Adeno-associated virus mutant capsids with improved lung tropism and uses thereof - Patents.com

Abstract

The present disclosure provides a mutant AAV capsid protein that confers tropism to lung cells, and a recombinant adeno-associated virus that comprises the mutant AAV capsid protein, and a pharmaceutical composition that comprises the same, and their use in delivering heterologous nucleic acid to lung cells for the treatment of lung disorders.The recombinant AAV virion that comprises the mutant AAV capsid protein described herein and its pharmaceutical composition can be used in delivering nucleic acid sequences to one or more cells of the lung for the treatment of lung disorders and diseases.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 317,450, filed March 7, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] Submit 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 incorporated herein by reference in its entirety. [Background technology]

[0003] Gene delivery vectors based on adeno-associated virus (AAV) have demonstrated promise in both preclinical disease models and recently in human clinical trials for several disease targets. Because wild-type AAV is non-pathogenic and has no etiological association with any known disease, AAV-based vectors are extremely safe. Furthermore, AAV offers the capacity for highly efficient gene delivery and sustained transgene expression in multiple tissues, including liver, muscle, lung, retina and brain.

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

[0005] AAVs have shown 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; A. M. Maguire et al., 2009; A. Maguire & Simonelli, 2008; Nathwani et al., 2011, 2014; Stroes et al., 2008). However, there are obstacles to gene delivery that may limit the utility of AAVs, such as anti-capsid immune responses, limited transduction of certain tissues, inability to target delivery to certain cell types, and relatively low packaging capacity. In many situations, there is insufficient mechanistic knowledge to effectively implement rational design in the ability to improve AAVs. As an alternative, directed evolution has emerged as a strategy to generate novel AAV variants that meet specific biomedical needs. Directed evolution strategies utilize genetic diversification and selection processes to allow the accumulation of beneficial mutations that gradually improve the function of biomolecules. In this process, the wild-type AAV cap gene is diversified using several different approaches to create a large gene library that is packaged to generate a library of viral particles, and then selective pressure is applied to isolate novel mutants that can overcome the gene delivery barrier.

[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 pig lung in vivo. These previous studies have demonstrated that AAV variants optimized for lung epithelial transduction can result in significant improvements in gene therapy treatment of CF when evaluated in the context of their chosen model. However, evaluation of these vectors demonstrated species differences between pig and human lung epithelium that result in limited transduction of human lung epithelium by vectors evolved for pig lung epithelium transduction.

[0007] There is a need in the art for the development of new AAV variants with improved delivery to human lung cells, such as human lung epithelium, for the treatment of pulmonary disorders. Summary of the Invention [Means for solving the problem]

[0008] Provided herein are mutant adeno-associated virus (AAV) capsid proteins having one or more modifications in amino acid sequence relative to the parent AAV capsid protein, which when present in an AAV virion confer increased infectivity of one or more types of lung cells relative to infectivity of the lung cells by AAV virions containing the unmodified parent AAV capsid protein. Also provided are recombinant AAV virions and pharmaceutical compositions thereof containing the mutant AAV capsid proteins described herein, methods of making the mutant rAAV capsid proteins and virions, and methods of using these rAAV capsid proteins and virions in research and clinical practice, for example, in the delivery of 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 disclosure, mutant adeno-associated virus (AAV) capsid proteins are provided which have one or more modifications in amino acid sequence relative to a parent AAV capsid which, when present in an AAV virion, confer increased infectivity of one or more types of lung cells (e.g., airway epithelial cells including, but not limited to, alveolar epithelial cells, bronchial (primary, secondary or tertiary) epithelial cells or tracheal epithelial cells, ciliated airway epithelial cells, alveolar epithelial type 1 (AECI) or type 2 (AECII) cells, smooth muscle or endothelial cells) relative to infectivity of the lung cells by AAV virions containing the parent AAV capsid protein that does not contain the amino acid sequence modifications.

[0010] In some aspects of the disclosure, recombinant AAV (rAAV) virions are provided, which comprise mutant capsid proteins as described herein, and the rAAV virions exhibit increased infectivity of one or more types of lung cells relative to the infectivity of lung cells by AAV virions comprising the corresponding unmodified parent AAV capsid proteins. In some embodiments, the rAAV virions exhibit increased infectivity of all lung cells relative to AAV virions comprising the parent AAV capsid proteins. In other embodiments, the rAAV virions exhibit increased infectivity of certain lung cell types but not other relative AAV virions comprising the parent AAV capsid proteins. In other words, the rAAV virions exhibit increased infectivity preferentially to certain lung cell types but not to other cell types, e.g., the rAAV exhibits increased infectivity preferentially to one or more lung upper airway cells but not to all cell types. In some aspects, the mutant capsid proteins described herein confer increased resistance to human AAV neutralizing antibodies to these rAAV virions.

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

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

[0013] Also provided is the use of rAAV virions comprising mutant capsid proteins as described herein in a method for delivering heterologous nucleic acid to a target cell (such as a lung cell) by contacting the target cell with the rAAV virion. In some embodiments, the target cell is in vivo, for example, in the lung of an individual in need of treatment for a lung disease. In other embodiments, the target cell is in vitro.

[0014] Also provided are methods of treating pulmonary disease by administering to a subject in need of such treatment an effective amount of a rAAV virion comprising a mutant capsid protein described herein, or a pharmaceutical composition comprising an effective amount of the rAAV virion.

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

[0016] In some embodiments, the mutant AAV capsid protein comprises an insertion of about 5 amino acids to about 20 amino acids (a "heterologous peptide", or "peptide insertion") within the GH loop of the capsid protein, preferably within a surface-exposed region of the GH loop, relative to the corresponding parent AAV capsid protein, and the mutant capsid protein, when present in an AAV virion, confers increased infectivity of retinal cells compared to the infectivity of retinal cells by AAV virions containing the corresponding parent AAV capsid protein. In some embodiments, the peptide is 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), ENRTSN (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), 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),The nucleic acid sequence of the present invention may comprise, consist essentially of, or consist of a sequence selected from the group consisting of 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 embodiments, the mutant AAV capsid protein comprises a peptide insertion within 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, and the mutant capsid protein, when present in an AAV virion, confers increased infectivity of a lung cell compared to the infectivity of the lung cell by an AAV virion comprising the corresponding parent AAV capsid protein. In some preferred embodiments, the mutant AAV capsid protein comprises a peptide insertion within 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, and ... further comprises one or more amino acid substitutions relative to the corresponding parent AAV capsid protein, and further comprises a peptide insertion within 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, and further comprises TSN (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), 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),The present invention relates to a method for the preparation of a VP1 polypeptide comprising: 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 comprising a V708I substitution for VP1 of AAV2.

[0018] Also disclosed herein are methods for the production and / or delivery of rAAVs that contain the mutant AAV capsids disclosed herein. Additionally, provided herein are kits that contain rAAVs that contain the mutant AAV capsids disclosed herein for use in the methods described herein.

[0019] In other embodiments, the AAV virion comprising the mutant capsid protein of the previous paragraph can incorporate any of the embodiments disclosed above or below. Indeed, it is understood that certain features of the invention that are described in the context of separate embodiments for clarity may be provided in combination in a single embodiment. Conversely, various features of the invention that are described in the context of a single embodiment for brevity may be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present invention are specifically embraced by the present invention and are disclosed herein as if each and every combination were individually and expressly disclosed herein. Moreover, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein as if each and every such subcombination were individually and expressly disclosed herein. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 is a diagram illustrating the directed evolution process used to identify mutant AAV capsids that confer improved airway transduction.

[0021] [Diagram 2] Figure 2 is a schematic of the model system decision tree. Decisions were based on sequencing analyses performed after rounds 2 and 3 of NHP delivery.

[0022] [Diagram 3] Figure 3a shows the estimated genetic diversity of the capsid library with a total diversity of over 1 billion variants. Figure 3b shows the productivity of the capsid library, all of which were produced at levels sufficient to produce material for the in vivo therapeutic vector evolution process. The administered viral genome (vg) represents the target dose and does not account for losses associated with the delivery device and route of administration.

[0023] [Figure 4] Figure 4 shows ddPCR quantification of viral genomes present in the trachea and primary, secondary and tertiary bronchi of NHPs administered the capsid library over rounds 1 to 3 of the selection process.

[0024] [Diagram 5]Figure 5 shows the frequency of "hits" in the round 3 sequencing analysis. The sequencing analysis was based on the total frequency within the sequenced population for each region. The frequencies in the left panel (trachea and primary bronchi) 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%. Frequencies in the right panel (secondary and tertiary bronchi) 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] Figures 6A-6B show ddPCR quantification of viral genomes present in human upper airway ALI cultures from administered capsid libraries in the absence and presence of human IVIG for selected rounds 4 (Figure 6a) and 5 (Figure 6b).

[0026] [Figure 7]Figure 7 shows the frequency of "hits" in the sequencing analysis of round 5. The sequencing analysis was based on the total frequency within the sequenced population for selection in the presence and absence of IVIG. The 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) other = 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] [Figure 8] Figure 8A-8C show AAV capsid transduction efficiency in pulmonary upper airway epithelial ALI cultures measured by reporter EGFP expression using fluorescence microscopy. Figure 8a-8b show human (Figure 8a) and NHP (Figure 8b) apical transduction of the six novel variants compared to AAV2, AAV5 and AAV101. Figure 8c shows human basal transduction of the 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] [Figure 9A]Figure 9A-9C Secondary analysis of the top novel capsids compared to AAV2 and AAV101 by immunocytochemistry (Figure 9a, Figure 9b) and ddPCR (Figure 9c). AAV capsid transduction efficiency in pulmonary upper airway epithelial ALI cultures measured by reporter EGFP expression using fluorescence microscopy (Figure 9a). Quantification of micrograph pixel intensity (Figure 9b). Transcript quantification after transduction (Figure 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. [Figure 9B] Figure 9A-9C Secondary analysis of the top novel capsids compared to AAV2 and AAV101 by immunocytochemistry (Figure 9a, Figure 9b) and ddPCR (Figure 9c). AAV capsid transduction efficiency in pulmonary upper airway epithelial ALI cultures measured by reporter EGFP expression using fluorescence microscopy (Figure 9a). Quantification of micrograph pixel intensity (Figure 9b). Transcript quantification after transduction (Figure 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. [Figure 9C] Figure 9A-9C Secondary analysis of the top novel capsids compared to AAV2 and AAV101 by immunocytochemistry (Figure 9a, Figure 9b) and ddPCR (Figure 9c). AAV capsid transduction efficiency in pulmonary upper airway epithelial ALI cultures measured by reporter EGFP expression using fluorescence microscopy (Figure 9a). Quantification of micrograph pixel intensity (Figure 9b). Transcript quantification after transduction (Figure 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] [Figure 10]Figures 10A-B show EGFP expression in human airway epithelial cell cultures following transduction with a specific AAV capsid carrying a nucleic acid containing an EGFP reporter gene operably linked to a CAG promoter in the presence or absence of mucus. Cultures were grown at an air-liquid interface for ≥30 days; 7 days post-infection; -mucus, no mucus; +mucus, mucus present; apical transduction; multiplicity of infection (MOI) of 25,000. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] definition

[0031] As used in this 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.

[0032] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements but do not exclude other elements. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements that are essential to the combination for the intended use. For example, a composition consisting essentially of the elements defined herein does not exclude trace contaminants from the isolation and purification methods, as well as pharma- ceutically acceptable carriers such as phosphate buffered saline, preservatives, and the like. "Consisting of" is intended to mean excluding trace elements of other components and substantial method steps for administering the compositions disclosed herein. Embodiments defined by each of these transition terms are within the scope of the present disclosure.

[0033] Adeno-associated viruses are nonpathogenic parvoviruses composed of a 4.7 kb single-stranded DNA genome within a nonenveloped icosahedral capsid. The genome contains three open reading frames (ORFs) flanked by inverted terminal repeats (ITRs) that function as viral origins of replication and packaging signals. The rep ORFs encode four nonstructural proteins that play roles in viral replication, transcriptional regulation, site-specific integration, and virion assembly. The cap ORF encodes three structural proteins (VP1-3) that assemble to form the 60-mer viral capsid. Finally, an ORF present as an alternative reading frame within the cap gene produces the assembly activating protein (AAP), a viral protein that localizes AAV capsid proteins to the nucleolus and functions in the capsid assembly process.

[0034] There are several naturally occurring ("wild type") serotypes and over 100 known variants of AAV, each of which differs in amino acid sequence, particularly within the hypervariable regions of the capsid protein, and therefore in gene delivery properties. AAV has not been associated with any human disease, making recombinant AAV attractive for clinical use.

[0035] For 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, AAV type 1 (AAV-1 or AAV1), AAV type 2 (AAV-2 or AAV2), AAV type 3A (AAV-3A or AAV3A), AAV type 3B (AAV-3B or AAV3B), AAV type 4 (AAV-4 or AAV4), AAV type 5 (AAV-5 or AAV5), AAV type 6 (AAV-6 or AAV6), AAV type 7 (AAV-7 or AAV7), AAV type 8 (AAV-8 or AAV8), AAV type 9 (AAV-9 or AAV9), AAV type 10 (AAV-10 or AAV10 or AAVrhlO), 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.

[0036] The genomic sequences of various serotypes of AAV, as well as the sequences of natural 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. For example, GenBank accession numbers 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), ... .__.See 006152.1 (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), the disclosures of which are incorporated herein by reference for teaching AAV nucleic acid and amino acid sequences. See, 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; Morris et al. (2004) Virology 33:375-383; International Patent Publications WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; and U.S. Patent No. 6,156,303.

[0037] 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 "mutant AAV capsid protein" or "AAV mutant" refers to an AAV capsid protein that comprises an amino acid sequence that contains at least one modification or substitution (including a deletion, insertion, point mutation, etc.) relative to the naturally occurring or "wild-type" AAV capsid protein sequence, e.g., as set forth in SEQ ID NOs:1-11 herein. The mutant AAV capsid protein may have about 80% or more identity to the amino acid sequence of the wild-type capsid protein, e.g., 85% or more identity, 90% or more identity, or 95% or more identity to the amino acid sequence of the wild-type capsid protein, e.g., 98% or 99% identity to the wild-type capsid protein. The mutant AAV capsid protein may not be a wild-type capsid protein.

[0038] 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.

[0039] For the purpose of the disclosure herein, the term "rAAV" is an abbreviation that refers to recombinant adeno-associated virus. "Recombinant" as applied to polynucleotide 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 virus is a virus particle that contains recombinant polynucleotide. Each of these terms includes the replica of the original polynucleotide construct and the progeny of the original virus construct.

[0040] The term "rAAV vector" encompasses rAAV virions, rAAV viral particles (e.g., infectious rAAV virions), which by definition contain an rAAV polynucleotide, and also encompasses polynucleotides encoding rAAV (e.g., single-stranded polynucleotides encoding rAAV (ss-rAAV); double-stranded polynucleotides encoding rAAV (ds-rAAV), e.g., plasmids encoding rAAV; etc.).

[0041] When an AAV virion contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene delivered to a target cell, an RNAi agent or a CRISPR agent delivered to a target cell, etc.), it is typically referred to as a "recombinant AAV (rAAV) virion" or "rAAV virus particle." Generally, the heterologous polynucleotide is flanked by at least one, and generally two, AAV inverted terminal repeat sequences (I'I'R).

[0042] The term "packaging" refers to a series of intracellular events that result in the assembly and encapsulation of AAV particles. AAV "rep" and "cap" genes refer to the polynucleotide sequences that code for the replication and encapsidation proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

[0043] 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. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpes viruses, and poxviruses such as vaccinia. Adenoviruses encompass several different subgroups, but adenovirus type 5 of subgroup C is the most commonly used. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and available from depositories such as the ATCC. Viruses of the Herpesviridae family include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV). Also available from depositories such as the ATCC.

[0044] The term "helper virus functions" refers to functions encoded in the helper virus genome that allow replication and packaging of AAV (along with other requirements for replication and packaging as described herein). As described herein, "helper virus functions" can be provided in several ways, including providing a helper virus or, for example, providing a polynucleotide sequence encoding the necessary functions to a producer cell in trans. For example, a plasmid or other expression vector containing a nucleotide sequence encoding one or more adenovirus proteins is transfected into a producer cell along with a rAAV vector.

[0045] The term "infectious" virus or virus particle is one that contains a properly assembled viral capsid and can deliver polynucleotide components to cells for which the viral species is tropic. This term does not necessarily imply the replication capacity of the virus. Assays for counting infectious virus particles are described in this disclosure and elsewhere in the art. Viral infectivity can be expressed as the 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, for example, Grainger et al. (2005) Mol. Ther. 11:S337 (describing TCID50 infectious titer assay); and Zolotukhin et al. (1999) Gene Ther. 6:973. See also the Examples.

[0046] The term "tropism" as used herein refers to preferential targeting by a virus (e.g., AAV) of cells of a particular host species or of a particular cell type within a host species. For example, a virus that can infect cells of the heart, lungs, liver, and muscles will have a broader (i.e., increased) tropism relative to a virus that can infect only cells of the lungs and muscles, and tropism may also include the dependency of the virus on a particular type of cell surface molecule of the host. For example, some viruses can only infect cells with surface glycosaminoglycans, while others can only infect cells with sialic acid (such dependency can be tested using various cell lines that are deficient in a particular class of molecules as potential host cells for viral infection). In some cases, the tropism of a virus represents the relative preference of the virus. For example, a first virus may be able to infect all cell types, but is much more successful at infecting those cells with surface glycosaminoglycans. If the second virus also favors the same characteristics (e.g., the second virus is also more successful in infecting those cells with surface glycosaminoglycans), the second virus can be considered to have a similar (or identical) tropism to the first virus, even if the absolute transduction efficiency is not similar. For example, the second virus may be more efficient than the first virus in infecting every given cell type tested, but if the relative preferences are similar (or identical), the second virus can still be considered to have a similar (or identical) tropism to the first virus. In some embodiments, the tropism of the virions containing the subject mutant AAV capsid proteins is unchanged relative to naturally occurring virions. In some embodiments, the tropism of the virions containing the subject mutant AAV capsid proteins is expanded (i.e., broadened) relative to naturally occurring virions. In some embodiments, the tropism of the virions containing the subject mutant AAV capsid proteins is reduced relative to naturally occurring virions.

[0047] The term (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 functions). In the case of AAV, replication-competence generally requires the presence of functional AAV packaging genes. In general, the rAAV vectors described herein are replication-incompetent in mammalian cells (particularly human cells) due to the lack of one or more AAV packaging genes. Typically, such rAAV vectors lack AAV packaging gene sequences to minimize the possibility that replication-competent AAV is generated by recombination between the AAV packaging genes and the incoming rAAV vector. In many embodiments, the rAAV vector preparations described herein contain at least some replication-competent AAV (rcAAV, also referred to as RCA) (e.g., 10 2 Approximately 1 rcAAV per rAAV particle, less than 10 4 Less than about 1 rcAAV per rAAV particle, less than about 1 rcAAV per 10 rAAV particles, less than about 1 rcAAV per 10 12 less than approximately 1 rcAAV per rAAV particle, or no rcAAV).

[0048] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. As used herein, the term polynucleotide refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment herein that includes a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

[0049] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, meaning that when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. Sequence similarity can be determined in several different ways. To determine sequence identity, sequences can be aligned using methods and computer programs including BLAST, available on the World Wide Web at nebi.nlm.nih.gov / BLAST / . Another alignment algorithm is PASTA, available in the Genetics Computing Group (GCC) package from Oxford Molecular Group, Inc., a wholly owned subsidiary of Madison, Wisconsin, USA. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA. Of particular interest are alignment programs that allow gaps in sequences. Smith-Waterman is one type of algorithm that allows gaps in sequence alignments. See Meth. Mol. Biol. 70:173-187 (1997). Also, the GAP program, which uses the Needleman and Wunsch alignment method, can be utilized to align sequences. See J. Mol. Biol. 48:443-453 (1970).

[0050] The term "gene" refers to a polynucleotide that performs some function in a cell. For example, a gene can include an open reading frame that can code for a gene product. One example of a gene product is a protein that is transcribed and translated from a gene. Another example of a gene product is an RNA that is transcribed but not translated, such as a functional RNA product, such as 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.

[0051] The term "gene expression product" or "gene product" refers to a molecule resulting from the expression of a particular gene, as defined above. Gene expression products include, for example, polypeptides, aptamers, interfering RNA, messenger RNA (mRNA), rRNA, tRNA, non-coding RNA (ncRNA), and the like.

[0052] 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 the structure called for by complementary pairing between two regions of an RNA molecule, forming a double-stranded region. RNA (dsRNA). siRNA "targets" a gene in that the nucleotide sequence of the duplex portion of the siRNA is complementary to the nucleotide sequence of the target gene. In some embodiments, the length of the siRNA duplex is less than 30 nucleotides. In some embodiments, 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 long. In some embodiments, "siRNA-mediated gene targeting" is achieved by the use of DNA-directed RNA interference (ddRNAI), a gene silencing technique that utilizes DNA constructs to activate the endogenous RNA interference (RNAi) pathway in animal cells. Such DNA constructs are designed to express self-complementary double-stranded RNA, typically short hairpin RNA (shRNA), that upon processing results in silencing of one or more target genes. Any RNA, including endogenous mRNA or viral RNA, can be silenced by designing a construct to express a double-stranded RNA complementary to a desired mRNA target. Thus, the RNA duplex portion of the siRNA agent can be part of a short hairpin structure called an shRNA. In addition to the duplex portion, the hairpin structure can include a loop portion located between the two sequences that form the duplex. The length of the loop can vary. In sonic embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. The hairpin structure can also include a 3' or 5' overhang portion. In some embodiments, the overhang is a 3' or 5' overhang that is 0, 1, 2, 3, 4 or 5 nucleotides in length.In general, the level of an expression product (e.g., mRNA, polypeptide, etc.) of a target gene is reduced by an siRNA agent (e.g., siRNA, shRNA, etc.) that comprises a specific double-stranded nucleotide sequence that is complementary to at least a 19-25 nucleotide long segment (e.g., a 20-21 nucleotide sequence) of the target gene transcript, including the 5' untranslated (UT) region, ORF, or 3'UT region. In some embodiments, the small interfering RNA is about 19-25 nt in length. See, for example, PCT Applications WO 0144895; WO 99 / 32619; WO 01 / 75164; WO 01 / 92513; WO 01 / 29058; WO 01 / 89304; WO 02 / 16620 and WO 02 / 29858; and U.S. Patent Publication No. 20040023390 for a description of siRNA technology. The siRNA and / or shRNA may be encoded by a nucleic acid sequence, and the nucleic acid sequence may also include a promoter.The nucleic acid sequence may also include a polyadenylation signal.In some embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal.

[0053] The term "antisense RNA" includes RNA that is complementary to gene expression products.For example, antisense RNA that targets a specific mRNA is an RNA-based agent (can be modified RNA) that is complementary to the mRNA (for example, by changing the stability of the RNA, changing the translation of the RNA, etc.), and the hybridization of antisense RNA to the mRNA changes the expression of the mRNA. "Antisense RNA" also includes the nucleic acid that codes for antisense RNA.

[0054] With respect to "CRISPR / Cas9 agents," the term "CRISPR" encompasses the clustered regularly interspaced short palindromic repeats / CRISPR-associated (Cas) system that evolved to provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNA (crRNA) to guide the silencing of invading nucleic acids. The Cas9 protein (or its functional equivalents and / or variants, i.e., Cas9-like proteins) naturally produces DNA endonuclease activity that depends on the association of the protein with two naturally occurring or synthetic RNA molecules (also called guide RNAs) called crRNA and tracrRNA. In some cases, the two molecules are covalently linked to form a single molecule (also called single guide RNA ("sgRNA")). Thus, the Cas9 or Cas9-like protein associates with a DNA-targeting RNA (this term encompasses both bi-molecule and mono-molecule guide RNA arrangements) that activates the Cas9 or Cas9-like protein and guides the protein to the target nucleic acid sequence.

[0055] If Cas9 or Cas9-like protein retains its native enzymatic function, it cleaves the target DNA to generate double-strand breaks, which can result in genomic alterations (i.e., edits: deletions, insertions (if donor polynucleotides are present), substitutions, etc.), thereby altering gene expression. Some mutants of Cas9 (which are encompassed by the term Cas9-like) have been engineered to have reduced DNA cleavage activity (in some cases, they cleave a single strand rather than both strands of the target DNA, but in other cases, they do not have significantly reduced DNA cleavage activity). Even Cas9-like proteins with reduced (no) DNA cleavage activity can be guided to target DNA and block RNA polymerase activity. Alternatively, Cas9 or Cas9-like proteins can be modified by fusing the VP64 transcription activation domain to the Cas9 protein and co-delivering the fusion protein with the MS2-P65-HSF1 helper protein and a single guide RNA that contains the MS2 RNA aptamer at the tetraloop and stem loop to form a synergistic activation mediator (Cas9-SAM) complex in cells that activates transcription. Thus, enzymatically inactive Cas9-like proteins can be targeted to specific locations in target DNA by DNA-targeting RNA to block or activate transcription of the target DNA. The term "CRISPR'Cas9 agent" as used herein encompasses all forms of CRISPR / Cas9 described above or known in the art.

[0056] Detailed information regarding CRISPR agents can be found, for example, in (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. 28; 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 No. 13 / 842,859 and PCT Application No. PCT / US13 / 32589, all of which are incorporated herein by reference in their entireties. Thus, the term "CRISPR agent" as used herein encompasses any agent comprising naturally occurring and / or synthetic sequences that can be used in a Cas9 system (e.g., a Cas9 or Cas9-like protein; any component of a DNA-targeting RNA, e.g., a crRNA-like RNA, a tracrRNA-like RNA, a single guide RNA, etc.; a donor polynucleotide, etc.).

[0057] "Zinc finger nuclease" (ZFN) refers to an artificial DNA endonuclease generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain. ZFNs can be engineered to target a desired DNA sequence, allowing the zinc finger nuclease to cleave a unique target sequence. When introduced into a cell, ZFNs can be used to edit target DNA within the cell (e.g., the cell's genome) by inducing a double-stranded break. For further information regarding the use of ZFNs, see, e.g., Asuri et al., Mol Ther. 2012 February;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 August;15(8):875-85; Takasu et al., Insect Biochem Mol 2010 October;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 Res. 2011 November;21(11):1638-40, all of which are incorporated by reference herein for their teachings related to ZFNs. The term "ZFN agent" encompasses zinc finger nucleases and / or polynucleotides comprising a nucleotide sequence encoding a zinc finger nuclease.

[0058] The term "transcription activator-like effector nuclease" or "TALEN" agent refers to a transcription activator-like effector nuclease (TALEN) that is an artificial DNA endonuclease generated by fusing a TAL (transcription activator-like) effector DNA binding domain to a DNA cleavage domain. TALENs can be rapidly engineered to bind to virtually any desired DNA sequence, and when introduced into a cell, TALENs can be used to edit target DNA in the cell (e.g., the genome of the cell) by inducing a double-strand break. For further information regarding the use of TALENs, see, for example, 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. All of which are incorporated herein by reference for their teachings related to TALENs. The term "TALEN agent" encompasses TALENs and / or polynucleotides comprising nucleotide sequences encoding TALENs.

[0059] The term "control element" or "control sequence" refers to a nucleotide sequence involved in molecular interactions that contribute to the functional regulation of a polynucleotide, including the duplication, replication, transcription, splicing, translation, or degradation of the polynucleotide. Regulation can affect the frequency, rate, or specificity of a process, and can 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 under certain conditions of binding RNA polymerase and initiating transcription of a coding region that is usually located downstream (3' direction) of the promoter. A promoter can be ubiquitously acting, i.e., active in many cell types, e.g., CAG or CMV promoters; or tissue or cell specific.

[0060] The term "operably linked" or "operably linked" refers to the juxtaposition of genetic elements, where the elements are in a relationship that allows them to operate in an expected manner. For example, a promoter is operably linked to a coding region if it helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and the coding region, so long as this functional relationship is maintained.

[0061] The term "expression vector" encompasses vectors that contain a polynucleotide region that encodes a polypeptide of interest and is used to effect expression of the protein in the intended target cell. Expression vectors may also contain control elements operably linked to the coding region to facilitate expression of the protein in the target. The combination of control elements and one or more genes to which they are operably linked for expression may be referred to as an "expression cassette", many of which are known and available in the art or can be readily constructed from components available in the art.

[0062] The term "heterologous" means that it is derived from a genotypically different entity from the rest of the entity being compared. For example, a polynucleotide introduced into a plasmid or vector derived from a different species by genetic engineering techniques is a heterologous polynucleotide. A promoter that is removed from its native coding sequence and operably linked to a coding sequence that is not naturally linked is a heterologous promoter. Thus, for example, a rAAV that comprises a heterologous nucleic acid sequence that encodes a heterologous gene product is a rAAV that comprises a polynucleotide that is not normally included in naturally occurring wild-type AAV, and the encoded heterologous gene product is a gene product that is not normally encoded by naturally occurring wild-type AAV.

[0063] The terms "genetic change" 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 by other than mitosis or division. The element may be heterologous to the cell, or may be an additional copy or improved version of an element already present in the cell. Genetic change may be performed by transfecting the cell with a recombinant plasmid or other polynucleotide via any process known in the art, such as, for example, electroporation, calcium phosphate precipitation, or contact with a polynucleotide-liposome complex. Genetic change may also be achieved by transduction or infection, for example, with a DNA or RNA virus or viral vector. Generally, the genetic element is introduced into a chromosome or minichromosome in the cell. Any change that changes the phenotype and / or genotype of the cell and its progeny is included in this term.

[0064] With respect to cell modification, the terms "genetically modified" or "transformed" or "transfected" or "transduced" with exogenous DNA (e.g., via a recombinant virus) refer to when such DNA is introduced into a cell. The presence of the exogenous DNA results in a permanent or transient genetic change. 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 common ancestor by mitosis. A "cell line" is a clone of a primary cell capable of stable growth in vitro for many generations.

[0065] As used herein, a cell is said to be "stably" modified, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during long-term culture of the cells in vitro and / or in vivo. Generally, such cells are "genetically" modified (genetically altered) in that a genetic change is introduced that is inheritable to the progeny of the modified cell.

[0066] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass modified amino acid polymers, such as disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling moiety. When discussed in relation to delivering polypeptides, such as antiangiogenic polypeptides, neuroprotective polypeptides, gene products to a mammalian subject, and compositions thereof, refer to the respective intact polypeptides, or any fragments or engineered derivatives thereof that retain the desired biochemical function of the intact protein. Similarly, references to nucleic acids encoding antiangiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for use in delivering gene products to a mammalian subject (which may be referred to as "transgenes" delivered to recipient cells) include polynucleotides encoding the intact polypeptides or any fragments or engineered derivatives that have the desired biochemical function.

[0067] 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 of the other components that may also be present when the substance or similar substance occurs in nature or is initially prepared. Thus, for example, an isolated substance may be prepared by using purification techniques to enrich it from a source mixture. Enrichment can be measured in absolute terms, such as weight per volume of solution, or may be measured with respect to a second potential interfering substance present in the source mixture. Increasing enrichment of the embodiments of the present disclosure is increasingly isolated. An isolated plasmid, nucleic acid, vector, virus, host cell, or other substance, in some embodiments, is purified, e.g., 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% or more pure.

[0068] 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 that it completely or partially prevents the disease or its symptoms, and / or it may be therapeutic in that it partially or completely cures the disease and / or the adverse effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes (a) preventing the onset of a disease in a subject who may be susceptible to or at risk of acquiring the disease (and / or the symptoms caused by the disease), but has not yet been diagnosed as having it, (b) inhibiting the disease (and / or the symptoms caused by the disease), i.e., arresting its development, and (c) relieving the disease (and / or the symptoms caused by the disease), i.e., causing the regression of the disease (and / or the 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 be directed to the treatment of a pulmonary disease.

[0069] The terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to, but are not limited to, humans; mammals, including 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.).

[0070] In some embodiments, the individual is a human who has been naturally exposed to AAV before and has anti-AAV antibodies (i.e., AAV neutralizing antibodies) as a result. In some embodiments, the individual is a human who has been previously administered an AAV vector (and may have anti-AAV antibodies as a result) and requires re-administration of the vector for the treatment of a different condition or for further treatment of the same condition. Many such therapeutic applications / disease targets exist, based on positive results in clinical trials involving, for example, AAV gene delivery to liver, muscle and retina (all tissues affected by neutralizing antibodies against this vehicle).

[0071] The term "effective amount" as used herein is an amount sufficient to produce a beneficial or desired clinical result. An effective amount can be administered in one or more administrations. For purposes of this disclosure, an effective amount of a compound (e.g., infectious rAAV virion) is an amount sufficient to alleviate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of a particular disease state (e.g., pulmonary disease) (and / or associated symptoms thereof). Thus, an effective amount of infectious rAAV virion is an amount of infectious rAAV virion that can effectively deliver heterologous nucleic acid to a target cell (or target cells) of an individual. An effective amount can be determined preclinically, for example, by detecting gene products (RNA, protein) encoded by the heterologous nucleic acid sequence in cells or tissues using techniques well understood in the art, such as RT-PCR, Western blotting, ELISA, fluorescent or other reporter readouts, etc. An effective amount can be determined clinically, for example, by detecting changes in the onset or progression of a disease using methods known in the art.

[0072] The term "directed evolution" refers to an in vitro and / or in vivo capsid engineering methodology that emulates natural evolution through iterative rounds of genetic diversification and selection processes, thereby accumulating beneficial mutations that gradually improve the function of a biomolecule. Directed evolution often involves an in vivo method called "biopanning" to select from a library AAV variants that have more efficient levels of infectivity for a cell or tissue type of interest.

[0073] The term "interfering RNA" encompasses both small interfering RNAs and microRNAs (miRNAs), including artificial miRNAs.

[0074] "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 IRES elements in multicistronic vectors. 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, the expression of 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 internal ribosome entry sites (IRES) or 2A peptide sequences.

[0075] By "increased resistance" is meant that the subject infectious rAAV virions exhibit increased infectivity in the presence of human anti-AAV antibodies. Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Thus, increased infectivity means an increase in the ratio of infectious viral particles to total viral particles. 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% of that in the absence of human anti-AAV antibodies. Viruses that require higher antibody concentrations to reduce the gene delivery efficiency to 50% of that in the absence of human anti-AAV antibodies are said to have increased resistance to antibody neutralization. Thus, a two-fold increase in resistance means a two-fold increase in the antibody concentration required to reduce the gene delivery efficiency to 50% of that 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.) greater resistance to human AAV neutralizing antibodies than the resistance exhibited by a wild-type AAV (e.g., AAV2 (wild-type AAV serotype 2)) or an AAV comprising a wild-type capsid protein.

[0076] A subject infectious rAAV virion may be said to exhibit increased transduction of mammalian cells in the presence of human AAV neutralizing 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.) greater transduction of mammalian cells in the presence of human AAV neutralizing antibodies than the transduction exhibited by a wild-type AAV (e.g., AAV2 (wild-type AAV serotype 2)) or an AAV comprising a wild-type capsid protein.

[0077] In some embodiments, a subject infectious rAAV virion exhibits reduced binding to a neutralizing antibody that binds to a wild-type AAV capsid protein. For example, a subject infectious rAAV virion may have reduced binding (e.g., reduced affinity) to a neutralizing antibody that binds to a wild-type AAV capsid protein by 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.) compared to the binding affinity of the antibody to the wild-type AAV capsid protein.

[0078] In some embodiments, the anti-AAV neutralizing antibody is about 10 -7 Less than M, about 5 x 10 -6 Less than M, about 10 -6 Less than M, about 5 x 10 -5 Less than M, about 10 -5Less than M, about 10 -4 It binds to the target infectious rAAV virion with an affinity of less than or equal to M.

[0079] Adeno-associated viruses (AAV) are a family of parvoviruses with a 4.7 kb single-stranded DNA genome contained within a non-enveloped capsid. The naturally occurring viral genome of AAV has two inverted terminal repeats (ITRs) that function as viral origins of replication and packaging signals flanking two primary open reading frames (ORFs): rep (encoding proteins that function in viral replication, transcriptional regulation, site-specific integration and virion assembly) and cap. The cap ORF encodes three structural proteins that assemble to form the 60-mer viral capsid. Many naturally occurring AAV variants and serotypes have been isolated, none of which are associated with human disease.

[0080] 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 postmitotic tissues for several years. For example, 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 Acad Sci 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 Natl Acad USA 1966;55:1467-74;Blacklow NR, Hoggan MD, Rowe W P.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-containing viruses(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 of capsid protein.Virology 2004;330:375-83;Flotte T R.Progress and prospects of gene therapy:recombinant adeno-associated virus(rAAV)vectors.Gene Ther 2004;11:805-10.

[0081] Recombinant AAV (interchangeably referred to herein as "AAV" or "rAAV") has shown promising results in a growing number of clinical trials. However, there are obstacles to gene delivery that may limit the usefulness of AAV, such as anti-capsid immune responses, low transduction of certain tissues, inability to target delivery to certain cell types, and relatively low carrying capacity. In many situations, there is insufficient mechanistic knowledge to effectively implement rational design in the ability to improve AAV. As an alternative, directed evolution has emerged as a strategy to create novel AAV variants that meet specific biomedical needs. Directed evolution strategies utilize genetic diversification and selection processes to allow the accumulation of beneficial mutations that gradually improve the function of biomolecules. In this process, wild-type AAV cap genes are diversified by several approaches to create large gene libraries that are packaged to generate libraries of viral particles, and then selective pressure is applied to isolate novel variants that can overcome gene delivery barriers. Importantly, the mechanistic basis underlying the gene delivery problem does not need to be known for directed evolution of function, thus accelerating the development of enhanced vectors.

[0082] Typically, the variants disclosed herein are generated by the use of an AAV library and / or library. Such one or more AAV libraries are generated by mutating the cop gene, which is a gene encoding the structural protein of the AAV capsid, by a series of directed evolution techniques known and readily available to those skilled in the art of viral genome engineering. For example, see 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, J.T. 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 See Then. 18, 367-378 (2007); and Koerber, J.T. et al. Mol. Ther. 17, 2088-2095 (2009). Such techniques include, but are not limited to: i) error-prone PCR to introduce random point mutations into the AAV cap open reading frame (ORF) at a predetermined and correctable rate; ii) in vitro or in vivo viral recombination or "DNA shuffling" to create random chimeras of AAV cap genes to obtain gene libraries with multiple AAV serotypes; iii) random peptide insertion into a predetermined site of the capsid by ligation of degenerate oligonucleotides in the cap ORF; iv) defined insertion of peptide-encoding 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 level of conservation of each amino acid arrangement among native AAV serotypes and mutants to generate a "loop swap library"; vi) random amino acid substitutions at degenerate arrangements among AAV serotypes to generate a library of ancestral mutants (Santiago-Ortiz et al., 2015); and combinations of such techniques.

[0083] DNA shuffling generates chimeras that combine the properties of their parents in unique, often beneficial ways. Some, however, may be virtually impossible to package, which reduces the diversity of the library. Library diversity enrichment is achieved by peptide insertion techniques, such as, but not limited to, iii-iv) above. Library diversity has also been concentrated by techniques such as v) above, where such concentration is directed at multiple hypervariable regions located on the surface-exposed loops of the AAV capsid. Many of these techniques generate mutant capsids in which only small regions of the capsid are mutated, but these techniques can be paired with additional mutagenesis strategies to modify the complete capsid.

[0084] Once one or more AAV libraries have been generated, the viruses are then packaged and purified such that each AAV particle is composed of a mutant capsid surrounding the cap gene that encodes the capsid. The variants of the library are then subjected to in vitro and / or in vivo selection pressure techniques that are known and readily available to those skilled in the art of AAV. 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 limited to, AAV variants may be selected using: i) affinity columns, where elution of different fractions yields variants with altered binding properties; ii) primary cells isolated from tissue samples or immortal cell lines that mimic the behavior of cells in the human body, resulting in AAV variants with improved efficiency and / or tissue specificity; iii) animal models that mimic the clinical gene therapy environment, resulting in AAV variants that successfully infect target tissues; iv) human xenograft models that produce AAV variants with infected transplanted human cells; and / or a combination of these selection techniques.

[0085] Once the viruses are selected, they may be recovered by known techniques, including but not limited to adenovirus-mediated replication, PCR amplification, next-generation sequencing and cloning, and then the viral clones are enriched through repeated rounds of selection techniques and AAV DNA is isolated to recover the selected mutant cap genes of interest. Such selected mutants can undergo further modification or mutation, thus serving as new starting points for further selection steps that iteratively increase AAV virus fitness, although in certain instances, successful capsids have been generated without further mutation.

[0086] The AAV mutants disclosed herein are at least partially generated by using in vivo directed evolution methodologies such as the above-mentioned techniques, including using primate lung screening after aerosol administration.Thus, the AAV mutant capsids disclosed herein contain one or more modifications of amino acid sequence that confer more efficient transduction of primate lung cells than corresponding parent AAV capsid protein.As used herein, "corresponding parent AAV capsid protein" refers to the AAV capsid protein of the same wild type or mutant AAV serotype as the target mutant AAV capsid protein, but does not contain one or more amino acid sequence modifications of the target mutant AAV capsid protein.

[0087] In some embodiments, a subject mutant AAV capsid protein comprises a heterologous peptide of about 5 amino acids to about 20 amino acids covalently inserted into the AAV capsid protein GH loop or loop IV relative to the corresponding parent AAV capsid protein. By "GH loop" or loop IV of an AAV capsid protein is meant the solvent accessible portion of the AAV capsid protein referred to in the art as the OH loop or loop IV. For the OH loop / loop IV of AAV capsids, 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. Thus, for example, the insertion site can be within about amino acids 411 to 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 within the corresponding amino acids of any variant thereof. Those skilled in the art will know that, based on a comparison of the amino acid sequences of the capsid proteins of various AAV serotypes, there are insertion sites within the capsid protein of any given AAV serotype that "correspond to amino acids of AAV2. See also Figure 6 of US Patent Application Publication No. 2019 / 0255192 for an alignment of wild-type AAV SEQ ID NOs: 1-11, providing the amino acid positions between and among the wild-type (naturally occurring) serotypes AAV1, AAV2, AAV3A, AAV3B, and AAV4-10, the entire contents of which are incorporated herein by reference.

[0088] In certain embodiments, the insertion site is a single insertion site between two adjacent amino acids located between amino acids 570-614 of VP1 of any wild-type AAV serotype or AAV variant, for example, the insertion site is between two adjacent amino acids located between 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, amino acids 581 and 582, amino acids 583 and 584, amino acids 584 and 585, amino acids 585 and 586, amino acids 586 and 587, amino acids 587 and 588, amino acids 588 and 589, or amino acids 589 and 590. The site of insertion 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 site of insertion can be between amino acids 590 and 591, amino acids 591 and 592, amino acids 592 and 593, amino acids 593 and 594, amino acids 594 and 595, amino acids 595 and 596, amino acids 596 and 597, amino acids 597 and 598, amino acids 598 and 599, or 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.

[0089] In some embodiments, the peptide insertions disclosed herein have 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 insertions disclosed herein include 1-4 spacer amino acids at the amino terminus (N-terminus) and / or carboxyl terminus (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 includes two spacer amino acids at the N-terminus and two spacer amino acids at the C-terminus. In other embodiments, the peptide insertion includes two spacer amino acids at the N-terminus and one spacer amino acid at the C-terminus.

[0090] In some embodiments, the inserted peptide is 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), ENRTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TNNSKPD (SEQ ID NO: 25), SKLTLNN (SEQ ID NO: 26), and the like. 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).

[0091] In other preferred embodiments, the inserted peptide is 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), ENRTSN (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), SPGATTNN (SEQ ID NO:39).In certain such embodiments, the inserted peptide is 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), 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), LATNNSKPDA (SEQ ID NO:54), LATNNSKPDA (SEQ ID NO:55), LATNNSKPDA (SEQ ID NO:56), LATNNSKPDA (SEQ ID NO:57), LATNNSKPDA (SEQ ID NO:58), LATNNSKPDA (SEQ ID NO:59), LATNNSKPDA (SEQ ID NO:60), LATNNSKPDA (SEQ ID NO:61), LATNNSKPDA (SEQ ID NO:62), LATNNSKPDA (SEQ ID NO:63), LATNNSKPDA (SEQ ID NO:64), LATNNSKPDA (SEQ ID NO:65), LATNNSKPDA (SEQ ID NO:66), LATNNSKPDA (SEQ ID NO:67), LATNNSKPDA (SEQ ID NO:68), LATNNSKPDA (SEQ ID NO:69), LATNNSKPDA (SEQ ID NO:69), LATNNSKPDA (SEQ ID NO:69), LATNNSKPDA (SEQ ID NO:70), L 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).

[0092] In some embodiments, a subject mutant AAV capsid protein does not contain any other amino acid sequence modification 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, a subject mutant AAV capsid protein contains 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), ENRTSSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), or a combination thereof. ), 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), 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), LA ENRTTSNA (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),and wherein the mutant AAV capsid does not contain other amino acid substitutions, insertions or deletions (i.e., the mutant AAV capsid protein contains the insertion and is otherwise identical to the corresponding AAV capsid protein. In other words, the mutant AAV capsid protein containing the insertion is otherwise identical to the parent AAV capsid protein into which the peptide was inserted). As another example, the subject mutant AAV capsid proteins are 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), ENRTSN (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), 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 ID NO:67), wherein the peptide insertion is located between amino acids 587 and 588 of VP1 of the AAV2 capsid, or between the corresponding amino acids of VP1 of another parent AAV, e.g., 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 and 591 of VP1 of AAV8 or AAV10, and the variant AAV capsid protein sequence is otherwise identical to the corresponding parent AAV capsid protein sequence, e.g., any one of SEQ ID NOs:1-12.

[0093] In other embodiments, a subject mutant AAV capsid protein comprises about 1 to about 100 amino acid substitutions or deletions, e.g., 1 to about 5, about 2 to about 4, about 2 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to 50, about 50 to 100 amino acid substitutions or deletions, relative to a parent AAV capsid protein, in addition to comprising a peptide insertion within the GH loop, e.g., as disclosed herein or known in the art. Thus, in some embodiments, a subject mutant capsid protein comprises an amino acid sequence having 85% or more, 90% or more, 95% or more, or 98% or more, e.g., 99% or more, sequence identity to a corresponding parent AAV capsid, e.g., a wild-type capsid protein set forth in SEQ ID NOs: 1-12. In some embodiments, a subject mutant 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 the AAV2 capsid protein (SEQ ID NO:2).

[0094] In further embodiments, the one or more amino acid substitutions are at or near the 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, 240, 250, 283, 312, 344, 347, 363, 370, 372, 376, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 420, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 441, 442, 443, 444, 445, 446, 447, 448, In some such embodiments, the one or more amino acid substitutions are at amino acid residues 68, 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 the corresponding amino acid residues in another AAV 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, T344S, and E347D in the VP1 capsid protein. , G376A, P399H, G406E, Q428H, P436H, N449D, P451Q, N469D, D472N, T491I, K532E, K544E, R585K, A591D, A593E, D594N, D608N, H641N, K688R, N705S, and V708I, or the corresponding amino acid residues in another AAV capsid protein.In other such embodiments, the one or more amino acid substitutions are made in the AAV2 numbered sequence prior to insertion of the peptide. and L735Q of the VP1 capsid protein, or the corresponding amino acid residues of another AAV capsid protein. In some preferred embodiments, a subject mutant 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, a subject mutant capsid protein comprises a V708I amino acid substitution to the AAV2 VP1 capsid protein (SEQ ID NO:2), and optionally further comprises one or more amino acid substitutions as described herein.

[0095] In some embodiments, the subject mutant AAV capsid proteins confer increased infectivity of one or more lung cell types to the rAAV and also confer increased resistance to human AAV neutralizing antibodies compared to the resistance exhibited by AAV2 (wild-type AAV serotype 2). In some cases, the 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 rAAV exhibits resistance to human AAV neutralizing antibodies that 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.) greater than the resistance exhibited by AAV2. In some cases, the rAAV exhibits increased transduction of one or more mammalian lung cell types in the presence of human AAV neutralizing antibodies compared to the transduction of mammalian cells exhibited by wild-type AAV serotype 2 (AAV2).

[0096] In a preferred embodiment, a mutant AAV capsid protein is provided, comprising a) a peptide insertion in the GH loop of the capsid protein, the peptide insertion being 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), ENRTSN (SEQ ID NO: 22), P QQDTTH (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), 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),and b) a peptide insertion comprising or consisting essentially of an amino acid sequence selected from LAKTPNLTSA (SEQ ID NO:65), LASNTPALSA (SEQ ID NO:66) and LASPGATTNA (SEQ ID NO:67); and b) one or more of the following amino acid substitutions compared to the amino acid sequence of AAV2 (SEQ ID NO:2), or the corresponding substitutions in another AAV parent serotype (i.e., other than AAV2), wherein the substituted amino acids are in the corresponding positions: M1L, L15P, P34A, N57, N61, N71, N81, N91, N101, N111, N121, N131, N141, N151, N162, N173, N184, N185, N186, N187, N188, N189, N190, N191, N192, N193, N194, N195, N196, N197, N198, N199, N200, N201, N202, N203, N204, N205, N206, N217, N220, N230, N240, N250, N261, N272, N283, N294, N295, N296, N207, N208, N2197, N2209, N231, N242, N253, N264, N275, N285, N297, N298, N209, N2010, N202, N203, N204, N205, N206, N217, N228, N231, N232, N233, N244, N245, N254 D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L1881, S196Y, G226E, G236V, I2401, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A5241, P535S, N551S, A593E, I698V, V708I, V719M, S721L, L735Q and combinations thereof are not naturally occurring. 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 the corresponding position in the capsid protein of another AAV serotype.

[0097] In particularly preferred embodiments, the mutant AAV capsid comprises, consists essentially of, or consists of a peptide insertion comprising the amino acid sequence HDITKNI (SEQ ID NO: 12) between amino acids 587 and 588 of VP1 of AAV2, or comprises, consists essentially of, or consists of the amino acid sequence LAHDITKNIA (SEQ ID NO: 40) between corresponding amino acids of VP1 of AAV2 or of another AAV capsid, 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 mutant 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 full length of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding parent AAV capsid. In particularly preferred embodiments, the mutant AAV capsid has an amino acid sequence that has 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 is 100% identical to the following amino acid sequence (AAV102): [ka]

[0098] In another embodiment, a mutant AAV capsid protein is provided, comprising: a) a peptide insertion located between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6 or AAV9, between amino acids 586 and 587 of AAV4, between amino acids 577 and 578 of AAV5, between amino acids 589 and 590 of AAV7, or between amino acids 590 and 591 of AAV8 or AAV10, wherein the peptide insertion comprises, consists essentially of, or consists of an amino acid sequence selected from LAHDITKNIA (SEQ ID NO:40) and HDITKNI (SEQ ID NO:12); and b) a peptide insertion located between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6 or AAV9, between amino acids 586 and 587 of AAV4, between amino acids 577 and 578 of AAV5, between amino acids 589 and 590 of AAV7, or between amino acids 590 and 591 of AAV8 or AAV10. or a valine to isoleucine substitution at amino acid 709 of AAV3B, an alanine to isoleucine substitution at sequence 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 a preferred embodiment, the mutant capsid protein comprises: a) a peptide insertion comprising an amino acid sequence comprising, consisting of, or essentially consisting of the amino acid sequence HDITKNI (SEQ ID NO: 12), which comprises, consists of, or consists essentially of the amino acid sequence LAHDITKNIA (SEQ ID NO: 40) between amino acids 587 and 588 of the AAV2 capsid; and b) an amino acid substitution from valine to isoleucine at amino acid 708 compared to the amino acid sequence of AAV2, wherein the mutant capsid protein comprises 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions.

[0099] In yet another embodiment, the mutant capsid protein a) comprises, consists of, or consists essentially of the amino acid sequence HDITKNI (SEQ ID NO: 12) between amino acids 587 and 588 of the AAV2 capsid, or comprises, consists essentially of, or consists of the amino acid sequence LAHDITKNIA (SEQ ID NO: 40), and contains a peptide insertion otherwise identical to the amino acid sequence of SEQ ID NO: 2.

[0100] In particularly preferred embodiments, the mutant AAV capsid comprises, consists essentially of, or consists of the amino acid sequence NQDYTKT (SEQ ID NO: 13), or comprises a peptide insertion comprising, consists essentially of, or consists of the amino acid sequence LANQDYTKTA (SEQ ID NO: 41) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acid of another AAV capsid, 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 in another AAV capsid. The mutant 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 full length of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding parent AAV capsid. In particularly preferred embodiments, the mutant AAV capsid has an amino acid sequence that has 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 is 100% identical to the following amino acid sequence (AAV103): [ka] [ka]

[0101] In another embodiment, a mutant AAV capsid protein is provided, comprising: a) a peptide insertion located between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6, or AAV9, between amino acids 586 and 587 of AAV4, between amino acids 577 and 578 of AAV5, between amino acids 589 and 590 of AAV7, or between amino acids 590 and 591 of AAV8 or AAV10, the peptide insertion comprising, consisting essentially of, or consisting of an amino acid sequence selected from LANQDYTKTA (SEQ ID NO:41) and NQDYTKT (SEQ ID NO:13). and b) a valine to isoleucine substitution at amino acid 709 of AAV3A or AAV3B, an alanine to isoleucine substitution at sequence 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 a preferred embodiment, the mutant capsid protein comprises: a) a peptide insertion comprising, consisting of, or essentially consisting of the amino acid sequence NQDYTKT (SEQ ID NO: 13), or comprising, consisting of, or essentially consisting of the amino acid sequence LANQDYTKTA (SEQ ID NO: 41) between amino acids 587 and 588 of the AAV2 capsid; and b) an amino acid substitution of valine to isoleucine at amino acid 708 compared to the amino acid sequence of AAV2, wherein the mutant capsid protein comprises 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions.

[0102] In yet another embodiment, the mutant capsid protein a) comprises a peptide insertion between amino acids 587 and 588 of the AAV2 capsid that comprises, consists essentially of, or consists of the amino acid sequence NQDYTKT (SEQ ID NO: 13), or the amino acid sequence LANQDYTKTA (SEQ ID NO: 41), and is otherwise identical to the amino acid sequence of SEQ ID NO: 2.

[0103] In particularly preferred embodiments, the mutant AAV capsid comprises, consists essentially of, or consists of the amino acid sequence DNTVTRS (SEQ ID NO: 14), or comprises a peptide insertion comprising, consists essentially of, or consists of the amino acid sequence LADNTVTRSA (SEQ ID NO: 42) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acid of another AAV capsid, 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 in another AAV capsid. The mutant 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 amino acid sequence set forth in SEQ ID NO: 2 or the full length of the corresponding parent AAV capsid. In particularly preferred embodiments, the mutant AAV capsid has an amino acid sequence that has 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 is 100% identical to the following amino acid sequence (AAV104): [ka]

[0104] In another embodiment, a mutant AAV capsid protein is provided, comprising: a) a peptide insertion located between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6, or AAV9, between amino acids 586 and 587 of AAV4, between amino acids 577 and 578 of AAV5, between amino acids 589 and 590 of AAV7, or between amino acids 590 and 591 of AAV8 or AAV10, the peptide insertion comprising, consisting of, or consisting essentially of an amino acid sequence selected from LADNTVTRSA (SEQ ID NO:42) and DNTVTRS (SEQ ID NO:14). and b) a valine to isoleucine substitution at amino acid 709 of AAV3A or AAV3B, an alanine to isoleucine substitution at sequence 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 a preferred embodiment, the mutant capsid protein comprises: a) a peptide insertion comprising, consisting of, or essentially consisting of the amino acid sequence DNTVTRS (SEQ ID NO: 14) or comprising, consisting of, or essentially consisting of the amino acid sequence LADNTVTRSA (SEQ ID NO: 42) between amino acids 587 and 588 of the AAV2 capsid; and b) an amino acid substitution of valine to isoleucine at amino acid 708 compared to the amino acid sequence of AAV2, wherein the mutant capsid protein comprises 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions.

[0105] In yet another embodiment, the mutant capsid protein a) comprises, consists of, or consists essentially of the amino acid sequence DNTVTRS (SEQ ID NO: 14) between amino acids 587 and 588 of the AAV2 capsid, or comprises, consists essentially of, or consists of the amino acid sequence LADNTVTRSA (SEQ ID NO: 42), and contains a peptide insertion otherwise identical to the amino acid sequence of SEQ ID NO: 2.

[0106] In particularly preferred embodiments, the mutant AAV capsid comprises, consists essentially of, or consists of the amino acid sequence SNSVQSI (SEQ ID NO: 15), or comprises a peptide insertion comprising, consists essentially of, or consists of the amino acid sequence LASNSVQSIA (SEQ ID NO: 43) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acid of another AAV capsid, 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 in another AAV capsid. The mutant 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 amino acid sequence set forth in SEQ ID NO: 2 or the full length of the corresponding parent AAV capsid. In particularly preferred embodiments, the mutant AAV capsid has an amino acid sequence that has 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 is 100% identical to the following amino acid sequence (AAV105): [ka]

[0107] In another embodiment, a mutant AAV capsid protein is provided, comprising: a) a peptide insertion located between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6, or AAV9, between amino acids 586 and 587 of AAV4, between amino acids 577 and 578 of AAV5, between amino acids 589 and 590 of AAV7, or between amino acids 590 and 591 of AAV8 or AAV10, the peptide insertion comprising, consisting of, or consisting essentially of an amino acid sequence selected from LASNSVQSIA (SEQ ID NO:43) and SNSVQSI (SEQ ID NO:15). and b) a valine to isoleucine substitution at amino acid 709 of AAV3A or AAV3B, an alanine to isoleucine substitution at sequence 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 a preferred embodiment, the mutant capsid protein comprises: a) a peptide insertion comprising, consisting of, or essentially consisting of the amino acid sequence SNSVQSI (SEQ ID NO: 15), or comprising, consisting of, or essentially consisting of the amino acid sequence LASNSVQSIA (SEQ ID NO: 43) between amino acids 587 and 588 of the AAV2 capsid; and b) an amino acid substitution of valine to isoleucine at amino acid 708 compared to the amino acid sequence of AAV2, wherein the mutant capsid protein comprises 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions.

[0108] In yet another embodiment, the mutant capsid protein a) comprises, consists of, or consists essentially of the amino acid sequence SNSVQSI (SEQ ID NO: 15) between amino acids 587 and 588 of the AAV2 capsid, or comprises, consists essentially of, or consists of the amino acid sequence LASNSVQSIA (SEQ ID NO: 43), and contains a peptide insertion otherwise identical to the amino acid sequence of SEQ ID NO: 2.

[0109] In particularly preferred embodiments, the mutant AAV capsid comprises, consists essentially of, or consists of the amino acid sequence NSTRHTD (SEQ ID NO: 16), or comprises a peptide insertion comprising, consists essentially of, or consists of the amino acid sequence LANSTRHTDA (SEQ ID NO: 44) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acid of another AAV capsid, 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 in another AAV capsid. The mutant 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 full length amino acid sequence set forth in SEQ ID NO: 2 or the corresponding parent AAV capsid. In particularly preferred embodiments, the mutant AAV capsid has an amino acid sequence that has 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 is 100% identical to the following amino acid sequence (AAV106): [ka] [ka]

[0110] In another embodiment, a mutant AAV capsid protein is provided, comprising: a) a peptide insertion located between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6, or AAV9, between amino acids 586 and 587 of AAV4, between amino acids 577 and 578 of AAV5, between amino acids 589 and 590 of AAV7, or between amino acids 590 and 591 of AAV8 or AAV10, the peptide insertion comprising, consisting of, or consisting essentially of an amino acid sequence selected from LANSTRHTDA (SEQ ID NO:44) and NSTRHTD (SEQ ID NO:16). and b) a valine to isoleucine substitution at amino acid 709 of AAV3A or AAV3B, an alanine to isoleucine substitution at sequence 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 a preferred embodiment, the mutant capsid protein comprises: a) a peptide insertion comprising, consisting of, or essentially consisting of the amino acid sequence NSTRHTD (SEQ ID NO: 16), or comprising, consisting of, or essentially consisting of the amino acid sequence LANSTRHTDA (SEQ ID NO: 44) between amino acids 587 and 588 of the AAV2 capsid; and b) an amino acid substitution of valine to isoleucine at amino acid 708 compared to the amino acid sequence of AAV2, wherein the mutant capsid protein comprises 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions.

[0111] In yet another embodiment, the mutant capsid protein a) comprises, consists of, or consists essentially of the amino acid sequence NSTRHTD (SEQ ID NO: 16) between amino acids 587 and 588 of the AAV2 capsid, or comprises, consists essentially of, or consists of the amino acid sequence LANSTRHTDA (SEQ ID NO: 44), and contains a peptide insertion otherwise identical to the amino acid sequence of SEQ ID NO: 2.

[0112] In particularly preferred embodiments, the mutant AAV capsid comprises, consists essentially of, or consists of the amino acid sequence TNRTSPD (SEQ ID NO:17), or comprises a peptide insertion comprising, consists essentially of, or consists of the amino acid sequence LATNRTSPDA (SEQ ID NO:45) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acid of another AAV capsid, 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 in another AAV capsid. The mutant 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 amino acid sequence set forth in SEQ ID NO:2 or the full length of the corresponding parent AAV capsid. In particularly preferred embodiments, the mutant AAV capsid has an amino acid sequence that has 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 is 100% identical to the following amino acid sequence (AAV107): [ka]

[0113] In another embodiment, a mutant AAV capsid protein is provided, comprising: a) a peptide insertion located between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6, or AAV9, between amino acids 586 and 587 of AAV4, between amino acids 577 and 578 of AAV5, between amino acids 589 and 590 of AAV7, or between amino acids 590 and 591 of AAV8 or AAV10, the peptide insertion comprising, consisting of, or consisting essentially of an amino acid sequence selected from LATNRTSPDA (SEQ ID NO:45) and TNRTSPD (SEQ ID NO:17). and b) a valine to isoleucine substitution at amino acid 709 of AAV3A or AAV3B, an alanine to isoleucine substitution at sequence 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 a preferred embodiment, the mutant capsid protein comprises: a) a peptide insertion comprising, consisting of, or essentially consisting of the amino acid sequence TNRTSPD (SEQ ID NO: 17), or comprising, consisting of, or essentially consisting of the amino acid sequence LATNRTSPDA (SEQ ID NO: 45) between amino acids 587 and 588 of the AAV2 capsid; and b) an amino acid substitution of valine to isoleucine at amino acid 708 compared to the amino acid sequence of AAV2, wherein the mutant capsid protein comprises 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions.

[0114] In yet another embodiment, the mutant capsid protein a) comprises, consists of, or consists essentially of the amino acid sequence TNRTSPD (SEQ ID NO: 17) between amino acids 587 and 588 of the AAV2 capsid, or comprises, consists essentially of, or consists of the amino acid sequence LATNRTSPDA (SEQ ID NO: 45), and contains a peptide insertion otherwise identical to the amino acid sequence of SEQ ID NO: 2.

[0115] In other embodiments, the mutant AAV capsid is 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), 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 or between amino acids 587 and 588 of VP1 of AAV2, or between the corresponding amino acids of another AAV capsid, selected from 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), LATNNS and further comprising one or more amino acid substitutions relative to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2).In some embodiments, the one or more amino acid substitutions comprise a V708I amino acid substitution at residue 708 relative to the amino acid sequence of AAV2 capsid (SEQ ID NO:2) or the corresponding residue in another AAV capsid. The mutant 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 full length of the amino acid sequence set forth in SEQ ID NO:2 or the corresponding parent AAV capsid.

[0116] In a related embodiment, a mutant AAV capsid protein is provided, comprising: a) a peptide insertion located between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6 or AAV9, between amino acids 586 and 587 of AAV4, between amino acids 577 and 578 of AAV5, between amino acids 589 and 590 of AAV7, or between amino acids 590 and 591 of AAV8 or AAV10, wherein the peptide insertion is selected from the group consisting of HDITKNI (SEQ ID NO: 12), NQDYTKT (SEQ ID NO: 13), DNTVTRS (SEQ ID NO: 14), SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO NSVQSI (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), ENRTSN (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), 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),and b) a peptide insertion comprising, consisting of, or consisting essentially of an amino acid sequence selected from 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). or 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 a preferred embodiment, the mutant capsid proteins are selected from the group consisting of a) 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), and / or a) IL-16 (SEQ ID NO: 32). or comprising, consisting of, or consisting essentially of an amino acid sequence selected from among amino acids 587 and 588 of the AAV2 capsid, such as LAISDQTKHA (SEQ ID NO: 46), LAQADTTKNA (SEQ ID NO: 47), between amino acids 587 and 588 of the AAV2 capsid;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), LAIIDA and b) an amino acid substitution of valine to isoleucine at amino acid 708 compared to the amino acid sequence of AAV2, wherein the mutant capsid protein comprises 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions.

[0117] In yet another embodiment, the mutant capsid protein is a) 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), VTTNN (SEQ ID NO: 31), VTTNN (SEQ ID NO: 32), VTTNN (SEQ ID NO: 33), VTTNN (SEQ ID NO: 34), VTTNN (SEQ ID NO: 35), VTTNN (SEQ ID NO: 36), VTTNN (SEQ ID NO: 37), VTTNN (SEQ ID NO: 38), VTTNN (SEQ ID NO: 39), VTTNN (SEQ ID NO: 40), VTTNN (SEQ ID NO: 41), VTTNN (SEQ ID NO: 42), VTTNN (SEQ ID NO: 43), VTTNN (SEQ ID NO: 44), VTTNN (SEQ ID NO: 45), VTTNN (SEQ ID NO: 46), VTTNN (SEQ ID NO: 47), VTTNN (SEQ ID NO: 48), VTTNN (SEQ ID NO: 49), VTTNN (SEQ ID NO: 50), VTTNN (SEQ ID NO: 51), VTTNN (SEQ ID NO: 52), VTTNN (SEQ ID NO: 53), VTTNN (SEQ ID NO: or comprising, consisting of, or consisting essentially of an amino acid sequence selected from the group consisting of LAISDQTKHA (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), and SPGATTN (SEQ ID NO:39), or a sequence between amino acids 587 and 588 of the AAV2 capsid. (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), 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 is otherwise identical to the amino acid sequence of SEQ ID NO:2.

[0118] In another embodiment, a mutant AAV capsid protein is provided that comprises 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 mutant AAV capsid protein comprises the following amino acid substitutions relative to AAV2: G586V, N587P, R588T, Q589, A591E, A593L, D594N, and T597G. The mutant AAV capsid protein may comprise the amino acid substitutions described above and may otherwise be 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.

[0119] In another embodiment, a mutant AAV capsid protein having improved tropism for one or more lung cells is provided that comprises a substitution 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 mutant AAV capsid protein comprises the following amino acid substitutions relative to AAV2: R588L, Q589A, A590P, A591D, T592F, A593T, D594T, V595L, N596D, and T597A. The mutant AAV capsid protein may comprise the amino acid substitutions described above and may otherwise be 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.

[0120] In another embodiment, a mutant AAV capsid protein having improved tropism for one or more lung cells is provided, comprising, consisting of, or consisting essentially of a substitution of amino acids 575-586 of wt AAV5 (SSTTAPATGTYN; SEQ ID NO: 76) of SEQ ID NO: 6 with an amino acid sequence selected from 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), and TGLYSPNDGGLS (SEQ ID NO: 88). The mutant AAV capsid protein may contain the amino acid substitutions described above and may otherwise be 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.

[0121] In another embodiment, a mutant AAV capsid protein having improved tropism for one or more lung cells is provided, comprising, consisting of, or consisting essentially of a substitution of amino acids 533 to 545 of wt AAV5 (PANPGTTATYLEG; SEQ ID NO: 89) of SEQ ID NO: 6 with an amino acid sequence selected from 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 related embodiments, a mutant AAV capsid protein having improved tropism for one or more lung cells is provided, which comprises a substitution of amino acids 533-544 of wt AAV5 of SEQ ID NO:6 (PANPGTTATYLE; SEQ ID NO:100) with the following amino acid sequence: LSTpFIVaGSGI (SEQ ID NO:101). The lowercase letters in each sequence correspond to amino acids in the wild-type AAV5 sequence. The mutant AAV capsid protein may contain the amino acid substitutions described above and may otherwise be 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.

[0122] The AAV mutants disclosed herein are generated by using in vivo directed evolution, including using primate lung screening after aerosol administration.In some embodiments, the mutant capsid proteins disclosed herein, when present in AAV virions, confer increased transduction of lung cells compared to the transduction of lung cells by AAV virions that contain corresponding parent AAV capsid proteins or wild-type AAV.For example, in some embodiments, the mutant capsid proteins disclosed herein, when present in AAV virions, confer more efficient transduction of primate lung cells than AAV virions that contain corresponding parent AAV capsid proteins or wild-type AAV capsid proteins, for example, lung cells take up more AAV virions that contain target mutant AAV capsid proteins than AAV virions that contain parent AAV capsid proteins or wild-type AAV. In some such embodiments, AAV mutant virions or mutant rAAV show 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 the transduction of lung cells by wild-type AAV virions or rAAVs that contain corresponding parent AAV capsid proteins.In certain such embodiments, the mutant capsid proteins disclosed herein, when present in AAV virions, confer a wider range of transduction of primate lung cells than AAV virions that contain corresponding parent AAV capsid proteins or wild-type AAV capsid proteins.In other words, mutant AAV virions transduce cell types that are not transduced by virions that contain corresponding parent AAV capsid proteins, and therefore more types of cells in the lung than corresponding parent AAV virions. In some embodiments, the AAV mutant virions preferentially transduce lung cells, e.g., the subject rAAV virions infect lung cells with 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, or more than 50-fold specificity over other lung cells or non-lung cells, e.g., extrapulmonary cells. In some embodiments, the transduced lung cells are upper airway cells. In some embodiments, the lung cells are upper airway 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 airway 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 efficiency of transduction, broader transduction, more preferential transduction, etc., can be easily assessed in vitro or in vivo by any number of methods in the art for measuring gene expression. For example, AAV may be packaged with a genome that includes an expression cassette that includes a reporter gene, e.g., a fluorescent protein, under the control of a ubiquitous or tissue-specific promoter, and the degree of transduction is assessed by detecting the fluorescent protein, e.g., by fluorescence microscopy. As another example, the AAV may be packaged with a genome that includes a barcoded nucleic acid sequence, and the degree of transduction assessed by detecting the nucleic acid sequence, for example, by PCR. As another example, the AAV may be packaged with a genome that includes an expression cassette that includes a therapeutic gene for treating a pulmonary disease, and the degree of transduction assessed by detecting treatment of the pulmonary disease in an affected patient to whom the AAV has been administered.

[0123] 1. A method of delivering a heterologous nucleic acid to a lung cell, comprising: treating the lung cell with one or more of the following: (i) 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), ENRTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TNNSKP D (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), LAHDITKNI A (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), LATN NSKPDA (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),Provided herein are methods that include contacting a rAAV virion comprising a capsid protein that comprises, consists of, or consists essentially of a peptide insert selected from LASNTPALSA (SEQ ID NO: 66) and LASPGATTNA (SEQ ID NO: 67) and (ii) a heterologous nucleic acid that comprises 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, the heterologous nucleic acid encodes a protein and / or a small interfering RNA. In some preferred embodiments, the lung cell is any cell of the lung or trachea. In other preferred embodiments, the lung cell is an airway epithelial cell, including but not limited to an alveolar epithelial cell, a bronchial (primary, secondary, or tertiary) epithelial cell, or a tracheal epithelial cell. In some preferred aspects, the lung cell is a ciliated airway epithelial cell. In some preferred aspects, the lung cell is an alveolar epithelial type 1 (AECI) or type 2 (AECII) cell. In other embodiments, the lung cell is a smooth muscle or endothelial cell. In other embodiments, the lung cell is a basal cell, a goblet cell, or an oocyte.

[0124] 1. A method of delivering a heterologous nucleic acid to the lung of a subject (e.g., a human subject), comprising: (i) administering to the lung of a subject a nucleic 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), ENRTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TN NSKPD (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), LAHDIT KNIA (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), LAT NNSKPDA (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),Also provided herein are methods that include administering to a subject an rAAV virion that includes a capsid protein that includes, consists of, or consists essentially of a peptide insert selected from LASNTPALSA (SEQ ID NO: 66) and LASPGATTNA (SEQ ID NO: 67), and (ii) a heterologous nucleic acid that includes a nucleotide sequence encoding one or more gene products. In some embodiments, the heterologous nucleic acid encodes a protein and / or a small interfering RNA. In related embodiments, a method of delivering a heterologous nucleic acid to the upper respiratory tract, nasopharynx, paranasal sinuses, oral / cheek region, and / or salivary glands of a subject (e.g., a human subject) includes ... 9), 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), L NTTKDI (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 ID NO:67); and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products. In related embodiments, the rAAV or pharmaceutical composition comprising the same is administered to the subject by pulmonary, intrabronchial, intranasal, intratracheal and / or intrabronchial administration. In a preferred embodiment, delivery of the heterologous nucleic acid to the lungs of the subject delivers one or more encoded gene products to the lungs of the subject. Uses of the gene product include, but are not limited to, increasing the level of a factor in a cell, increasing the level of a factor in adjacent cells by secretion of the factor, decreasing the level of a factor in a cell, or decreasing the level of a factor in adjacent cells by secretion of the factor. The gene product can be designed to complement the level of a missing or defective gene product (e.g., the gene product can be a therapeutic replacement gene), decrease the level of a defective gene product (e.g., the gene product can be an interfering RNA such as an siRNA or miRNA that decreases the expression of a defective gene product), introduce a new supporting gene product, complement the level of a supporting gene product, decrease the level of an interfering gene product, or both decrease the level of an interfering gene product and introduce or complement the level of a supporting gene product.

[0125] 23. A method for treating a pulmonary disease, comprising administering to a subject in need thereof: (i) 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), ENRTSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TN NSKPD (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), LAHDIT KNIA (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), LAT NNSKPDA (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),Also provided herein is a method of administering a therapeutically effective amount of a recombinant AAV (rAAV) comprising a capsid protein comprising, consisting of, or consisting essentially of a peptide insert selected from LASNTPALSA (SEQ ID NO: 66) and LASPGATTNA (SEQ ID NO: 67), and (ii) a heterologous nucleic acid having a nucleotide sequence encoding one or more gene products, the one or more gene products being operably linked to a promoter. Use of a recombinant AAV (rAAV) comprising: (i) 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), ENRTSN (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), SNSAHI T (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),Also provided herein is a use of a recombinant AAV (rAAV) comprising a capsid protein comprising, consisting of, or consisting essentially of a peptide insert selected from 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 operably linked to a promoter, for the treatment of a pulmonary disease. (i) use of a recombinant AAV (rAAV) comprising: (i) 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), ENRTSN (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), 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), LANH Also provided herein is the use of a recombinant AAV (rAAV) comprising a capsid protein comprising, consisting of, or consisting essentially of a peptide insert selected from ISQTNA (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 operably linked to a promoter in the manufacture of a medicament for the treatment of a pulmonary disease. In some aspects, the heterologous nucleic acid comprises a nucleotide sequence encoding multiple gene products, in which the expression of the multiple (e.g., 2) gene products may be mediated by multiple (e.g., 2) independent promoters or may be mediated by a single promoter, and the multiple transgenes are separated by an internal ribosome entry site (IRES) or 2A peptide sequence. In a preferred embodiment, the heterologous nucleic acid encodes a therapeutic protein and / or a therapeutic small interfering RNA. In related embodiments, the gene product delivered by the rAAV reduces the level of an interfering gene product and / or induces or complements the level of a supporting gene product.

[0126] Pulmonary diseases that can be treated using the mutant rAAV vectors or virions and / or methods disclosed herein include, but are not limited to, monogenic diseases, complex genetic diseases, acquired diseases, and traumatic injuries. In some embodiments, the pulmonary disease is chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), cystic fibrosis, pulmonary arterial 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, aspergillosis, coccidiosis, 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, The disease is selected from acute respiratory distress syndrome (ARDS), pneumoconiosis (including black lung disease, asbestosis, and silicosis), interstitial lung disease (including sarcoidosis and autoimmune diseases), lung, pleural effusion, pleuritis, mesothelioma, pneumothorax, acute bronchitis, bronchiolitis (including bronchiolitis obliterans), sudden death syndrome, sleep apnea, bronchial dysplasia, occult pulmonary dysplasia, organizing pneumonia, e-cigarette or vape 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).

[0127] In some embodiments, genes that can be targeted for the treatment of IPF include, but are not limited to, SFTPA1 (surfactant A1) and Caveolin-1. Genes that can be targeted for the treatment of COPD include, but are not limited to, alpha-1-antitrypsin, alpha-1-antichymotrypsin, alpha-1-macroglobulin, matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 12 (MMP12), microsomal epoxide hydrolase, CYP1A1, glutathione S-transferase, heme oxygenase-1, TGF-beta-1, TNF-alpha, IL-1 complex, IL-8, IL-13, human leukocyte antigen (HLA-B7 and Bw16), vitamin D binding protein, and / or genes encoding beta-2-adrenergic receptor or biologically active parts thereof.

[0128] 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) comprising: (i) a subject mutant AAV capsid protein; and (ii) a pharmaceutical composition comprising a heterologous nucleic acid or rAAV comprising a nucleotide sequence encoding one or more gene products operably linked to one or more promoters, wherein the gene products knockdown, modify, and / or overexpress viral gene products or host cell genes to reduce or eliminate viral pathogenicity or replication in either the lungs or nasopharynx, and / or express neutralizing antibodies against epitopes on the virus.

[0129] In some aspects, a method for treating IPF is provided, comprising administering to a subject in need thereof a therapeutically effective amount of a recombinant AAV (rAAV) or a pharmaceutical composition comprising an rAAV, comprising (i) a subject mutant 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. In some preferred embodiments, a rAAV is provided for treating IPF, comprising a subject mutant AAV capsid protein and a nucleic acid comprising a nucleotide sequence encoding SFTPA1 and / or Caveolin-1 or a biologically active portion thereof.

[0130] In some aspects, a method of treating COPD is provided, comprising administering to a subject in need thereof a therapeutically effective amount of a recombinant AAV (rAAV) or a pharmaceutical composition comprising the rAAV, comprising (i) a subject mutant 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. In some preferred embodiments, a rAAV is provided for the treatment of COPD, the rAAV comprising a subject mutant AAV capsid protein and a nucleic acid comprising a nucleotide sequence encoding alpha-1-antitrypsin or a biologically active portion thereof.

[0131] In some preferred embodiments, rAAV comprising the subject mutant AAV capsid and the nucleic acid encoding CFTR or a biologically active portion thereof is provided for treating cystic fibrosis or a lung disease associated therewith as described herein, or for use in manufacturing 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 natural human CFTR protein and has the following sequence or at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical thereto: [ka] [ka] [ka]

[0132] In preferred embodiments, the nucleotide sequence encoding human CFTR or a biologically active 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: [ka] [ka] [ka]

[0133] Preferably, the nucleotide sequence encoding CFTR or a biologically active portion thereof is operably linked 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 linked to a nucleotide sequence encoding human CFTR or a biologically active portion thereof. In other aspects, the promoter is a tissue-specific promoter, preferably, the promoter directs preferential expression of the nucleic acid in lung cells, and is operably linked to a nucleotide sequence encoding human CFTR or a biologically active portion thereof. In a preferred embodiment, the promoter is a truncated CMVie promoter, and is operably linked to a nucleotide sequence encoding human CFTR or a biologically active portion thereof. In a particularly preferred embodiment, the CMVie promoter is CMV173, which has the following sequence or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto: [ka]

[0134] In particularly preferred embodiments, the rAAV vector comprises: (i) 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), ENRTSSN (SEQ ID NO: 22), PQQDTTH (SEQ ID NO: 23), NATNHVI (SEQ ID NO: 24), TNNSKPD (SEQ ID NO: 25), TNNSKPD (SEQ ID NO: 26), TNNSKPD (SEQ ID NO: 27), TNNSKPD (SEQ ID NO: 28), TNNSKPD (SEQ ID NO: 29), TNNSKPD (SEQ ID NO: 30), TNNSKPD (SEQ ID NO: 31), TNNSKPD (SEQ ID NO: 32), TNNSKPD (SEQ ID NO: 33), TNNSKPD (SEQ ID NO: 34), TNNSKPD (SEQ ID NO: 35), TNNSKPD (SEQ ID NO: 36), TNNSKPD (SEQ ID NO: 37), TNNSKPD (SEQ ID NO: 38), TNNSKPD (SEQ ID NO: 39), TNNSKPD (SEQ ID NO: 40), TNNSKPD (SEQ ID NO: 41), TNNSKPD (SEQ ID NO: 42), TNNSKPD (SEQ ID NO: 43), TNNSKPD (SEQ ID NO: 44), TNNSKPD (SEQ ID NO: 45), TNNSKPD (SEQ ID NO: 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), 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), LATNN SKPDA (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),(ii) a nucleic acid comprising from 5' to 3': (a) an AAV2 long 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 long terminal repeat sequence.

[0135] In some embodiments, a subject is administered an amount of rAAV effective to ameliorate one or more characteristics of cystic fibrosis, non-limiting examples of which include inflammation of the upper and lower respiratory tract, abnormal epithelial cytokine signaling, and elevated IgE levels. In other embodiments, a method for treating pulmonary disease associated with cystic fibrosis, including, but not limited to, upper respiratory tract disease, lower respiratory tract disease, nasopharyngeal disease, sinusitis, and / or salivary disease associated with cystic fibrosis, comprising administering to the subject an amount of rAAV effective to ameliorate one or more characteristics of ... the method comprising administering to the subject an amount of rAAV effective to ameliorate one or more characteristics of cystic fibrosis, the non-limiting examples of which include, but are not limited to, upper respiratory tract disease, lower respiratory tract disease, nasopharyngeal disease, sinusitis, and / or salivary disease associated with cystic fibrosis, the method comprising administering to the subject an amount of rAAV effective to ameliorate one or more characteristics of cystic fibrosis, the non-limiting examples of which are: (i) 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 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), Row number 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), LAISD QTKHA (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),Provided herein are methods that include administering to a subject a therapeutically effective amount of an infectious rAAV comprising: (i) a capsid protein that comprises, consists of, or consists essentially of a peptide insert selected from 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 nucleic acid operably linked to a promoter, the nucleic acid 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.

[0136] The rAAV gene therapy vectors of the invention comprising a subject mutant capsid protein can be administered to a patient by a variety of means to achieve and maintain therapeutically effective levels of a gene product (e.g., CFTR or a biologically active portion thereof) in target cells (e.g., upper respiratory tract cells). In some embodiments, an infectious rAAV is administered to a subject (e.g., a subject with cystic fibrosis) in one or more doses, each dose being about 1×10 13 ~Approx. 1×10 15 Vector genome (vg), approximately 1 × 10 13 ~Approx. 1×10 14 vg, approx. 1×10 14 ~Approx. 1×10 15 vg, or approximately 1 × 10 15 ~Approx. 5×10 15 In some preferred embodiments, each dose contains about 1×10 14 vg or approximately 1×10 15 In another embodiment, the rAAV comprises about 10 12 ~10 14 The subject is administered at least one dose of vector genome (vg) / kg. In a related embodiment, the subject is administered about 1×10 11~Approx. 1×10 14 vg / kg, approximately 1×10 12 ~Approx. 9×10 13 vg / kg, approximately 1×10 12 vg / kg ~ approx. 9×10 12 vg / kg, preferably about 2×10 12 vg / kg ~ approx. 3×10 12 vg / kg, more preferably about 2.6×10 12 vg / kg, approx. 2.7×10 12 vg / kg, approx. 2.8×10 12 vg / kg, approximately 2.9×10 12 vg / kg, approx. 3.0×10 12 vg / kg or approximately 3.1 × 10 12 vg / kg is administered.

[0137] In some embodiments, the treatment involves administration of no more than a single dose to the subject and is effective to achieve a durable and sustained therapeutic concentration of the gene product (e.g., CFTR or a biologically active portion thereof). In related embodiments, the treatment involves administration of about 1×10 rAAV comprising a heterologous nucleic acid encoding a subject mutant capsid protein and a gene product. 13 ~Approx. 1×10 15 This includes administration of plaque forming units (pfu), viral particles (vp) or viral genomes (vg) to a human (e.g., a human with cystic fibrosis) in a single dose or less by inhalation. In other aspects, the dosing treatment can be a multiple dose schedule. Methods for administering AAV vectors to humans have been previously described by Kay et al. (2000, Nat Genet 24:257-261), the entire contents of which are incorporated herein by reference. In some preferred embodiments, the infectious rAAV is administered to a subject by pulmonary, intrabronchial, intranasal, intratracheal and / or intrabronchial administration. In some preferred embodiments, the infectious rAAV is administered by inhalation of an aerosol suspension (e.g., via a nebulizer) containing the rAAV.

[0138] For purposes of the present invention, the disclosure provides an isolated nucleic acid comprising a nucleotide sequence encoding a subject mutant AAV capsid protein as described above. The isolated nucleic acid can be an AAV vector, such as a recombinant AAV vector.

[0139] The disclosure herein further provides host cells, such as, but not limited to, isolated (genetically modified) host cells comprising the subject nucleic acid. The host cells according to the invention disclosed herein can be isolated cells, such as cells from an in vitro cell culture. Such host cells are useful for producing the subject rAAV mutant virions, as described herein. In one embodiment, such host cells are stably genetically modified with the nucleic acid. In other embodiments, the host cells are transiently genetically modified with the nucleic acid. Such nucleic acids are stably or transiently introduced into the host cells using established techniques, including, but not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, and the like. For stable transformation, the nucleic acid will generally further include a selection marker, such as any of several well-known selection markers, such as neomycin resistance. Such host cells are generated by introducing the nucleic acid into any of a variety of cells, such as mammalian cells, such as mouse cells and primate cells (e.g., human cells). Exemplary mammalian cells include, but are not limited to, primary cells and cell lines, and exemplary cell lines include 293 cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1 / 2 fibroblasts, CHO cells, and the like, and exemplary host cells include, without limitation, 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 These include 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCL1.3), human embryonic kidney (HEK) cells (ATCC No., CRL1573), HLHepG2 cells, and the like.The host cell can also be made using baculovirus to infect insect cells such as Sf9 cells to produce AAV (see, for example, U.S. Patent No. 7,271,002; U.S. Patent Application No. 12 / 297,958). In some embodiments, the genetically modified host cell comprises a nucleic acid comprising a nucleotide sequence encoding one or more AAV rep proteins in addition to a nucleic acid comprising a nucleotide sequence encoding a mutant AAV capsid protein, as described above. In other embodiments, the host cell further comprises a rAAV mutant vector. The rAAV mutant virion can be produced using such a host cell. Methods for producing rAAV virions are described, for example, in U.S. Patent Application Publication No. 2005 / 0053922 and U.S. Patent Application Publication No. 2009 / 0202490.

[0140] In some embodiments of the mutant 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, the cytomegalovirus promoter (CMV) (Stinski et al., (1985) Journal of Virology 55(2):431-441), the CMV early enhancer / chicken p-actin (CBA) promoter / rabbit β-globin intron (CAG) (Miyazaki et al., (1989) Gene 79(2):269-277, CAG ... 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 examples, 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 examples, the nucleotide sequence encoding the gene product of interest is operably linked to a lung-specific regulatory element, e.g., a regulatory element that confers selective expression of the operably linked gene in lung cells. Lung-specific promoters include, but are not limited to, the surfactant protein B (SPB) gene promoter and the surfactant protein C (SPC) promoter.

[0141] Also provided herein is a pharmaceutical composition comprising: a) a rAAV comprising a heterologous nucleic acid encoding a subject mutant AAV capsid protein and one or more gene products; and b) a pharma- ceutically 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, the pharma- ceutically 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 undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts may include, for example, mineral acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, and the like, and salts of organic acids such as acetate, propionate, malonate, benzoate, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances, and the like, may be present in such vehicles. A wide variety of pharma- ceutically acceptable excipients are known in the art and need not be discussed at length here. Pharmaceutically acceptable excipients can be found, for example, in A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds., 7th ed. th eds., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3 rd and is described in detail in various publications, including ed. Amer. Pharmaceutical Assoc.

[0142] In some embodiments, the pharmaceutical composition comprises 1×10 8 ~1×10 15of vector particles or vector genomes, 1 × 10 10 ~1×10 13 vector particles or vector genomes, or approximately 1 × 10 10 , about 2×10 10 , 3×10 10 , about 4×10 10 , about 5×10 10 , about 6×10 10 , about 7×10 10 , about 8×10 10 , about 9×10 10 , about 1×10 11 , about 2×10 11 , about 3×10 11 , about 4×10 11 , about 5×10 11 , about 6×10 11 , about 7×10 11 , about 8×10 11 , about 9×10 11 , about 1×10 12 , about 2×10 12 , about 3×10 12 , about 4×10 12 , about 5×10 12 , about 6×10 12 , about 7×10 12 , about 8×10 12 , about 9×10 12 Or about 1 x 10 13 In some embodiments, the pharmaceutical composition comprises about 1×10 vector particles or vector genomes. 11 ~Approx. 1×10 12 The vector particles or vector genomes include: EXAMPLES

[0143] The following examples illustrate preferred embodiments of the invention and are not intended to limit its scope in any way. Although the invention has been explained in relation to its preferred embodiments, various modifications thereof will become apparent to those skilled in the art from reading the present application.

[0144] Example 1 Directed evolution screening was used to identify AAV capsid variants capable of conferring more efficient transduction of primate lungs and improved gene delivery efficiency to upper airway cells of primate lungs following intratracheal aerosol administration to non-human primates (NHPs). The selection process incorporated delivery to NHP lungs in vivo and the use of human lung cultures in vitro.

[0145] method

[0146] Therapeutic Vector Evolution

[0147] A directed evolution process was applied to discover AAV capsid variants capable of broadly transducing upper airway cells in primate lungs after aerosol administration (Figure 1). Briefly, using various molecular biology techniques and several different AAV serotypes as templates, a library of approximately 1 billion unique synthetic variant AAV capsid sequences was created from 37 different proprietary sub-libraries. The library was packaged in HEK293T cells to generate viral particles such that each viral particle was composed of a synthetic capsid shell surrounding a viral genome encoding the same capsid. The variants in 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 DNA was harvested from lung tissue or cell culture, the genomes of capsids amplified from the tissue were packaged as described above as the starting library for the next round of selection. This procedure was performed for a total of 5 cycles.

[0148] A motif was declared a “hit” when certain selection criteria were met: (1) the motif represents a certain percentage of the sequenced population in two or more successive rounds of selection, or (2) the motif represents a certain percentage of the sequenced population in one or more rounds of selection.

[0149] The time point at which the model system transitioned from in vivo NHP to in vitro proximal airway organotypic cultures was based on the results of sequencing analyses performed after the second and third rounds of NHP delivery (Figure 2). Briefly, the transition to the in vitro human ALI model system occurred when: (1) the most frequent hits represented a certain percentage of the sequenced population and (2) less than a certain percentage of the sequenced clones represented unique sequences.

[0150] Selection was considered complete when the following selection completion criteria were met: fewer than two new “hits” were identified in the round, (1) the combined “hits” represented a specific percentage of the sequenced population, and / or (2) a single “hit” represented a specific percentage of the population.

[0151] Cell line and library generation

[0152] 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. Viral libraries were generated in HEK293T cells using triple transfection and iodixanol gradient centrifugation. 19、21 Virus was purified by Amicon filtration and DNase-resistant genome titers were determined by quantitative PCR (qPCR) as previously described. 19、21

[0153] Intratracheal injection and tissue collection

[0154] For each round of selection, one male cynomolgus monkey (Macaca fascicularis), 4-6 years old and weighing 5.5-7.1 kg, was administered. Animals were anesthetized with 10 mg / kg ketamine and 15 μg / kg dexmedetomidine delivered intramuscularly (IM). 5 mL of library was precomplexed 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 of the tube positioned at clavicle level (approximately 5 cm above the tracheal carina), and its position confirmed by fluoroscopy. Animals were positioned in a chair in a sitting position for administration.

[0155] For the first round of selection, a nebulizer device was connected to the distal end of the endotracheal tube and a Byrd respirator was used to deliver breaths at a rate of 15 ± 1 breaths / min at a pressure of 20 cmH2O. For the second and third rounds of selection, an AeroEclipse II nebulizer device was connected to the distal end of the endotracheal tube and a Byrd respirator was used to deliver breaths at a rate of 12–24 breaths / min at a pressure of 15–20 cmH2O. After completion of dosing, each animal was extubated and administered 0.15 mg / kg atipamezole IM to reverse sedation. Animals were visually monitored until fully recovered from anesthesia before being returned to their home cages.

[0156] Euthanasia was performed by trained veterinary staff using 100 mg / kg sodium pentobarbital delivered intravenously on day 15±1. Lungs including tracheas were removed and dissected as detailed below. DNA was isolated from upper respiratory tract cells and stored at −20° C. until viral genome amplification.

[0157] Upper airway epithelial cell isolation

[0158] 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. Excessive 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 reduce 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 h, with inversion of the tube twice a day. The enzymes were inactivated by the addition of FBS to 10% of the total volume. The pieces were then cut lengthwise and the epithelial lining scraped away from the cartilage. The cells were harvested and centrifuged at 300 × g for 10 min. The cell pellet was rinsed twice with PBS. The pellet was resuspended in airway epithelial cell basal medium with supplement (ATCC, Manassas, VA) containing 5% FBS, and the cells were plated onto tissue culture-treated 10 cm dishes to allow fibroblast adhesion and further refine cell isolation to airway epithelial cells. After 2-4 h of seeding, the medium containing non-adherent cells was collected and centrifuged at 300 x g for 10 min. Pelleted cells were either lysed to extract library DNA or resuspended in PBS, counted, and plated for another round of in vitro selection.

[0159] In vitro library transduction

[0160] Human ALI cultures were transduced 30 days after seeding on human placental collagen type IV (Sigma). On the day of transduction, three inserts were incubated with trypsin-EDTA 0.05% (ThermoFisher) for 10 min at 37 °C. Trypsin was inactivated with defined trypsin inhibitor (ThermoFisher). Cells were harvested from the inserts and counted with a hemocytometer. The average cell number per insert was determined and used to calculate the total viral genomes required per insert. Mucus produced by the cultures was removed prior to viral transduction by washing with PBS. A multiplicity of infection (MOI) of 50,000 for round 4 and 10,000, 25,000 and 50,000 for round 5 were used. Each round was performed in two parallel sets, one where the library was precomplexed with a 1:10 dilution of human IVIG for 30 min prior to cell administration and one without IVIG precomplexation. Cells were exposed apically with the library for 24 h. Four days after infection, DNA was isolated from the cultures and stored at -20°C until viral genome amplification.

[0161] DNA quantification, DNA amplification and sequencing analysis

[0162] DNA was isolated from the above cells using the DNeasy Blood & Tissue kit (Qiagen). The AAV library genome was quantified by digital droplet polymerase chain reaction (ddPCR). The AAV mutant cap genes were amplified by PCR. The cap genes were inserted into the pSub2 library packaging plasmid using NotI and HindIII. The cap genes were then sequenced by a third-party DNA sequencing facility. The sequencing files were analyzed using Geneious software (Biomatters).

[0163] result

[0164] Pilot study of delivery device parameters and upper airway cell isolation

[0165] Two delivery devices, an in-house nebulizer from Valley Biosystem and an AeroEclipse II breath-actuated nebulizer (Trudell Medical International), were used to allow downstream compatibility with multiple clinically mobile devices. Both delivery devices were evaluated in a pilot study delivering Evans Blue dye to ensure ventilation parameters were adequately distributed to all lobes and alveolar sacs. The Valley Biosystem in-house nebulizer demonstrated good distribution to all lobes, including the alveolar compartment, with more intense dye observed in the dependent lobes. In parallel with the first round of selection, additional method development and airway delivery device testing was performed to successfully adapt and develop the AeroEclipse II breath-actuated nebulizer to deliver aerosolized AAV particles to intubated NHPs. Throughout the dye distribution study, excellent independent delivery to all six NHP lobes, including both lobes, was demonstrated. The AeroEclipse II nebulizer was then used for selection rounds 2 and 3.

[0166] A total of six NHP lungs were used to optimize the upper airway cell isolation protocol. Protocol optimization resulted in high yields and purity of cells from the trachea and primary, secondary, and tertiary bronchi isolated from all three sets of NHP lungs used during therapeutic vector evolution.

[0167] Library generation and construction

[0168] All 37 capsid libraries were synthesized, produced, and characterized prior to the initiation of round 1 of the therapeutic vector evolution program. As shown in Figure 3a, the diversity of the plasmid libraries was approximately 1 × 10 per library for a total diversity of over one billion genetic variant sequences. 6 ~1×10 8The total library was estimated to contain over 100 unique variants. This represents a high-quality, highly diverse starting library of AAV variants. We then completed the generation of individual libraries to generate sufficient material for the first round of selection. As shown in Figure 3b, all libraries were produced at sufficient levels to produce material for in vivo therapeutic vector evolution selection. After generation and prior to library administration, sequences from all libraries were assessed for the presence of non-functional mutations (i.e., frameshift mutations or stop codons) and the frequency of unique sequences by Sanger sequencing. Most libraries had minimal frequencies of non-functional mutations, and all libraries were incorporated into the first round of administration to NHPs.

[0169] Aerosol delivery to NHP lungs

[0170] For the first round of selection, all libraries were combined and successfully administered to a single NHP by single-dose aerosol administration using an in-house nebulizer system from Valley Biosystems. Prior to administration, the library was incubated with 1.75 mg / mL human IVIG, which represents a high but physiologically relevant lung mucus concentration of human NAbs. 12 The administered library dose of 100x vg represents a dose approximately 100x lower than the current maximum feasible dose based on manufacturing considerations. This therefore represents a stringent selection pressure to enable the discovery of vectors capable of transducing upper airway cells in the trachea and bronchi. NHP lungs were harvested 2 weeks after administration. Cells were isolated from the lungs in separate trachea / primary bronchus and secondary / tertiary bronchus samples, and DNA was isolated from the cells. High total cell yields and high populations of acetylated tubulin+ cells were obtained from both regions. This process was repeated for two more rounds using progressively lower doses of the viral library.

[0171] Following DNA isolation from each round of selection, the AAV viral library genomes were quantified by ddPCR to confirm successful localization of the library vector to the cell type of interest. There was a dose-dependent decrease in the amount of viral genome present in the upper respiratory tract, corresponding to the lower doses administered in each round (Figure 4).

[0172] In addition to quantification by ddPCR, amplification of the capsid gene from tissues represents successful localization of the library vector to the cell type of interest. Amplified capsids from each round of selection in NHPs were cloned into an AAV library packaging plasmid for sequence analysis and to initiate subsequent rounds of selection. Sequencing was performed on individual clones in the library to determine the frequency of variants in the population. Sequencing was performed on a minimum of 90 clones from tracheal / primary bronchial and secondary / tertiary bronchial samples from each round. Variants were assessed for the presence of motifs in the sequencing data. Variants were grouped into motifs based on the presence of unifying variations occurring in multiple sequences (e.g., specific point mutations or specific peptide insertion sequences at consistent positions in the capsid). A motif was designated as a hit if it represented a certain percentage of the population sequenced in two or more consecutive rounds of selection or a certain percentage of the population sequenced in one or more rounds of selection.

[0173] Two motifs (point mutant #1, peptide insertion #1 (SEQ ID NO: 40) in the trachea / primary bronchus region and one motif (peptide insertion #2 (SEQ ID NO: 41) in the secondary / tertiary bronchus region) 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 to be monitored to determine whether either or both could be designated as hits after additional in vitro rounds of therapeutic vector evolution. Point mutation #1 in the tracheal / 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 specific percentage of the sequenced AAV population in that region (Figure 5). Within the total sequenced population, a specific percentage of the sequenced AAV clones represented unique AAV sequences. Therefore, based on the sequencing analysis performed in round 3 of the Therapeutic Vector Evolution Program, the model system was transitioned from in vivo NHP to in vitro proximal airway organotypic cultures for the remainder of the selection process.

[0174] Apical delivery into human ALI cultures

[0175] For rounds 4 and 5 of in vitro selection, cells from three human lung donors were pooled and cultured in multilayer air-liquid interface (ALI) culture in a transwell system. 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 immunocytochemistry 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).

[0176] The AAV library was administered to the apical side of the proximal airway organotypic culture system for rounds 4 and 5 at MOIs of 50,000 and 10,000, respectively. Similar to the in vivo portion of the selection, after isolating DNA from each round in vitro, the AAV viral library genome was quantified by ddPCR to confirm successful localization of the library vector to the cell type of interest. Unlike the in vivo portion of the selection, no dose-dependency was observed between rounds, likely due to the high MOI used in vitro (Figure 6a). Furthermore, preincubation with human IVIG did not appear to significantly affect genome presence after transduction (Figure 6b).

[0177] As previously described, amplification of capsid genes from tissues represents successful localization of the library vector to the cell type of interest. Amplified capsids from each in vitro selection round were recloned into AAV library packaging plasmids for sequence analysis and to initiate subsequent selection rounds. Sequencing was again performed on individual clones in the library to determine the frequency of mutants in the population. A minimum of 89 clones from the absence and presence of human IVIG were sequenced for each round.

[0178] The hits identified during the in vivo portion of the selection (point mutant #1, peptide insertion #1, peptide insertion #2) remained at relatively high frequency after two rounds of in vitro selection (Figure 7). One of the mutants of interest (peptide insertion #3) dropped in frequency and never met the criteria to be designated as a hit. The other mutant (peptide insertion #4) monitored after round 3 met the criteria to be nominated as a hit after round 4, but also dropped in frequency in round 5 (Figure 7). Following round 4, two additional motifs (peptide insertion #5 (SEQ ID NO: 44), peptide insertion #6 (SEQ ID NO: 45)) were designated as hits. No new hits were identified in round 5, and the combined hits represented more than 50% of the sequenced population (Figure 7). Therefore, based on the sequencing analysis performed in round 5 of the therapeutic vector evolution program, the discovery program was considered complete.

[0179] result

[0180] AAV capsids were successfully amplified from isolated upper airway cell populations isolated from the trachea and primary, secondary and tertiary bronchi from three consecutive rounds of selection in NHPs after one round of intratracheal aerosol administration of the library using a nebulizer. Human in vitro ALI cultures were utilized for the subsequent two rounds of selection. Following successful amplification of viral genomes from NHP lungs or human ALI cultures, sequencing was performed on individual clones within the library. Individual sequences were grouped into motifs based on the presence of unifying variations and assessed based on frequency and diversity of variations within the sequencing analysis. After analysis, six mutant sequences emerged as hits and were recommended for further characterization. These six mutants (AAV102-AAV107, SEQ ID NOs: 68-73) are AAV2-based capsids, each containing a peptide inserted in the loop region and a V708I amino acid substitution.

[0181] A summary of the frequency of peptide insertion motifs for each of the above selection rounds is shown in Table 1 below. [Table 1-1] [Table 1-2]

[0182] Sequenced clones identified from the directed evolution screen included mutant capsids with only the peptide insertion (e.g., SEQ ID NO: 40, 41, 42, 43, 44 or 45) and otherwise identical to SEQ ID NO: 2, as well as mutant capsids with peptide inserts in combination with various amino acid substitutions. A summary of the amino acid substitutions (numbering relative to SEQ ID NO: 2) that were tolerated in combination with the inserted peptides of Table 1 in the sequenced clones identified from the directed evolution screen is provided in Table 2 (in each case the mutant AAV capsid protein is otherwise identical to SEQ ID NO: 2): [Table 2-1] [Table 2-2]

[0183] Example 2 Six mutant capsid sequences identified above (AAV102-AAV107; SEQ ID NOs: 68-73) that exhibit a preference for upper airway epithelial cell transduction were characterized in vitro in non-human primate (NHP) and human ex vivo pulmonary upper airway air-liquid interface (ALI) cultures at various multiplicities of infection (MOI).

[0184] Briefly, recombinant AAVs (rAAVs) were produced containing the cap mutant sequences of AAV102-AAV107, each of which contains a reporter cassette with a ubiquitous promoter (CMV enhancer, chicken beta-actin promoter and rabbit beta-globin splice acceptor site, CAG) driving EGFP. Transduction of six mutant AAV capsids, AAV101, and the native serotypes AAV2 and AAV5, was evaluated in vitro at four multiplicities of infection, 12,500, 25,000, 50,000 and 100,000.

[0185] A first generation capsid mutant (AAV101) engineered for enhanced transduction of airway epithelia in in vitro organotypic ALI cultures has been shown to be superior to naturally occurring AAV capsids (see SEQ ID NO: 12 in U.S. Patent Application Publication No. 2021 / 0395772, the contents of which are incorporated herein by reference). However, further discovery was undertaken to identify vectors that could efficiently transduce both in vitro and in vivo at levels sufficient to achieve clinical benefit in cystic fibrosis (CF).

[0186] method

[0187] AAV manufacturing

[0188] rAAVs containing mutant capsid proteins AAV101-AAV107 and native serotypes AAV2 and AAV5, respectively, were produced in a triple plasmid transfection process utilizing commercially available transfection reagents and human embryonic kidney 293 (HEK293) cells cultured in flatware. The transfected cells were harvested with the supernatant, followed by nuclease treatment and clarification through a 0.2 μm filter. The clarified harvest was purified using affinity chromatography (AVB Sepharose HP, Cytiva Life Sciences) and buffer exchanged into DPBS containing 0.005% Pluronic® F68. After 0.2 μm filtration, the drug substance (BDS) was dispensed into cryovials (Corning) and stored at -80°C.

[0189] Digital droplet PCR (ddPCR) for AAV titer analysis

[0190] Viral genome titers are determined by ddPCR. Test samples are diluted, DNase treated, and then further diluted in DPBS containing 0.02% Pluronic F68, ddPCR Supermix, and primers / FAM-labeled probes corresponding to SV40PolyA sequences. 20 μL of sample is dispensed into droplets using a Bio-Rad Automated Droplet Generator, subjected to PCR, and then read on a Bio-Rad QX200 Droplet Reader, which measures each droplet individually for fluorescent signal. Data is analyzed using Bio-Rad QuantaSoft software, which provides absolute quantification of target sequences using Poisson statistical analysis of positive and negative droplets. A no-template control is used to set a negative baseline for the samples. An internal ddPCR reference standard virus is used as the reference standard control.

[0191] Isolation and culture of upper airway epithelial cells

[0192] Airway epithelial cells were isolated from the trachea and primary-tertiary bronchi of NHPs and human donor lungs rejected from transplants (Donor Network West) according to published protocols (Karp et al., 2002). Isolated cells were frozen and stored in liquid nitrogen.

[0193] Transwell insert (0.4 μm, 0.32 cm 2 Airway epithelial cells were thawed on human placental collagen IV (60 μg / mL, MilliporeSigma, Burlington, MA) in Airway Epithelial Cell Basal Medium with growth supplements in a Bronchial Epithelial Cell Growth Kit (ATCC, Manassas, VA) on a 100-well plate (Corning, Corning, NY). Two days after seeding, medium was aspirated from the inserts and basal chambers. The basal chambers were supplemented with PneumaCult ALI basal medium provided with growth supplements (Stem Cell Technologies, Vancouver, Canada). Basal medium was replaced every 2–3 days. The liquid on the apical side of the transwell was aspirated daily until an air-liquid interface (ALI) was achieved. Cells were allowed to mature for 30 days before cell characterization and transduction.

[0194] AAV transduction

[0195] After at least 30 days in culture, cells were washed extensively to remove accumulated mucus. Three inserts of each species were harvested and cells were counted. Based on the AAV titer and average cell number of each species, the virus volume was diluted to reach MOIs of 12,500, 25,000, 50,000 and 100,000. Volumes were equalized with diluent to obtain a transduction volume of 100 μl per insert for each species. Human cells were transduced apically and basally, and NHP cells were transduced apically with AAV after mucus washing (n=1 / condition). 24 hours after infection, the fluid on the inserts was aspirated and the basal compartment medium was replaced. Cells were fixed for immunocytochemistry 7 days after infection.

[0196] immunocytochemistry

[0197] Cells were fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were then imaged using a Zeiss Axio Observer D.1 fluorescent microscope to visualize EGFP overlooking the cells. Inserts containing cells were then embedded in optimal cutting temperature medium (OCT, ThermoFisher, Waltham, MA) and sectioned into 20 μm slices on a CryoStar cryostat. Slides containing cross sections of inserts were blocked for 1 h at room temperature in 2% goat serum, 5% bovine serum albumin in 0.02% Triton X-100 in PBS. Slides were then incubated with primary antibody for 2 h at room temperature, washed three times with 0.02% Triton X-100, and incubated with secondary antibody for 1 h at room temperature. Slides were then counterstained with DAPI (nuclear stain) for 5 min at room temperature, washed three times, and sealed with a coverslip using Prolong Gold Anti-fade mountant (ThermoFisher). The cells were then imaged using a Zeiss Axio Observer D.1 fluorescent microscope.

[0198] Digital droplet polymerase chain reaction (ddPCR) for AAV transcript detection

[0199] RNA was extracted from each insert using the Qiagen RNeasy Plus Mini Kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). RNA was quantified and 2 μg of cDNA was made using the iScript cDNA Synthesis Kit according to the manufacturer's instructions (total volume 40 μL, Bio-Rad, Hercules, CA). Dilutions of cDNA were made 1:10 and 1:100 in water to a total volume of 50 μL and 2 μL was added per assay plate well. A master mix was made with 2× Supermix ddPCR™ (without dUTP, Bio-Rad) and ddPCR-compatible primer / probe mix (HPRT1 HEK-Housekeeper, 20× ID Bio-Rad and SV40 FAM ID (Furuta-Hanawa, Yamaguchi & Uchida, 2019)) up to a total volume of 18 μL per well. The SV40 primers / probe are a surrogate for measuring EGFP transgene transcript levels since the SV40 polyadenylation site is included as part of the virally delivered transgene cassette. The master mix and cDNA were combined in a ddPCR™ 96-well assay plate and loaded into the QX200™ AutoDG Droplet Digital™ PCR System according to the manufacturer's instructions. Droplets were generated and the plate transferred to a C1000 Touch Thermal Cycler for PCR. ThermoCycler conditions were as follows: 94°C 10 min, 94°C 30 sec, 60°C 1 min (steps 2-3 repeated 39x), 98°C 10 min, hold at 12°C. The plate was then transferred to a QX200™ Droplet Reader with parameters set to read on the FAM and HEK channels. Data was analyzed with QuantaSoft software with a threshold of 3000 and then transferred to an Excel file where further analysis was completed. The lowest dilution with a viable read was used to generate a graph using the ratio of SV40 to housekeeper values.

[0200] Mean fluorescence intensity calculation

[0201] For each well of transduced ALI, three images were taken per well using a Zeiss Axio Observer D.1 fluorescent microscope with a 10x objective. Imaged areas were selected in a consistent and unbiased pattern per well. The exposure time for each image was set using a standard AAV2 MOI of 25,000 transduced wells. After imaging, all images were processed using ImageJ software. After subtracting background fluorescence, MFI values ​​were obtained using the "Measure Mean Gray Value" tool. The average of the three images from each well was taken to find 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 graph (Figure 3b).

[0202] result

[0203] AAV production of novel capsids

[0204] rAAV containing mutant capsid proteins and wild-type serotype were produced by triple transfection in HEK293 cells cultured on CellSTACK and purified using affinity chromatography. Upstream productivity (reported in units of log viral genomes (vg) per square centimeter of cell culture surface area) was measured after supernatant and cell harvest by ddPCR analysis (Table 1, Upstream Productivity). All novel capsids were capable of packaging GFP payloads, demonstrating productivity comparable to other AAV vectors produced using flatware production processes. Downstream yields (reported as % of vg recovered) were determined by ddPCR after affinity purification and buffer exchange (Table 3, Downstream Yield). Initial packaging and yield data for AAV101-EGFP are also shown in Table 3 for productivity and yield comparison. [Table 3-1] [Table 3-2]

[0205] Transduction efficiency of novel AAV mutants

[0206] The transduction efficiency of six novel variants identified in Example 1: AAV2, AAV5 and AAV101 was compared to the AAV control. 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.

[0207] For apically transduced human cells, minimal EGFP expression was detected in AAV2 and AAV5 controls. Among the six novel variants, the transduction efficiency of AAV102 and AAV103 was higher at lower doses than AAV2, AAV5 and AAV101 when analyzed by fluorescence microscopy (Fig. 8a). AAV103 transduction at MOI 100,000 was low, likely due to inefficient mucosal washing of the well. AAV106 transduction was higher than AAV2 and AAV5.

[0208] In apically transduced NHP cells, the EGFP pattern was variable within the cells. Enhanced transduction around the outer edge of the transwell was repeatedly observed in a ring pattern with limited transduction in the center of the transwell. Overall, the novel 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 was not as clear as in the human cultures, likely due to edge effects. All images shown for NHP transduction capture part of the edge of the well to visualize transduction.

[0209] In basally transduced human cells, a different pattern emerged. AAV104 had higher transduction efficiency than AAV2, AAV5 and AAV101 (Fig. 8c). AAV102 and AAV103 did not show as good transduction upon apical administration. Overall, basal transduction was lower than apical transduction. The relative lack of transduction after basal administration compared to apical administration is not surprising, given that these capsid variants were selected by apical administration to NHPs in vivo and human ALI in vivo. NHP in vitro cultures were not basally transduced.

[0210] The cell culture composition of human ex vivo lung epithelial airway cultures was assessed 30 days post-thaw by immunocytochemistry analysis using antibodies against acetylated tubulin (Ac-tub; ciliated cells), cytokeratin 5 (KRT5; basal cells), and mucin (MUC5ac; goblet cells). Cross-sectional analysis of human apically and basally transduced cells was completed with inconclusive results for specific cell variant tropism within this ALI system (data not shown). Further analysis is required to properly understand the specific tropism of each capsid within this heterogeneous culture system. Further studies in vivo may also help to better characterize cell-specific transduction for each capsid.

[0211] Follow-up studies were completed to further examine AAV102 and AAV103, the best performing variants from the initial apical transduction screen in human ALI cultures, compared to AAV2 and AAV101. Immunocytochemistry analysis showing EGFP is shown in Figure 9a. Mean fluorescence intensity was averaged and graphed from data from three images per well, three wells per condition (Figure 9b). Finally, transcription levels of each variant were determined by digital droplet PCR (Figure 9c). All three analyses confirmed that AAV102 and AAV103 outperformed AAV2 in transduction efficiency of ex vivo airway cultures when transduced apically at a low MOI.

[0212] 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 at the air-liquid interface for at least 30 days. Cells were transduced with AAV102-AAV107 or control capsids carrying an EGFP reporter gene driven by a CAG promoter (MOI=25,000) in the presence or absence of mucus. Seven days after transduction, images of EGFP were taken as representative of the transduction efficiency of the capsids. Each of the capsids AAV102-AAV107 showed stronger transduction efficiency when transduced in the absence of mucus compared to the wild-type AAV serotype and compared to the AAV101 control (Figures 10A and 10B, top panels). Transduction in the presence of mucus reduced the transduction efficiency of each capsid (except AAV5, which showed no transduction in the absence of mucus) (compare the top panel with the bottom panel in Figure 10A and Figure 10B).

[0213] In vitro analysis of six top AAV capsid mutants identified by selection in NHPs using aerosolized delivery technology and apical administration to human ex vivo upper airway epithelial ALI cultures according to Example 1 demonstrates that rAAVs with mutant capsid proteins are superior in transducing human and NHP ex vivo upper airway epithelial ALI cultures compared to AAV101 and wild-type AAV serotypes. Different mutants showed preferences for transduction depending on the route of administration. AAV102 and AAV103 apically transduced human in vitro upper airway epithelial ALI cultures better than AAV101 and WT serotypes at lower MOI. In basally transduced human cells, AAV104 had the highest transduction efficiency.

[0214] Example 3 The novel AAV capsid mutants will be examined in neutralizing antibody screens and in vivo vector characterization in NHPs.

[0215] While the materials and methods of the present invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that variations can be made in the methods described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention.

Claims

1. A mutant adeno-associated virus (AAV) capsid protein comprising a peptide insertion into a corresponding parent AAV capsid protein, wherein the peptide insertion comprises an amino acid sequence selected from HDITKNI (SEQ ID NO: 12), DNTVTRS (SEQ ID NO: 14), NSTRHTD (SEQ ID NO: 16), NQDYTKT (SEQ ID NO: 13), SNSVQSI (SEQ ID NO: 15), and TNRTSPD (SEQ ID NO: 17).

2. The mutant AAV capsid protein according to claim 1, 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 in the corresponding arrangement in a capsid protein of another AAV serotype, preferably between amino acids 587 and 588 of VP1 of AAV2 (SEQ ID NO: 2), or between amino acids 588 and 589 of VP1 of AAV2 (SEQ ID NO: 2), or in the corresponding arrangement in the capsid protein of another AAV serotype.

3. The aforementioned peptide insertion is Y １ Y ２ SNSVQSIY ３ and Y １ Y ２ NSTRHTDY ３ It includes an amino acid sequence selected from Y １ -Y ３ Each of is independently selected from Ala, Leu, Gly, Ser, Thr, and Pro, and preferably the peptide insertion comprises an amino acid sequence selected from LAHDITKNIA (SEQ ID NO: 40), LADNTVTRSA (SEQ ID NO: 42), LANSTRHTDA (SEQ ID NO: 44), LANQDYTKTA (SEQ ID NO: 41), LASNSVQSIA (SEQ ID NO: 43), and LATNRTSPDA (SEQ ID NO: 45), the mutant AAV capsid protein according to claim 1.

4. The mutant AAV capsid protein further comprises one or more amino acid substitutions for VP1 of AAV2 (SEQ ID NO: 2), or one or more corresponding substitutions in the capsid protein of another AAV serotype, wherein the one or more amino acid substitutions are 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, T344S, E347D, A mutant AAV capsid protein according to claim 1, selected from the group consisting of G376A, P399H, G406E, Q428H, P436H, N449D, P451Q, N469D, D472N, T491I, K532E, K544E, R585K, A591D, A593E, D594N, D608N, H641N, K688R, N705S, and V708I, numbered according to Sequence ID No. 2, preferably the mutant AAV capsid protein comprises a V708I amino acid substitution for the corresponding arrangement in the capsid protein of VP1 of AAV2 (Sequence ID No. 2) or another AAV serotype.

5. The mutant AAV capsid protein according to claim 1, wherein the mutant AAV capsid protein is at least 95%, at least 98%, or at least 99% identical to the amino acid sequence described in any one of SEQ ID NOs. 68, 70, 72, 69, 71, and 73.

6. The mutant AAV capsid protein according to claim 5, comprising the amino acid sequence described in any one of the above sequence numbers 68, 70, 72, 69, 71, and 73.

7. The mutant AAV capsid according to claim 1, wherein the capsid protein confers increased infectivity of lung cells to infectious rAAV virions compared to the infectivity of lung cells by AAV virions containing wild-type AAV capsid protein, and preferably, the capsid protein further confers increased resistance to neutralization by neutralizing antibodies to infectious rAAV virions compared to AAVs containing the corresponding parental AAV capsid protein.

8. An isolated nucleic acid comprising a nucleotide sequence encoding the mutant AAV capsid protein according to any one of claims 1 to 7.

9. An infectious recombinant AAV (rAAV) virion comprising the mutant AAV capsid protein described in any one of claims 1 to 7.

10. The rAAV virion according to claim 9, further comprising heterogeneous nucleic acids including nucleotide sequences encoding one or more gene products, preferably the one or more gene products being operably linked to an expression control sequence.

11. The rAAV virion according to claim 10, wherein the gene product is a protein, a small interfering RNA, a microRNA, a short hairpin RNA, or an antisense RNA.

12. The rAAV according to claim 11, wherein the heterogeneous nucleic acid comprises a nucleotide sequence encoding CFTR or a biologically active portion thereof, and SERPINA1 or a biologically active portion thereof.

13. A host cell containing rAAV as described in claim 11.

14. A pharmaceutical composition comprising the rAAV described in claim 12 and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition according to claim 14, for use in the treatment of cystic fibrosis or alpha-antitrypsin deficiency, wherein the heterogeneous nucleic acid comprises a nucleotide sequence encoding CFTR or a biologically active portion thereof, and SERPINA1 or a biologically active portion thereof.

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