WO2017201121A1

WO2017201121A1 - Recombinant aav for gene therapy in lungs

Info

Publication number: WO2017201121A1
Application number: PCT/US2017/033020
Authority: WO
Inventors: George Vladimirovich ASLANDIDI; Yanerys M. COLON-CORTES; Mutasim N. ABU-HASAN
Original assignee: University Of Florida Research Foundation, Inc.
Priority date: 2016-05-17
Filing date: 2017-05-17
Publication date: 2017-11-23

Abstract

Gene therapy is an attractive approach to treat genetic lung disorders, particular cystic fibrosis (CF), by treating the cause of the disease by restoring the defective gene. Herein are disclosed compositions of variant adeno-associated virus (AAV) with mutations in capsid protein and methods of delivering a gene to lung tissue of a host using the variant AAV.

Description

RECOMBINANT AAV FOR GENE THERAPY IN LUNGS

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 62/337,847, filed on May 17, 2016, entitled "RECOMBINANT AAV FOR GENE THERAPY IN LUNGS," the entire contents of which are incorporated by reference herein.

BACKGROUND

Gene therapy is an attractive approach to treat genetic lung disorders, particular cystic fibrosis (CF), by treating the cause of the disease by restoring the defective gene. To this end, viral vectors for lung-directed gene therapy or more specifically to particular cells in the lung (e.g., bronchial epithelial cells) would be beneficial.

SUMMARY

Aspects of the present application are related to the plasticity of the adeno-associated virus (AAV) capsid that can tolerate modifications that help overcome several limitations associated with wild-type (WT) AAV particle-based gene delivery to cells in the lung (e.g., airway epithelial cells). These limitations include poor transduction of airway cells, poor vector immunogenicity, and poor integration of an expression cassette into a host genome, which is important considering that lung epithelial cells, depending on the disease or condition, involves a higher turnover or shedding of cells compared to cells of other organs, e.g., liver cells.

Furthermore, depending on the respiratory disease or condition, e.g., cystic fibrosis, AAV particles may not effectively penetrate mucus found in the airway.

Accordingly, provided herein is a recombinant AAV (rAAV) particle comprising a nucleic acid encoding a gene (e.g., a therapeutic gene for treating a lung or respiratory condition), and a capsid protein having a mutation that promotes delivery of the rAAV particle to lung tissue. A rAAV particle with one or more mutations (e.g., 2, 3, 4 or more) is also referred herein as a variant rAAV particle or vector.

In some embodiments, a variant rAAV particle has a capsid protein comprising more than one mutation (e.g., 2, 3, 4, or 5 or more mutations) that promote delivery to lung tissue.

In some embodiments, a variant rAAV particle is of serotype 1, 2, 5, 6 or serotypes 5 and 6. AAV5 as a better packaging capacity compared to some other AAV serotypes. AAV6 is better at infecting lung cells compared to some other AAV serotypes. Therefore, it can be useful to create chimeric rAAV particles that comprise capsid sequences from both AAV5 and AAV6. In some embodiments, a rAAV particle as disclosed herein is of serotype 5 or 6. In some embodiments, a rAAV particle as disclosed herein is of serotype 5. In some embodiments, a rAAV particle as disclosed herein is of serotype 6. In some embodiments, a capsid protein of any one of the rAAV particles disclosed herein comprises VP1, VP2 and/or VP3. Capsid proteins VP1, VP2 and VP3 in a rAAV particle as disclosed here may be of the same serotype, e.g., all from AAV5, or from two or more serotypes (e.g., from serotype 5 and 6).

In some embodiments of any one of the rAAV particles described herein, a mutation is at an amino acid residue that is exposed on the outside surface of the AAV particle. In some embodiments, a mutation is at a tyrosine, serine or threonine of wild-type AAV capsid protein. In some embodiments, a mutation is located in an IH loop of the capsid as depicted in FIG. 7.

In some embodiments, a rAAV particle is rAAV5, i.e., of serotype 5, and has a mutation at one or more of the following positions: S651, S485, Y436 or Y719. In some embodiments, a rAAV5 particle has one or more of the following mutations in a capsid protein: S651V, S485V, Y436F and Y719F (e.g., 2, 3 or 4 of these mutations).

In some embodiments, a rAAV particle is rAAV6, i.e., of serotype 6, and has a mutation at one or more of the following positions: S663, T492, Y705 or Y731F. In some embodiments, a rAAV6 particle has one or more of the following mutations in a capsid protein: S663V, T492V, Y705F and Y73 IF (e.g., 2, 3 or 4 of these mutations).

Some embodiments of any one of the rAAV particles disclosed herein comprise a mutation that is a substitution of a hydrophilic amino acid selected from the group consisting of Arg, Asn, Glu and Pro, to a hydrophobic amino acid selected from the group consisting of Ala, Val, Thr, Phe, Trp, Leu and Iso.

In some embodiments, a mutation results in an rAAV particle that is more effectively transduced than a wild-type AAV particle (e.g., of the same serotype). In some embodiments, a mutation results in an rAAV particle that has a higher packaging capacity than a wild-type AAV particle (e.g., of the same serotype). In some embodiments, a mutation results in an rAAV particle that more effectively penetrates mucus than a wild-type AAV particle (e.g., of the same serotype). In some embodiments, a mutation results in a rAAV particle that more effectively integrates into a host genome than a wild-type AAV particle (e.g., of the same serotype). In some embodiments, a mutation results in a rAAV particle that is less immunogenic compared to wild-type rAAV particle (e.g., of the same serotype). It is to be understood that for a chimeric rAAV particle or more than one serotype, the comparison between a variant rAAV particle and a wild-type particle can be of any of the serotypes from which the variant rAAV particle is derived. For example, a variant rAAV particle of serotypes 5 and 6 may be less immunogenic compared to either wild-type rAAV5 or wild-type rAAV6.

For delivery of an rAAV particle as disclosed herein, it may be useful to formulate the particles in a composition such that desirable characteristics are provided, e.g., stability during storage and during and after administration to a subject, and sterility. In certain aspects, this disclosure provides a composition comprising any one of the rAAV particles described herein. In some embodiments, a composition comprises a pharmaceutically acceptable carrier. In some embodiments, a composition may comprise more than one of the particles as disclosed herein, e.g., to deliver more than one therapeutic genes, or to target more than one respiratory cell type (e.g., bronchial epithelial cell and glandular cell).

Provided herein is also a method of delivering a therapeutic gene to lung tissue of a host, the method comprising delivering any one of the rAAV particles disclosed herein or any one of the compositions disclosed herein. In some embodiments, a lung tissue that is targeted for gene delivery is bronchial epithelium or tracheal epithelium.

In some embodiments, a host is a mammal, e.g., a human or a mouse.

In some embodiments, a rAAV particle or composition is administered by nebulization.

In some embodiments, a subject suffers from or has cystic fibrosis. In some

embodiments, a therapeutic protein encoded by a therapeutic gene is Cystic fibrosis

Transmembrane Conductance regulator (CFTR). In some embodiments, a subject has cystic fibrosis (CF) and a therapeutic protein delivered is CFTR. In some embodiments, CFTR that is delivered is wild-type CFTR having the amino acid sequence SEQ ID NO: 12. In some embodiments, CFTR that is delivered is a truncated version of wild-type CFTR. For example, in some embodiments, a CFTR that is delivered is CFTRA264, which has the amino acid sequence of SEQ ID NO: 14. In some embodiments, a CFTR is CFTRAR, which has the amino acid sequence of SEQ ID NO: 12 without amino acids 708-759. Therefore, as defined herein, a CFTR (or nucleic acid that encodes CFTR) that is delivered using any one of the variant rAAV particles disclosed herein may be full-length wild-type CFTR or a truncated version of CFTR.

In some embodiments, a therapeutic gene encodes an shRNA or siRNA for gene silencing, or genome editing proteins (e.g., include Zinc Finger Nucleases (ZFNs), Transcription Activator-like Efforcot Nuclease (TALENs), CRISPR/Cas proteins, and/or meganucleases). In some aspects, provided herein is a method of expressing a gene of interest in a cell derived from a lung. Lung cells can be infected with rAAV particles comprising a gene of interest for various reasons, e.g., to study a gene, or to infect cells under a clinical setting so that they can be administered to a subject. In some embodiments, a method of expressing a gene of interest in a lung cells comprises infecting the cell with any one of the rAAV particles or compositions disclosed herein. In some embodiments, a cell is of a cell-line (e.g., A549 cells or BEAS-2B cells). In some embodiments, a cell is isolated from a lung (e.g., bronchia and/or trachea) of a donor subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. It is to be understood that the data illustrated in the drawings in no way limit the scope of the disclosure.

FIGs. 1A to IB show schematics of a model AAV capsid (FIG. 1A) and a model of intracellular trafficking (FIG. IB).

FIGs. 2A to 2B are graphs showing transduction efficiency of GFP after infecting airway epithelial cells with AAV particles comprising a gene encoding GFP. FIG. 2A shows transduction efficiency of multiple mutants of surface-exposed tyrosine, serine, and threonine residues in different self-complementary AAV-GFP capsid serotypes in cystic fibrosis airway epithelial cells. FIG. 2B shows transduction efficiency of multiple mutants of surface-exposed tyrosine, serine, and threonine residues in different self-complementary AAV-GFP capsid serotypes in normal airway epithelial cells. Cells were infected with an MOI of 1000 vgs/cell. Quantitation of the transduction efficiency was estimated by fluorescence intensity per visual field. M2: Y436-719F; M3: Y705-731F+T492V, M4: Y444-500-730F+T491V; 663: S663V.

FIG. 3 is a graph showing an integration of the expression cassette into a host genome. Cells were passaged multiple times during the experiment.

FIG. 4 shows the packaging efficiency of AAV serotypes using different expression cassette sizes and shows the SDS PAGE analysis of AAV capsid integrity.

FIG. 5 is an image showing AAV-M3 -mediated luciferase expression in a mouse model.

FIG. 6 is a graph showing activity of CI- channels in cells cultured in an Ussing chamber. FIG. 7 shows an amino acid alignment of AAV 5 (SEQ ID NO: 5) and 6 (SEQ ID NO: 6) serotype capsids. The highlighted regions in dark grey show amino acid residues that comprise IH loops.

FIG. 8 shows distribution of viral DNA in the nuclei and cytoplasm of human airway epithelial cells grown under submerged cultures after infection with wild-type and mutant AAV6 particles, using qPCR. Data are presented as percentage of the total amount of viral genome in the nuclear and cytoplasmic fractions.

FIG. 9 shows persistence of luciferase expression up to 216 days in mice infected with wild-type and AAV6-Y705-731F+T492V mutant (AAV6-M3) particles comprising a gene encoding luciferase.

FIG. 10 shows data from an experiment in which mice were infected with wild-type and AAV6-Y705-731F+T492V mutant (AAV6-M3) particles comprising a gene encoding luciferase, and luciferase activity measured for 156 days post infection.

DETAILED DESCRIPTION

Several different methods of delivering genes to lung tissue have been tried without much success. These methods include delivery via liposomes, nanoparticles and certain viral vectors. Aspects of the present application are related to the plasticity of the adeno-associated virus (AAV) capsid that can tolerate modifications that help overcome several limitations associated with wild-type (WT) AAV particles, including poor transduction of airway cells, integration of an expression cassette into a host genome, vector immunogenicity, and mucus penetration. To overcome these limitations, several mutations in the AAV capsid protein can be used (see Table 1). FIGs. 1A to IB show schematics of a model AAV capsid (FIG. 1A) and a model of intracellular trafficking (FIG. IB).

Table 1 provides a summary of advantages of using variant AAV vectors for targeting lung cells (e.g., airway epithelial cells). The "Problem" column describes current drawbacks associated with wild-type or naturally occurring AAV vectors, and the "Solution" column describes examples of ways in which variant AAV vectors (also referred to herein as "variant AAV particles") overcome the problems, "variant AAV vectors" or "variant AAV particles" refer to AAV vectors or particles having a different amino acid compared to the wild-type AAV sequence of the serotype in question. For example, a variant rAAV particle designated AAV6- S663V is a variant of wild-type AAV6 particles in that the serine at position 663 of the AAV6 capsid is a valine. Table 1. Summary of certain AAV capsid variants that can be used to overcome major challenges for efficient lung gene transfer (for example in patients with cystic fibrosis).

The natural plasticity of AAV structural components were utilized to bioengineer viral capsids with distinct properties. Herein are disclosed capsid-variant recombinant AAV particles, compositions thereof, and methods useful for gene delivery to lung tissue, as well as cells in a laboratory or clinical setting.

Variant AAV particles

In some aspects, this application provides a recombinant AAV (rAAV) particle comprising i) a nucleic acid encoding a therapeutic gene for treating a lung condition, and ii) a capsid protein having a mutation that promotes delivery of the rAAV particle to lung tissue. A rAAV particle with one or more mutations (e.g., 2, 3 or 4 or more) in the capsid protein/s is also referred to herein as a variant rAAV particle or vector, or a variant rAAV particle or vector.

In some embodiments, the capsid protein has more than one (e.g., 2, 3 or 4 or more) mutations that promote delivery of the rAAV particle to lung tissue.

In some embodiments, a rAAV particle is of any serotype (e.g., 1, 2, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13). In some embodiments, a rAAV particle is a chimeric rAAV particle, also referred to as a mosaic or hybrid particle. Chimeric rAAV particles have capsid proteins from one or more serotypes. For example, a rAAV particle may have VP1 protein of serotype 5, VP2 protein of serotype 2 and VP3 protein of serotype 3. Alternatively, a capsid protein (e.g., VP1) may have an amino acid sequence derived from more than one serotype. Kohlbremer et al. (Mol Ther, 2005, 12(6): 1217) and Choi et al. (Curr Gene Ther, 2005, 5(3): 299) describe methods of making chimeric rAAV particles, each of which are incorporated herein by reference in their entirety. AAV5 as a better packaging capacity compared to some other AAV serotypes. AAV6 is better at infecting lung cells compared to some other AAV serotypes. Accordingly, in some embodiments, a rAAV is a chimera of serotypes 5 and 6. In some embodiments, a rAAV has core elements from serotype 5 and one or more IH loops from serotype 6. In some

embodiments, modification of AAV produces a chimeric AAV (e.g. , AAV6/AAV5) in which core structural components are derived from AAV5 and loops which interact with cellular receptors and/or are involved in intracellular trafficking are derived from AAV6. AAV6 particles transduce airway epithelial cells better than AAV5. However, AAV5 show better packaging capacity compared to AAV6.

In some embodiments, mutagenesis on several surface exposed capsid tyrosine (Y), serine (S), and/or threonine (T) residues allows the rAAV particles to escape proteasome degradation and increases the number of viral copies accumulating in the nucleus.

In some embodiments, a mutation is at an amino acid residue that is exposed on the outside surface of the rAAV particle. In some embodiments, a capsid protein comprises VP1, VP2 and/or VP3.

In some embodiments, a mutation is at a tyrosine, serine or threonine of wild-type AAV. In some embodiments, a mutation is located in an IH loop of the capsid as depicted in FIG. 7. In some embodiments, a mutation lies in the following regions of AAV5 or AAV6 sequences: amino acids 491-501, amino acids 716 to 731, and amino acids 700 to 751 as depicted in FIG. 7. In some embodiments, a mutation is at a position that is marked by an asterisk (*), 0 or °/₀ (see FIG. 7)_. In some embodiments, a mutation is at S663 and/or S651. In some embodiments, a mutation is at S663 and/or S651 on AAV5 or AAV6. In some embodiments, a mutation is a substitution of a hydrophilic amino acid selected from the group consisting of Arg, Asn, Glu and Pro, to a hydrophobic amino acid selected from the group consisting of Ala, Val, Thr, Phe, Trp, Leu and Iso.

In some embodiments, a rAAV particle as disclosed herein is of serotype 5 or has capsid protein of serotype 5. In some embodiments, a rAAV5 particle has a mutation at one of the following capsid protein amino acids: S651, S485, Y436 or Y719. In some embodiments, a rAAV5 particle has one or more of the following mutations: S651V, S485V, Y436F and Y719F (e.g., 2, 3 or 4 of these mutations).

In some embodiments, a rAAV particle as disclosed herein is of serotype 6 or has capsid protein of serotype 6. In some embodiments, a rAAV6 particle has a mutation at one of the following capsid protein amino acids: S663, T492, Y705 or Y731F. In some embodiments, a rAAV6 particle has one or more of the following mutations: S663V, T492V, Y705F and Y731F (e.g., 2, 3 or 4 of these mutations). Sayroo et al.(Gene Therapy, 2016, 23(1): 18), Rosario et al. (Mol Ther Methods Clin Dev, 2016, 3: 16026) and Pandya et al. (Immunology and Cell Biol, 2014, 92(2): 116-23), the entire contents of each of which are incorporated herein by reference, provide examples of AAV mutations for targeting specific tissue types.

In some embodiments, a mutation results in an rAAV particle that is more effectively transduced than wild-type AAV particles. In some embodiments, a mutation results in an rAAV particle that has a higher packaging capacity compared to wild-type AAV particles. In some embodiments, a mutation results in an rAAV particle that more effectively penetrates mucus (e.g. , mucus in the airways of a CF patient) compared to wild-type AAV particles. In some embodiments, a mutation results in an rAAV particle that more effectively integrates into a host genome compared to wild-type AAV particles. In some embodiments, a mutation results in a rAAV particle that is less immunogenic compared to wild-type rAAV particle (e.g., of the same serotype). It is to be understood that for a chimeric rAAV particle or more than one serotype, the comparison between a variant rAAV particle and a wild-type particle can be of any of the serotypes from which the variant rAAV particle is derived. For example, a variant rAAV particle of serotypes 5 and 6 may be less immunogenic compared to either wild-type rAAV5 or wild-type rAAV6. In some embodiments, a mutation is made to include amino acid

substitutions that are non-polar and hydrophobic to increase penetration through mucus. In some embodiments, a mutation is made to include a valine or phenylalanine to increase penetration through mucus. There are several respiratory diseases that are characterized with an overproduction of mucus, e.g., CF. Mucus hyperplasia is also an important feature of asthma.

Non-limiting examples of wild-type AAV capsid protein sequences are provided below. In some embodiments, one or more sequence variations described herein can be introduced into one of these capsid sequences and used to prepare rAAV for delivering recombinant nucleic acid to a patient (for example to the airways of a patient).

Example of AAV1 capsid protein sequence

1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY

51 KYLGPFNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF

101 QERLQEDTSF GGNLGRAVFQ AKKRVLEPLG LVEEGAKT AP GKKRPVEQSP

151 QEPDSSSGIG KTGQQPAKKR LNFGQTGDSE S VPDPQPLGE PPATPAAVGP 201 TTMASGGGAP MADNNEGADG VGNASGNWHC DSTWLGDRVI TTSTRTWALP

251 TYNNHLYKQI SS ASTGASND NHYFGYSTPW GYFDFNRFHC HFSPRDWQRL

301 INNNWGFRPK RLNFKLFNIQ VKEVTTNDGV TTIANNLTST VQVFSDSEYQ

351 LPYVLGSAHQ GCLPPFPADV FMIPQYGYLT LNNGSQAVGR SSFYCLEYFP

401 SQMLRTGNNF TFSYTFEEVP FHSSYAHSQS LDRLMNPLID QYLYYLNRTQ

451 NQSGS AQNKD LLFSRGSPAG MS VQPKNWLP GPCYRQQRVS KTKTDNNNSN

501 FTWTGASKYN LNGRESIINP GT AMASHKDD EDKFFPMSGV MIFGKES AGA

551 SNTALDNVMI TDEEEIKATN PVATERFGTV AVNFQSSSTD PATGDVHAMG

601 ALPGMVWQDR DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KNPPPQILIK

651 NTPVPANPPA EFS ATKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEVQ

701 YTSNYAKSAN VDFTVDNNGL YTEPRPIGTR YLTRPL (SEQ ID NO: 1)

Example of AAV2 capsid protein sequence

1 MAADGYLPDW LEDTLSEGIR QWWKLKPGPP PPKPAERHKD DSRGLVLPGY

51 KYLGPFNGLD KGEPVNEADA AALEHDKAYD RQLDSGDNPY LKYNHADAEF

101 QERLKEDTSF GGNLGRAVFQ AKKRVLEPLG LVEEPVKTAP GKKRPVEHSP

151 VEPDSSSGTG KAGQQPARKR LNFGQTGDAD S VPDPQPLGQ PPAAPSGLGT

201 NTMATGSGAP MADNNEGADG VGNSSGNWHC DSTWMGDRVI TTSTRTWALP

251 TYNNHLYKQI SSQSGASNDN HYFGYSTPWG YFDFNRFHCH FSPRDWQRLI

301 NNNWGFRPKR LNFKLFNIQV KEVTQNDGTT TIANNLTSTV QVFTDSEYQL

351 PYVLGSAHQG CLPPFPADVF MVPQYGYLTL NNGSQAVGRS SFYCLEYFPS

401 QMLRTGNNFT FSYTFEDVPF HSSYAHSQSL DRLMNPLIDQ YLYYLSRTNT

451 PSGTTTQSRL QFSQAGASDI RDQSRNWLPG PCYRQQRVSK TS ADNNNSEY

501 SWTGATKYHL NGRDSLVNPG PAMASHKDDE EKFFPQSGVL IFGKQGSEKT

551 NVDIEKVMIT DEEEIRTTNP VATEQYGSVS TNLQRGNRQA ATADVNTQGV

601 LPGMVWQDRD VYLQGPIWAK IPHTDGHFHP SPLMGGFGLK HPPPQILIKN

651 TPVPANPSTT FS AAKFASFI TQYSTGQVS V EIEWELQKEN SKRWNPEIQY

701 TSNYNKSVNV DFTVDTNGVY SEPRPIGTRY LTRNL (SEQ ID NO: 2)

Example of AAV3a capsid protein sequence

1 MAADGYLPDW LEDNLSEGIR EWWALKPGVP QPKANQQHQD NRRGLVLPGY

51 KYLGPGNGLD KGEPVNEADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF 101 QERLQEDTSF GGNLGRAVFQ AKKRILEPLG LVEEAAKT AP GKKGAVDQSP 151 QEPDSSSGVG KSGKQPARKR LNFGQTGDSE S VPDPQPLGE PPAAPTSLGS 201 NTMASGGGAP MADNNEGADG VGNSSGNWHC DSQWLGDRVI TTSTRTWALP 251 TYNNHLYKQI SSQSGASNDN HYFGYSTPWG YFDFNRFHCH FSPRDWQRLI 301 NNNWGFRPKK LSFKLFNIQV RGVTQNDGTT TIANNLTSTV QVFTDSEYQL

351 PYVLGSAHQG CLPPFPADVF MVPQYGYLTL NNGSQAVGRS SFYCLEYFPS

401 QMLRTGNNFQ FSYTFEDVPF HSSYAHSQSL DRLMNPLIDQ YLYYLNRTQG

451 TTSGTTNQSR LLFSQAGPQS MSLQARNWLP GPCYRQQRLS KTANDNNNSN

501 FPWT A AS KYH LNGRDSLVNP GPAMASHKDD EEKFFPMHGN LIFGKEGTTA

551 SNAELDNVMI TDEEEIRTTN PVATEQYGTV ANNLQSSNTA PTTGTVNHQG

601 ALPGMVWQDR DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK

651 NTPVPANPPT TFSPAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ

701 YTSNYNKSVN VDFTVDTNGV YSEPRPIGTR YLTRNL (SEQ ID NO: 3)

Example of AAV4 capsid protein sequence

1 MTDGYLPDWL EDNLSEGVRE WWALQPGAPK PKANQQHQDN ARGLVLPGYK

51 YLGPGNGLDK GEPVNAADAA ALEHDKAYDQ QLKAGDNPYL KYNHADAEFQ

101 QRLQGDTSFG GNLGRAVFQA KKRVLEPLGL VEQAGETAPG KKRPLIESPQ

151 QPDSSTGIGK KGKQPAKKKL VFEDETGAGD GPPEGSTSGA MSDDSEMRAA

201 AGGAAVEGGQ GADGVGNASG DWHCDSTWSE GHVTTTSTRT WVLPTYNNHL

251 YKRLGESLQS NTYNGFSTPW GYFDFNRFHC HFSPRDWQRL INNNWGMRPK

301 AMRVKIFNIQ VKEVTTSNGE TTVANNLTST VQIFADSSYE LPYVMDAGQE

351 GSLPPFPNDV FMVPQYGYCG LVTGNTSQQQ TDRNAFYCLE YFPSQMLRTG

401 NNFEITYSFE KVPFHSMYAH SQSLDRLMNP LIDQYLWGLQ STTTGTTLNA

451 GTATTNFTKL RPTNFSNFKK NWLPGPSIKQ QGFSKTANQN YKIPATGSDS

501 LIKYETHSTL DGRWSALTPG PPMATAGPAD SKFSNSQLIF AGPKQNGNTA

551 TVPGTLIFTS EEELAATNAT DTDMWGNLPG GDQSNSNLPT VDRLTALGAV

601 PGMVWQNRDI YYQGPIWAKI PHTDGHFHPS PLIGGFGLKH PPPQIFIKNT

651 PVPANPATTF SSTPVNSFIT QYSTGQVS VQ IDWEIQKERS KRWNPEVQFT

701 SNYGQQNSLL WAPDAAGKYT EPRAIGTRYL THHL (SEQ ID NO: 4)

Example of AAV5 capsid protein sequence

1 MSFVDHPPDW LEEVGEGLRE FLGLEAGPPK PKPNQQHQDQ ARGLVLPGYN

51 YLGPGNGLDR GEPVNRADEV AREHDIS YNE QLEAGDNPYL KYNHADAEFQ

101 EKLADDTSFG GNLGKAVFQA KKRVLEPFGL VEEGAKTAPT GKRIDDHFPK

151 RKKARTEEDS KPSTSSDAEA GPSGSQQLQI PAQPASSLGA DTMS AGGGGP

201 LGDNNQGADG VGNASGDWHC DSTWMGDRVV TKSTRTWVLP SYNNHQYREI

251 KSGSVDGSNA NAYFGYSTPW GYFDFNRFHS HWSPRDWQRL INNYWGFRPR

301 SLRVKIFNIQ VKEVTVQDST TTIANNLTST VQVFTDDD YQ LPYV VGNGTE

351 GCLPAFPPQV FTLPQYGYAT LNRDNTENPT ERSSFFCLEY FPSKMLRTGN 401 NFEFTYNFEE VPFHSSFAPS QNLFKLANPL VDQYLYRFVS TNNTGGVQFN

451 KNLAGRYANT YKNWFPGPMG RTQGWNLGSG VNRASVSAFA TTNRMELEGA

501 SYQVPPQPNG MTNNLQGSNT YALENTMIFN SQPANPGTTA TYLEGNMLIT

551 SESETQPVNR VAYNVGGQMA TNNQSSTTAP ATGTYNLQEI VPGSVWMERD

601 VYLQGPIWAK IPETGAHFHP SPAMGGFGLK HPPPMMLIKN TPVPGNITSF

651 SDVPVSSFIT QYSTGQVTVE MEWELKKENS KRWNPEIQYT NNYNDPQFVD

701 FAPDSTGEYR TTRPIGTRYL TRPL (SEQ ID NO: 5)

Example of AAV6 capsid protein sequence

1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY

51 KYLGPFNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF

101 QERLQEDTSF GGNLGRAVFQ AKKRVLEPFG LVEEGAKTAP GKKRPVEQSP

151 QEPDSSSGIG KTGQQPAKKR LNFGQTGDSE S VPDPQPLGE PPATPAAVGP

201 TTMASGGGAP MADNNEGADG VGNASGNWHC DSTWLGDRVI TTSTRTWALP

251 TYNNHLYKQI SS ASTGASND NHYFGYSTPW GYFDFNRFHC HFSPRDWQRL

301 INNNWGFRPK RLNFKLFNIQ VKEVTTNDGV TTIANNLTST VQVFSDSEYQ

351 LPYVLGSAHQ GCLPPFPADV FMIPQYGYLT LNNGSQAVGR SSFYCLEYFP

401 SQMLRTGNNF TFSYTFEDVP FHSSYAHSQS LDRLMNPLID QYLYYLNRTQ

451 NQSGS AQNKD LLFSRGSPAG MS VQPKNWLP GPCYRQQRVS KTKTDNNNSN

501 FTWTGASKYN LNGRESIINP GTAMASHKDD KDKFFPMSGV MIFGKESAGA

551 SNTALDNVMI TDEEEIKATN PVATERFGTV AVNLQSSSTD PATGDVHVMG

601 ALPGMVWQDR DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK

651 NTPVPANPPA EFS ATKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEVQ

701 YTSNYAKSAN VDFTVDNNGL YTEPRPIGTR YLTRPL (SEQ ID NO: 6)

Example of AAV7 capsid protein sequence

1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD NGRGLVLPGY

51 KYLGPFNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF

101 QERLQEDTSF GGNLGRAVFQ AKKRVLEPLG LVEEGAKTAP AKKRPVEPSP

151 QRSPDSSTGI GKKGQQPARK RLNFGQTGDS ES VPDPQPLG EPPAAPSSVG

201 SGTVAAGGGA PMADNNEGAD GVGNASGNWH CDSTWLGDRV ITTSTRTWAL

251 PTYNNHLYKQ ISSETAGSTN DNTYFGYSTP WGYFDFNRFH CHFSPRDWQR

301 LINNNWGFRP KKLRFKLFNI QVKEVTTNDG VTTIANNLTS TIQVFSDSEY

351 QLPYVLGSAH QGCLPPFPAD VFMIPQYGYL TLNNGSQSVG RSSFYCLEYF

401 PSQMLRTGNN FEFSYSFEDV PFHSSYAHSQ SLDRLMNPLI DQYLYYLART

451 QSNPGGTAGN RELQFYQGGP STMAEQAKNW LPGPCFRQQR VSKTLDQNNN 501 SNFAWTGATK YHLNGRNSLV NPGVAMATHK DDEDRFFPSS GVLIFGKTGA

551 TNKTTLENVL MTNEEEIRPT NPVATEEYGI VSSNLQAANT AAQTQVVNNQ

601 GALPGM V WQN RD V YLQGPIW AKIPHTDGNF HPSPLMGGFG LKHPPPQILI

651 KNTPVPANPP EVFTPAKFAS FITQYSTGQV SVEIEWELQK ENSKRWNPEI

701 QYTSNFEKQT GVDFAVDSQG VYSEPRPIGT RYLTRNL (SEQ ID NO: 7)

Example of AAV8 capsid protein sequence

1 MAADGYLPDW LEDNLSEGIR EWWALKPGAP KPKANQQKQD DGRGLVLPGY

51 KYLGPFNGLD KGEPVNAADA AALEHDKAYD QQLQAGDNPY LRYNHADAEF

101 QERLQEDTSF GGNLGRAVFQ AKKRVLEPLG LVEEGAKT AP GKKRPVEPSP

151 QRSPDSSTGI GKKGQQPARK RLNFGQTGDS ES VPDPQPLG EPPAAPSGVG

201 PNTMAAGGGA PMADNNEGAD GVGSSSGNWH CDSTWLGDRV ITTSTRTWAL

251 PTYNNHLYKQ ISNGTSGGAT NDNTYFGYST PWGYFDFNRF HCHFSPRDWQ

301 RLINNNWGFR PKRLSFKLFN IQVKEVTQNE GTKTIANNLT STIQVFTDSE

351 YQLPYVLGSA HQGCLPPFPA DVFMIPQYGY LTLNNGSQAV GRSSFYCLEY

401 FPSQMLRTGN NFQFTYTFED VPFHSSYAHS QSLDRLMNPL IDQYLYYLSR

451 TQTTGGTANT QTLGFSQGGP NTMANQAKNW LPGPCYRQQR VSTTTGQNNN

501 SNFAWT AGTK YHLNGRNSLA NPGIAMATHK DDEERFFPSN GILIFGKQNA

551 ARDNADYSDV MLTSEEEIKT TNPVATEEYG IVADNLQQQN TAPQIGTVNS

601 QGALPGM V WQ NRD V YLQGPI W AKIPHTDGN FHPSPLMGGF GLKHPPPQIL

651 IKNTPVPADP PTTFNQSKLN SFITQYSTGQ VSVEIEWELQ KENSKRWNPE

701 IQYTSNYYKS TSVDFAVNTE GVYSEPRPIG TRYLTRNL (SEQ ID NO: 8)

Example of AAV9 capsid protein sequence

1 MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NARGLVLPGY

51 KYLGPGNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF

101 QERLKEDTSF GGNLGRAVFQ AKKRLLEPLG LVEEAAKT AP GKKRPVEQSP

151 QEPDSSAGIG KSGAQPAKKR LNFGQTGDTE SVPDPQPIGE PPAAPSGVGS

201 LTMASGGGAP VADNNEGADG VGSSSGNWHC DSQWLGDRVI TTSTRTWALP

251 TYNNHLYKQI SNSTSGGSSN DNAYFGYSTP WGYFDFNRFH CHFSPRDWQR

301 LINNNWGFRP KRLNFKLFNI QVKEVTDNNG VKTIANNLTS TVQVFTDSDY

351 QLPYVLGSAH EGCLPPFPAD VFMIPQYGYL TLNDGSQAVG RSSFYCLEYF

401 PSQMLRTGNN FQFSYEFENV PFHSSYAHSQ SLDRLMNPLI DQYLYYLSKT

451 INGSGQNQQT LKFSVAGPSN MAVQGRNYIP GPS YRQQRVS TTVTQNNNSE

501 FAWPGASSWA LNGRNSLMNP GPAMASHKEG EDRFFPLSGS LIFGKQGTGR

551 DNVDADKVMI TNEEEIKTTN PVATES YGQV ATNHQSAQAQ AQTGWVQNQG 601 ILPGMVWQDR DVYLQGPIWA KIPHTDGNFH PSPLMGGFGM KHPPPQILIK

651 NTPVPADPPT AFNKDKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ

701 YTSNYYKSNN VEFAVNTEGV YSEPRPIGTR YLTRNL (SEQ ID NO: 9)

Example of AAV10 capsid protein sequence

1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY

51 KYLGPFNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF

101 QERLQEDTSF GGNLGRAVFQ AKKRVLEPLG LVEEGAKT AP GKKRPVEPSP

151 QRSPDSSTGI GKKGQQPAKK RLNFGQTGDS ESVPDPQPIG EPPAGPSGLG

201 SGTMAAGGGA PMADNNEGAD GVGSSSGNWH CDSTWLGDRV ITTSTRTWAL

251 PTYNNHLYKQ ISNGTSGGST NDNTYFGYST PWGYFDFNRF HCHFSPRDWQ

301 RLINNNWGFR PKRLNFKLFN IQVKEVTQNE GTKTIANNLT STIQVFTDSE

351 YQLPYVLGSA HQGCLPPFPA DVFMIPQYGY LTLNNGSQAV GRSSFYCLEY

401 FPSQMLRTGN NFEFSYQFED VPFHSSYAHS QSLDRLMNPL IDQYLYYLSR

451 TQSTGGTAGT QQLLFSQAGP NNMS AQAKNW LPGPCYRQQR VSTTLSQNNN

501 SNFAWTGATK YHLNGRDSLV NPGVAMATHK DDEERFFPSS GVLMFGKQGA

551 GKDNVDYSS V MLTSEEEIKT TNPVATEQYG VVADNLQQQN AAPIVGAVNS

601 QGALPGM V WQ NRD V YLQGPI W AKIPHTDGN FHPSPLMGGF GLKHPPPQIL

651 IKNTPVPADP PTTFSQAKLA SFITQYSTGQ VSVEIEWELQ KENSKRWNPE

701 IQYTSNYYKS TNVDFAVNTD GTYSEPRPIG TRYLTRNL (SEQ ID NO: 10)

There may be several reasons or mechanisms for the promotion of delivery of an rAAV particle as disclosed herein to lung tissue (see Table 1). For example, a mutation in specific tyrosine, threonine or serine allows escape of the recombinant vector from proteosomal degradation, resulting in a higher level of rAAV in the cell for gene delivery. Escape from proteosomal degradation can also lead to a decrease in presentation by antigen-presenting cells. In some embodiments, a mutation or a set of mutations in a variant rAAV particle (e.g., AAV6- Y705-731F+T492V) leads to a higher quantity in the nucleus, which is associated with a greater rate of integration of the recombinant gene into the genome of the host. This is especially useful because there is a high turnover of cells or shedding of cells in the airway, especially under certain conditions (e.g., asthma). In some embodiments, a mutation or a set of mutations in a variant rAAV particle (e.g., to increase hydrophobicity of a capsid surface) results in better penetration of the mucus layer in the airway, thereby increasing the contact between the cells and the rAAV particles. In addition to a mutation that affects the delivery of rAAV particles to target lung cells, a mutation as contemplated herein may lower the dose that is effective to achieve expression of a therapeutic gene of interest, and thereby lower the production costs of deliverable rAAV in a clinical setting. A mutation in a variant rAAV may lower the effective dose compared to wild- type rAAV by 2-99% (e.g., 2-5, 5-10, 5- 20, 5-50, 10-30, 10-50, 10-99, 20-80, or 80-99%). Manufacturing of rAAV particles for use in clinical settings is described by Clement and Grieger (Mol Ther Methods Clin Dev. 2016; 3: 16002), and Kotin and Snyder (Hum Gene Ther, 2017 Apr;28(4):350-360), each of which is incorporated by reference herein in its entirety. It is to be understood that the activities and/or properties (e.g., transduction of cells, mucus penetration, packaging capacity or integration of nucleic acid comprises by the rAAV to host genome) of disclosed variant rAAV particles is compared to that the activities and/or properties of wild-type AAV particles. For example, mutations Y705F, Y731F and T492V in rAAV6 promotes delivery of the rAAV particle to lung tissue, compared to wild-type rAAV6. The differences between activities and/or properties of variant rAAV and wild-type rAAV may be up to 99% (e.g., up to 99%, up to 90% up to 80%, up to 60%, up to 40%, up to 20%, 2-5, 5-10, 5- 20, 5-50, 10-30, 10-50, 10-99, 20-80, or 80-99%).

Compositions

In some aspects, this disclosure provides a composition comprising any one of the variant rAAV particles described herein, for example, an rAAV particle comprising i) a nucleic acid encoding a therapeutic gene for treating a lung condition, and ii) a capsid protein having a mutation that promotes delivery of the rAAV particle to lung tissue.

In some embodiments, a composition of recombinant rAAV particles comprises a buffer or salt or other pharmaceutically acceptable carriers. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the rAAV particle is administered. Such

pharmaceutical carriers can be sterile liquids, or a solid (e.g., one which can be aerosolized).

In some embodiments, a composition of rAAV particles as contemplated herein is formulated to facilitate nebulization or spraying of the formulation into the airway of a subject.

In some embodiments, a formulation as disclosed herein is optimized for inhalation. Methods for formulating a composition for nebulization or inhalation are known in the art, see e.g.,

WO2006079841 Al, Patlolla et al. (J Control Release. 2010 Jun 1; 144(2): 233-241), Umerska et al. (Int J Pharm. 2015 Sep 30;493(l-2):224-32), and Rahimpour et al. (Drug Discov Today.

2014 May;19(5):618-26), the contents of each of which is incorporated by reference herein in their entirety. Techniques related to aerosol-mediated delivery of AAV formulations are also known in the art, see e.g., MacLoughlin et al. (Hum Gene Ther. 2015 Jan 1; 26(1): 36-46), Moss et al. (Chest. 2004 Feb;125(2):509-21), and Flotte and Laube (Chest. 2001 Sep; 120(3

Suppl): 124S-131S.), the contents of each of which is incorporated by reference herein in their entirety.

In some embodiments, a carrier can be a solvent or dispersion medium containing, for example, water, saline, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and/or by the use of surfactants.

A composition may further optionally comprise a liposome, a lipid, a lipid complex, a microsphere, a microparticle, a nanosphere, or a nanoparticle.

In some embodiments, a composition of rAAV particles as contemplated herein is formulated for intravenous, intramuscular, intravitreal, subretinal, subcutaneous or

intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see e.g.,

"Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by, e.g., FDA Office of Biologies standards.

In some embodiments, a composition is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Typically, such compositions may contain at least about 0.1% of the therapeutic agent (e.g., rAAV particle) or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation. Naturally, the amount of therapeutic agent(s) (e.g., rAAV particle) in each therapeutically-useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

Methods of delivering a gene to cell-lines or cells derived from a subject

In some aspects, provided herein are methods of delivering a gene (e.g., a therapeutic gene, or a gene the function of which is to be studied) to a cell. A cell may be of a particular cell line, e.g., A549,BEAS-2B cells, CRL-4011 or CRL-4013 cells. In some embodiments, a cell is a primary cell isolated from a lung of a subject (e.g., a mouse or human).

In some embodiments, lung cells are native to the lung (e.g., cells that develop there such as cells in the alveoli or trachea). In some embodiments, lung cells are cells that move into or infiltrate the lung (e.g., immune cells such as basophils or mast cells).

A lung cell may be of the airway or of the lung parenchyma. Cells of the airway may be of the extrathoracic (or superior) airway (e.g., of the supraglottic, glottis or infraglottic regions) or the intrathoracic (or inferior) airway (e.g., of the trachea, mainstem bronchi or multiple bronchial generations). Cells of the lung parenchyma may be of the lobes or of the segments. In some embodiments, a lung cell is a pseudostratified columnar epithelium cell (e.g., ciliated airway epithelial cell, goblet (or mucus) airway epithelial cells, or a basal airway epithelial cell. Basal cells in the respiratory epithelium are small, nearly cuboidal cells that can differentiate into other cells types found within the epithelium. In some embodiments, an airway epithelial cells is squamous, cuboidal or columnar. In some embodiments, a lung cells is an alveolar cell (e.g., a type I alveolar cell or type II alveolar cell).

Lung cells can also be of the pulmonary vasculature, lymphatic system or muscle cells (e.g., smooth muscle cells). In some embodiments, lung cells are lung fibroblasts.

In some embodiments, different lung cells (e.g., mucus/goblet airway epithelial cells, ciliary airway epithelial cells and basal cells) are cultured together.

In some embodiments, a cell is isolated from the lung (e.g., bronchia and/or trachea) of a donor subject. In some embodiments, a cell is isolated by brushing of the surface of the bronchia and/or trachea of a subject under the influence of an anesthetic.

In some embodiments, a cell is cultured in a laboratory dish under submerged cultures.

In some embodiments, a cell (e.g., a primary airway epithelial cell) is cultured on an air- liquid interface and allowed to differentiate into several different types of airway epithelial cells (e.g., ciliated cells or mucus producing cells). In some embodiments, rAAV particles are introduced in the air-compartment. In some embodiments, rAAV particles are introduced in the liquid compartment. rAAV particles as described herein may be introduced to cells for a finite period of time (e.g., 5min-5 hours, 15 mintutes-2 hours, or 30 minutes- lh). In some

embodiments, cells are subsequently washed out after infection to remove rAAV particles. In some embodiments, rAAV particles are introduced to cells in as small as volume as possible to allow the most contact of viral particles to cells. In some embodiments, the temperature and/or mixing conditions at which infection is carried out is optimized for maximal infection.

Methods of delivering a therapeutic gene to lung tissue of a host

In some aspects, the disclosure provides a method of delivering a therapeutic gene to lung tissue of a host, the method comprising delivering an rAAV particle comprising i) a nucleic acid encoding a therapeutic gene for treating a lung condition, and ii) a capsid protein having a mutation that promotes delivery of the rAAV particle to lung tissue, or a composition comprising such rAAV particles, to a host. Herein, "host" and "subject" are used

interchangeably.

In some embodiments, a host is a mammal (e.g., a human, a mouse, a rat, a pig, a hamster, a dog, a cat, a horse or a cow).

In some embodiments, a composition of variant rAAV particles as described herein can be administered using any of the following methods/routes of administration. In some embodiments, a rAAV particle or a composition comprising a rAAV particle is administered by nebulization or injection into the trachea of a host. In some embodiments, a rAAV particle or a composition comprising a rAAV particle is administered by intra-tracheal intubation. In some embodiments, a rAAV particle or a composition comprising a rAAV particle is administered by liquid instillation. In some embodiments, a rAAV particle or a composition comprising a rAAV particle as disclosed herein is administered by inhalation. In some embodiments, a composition of rAAV particles as contemplated herein is formulated to facilitate nebulization or spraying of the formulation into the airway of a subject. Strategies of delivering drugs (e.g., rAAV particles) to specific regions of the lung are known in the art, see e.g., Patil and Sarasija (Lung India. 2012 Jan-Mar; 29(1): 44-49), Fuerst

diseases/better-way- arget-drog'-deliveiy-lungs), Gronenberg et al. (Respir Med. 2003

Apr;97(4):382-7), the contents of each of which are incorporated herein by reference in their entirety. Methods for formulating a composition for nebulization or inhalation are known in the art, as described above. In some embodiments, different parts of the lung are targeted for AAV delivery, e.g., the nose, trachea, bronchia, or alveolar sacs. In some embodiments, different types of lung cells are targeted, e.g., nasal cells, tracheal cells, bronchial cells or alveolar cells.

In some embodiments, lung cells targeted for gene delivery are native to the lung (e.g., cells that develop there such as cells in the alveoli or trachea). In some embodiments, lung cells are cells that move into or infiltrate the lung (e.g., immune cells such as basophils or mast cells).

A lung cell may be of the airway or of the lung parenchyma. Cells of the airway may be of the extrathoracic (or superior) airway (e.g., of the supraglottic, glottis or infraglottic regions) or the intrathoracic (or inferior) airway (e.g., of the trachea, mainstem bronchi or multiple bronchial generations). In some embodiments, a lung cell is a pseudostratified columnar epithelium cell (e.g., ciliated airway epithelial cell, goblet (or mucus) airway epithelial cells, or a basal airway epithelial cell. In some embodiments, an airway epithelial cells is squamous, cuboidal or columnar. In some embodiments, a lung cells is an alveolar cell (e.g., a type I alveolar cell or type II alveolar cell).

In some embodiments, different lung cells (e.g., mucus/goblet airway epithelial cells, ciliary airway epithelial cells and basal cells) are targeted for gene delivery (e.g., those that are found in the same location of the cells such as airway epithelial cells and underlying muscles cells, or two different types of airway epithelial cells).

In some embodiments, airway epithelial cells are targeted for gene delivery (e.g., nasal epithelial cells, tracheal epithelial cells or bronchial epithelial cells).

In some embodiments, a host is a patient (e.g., a patient suffering from a disease, e.g., a pulmonary disease). In some embodiments, the disease to be treated is CF. CF is an autosomal recessive disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Non-pathogenic adeno-associated virus (AAV) particles comprising expression vectors are attractive delivery vehicles for gene therapy for CF and currently used in a number of clinical trials for single gene disorders such as hemophilia B, muscular dystrophy, and ocular diseases. However, there are several challenges that remain unresolved such as poor transduction efficiency of airway epithelial cells (AEC), mucus penetration and low integration frequency of the expression cassette into the host genome.

Aspects of the present application can be useful to address one or more of these challenges. A therapeutic gene is one that encodes a RNA or protein that provides therapeutic benefit in a cell or tissue of a subject. In some embodiments, a therapeutic gene encodes an shRNA or siRNA for gene silencing, or genome editing proteins (e.g., include Zinc Finger Nucleases (ZFNs), TALENs, CRISPR/Cas proteins, and/or meganucleases).

In some embodiments, a therapeutic protein encoded by a therapeutic gene is CFTR. In some embodiments, the CFTR is encoded by the nucleic acid of SEQ ID NO: 11. In some embodiments, the amino acid sequence of CFTR is SEQ ID NO: 12. Truncated versions of CFTR are also contemplated herein. For example, in some embodiments, the CFTR is

CFTRA264 and is encoded by the nucleic acid of SEQ ID NO: 13. In some embodiments, the amino acid sequence of CFTRA264 is SEQ ID NO: 14. In some embodiments, an alternative deletion variant of CFTR can be used, e.g., CFTRAR, which has a deletion in the regulatory domain (Δ708-759; Vidovic et al., Am J Respir Crit Care Med Vol 193, 3: 288-298, Feb 1, 2016).

Nucleic acid sequence of full length CFTR

atgcagaggtcgcctctggaaaaggccagcgttgtctccaaactttttttcagctggaccagaccaattttgaggaaaggatacagacagcg cctggaattgtcagacatataccaaatcccttctgttgattctgctgacaatctatctgaaaaattggaaagagaatgggatagagagctggct tcaaagaaaaatcctaaactcattaatgcccttcggcgatgttttttctggagatttatgttctatggaatctttttatatttaggggaagtcaccaa agcagtacagcctctcttactgggaagaatcatagcttcctatgacccggataacaaggaggaacgctctatcgcgatttatctaggcatag gcttatgccttctctttattgtgaggacactgctcctacacccagccatttttggccttcatcacattggaatgcagatgagaatagctatgtttag tttgatttataagaagactttaaagctgtcaagccgtgttctagataaaataagtattggacaacttgttagtctcctttccaacaacctgaacaaa tttgatgaaggacttgcattggcacatttcgtgtggatcgctcctttgcaagtggcactcctcatggggctaatctgggagttgttacaggcgtc tgccttctgtggacttggtttcctgatagtccttgccctttttcaggctgggctagggagaatgatgatgaagtacagagatcagagagctggg aagatcagtgaaagacttgtgattacctcagaaatgattgaaaatatccaatctgttaaggcatactgctgggaagaagcaatggaaaaaatg attgaaaacttaagacaaacagaactgaaactgactcggaaggcagcctatgtgagatacttcaatagctcagccttcttcttctcagggttct ttgtggtgtttttatctgtgcttccctatgcactaatcaaaggaatcatcctccggaaaatattcaccaccatctcattctgcattgttctgcgcatg gcggtcactcggcaatttccctgggctgtacaaacatggtatgactctcttggagcaataaacaaaatacaggatttcttacaaaagcaagaa tataagacattggaatataacttaacgactacagaagtagtgatggagaatgtaacagccttctgggaggagggatttggggaattatttgag aaagcaaaacaaaacaataacaatagaaaaacttctaatggtgatgacagcctcttcttcagtaatttctcacttcttggtactcctgtcctgaaa gatattaatttcaagatagaaagaggacagttgttggcggttgctggatccactggagcaggcaagacttcacttctaatggtgattatggga gaactggagccttcagagggtaaaattaagcacagtggaagaatttcattctgttctcagttttcctggattatgcctggcaccattaaagaaa atatcatctttggtgtttcctatgatgaatatagatacagaagcgtcatcaaagcatgccaactagaagaggacatctccaagtttgcagagaa agacaatatagttcttggagaaggtggaatcacactgagtggaggtcaacgagcaagaatttctttagcaagagcagtatacaaagatgctg atttgtatttattagactctccttttggatacctagatgttttaacagaaaaagaaatatttgaaagctgtgtctgtaaactgatggctaacaaaact aggattttggtcacttctaaaatggaacatttaaagaaagctgacaaaatattaattttgcatgaaggtagcagctatttttatgggacattttcag aactccaaaatctacagccagactttagctcaaaactcatgggatgtgattctttcgaccaatttagtgcagaaagaagaaattcaatcctaact gagaccttacaccgtttctcattagaaggagatgctcctgtctcctggacagaaacaaaaaaacaatcttttaaacagactggagagtttggg gaaaaaaggaagaattctattctcaatccaatcaactctatacgaaaattttccattgtgcaaaagactcccttacaaatgaatggcatcgaag aggattctgatgagcctttagagagaaggctgtccttagtaccagattctgagcagggagaggcgatactgcctcgcatcagcgtgatcag cactggccccacgcttcaggcacgaaggaggcagtctgtcctgaacctgatgacacactcagttaaccaaggtcagaacattcaccgaaa gacaacagcatccacacgaaaagtgtcactggcccctcaggcaaacttgactgaactggatatatattcaagaaggttatctcaagaaactg gcttggaaataagtgaagaaattaacgaagaagacttaaaggagtgcttttttgatgatatggagagcataccagcagtgactacatggaac acataccttcgatatattactgtccacaagagcttaatttttgtgctaatttggtgcttagtaatttttctggcagaggtggctgcttctttggttgtgc tgtggctccttggaaacactcctcttcaagacaaagggaatagtactcatagtagaaataacagctatgcagtgattatcaccagcaccagtt cgtattatgtgttttacatttacgtgggagtagccgacactttgcttgctatgggattcttcagaggtctaccactggtgcatactctaatcacagt gtcgaaaattttacaccacaaaatgttacattctgttcttcaagcacctatgtcaaccctcaacacgttgaaagcaggtgggattcttaatagatt ctccaaagatatagcaattttggatgaccttctgcctcttaccatatttgacttcatccagttgttattaattgtgattggagctatagcagttgtcgc agttttacaaccctacatctttgttgcaacagtgccagtgatagtggcttttattatgttgagagcatatttcctccaaacctcacagcaactcaaa caactggaatctgaaggcaggagtccaattttcactcatcttgttacaagcttaaaaggactatggacacttcgtgccttcggacggcagcctt actttgaaactctgttccacaaagctctgaatttacatactgccaactggttcttgtacctgtcaacactgcgctggttccaaatgagaatagaa atgatttttgtcatcttcttcattgctgttaccttcatttccattttaacaacaggagaaggagaaggaagagttggtattatcctgactttagccat gaatatcatgagtacattgcagtgggctgtaaactccagcatagatgtggatagcttgatgcgatctgtgagccgagtctttaagttcattgac atgccaacagaaggtaaacctaccaagtcaaccaaaccatacaagaatggccaactctcgaaagttatgattattgagaattcacacgtgaa gaaagatgacatctggccctcagggggccaaatgactgtcaaagatctcacagcaaaatacacagaaggtggaaatgccatattagagaa catttccttctcaataagtcctggccagagggtgggcctcttgggaagaactggatcagggaagagtactttgttatcagcttttttgagactac tgaacactgaaggagaaatccagatcgatggtgtgtcttgggattcaataactttgcaacagtggaggaaagcctttggagtgataccacag aaagtatttattttttctggaacatttagaaaaaacttggatccctatgaacagtggagtgatcaagaaatatggaaagttgcagatgaggttgg gctcagatctgtgatagaacagtttcctgggaagcttgactttgtccttgtggatgggggctgtgtcctaagccatggccacaagcagttgat gtgcttggctagatctgttctcagtaaggcgaagatcttgctgcttgatgaacccagtgctcatttggatccagtaacataccaaataattagaa gaactctaaaacaagcatttgctgattgcacagtaattctctgtgaacacaggatagaagcaatgctggaatgccaacaatttttggtcataga agagaacaaagtgcggcagtacgattccatccagaaactgctgaacgagaggagcctcttccggcaagccatcagcccctccgacagg gtgaagctctttccccaccggaactcaagcaagtgcaagtctaagccccagattgctgctctgaaagaggagacagaagaagaggtgcaa gatacaaggctttaga (SEQ ID NO: 11) Amino acid sequence of full length CFTR

mqrsplekasvvsklffswtrpilrkgyrqrlelsdiyqipsvdsadnlse

vtkavqplllgriiasydpdnkeersiaiylgiglcllfivrtlllhpaifgm^

nkfdeglalahfvwiaplqvallmgliwellqasafcglgflivlalfqaglgmimmkyrdqragkiserlvitsemieniqsvka eeamekmienlrqtelkltrkaayvryfnssafffsgffvvflsv

iqdflqkqeyktleynltttevvmenvtafweegfgelfekakqnnnnrktsngddslffsnfsllgtpvlkdinfkiergqllavagstg agktsllmvimgelepsegkikhsgrisfcsqfswimpgtikeniifgvsydeyryrsvikacqleediskfaekdnivlgeggitlsgg qrarislaravykdadlylldspfgyldvltekeifescvcklmanktrilvtskmeh^

mgcdsfdqfsaermsiltetlhrfslegdapvswtetldcqsfkqtgefgelfl-knsilnpinsirkfsivqktp

slvpdseqgeailprisvistgptlqarrrqsvlnlmthsvnqgqnihrkttastrkvslapqanlteldiysrrlsqetgleiseeineedlke cffddmesipavttwntylryitvhkslifvliwclviflaevaaslvvlwllgntplqdkgnsthsrnnsyaviitstssyyvfyiyvgva dtllamgffrglplvhtlitvskilhhkmlhsvlqapmstlntlkaggilnrfskdiailddllpltifdfiqlllivig

vpvivafimlrayflqtsqqlkqlesegrspifthlvtslkglwtlra

vtfisilttgegegrvgiiltlamnimstlqwavnssidvdslmrsvsrvfkfidmptegkptkstkpykngqlskvmiiens iwpsggqmtvkdltakyteggnailenisfsispgqrvgllgrtgsgkstllsaflrllntegeiqidgvswdsitlqqwrkafgvipqkvf ifsgtfrknldpyeqwsdqeiwkvadevglrsvieqfpgkldfvlvdggcvlshghkqlmclarsvlskakillldepsahldpvtyqii rrtlkqafadctvilcehrieamlecqqflvieenkvrqydsiqkllnerslfrqaispsdrvklfphrnsskckskpqiaalkeeteeevqd trl* (SEQ ID NO: 12)

Nucleic acid sequence of CFTRA264

atgatcgagaacatccaatctgttaaggcatactgctgggaagaagcaatggaaaaaatgattgaaaacttaagacaaacagaactgaaac tgactcggaaggcagcctatgtgagatacttcaatagctcagccttcttcttctcagggttctttgtggtgtttttatctgtgcttccctatgcacta atcaaaggaatcatcctccggaaaatattcaccaccatctcattctgcattgttctgcgcatggcggtcactcggcaatttccctgggctgtac aaacatggtatgactctcttggagcaataaacaaaatacaggatttcttacaaaagcaagaatataagacattggaatataacttaacgactac agaagtagtgatggagaatgtaacagccttctgggaggagggatttggggaattatttgagaaagcaaaacaaaacaataacaatagaaaa acttctaatggtgatgacagcctcttcttcagtaatttctcacttcttggtactcctgtcctgaaagatattaatttcaagatagaaagaggacagtt gttggcggttgctggatccactggagcaggcaagacttcacttctaatgatgattatgggagaactggagccttcagagggtaaaattaagc acagtggaagaatttcattctgttctcagttttcctggattatgcctggcaccattaaagaaaatatcatctttggtgtttcctatgatgaatatagat acagaagcgtcatcaaagcatgccaactagaagaggacatctccaagtttgcagagaaagacaatatagttcttggagaaggtggaatcac actgagtggaggtcaacgagcaagaatttctttagcaagagcagtatacaaagatgctgatttgtatttattagactctccttttggatacctaga tgttttaacagaaaaagaaatatttgaaagctgtgtctgtaaactgatggctaacaaaactaggattttggtcacttctaaaatggaacatttaaa gaaagctgacaaaatattaattttgcatgaaggtagcagctatttttatgggacattttcagaactccaaaatctacagccagactttagctcaa aactcatgggatgtgattctttcgaccaatttagtgcagaaagaagaaattcaatcctaactgagaccttacaccgtttctcattagaaggagat gctcctgtctcctggacagaaacaaaaaaacaatcttttaaacagactggagagtttggggaaaaaaggaagaattctattctcaatccaatc aactctatacgaaaattttccattgtgcaaaagactcccttacaaatgaatggcatcgaagaggattctgatgagcctttagagagaaggctgt ccttagtaccagattctgagcagggagaggcgatactgcctcgcatcagcgtgatcagcactggccccacgcttcaggcacgaaggagg cagtctgtcctgaacctgatgacacactcagttaaccaaggtcagaacattcaccgaaagacaacagcatccacaggaaaagtgtcactgg cccctcaggcaaacttgactgaactggatatatattcaagaaggttatctcaagaaactggcttggaaataagtgaagaaattaacgaagaa gacttaaaggagtgcctttttgatgatatggagagcataccagcagtgactacatggaacacataccttcgatatattactgtccacaagagct taatttttgtgctaatttggtgcttagtaatttttctggcagaggtggctgcttctttggttgtgctgtggctccttggaaacactcctcttcaagaca aagggaatagtactcatagtagaaataacagctatgcagtgattatcaccagcaccagttcgtattatgtgttttacatttacgtgggagtagcc gacactttgcttgctatgggattcttcagaggtctaccactggtgcatactctaatcacagtgtcgaaaattttacaccacaaaatgttacattct gttcttcaagcacctatgtcaaccctcaacacgttgaaagcaggtgggattcttaatagattctccaaagatatagcaattttggatgaccttctg cctcttaccatatttgacttcatccagttgttattaattgtgattggagctatagcagttgtcgcagttttacaaccctacatctttgttgcaacagtg ccagtgatagtggcttttattatgttgagagcatatttcctccaaacctcacagcaactcaaacaactggaatctgaaggcaggagtccaatttt cactcatcttgttacaagcttaaaaggactatggacacttcgtgccttcggacggcagccttactttgaaactctgttccacaaagctctgaattt acatactgccaactggttcttgtacctgtcaacactgcgctggttccaaatgagaatagaaatgatttttgtcatcttcttcattgctgttaccttca tttccattttaacaacaggagaaggagaaggaagagttggtattatcctgactttagccatgaatatcatgagtacattgcagtgggctgtaaa ctccagcatagatgtggatagcttgatgcgatctgtgagccgagtctttaagttcattgacatgccaacagaaggtaaacctaccaagtcaac caaaccatacaagaatggccaactctcgaaagttatgattattgagaattcacacgtgaagaaagatgacatctggccctcagggggccaa atgactgtcaaagatctcacagcaaaatacacagaaggtggaaatgccatattagagaacatttccttctcaataagtcctggccagagggt gggcctcttgggaagaactggatcagggaagagtactttgttatcagcttttttgagactactgaacactgaaggagaaatccagatcgatgg tgtgtcttgggattcaataactttgcaacagtggaggaaagcctttggagtgataccacagaaagtatttattttttctggaacatttagaaaaaa cttggatccctatgaacagtggagtgatcaagaaatatggaaagttgcagatgaggttgggctcagatctgtgatagaacagtttcctggga agcttgactttgtccttgtggatgggggctgtgtcctaagccatggccacaagcagttgatgtgcttggctagatctgttctcagtaaggcgaa gatcttgctgcttgatgaacccagtgctcatttggatccagtaacataccaaataattagaagaactctaaaacaagcatttgctgattgcacag taattctctgtgaacacaggatagaagcaatgctggaatgccaacaatttttggtcatagaagagaacaaagtgcggcagtacgattccatcc agaaactgctgaacgagaggagcctcttccggcaagccatcagcccctccgacagggtgaagctctttccccaccggaactcaagcaag tgcaagtctaagccccagattgctgctctgaaagaggagacagaagaagaggtgcaagatacaaggctttag (SEQ ID NO: 13)

Amino acid sequence of CFTRA264

mieniqsvkaycweeamekmienlrqtelkltrkaayvryfnssafffsgffvvflsvlpyalikgiilrkifttisfc

avqtwydslgainkiqdflqkqeyktleynltttevvmenvtafweegfgelfekakqnnnnrktsngddslffsnfsllgtpvlkdinf kiergqllavagstgagktsllmmimgelepsegkikhsgrisfcsqfswi

dnivlgeggitlsggqrarislaravykdadlylldspfgyldvltekeifescvcklmanktrilvtskmehlkkadkililhegssyfygt fselqnlqpdfssklmgcdsfdqfsaermsiltetlhrfslegdapvswtet^ ngieedsdeplerrlslvpdseqgeailprisvistgptlqarrrqsvlnlmthsvnqgqnihrkttastgkvslapqanlteld gleiseeineedlkeclfddmesipavttwntylryi

ssyyvfyiyvgvadtllamgffrglplvhtlitvskilhhkmm^

vvavlqpyifvatvpvivafimlrayllqtsqqlkqlesegrspifthlvtslkglwtlrafgrqpyfetlflikalnlhtanwfl mriemifviffiavtfisilttgegegrvgiiltlamnimsti^

miienshvldcddiwpsggqmtvkdltakyteggnailenisfsispgqrvgllgrtgsgkstllsailrllntegei

wrkafgvipqkvfifsgtfrknldpyeqwsdqeiwkvadevglrsvieqfpgkldfvlvdggcvlshghkqlmclarsvlskakillld epsahldpvtyqiirrtlkqafadctvilcehrieamlecqqllvieenkvrqydsiqkllnerslfrqaispsdrvklfphm iaalkeeteeevqdtrl* (SEQ ID NO: 14)

As contemplated herein, a truncated version of CFTR retains at least 30% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%) of the chloride channel function as wild-type CFTR. Various methods for measuring the chloride channel function of CFTR in vitro are known in the art. For example, CFTR function can be assessed using ionic current measurement (e.g., using patch-clamp, two electrode voltage-clamp or an Ussing chamber), membrane potential measurements or chloride flux assays. Moran and Zegarra-Moran (J Cyst Fibros. 2008 Nov;7(6):483-94) discuss these methods and cellular systems for measuring CFTR function. Vijftigschild and Beekman (Cytometry A. 2013 Jun;83(6):576-84) discuss a cytometric method using a fluorescence sensor to measure CFTR function. The Cystic Fibrosis Foundation also provides information about CFTR assays on their website (https://www.cff.org/Research/Researcher-Resources/Tools-and- Resources/CFTR-Assays/ ). All of the references providing methods of measuring CFTR function are incorporated herein by reference in their entirety.

Grieger and Samulki (J Virol. 2005 Aug;79(15):9933-44) discuss packaging variant forms of CFTR into AAV particles, which is incorporated herein by reference in its entirety.

In some embodiments, a disease to be treated by delivering a therapeutic gene to lung tissue of a host is cystic fibrosis. In some embodiments, a CFTR gene is delivered to lung tissue of a host having cystic fibrosis.

In some embodiments, other genes can be delivered, for example to treat one or more other lung or airway conditions. For example, any one of the methods and variant rAAV particles described herein can be used to treat a host or subject with asthma by delivery of one or more immunomodulatory genes (e.g., IL4 or IL12), or silencing of genes, e.g., using shRNA. For example, ADAM33 can be silenced for the treatment of asthma. In some embodiments, any one of the methods and variant rAAV particles described herein can be used to treat a host or subject with alpha- 1 antitrypsin deficiency by delivery of alpha- 1 antitrypsin to the host's or subject's lung.

It is also contemplated herein to deliver nucleic acid for the use of gene editing (e.g., a nuclease or other protein for gene editing).

The terms "deliver" and "administer" are used interchangeably herein. In some embodiments, "administering" or "administration" means providing a material to a subject in a manner that is pharmacologically useful.

To "treat" a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. The compositions described above or elsewhere herein are typically administered to a subject in an effective amount, that is, an amount capable of producing a desirable result. The desirable result will depend upon the active agent being administered. For example, an effective amount of rAAV particles may be an amount of the particles that are capable of transferring an expression construct to a host organ, tissue, or cell. A therapeutically acceptable amount may be an amount that is capable of treating a disease, e.g., CF or asthma. As is well known in the medical and veterinary arts, dosage for any one subject depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, the active ingredient(s) in the composition, time and route of administration, general health, and other drugs being administered concurrently.

In some embodiments, the concentration of rAAV particles administered to a subject may be on the order ranging from 10⁶ to 10¹⁴ particles/ml or 10³ to 10¹⁵ particles/ml, or any values therebetween for either range, such as for example, about 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹,

10 12 , 1013 , or 101¹4" particles/ml. In some embodiments, rAAV particles of a higher concentration than 10 13 particles/ml are administered. In some embodiments, the concentration of rAAV particles administered to a subject may be on the order ranging from 10⁶ to 10¹⁴ vector genomes(vgs)/ml or 10³ to 10¹⁵ vgs/ml, or any values therebetween for either range (e.g., 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, or 10¹⁴ vgs/ml). In some embodiments, rAAV particles of higher concentration than 10 13 vgs/ml are administered. The rAAV particles can be

administered as a single dose, or divided into two or more administrations as may be required to achieve therapy of the particular disease or disorder being treated. In some embodiments, 0.0001 ml to 10 mis are delivered to a subject. In some embodiments, the number of rAAV particles administered to a subject may be on the order ranging from 10⁶-10¹⁴ vg/kg, or any values therebetween (e.g., 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, or 10¹⁴ vgs/mg). In some embodiments, the dose of rAAV particles administered to a subject may be on the order ranging from 10¹²-10¹⁴ vgs/kg. In some embodiments, the volume of rAAVrh. lO composition delivered to a subject (e.g., via one or more routes of administration as described herein) is 0.0001 mL to 10 mis.

Lung conditions

Respiratory diseases or ailments, which are referred to herein as lung conditions, may be an obstructive condition (e.g., emphysema, bronchitis or asthma attacks), a restrictive condition (e.g., fibrosis, sarcoidosis, alveolar damage, pleural effusion), a vascular disease (e.g., pulmonary edema, pulmonary embolism or pulmonary hypertension), or an infectious or environmental disease (e.g., pneumonia, tuberculosis, or irritation cause by asbestosis and particulate pollutants). A respiratory tract infection may be of the upper respiratory tract (e.g., sinusitis, tonsillitis, otitis media, pharyngitis or laryngitis) or the lower respiratory tract (e.g., pneumonia). A lung condition may be COPD, chronic bronchitis, emphysema, asthma, pneumonia. A lung condition may be one of the upper respiratory tract, trachea, bronchi, bronchioles, alveoli, pleura and pleural cavity, or the nerves and muscles of breathing.

Respiratory diseases range from mild and self-limiting, such as the common cold, to life- threatening entities like bacterial pneumonia, pulmonary embolism, acute asthma and lung cancer. In some embodiments, a lung condition is a cancer or caused by a tumor. In some embodiments, a lung cancer is small cell lung cancer, non-small cell lung cancer (e.g., adenocarcinoma of the lung, squamous cell carcinoma of the lung, large cell lung carcinoma), carcinoid, Kaposi's sarcoma, melanoma, lymphoma, head and neck cancer or pleural mesothelioma. In some embodiments, a pulmonary vascular disease of the lung is pulmonary embolism, pulmonary arterial hypertension, pulmonary edema or pulmonary hemorrhage. In some embodiments, a lung condition is a neonatal disease (e.g., pulmonary hyperplasia or infant respiratory distress syndrome).

Administration of cells infected with variant AAV particles into a host

In some embodiments, cells in the lung of a subject (e.g., airway epithelial cells) are obtained (e.g., from the subject) and modified ex vivo (e.g., by infection with any one of the rAAV particles disclosed herein) before reintroducing the cells to the subject. In some embodiments, cells that are modified ex vivo and introduced to a subject are from another subject (e.g., allogeneic cells). In some embodiments, cells are infected with any one of the variant rAAV particles as disclosed herein, which are then administered to a host or subject in need of a transgene that is expressed by the administered cells.

In some embodiments, a lung cell that is modified ex vivo and introduced in a subject is a lung cell of the airway or of the lung parenchyma. In some embodiments, a lung cell that is modified ex vivo and introduced in a subject is a pseudostratified columnar epithelium cell (e.g., ciliated airway epithelial cell, goblet (or mucus) airway epithelial cells, or a basal airway epithelial cell. In some embodiments , an airway epithelial cell that is isolated from a subject and modified ex vivo is squamous, cuboidal or columnar. In some embodiments, a lung cell is an alveolar cell (e.g., a type I alveolar cell or type II alveolar cell).

Lung cells that are modified ex vivo and introduced in a subject can also be of the pulmonary vasculature, lymphatic system or muscle cells (e.g., smooth muscle cells). In some embodiments, lung cells are lung fibroblasts. In some embodiments, different lung cells (e.g., mucus/goblet airway epithelial cells, ciliary airway epithelial cells and basal cells) are cultured together and administered to a subject together. In some embodiments, different lung cells are cultured together, differentiated to a predominant cell type ex vivo and then administered to a subject.

In some embodiments, cells infected with a variant rAAV particle and administered to a subject are stem cells or progenitor cells for lung cells (e.g., airway epithelial cells). In some embodiments, cells infected with a variant rAAV particle and administered to a subject are autologous or allogeneic. In some embodiments, cells infected with a variant rAAV particle and then administered to a subject are induced pluripotent stem cell (iPSC) lines. For example, Crane et al. (Stem Cell Reports. 2015 Apr 14;4(4):569-77) describe derivation of iPSCs from skin fibroblasts from patients diagnosed with CF. rAAV particles, preparations, and nucleic acid vectors

The wild-type AAV genome is a single- stranded deoxyribonucleic acid (ssDNA), either positive- or negative-sensed. The genome comprises two inverted terminal repeats (ITRs), one at each end of the DNA strand, and two open reading frames (ORFs): rep and cap between the ITRs. The rep ORF comprises four overlapping genes encoding Rep proteins required for the AAV life cycle: Rep78, Rep68, Rep52 and Rep40. The cap ORF comprises overlapping genes encoding capsid proteins: VP1, VP2 and VP3, which interact together to form the viral capsid. VP1, VP2 and VP3 are translated from one mRNA transcript, which can be spliced in two different manners: either a longer or shorter intron can be excised resulting in the formation of two isoforms of mRNAs: a -2.3 kb- and a -2.6 kb-long mRNA isoform. The capsid forms a supramolecular assembly of approximately 60 individual capsid protein subunits into a non- enveloped, T- l icosahedral lattice capable of protecting the AAV genome. The mature capsid is composed of VP1, VP2, and VP3 (molecular masses of approximately 87, 73, and 62 kDa respectively) in a ratio of about 1 : 1 : 10. rAAV particles may comprise VP1, VP2 and/or VP3. rAAV particles may comprise a nucleic acid vector, which may comprise at a minimum: (a) one or more heterologous nucleic acid regions comprising a sequence encoding a protein or polypeptide of interest or an RNA of interest (e.g. , a siRNA or microRNA), and (b) one or more regions comprising inverted terminal repeat (ITR) sequences (e.g. , wild-type ITR sequences or engineered ITR sequences) flanking the one or more nucleic acid regions (e.g. , heterologous nucleic acid regions). Herein, heterologous nucleic acid regions comprising a sequence encoding a protein of interest or RNA of interest are referred to as genes of interest. In some embodiments, the nucleic acid vector is between 4kb and 5kb in size (e.g. , 4.2 to 4.7 kb in size). Any nucleic acid vector described herein may be encapsidated by a viral capsid, such as an AAV5 or AAV6 capsid or any other serotype, which may comprise a modified capsid protein as described herein. In some embodiments, the nucleic acid vector is circular. In some

embodiments, the nucleic acid vector is single-stranded. In some embodiments, the nucleic acid vector is double-stranded. In some embodiments, a double-stranded nucleic acid vector may be, for example, a self-complimentary vector that contains a region of the nucleic acid vector that is complementary to another region of the nucleic acid vector, initiating the formation of the double-strandedness of the nucleic acid vector.

Accordingly, in some embodiments, a rAAV particle or rAAV preparation containing such particles comprises a viral capsid and a nucleic acid vector, which is encapsidated by the viral capsid. In some embodiments, the nucleic acid vector comprises (1) one or more heterologous nucleic acid regions comprising a sequence encoding an RNA, protein or polypeptide of interest, (2) one or more nucleic acid regions comprising a sequence that facilitates expression of the heterologous nucleic acid region (e.g. , a promoter), and (3) one or more nucleic acid regions comprising a sequence that facilitate integration of the heterologous nucleic acid region (optionally with the one or more nucleic acid regions comprising a sequence that facilitates expression) into the genome of the subject. In some embodiments, viral sequences that facilitate integration comprise Inverted Terminal Repeat (ITR) sequences. In some embodiments, the nucleic acid vector comprises one or more heterologous nucleic acid regions comprising a sequence encoding an RNA, protein or polypeptide of interest operably linked to a promoter, wherein the one or more heterologous nucleic acid regions are flanked on each side with an ITR sequence. Such a nucleic acid vector is herein also referred to as AAV- ITR containing one or more genes of interest. In some embodiments, the nucleic acid vector comprises (1) one or more heterologous nucleic acid regions comprising a sequence encoding an RNA, protein or polypeptide of interest, (2) one or more nucleic acid regions comprising a sequence that facilitates expression of the heterologous nucleic acid region (e.g. , a promoter), and (3) one or more nucleic acid regions comprising a sequence that facilitate integration of the heterologous nucleic acid region (optionally with the one or more nucleic acid regions comprising a sequence that facilitates expression) into the genome of the subject. In some embodiments, viral sequences that facilitate integration comprise Inverted Terminal Repeat (ITR) sequences of a first serotype. In some embodiments, the nucleic acid vector comprises one or more heterologous nucleic acid regions comprising a sequence encoding an RNA, protein or polypeptide of interest operably linked to a promoter, wherein the one or more heterologous nucleic acid regions are flanked on each side with an ITR sequence of a first serotype. In some embodiments, viral sequences that facilitate integration comprise Inverted Terminal Repeat (ITR) sequences. The ITR sequences can be derived from any AAV serotype (e.g. , serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) or can be derived from more than one serotype.

ITR sequences and plasmids containing ITR sequences are known in the art and commercially available (see, e.g., products and services available from Vector Biolabs,

Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, MA; and Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Kessler PD, Podsakoff GM, Chen X, McQuiston SA, Colosi PC, Matelis LA, Kurtzman GJ, Byrne BJ. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24): 14082-7; and Curtis A. Machida. Methods in Molecular Medicine™. Viral Vectors for Gene TherapyMethods and Protocols. 10.1385/1-59259-304-6:201 © Humana Press Inc. 2003. Chapter 10. Targeted Integration by Adeno- Associated Virus. Matthew D. Weitzman, Samuel M. YoungJr., Toni Cathomen and Richard Jude Samulski; U.S. Pat. Nos. 5, 139,941 and 5,962,313, all of which are incorporated herein by reference).

Genebank reference numbers for sequences of AAV serotypes 1, 2, 3, 3B, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 are listed in patent publication WO2012064960, which is incorporated herein by reference in its entirety.

In some embodiments, the nucleic acid vector comprises one or more regions comprising a sequence that facilitates expression of the nucleic acid (e.g. , the heterologous nucleic acid), e.g. , expression control sequences operatively linked to the nucleic acid. Numerous such sequences are known in the art. Non-limiting examples of expression control sequences include promoters, insulators, silencers, response elements, introns, enhancers, initiation sites, termination signals, and poly(A) tails. Any combination of such control sequences is contemplated herein (e.g. , a promoter and an enhancer).

To achieve appropriate expression levels of the protein or polypeptide of interest, any of a number of promoters suitable for use in the selected host cell may be employed. The promoter may be, for example, a constitutive promoter, tissue-specific promoter, inducible promoter, or a synthetic promoter.

For example, constitutive promoters of different strengths can be used. A nucleic acid vector described herein may include one or more constitutive promoters, such as viral promoters or promoters from mammalian genes that are generally active in promoting transcription. Non- limiting examples of constitutive viral promoters include the Herpes Simplex virus (HSV), thymidine kinase (TK), Rous Sarcoma Virus (RSV), Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV), Ad ElA,cytomegalovirus (CMV) and MND promoters. In some embodiments, an MND promoter contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus. Non-limiting examples of constitutive mammalian promoters include various housekeeping gene promoters, as exemplified by the β-actin promoter (e.g., chicken β-actin promoter) and human elongation factor- 1 a (EF-la) promoter.

Inducible promoters and/or regulatory elements are also be contemplated for achieving appropriate expression levels of the protein or polypeptide of interest. Non-limiting examples of suitable inducible promoters include those from genes such as cytochrome P450 genes, heat shock protein genes, metallothionein genes, and hormone-inducible genes, such as the estrogen gene promoter. Another example of an inducible promoter is the tetVP16 promoter that is responsive to tetracycline.

Tissue- specific promoters and/or regulatory elements are also contemplated herein. Non-limiting examples of such promoters that may be used include airway epithelial cell- specific promoters. For example, a FOXJ1 promoter can be used to target expression of a transgene in airway epithelial cells that are ciliated (see e.g., Zhang et al., Am J Respir Cell Mol Biol. 2007 May; 36(5): 515-519; Ostrowski et al., Mol Ther. 2003 Oct;8(4):637-45). Rawlins and Perl (Am J Respir Cell Mol Biol. 2012 Mar;46(3):269-82) also discuss lung specific promoters, the contents of which are incorporated herein by reference in their entirety.

Synthetic promoters are also contemplated herein. A synthetic promoter may comprise, for example, regions of known promoters, regulatory elements, transcription factor binding sites, enhancer elements, repressor elements, and the like.

An rAAV particle or particle within an rAAV preparation may be of any AAV serotype, including any derivative or pseudotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 2/1, 2/5, 2/8, 2/9, 3/1, 3/5, 3/8, or 3/9). As used herein, the serotype of an rAAV viral vector (e.g. , an rAAV particle) refers to the serotype of the capsid proteins of the recombinant virus. Non-limiting examples of derivatives and pseudotypes include rAAV2/l, rAAV2/5, rAAV2/8, rAAV2/9, AAV2-AAV3 hybrid, AAVrh. lO, AAVhu.14, AAV3a/3b, AAVrh32.33, AAV-HSC 15, AAV- HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC 15/17,

AAVM41, AAV9.45, AAV6(Y445F/Y731F), AAV2.5T, AAV-HAE1/2, AAV clone 32/83, AAVShHIO, AAV2 (Y->F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, and AAVr3.45. Such AAV serotypes and derivatives/pseudotypes, and methods of producing such

derivatives/pseudotypes are known in the art (see, e.g., Mol Ther. 2012 Apr;20(4):699-708. doi: 10.1038/mt.2011.287. Epub 2012 Jan 24. The AAV vector toolkit: poised at the clinical crossroads. Asokan Al, Schaffer DV, Samulski RJ.). In some embodiments, the rAAV particle is a pseudotyped rAAV particle, which comprises (a) a nucleic acid vector comprising ITRs from one serotype (e.g., AAV2, AAV3) and (b) a capsid comprised of capsid proteins derived from another serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10). Methods for producing and using pseudotyped rAAV vectors are known in the art (see, e.g. , Duan et al., J. Virol., 75:7662-7671, 2001 ; Halbert et al., J. Virol., 74: 1524- 1532, 2000; Zolotukhin et al., Methods, 28: 158- 167, 2002; and Auricchio et al., Hum. Molec. Genet, 10:3075-3081, 2001).

Methods of producing rAAV particles and nucleic acid vectors are described herein. Other methods are also known in the art and commercially available (see, e.g. , Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158-167; and U.S. Patent Publication Numbers US20070015238 and US20120322861, which are incorporated herein by reference; and plasmids and kits available from ATCC and Cell Biolabs, Inc.). For example, a plasmid comprising a gene of interest may be combined with one or more helper plasmids, e.g. , that contain a rep gene (e.g. , encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene (encoding VPl, VP2, and/or VP3, including a modified VP2 region as described herein), and transfected into recombinant cells such that the rAAV particle can be packaged and subsequently purified.

In some embodiments, the packaging is performed in a helper cell or producer cell, such as a mammalian cell or an insect cell. Non-limiting examples of mammalian cells include, but are not limited to, HEK293 cells, COS cells, HeLa cells, BHK cells, or CHO cells (see, e.g., ATCC® CRL-1573™, ATCC® CRL-1651™, ATCC® CRL-1650™, ATCC® CCL-2, ATCC® CCL-10™, or ATCC® CCL-61™). Non-limiting examples of insect cells include, but are not limited to Sf9 cells (see, e.g., ATCC® CRL-1711™). A helper cell may comprises rep and/or cap genes that encode the Rep protein and/or Cap proteins for use in a method described herein. In some embodiments, packaging is performed in vitro.

In some embodiments, a plasmid comprising the gene of interest is combined with one or more helper plasmids, e.g., that contain a rep gene of a first serotype and a cap gene of the same serotype or a different serotype, and transfected into helper cells such that the rAAV particle is packaged.

In some embodiments, the one or more helper plasmids include a first helper plasmid comprising a rep gene and a cap gene, and a second helper plasmid comprising one or more of the following helper genes: Ela gene, Elb gene, E4 gene, E2a gene, and VA gene. For clarity, helper genes are genes that encode helper proteins Ela, Elb, E4, E2a, and VA. In some embodiments, the cap gene is modified such that one or more of the proteins VPl, VP2 and VP3 do not get expressed. In some embodiments, the cap gene is modified such that VP2 does not get expressed. Methods for making such modifications are known in the art (Lux et al. (2005), J Virology, 79: 11776-87)

Helper plasmids, and methods of making such plasmids, are known in the art and are also commercially available (see, e.g., pDF6, pRep, pDM, pDG, pDPlrs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E/R585E), and pDP8.ape plasmids from PlasmidFactory, Bielefeld, Germany; other products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, Ca; and Addgene,

Cambridge, MA; pxx6; Grimm et al. (1998), Novel Tools for Production and Purification of Recombinant Adeno associated Virus Vectors, Human Gene Therapy, Vol. 9, 2745-2760; Kern, A. et al. (2003), Identification of a Heparin-Binding Motif on Adeno-Associated Virus Type 2 Capsids, Journal of Virology, Vol. 77, 11072-11081.; Grimm et al. (2003), Helper Virus-Free, Optically Controllable, and Two-Plasmid-Based Production of Adeno-associated Virus Vectors of Serotypes 1 to 6, Molecular Therapy, Vol. 7, 839-850; Kronenberg et al. (2005), A Conformational Change in the Adeno-Associated Virus Type 2 Capsid Leads to the Exposure of Hidden VP1 N Termini, Journal of Virology, Vol. 79, 5296-5303; and Moullier, P. and Snyder, R.O. (2008), International efforts for recombinant adeno-associated viral vector reference standards, Molecular Therapy, Vol. 16, 1185-1188). Plasmids that encode wild-type AAV coding regions for specific serotypes are also know and available. For example, pSub201 is a plasmid that comprises the coding regions of the wild-type AAV2 genome (Samulski et al. (1987), J Virology, 6:3096-3101).

A non-limiting example of a rAAV particle production method is described next. One or more helper plasmids are produced or obtained, which comprise rep and cap ORFs for the desired AAV serotype and the adenoviral VA, E2A (DBP), and E4 genes under the

transcriptional control of their native promoters. In some embodiments, the one or more helper plasmids comprise rep genes, cap genes, and optionally one or more of the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. In some embodiments, the one or more helper plasmids comprise cap ORFs (and optionally rep ORFs) for the desired AAV serotype and the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. A cap ORF may also comprise one or more modifications to produce a modified capsid protein as described herein. HEK293 cells

(available from ATCC®) are transfected via CaP0₄-mediated transfection, lipids or polymeric molecules such as Polyethylenimine (PEI) with the helper plasmid(s) and a plasmid containing a nucleic acid vector described herein. HEK293 cells are then incubated for at least 60 hours to allow for rAAV particle production. Alternatively, HEK293 cells are transfected via methods described above with AAV-ITR containing one or more genes of interest, a helper plasmid comprising genes encoding Rep and Cap proteins, and co-infected with a helper virus. Helper viruses are viruses that allow the replication of AAV. Non-limiting examples of helper virus are adenovirus and herpesvirus.

Alternatively, Sf9-based producer stable cell lines can be infected with a single recombinant baculovirus containing the nucleic acid vector. As a further alternative, HEK293 or BHK cell lines can be infected with a Herpes Simplex Virus (HSV) containing the nucleic acid vector and optionally one or more helper HSVs containing rep and cap ORFs as described herein and the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. HEK293, BHK, or Sf9 cells are then incubated for at least 60 hours to allow for rAAV particle production. The rAAV particles can then be purified using any method known in the art or described herein, e.g. , by iodixanol step gradient, CsCl gradient, chromatography, or polyethylene glycol (PEG) precipitation.

EXAMPLES

Example 1 : Recombinant AAV for effective gene therapy in bronchial epithelium

The data described below show how variant rAAV particles comprising expression vectors encoding a therapeutic gene (e.g., CFTR for CF) or a detectable marker (e.g. , GFP or luciferase) can be used for gene delivery or gene therapy to human airway epithelial cells (AECs) that have been isolated from healthy subjects or subjects with CF.

First, the transduction efficiency of several capsid-variant AAV serotype vectors expressing green-fluorescence protein (AAV-GFP) in normal and CF donor-derived (CRL- 4013) human AEC were evaluated. In these experiments, cells from CF donor and normal subjects were infected with either wild-type or mutant AAV1, AAV2, AAV5 or AAV6 comprising nucleic acid encoding GFP. Thereafter, GFP expression was assessed by measuring fluorescence intensity. Data indicated that capsid-variant AAV6 vectors transduce both CF- donor derived and normal human subject-derived cells at 2-3-fold higher than other AAV serotypes (FIG. 2A and 2B). (AAV6>AAV2>AAV5>AAV1). Similar results were achieved on primary human AEC from two different CF donors. Furthermore, AAV6-Y705-731F+T492V (M3) and AAV-S663V (663) capsid variants performed better than the wild-type AAV6 particles.

Next, the following hypothesis was tested: an increased viral copy number in the nucleus would allow a higher frequency of integration of nucleic acid carrying the GFP gene. The frequency of integration was determined by flow cytometry analysis on a number of GFP positive AEC persistent in culture for ten passages. Results from these experiments suggest that capsid-variant AAV6 vectors integrate with approximately 2-fold higher frequency compared to WT-AAV6 (FIG. 3).

It was found that the packaging capacity of AAV5 was approximately 5-fold higher than AAV6 (FIG. 4 top panel), and that AAV capsid integrity was intact after delivery (FIG. 4 bottom panel).

It was also hypothesized that a higher degree of viral DNA detected in the nuclei as compared to the cytoplasm represents a higher degree of integration into the genome of the host. Accordingly, experiments were carried out in which airway epithelial cells (2xE6) grown under submerged culture conditions were infected by various variant and wild-type AAV6 particles. After infection, cells were collected at 18h and 48h post- infection and nuclear and cytoplasmic fractions were separated with a 2-step lysis and centrifugation protocol. Viral DNA was detected by qPCR analysis with chicken β-actin promoter- specific primers. For each pair-wise set of samples, the fold change in viral DNA was calculated as fold change 2^A(CT_cyt0pia_Sm- CT_nuci_eus). FIG. 8 shows data from these experiments. The percentage of nuclear viral DNA was higher in cells infected with AAV6- Y705-731F+T492V (M3) particles compared to cells infected with wild-type AAV6 or AAV6-S663V particles.

FIGs. 5 and 10 demonstrates successful in vivo delivery of nucleic acid encoding luciferase comprised by variant AAV particles, and subsequent luciferase expression. Mice were anesthetized and then intubated by a guiding wire threaded through a catheter into the trachea. A precision syringe or blunt needle containing liquid suspension/air cushion was inserted to the catheter. 50 μΐ of AAV (5 x 10¹⁰ vg/mouse) comprising nucleic acid encoding luciferase was delivered to the lung. Luciferase activity was analyzed two weeks post injection using a Xenogen IVIS Lumina System (Caliper Life Sciences).

FIGs. 9 and 10 show that the expression of an encoded nucleic acid persists longer in mice that are infected with a variant or mutant AAV6 particle comprising the nucleic acid, compared to wild-type AAV6 particles comprising the nucleic acid. It can be seen in both FIGs. 9 and 10 that there is more luciferase expression over time in mice infected with AAV6- Y705- 731F+T492V particles comprising nucleic acid encoding luciferase, compared to wild-type AAV6 particles comprising nucleic acid encoding luciferase.

FIG 6. shows that transduction of CF AECs with AAV particles comprising nucleic acid encoding CFTR results in functional benefit of the CFTR chloride channel. AEC were seated to snapwell and infected with AAV particles comprising nucleic acid encoding A264CFTR at day 3 after reaching 90-100% confluency. Differentiated and infected cells were grown in snapwell at air-liquid interface for 2-3 weeks. Trans-epithelial electrical resistance (TEER) was measured using EVOM2 epithelial volt-Ohm meter with STX3 chopstick type electrodes as an indication of epithelial integrity of the tight junctions. Upon reaching maximum TEER of 500-700 Ohm- cm , cells were then used for evaluation of chloride channel activity in Ussing chamber. Trans- epithelial short circuit currents were then measured with an epithelial voltage clamp and a self- contained Ussing chamber system. As can be seen in FIG. 6, the trans-epithelial short circuit currents, which are a measure of chloride channel function, are much improved in cells transfected with AAV particles comprising nucleic acid encoding A264CFTR, compared to uninfected cells.

The data described above show that certain AAV5 and AAV6 capsid variants can increase AAV transduction efficiency. For example, variants can include ones that are mutated in serine residue S663 and/or S651, which is located in IH loop of the capsid which can have a significant effect on vector activity (FIG. 7).

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding,"

"composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., "comprising") are also contemplated, in alternative embodiments, as "consisting of and "consisting essentially of the feature described by the open-ended transitional phrase. For example, if the disclosure describes "a composition comprising A and B", the disclosure also contemplates the alternative embodiments "a composition consisting of A and B" and "a composition consisting essentially of A and B".

Claims

CLAIMS What is claimed is:

1. A recombinant AAV (rAAV) particle comprising:

a nucleic acid encoding a therapeutic gene for treating a lung condition, and

a capsid protein having a mutation that promotes delivery of the rAAV particle to lung tissue.

2. The rAAV particle of claim 1, wherein the capsid protein comprises a second mutation that promotes delivery of the rAAV particle to lung tissue.

3. The rAAV particle of claims 1 or 2, wherein the AAV particle is of serotype 1, 2, 5, 6 or serotype 5 and 6.

4. The rAAV particle of any one of the preceding claims, wherein the AAV particle is of serotype 5 or 6.

5. The rAAV particle of claim 4, wherein the AAV particle is of serotype 5.

6. The rAAV particle of claim 4, wherein the AAV particle is of serotype 6.

7. The rAAV particle of any one of the preceding claims, wherein the mutation is at an amino acid residue that is exposed on the outside surface of the AAV particle.

8. The rAAV particle of any one of the preceding claims, wherein the capsid protein comprises VP1, VP2 and/or VP3.

9. The rAAV particle of any one of the preceding claims, wherein the mutation is at a tyrosine, serine or threonine of wild-type AAV capsid protein.

10. The rAAV particle of any one of the preceding claims, wherein the mutation is located in an IH loop of the capsid as depicted in FIG. 7.

11. The rAAV particle of claim 5, wherein the mutation is at S651, S485, Y436 or Y719.

12. The rAAV particle of claim 11, wherein the mutation is one or more of the following: S651V, S485V, Y436F and Y719F.

13. The rAAV particle of claim 6, wherein the mutation is at S663, T492, Y705 or Y731.

14. The rAAV particle of claim 13, wherein the mutation is one or more of the following: S663V, T492V, Y705F and Y731F.

15. The rAAV particle of any one of claims 1-8, wherein the mutation is a substitution of a hydrophilic amino acid selected from the group consisting of Arg, Asn, Glu and Pro, to a hydrophobic amino acid selected from the group consisting of Ala, Val, Thr, Phe, Trp, Leu and Iso.

16. The rAAV particle of any one of the preceding claims, wherein the mutation results in an rAAV particle that is more effectively transduced into lung cells than a wild-type AAV particle.

17. The rAAV particle of any one of the preceding claims, wherein the mutation results in an rAAV particle that has a higher packaging capacity compared to a wild-type AAV particle.

18. The rAAV particle of any one of the preceding claims, wherein the mutation results in an rAAV particle that more effectively penetrates mucus compared to a wild-type AAV particle.

19. The rAAV particle of any one of the preceding claims, wherein the mutation results in an rAAV particle that more effectively integrates into a host genome compared to a wild-type AAV particle.

20. The rAAV particle of any one of the preceding claims, wherein the results in a rAAV particle that is less immunogenic compared to a wild-type rAAV particle.

21. The rAAV particle of any one of the preceding claims, wherein the lung condition is selected from the group consisting of an obstructive condition, a restrictive condition, a vascular disease and an infectious or environmental disease.

22. A composition comprising the rAAV particle of any one of the preceding claims.

23. A method of delivering a therapeutic gene to lung tissue of a host, the method comprising delivering the rAAV particle of any of claims 1-21, or the composition of claim 20, to the host.

24. The method of claim 23, wherein the lung tissue is bronchial epithelium or tracheal epithelium.

25. The method of claim 23 or 24, wherein the host is a mammal.

26. The method of claim 25, wherein the mammal is a human.

27. The method of any one of the claims 23-26, wherein the rAAV particle or composition is administered by nebulization.

28. The method of any one of the claims 23-27, wherein a therapeutic protein encoded by the therapeutic gene is Cystic fibrosis transmembrane conductance regulator (CFTR).

29. The method of claim 28, wherein the amino acid sequence of CFTR is SEQ ID NO: 12.

30. The method of claim 28, wherein the CFTR is CFTRA264, which has the amino acid sequence of SEQ ID NO: 14.

31. The method of claim 29, wherein the CFTR is CFTRAR, which has the amino acid sequence of SEQ ID NO: 12 without amino acids 708-759.

32. The method of any one of claims 23-30, wherein the host has cystic fibrosis.

33. The method of claims 23-27, wherein the therapeutic gene encodes an shRNA or siRNA for gene silencing, or a genome editing protein selected from the group consisting of a Zinc Finger Nuclease (ZFN), a TALEN, a CRISPR/Cas proteins and a meganuclease.

34. A method of expressing a gene of interest in a cell derived from a lung, the method comprising infecting the cell with an rAAV particle of any one of claims 1-21, or the composition of claim 22.