ ID NO:53), LASKLTLNNA (SEQ ID NO:54), LAVTAGMGAA (SEQ ID NO:55), LAPNSTTNNA (SEQ ID NO:56), LANSTSRIDA (SEQ ID NO:57), LAVASHTNNA (SEQ ID NO:58), LARSHQEIPA (SEQ IDNO:59), LALNTTKDIA (SEQ ID NO:60), LAIIDATKNA (SEQ ID NO:61), LAHISQTNA (SEQ ID NO:62), LASNSAHITA (SEQ ID NO:63), LASTHQSNNA (SEQ ID NO:64), LAKTPNLTSA (SEQ IDNO:65), LASNTPALSA (SEQ ID NO:66) and LASPGATTNA (SEQ ID NO:67), and (ii) from 5' to 3' comprising: (a) an AAV2 terminal repeat sequence (b) a promoter (c) a nucleotide sequence encoding a human cystic fibrosis transmembrane conductance regulator (CFTR) protein or a biologically active truncated CFTR protein lacking amino acids 708-759 of the human CFTR protein sequence (d) a polyadenylation sequence and (e) an AAV2 terminal repeat sequence.

[0125] In some aspects, an amount of rAAV is administered to a subject that is effective to improve one or more characteristics of cystic fibrosis, non-limiting examples of which include upper and lower airway inflammation, aberrant epithelial cytokine signaling, and elevated IgE levels. In other aspects, provided herein are methods for treating lung diseases associated with cystic fibrosis, including but not limited to upper respiratory tract diseases, lower respiratory tract diseases, nasopharyngeal diseases, sinusitis and / or salivary diseases associated with cystic fibrosis, comprising administering to a subject a therapeutically effective amount of an infectious rAAV, the infectious rAAV comprising (i) a capsid protein comprising, consisting of, or consisting essentially of a peptide insertion selected from the group consisting of HDITKNI (SEQ ID NO: 12), NQDYTKT (SEQ ID NO: 13), DNTVTRS (SEQ ID NO: 14), SNSVQSI (SEQ ID NO: 15), NSTRHTD (SEQ ID NO: 16), TNRTSPD (SEQ ID NO: 17), ISDQTKH (SEQ ID NO: 18), QADTTKN (SEQ ID NO: 19), NAVKTDF (SEQ ID NO: 20), TNQTLSA (SEQ ID NO: 21), ENRTTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NO:23), NATNHVI (SEQ ID NO:24), TNNSKPD (SEQ ID NO:25), SKLTLNN (SEQ ID NO:26), VTAGMGA (SEQ ID NO:27), PNSTTNN (SEQ ID NO:28), NTSSRID (SEQ ID NO:29), VASHTNN (SEQ ID NO:30), RSHQEIP (SEQ ID NO:31), LNTTKDI (SEQ ID NO:32), IIDATKN (SEQ ID NO:33), NHISQTN (SEQ ID NO:34), SNSAHIT (SEQ ID NO:35), STHQSNN (SEQ ID NO:36), KTPNLTS (SEQ ID NO:37), SNTPALS (SEQ ID NO:38), SPGATTN (SEQ ID NO:39), LAHDITKNIA (SEQ ID NO:40), LANQDYTKTA (SEQ ID NO:41), LADNTVTRSA (SEQ ID NO:42), LASNSVQSIA (SEQ ID NO:43), LANSTRHTDA (SEQ ID NO:44), LATNRTSPDA (SEQ IDNO:45), LAISDQTKHA (SEQ ID NO:46), LAQADTTKNA (SEQ ID NO:47), LANAVKTDFA (SEQ ID NO:48), LATNQTLSAA (SEQ ID NO:49), LAENRTTSNA (SEQ ID NO:50), LAPQQDTTHA (SEQ ID NO:51), LANATNHVIA (SEQ IDNO: 52), LATNNSKPDA (SEQ ID NO: 53), LASKLTLNNA (SEQ ID NO: 54), LAVTAGMGAA (SEQ ID NO: 55), LAPNSTTNNA (SEQ ID NO: 56), LANSTSRIDA (SEQ ID NO: 57), LAVASHTNNA (SEQ ID NO: 58), LARSHQEIPA (SEQ ID NO:59), LALNTTKDIA (SEQ ID NO:60), LAIIDATKNA (SEQ ID NO:61), LAHISQTNA (SEQ ID NO:62), LASNSAHITA (SEQ ID NO:63), LASTHQSNNA (SEQ ID NO:64), LAKTPNLTSA (SEQ ID NO:65), LASNTPALSA (SEQ ID NO:66) and LASPGATTNA (SEQ ID NO:67), and (ii) a nucleic acid comprising a nucleotide sequence encoding a human cystic fibrosis transmembrane conductance regulator (CFTR) protein or a biologically active truncated CFTR protein lacking amino acids 708-759 of the human CFTR protein sequence operably linked to a promoter.

[0126] The rAAV gene therapy vectors of the invention comprising the subject variant capsid proteins can be administered to a patient in a variety of ways to achieve and maintain therapeutically effective levels of gene product (e.g., CFTR or a biologically active portion thereof) in target cells (e.g., upper respiratory tract cells). In some aspects, infectious rAAV is administered to a subject (e.g., a subject with cystic fibrosis) in one or more doses, each dose comprising about 1x10 13 - Approx. 1x10 15 Vector genome (vg), about 1x10 13 - Approx. 1x10 14 vg, about 1x10 14 - Approx. 1x10 15 vg or about 1x10 15 - Approx. 5x10 15 vg. In some preferred aspects, each dose contains about 1x1014 vg or about 1x10 15 In other aspects, at least one dose of about 10 12 -10 14 Vector genomes (vg) / kg of rAAV to treat lung disease. In a related aspect, about 1x10 11 - Approx. 1x10 14 vg / kg, about 1x10 12 - Approx. 9x10 13 vg / kg, about 1x10 12 vg / kg-approx. 9x10 12 vg / kg, preferably about 2x10 12 vg / kg-approx. 3x10 12 vg / kg, more preferably about 2.6x10 12 vg / kg, about 2.7x10 12 vg / kg, about 2.8x10 12 vg / kg, about 2.9x10 12 vg / kg about 3.0x10 12 vg / kg or about 3.1x10 12 vg / kg.

[0127] In some aspects, treatment comprises administering to a subject no more than a single dose and effective to achieve sustained and maintained therapeutic concentrations of a gene product (e.g., CFTR or a biologically active portion thereof). In a related aspect, treatment comprises administering to a human (e.g., a human with cystic fibrosis) a dose of about 1 x 10 13 - Approx. 1x10 15 Plaque forming units (pfu), viral particles (vp) or viral genomes (vg) of rAAV are administered in no more than a single dose, and the rAAV comprises a heterologous nucleic acid of a subject variant capsid protein and a coding gene product. In other aspects, the dosage treatment can be a multiple dose schedule. Kay et al. have previously described methods for administering AAV vectors to people (2000, Nat Genet 24: 257-261), the entire contents of which are incorporated herein by reference. In some preferred embodiments, infectious rAAV is administered to a subject by pulmonary, intrabronchial, intranasal, intratracheal and / or intrabronchial administration. In some preferred embodiments, infectious rAAV is administered by inhaling an aerosol suspension comprising rAAV (e.g., via a nebulizer).

[0128] For the purposes of the present invention, the disclosure herein provides an isolated nucleic acid comprising a nucleotide sequence encoding a subject variant AAV capsid protein as described above. The isolated nucleic acid can be an AAV vector, such as a recombinant AAV vector.

[0129] The disclosure herein further provides host cells, such as, but not limited to, isolated (genetically modified) host cells comprising subject nucleic acids. The host cells according to the present invention disclosed herein can be isolated cells, such as cells from in vitro cell cultures. As described herein, such host cells are used to produce subject rAAV variant virions. In one embodiment, such host cells are stably genetically modified with nucleic acids. In other embodiments, host cells are transiently genetically modified with nucleic acids. Such nucleic acids are stably or transiently introduced into host cells using established techniques, including but not limited to electroporation, calcium phosphate precipitation, liposome-mediated transfection, etc. For stable transformation, nucleic acids will generally further include a selection marker, such as, any of several well-known selection markers, such as neomycin resistance, etc. Such host cells are produced by introducing nucleic acids into any of a variety of cells, such as mammalian cells, including, for example, mouse cells and primate cells (e.g., human cells). Exemplary mammalian cells include, but are not limited to, primary cells and cell lines, wherein exemplary cell lines include, but are not limited to, 293 cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1 / 2 fibroblasts, CHO cells, and the like. Exemplary host cells include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 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), RAT1 cells, mouse L cells (ATCC No. CCL1.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like. Baculovirus can also be used to infect insect cells such as Sf9 cells to prepare host cells that produce AAV (see, e.g., U.S. Patent No. 7,271,002; U.S. Patent Application Serial No. 12 / 297,958). In some embodiments, in addition to the nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein as described above, the genetically modified host cell comprises a nucleic acid comprising a nucleotide sequence encoding one or more AAV rep proteins. In other embodiments, the host cell further comprises a rAAV variant vector.Such host cells can be used to produce rAAV variant virions. Methods of producing rAAV virions are described in, for example, U.S. Patent Publication No. 2005 / 0053922 and U.S. Patent Publication No. 2009 / 0202490.

[0130] In some embodiments of the variant rAAV vectors disclosed herein, the nucleotide sequence encoding the gene product of interest is operably linked to a constitutive promoter. Suitable constitutive promoters include, for example, cytomegalovirus promoter (CMV) (Stinski et al., (1985) Journal of Virology 55(2):431-441), CMV early enhancer / chicken p-actin (CBA) promoter / rabbit β-globin intron (CAG) (Miyazaki et al. (1989) Gene 79(2):269-277, C SB (Jacobson et al. (2006) Molecular Therapy 13(6):1074-1084), human elongation factor 1α promoter (EF1α) (Kim et al. (1990) Gene 91(2):217-223), human phosphoglycerate kinase promoter (PGK) (Singer-Sam et al. (1984) Gene 32(3):409-417, mitochondrial heavy chain promoter (Loderio et al. (2012) PNAS 109(17):6513-6518), ubiquitin promoter (Wulff et al. (1990) FEBS Letters 261:101-105). In other embodiments, the nucleotide sequence encoding the gene product of interest is operably linked to an inducible promoter. In some cases, the nucleotide sequence encoding the gene product of interest is operably linked to a tissue-specific or cell-type-specific regulatory element. For example, in some cases, the nucleotide sequence encoding the gene product of interest is operably linked to a lung-specific regulatory element, for example, a regulatory element that confers selective expression of the operably linked gene in lung cells. Lung-specific promoters include, but are not limited to, surfactant protein B (SPB) gene promoter and surfactant protein C (SPC) promoter.

[0131] Also provided herein is a pharmaceutical composition comprising: a) rAAV comprising a subject variant AAV capsid protein and a heterologous nucleic acid encoding one or more gene products; and b) a pharmaceutically acceptable carrier, diluent, excipient or buffer. In some preferred embodiments, the nucleic acid comprises a nucleotide sequence encoding a therapeutic gene and / or encoding an interfering RNA. In some embodiments, a pharmaceutically acceptable carrier, diluent, excipient or buffer is suitable for use in a human or non-human patient. Such excipients, carriers, diluents and buffers include any pharmaceutical agent that can be administered without excessive toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts may be included, such as inorganic acid salts, such as hydrochlorides, hydrobromides, phosphates, sulfates, etc.; and salts of organic acids, such as acetates, propionates, malonates, benzoates, etc. In addition, auxiliary substances such as wetting agents or emulsifiers, surfactants, pH buffer substances, etc. may be present in such a vehicle. A variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been described in detail in a variety of publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HC Ansel et al., ed., 7th edition, Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AK Kibbe et al., ed., 3rd edition Amer. Pharmaceutical Assoc..

[0132] In some embodiments, the pharmaceutical composition comprises 1x10 8 -1x10 15 Vector particles or vector genomes, 1x10 10 -1x10 13 Vector particles or vector genomes, or about 1x10 10 , about 2x10 10 , 3x10 10 , about 4x10 10 , about 5x10 10 , about 6x10 10 , about 7x10 10 , about 8x10 10 , about 9x10 10, about 1x10 11 , about 2x10 11 , about 3x10 11 , about 4x10 11 , about 5x10 11 , about 6x10 11 , about 7x10 11 , about 8x10 11 , about 9x10 11 , about 1x10 12 , about 2x10 12 , about 3x10 12 , about 4x10 12 , about 5x10 12 , about 6x10 12 , about 7x10 12 , about 8x10 12 , about 9x10 12 or about 1x10 13 Vector particles or vector genomes. In some aspects, the pharmaceutical composition comprises about 1x10 11 - Approx. 1x10 12 Vector particle or vector genome. Example The following examples illustrate preferred embodiments of the present invention, but are not intended to limit the scope of the present invention in any way. Although the present invention has been described with respect to its preferred embodiments, various modifications thereof will be apparent to those skilled in the art upon reading this application.

[0134] Example 1 Directed evolution screening was used to identify AAV capsid variants that confer more efficient primate lung transduction and enhanced gene delivery efficiency to primate lung upper airway cells following intratracheal aerosol administration to nonhuman primates (NHPs). The selection process included the use of delivery to NHP lungs in vivo and human lung cultures in vitro.

[0135] method Evolution of therapeutic vectors A directed evolution process was used to discover AAV capsid variants that were able to broadly transduce upper airway cells in primate lungs following aerosol administration ( Figure 1). In brief, a library of approximately 1 billion unique synthetic variant AAV capsid sequences was generated from 37 different proprietary sub-libraries using various molecular biology techniques and several different AAV serotypes as templates. The library was packaged in HEK293T cells to produce viral particles so that each viral particle consisted of a synthetic capsid shell surrounding a viral genome encoding the same capsid. The variants within the library were then subjected to in vivo and in vitro selection pressure techniques in NHP and human cell cultures to mimic clinical gene therapy treatments. All synthetic libraries were injected for the first round of selection. After harvesting DNA from lung tissue or cell culture, the capsid genome amplified from the tissue was then packaged as described above as the starting library for the next round of selection. The process was performed for a total of five cycles.

[0136] A motif is declared a "hit" when certain selection criteria are met: (1) the motif represents a certain percentage of the sequenced population in two or more consecutive rounds of selection, or (2) the motif represents a certain percentage of the sequenced population in one or more rounds of selection.

[0137] The point at which the model system was switched from in vivo NHP to in vitro proximal airway organotypic culture was based on the results of sequencing analyses performed after the second and third rounds of NHP delivery ( Figure 2 Briefly, transition to an in vitro human ALI model system occurs when (1) the most frequent hits represent a certain percentage of the sequenced population, and (2) less than a certain percentage of the sequenced clones represent unique sequences.

[0138] Selection was considered complete when the following selection completion criteria were met: fewer than two new “hits” were identified in the round, and (1) the combined “hits” represented a certain percentage of the sequenced population and / or (2) a single “hits” represented a certain percentage of the population.

[0139] Cell lines and library production HEK293T cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA) and 1% penicillin / streptomycin (Invitrogen, Carlsbad, CA) at 37°C and 5% CO2. Virus libraries were generated in HEK293T cells using triple transfection and purified by iodixanol gradient centrifugation. 19，21 Viruses were purified by filtration with Amicon. DNase-resistant genomic titers were determined via quantitative PCR (qPCR) as described previously. 19，21 Intratracheal injection and tissue harvest For each round of selection, a single male cynomolgus monkey (Macaca fascicularis) aged 4-6 years and weighing 5.5-7.1 kg was dosed. The animals were anesthetized with 10 mg / kg ketamine and 15 μg / kg dexmedetomidine delivered intramuscularly (IM). 5 mL of the library was pre-compounded with 1.75 mg / mL human intravenous immunoglobulin (IVIG) and administered as described below. Each animal was intubated with a 5 mm endotracheal tube with the tip at the level of the clavicle (approximately 5 cm above the carina) and its position was confirmed by fluoroscopy. The animals were placed in a chair in a sitting position for administration.

[0140] For the first round of selection, the nebulizer device was connected to the distal end of the endotracheal tube, and a bird brand respirator was used to deliver respiration at a speed of 15 ± 1 breaths / minute with a pressure of 20 cm H2O. For the second and third rounds of selection, the AeroEclipse II nebulizer device was connected to the distal end of the endotracheal tube, and a bird brand respirator was used to deliver respiration at a speed of 12-14 breaths / minute with a pressure of 15-20 cm H2O. After administration, each animal was extubated and IM received 0.15 mg / kg atipamezole to reverse sedation. Before being placed back in its home cage, the animals were visually monitored until they fully recovered from anesthesia.

[0141] Euthanasia was performed on day 15 ± 1 by trained veterinary staff using 100 mg / kg sodium pentobarbital delivered intravenously. Lungs including trachea were removed and dissected as detailed below. DNA was isolated from upper respiratory tract cells and stored at -20°C until viral genome amplification.

[0142] Upper respiratory epithelial cell isolation NHP or human lungs were flushed with phosphate buffered saline (PBS; Gibco, Carlsbad, CA) to remove excess mucosal secretions and residual blood. The trachea and primary, secondary and tertiary bronchi were separated from the parenchymal lung tissue. Excess supporting tissue and lymph nodes were removed. The trachea and bronchi were cut into 2-4 cm pieces and placed in an enzyme solution to release epithelial cells (pronase, 1.4 mg / mL and DNase, 0.1 mg / mL). The trachea and primary bronchi were treated together, and the secondary and tertiary bronchial cell solutions were incubated at 4°C for 48 hours, with the tubes inverted twice a day. The enzymes were inactivated by adding up to 10% of the total volume of FBS. The sections were then cut longitudinally and the epithelial layer was scraped off the cartilage. The cells were collected and centrifuged at 300 x g for 10 minutes. The cell pellet was rinsed twice with PBS. The pellet was resuspended in respiratory epithelial cell basal medium (ATCC, Manassas, Virginia) with supplements containing 5% FBS, and the cells were plated into 10 cm dishes treated with tissue culture to allow fibroblasts to adhere to the wall, thereby further purifying the cell isolate into respiratory epithelial cells. Two to four hours after inoculation, the medium with non-adherent cells was collected and centrifuged at 300 x g for 10 minutes. The pelleted cells were lysed to extract library DNA or resuspended in PBS, counted and inoculated for in vitro selection rounds.

[0143] In vitro library transduction Human ALI cultures were inoculated on human placental collagen type IV (Sigma) and transduced 30 days later. On the day of transduction, three inserts were incubated with trypsin-EDTA 0.05% (ThermoFisher) at 37°C for 10 minutes. Trypsin was inactivated using Defined Trypsin Inhibitor (ThermoFisher). Cells were collected from the chambers and counted on a hemocytometer. The average number of cells per chamber was determined and used to calculate the total viral genome required for each chamber. Before viral transduction, mucus produced by the culture was removed by washing with PBS. A multiplicity of infection (MOI) of 50,000 was used for the 4th round, and 10,000, 25,000 and 50,000 for the 5th round. Each round was run in two parallel groups, one group pre-compounded the library with a 1:10 dilution of human IVIG for 30 minutes before cell administration, and the other group did not pre-compound IVIG. Cells were exposed to the library tips for 24 hours. Four days after infection, DNA was isolated from the cultures and stored at -20°C until viral genome amplification.

[0144] DNA quantification, DNA amplification and sequencing analysis DNA was isolated from the above cells using a DNeasy Blood & Tissue kit (Qiagen). The AAV library genome was quantified by digital droplet polymerase chain reaction (ddPCR). The AAV variant cap gene was amplified by PCR. The cap gene was inserted into the pSub2 library packaging plasmid using NotI and HindIII. The Cap gene was then sequenced by a third-party DNA sequencing agency. The sequencing files were analyzed using Geneious software (Biomatters).

[0145] result Pilot study of delivery device parameters and upper airway cell isolation Two delivery devices, Valley Biosystem's internal nebulizer and AeroEclipse II breath-driven nebulizer (Trudell Medical International), were used to enable downstream compatibility with a variety of clinically translatable devices. Two delivery devices were evaluated in a pilot study of delivering Evans blue dye to ensure that ventilation parameters resulted in adequate distribution to all lobes and alveolar sacs. Valley Biosystem's internal nebulizer showed good distribution to all lobes including the alveolar compartment, with thicker dye observed in the dependent lobe. While the first round of selection, additional method development and respiratory delivery device testing were performed to successfully transform and develop the AeroEclipse II breath-driven nebulizer to deliver aerosolized AAV particles to intubated NHPs. Through dye distribution studies, excellent, non-dependent delivery to all six NHP lobes including two anterior lobes was demonstrated. AeroEclipse II nebulizer was then used for the 2nd and 3rd rounds of selection.

[0146] The upper airway cell isolation protocol was optimized using a total of 6 NHP lungs. Protocol optimization resulted in high yield and purity of cells from trachea and primary, secondary, and tertiary bronchi isolated from all three sets of NHP lungs used during therapeutic vector evolution.

[0147] Library generation and production Before the initiation of round 1 of the therapeutic vector evolution program, all 37 capsid libraries were synthesized, manufactured, and characterized. As shown in FIG3a , the diversity of the plasmid libraries was estimated to include approximately 1×10 6 To>1x10 8Unique variants, reaching the total diversity of> 1 billion genetic variation sequences. This represents a high-quality, highly diverse AAV variant starting library. Next, complete the production of each individual library, to produce enough materials for the first round of selection. As shown in Figure 3b, all libraries are manufactured at a level sufficient to produce materials for in vivo therapeutic vector evolution selection. After production and before the library is used, the presence of non-functional mutations (that is, frameshift mutations or stop codons) from the sequences of all libraries and the frequency of unique sequences are assessed by Sanger sequencing. Most libraries have a minimum frequency of non-functional mutations, and all libraries are included in the first round of use to NHP.

[0148] Aerosol delivery to NHP lungs For the first round of selection, all libraries were combined and successfully administered to a single NHP via a single-dose aerosol administration using Valley Biosystems' in-house nebulizer system. Prior to administration, the library was incubated with 1.75 mg / mL human IVIG. This represents a higher but physiologically relevant lung mucus concentration of human NAbs. The 1.7x10 12 The library dose of vg represents a dose of about 1 / 100 of the current maximum feasible dose based on manufacturing considerations. Therefore, this represents a strict selection pressure to enable the discovery of vectors capable of transducing upper respiratory tract cells in the trachea and bronchi. NHP lungs were harvested two weeks after administration. Cells were isolated from lungs in separate trachea / primary bronchus and secondary / tertiary bronchus samples, and DNA was isolated from the cells. High total cell yields and high acetylated tubulin+ cell populations were obtained from both regions. The process was repeated for two additional rounds using progressively lower viral library doses.

[0149] After DNA isolation from each round of selection, AAV viral library genomes were quantified by ddPCR to confirm successful targeting of the library vectors to the cell type of interest. There was a dose-dependent decrease in the amount of viral genomes present in the upper respiratory tract, corresponding to the lower doses administered with each round ( Figure 4 ).

[0150] In addition to being quantified by ddPCR, capsid gene amplification from tissues represents that the library vector is successfully positioned to the cell type of interest. The capsids selected for each round of amplification from NHPs are cloned into AAV library packaging plasmids for sequence analysis and initiation of subsequent selection rounds. Each clone in the library is sequenced to determine the frequency of variants within the population. A minimum of 90 clones from trachea / primary bronchus and secondary / tertiary bronchial samples are sequenced in each round. The presence of motifs in the sequencing data of the variants is assessed. Based on the presence of uniform variations (e.g., specific point mutations or specific peptide insertion sequences in a consistent position in the capsid) that appear in multiple sequences, variants are grouped into motifs. If the motif represents a certain percentage of a sequencing population in two or more consecutive rounds of selection or a certain percentage of a sequencing population in one or more rounds of selection, the motif is designated as a hit.

[0151] Two motifs in the trachea / primary bronchus region (point mutation #1, peptide insertion #1 (SEQ ID NO:40) and one motif in the secondary / tertiary bronchus region (peptide insertion #2 (SEQ ID NO:41) were designated as hits after round 3 in vivo ( Figure 5 In addition, two additional motifs of interest (peptide insertion #3 (SEQ ID NO:42), peptide insertion #4 (SEQ ID NO:43) were identified as motifs that will be monitored to determine if one or both can be designated as hits after further in vitro therapeutic vector evolution rounds. Point mutation #1 in the trachea / primary bronchus region and peptide insertion #2 in the secondary / tertiary bronchus region were the most frequent hits in each region, and each of these hits represented a percentage of the sequenced AAV population in that region ( Figure 5 ). A certain percentage of the sequenced AAV clones represented unique AAV sequences in the total sequenced population. Therefore, based on the sequencing analysis performed in round 3 of the therapeutic vector evolution program, the model system was switched from in vivo NHP to in vitro proximal airway organotypic culture for the remainder of the selection process.

[0152] Apical delivery to human ALI cultures For in vitro selection rounds 4 and 5, cells from three human lung donors were pooled and cultured in a transwell system as a multilayered air-liquid interface (ALI) culture. The cells formed striated layers and produced mucus. Prior to library transduction, the cell culture composition of human ex vivo lung epithelial airway cultures was assessed 30 days after thawing by immunocytochemical analysis using antibodies against acetylated tubulin (a marker for ciliated cells), cytokeratin 5 (a marker for basal cells), and mucin (a marker for goblet cells).

[0153] For rounds 4 and 5, the AAV library was administered to the apical side of the proximal airway organotypic culture system at an MOI of 50,000 and 10,000, respectively. Similar to the in vivo selection section, the genome of the AAV viral library was quantified by ddPCR after DNA isolation from each round in vitro to confirm successful targeting of the library vector to the cell type of interest. Unlike the in vivo selection section, no dose-dependency was observed between rounds ( Figure 6 a), which may be due to the high MOIs used in vitro. In addition, preincubation with human IVIG did not appear to have a significant effect on the presence of the genome after transduction ( Figure 6 b).

[0154] As before, the capsid gene amplification from the tissue represents that the library vector is successfully positioned to the cell type of interest. The capsid amplified from each round of in vitro selection is cloned into the AAV library packaging plasmid again for sequence analysis and to initiate subsequent selection rounds. Each clone in the library is sequenced again to determine the frequency of variants in the colony. Each round is sequenced from a minimum of 89 clones with and without human IVIG conditions.

[0155] The hits identified during the in vivo selection portion (point mutation #1, peptide insertion #1, peptide insertion #2) remained at relatively high frequencies after two rounds of in vitro selection ( Figure 7 ). One of the variants of interest (peptide insertion #3) decreased in frequency and never met the criteria for being designated as a hit. Another variant monitored after round 3 (peptide insertion #4) did meet the criteria for being designated as a hit after round 4, but its frequency also decreased in round 5 ( Figure 7 After round 4, two additional motifs (peptide insertion #5 (SEQ ID NO:44), peptide insertion #6 (SEQ ID NO:45) were assigned as hits. No new hits were identified in round 5, and the combined hits represented greater than 50% of the sequenced population ( Figure 7 ). Therefore, based on the sequencing analysis performed on round 5 of the therapeutic vector evolution program, the discovery program was considered complete.

[0156] result After a single round of intratracheal aerosol administration of the library using a nebulizer, AAV capsids were successfully amplified from isolated upper respiratory tract cell populations from trachea and primary, secondary and tertiary bronchi isolated from three sequential rounds of selection in NHPs. Human in vitro ALI cultures were used for two subsequent rounds of selection. After successful amplification of the viral genome from NHP lungs or human ALI cultures, individual clones in the library were sequenced. Each sequence was grouped into motifs based on the presence of uniform variations and evaluated based on the frequency and diversity of variations in sequencing analysis. After analysis, six variant sequences emerged as hits designated for further characterization. These six variants (AAV102-AAV107, SEQ ID Nos: 68-73) are capsids based on AAV2, each of which contains a peptide inserted into the loop region and a V708I amino acid substitution.

[0157] Table 1 below provides a summary of the frequencies of peptide insertion motifs for each of the selection rounds described above: Table 1 T / 1° = trachea / primary bronchus 2 / 3° = Secondary / tertiary bronchus IVIG = human intravenous immunoglobulin (1:10) Sequenced clones identified from directed evolution screens include variant capsids that have only peptide insertions (e.g., SEQ ID No: 40, 41, 42, 43, 44, or 45) and are otherwise identical to SEQ ID NO: 2, as well as variant capsids that have peptide insertions in combination with various amino acid substitutions. Table 2 provides a summary of amino acid substitutions (numbered relative to SEQ ID NO: 2) tolerated in sequenced clones identified from directed evolution screens in combination with the inserted peptides of Table 1 (in each case, the variant AAV capsid protein is otherwise identical to SEQ ID NO: 2): Table 2 Example 2 The six variant capsid sequences identified above that exhibited a preference for transduction of upper respiratory epithelial cells (AAV102-AAV107; SEQ ID Nos:68-73) were characterized in vitro at a range of multiplicities of infection (MOIs) in non-human primate (NHP) and human ex vivo lung upper respiratory tract air-liquid interface (ALI) cultures.

[0158] Briefly, recombinant AAV (rAAV) were produced, each containing cap variant sequences of AAV102-AAV107 containing a reporter gene cassette with a ubiquitous promoter (CMV enhancer, chicken β-actin promoter and rabbit β-globin splice acceptor site, CAG) driving EGFP. Transduction of six variant AAV capsids, AAV101 and native serotypes AAV2 and AAV5 was evaluated in vitro at four multiplicity of infection (12,500, 25,000, 50,000 and 100,000).

[0159] A first generation capsid variant (AAV101) engineered for enhanced transduction of respiratory epithelium in in vitro organotypic ALI cultures has been shown to outperform naturally occurring AAV capsids (see SEQ ID NO: 12 of U.S. Patent Publication No. 2021 / 0395772, the contents of which are incorporated herein by reference). However, additional discoveries were made to identify vectors that can effectively transduce both in vitro and in vivo at levels sufficient to achieve clinical benefit in cystic fibrosis (CF).

[0160] method AAV Manufacturing rAAV containing each of the variant capsid proteins AAV101-AAV107 and the native serotypes AAV2 and AAV5 was produced using a triple plasmid transfection method using commercially available transfection reagents and human embryonic kidney 293 (HEK293) cells cultured in shallow dishes. The transfected cells were harvested along with the supernatant and then treated with nuclease and clarified through a 0.2 μm filter. The clarified harvest was purified using affinity chromatography (AVB Sepharose HP, Cytiva Life Sciences) and its buffer was exchanged into DPBS with 0.005% Pluronic F68. After 0.2 μm filtration, the bulk drug substance (BDS) was aliquoted into frozen vials (Corning) and stored at -80°C.

[0161] Digital droplet PCR (ddPCR) for AAV titer analysis Viral genome titers were determined by ddPCR. Test samples were diluted and treated with DNase, then further diluted into DPBS with 0.02% Pluronic F68, ddPCR Supermix, and primer / FAM-labeled probes corresponding to the SV40 PolyA sequence. 20 μL of sample was divided into droplets using a Bio-Rad automatic droplet generator, subjected to PCR, and then read on a Bio-Rad QX200 droplet reader, which measures the fluorescence signal of each droplet individually. Data were analyzed using Bio-Rad QuantaSoft software, which uses Poisson statistical analysis of positive and negative droplets to provide absolute quantification of target sequences. No template control was used to set the negative baseline of the sample. An internal ddPCR reference standard virus was used as a reference standard control.

[0162] Upper respiratory epithelial cell isolation and culture Airway epithelial cells were isolated from trachea and primary to tertiary bronchi from transplant-rejected NHPs and human donor lungs (DonorNetwork West) according to published protocols (Karp et al., 2002). The isolated cells were frozen and stored in liquid nitrogen.

[0163] Airway epithelial cells were thawed and placed into transwell chambers (0.4 μm, 0.32 cm 2 , Corning, Corning, NY) on human placental collagen IV (60 μg / mL, MilliporeSigma, Burlington, MA) in respiratory epithelial cell basal medium with growth supplements in a bronchial epithelial cell growth kit (ATCC, Manassas, VA). Two days after inoculation, the medium was aspirated from the cuvette and basal chambers. The basal chamber was supplemented with PneumaCult ALI basal medium (Stem Cell Technologies, Vancouver, Canada) with provided growth supplements. The basal medium was replaced every 2-3 days. The liquid on the top side of the transwell was aspirated every day until an air-liquid interface (ALI) was obtained. The cells were allowed to mature for 30 days before cell characterization and transduction.

[0164] AAV transduction After at least 30 days in culture, the cells were washed thoroughly to remove accumulated mucus. Three chambers of each species were harvested and cell counts were performed. Based on the AAV titer and the average cell count of each species, the virus volume was diluted to reach an MOI of 12,500, 25,000, 50,000, and 100,000. The volume was balanced using diluent to obtain a transduction volume of 100 μl per chamber of each species. Human cells were transduced apically and basally, and NHP cells were transduced apically with AAV after mucus washing, n=1 for each condition. The liquid on the chamber was aspirated 24 hours after infection and the basal chamber medium was replaced. Cells were fixed seven days after infection for immunocytochemistry.

[0165] Immunocytochemistry Cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature. Cells were then imaged using a Zeiss Axio Observer D.1 fluorescence microscope to visualize EGFP viewed from top to bottom on the cells. The chambers containing the cells were then embedded in optimal cutting temperature medium (OCT, ThermoFisher, Waltham, MA) and sectioned at 20 μm on a CryoStar cryostat. Slides containing chamber cross sections were blocked in 2% goat serum, 5% bovine serum albumin, and 0.02% triton X-100 in PBS for 1 hour at room temperature. The slides were then incubated with the primary antibody for 2 hours at room temperature, washed three times with 0.02% triton X-100, and incubated with the secondary antibody for 1 hour at room temperature. The slides were then counterstained with DAPI (nuclear stain) for five minutes at room temperature, washed three times, and sealed with a coverslip using Prolong Gold antifade dipsticks (ThermoFisher). The cells were then imaged using a Zeiss AxioObserver D.1 fluorescence microscope.

[0166] Digital droplet polymerase chain reaction (ddPCR) for AAV transcript detection RNA was extracted from each chamber using the Qiagen RNeasy Plus Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA was quantified and 2 μg of cDNA was generated using the iScript cDNA synthesis kit according to the manufacturer's instructions (40 μL total volume, Bio-Rad, Hercules, CA). Dilutions of cDNA were prepared at 1:10 and 1:100 in water to a total volume of 50 μL, and 2 μL was added to each assay plate well. The master mix consisted of 2× Supermix ddPCR up to a total volume of 18 μL per well. TM(without dUTPs, Bio-Rad) and a ddPCR-compatible primer / probe mix (HPRT1HEK-Housekeeper, 20× ID Bio-Rad and SV40 FAM ID (Furuta-Hanawa, Yamaguchi, & Uchida, 2019)) were prepared. The SV40 primer / probe is an alternative for measuring EGFP transgene transcript levels because the SV40 polyadenylation site is included as part of the virally delivered transgene cassette. The master mix (Mastermix) and cDNA were combined in ddPCR according to the manufacturer's instructions. TM 96-well assay plates and loaded into the QX200 TM AutoDG Droplet Digital TM PCR system. Droplets were generated and the plate was transferred to a C1000 Touch thermal cycler to run PCR. The ThermoCycler conditions were as follows: 94°C for 10 minutes, 94°C for 30 seconds, 60°C for 1 minute (repeat steps 2-3, 39x), 98°C for 10 minutes, hold at 12°C. The plate was then transferred to a QX200 TM The PCR products were analyzed in the QuantaSoft software at a threshold of 3000 and then transferred to an Excel file where further analysis was done. Using the lowest dilution with a viable read, a graph was created using the ratio of SV40 to housekeeping values.

[0167] Mean fluorescence intensity calculation For each ALI transduced well, 3 images were taken per well using the 10× objective of a Zeiss Axio Observer D.1 fluorescence microscope. The imaging area was selected in a consistent and unbiased pattern from one well to another. The exposure time for each image was set using the standard AAV2 MOI 25,000 transduction well. After imaging, all images were processed using ImageJ software. Background fluorescence was subtracted, and the “Measure Mean Gray Value” tool was then used to generate MFI values. The three images for each well were averaged to obtain the final MFI value for each well. Each well within a condition was then averaged, and the standard deviation was calculated to generate the final chart (Figure 3b).

[0168] result AAV production of novel capsids rAAV containing variant capsid proteins and wild-type serotypes were manufactured by triple transfection in HEK293 cells cultured on CellSTACKs and purified using affinity chromatography. After supernatant and cell harvest, upstream productivity (reported in units of log viral genome (vg) / square centimeter of cell culture surface area) was measured by ddPCR analysis (Table 1, upstream productivity). All new capsids were able to package GFP payloads and showed productivity similar to that of other AAV vectors produced using a shallow dish production process. After affinity purification and buffer exchange, downstream yields (reported as % of recovered vgs) were determined by ddPCR (Table 3, downstream yields). Table 3 also shows the initial packaging and yield data of AAV101-EGFP for productivity and yield comparison.

[0169] Table 3 *relative to AAV2 capsid of SEQ ID NO: 2 Transduction efficiency of new AAV variants The transduction efficiency of the six new variants identified in Example 1 was compared to AAV controls: AAV2, AAV5, and AAV101. Each capsid contained a cassette with a ubiquitous promoter (CAG) driving EGFP. Human and NHP upper airway epithelial cells were transduced after mucus removal. Seven days after infection, cells were imaged top-down using an epifluorescence microscope to visualize EGFP.

[0170] For apical transduced human cells, minimal EGFP expression was detected in AAV2 and AAV5 controls. As analyzed by fluorescence microscopy, among the six new variants, AAV102 and AAV103 transduction efficiencies were higher than AAV2, AAV5, and AAV101 at lower doses (Figure 8a). AAV103 transduction at MOI 100,000 was low, likely due to ineffective mucosal washing of the wells. AAV106 transduction was higher than AAV2 and AAV5.

[0171] In the NHP cells transduced at the top, the EGFP pattern was variable in the hole (intrawell). The enhanced transduction of the ring pattern was repeatedly observed around the outer edge of the transwell, while the transduction was limited in the center of the transwell. In general, the new variants showed stronger transduction efficiency than AAV2, AAV5 and AAV101. AAV102 and AAV104 expressed the highest levels of EGFP (Figure 8b). The NHP transduction pattern is not as obvious as in human culture, which may be attributed to the edge effect. For all images shown for NHP transduction, a portion of the edge of the hole was taken to show the transduction.

[0172] For basal transduction of human cells, a different pattern emerged. AAV104 had the highest transduction efficiency, higher than AAV2, AAV5 and AAV101 (Fig. 8c). AAV102 and AAV103 did not show the same superior transduction as when they were apically applied. In general, basal transduction appears to be inferior to apical transduction. Considering that these capsid variants were selected by apical application in NHP in vivo and in human ALI, the relative lack of transduction after basal application compared to apical application is not surprising. NHP in vitro cultures were not transduced by basal application.

[0173] Cell culture composition of human ex vivo lung epithelial airway cultures was assessed by immunocytochemical analysis 30 days after thawing using antibodies against acetylated tubulin (Ac-tub; ciliated cells), cytokeratin 5 (KRT5; basal cells), and mucin (MUC5ac; goblet cells). Cross-sectional analysis of human apical and basal transduced cells was completed and yielded inconclusive results regarding the tropism of specific cell variants within the ALI system (data not shown). More analysis is needed to properly understand the specific tropism of each capsid in the heterogeneous culture system. Additional in vivo studies may also help to better characterize the cell-specific transduction of each capsid.

[0174] Follow-up studies were completed to further examine AAV102 and AAV103, the best performing variants from the initial apical transduction screen in human ALI culture compared to AAV2 and AAV101. Figure 9a shows an immunocytochemical analysis depicting EGFP. The mean fluorescence intensity was averaged and graphed based on data from three images per well and three wells per condition (Figure 9b). Finally, digital droplet PCR determined the transcript levels of each variant (Figure 9c). All three analyses confirmed that AAV102 and AAV103 were superior to AAV2 in the transduction efficiency of respiratory cultures in vitro ex vivo when apical transduction was performed at low MOIs.

[0175] Additional follow-up studies were completed to compare apical transduction of human airway epithelial cells in ALI cultures by each of AAV102-AAV107 compared to AAV2, AAV5, AAV6, and AAV101 in the presence and absence of mucus. Briefly, human lung epithelial cell cultures were grown in an air-liquid interface for at least 30 days. Cells were transduced with AAV102-AAV107 or control capsids carrying an EGFP reporter gene driven by the CAG promoter in the presence or absence of mucus (MOI = 25,000). Seven days after transduction, images of EGFP were taken as a proxy for the transduction efficiency of the capsids. When transduced in the absence of mucus, each of the capsids AAV102-AAV107 demonstrated enhanced transduction efficiency compared to the wild-type AAV serotype, and compared to the AAV101 control ( Fig. 10A and 10B , top panel). Transduction in the presence of mucus reduced the transduction efficiency of each capsid (except AAV5, which showed no transduction in the absence of mucus) ( Fig. 10A and 10B Compare the top graph with the bottom graph).

[0176] In vitro analysis of six of the top AAV capsid variants identified by selection in NHPs using aerosol delivery technology and apical administration to human ex vivo upper airway epithelial ALI cultures according to Example 1 demonstrated that rAAVs with variant capsid proteins were superior in transducing human and NHP ex vivo upper airway epithelial ALI cultures compared to AAV101 and wild-type AAV serotypes. Different variants showed a preference for transduction that depended on the route of administration. AAV102 and AAV103 were better at apical transduction of human ex vivo upper airway epithelial ALI cultures at lower MOIs than AV101 and WT serotypes. In basally transduced human cells, AAV104 had the highest transduction efficiency.

[0177] Example 3 New AAV capsid variants were examined in neutralizing antibody screening within NHPs and in vivo vector characterization.

[0178] Although the materials and methods of the present invention have been described according to preferred embodiments, it will be apparent to those skilled in the art that changes may be applied to the methods described herein without departing from the concept, spirit and scope of the present invention. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present invention.

Claims

1. A variant adeno-associated virus (AAV) capsid protein comprising a peptide insertion relative to a corresponding parent AAV capsid protein, wherein the peptide insertion comprises an amino acid sequence selected from the group consisting of DNTVTRS (SEQ ID NO: 14), SNSVQSI (SEQ ID NO: 15), and NSTRHTD (SEQ ID NO: 16).

2. The variant AAV capsid protein of claim 1, wherein the insertion site is located within the GH-loop of the capsid protein.

3. The variant AAV capsid protein according to claim 2, wherein the insertion site is located between amino acids 570 and 611 of VP1 of AAV2 (SEQ ID NO: 2) or between two adjacent amino acids at the corresponding position in the capsid protein of another AAV serotype, preferably, wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 (SEQ ID NO: 2) or between amino acids corresponding to amino acids 588 and 589 of VP1 of AAV2 (SEQ ID NO: 2) or at the corresponding position in the capsid protein of another AAV serotype.

4. The variant AAV capsid protein of any one of claims 1-3, wherein the peptide insertion comprises an amino acid sequence selected from Y1Y2DNTVTRSY3 Y1Y2SNSVQSIY3 and Y1Y2NSTRHTDY3, wherein each of Y1-Y3 is independently selected from Ala, Leu, Gly, Ser, Thr and Pro.

5. The variant AAV capsid protein of claim 4, wherein the peptide insertion comprises an amino acid sequence selected from LADNTVTRSA (SEQ ID NO: 42), LASNSVQSIA (SEQ ID NO: 43), and LANSTRHTDA (SEQ ID NO: 44).

6. The variant AAV capsid protein of any one of claims 1-5, wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 (SEQ ID NO: 2) or a corresponding position in a capsid protein of another AAV serotype.

7. The variant AAV capsid protein of any one of claims 1-6, wherein the variant AAV capsid protein further comprises one or more amino acid substitutions relative to VP1 of AAV2 (SEQ ID NO: 2) or one or more corresponding substitutions in a capsid protein of another AAV serotype, wherein the one or more amino acid substitutions are optionally selected from Y6F, S16Y, G18E, P30L, R37L, H38Q, V65A, L91I, E99D, R103L, R103C, S109T, V118A, Q120H, E133D, E134Q, P135A, V136G, K137E, T138R, T200 I, D213Y, G220R, P250S, D283E, N312K, T344S, E347D, G376A, P399H, G406E, Q428H, P436H, N449D, P451Q, N469D, D472N, T491I, K532E, K544E, R585K, A591D, A593E, D594N, D608N, H641N, K688R, N705S and V708I, numbered consistently with SEQ ID NO:

2.

8. The variant AAV capsid protein of claim 7, wherein the variant AAV capsid protein comprises a V708I amino acid substitution relative to VP1 of AAV2 (SEQ ID NO: 2) or a corresponding position in a capsid protein of another AAV serotype.

9. The variant AAV capsid protein of any one of claims 1-8, wherein the variant AAV capsid protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:

2.

10. The variant AAV capsid protein of any one of claims 1-9, wherein the variant AAV capsid protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence shown in any one of SEQ ID Nos:70-72.

11. The variant AAV capsid protein of any one of claims 1-10, wherein the AAV capsid protein comprises the amino acid sequence shown in any one of SEQ ID Nos: 70-72.

12. The variant AAV capsid of any one of claims 1-11, wherein the capsid protein confers increased infectivity of infectious rAAV virions to lung cells compared to the infectivity of AAV virions comprising wild-type AAV capsid protein to lung cells.

13. The variant AAV capsid protein of claim 12, wherein the capsid protein further confers increased resistance to neutralization by neutralizing antibodies to infectious rAAV virions compared to AAV comprising the corresponding parent AAV capsid protein.

14. An isolated nucleic acid comprising a nucleotide sequence encoding the variant AAV capsid protein according to any one of claims 1-13.

15. An infectious recombinant AAV (rAAV) virion comprising a variant AAV capsid protein according to any one of claims 1-13.

16. The rAAV virion of claim 15, further comprising a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products.

17. The rAAV virion of claim 16, wherein the gene product is a protein, a small interfering RNA, a microRNA, a short hairpin RNA, or an antisense RNA.

18. The rAAV virion of claim 16 or 17, wherein the nucleotide sequence encoding one or more gene products is operably linked to an expression control sequence.

19. The rAAV of claim 18, wherein the expression control sequence comprises a ubiquitous promoter.

20. The rAAV of claim 18, wherein the expression control sequence comprises a tissue-specific promoter.

21. The rAAV of any one of claims 16-20, wherein the heterologous nucleic acid comprises a nucleotide sequence encoding CFTR or a biologically active portion thereof.

22. A host cell comprising the rAAV according to any one of claims 15-21.

23. A pharmaceutical composition comprising the rAAV according to any one of claims 15-21 and a pharmaceutically acceptable carrier.

24. A method for delivering a heterologous nucleic acid to a lung cell, comprising contacting the lung cell with an rAAV virion, the rAAV virion comprising (i) a variant capsid protein comprising a peptide insertion selected from the group consisting of: HDITKNI (SEQ ID NO: 12), NQDYTKT (SEQ ID NO: 13), DNTVTRS (SEQ ID NO: 14), SNSVQSI (SEQ ID NO: 15), NSTRHTD (SEQ ID NO: 16), TNRTSPD (SEQ ID NO: 17), LAHDITKNIA (SEQ ID NO: 40), LANQDYTKTA (SEQ ID NO: 41), LADNTVTRSA (SEQ ID NO: 42), LASNSVQSIA (SEQ ID NO: 43), LANSTRHTDA (SEQ ID NO: 44), LATNRTSPDA (SEQ ID NO: 45), NO:45) and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products, preferably, wherein the rAAV virion is administered to a mammalian subject via pulmonary, intrabronchial, intranasal, intratracheal and / or intrabronchial administration.

25. The method of claim 24, wherein the peptide insertion comprises an amino acid sequence selected from the group consisting of Y1Y2HDITKNIY3, Y1Y2NQDYTKTY3, Y1Y2DNTVTRSY3, Y1Y2SNSVQSIY3, Y1Y2NSTRHTDY3 and Y1Y2TNRTSPDY3, wherein each of Y1-Y3 is independently selected from Ala, Leu, Gly, Ser, Thr and Pro.

26. The method of claim 25, wherein the peptide insertion comprises an amino acid sequence selected from the group consisting of LAHDITKNIA (SEQ ID NO:40), LANQDYTKTA (SEQ ID NO:41), LADNTVTRSA (SEQ ID NO:42), LASNSVQSIA (SEQ ID NO:43), LANSTRHTDA (SEQ ID NO:44), LATNRTSPDA (SEQ ID NO:45).

27. The method according to any one of claims 24-26, wherein the insertion site is located within the GH loop of the capsid protein, preferably between amino acids 570 and 611 of VP1 of AAV2 (SEQ ID NO: 2) or a corresponding position in the capsid protein of another AAV serotype, preferably, wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 (SEQ ID NO: 2) or between amino acids corresponding to amino acids 588 and 589 of VP1 of AAV2 (SEQ ID NO: 2) or a corresponding position in the capsid protein of another AAV serotype, more preferably between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 (SEQ ID NO: 2) or a corresponding position in the capsid protein of another AAV serotype.

28. The method of any one of claims 24-26, wherein the variant AAV capsid protein further comprises one or more amino acid substitutions relative to VP1 of AAV2 (SEQ ID NO: 2) or one or more corresponding substitutions in a capsid protein of another AAV serotype, wherein the one or more amino acid substitutions are optionally selected from Y6F, S16Y, G18E, P30L, R37L, H38Q, V65A, L91I, E99D, R103L, R103C, S109T, V118A, Q120H, E133D, E134Q, P135A, V136G, K137E, T138R, T200 I, D213Y, G220R, P250S, D283E, N312K, T344S, E347D, G376A, P399H, G406E, Q428H, P436H, N449D, P451Q, N469D, D472N, T491I, K532E, K544E, R585K, A591D, A593E, D594N, D608N, H641N, K688R, N705S and V708I, numbered consistently with SEQ ID NO:

2.

29. The method of claim 28, wherein the variant AAV capsid protein comprises a V708I amino acid substitution relative to VP1 of AAV2 (SEQ ID NO: 2) or a corresponding position in a capsid protein of another AAV serotype.

30. The method of any one of claims 24-29, wherein the variant AAV capsid protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:

2.

31. The method of any one of claims 24-30, wherein the variant AAV capsid protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence shown in any one of SEQ ID Nos: 70-72.

32. The method of any one of claims 24-30, wherein the variant AAV capsid protein comprises or consists of an amino acid sequence shown in any one of SEQ ID Nos: 70-72.

33. The method of any one of claims 24-32, wherein the lung cell is an upper respiratory epithelial cell, an alveolar epithelial cell, a primary, secondary or tertiary bronchial epithelial cell, a tracheal epithelial cell, a ciliated respiratory epithelial cell, an alveolar epithelial type 1 (AECI) or type 2 (AECII) cell, a smooth muscle cell, or an endothelial cell.

34. A method for treating a lung disease, comprising administering to a subject in need thereof a therapeutically effective amount of rAAV virions comprising (i) a variant capsid protein comprising a peptide insertion selected from the group consisting of HDITKNI (SEQ ID NO: 12), NQDYTKT (SEQ ID NO: 13), DNTVTRS (SEQ ID NO: 14), SNSVQSI (SEQ ID NO: 15), NSTRHTD (SEQ ID NO: 16), TNRTSPD (SEQ ID NO: 17), LAHDITKNIA (SEQ ID NO: 40), LANQDYTKTA (SEQ ID NO: 41), LADNTVTRSA (SEQ ID NO: 42), LASNSVQSIA (SEQ ID NO: 43), LANSTRHTDA (SEQ ID NO: 44), LATNRTSPDA (SEQ ID NO: 45), and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products, or a pharmaceutical composition comprising the rAAV virions and a pharmaceutically acceptable carrier.

35. The method of claim 34, wherein the pulmonary disease is selected from the group consisting of chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), cystic fibrosis, pulmonary hypertension, pulmonary hypertension, lung cancer (primary, secondary and metastatic), surfactant deficiency, viral and / or bacterial infections, acute bronchitis, pneumonia (including viral, bacterial and fungal pneumonia), respiratory tract infections (including pharyngitis, croup, aspergillus, coccidiomycosis, Hantavirus pulmonary syndrome and histoplasmosis), chemical and hypersensitivity pneumonitis, tuberculosis and other mycobacterial infections (including but not limited to Mycobacterium avium), sarcoidosis, respiratory syncytial virus, pulmonary edema, Acute respiratory distress syndrome (ARDS), pneumoconiosis (including black lung disease, asbestosis, and silicosis), interstitial lung disease (including sarcoidosis and autoimmune diseases), pulmonary embolism, pleural effusion, pleurisy, mesothelioma, pneumothorax, acute bronchitis, bronchiolitis (including bronchiolitis obliterans), sudden infant death syndrome, sleep apnea, bronchiectasis, bronchopulmonary dysplasia, cryptogenic organizing pneumonia, electronic cigarette use-associated lung injury (EVALI), Middle East respiratory syndrome (MERS), primary ciliary dyskinesia, severe acute respiratory syndrome (SARS), alpha-1-antitrypsin deficiency, asthma, interstitial lung disease, and COVID-19 (coronavirus disease 2019).

36. The method of claim 35, wherein the lung disease is COPD.

37. The method of claim 36, wherein the heterologous nucleic acid comprises a nucleotide sequence encoding a gene for alpha-1-antitrypsin, alpha-1-antichymotrypsin, alpha-1-macroglobulin, matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 12 (MMP12), microsomal epoxidase, CYP1A1, glutathione S-transferase, heme oxygenase-1, TGF-β-1, TNF-α, IL-1 complex, IL-8, IL-13, human leukocyte antigens (HLA-B7 and Bw16), vitamin D binding protein, and / or beta-2-adrenergic receptor, or a biologically active portion thereof.

38. The method of claim 35, wherein the lung disease is IPF.

39. The method of claim 38, wherein the heterologous nucleic acid comprises a nucleotide sequence encoding SFTPA1 (Surfactant A1) and caveolin-1 or a biologically active portion thereof.

40. The method of claim 35, wherein the lung disease is alpha-1 -antitrypsin deficiency.

41. The method of claim 40, wherein the heterologous nucleic acid comprises a nucleotide sequence encoding alpha-1-antitrypsin or a biologically active portion thereof.

42. The method of claim 35, wherein the lung disease is cystic fibrosis.

43. The method of claim 42, wherein the heterologous nucleic acid comprises a nucleotide sequence selected from SEQ ID NO: 30 or SEQ ID NO: 31, or a sequence at least 80%, at least 90%, at least 95% or at least 99% identical thereto.

44. The method of any one of claims 34-43, wherein the rAAV or pharmaceutical composition is administered to the subject by inhalation.

45. The method of claim 44, wherein the rAAV or pharmaceutical composition is administered to the subject by inhalation of an aerosol suspension comprising the rAAV, preferably wherein the aerosol suspension is generated by a liquid nebulizer.

46. ​​An infectious rAAV comprising (i) a variant capsid protein comprising a peptide insertion selected from the group consisting of HDITKNI (SEQ ID NO: 12), NQDYTKT (SEQ ID NO: 13), DNTVTRS (SEQ ID NO: 14), SNSVQSI (SEQ ID NO: 15), NSTRHTD (SEQ ID NO: 16), TNRTSPD (SEQ ID NO: 17), LAHDITKNIA (SEQ ID NO: 40), LANQDYTKTA (SEQ ID NO: 41), LADNTVTRSA (SEQ ID NO: 42), LASNSVQSIA (SEQ ID NO: 43), LANSTRHTDA (SEQ ID NO: 44), LATNRTSPDA (SEQ ID NO: 45) and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene selected from the group consisting of CFTR or a biologically active portion thereof and SERPINA1 or a biologically active portion thereof.

47. The infectious rAAV of claim 46, wherein the nucleotide sequence encodes CFTR or a biologically active portion thereof operably linked to a ubiquitous promoter.

48. The infectious rAAV of claim 47, wherein the nucleotide sequence is selected from SEQ ID NO: 102 or SEQ ID NO: 103 or a sequence that is at least 80%, at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 102 or SEQ ID NO:

103.

49. The infectious rAAV of claim 46, wherein the nucleotide sequence encodes alpha-1-antitrypsin and is operably linked to a ubiquitous promoter.

50. A pharmaceutical composition comprising the rAAV according to any one of claims 46-49 and a pharmaceutically acceptable carrier.

51. The variant AAV capsid protein of any one of claims 1-13, wherein the variant AAV capsid protein confers increased resistance, preferably at least 1.5-fold increased resistance, to neutralization by neutralizing antibodies to infectious rAAV virions compared to AAV comprising the corresponding parent AAV capsid protein, preferably wherein the parent AAV capsid protein is a capsid protein of serotype 2.

52. A variant adeno-associated virus (AAV) capsid protein comprising a peptide insertion relative to a corresponding parent AAV capsid protein,wherein the peptide is inserted into an amino acid sequence selected from the following: QADTTKN (SEQ ID NO: 19), NAVKTDF (SEQ ID NO: 20), TNQTLSA (SEQ ID NO: 21), ENRTTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TNNSKPD (SEQ ID NO: 25), SKLTLNN (SEQ ID NO: 26), VTAGMGA (SEQ ID NO: 27), PNSTTNN (SEQ ID NO: 28), NSTSRID (SEQ ID NO: 29), VASHTNN (SEQ ID NO: 30), RSHQEIP (SEQ ID NO: 31), LNTTKDI (SEQ ID NO: 32), IIDATKN (SEQ ID NO: 33), NHISQTN (SEQ ID NO: 34), SNSAHIT (SEQ ID NO: 35), STHQSNN (SEQ ID NO: 36), KTPNLTS (SEQ ID NO: 37), SNTPALS (SEQ ID NO: 38), SPGATTN (SEQ ID NO: 39), LAQADTTKNA (SEQ ID NO: 47), LANAVKTDFA (SEQ ID NO: 48), LATNQTLSAA (SEQ ID NO: 49), LAENRTTSNA (SEQ ID NO: 50), LAPQQDTTHA (SEQ ID NO: 51), LANATNHVIA (SEQ ID NO: 52), LATNNSKPDA (SEQ ID NO: 53), LASKLTLNNA (SEQ ID NO: 54), LAVTAGMGAA (SEQ ID NO: 55), LAPNSTTNNA (SEQ ID NO: 56), LANSTSRIDA (SEQ ID NO: 57), LAVASHTNNA (SEQ ID NO: 58), LARSHQEIPA (SEQ ID NO: 59), LALNTTKDIA (SEQ ID NO: 60), LAIIDATKNA (SEQ ID NO: 61), LANHISQTNA (SEQ ID NO: 62), LASNSAHITA (SEQ ID NO: 63), LASTHQSNNA (SEQ ID NO: 64), LAKTPNLTSA (SEQ ID NO: 65), LASNTPALSA (SEQ ID NO: 66) and LASPGATTNA (SEQ ID NO: 67).

53. A variant AAV capsid protein according to claim 52, wherein the peptide insertion comprises an amino acid sequence selected from Y1Y2DNTVTRSY3Y1Y2SNSVQSIY3 and Y1Y2NSTRHTDY3, wherein each of Y1-Y3 is independently selected from Ala, Leu, Gly, Ser, Thr and Pro.

54. A variant AAV capsid protein according to claim 52 or 53, wherein the insertion site is located within the GH loop of the capsid protein, preferably between amino acids 570 and 611 of VP1 of AAV2 (SEQ ID NO: 2) or the corresponding position in the capsid protein of another AAV serotype, more preferably between amino acids 587 and 588 or 588 and 589 corresponding to VP1 of AAV2 (SEQ ID NO: 2) or the corresponding position in the capsid protein of another AAV serotype.

55. The variant AAV capsid protein of any one of claims 52-54, wherein the variant AAV capsid protein further comprises one or more amino acid substitutions relative to VP1 of AAV2 (SEQ ID NO: 2) or one or more corresponding substitutions in a capsid protein of another AAV serotype, wherein the one or more amino acid substitutions are optionally selected from Y6F, S16Y, G18E, P30L, R37L, H38Q, V65A, L91I, E99D, R103L, R103C, S109T, V118A, Q120H, E133D, E134Q, P135A, V136G, K137E, T138R, T200 I, D213Y, G220R, P250S, D283E, N312K, T344S, E347D, G376A, P399H, G406E, Q428H, P436H, N449D, P451Q, N469D, D472N, T491I, K532E, K544E, R585K, A591D, A593E, D594N, D608N, H641N, K688R, N705S and V708I, numbered consistently with SEQ ID NO:

2.

56. The variant AAV capsid protein of claim 54, wherein the variant AAV capsid protein comprises a V708I amino acid substitution relative to VP1 of AAV2 (SEQ ID NO: 2) or a corresponding position in a capsid protein of another AAV serotype.

57. The variant AAV capsid protein of any one of claims 52-56, wherein the variant AAV capsid protein is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO:

2.

58. The variant AAV capsid of any one of claims 52-57, wherein the capsid protein confers increased infectivity of infectious rAAV virions to lung cells compared to the infectivity of AAV virions comprising wild-type AAV capsid proteins to lung cells.

59. The variant AAV capsid protein of claim 58, wherein the capsid protein further confers increased resistance to neutralization by neutralizing antibodies to infectious rAAV virions compared to AAV comprising the corresponding parent AAV capsid protein.

60. An isolated nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein according to any one of claims 52-59.

61. An infectious recombinant AAV (rAAV) virion comprising a variant AAV capsid protein according to any one of claims 52-59.

62. The rAAV virion of claim 61, further comprising a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products.

63. The rAAV virion of claim 62, wherein the gene product is a protein, a small interfering RNA, a microRNA, a short hairpin RNA, or an antisense RNA.

64. The rAAV virion of claim 62 or 63, wherein the nucleotide sequence encoding one or more gene products is operably linked to an expression control sequence.

65. The rAAV of claim 64, wherein the expression control sequence comprises a ubiquitous promoter.

66. The rAAV of claim 64, wherein the expression control sequence comprises a tissue-specific promoter.

67. The rAAV of any one of claims 62-66, wherein the heterologous nucleic acid comprises a nucleotide sequence encoding CFTR or a biologically active portion thereof.

68. A host cell comprising the rAAV according to any one of claims 61-67.

69. A pharmaceutical composition comprising the rAAV according to any one of claims 62-67 and a pharmaceutically acceptable carrier.

70. A method for delivering a heterologous nucleic acid to lung cells, comprising contacting the lung cells with an rAAV according to any one of claims 62-67, preferably, wherein the rAAV virions are administered to a mammalian subject via pulmonary, intrabronchial, intranasal, intratracheal and / or intrabronchial administration.

71. The method of claim 70, wherein the lung cell is an upper respiratory epithelial cell, an alveolar epithelial cell, a primary, secondary or tertiary bronchial epithelial cell, a tracheal epithelial cell, a ciliated respiratory epithelial cell, an alveolar epithelial type 1 (AECI) or type 2 (AECII) cell, a smooth muscle cell, or an endothelial cell.

72. A method of treating a pulmonary disorder by administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 69.

73. The method of claim 72, wherein the pharmaceutical composition is administered to the subject by inhalation.

74. The method according to claim 73, wherein the pharmaceutical composition is administered to the subject by inhalation of an aerosol suspension comprising the rAAV, preferably wherein the aerosol suspension is generated by a liquid nebulizer.

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