Combination therapy with adeno-associated virus and CFTR modulator(s) for treatmet of cftr

Abstract

Provided herein are pharmaceutical combinations comprising recombinant adeno-associated virus (AAV) and CFTR modulator(s) for the treatment of cystic fibrosis.

Description

COMBINATION THERAPY WITH ADENO-ASSOCIATED VIRUS AND CFTR MODULATOR(S) FOR TREATMET OF CFTRCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 698,914, filed September 25, 2024, the entire contents of each of which are incorporated herein by reference.SEQUENCE LISTING SUBMISSION VIA EFS-WEB

[0002] A computer readable XML file, entitled “090400-5031 WO Sequence Listing” created on September 19, 2025, with a file size of 114,783 bytes contains the sequence listing for this application and is hereby incorporated by reference in its entirety.BACKGROUND OF THE INVENTION

[0003] Current treatments for cystic fibrosis (CF) include CFTR modulator therapy, which attempts to correct the CFTR malfunctions, and gene therapy.

[0004] CFTR modulators offer improvements for many CF patients; however, despite the recent successes of CFTR channel modulators an unmet need remains for those patients unable to tolerate side effects of ion channel modulator therapy, or subsets still lacking disease-modifying treatment options such as patients affected by homozygous Class I mutations.

[0005] Since the cloning of the CFTR gene in 1989, there has been significant interest in the possibility of gene therapy as a treatment for CF. However, aerosol delivery of genes to the lung using AAV has not been successful. As of 2024, clinical trials with AAV2-based gene therapy have failed to detect transgene expression and no improvement in lung function (measured as ppFEVl) was observed. In addition, the ability to administer conventional viral vectors repeatedly, mandatory for the life-long treatment of a self-renewing epithelium, is limited, because of patients' adaptive immune responses, which prevent successful repeat administration.

[0006] There is accordingly a need for new and effective therapies for CF, particularly for patients who are CF modulator-insensitive or intolerant, or who lack disease-modifying treatment options.SUMMARY OF THE INVENTION

[0007] In some embodiments, combination therapies for the treatment of cystic fibrosis (CF) are provided, comprising administering a therapeutic combination to a subject with cystic fibrosis, wherein the therapeutic combination comprises a recombinant adeno- associated virus (rAAV) and one or more CFTR modulators.

[0008] In related embodiments, a method for the treatment of CF is provided, comprising administering to a subject in need thereof, a therapeutically effective amount of each of (A) a pharmaceutical composition comprising an rAAV vector, said rAAV vector comprising a capsid comprising a capsid protein comprising the amino acid sequence as set forth as SEQ ID NO: 12 or an amino acid sequence at least 90% identical to SEQ ID NO: 12 and comprising a Thr at amino acid 469 and an Ala at amino acid 598 based on the amino acid numbering set forth in SEQ ID NO: 12 and a heterologous nucleic acid comprising a nucleotide sequence encoding a cystic fibrosis transmembrane conductance regulator (CFTR protein) gene product operably linked to a promoter and (B) one or more CFTR modulators. In preferred embodiments, the pharmaceutical composition is formulated as an aerosol.

[0009] In related embodiments, the use of a pharmaceutical composition comprising an rAAV vector, said rAAV vector comprising a capsid comprising a capsid protein comprising the amino acid sequence as set forth as SEQ ID NO: 12 or an amino acid sequence at least 90% identical to SEQ ID NO: 12 and comprising a Thr at amino acid 469 and an Ala at amino acid 598 based on the amino acid numbering set forth in SEQ ID NO: 12 and a heterologous nucleic acid comprising a nucleotide sequence encoding a cystic fibrosis transmembrane conductance regulator (CFTR protein) gene product operably linked to a promoter, in the treatment of CF is provided, wherein the method further comprises administration of one or more CFTR modulator(s).

[0010] In other related embodiments, the use of a pharmaceutical composition comprising an rAAV vector, said rAAV vector comprising a capsid comprising a capsid protein comprising the amino acid sequence as set forth as SEQ ID NO: 12 or an amino acid sequence at least 90% identical to SEQ ID NO: 12 and comprising a Thr at amino acid 469 and an Ala at amino acid 598 based on the amino acid numbering set forth in SEQ ID NO: 12 and a heterologous nucleic acid comprising a nucleotide sequence encoding a cystic fibrosis transmembrane conductance regulator (CFTR protein) gene product operably linked to a promoter, in the manufacture of a medicament for treatment of CF is provided, wherein the method further comprises administration of one or more CFTR modulator(s).

[0011] In some aspects, the subject in need of treatment with the combination therapy is administered an amount of a pharmaceutical composition that is effective to achieve a target clinical delivered dose of about 5x1013vector genomes (vg) to about 4.7x1014vg, preferably about 6x1013vg, about 1.2x1014vg or about 2.3x1014vg to the lungs of the subject.Preferably, the pharmaceutical composition is suitable for aerosolization and is administered to the subject by a nebulizer, e.g. a breath-actuated or vibrating mesh nebulizer. Although any nebulizer or pressurized metered dosage device may be used to achieve such a targetclinical delivered dose (e.g., based on the unit dose of rAAV present in the pharmaceutical composition prior to aerosolization and the overall efficiency and percentage of aerosolized particles in the respirable range (5.4-1 mm)), preferably a breath-actuated nebulizer is used, more preferably an AeroEclipse II BAN nebulizer. In some aspects, the subject is a primate (e.g., human).

[0012] In some aspects, the subject is administered a pharmaceutical composition comprising a unit dose of about 8x1013vg to about 3x1014vg, preferably about 8x1013vg, about 1.6x1014vg or about 3x1014vg of the rAAV. Preferably, the pharmaceutical composition is suitable for aerosolization and is administered by a breath-actuated nebulizer, more preferably by an AeroEclipse II BAN nebulizer. In other aspects, the subject is administered a pharmaceutical comprising a unit dose of about 5x10'4viral genomes (vg) or about 2.5x1014vg or about 1.25x1014of the rAAV. In preferred aspects, the pharmaceutical composition is formulated as an aerosol.In some preferred aspects, the pharmaceutical composition of the combination comprises an rAAV, said rAAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO: 12 and (ii) a nucleic acid comprising from 51to 31: (a) an AAV2 terminal repeat (b) a promoter (c) a nucleotide sequence encoding a human cystic fibrosis transmembrane conductance regulator (CFTR) protein or a biologically active truncated CFTR protein lacking amino acids 708-759 of the human CFTR protein sequence (d) a polyadenylation sequence and (e) an AAV2 terminal repeat.

[0013] In other preferred aspects, a primate (e.g., human) in need of treatment for cystic fibrosis is administered an amount of a pharmaceutical composition comprising an rAAV comprising a capsid comprising a capsid protein of SEQ ID NO: 12 or comprising an amino acid sequence at least 90% identical thereto and a nucleic acid encoding a CFTR or biologically active fragment thereof, that is effective to achieve a delivered dose of about 5x1013vector genomes (vg) to about 4.7x1014vg, preferably about 6x1013vg, about 1.2x1014vg or about 2.3x1014vg, to the primate, wherein the pharmaceutical composition is administered to the primate by a nebulizer, e.g. a breath-actuated or vibrating mesh nebulizer.

[0014] In related embodiments, the nucleotide sequence encoding human CFTR or a biologically active portion thereof encodes a native human CFTR protein and has the following sequence or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical thereto:ATGCAGAGGTCGCCTCTGGAAAAGGCCAGCGTTGTCTCCAAACTTTTTTTCAGCT GGACCAGACCAATTTTGAGGAAAGGATACAGACAGCGCCTGGAATTGTCAGACA TATACCAAATCCCTTCTGTTGATTCTGCTGACAATCTATCTGAAAAATTGGAAAG AGAATGGGATAGAGAGCTGGCTTCAAAGAAAAATCCTAAACTCATTAATGCCCTTCGGCGATGTTTTTTCTGGAGATTTATGTTCTATGGAATCTTTTTATATTTAGGGGAAGTCACCAAAGCAGTACAGCCTCTCTTACTGGGAAGAATCATAGCTTCCTATGACCCGGATAACAAGGAGGAACGCTCTATCGCGATTTATCTAGGCATAGGCTTATGCCTTCTCTTTATTGTGAGGACACTGCTCCTACACCCAGCCATTTTTGGCCTTCATCACATTGGAATGCAGATGAGAATAGCTATGTTTAGTTTGATTTATAAGAAGACTTTAAAGCTGTCAAGCCGTGTTCTAGATAAAATAAGTATTGGACAACTTGTTAGTCTCCTTTCCAACAACCTGAACAAATTTGATGAAGGACTTGCATTGGCACATTTCGTGTGGATCGCTCCTTTGCAAGTGGCACTCCTCATGGGGCTAATCTGGGAGTTGTTACAGGCGTCTGCCTTCTGTGGACTTGGTTTCCTGATAGTCCTTGCCCTTTTTCAGGCTGGGCTAGGGAGAATGATGATGAAGTACAGAGATCAGAGAGCTGGGAAGATCAGTGAAAGACTTGTGATTACCTCAGAAATGATTGAAAATATCCAATCTGTTAAGGCATACTGCTGGGAAGAAGCAATGGAAAAAATGATTGAAAACTTAAGACAAACAGAACTGAAACTGACTCGGAAGGCAGCCTATGTGAGATACTTCAATAGCTCAGCCTTCTTCTTCTCAGGGTTCTTTGTGGTGTTTTTATCTGTGCTTCCCTATGCACTAATCAAAGGAATCATCCTCCGGAAAATATTCACCACCATCTCATTCTGCATTGTTCTGCGCATGGCGGTCACTCGGCAATTTCCCTGGGCTGTACAAACATGGTATGACTCTCTTGGAGCAATAAACAAAATACAGGATTTCTTACAAAAGCAAGAATATAAGACATTGGAATATAACTTAACGACTACAGAAGTAGTGATGGAGAATGTAACAGCCTTCTGGGAGGAGGGATTTGGGGAATTATTTGAGAAAGCAAAACAAAACAATAACAATAGAAAAACTTCTAATGGTGATGACAGCCTCTTCTTCAGTAATTTCTCACTTCTTGGTACTCCTGTCCTGAAAGATATTAATTTCAAGATAGAAAGAGGACAGTTGTTGGCGGTTGCTGGATCCACTGGAGCAGGCAAGACTTCACTTCTAATGGTGATTATGGGAGAACTGGAGCCTTCAGAGGGTAAAATTAAGCACAGTGGAAGAATTTCATTCTGTTCTCAGTTTTCCTGGATTATGCCTGGCACCATTAAAGAAAATATCATCTTTGGTGTTTCCTATGATGAATATAGATACAGAAGCGTCATCAAAGCATGCCAACTAGAAGAGGACATCTCCAAGTTTGCAGAGAAAGACAATATAGTTCTTGGAGAAGGTGGAATCACACTGAGTGGAGGTCAACGAGCAAGAATTTCTTTAGCAAGAGCAGTATACAAAGATGCTGATTTGTATTTATTAGACTCTCCTTTTGGATACCTAGATGTTTTAACAGAAAAAGAAATATTTGAAAGCTGTGTCTGTAAACTGATGGCTAACAAAACTAGGATTTTGGTCACTTCTAAAATGGAACATTTAAAGAAAGCTGACAAAATATTAATTTTGCATGAAGGTAGCAGCTATTTTTATGGGACATTTTCAGAACTCCAAAATCTACAGCCAGACTTTAGCTCAAAACTCATGGGATGTGATTCTTTCGACCAATTTAGTGCAGAAAGAAGAAATTCAATCCTAACTGAGACCTTACACCGTTTCTCATTAGAAGGAGATGCTCCTGTCTCCTGGACAGAAACAAAAAAACAATCTTTTAAACAGACTGGAGAGTTTGGGGAAAAAAGGAAGAATTCTATTCTCAATCCAATCAACTCTATACGAAAATTTTCCATTGTGCAAAAGACTCCCTTACAAATGAATGGCATCGAAGAGGATTCTGATGAGCCTTTAGAGAGAAGGCTGTCCTTAGTACCAGATTCTGAGCAGGGAGAGGCGATACTGCCTCGCATCAGCGTGATCAGCACTGGCCCCACGCTTCAGGCACGAAGGAGGCAGTCTGTCCTGAACCTGATGACACACTCAGTTAACCAAGGTCAGAACATTCACCGAAAGACAACAGCATCCACACGAAAAGTGTCACTGGCCCCTCAGGCAAACTTGACTGAACTGGATATATATTCAAGAAGGTTATCTCAAGAAACTGGCTTGGAAATAAGTGAAGAAATTAACGAAGAAGACTTAAAGGAGTGCTTTTTTGATGATATGGAGAGCATACCAGCAGTGACTACATGGAACACATACCTTCGATATATTACTGTCCACAAGAGCTTAATTTTTGTGCTAATTTGGTGCTTAGTAATTTTTCTGGCAGAGGTGGCTGCTTCTTTGGTTGTGCTGTGGCTCCTTGGAAACACTCCTCTTCAAGACAAAGGGAATAGTACTCATAGTAGAAATAACAGCTATGCAGTGATTATCACCAGCACCAGTTCGTATTATGTGTTTTACATTTACGTGGGAGTAGCCGACACTTTGCTTGCTATGGGATTCTTCAGAGGTCTACCACTGGTGCATACTCTAATCACAGTGTCGAAAATTTTACACCACAAAATGTTACATTCTGTTCTTCAAGCACCTATGTCAACCCTCAACACGTTGAAAGCAGGTGGGATTCTTAATAGATTCTCCAAAGATATAGCAATTTTGGATGACCTTCTGCCTCTTACCATATTTGACTTCATCCAGTTGTTATTAATTGTGATTGGAGCTATAGCAGTTGTCGCAGTTTTACAACCCTACATCTTTGTTGCAACAGTGCCAGTGATAGTGGCTTTTATTATGTTGAGAGCATATTTCCTCCAAACCTCACAGCAACTCAAACAACTGGAATCTGAAGGCAGGAGTCCAATTTTCACTCATCTTGTTACAAGCTTAAAAGGACTATGGACACTTCGTGCCTTCGGACGGCAGCCTTACTTTGAAACTCTGTTCCACAAAGCTCTGAATTTACATACTGCCAACTGGTTCTTGTACCTGTCAACACTGCGCTGGTTCCAAATGAGAATAGAAATGATrTTTGTCATCTTCTTCATTGCTGTTACCTTCATTTCCATTnAACAACAGGAGAAGGAGAAGGAAGAGTTGGTATTATCCTGACTTTAGCCATGAATATCATGAGTACATTGCAGTGGGCTGTAAACTCCAGCATAGATGTGGATAGCTTGATGCGATCTGTGAGCCGAGTCTTTAAGTTCATTGACATGCCAACAGAAGGTAAACCTACCAAGTCAACCAAACCATACAAGAATGGCCAACTCTCGAAAGTTATGATTATTGAGAATTCACACGTGAAGAAAGATGACATCTGGCCCTCAGGGGGCCAAATGACTGTCAAAGATCTCACAGCAAAATACACAGAAGGTGGAAATGCCATATTAGAGAACATTTCCTTCTCAATAAGTCCTGGCCAGAGGGTGGGCCTCTTGGGAAGAACTGGATCAGGGAAGAGTACTTTGTTATCAGCTTTTTTGAGACTACTGAACACTGAAGGAGAAATCCAGATCGATGGTGTGTCTTGGGATTCAATAACTTTGCAACAGTGGAGGAAAGCCTTTGGAGTGATACCACAGAAAGTATTTATTTTTTCTGGAACATTTAGAAAAAACTTGGATCCCTATGAACAGTGGAGTGATCAAGAAATATGGAAAGTTGCAGATGAGGTTGGGCTCAGATCTGTGATAGAACAGTTTCCTGGGAAGCTTGACTTTGTCCTTGTGGATGGGGGCTGTGTCCTAAGCCATGGCCACAAGCAGTTGATGTGCTTGGCTAGATCTGTTCTCAGTAAGGCGAAGATCTTGCTGCTTGATGAACCCAGTGCTCATTTGGATCCAGTAACATACCAAATAATTAGAAGAACTCTAAAACAAGCATTTGCTGATTGCACAGTAATTCTCTGTGAACACAGGATAGAAGCAATGCTGGAATGCCAACAATTTTTGGTCATAGAAGAGAACAAAGTGCGGCAGTACGATTCCATCCAGAAACTGCTGAACGAGAGGAGCCTCTTCCGGCAAGCCATCAGCCCCTCCGACAGGGTGAA GCTCTTTCCCCACCGGAACTCAAGCAAGTGCAAGTCTAAGCCCCAGATTGCTGCT CTGAAAGAGGAGACAGAAGAAGAGGTGCAAGATACAAGGCTTTAG (SEQ ID NO:42).

[0015] In preferred embodiments, the nucleotide sequence encoding human CFTR or a biologically activated truncated CFTR protein comprises the following nucleotide sequence or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical thereto:ATGCAGCGCAGCCCACTGGAGAAGGCAAGCGTGGTGTCCAAGCTGTTCTTTTCCT GGACCAGGCCTATCCTGAGGAAGGGATACAGGCAGCGGCTGGAGCTGAGCGACA TCTATCAGATCCCTTCTGTGGACAGCGCCGATAATCTGTCCGAGAAGCTGGAGAG AGAGTGGGATAGGGAGCTGGCCTCTAAGAAGAACCCAAAGCTGATCAATGCCCTGCGGAGATGCTTCTTTTGGCGGTTCATGTTCTACGGCATCTTCCTGTATCTGGGCG AGGTGACCAAGGCCGTGCAGCCACTGCTGCTGGGCAGAATCATCGCCTCTTACGA CCCCGATAACAAGGAGGAGAGGAGCATCGCCATCTATCTGGGCATCGGCCTGTG CCTGCTGTTTATCGTGAGGACACTGCTGCTGCACCCAGCCATCTTCGGCCTGCACC ACATCGGCATGCAGATGAGAATCGCCATGTTCAGCCTGATCTACAAGAAGACCCT GAAGCTGAGCTCCAGGGTGCTGGACAAGATCTCCATCGGCCAGCTGGTGTCCCTG CTGTCTAACAATCTGAACAAGTTTGATGAGGGACTGGCCCTGGCACACTTCGTGT GGATCGCACCACTGCAGGTGGCCCTGCTGATGGGCCTGATCTGGGAGCTGCTGCA GGCAAGCGCCTTTTGCGGACTGGGCTTCCTGATCGTGCTGGCCCTGTTCCAGGCA GGACTGGGACGCATGATGATGAAGTACAGAGACCAGAGGGCCGGCAAGATCTCT GAGCGGCTGGTCATCACCAGCGAGATGATCGAGAACATCCAGTCCGTGAAGGCC TATTGTTGGGAGGAGGCCATGGAGAAGATGATCGAGAATCTGCGCCAGACAGAG CTGAAGCTGACCAGAAAGGCCGCCTACGTGAGGTACTTCAACTCTAGCGCCTTCT TTT1CTCTGGCTTTTTCGTGGTGTTCCTGAGCGTGCTGCCATACGCCCTGATCAAG GGCATCATCCTGCGGAAGATCTTTACCACAATCTCCTTCTGCATCGTGCTGAGAA TGGCCGTGACAAGGCAGTTTCCCTGGGCCGTGCAGACCTGGTATGACTCTCTGGG CGCCATCAATAAGATCCAGGATTTCCTGCAGAAGCAGGAGTACAAGACACTGGA GTATAACCTGACCACAACCGAGGTGGTCATGGAGAATGTGACCGCCTTCTGGGA GGAGGGCTTTGGCGAGCTGTTCGAGAAGGCCAAGCAGAACAATAACAATCGCAA GACATCTAACGGCGACGATAGCCTGTTTTTCAGCAATTTTTCCCTGCTGGGCACCC CCGTGCTGAAGGACATCAACTTCAAGATCGAGAGGGGACAGCTGCTGGCAGTGG CAGGCTCCACAGGCGCCGGCAAGACCTCTCTGCTGATGATGATCATGGGCGAGCTGGAGCCAAGCGAGGGCAAGATCAAGCACTCCGGCCGGATCTCTTTTTGCAGCCA GTTCTCCTGGATCATGCCCGGCACCATCAAGGAGAATATCATCTTTGGCGTGTCCTACGATGAGTACAGATATAGGTCTGTGATCAAGGCCTGTCAGCTGGAGGAGGACATCAGCAAGTTCGCCGAGAAGGATAACATCGTGCTGGGCGAGGGCGGCATCACACTGAGCGGAGGACAGAGGGCAAGGATCTCCCTGGCCAGAGCCGTGTACAAGGACGCCGATCTGTATCTGCTGGACAGCCCCTTTGGCTATCTGGATGTGCTGACCGAGAAGGAGATCTTCGAGTCCTGCGTGTGCAAGCTGATGGCCAATAAGACAAGGATCCTGGTGACCTCTAAGATGGAGCACCTGAAGAAGGCCGACAAGATCCTGATCCTGCACGAGGGCTCCTCTTACTTTTATGGCACATTCAGCGAGCTGCAGAATCTGCAGCCTGACTTCAGCTCCAAGCTGATGGGCTGTGACTCCTTTGATCAGTTCTCTGCCGAGAGGCGCAACTCCATCCTGACAGAGACCCTGCACAGATTCTCTCTGGAGGGCGACGCACCCGTGAGCTGGACAGAGACCAAGAAGCAGTCCTTTAAGCAGACCGGCGAGTTCGGCGAGAAGAGGAAGAATTCTATCCTGAACCCTATCAATAGCACACTGCAGGCCCGGAGAAGGCAGTCTGTGCTGAACCTGATGACCCACAGCGTGAACCAGGGCCAGAATATCCACAGAAAGACAACCGCCAGCACAAGGAAGGTGTCCCTGGCACCTCAGGCAAACCTGACCGAGCTGGACATCTACTCCCGCCGGCTGTCTCAGGAGACCGGACTGGAGATCTCTGAGGAGATCAATGAGGAGGATCTGAAGGAGTGCTTTTTCGACGATATGGAGAGCATCCCAGCCGTGACAACCTGGAACACATACCTGCGCTATATCACCGTGCACAAGTCCCTGATCTTTGTGCTGATCTGGTGTCTGGTCATCTTCCTGGCAGAGGTGGCAGCATCTCTGGTGGTGCTGTGGCTGCTGGGCAACACACCCCTGCAGGACAAGGGCAATTCTACCCACAGCCGCAACAATTCCTACGCCGTGATCATCACATCTACCTCTAGCTACTACGTGTTCTACATCTATGTGGGCGTGGCCGATACACTGCTGGCCATGGGCTTTTTCCGGGGCCTGCCCCTGGTGCACACACTGATCACCGTGAGCAAGATCCTGCACCACAAGATGCTGCACAGCGTGCTGCAGGCCCCTATGTCCACACTGAACACCCTGAAGGCCGGCGGCATCCTGAATCGGTTTTCCAAGGACATCGCCATCCTGGACGATCTGCTGCCTCTGACCATCTTTGATTTCATCCAGCTGCTGCTGATCGTGATCGGAGCAATCGCAGTGGTGGCCGTGCTGCAGCCTTACATCTTCGTGGCCACAGTGCCAGTGATCGTGGCCTTTATCATGCTGCGCGCCTATTTCCTGCAGACCAGCCAGCAGCTGAAGCAGCTGGAGAGCGAGGGCCGGTCCCCTATCTTTACACACCTGGTGACCTCCCTGAAGGGACTGTGGACACTGAGGGCCTTCGGCCGGCAGCCATACTTTGAGACCCTGTTCCACAAGGCCCTGAACCTGCACACAGCCAATTGGTTTCTGTATCTGAGCACCCTGCGCTGGTTTCAGATGCGGATCGAGATGATCTTCGTGATCTTTTTCATCGCCGTGACCTTCATCTCCATCCTGACAACCGGAGAGGGAGAGGGAAGAGTGGGAATCATCCTGACACTGGCCATGAACATCATGTCTACCCTGCAGTGGGCCGTGAATTCCTCTATCGACGTGGATAGCCTGATGAGATCTGTGAGCAGGGTGTTTAAGTTCATCGACATGCCCACAGAGGGCAAGCCTACAAAGAGCACCAAGCCATACAAGAACGGCCAGCTGTCCAAAGTGATGATCATCGAGAATTCTCACGTGAAGAAGGACGATATCTGGCCATCCGGAGGACAGATGACCGTGAAGGATCTGACAGCCAAGTATACCGAGGGCGGCAACGCCATCCTGGAGAATATCTCCTTTTCTATCAGCCCTGGACAGAGGGTGGGACTGCTGGGACGGACAGGCTCCGGCAAGTCTACCCTGCTGAGCGCCTT CCTGAGGCTGCTGAATACAGAGGGCGAGATCCAGATCGACGGCGTGAGCTGGGA TTCCATCACCCTGCAGCAGTGGAGAAAGGCCTTTGGCGTGATCCCTCAGAAGGTG TTTATCTTCTCCGGCACCTTCAGGAAGAACCTGGACCCATACGAGCAGTGGTCTG ATCAGGAGATCTGGAAGGTGGCCGACGAAGTGGGCCTGAGATCTGTGATCGAGC AGTTTCCAGGCAAGCTGGACTTCGTGCTGGTGGATGGAGGATGCGTGCTGAGCCA CGGACACAAGCAGCTGATGTGCCTGGCCAGGTCTGTGCTGAGCAAGGCCAAGAT CCTGCTGCTGGACGAGCCAAGCGCCCACCTGGATCCCGTGACATACCAGATCATC AGAAGGACCCTGAAGCAGGCCTTTGCCGATTGCACCGTGATCCTGTGCGAGCACC GCATCGAGGCCATGCTGGAGTGCCAGCAGTTCCTGGTCATCGAGGAGAACAAGG TGCGGCAGTATGACAGCATCCAGAAGCTGCTGAATGAGCGGAGCCTGTTTCGGC AGGCCATCTCCCCCTCTGATCGCGTGAAGCTGTTCCCTCACCGGAACAGCTCCAA GTGTAAGTCCAAGCCCCAGATCGCCGCCCTGAAGGAGGAGACAGAGGAGGAGGT GCAGGACACCAGACTGTGA (SEQ IDNO:43)

[0016] SEQ ID NO:43 is a nucleotide sequence that has been codon optimized for expression in humans and encodes a biologically active truncated human CFTR protein lacking amino acids 708-759.

[0017] In some aspects, the promoter is a constitutive promoter, optionally a truncated cytomegalovirus immediate / early (CMVie) enhancer / promoter and is operably linked to the nucleotide sequence encoding the human CFTR or biologically active portion thereof.

[0018] In other aspects, the promoter is a tissue specific promoter, preferably wherein the promoter directs preferential expression of the nucleic acid in a lung cell, and is operably linked to the nucleotide sequence encoding the human CFTR or biologically active portion thereof.

[0019] In preferred embodiments, the promoter is a truncated CMVie promoter and is operably linked to the nucleotide sequence encoding human CFTR or a biologically active portion thereof. In a particularly preferred embodiment, the CMVie promoter is CMV173 having the following sequence or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto:ACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTG GCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACG CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTA GTGAACCGT (SEQ ID NO:44)

[0020] In a preferred embodiment, the pharmaceutical composition of the combination comprises an rAAV vector, said rAAV vector comprising (i) a capsid comprising a capsidprotein of SEQ ID NO: 12 and (ii) a nucleic acid comprising from 5' to 3': (a) an AAV2 terminal repeat (b) a CMV173 promoter of SEQ ID NO:44 or sequence at least 80% identical thereto (c) a nucleotide sequence encoding a human cystic fibrosis transmembrane conductance regulator (CFTR) protein or a biologically active truncated CFTR protein lacking amino acids 708-759 of the human CFTR protein sequence of SEQ IDNO:43 or sequence at least 80% identical thereto (d) a polyadenylation sequence and (e) an AAV2 terminal repeat.

[0021] In a particularly preferred embodiment, the pharmaceutical composition of the combination comprises an rAAV vector, said rAAV vector comprising (i) a capsid comprising a capsid protein of SEQ ID NO: 12 and (ii) a nucleic acid comprising from 5* to 3' the following nucleotide sequence (SEQ ID NO:45) or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical thereto:TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGG TCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCA GAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACTCACGGGG ATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATC AACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGG TAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAG AATTCTCGAGTGATCGAAAGAGCCTGCTAAAGCAAAAAAGAAGTCACCATGCAG CGCAGCCCACTGGAGAAGGCAAGCGTGGTGTCCAAGCTGTTCTTTTCCTGGACCA GGCCTATCCTGAGGAAGGGATACAGGCAGCGGCTGGAGCTGAGCGACATCTATC AGATCCCTTCTGTGGACAGCGCCGATAATCTGTCCGAGAAGCTGGAGAGAGAGT GGGATAGGGAGCTGGCCTCTAAGAAGAACCCAAAGCTGATCAATGCCCTGCGGA GATGCTTCnTTGGCGGTTCATGTTCTACGGCATCTTCCTGTATCTGGGCGAGGTG ACCAAGGCCGTGCAGCCACTGCTGCTGGGCAGAATCATCGCCTCTTACGACCCCG ATAACAAGGAGGAGAGGAGCATCGCCATCTATCTGGGCATCGGCCTGTGCCTGCT GTTTATCGTGAGGACACTGCTGCTGCACCCAGCCATCTTCGGCCTGCACCACATC GGCATGCAGATGAGAATCGCCATGTTCAGCCTGATCTACAAGAAGACCCTGAAG CTGAGCTCCAGGGTGCTGGACAAGATCTCCATCGGCCAGCTGGTGTCCCTGCTGT CTAACAATCTGAACAAGTTTGATGAGGGACTGGCCCTGGCACACTTCGTGTGGAT CGCACCACTGCAGGTGGCCCTGCTGATGGGCCTGATCTGGGAGCTGCTGCAGGCA AGCGCCTTTTGCGGACTGGGCTTCCTGATCGTGCTGGCCCTGTTCCAGGCAGGAC TGGGACGCATGATGATGAAGTACAGAGACCAGAGGGCCGGCAAGATCTCTGAGC GGCTGGTCATCACCAGCGAGATGATCGAGAACATCCAGTCCGTGAAGGCCTATTG TTGGGAGGAGGCCATGGAGAAGATGATCGAGAATCTGCGCCAGACAGAGCTGAA GCTGACCAGAAAGGCCGCCTACGTGAGGTACTTCAACTCTAGCGCCTTCTTmCTCTGGCTTTTTCGTGGTGTTCCTGAGCGTGCTGCCATACGCCCTGATCAAGGGCATCATCCTGCGGAAGATCTTTACCACAATCTCCTTCTGCATCGTGCTGAGAATGGCCGTGACAAGGCAGTTTCCCTGGGCCGTGCAGACCTGGTATGACTCTCTGGGCGCCATCAATAAGATCCAGGATTTCCTGCAGAAGCAGGAGTACAAGACACTGGAGTATAACCTGACCACAACCGAGGTGGTCATGGAGAATGTGACCGCCTTCTGGGAGGAGGGCTTTGGCGAGCTGTTCGAGAAGGCCAAGCAGAACAATAACAATCGCAAGACATCTAACGGCGACGATAGCCTGTTTTTCAGCAATTTTTCCCTGCTGGGCACCCCCGTGCTGAAGGACATCAACTTCAAGATCGAGAGGGGACAGCTGCTGGCAGTGGCAGGCTCCACAGGCGCCGGCAAGACCTCTCTGCTGATGATGATCATGGGCGAGCTGGAGCCAAGCGAGGGCAAGATCAAGCACTCCGGCCGGATCTCTTTTTGCAGCCAGTTCTCCTGGATCATGCCCGGCACCATCAAGGAGAATATCATCTTTGGCGTGTCCTACGATGAGTACAGATATAGGTCTGTGATCAAGGCCTGTCAGCTGGAGGAGGACATCAGCAAGTTCGCCGAGAAGGATAACATCGTGCTGGGCGAGGGCGGCATCACACTGAGCGGAGGACAGAGGGCAAGGATCTCCCTGGCCAGAGCCGTGTACAAGGACGCCGATCTGTATCTGCTGGACAGCCCCTTTGGCTATCTGGATGTGCTGACCGAGAAGGAGATCTTCGAGTCCTGCGTGTGCAAGCTGATGGCCAATAAGACAAGGATCCTGGTGACCTCTAAGATGGAGCACCTGAAGAAGGCCGACAAGATCCTGATCCTGCACGAGGGCTCCTCTTACTTT1ATGGCACATTCAGCGAGCTGCAGAATCTGCAGCCTGACTTCAGCTCCAAGCTGATGGGCTGTGACTCCTTTGATCAGTTCTCTGCCGAGAGGCGCAACTCCATCCTGACAGAGACCCTGCACAGATTCTCTCTGGAGGGCGACGCACCCGTGAGCTGGACAGAGACCAAGAAGCAGTCCTTTAAGCAGACCGGCGAGTTCGGCGAGAAGAGGAAGAATTCTATCCTGAACCCTATCAATAGCACACTGCAGGCCCGGAGAAGGCAGTCTGTGCTGAACCTGATGACCCACAGCGTGAACCAGGGCCAGAATATCCACAGAAAGACAACCGCCAGCACAAGGAAGGTGTCCCTGGCACCTCAGGCAAACCTGACCGAGCTGGACATCTACTCCCGCCGGCTGTCTCAGGAGACCGGACTGGAGATCTCTGAGGAGATCAATGAGGAGGATCTGAAGGAGTGCTTTTTCGACGATATGGAGAGCATCCCAGCCGTGACAACCTGGAACACATACCTGCGCTATATCACCGTGCACAAGTCCCTGATCTTTGTGCTGATCTGGTGTCTGGTCATCTTCCTGGCAGAGGTGGCAGCATCTCTGGTGGTGCTGTGGCTGCTGGGCAACACACCCCTGCAGGACAAGGGCAATTCTACCCACAGCCGCAACAATTCCTACGCCGTGATCATCACATCTACCTCTAGCTACTACGTGTTCTACATCTATGTGGGCGTGGCCGATACACTGCTGGCCATGGGCl 111 rCCGGGGCCTGCCCCTGGTGCACACACTGATCACCGTGAGCAAGATCCTGCACCACAAGATGCTGCACAGCGTGCTGCAGGCCCCTATGTCCACACTGAACACCCTGAAGGCCGGCGGCATCCTGAATCGGTTTTCCAAGGACATCGCCATCCTGGACGATCTGCTGCCTCTGACCATCTTTGATTTCATCCAGCTGCTGCTGATCGTGATCGGAGCAATCGCAGTGGTGGCCGTGCTGCAGCCTTACATCTTCGTGGCCACAGTGCCAGTGATCGTGGCCTTTATCATGCTGCGCGCCTATTTCCTGCAGACCAGCCAGCAGCTGAAGCAGCTGGAGAGCGAGGGCCGGTCCCCTATCTTTACACACCTGGTGACCTCCC TGAAGGGACTGTGGACACTGAGGGCCTTCGGCCGGCAGCCATACTTTGAGACCCT GTTCCACAAGGCCCTGAACCTGCACACAGCCAATTGGTTTCTGTATCTGAGCACC CTGCGCTGGTTTCAGATGCGGATCGAGATGATCTTCGTGATCTTTTTCATCGCCGT GACCTTCATCTCCATCCTGACAACCGGAGAGGGAGAGGGAAGAGTGGGAATCAT CCTGACACTGGCCATGAACATCATGTCTACCCTGCAGTGGGCCGTGAATTCCTCT ATCGACGTGGATAGCCTGATGAGATCTGTGAGCAGGGTGTTTAAGTTCATCGACA TGCCCACAGAGGGCAAGCCTACAAAGAGCACCAAGCCATACAAGAACGGCCAGC TGTCCAAAGTGATGATCATCGAGAATTCTCACGTGAAGAAGGACGATATCTGGCC ATCCGGAGGACAGATGACCGTGAAGGATCTGACAGCCAAGTATACCGAGGGCGG CAACGCCATCCTGGAGAATATCTCCTTTTCTATCAGCCCTGGACAGAGGGTGGGA CTGCTGGGACGGACAGGCTCCGGCAAGTCTACCCTGCTGAGCGCCTTCCTGAGGCTGCTGAATACAGAGGGCGAGATCCAGATCGACGGCGTGAGCTGGGATTCCATCA CCCTGCAGCAGTGGAGAAAGGCCTTTGGCGTGATCCCTCAGAAGGTGTTTATCTT CTCCGGCACCTTCAGGAAGAACCTGGACCCATACGAGCAGTGGTCTGATCAGGA GATCTGGAAGGTGGCCGACGAAGTGGGCCTGAGATCTGTGATCGAGCAGTTTCC AGGCAAGCTGGACTTCGTGCTGGTGGATGGAGGATGCGTGCTGAGCCACGGACA CAAGCAGCTGATGTGCCTGGCCAGGTCTGTGCTGAGCAAGGCCAAGATCCTGCTG CTGGACGAGCCAAGCGCCCACCTGGATCCCGTGACATACCAGATCATCAGAAGG ACCCTGAAGCAGGCCTTTGCCGATTGCACCGTGATCCTGTGCGAGCACCGCATCGAGGCCATGCTGGAGTGCCAGCAGTTCCTGGTCATCGAGGAGAACAAGGTGCGGC AGTATGACAGCATCCAGAAGCTGCTGAATGAGCGGAGCCTGTTTCGGCAGGCCA TCTCCCCCTCTGATCGCGTGAAGCTGTTCCCTCACCGGAACAGCTCCAAGTGTAA GTCCAAGCCCCAGATCGCCGCCCTGAAGGAGGAGACAGAGGAGGAGGTGCAGG ACACCAGACTGTGAAATAAAACATCTTTATTTTCATTACATCTGTGTGTTGGTTTT TTGTGTGAACAACGGCCGGCCGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCC TCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGG CCAA (SEQ ID NO: 45).

[0022] In a particularly preferred embodiment, a primate (e.g., human) in need of treatment for cystic fibrosis is administered an amount of a pharmaceutical composition comprising an rAAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO: 12 and (ii) a nucleic acid comprising from 5' to 3' the nucleotide sequence set forth as SEQ ID NO:45 or a sequence at least 80%, identical thereto, that is effective to achieve a delivered dose of about 5x1013vg to about 4.7x1014vg, preferably about 6x1013vg, about 1 ,2x1014vg or about 2.3x1014vg, to the primate, wherein the primate is also administered one or moreCFTR modulators. Preferably, the pharmaceutical composition and the one or more CFTR modulators are administered to the primate such that the therapeutic effect of the CFTR modulator and the CFTR gene product should overlap for at least a portion of the duration, so that the overall therapeutic effect of the combination therapy is attributable in part to the combined, additive, enhanced or synergistic effects of the combination therapy.

[0023] In some embodiments, the one or more CFTR modulators of the combination comprise one or more CFTR modulators selected from the group consisting of ivacaftor, lumacaftor, tezacaftor, elexacaftor and a combination thereof. In preferred embodiments, the one or more CFTR modulators comprises ivacaftor. In some aspects, the one or more CFTR modulators comprises Kalydeco® (ivacaftor), Orkambi® (lumacaftor / ivacaftor), Symdeko® (tezacaftor / ivacaftor) and / or Trikafta® (elexacaftor / tezacaftor / ivacaftor). In other embodiments, the one or more CFTR modulators comprises Alyftrek® (vanzacaftor / tezacaftor / deutivacaftor).

[0024] In some preferred aspects, the use of a pharmaceutical combination comprising (A) a pharmaceutical composition comprising an rAAV vector, said rAAV vector comprising a capsid comprising a capsid protein comprising the amino acid sequence as set forth as SEQ ID NO: 12 or an amino acid sequence at least 90% identical to SEQ ID NO: 12 and comprising a Thr at amino acid 469 and an Ala at amino acid 598 based on the amino acid numbering set forth in SEQ ID NO: 12 and a heterologous nucleic acid comprising a nucleotide sequence encoding a cystic fibrosis transmembrane conductance regulator (CFTR protein) gene product operably linked to a promoter and (B) ivacaftor and optionally one or more additional CFTR modulators (e.g., Trikafta®), for the treatment of cystic fibrosis is provided.

[0025] In other preferred aspects, the use of a pharmaceutical combination comprising (A) a pharmaceutical composition comprising an rAAV vector, said rAAV vector comprising a capsid comprising a capsid protein comprising the amino acid sequence as set forth as SEQ ID NO: 12 or an amino acid sequence at least 90% identical to SEQ ID NO: 12 and comprising a Thr at amino acid 469 and an Ala at amino acid 598 based on the amino acid numbering set forth in SEQ ID NO: 12 and a heterologous nucleic acid comprising a nucleotide sequence encoding a cystic fibrosis transmembrane conductance regulator (CFTR protein) gene product operably linked to a promoter and (B) tezacaftor and optionally one or more additional CFTR modulators (e.g., Alyftrek®), for the treatment of cystic fibrosis is provided.

[0026] The pharmaceutical composition and the CFTR modulator may be administered simultaneously or sequentially to a subject with CFTR. The pharmaceutical composition may be administered by inhalation; and / or the CFTR modulator may be administered orally.

[0027] The pharmaceutical composition may be administered as a single dose or as multipledoses. In some aspects, the method of treating cystic fibrosis comprises a single administration of the unit dose of the pharmaceutical composition to the primate (e.g., a unit dose effective to achieve a total clinical delivered dose of about 5x1013vg to about 4.7x1014vg, preferably about 6x1013vg, about 1.2x1014vg or about 2.3x1014vg to the primate) with no subsequent administrations for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or at least 12 months.

[0028] . The pharmaceutical composition may be administered multiple times as a unit dose with an interval of less than or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or less than or about 12 months between administrations. The pharmaceutical composition may be administered to the subject about once per month, about once every three months, about once every six months, about once per year, about once per two years, about once per three years, about once per for years, or about once per five years.

[0029] In some aspects, the CFTR modulator is administered at a concentration used for monotherapy of each modulator or lower. In related aspects, the at least one CFTR modulator is administered at least once per day, preferably about evety twelve hours. In preferred embodiments, the CFTR modulator is administered orally to the subject.

[0030] In some embodiments, a combination therapy for treating CF as herein described further comprises administration of a corticosteroid to the subject before, simultaneous with, or after administration of the pharmaceutical composition (the pharmaceutical composition preferably administered in an amount to achieve a total clinical delivered dose of about 5x1013vg to about 4.7x1014vg, preferably about 6x1013vg, about 1.2x1014vg or about 2.3x1014vg) gof an rAAV comprising a capsid comprising a capsid protein of SEQ ID NO: 12 or comprising an amino acid sequence at least 90% identical thereto and a nucleic acid encoding a CFTR or biologically active fragment thereof). In some embodiments, the pharmaceutical composition and the corticosteroid may be administered simultaneously. In some embodiments, the pharmaceutical composition and the corticosteroid may be administered sequentially. In some embodiments, the pharmaceutical composition and the corticosteroid may be administered separately. In some embodiments, the corticosteroid is selected from prednisolone, dexamethasone, hydrocortisone, methylprednisolone, betamethasone, cortisone, budesonide, and triamcinolone. In some embodiments, the corticosteroid is prednisolone. In some embodiments, the corticosteroid is prednisone or a corticosteroids equivalent to prednisone.

[0031] In some embodiments, the corticosteroid is administered at a dose of about 10 mg per day to about 100 mg per day. In some embodiments, the corticosteroid is administered at a dose of about 10 mg per day to about 60 mg per day. In some embodiments, the corticosteroid is administered at a dose of about 10 mg per day, about 20 mg per day, about 30 mg per day and / or about 40 mg per day. In some embodiments, the corticosteroid is administered once per day according to a tapered schedule in which the dose of corticosteroid decreases over the administration period. In some aspects, the dose of corticosteroid is reduced eveiy week (e.g., a reduction of about 10 mg every week).

[0032] In some preferred embodiments, a first dose of the corticosteroid is administered to the primate about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day prior to administering the pharmaceutical composition. In some embodiments, a first dose of prednisolone is administered at about 1 day prior to administration of the pharmaceutical composition. In some embodiments, the corticosteroid is administered once per day for an administration period comprising at least one, at least two, at least three, or at least four weeks. In some preferred embodiments, the prednisone or a corticosteroids equivalent to prednisone is administered once per day for an administration period of about four weeks, wherein the first dose of prednisone or a corticosteroids equivalent to prednisone is administered about 1 day prior to administration of the pharmaceutical composition. In some particularly preferred embodiments, prednisone or a corticosteroids equivalent to prednisone is administered according to the following schedule (QD - one a day):40 mg prednisone or a corticosteroids equivalent to 40 mg prednisone QD *7 days, followed by30 mg prednisone or a corticosteroids equivalent to 30 mg prednisone QD *7 days, followed by20 mg prednisone or a corticosteroids equivalent to 20 mg prednisone QD *7 days, followed by10 mg prednisone or a corticosteroids equivalent to 10 mg prednisone QD x7 days

[0033] In some aspects, the subject need of treatment for cystic fibrosis is a primate. In preferred aspects, the primate is a human. In some aspects, the non-human primate or human in need of treatment has pre-existing antibodies to an rAAV comprising a capsid protein of SEQ ID NO:12.

[0034] In related aspects, a subject in need of treatment for cystic fibrosis demonstrates an improvement in lung function at least 4, 5, 6, 7, 8, 9, 10, 11 or 12 months following acombination therapy as herein described relative to a pre-administration baseline, preferably wherein lung function is measured by improved percent predicted forced expiratory volume (ppFEV). In other aspects, the subject demonstrates an improvement in one or more respiratory symptoms at least 4, 5, 6, 7, 8, 9, 10, 11 or 12 months following administration of the pharmaceutical combination relative to a pre-administration baseline, preferably as measured by the Cystic Fibrosis Questionnaire-Revised (CFQ-R).

[0035] In other aspects, a method for delivering a heterologous nucleic acid to a lung cell in a primate is provided, the method comprising administering a pharmaceutical composition to the primate, wherein the pharmaceutical composition is administered in an amount effective to achieve a delivered dose of about 5x1013vg to about 4.7x1014vg, preferably about 6x1013vg, about 1.2x1014vg or about 2.3x1014vg in the lung of the primate and wherein the pharmaceutical composition comprises a recombinant adeno-associated virus (rAAV) vector, said rAAV vector comprising (i) a capsid comprising a capsid protein comprising the amino acid sequence as set forth as SEQ ID NO: 12 or an amino acid sequence at least 90% identical to SEQ ID NO: 12 and comprising a Thr at amino acid 469 and an Ala at amino acid 598 based on the amino acid numbering set forth in SEQ ID NO: 12 and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a cystic fibrosis transmembrane conductance regulator (CFTR protein) gene product operably linked to a promoter. In preferred aspects, the composition is formulated as an aerosol.

[0036] In other aspects, a method for delivering a heterologous nucleic acid to a lung cell in a primate is provided, the method comprising administering to the primate an amount of a pharmaceutical composition comprising an rAAV comprising a capsid comprising a capsid protein of SEQ ID NO: 12 or comprising an amino acid sequence at least 90% identical thereto and a nucleic acid encoding a CFTR or biologically active fragment thereof operably linked to a promoter, that is effective to achieve a delivered dose of about 6x1013vg to about 2.3x1014vg, preferably about 6x1013vg, 1.2x101+vg or 2.3x1014vg, to the primate, preferably wherein the pharmaceutical composition is administered to the primate by a nebulizer, more preferably an AeroEclipse II BAN nebulizer.

[0037] In some aspects, the heterologous nucleic acid is delivered to an airway epithelial cell, a smooth muscle cell, and / or an endothelial cell in the primate, preferably wherein a therapeutic level of the encoded CFTR protein is detected in the airway epithelial cell, smooth cell and / or endothelial cell. In some aspects, the airway epithelial cell is a basal cell, a goblet cell or a cilia celL.In related aspects, the airway epithelial cell is a lung alveolar epithelial type 1 (AECI), a lung alveolar type 2 (AECII) cell, a bronchial epithelial cell or a tracheal epithelial cell.

[0038] In some aspects, at least 90%, at least 95%, at least 98% or about 100% of the airway epithelial cells in the primate express the CFTR gene product at least 3 weeks or at least 4 weeks after administering the pharmaceutical composition to the primate. Preferably the CFTR gene product is a human CFTR protein or a biologically active truncated CFTR protein lacking amino acids 708-759 of the human CFTR protein sequence and the promoter is a constitutive promoter, preferably a CMV173 promoter.

[0039] In other aspects, a combination therapy as described herein provides a method for treating cystic fibrosis and / or a lung disease associated with cystic fibrosis, including but not limited to upper airway disease, lower airway disease, nasopharyngeal disease, sinusitis and / or salivary disease associated with cystic fibrosis, comprising administering to the subject a pharmaceutical composition as herein described, wherein the pharmaceutical composition comprises about 5x1014vg or about 2.5x1014vg or about 1.25x1014vg or about 8x1013vgto about 3x1014vg, preferably about 8x1013vg, about 1.6x1014vg or about 3x1014vg of the rAAV). Preferably the subject is a human or non-human primate. In related aspects, the methods comprise administering to a primate with cystic fibrosis an amount of a pharmaceutical composition comprising an rAAV effective to provide a delivered dose of about 5x1013vg to about 4.7x1014vg, preferably about 6x1013vg, about 1.2x1014vg or about 2.3x1014vg of the rAAV to the primate.

[0040] In some aspects, a primate (e.g., a human or non-human primate) is administered (i) one or more CFTR modulators and (ii) a pharmaceutical composition as herein described, wherein the pharmaceutical composition is administered by any of a variety of means, preferably as an aerosol, to achieve and maintain a therapeutically effective level of CFTR or a portion thereof for the treatment of cystic fibrosis or lung disease associated therewith for at least about 6 months, at least about one year, at least about 18 months, at least about two years, at least about 30 months, at least about three years or at least about 42 months, at least about 48 months or at least about four years.

[0041] In some aspects, the combination therapy comprises no more than a single dose administration of the pharmaceutical composition to the subject (e.g., a human with cystic fibrosis) and is effective to achieve a durable and maintained therapeutic concentration of CFTR or biologically active portion thereof. In related aspects, the treatment comprises no more than a single delivered dose of about 5x1013vg to about 4.7x1014vg, preferably about 6x1013vg, about 1 .2x1014vg or about 2,3x1014vg, of an rAAV comprising a capsid comprising a capsid protein of SEQ ID NO: 12 or comprising an amino acid sequence at least 90% identical thereto and a nucleic acid encoding a CFTR or biologically active fragment thereof). In some aspects, the subject is administered a single unit dose of a pharmaceuticalcomposition suitable for aerosolization comprising about 5x1014or about 2.5x1014or about 1.25x10]4or about 8x1013to about 3x1014, preferably about 3x1014or about 1.6x1014virus particles (vp) or virus genomes (vg) of rAAV comprising a capsid protein of SEQ ID NO: 12 and a nucleic acid of SEQ ID NO:45. In other aspects, the dosage treatment may be a multiple dose schedule. In some aspects, the subject receives a first administration of the pharmaceutical composition and receives at least a second administration of the pharmaceutical composition at least one year after receiving the first administration and within 5, 4, 3, or 2 years of receiving the first administration.

[0042] Methods pertaining to the administration of AAV vectors to humans have been previously described by Kay et al. (2000, Nat Genet 24:257-261), the entire content of which is incorporated herein by reference. In some preferred embodiments, the pharmaceutical composition (comprising infectious rAAV) is administered to the subject by pulmonary, endobronchial, intranasal, intratracheal, and / or intrabronchial administration. In some preferred embodiments, the pharmaceutical composition is administered using a nebulizer.

[0043] In some embodiments, the pharmaceutical composition of the combination is suitable for inhalation and comprises at least one unit dose of (i) about 5x10]4vg or about 2.5x1014vg or about 1 ,25x1014vg of an rAAV, more preferably comprises about 8x1013to about 3x1014vg, or about 8x1013vg, about 1.6x1014vg or about 3x1014vg of an rAAV, the rAAV comprising (a) a capsid comprising a capsid protein of SEQ ID NO: 12 and (b) a nucleic acid comprising a nucleotide sequence encoding a CFTR gene produces) in (ii) a buffer comprising about 10 mM to about 50 mM citrate, about 70 mM to about 150 mMNaCl and optionally a surfactant, preferably a non-ionic surfactant such as Pluronic F-68, more preferably about 0.005% Pluronic F68, and having pH of between 5 and 7, preferably having a pH of about 6.0. In some preferred aspects, the pharmaceutical composition comprises about 20 mM to about 50 mM citrate, about 85 mM to about 125 mM NaCl and about 0.005% Pluronic F68 and has a pH of about 6.0. In some particularly preferred aspects, the pharmaceutical composition of the combination comprises about 20 mM citrate, about 125 mM NaCl and about 0.005% Pluronic F68 and has a pH of about 6.0. In preferred embodiments, the pharmaceutical composition of the combination comprises at least one unit dose of about 5x1014vg or about 2.5x1014vg or about 1.25x1014vg of an rAAV, more preferably about 8x1013to about 3x10,4vg, or about 8x1013vg, about 1.6x1014vg or about 3x1014vg of an rAAV, the rAAV comprising (a) a capsid comprising a capsid protein of SEQ ID NO: 12 and (b) a nucleic acid comprising a nucleotide sequence encoding a human cystic fibrosis transmembrane conductance regulator (CFTR) protein or a biologically active truncated CFTR protein lacking amino acids 708-759 of the human CFTR protein sequenceoperably linked to a promoter. In related embodiments, the rAAV comprises a nucleic acid comprising or consisting of the nucleotide sequence of SEQ ID NO:45.

[0044] In some embodiments, the pharmaceutical composition is formulated as a liquid / suspension suitable for aerosolized delivery. In related embodiments, the pharmaceutical composition is formulated as an aerosol and / or is an inhaled dosage form.

[0045] In some embodiments, the patient to be administered a combination therapy as herein described is a human. In other aspects, the combination therapy is for administration to a human cystic fibrosis patient who is eligible and / or are receiving a stable dose of a CFTR modulator therapy at the time the combination therapy is initiated. In other aspects, the combination therapy is for administration to a human cystic fibrosis patient who is ineligible, refractory, not tolerant to, or who has discontinued treatment with one or more CFTR modulators. In other aspects, the combination therapy is for administration to a human cystic fibrosis patient who has previously had an incomplete response to one or more CFTR modulators. CF patients who previously did not respond to CFTR modulators may nevertheless benefit from the combination therapy as CFTR modulators may modulate the rAAV-delivered CFTR gene, as the binding sites are intact.

[0046] In some cases, the rAAV exhibits increased resistance to human AAV neutralizing antibodies compared to the resistance exhibited by AAV2 (wild type AAV serotype 2). In some cases, the rAAV exhibits at least about 1.5-fold (e.g., at least about 3- fold, at least about 5-fold, at least about 10-fold, at least about 30-fold, etc.) greater resistance to human AAV neutralizing antibodies than the resistance exhibited by AAV2. In some cases, the rAAV exhibits increased transduction of mammalian cells in the presence of human AAV neutralizing antibodies compared to the transduction of mammalian cells exhibited by wild type AAV serotype 2 (AAV2). In some cases, the mammalian cells are liver cells, pancreatic cells, skeletal muscle cells, heart muscle cells, fibroblasts, retinal cells, synovial joint cells, lung cells, T cells, neurons, glial cells, stem cells (e.g., hematopoietic stem cells, hematopoietic progenitor cells, neural stem cells, neural progenitor cells, neural crest stem cells, embryonic stem cells, induced pluripotent stem cells (iPS cells), mesenchymal stem cells, mesodermal stem cells, liver stem cells, pancreatic stem cells, pancreatic progenitor cells, muscle stem cells, retinal stem cells, and the like), endothelial cells, or cancer cells. In some cases, the heterologous nucleic acid comprises an RNA interfering agent. In some cases, the heterologous nucleic acid comprises a nucleotide sequence encoding a polypeptide.

[0047] In some cases, the encoded variant AAV capsid protein (encoded by an isolated nucleic acid) confers to an infectious recombinant adeno-associated virus (rAAV) virion an increased resistance to human AAV neutralizing antibodies compared to the resistanceexhibited by AAV2 (wild type AAV serotype 2). In some cases, increased resistance is at least about 1.5-fold (e.g., at least about 3-fold, at least about 5-fold, at least about 10-fold, at least about 30-fold, etc.) greater than the resistance exhibited by AAV2. In some cases, the encoded variant AAV capsid protein (encoded by an isolated nucleic acid) confers to an infectious recombinant adeno-associated virus (rAAV) virion an increased transduction of mammalian cells in the presence of human AAV neutralizing antibodies compared to the transduction exhibited by AAV2.DESCRIPTION OF THE DRAWINGS

[0048] FIGS. 1A-B depict directed Evolution of AAV for Enhanced Antibody Evasion.

[0049] FIGS. 2A-B depict the neutralization profiles of antibody evading variants using human IVIG.

[0050] FIGS. 3A-C depict the neutralization profiles of antibody evading variants using human sera acquired from individuals that were excluded from hemophilia B clinical trials due to the presence of high neutralizing antibody titers against AAV.

[0051] FIGS. 4A-B depict the amino acid sequences of loop-swap / shuffle and saturation mutagenesis clones.

[0052] FIG. 5 demonstrates the in vitro tropism of AAV variants.

[0053] FIGS. 6A-B show in vivo localization and neutralization of novel AAV variants.

[0054] FIGS. 7A-D demonstrate the generation of human antibody evaders.

[0055] FIGS. 8A-I depict the capsid protein sequence of Shuffle 100-1 (SEQ ID NO: 11) aligned with the wild type capsid protein sequences of AAV1 -9 (SEQ ID NOs: 1-9).

[0056] FIGS. 9A-I depict the capsid protein sequence of Shuffle 100-3 (SEQ ID NO: 12) aligned with the wild type capsid protein sequences of AAV1-9 (SEQ ID NOs: 1-9).

[0057] FIGS. 10A-I depict the capsid protein sequence of Shuffle 100-7 (SEQ ID NO: 13) aligned with the wild type capsid protein sequences of AAV1-9 (SEQ ID NOs: 1-9).

[0058] FIG. 11 shows the neutralizing antibody titers of library clones and parent serotypes in immunized mouse sera.

[0059] FIG. 12 illustrates the directed evolution process utilized to identify capsid variant “Aid” (comprising a capsid protein of SEQ IDNO:12) with enhanced gene delivery to the lung in the presence of human neutralizing antibodies.

[0060] FIGs. 13A-B Figure 13A illustrates estimated genetic diversity of the capsid libraries used for the directed evolution process. The total diversify of the libraries is > 1billion genetic variants. Figure 13B illustrates productivity of the capsid libraries. All capsid libraries were manufactured at a level sufficient to produce material for the in vivo Therapeutic Vector Evolution program study. The viral genomes (vg) administered represent the target dose, not accounting for losses associated with the delivery device and route of administration.

[0061] FIGs. 14A-B Fig.l4A illustrates external PCR amplification of viral genomes from the isolated AT II cells following a) AeroProbe® administration or b) nebulizer administration from the first round of selection. Bands within blue boxes represent successful amplification of viral genomes. Temperature gradient represents annealing temperatures used during PCR corresponding to each lane of the gel. Fig. 14B illustrates Internal PCR amplification of viral genomes from the isolated AT II cells following a) AeroProbe® administration or b) nebulizer administration from the first round of selection. Bands within blue boxes represent successful amplification of viral genomes.

[0062] FIGs. 15A- 15B Fig. 15A illustrates frequency of chimera motif within sequencing analysis for the study. Sequencing analysis is based on total frequency within sequenced population for both AeroProbe and Nebulizer delivery devices. Fig. 15B illustrates frequency of Al 01 variant within chimera motif for the study. Sequencing analysis based on total frequency within sequenced population for both AeroProbe and Nebulizer delivery devices.

[0063] FIG. 16 Lung Sampling Schematic (Examples 3 and 7). Schematic representation of trachea and lung sampling. Circles in right lung represent adjacent samples obtained for DNA and protein isolation. Samples oriented along the long and short axis for tissue sectioning are represented by squares.

[0064] FIG. 17 Variant Capsid (comprising a capsid protein of SEQ ID NO: 12) Transduction with NHP Serum Samples at 1: 10 Serum Dilution. Serum samples from NHPs eligible for study inclusion were analyzed for the presence of anti-AAV neutralizing antibodies. Transduction in the presence of a 1:10 serum dilution (compared to transduction in the absence of serum) is reported for all NHP. NHP selected for study inclusion are denoted by yellow bars. Error bars - Standard Deviation, n ™ 3 (internal replicates).

[0065] FIG. 18 Variant Capsid-Mediated Genome Biodistribution. Quantification of viral genomes in the lung and additional systemic organs by qPCR using primers and probe against the EGFP transgene. Viral genomes were detected in all 48 samples (n “ 16 samples per NHP; n = 3 NHP). All samples tested from skeletal muscle (triceps brachii, vastus lateralis), diaphragm, kidney, spleen, brain and spinal cord were below the lower limit of quantification. Mean ± standard error; n = 3 NHP (n = 16 biopsy sites per lung per NHP, n =10 biopsy sites per liver per NHP, n = 15 biopsy sites per heart per NHP, n = 9 biopsy sites per skeletal muscle per NHP, n = 2 samples per kidney per NHP, n = 1 sample per spleen per NHP, n = 8 biopsy sites per brain per NHP, n - 3 biopsy sites per spinal cord per NHP).

[0066] FIG. 19 Variant Capsid-Mediated Protein Expression in Lungs. Quantification ofEGFP protein expression in the lung by ELISA against the EGFP protein. EGFP expression was observed in all 48 lung samples (n = 16 samples per NHP; n - 3 NHP). EGFP expression was observed in 10 liver samples that were positive for viral genomes (n = 10 samples per NHP; n = 3 NHP). Mean + standard error.

[0067] FIG. 20 Variant Capsid-Mediated Protein Localization in Lung. Representative images of EGFP expression in the trachea (a-b), bronchi (c, e, g), and alveoli (d, f, h) of NHP V002969. Sections denoted by white boxes in trachea (b), alveoli (d), and bronchi (e) are provided as magnified images in i, j, and k, respectively. Approximate locations of images are denoted by magenta boxes on the schematic diagram. EGFP expression is detected by an anti- GFP antibody (red) in all images. Nuclei were counterstained with DAPI (blue).

[0068] FIGS. 21 A-2 ID. Alveolar Epithelial Type 2 Non-Human Primate CellCharacterization. NHP AECII cells were over 90% LysoTracker positive, shown by fluorescent microscopy (FIG. 17A) and quantified by flow cytometry (FIG. 2 IB). Surfactant protein C, a mature marker of AECII cells was evident on day 1 and day 5 after seeding (FIG. 21 C). AECII cells decreased their proliferation rate over time in culture shown by EdU incorporation (FIG. 21D). EdU- 5-Ethynyl-2'-deoxyuridine, Error bars-Standard Deviation, n=3 internal replicates.

[0069] FIGS. 22A-22B. Non-Human Primate Alveolar Epithelial Type 2 Cell Vector Characterization. The rAAV with capsid comprising capsid protein of SEQ ID NO: 12 (4D- A101) capsid showed a higher transduction rate than the AAV5 capsid, both carrying CAG- eGFP in ALI cultures of AECII NHP cells. Quantification of eGFP positive cells by flow cytometry (Figure 22A). Representative ICC images of eGFP positive cells (Figure 22B). Post-infection time of 3 days, 5 total days in culture. Error bars=Standard Deviation, n-3 internal replicates. Student’s t-test, p<0.05 compared to AAV5.

[0070] FIGS. 23A-23D. Alveolar Epithelial Type 2 Human Cell Characterization.Human AECII cells were around 80% LysoTracker positive until day 11 in culture when they decreased to 50%, shown by fluorescent microscopy (FIG. 23A) and quantified by flow cytometry (FIG. 23B). Surfactant protein C, a mature marker of AECII cells was evident on day 5 and day 11 after seeding (FIG. 23C). AECII cells decreased their proliferation rate over time in culture shown by EdU incorporation (FIG. 23D), EdU= 5-Ethynyl-2'-deoxyuridine, Error bars=Standard Deviation, n=3 internal replicates.

[0071] FIG. 24 Human Alveolar Epithelial Type 2 Ceil Vector Characterization. Capsid comprising a capsid protein of SEQ ID NO: 12 (4D-A101) showed a higher transduction rate than the AAV5 capsid, both carrying CAG-eGFP in AL1 cultures of AECII human cells. Representative ICC images of eGFP positive cells. Post-infection time of 6 and 10 days, 7 and 11 total days in culture.

[0072] FIG. 25 In Vitro Neutralization Profiles of Wild-Type AAV 1 , AAV2, AAV5, AAV8, AAV9 and rAAV comprising capsid comprising capsid protein of SEQ ID NO: 12 (4D-A101). rAAV comprising capsid comprising capsid protein of SEQ ID NO: 12 showed superior ability to avoid AAV neutralizing antibodies in human IVIG compared to wild type AAV. AAV.CAG.Luciferase vectors were incubated with dilutions of IVIG prior to infection of 2V6.11 cells at a MOI of 1,000. Vectors capable of evading antibodies transduced the cells, and luciferase activity was measured 48 hours post infection. IVIG™ intravenous immunoglobin, Error bars“Standard Deviation, n™3, internal replicates. * p < 0.05 for 4D- A101 vs AAV1, AAV2, AAV8, and AAV9,fp < 0.05 for 4D-A101 vs AAV5.

[0073] FIG. 26 Graph of net charge vs. pH for A 101 VP1 and VP3 capsid proteins.

[0074] FIG. 27 Graph of Al 01-GFP pH Solubility After I -day Storage at RoomTemperature.

[0075] FIGs. 28A-C. Transduction Leads to Robust Protein Expression and Membrane Localization in HEK2v6.11 Cells. HEK2v6.11 were transduced with recombinant AAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO: 12 and (ii) a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:45 and probed by western blot (Figure 28A) with anti-CFTR antibody (Figure 28A). Representative images (Figure 28B) show cells analyzed by immunocytochemistry, anti-CFTR (red), F-actin (green), DAPI, nuclear (Blue). Scale bars are 100 pM (Figure 28B) and 25 pM (Figure 28C).

[0076] FIGs. 29 A-B Transduction of 16HBE 14o-G542X cells with recombinant AAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO: ] 2 and (ii) a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:45. Reverse transcription- ddPCR (RT-ddPCR) digital droplet PCR (ddPCR) was performed on RNA extracted from the HBE cultures following transduction with recombinant AAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO: 12 and (ii) a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:45 at increasing MOIs (Figure 29 A). Exogenous CFTRAR transcript levels were determined and quantified as copies / pL above a set threshold and plotted on a linear scale. BLQ, below the limit of quantification. NT, nontransduced. Immunocytochemistry of HBE cultures following transduction at MOIs of 35,000 and 50,000 (Figure 29B). Blue is DAPI and red is CFTR protein. Scale is 100 pm.

[0077] FIG. 30 Transduction of healthy ex vivo ALI lung cultures with recombinant AAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO: 12 and (ii) a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO;45. ddPCR was performed on cDNA prepared from RNA extracted from the cultures following transduction with the rAAV. Two primer / probe sets were created to specifically differentiate the codon optimized human CFTRAR transgene from the endogenous human CFTR gene. Quantification analyzed the number of droplets, above the set threshold, containing the transcript of the primer / probe set examined. BLQ, below the limit of quantification. NT, nontransduced.

[0078] FIG. 31 4D-A101 Transduction with NHP Serum Samples at 1 : 10 SerumDilution. Serum samples from NHP eligible for study inclusion were analyzed for the presence of anti-AAV neutralizing antibodies to the capsid of SEQ ID NO: 12. Transduction in the presence of a 1:10 serum dilution (compared to transduction in the absence of serum) is reported for all NHP. Error bars = Standard Deviation, n = 3 (internal replicates).

[0079] FIGs. 32A-C. Quantification of viral genomes by qPCR using primers and probe against the CFTRAR transgene. Figure 32A, viral genomes were robustly detected in lung samples distributed throughout the right lung. Figure 32B, individual animal lung samples are denoted by approximate region and lung lobe: alveoli (green), primary / secondary bronchi (blue), tertiary / lower bronchi (red), cranial lobe (circle), middle lobe (square), caudal lobe (triangle), accessory lobe (diamond). Figure 32C, viral genomes quantified in 3 x 1013vg dosed animals demonstrate that all samples tested from heart, liver, brain, skeletal muscle (triceps brachii, vastus lateralis, diaphragm), spinal cord, pancreases, kidney, and testis were below the lower limit of quantification. All three animals had detectable viral genomes in the tracheobronchial (TB) lymph node, and one animal had detectable viral genomes in the spleen. Mean ±SD.

[0080] FIGs. 33A-B Transgene Transcript Expression in Lungs. Quantification of CFTRAR transcript by RT-qPCR using primers and probe against the CFTR transgene comprised within the heterologous nucleic acid of SEQ ID NO:45. Figure 33 A, transcripts were detected in the right lung samples distributed throughout the lobes in 3 x 1013vg dosed animals, all vehicle animals were BLQ. Figure 33B, individual animal lung samples dosed with 3 x 1013vg are denoted by approximate region and lung lobe: alveoli (green), primary / secondary bronchi (blue), tertiary / lower bronchi (red), cranial lobe (circle), middle lobe (square), caudal lobe (triangle), accessory lobe (diamond). Mean ±SD.

[0081] FIGs. 34A-B Protein Expression in Lungs. CFTR protein expression in the lung by immunohistochemistry staining. Figure 34A, CFTR expression in tracheal epithelium,bronchial epithelium, and alveoli sections of each treatment group, representative images. Figure 34B, CFTR protein expression in the tracheal epithelium, bronchial epithelium, and alveoli sections of 3 x IO’3vg (of recombinant AAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO: 12 and (ii) a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:45) treated animals (individual animals shown), representative images.

[0082] FIG. 35 Graph of AlOl-Luc Solubility vs. pH

[0083] FIG. 36 Graphic illustrating a multicenter, open-label Phase 1 clinical dose exploration study evaluating the safely, biodistribution, and biological activity of recombinant AAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO: 12 and (ii) a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:45 in adults with cystic fibrosis lung disease. Endobronchial biopsy (at one month) measured rAAV transgene and protein expression. CFTR = cystic fibrosis transmembrane conductance regulator. SRT = safety review team.

[0084] FIG. 37 Graphic illustrating the bronchoscopic sampling plan for the clinical trial. Cell and tissue samples for biomarker analyses were collected 4-8 weeks after administration of recombinant AAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO: 12 and (ii) a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:45. Bronchial brushings and endobronchial biopsies were performed according to standard institutional procedures. A total of 4 bronchial brushings were performed in subsegmental airways in the right (n-2) and left (n=2) lower lobe basilar segments. Following completion of the bronchial brushings, endobronchial biopsies were performed at a total of 4 sites.

[0085] FIG. 38 Qualitative and semi-quantitative analysis of CFTR expression in tissue samples from endobronchial biopsies and commercially acquired control samples.Visiopharm® machine-learning image analysis software was used to quantify the distribution and intensity of immunohistochemistry staining in bronchial epithelial cells.

[0086] FIG. 39 Demographics and baseline characteristics of patients enrolled in the clinical trial. As of the interim data analysis on 28 May 2024, a total of 10 participants had enrolled in the Phase 1 dose exploration study, including 3 participants in the low dose cohorts (5x1014vg and 2.5x1014vg).

[0087] FIG. 40 Safety outcomes following administration of recombinant AAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO: 12 and (ii) a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:45 at doses ranging from 2.5x1014vg to lx1015vg (n=6) in the clinical trial. Interim analysis of safety outcomes(duration of follow up, 1-25 months) showed that the rAAV was generally well tolerated. No dose-limiting toxicities or treatment-related serious adverse events were observed. Grade 1 (mild) transient increased sputum was reported in a single participant in the 5x1014vg group; no other clinically significant treatment-related adverse events were reported.

[0088] FIG. 41 CFTRDR RNA expression in bronchial epithelial cells 4-8 weeks after administration of recombinant AAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO: 12 and (ii) a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:45. Evaluation CFTRDR RNA expression by in situ hybridization demonstrated dose-dependent transgene expression. The proportion of CFTRDR RNA-positive epithelial cells ranged from 14% at the lowest tested dose to 53% at the highest tested dose. CFTRDR RNA was not detected in commercially acquired control samples from human CF and non-CF lungs. Quantification by Visiopharm® Al Machine Learning Analysis. *Attempts to genotype commercial CF samples yielded results for 13 / 35 samples; of these, a majority were AF5O8 homozygous mutations. CFTR, cystic fibrosis transmembrane conductance regulator; ISH, in situ hybridization.

[0089] FIG. 42 recombinant AAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO: 12 and (ii) a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:45-mediated CFTR protein expression in bronchial epithelial cells 4-8 weeks after administration of a single aerosolized dose. Evaluation of CTFR protein expression by immunohistochemistry demonstrated widespread expression at all tested doses. The proportion of CFTR-positive epithelial cells ranged from 94% to 100% in lung samples from rAAV-treated study participants compared to 18% and 44% respectively in CF (n=35) and non-CF (n=10) control samples (left panel). Quantitative assessment of staining intensity by machine-learning image analysis showed a >3-fold increase in mean intensity scores in rAAV-treated study participants compared to non-CF controls (right panel). *H-score. Quantification by Visiopharm Al Machine Learning Analysis.4Attempts to genotype commercial CF samples yielded results for 13 / 35 samples; of these, a majority were AF508 homozygous mutations. IHC, immunohistochemistry.

[0090] FIG. 43 Representative images of endobronchial biopsy samples obtained from the left secondary carina (top row) and right middle lobe (bottom row) 4-8 weeks after administration of recombinant AAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO: 12 and (ii) a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:45 and control lung samples obtained from human donors (below) showing widespread and consistent (100% of samples) CFTR protein expression. Assessment of CFTR expression by immunohistochemistiy showed robust and widespread expression in bronchial epithelial cells at all tested doses. At the highest tested dose (2x1015vg), positivestaining for CFTR protein was also observed in the interstitium. 16 of 16 endobronchial biopsy samples were positive for CFTR Protein by 1HC 4-8 Weeks After Dosing with recombinant AAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO: 12 and (ii) a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:45.* Representative images, endobronchial biopsy samples obtained from the left secondary carina (row 1) and right middle lobe (row 2). Endobronchial biopsy performed at Week 8.

[0091] FIG. 44 CFTR protein expression observed in multiple airway cell types. CFTR protein expression and localization (IHC) following administration of recombinant AAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO: 12 and (ii) a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:45 was observed in multiple airway cell types, including basal cells, goblet cells, and columnar ciliated cells (left panel). Expression was properly localized to the apical region (right panel). *Image from Cohort 1 (lxl 015vg) participant, images from Cohort 2 (2x1015vg) participants. CFTR, cystic fibrosis transmembrane conductance regulator. IHC, immunohistochemistry.

[0092] FIG. 45 Assessment of CFTR&R RNA expression 3 days post transduction of recombinant AAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO: 12 and (ii) a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:45, with and without ETI treatment. Cells were transduced at an MOI of 100,000. Results are normalized to endogenous P-actin levels. No significant difference was observed when comparing ETI treated and non-treated groups. ETI, elexacaftor, tezacaftor, ivacaftor; MOI: multiplicity of infection (vg / cell). Error bars - standard deviation; n~6.

[0093] FIG. 46 CFTR channel function in CF AF50S / AF50S (Panels A-Q and AF508 / G524X (Panels D-F) ALI airway epithelial cultures treated with E / T / I (2 pM VX-445, 3 pM VX-661, 0.1 pM VX-770) for 24 hours followed by transduction with recombinant AAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO: 12 and (ii) a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:45 (MOI 1x106) compared to either E / T / I or the rAAV alone. (Panels A, D) Representative / eqtracings, (Panels B, E) mean +SEM transepithelial current area under the curve after forskolin stimulation, and (Panels C, F) mean +SD transepithelial electrical resistance. *P<0.05, **P<0.01, ***P<0.001, ****p<0.0001 by one-way ANOVA with Bonferroni’s multiple comparisons. N=3 separate experiments. ALI-air liquid interface;E / T / I-elexacaftor / tezacaftor / ivacaftor; NT=non-transduced, MOI=multiplicity of infection; SD=standard deviation; SEM=standard error of the mean.|0094] FIGs. 47A-B Fig. 47A: Transgene expression kinetics following a single aerosolized dose of 3x 1013vg of rAAV comprising (i) a capsid comprising a capsid protein ofSEQ ID NO: 12 and (ii) a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:45. Proportion of NHP lung samples with CFTRAR transgene expression above the limit of quantification; bar represents mean value; n=2 NHPs per timepoint (n-32 samples per lung). Fig. 47B: rAAV (3 x 1013vg) transgene expression at 4 weeks in NHPs with high seropositivity for Al 01 (capsid) Nabs (Fig. 47B); bars (right) represent mean +SD number of copies in NAb-positive NHPs (n~5) and NAb-negative NHPs (n=2) at 4 weeks in Fig. 47A.DETAILED DESCRIPTION OF THE INVENTION

[0095] DEFINITIONS

[0096] Adeno-associated virus is a nonpathogenic parvovirus composed of a 4.7 kb single-stranded DNA genome within a non-enveloped, icosahedral capsid. “AAV” is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The genome contains three open reading frames (ORF) flanked by inverted terminal repeats (ITR) that function as the viral origin of replication and packaging signal. The rep ORF encodes four nonstructural proteins that play roles in viral replication, transcriptional regulation, site-specific integration, and virion assembly. The cap ORF encodes three structural proteins (VP 1-3) that assemble to form a 60-mer viral capsid. Finally, an ORF present as an alternate reading frame within the cap gene produces the assemblyactivating protein (AAP), a viral protein that localizes AAV capsid proteins to the nucleolus and functions in the capsid assembly process.

[0097] There are several naturally occurring serotypes and over 100 variants of AAV, each of which differs in amino acid sequence, particularly within the hypervariable regions of the capsid proteins, and thus in their gene delivery properties. No AAV has been associated with any human disease, making recombinant AAV attractive for clinical applications.

[0098] The term “AAV” as used herein covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. The term “AAV” includes AAV type 1 (AAV-1 or AAV1), AAV type 2 (AAV-2 or AAV2), AAV type 3 (AAV-3 or AAV3), AAV type 4 (A AV-4 or AAV4), AAV type 5 (A AV-5 or AAV5), AAV type 6 (AAV-6 or AAV6), AAV type 7 (AAV-7 or AAV7), AAV type 8 (AAV-8 or AAV8), AAV type 9 (AAV-9 or AAV9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. “Primate AAV” refers to AAV that infect primates, “non-primate AAV” refers to AAV that infect non-primate mammals, “bovine AAV” refers to AAV that infect bovine mammals, etc.J0099] The term “4D-A101” or “A101” as used herein refers to an AAV capsid comprising a capsid protein of SEQ ID NO: 12.

[0100] The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Numbers NC...002077.1 (AAV-1), AF063497.1 (AAV-1), NC— 001401.2 (AAV-2), AF043303.1 (AAV-2), J01901.1 (AAV-2), U48704.1 (AAV-3), NC 001729.1 (AAV-3), NC....001829.1 (AAV-4), U89790.1 (AAV-4), NC— 006152,1 (AAV-5), AF085716.1 (AAV-5), AF028704.1 (AAV-6), NC 006260.1 (AAV-7), AF513851 .1 (AAV- 7), AF513852.1 (AAV-8) NC..006261.1 (AAV-8), and AY530579.1 (AAV-9); the disclosures of which are incorporated by reference herein for teaching AAV nucleic acid and amino acid sequences. See also, e.g., Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71 :6823; Chiorini et al. (1999) J. Virology 73: 1309; Bantel-Schaal et al. (1999) J Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et al., (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nal. Acad.Sci. USA 99:11854; Moris et al. (2004) Virology 33:375-383; international patent publications WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; and U.S. Pat. No. 6,156,303.

[0101] The sequences of naturally existing cap (capsid) proteins associated with AAV serotypes are known in the art and include: AAV1 (SEQ ID NO: 1), AAV2 (SEQ ID NO: 2), AAV3 (SEQ ID NO: 3), AAV4 (SEQ ID NO: 4), AAV5 (SEQ ID NO: 5), AAV6 (SEQ ID NO: 6), AAV7 (SEQ ID NO: 7), AAV8 (SEQ ID NO: 8), and AAV9 (SEQ ID NO: 9). The term “variant AAV capsid protein” is a an AAV capsid protein comprising an amino acid sequence that includes at least one substitution (including deletion, insertion, etc.) relative to one of the naturally existing AAV capsid protein sequences set forth in SEQ ID NOs:l-9.

[0102] An “AAV virion” or “AAV viral particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated AAV polynucleotide.

[0103] “Recombinant,” as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature. A recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.

[0104] If an AAV virion comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome, e.g., a transgene to be delivered to a target cell, an RNAi agent or CRISPR agent to be delivered to a target cell, etc.), it is typically referred to as a “recombinant AAV (rAAV) virion” or an “rAAV viral particle.” In general, theheterologous polynucleotide is flanked by at least one, and generally by two, AAV inverted terminal repeat sequences (ITRs).[001051 The term “rAAV vector” encompasses rAAV virions (i.e., rAAV viral particles)(e.g,, an infectious rAAV virion), which by definition include an rAAV polynucleotide; and also encompasses polynucleotides encoding rAAV (e.g., a single stranded polynucleotide encoding rAAV (ss-rAAV); a double stranded polynucleotide encoding rAAV (ds-rAAV), e.g., plasmids encoding rAAV; and the like).

[0106] “Packaging” refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle.

[0107] AAV “rep” and “cap” genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV “packaging genes.”

[0108] A “helper virus” for AAV refers to a virus that allows AAV (e.g. wild-type AAV) to be replicated and packaged by a mammalian cell. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein- Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC.

[0109] “Helper virus function(s)” refers to function(s) encoded in a helper virus genome which allow AAV replication and packaging (in conjunction with other requirements for replication and packaging described herein). As described herein, “helper virus function” may be provided in a number of ways, including by providing helper virus or providing, for example, polynucleotide sequences encoding the requisite function(s) to a producer cell in trans. For example, a plasmid or other expression vector comprising nucleotide sequences encoding one or more adenoviral proteins is transfected into a producer cell along with an rAAV vector.

[0110] An “infectious” virus or viral particle is one that comprises a competently assembled viral capsid and is capable of delivering a polynucleotide component into a cell for which the viral species is tropic. The term does not necessarily imply any replication capacity of the virus. Assays for counting infectious viral particles are described elsewhere in this disclosure and in the art. Viral infectivity can be expressed as the ratio of infectious viralparticles to total viral particles. Methods of determining the ratio of infectious viral particle to total viral particle are known in the art. See, e.g., Grainger et al. (2005) Mol. Ther. 11 : S337 (describing a TCID50 infectious titer assay); and Zolotukhin et al. (1999) Gene Ther. 6:973. See also the Examples.

[0111] The term “tropism” as used herein refers to the preferential targeting of specific host species or specific cell types within a host species by a virus (e.g., an AAV), For example, a virus that can infect cells of the heart, lung, liver, and muscle has a broader (i.e., increased) tropism relative to a virus that can infect only lung and muscle cells. Tropism can also include the dependence of a virus on particular types of cell surface molecules of the host. For example, some viruses can infect only cells with surface glycosaminoglycans, while other viruses can infect only cells with sialic acid (such dependencies can be tested using various cells lines deficient in particular classes of molecules as potential host cells for viral infection). In some cases, the tropism of a virus describes the virus's relative preferences. For example, a first virus may be able to infect all cell types but is much more successful in infecting those cells with surface glycosaminoglycans. A second virus can be considered to have a similar (or identical) tropism as the first virus if the second virus also prefers the same characteristics (e.g., the second virus is also more successful in infecting those cells with surface glycosaminoglycans), even if the absolute transduction efficiencies are not similar. For example, the second virus might be more efficient than the first virus at infecting every given cell type tested, but if the relative preferences are similar (or identical), the second virus can still be considered to have a similar (or identical) tropism as the first virus. In some embodiments, the tropism of a virion comprising a subject variant AAV capsid protein is not altered relative to a naturally occurring virion. In some embodiments, the tropism of a virion comprising a subject variant AAV capsid protein is expanded (i.e., broadened) relative to a naturally occurring virion. In some embodiments, the tropism of a virion comprising a subject variant AAV capsid protein is reduced relative to a naturally occurring virion.

[0112] A “replication-competent” virus (e.g. a replication-competent AAV) refers to a phenotypically wild-type virus that is infectious, and is also capable of being replicated in an infected cell (i.e. in the presence of a helper virus or helper virus functions). In the case of AAV, replication competence generally requires the presence of functional AAV packaging genes. In general, rAAV vectors as described herein are replication-incompetent in mammalian cells (especially in human cells) by virtue of the lack of one or more AAV packaging genes. Typically, such rAAV vectors lack any AAV packaging gene sequences in order to minimize the possibility that replication competent AAV are generated by recombination between AAV packaging genes and an incoming rAAV vector. In many embodiments, rAAV vector preparations as described herein are those which contain few ifany replication competent AAV (rcAAV, also referred to as RCA) (e.g., less than about 1 rcAAV per 102rAAV particles, less than about 1 rcAAV per 104rAAV particles, less than about 1 rcAAV per 10srAAV particles, less than about 1 rcAAV per 1012rAAV particles, or no rcAAV),

[0113] The term “polynucleotide” refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment herein that comprises a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.[001141 A polynucleotide or polypeptide has a certain percent “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Of particular interest are alignment programs that permit gaps in the sequence. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J Mol. Biol. 48: 443-453 (1970)

[0115] A “gene” refers to a polynucleotide that performs a function of some kind in the cell. For example, a gene can contain an open reading frame that is capable of encoding a particular protein after being transcribed and translated. On the other hand a gene can encode a functional RNA product that is not translated (e.g., an aptamer, an interfering RNA, a ribosomal RNA (rRNA), a transfer RNA (tRNA), etc.).

[0116] A “gene expression product” or “gene product” is a molecule resulting from expression of a particular gene, as defined above. Gene expression products include, e.g., a polypeptide, an aptamer, an interfering RNA, a messenger RNA (mRNA), an rRNA, a tRNA, a non-coding RNA (ncRNA), and the like.

[0117] An “RNA interfering agent” or “RNAi agent” encompasses any agent (or a polynucleotide encoding such an agent) that can be used to change the expression of a gene (as defined above). Examples of RNAi agents known to one of ordinary skill in the art include, but are not limited to, (i) siRNA agents; (ii) antisense RNA; (iii) CRISPR agents; (iv) Zinc finger nuclease agents, and (v) Transcription activator-like effector nuclease (TALEN) agents.

[0118] (i) an siRNA agent (“small interfering” or “short interfering RNA” (or siRNA)) is an RNA duplex of nucleotides that is targeted to a gene interest (a “target gene”). An “RNA duplex” refers to the structure formed by the complementary pairing between two regions of a RNA molecule, forming a region of double stranded RNA (dsRNA). siRNA is “targeted” to a gene in that the nucleotide sequence of the duplex portion of the siRNA is complementary to a nucleotide sequence of the targeted gene. In some embodiments, the length of the duplex of siRNAs is less than 30 nucleotides. In some embodiments, the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotides in length. In some embodiments, the length of the duplex is 19-25 nucleotides in length. The RNA duplex portion of the siRNA can be part of a hairpin structure. siRNA agents that contain a hairpin can also be referred to as “shRNA (short hairpin RNA) agents.” In addition to the duplex portion, the hairpin structure may contain a loop portion positioned between the two sequences that form the duplex. The loop can vary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. The hairpin structure can also contain 3' or 5' overhang portions. In some embodiments, the overhang is a 3' or a 5' overhang 0, 1, 2, 3, 4 or 5 nucleotides in length. In general, the level of expression product (e.g., mRNA, polypeptide, etc.) of a target gene is reduced by an siRNA agent (e.g., an siRNA, an shRNA, etc.) that contains specific double stranded nucleotide sequences that are complementary to at least a 19-25 nucleotide long segment (e.g., a 20-21 nucleotide sequence) of the target gene transcript, including the 5' untranslated (UT) region, the ORF, or the 3' UT region. In some embodiments, short interfering RNAs are about 19-25 nt in length. See, e.g., PCT applications WOO / 44895, WO99 / 32619, WO01 / 75164, WOOl / 92513, W001 / 29058, W001 / 89304, W002 / 16620, and WO02 / 29858; and U.S. Patent Publication No. 20040023390 for descriptions of siRNA technology. The siRNA and / or shRNA can be encoded by a nucleic acid sequence, and the nucleic acid sequence can also include a promoter. The nucleic acidsequence can also include a polyadenylation signal. In some embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal.

[0119] (ii) antisense RNA is RNA that is complementary to a gene expression product. For example, an antisense RNA targeted to a specific inRNA is an RNA-based agent (or can be a modified RNA) that is complementary to the mRNA, where hybridization of the antisense RNA to the mRNA alters the expression of the mRNA (e.g., via altering the stability of the RNA, altering the translation of the RNA, etc.). Also included in “antisense RNA” are nucleic acids encoding an antisense RNA.

[0120] (iii) CRISPR agents. CRISPR (Clustered regularly interspaced short palindromic repeats) / CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. The Cas 9 protein (or functional equivalent and / or variant thereof, i.e., Cas9-like protein) naturally contains DNA endonuclease activity that depends on association of the protein with two naturally occurring or synthetic RNA molecules called crRNA and tracrRNA (also called guide RNAs). In some cases, the two molecules are covalently linked to form a single molecule (also called a single guide RNA (“sgRNA”)). Thus, the Cas9 or Cas9-like protein associates with a DNA-targeting RNA (which term encompasses both the two-molecule guide RNA configuration and the single-molecule guide RNA configuration), which activates the Cas9 or Cas9-like protein and guides the protein to a target nucleic acid sequence. If the Cas9 or Cas9-like protein retains its natural enzymatic function, it will cleave target DNA to create a double-strand break, which can lead to genome alteration (i.e., editing: deletion, insertion (when a donor polynucleotide is present), replacement, etc.), thereby altering gene expression. Some variants of Cas9 (which variants are encompassed by the term Cas9-like) have been altered such that they have a decreased DNA cleaving activity (in some cases, they cleave a single strand instead of both strands of the target DNA, while in other cases, they have severely reduced to no DNA cleavage activity). Cas9-like proteins with decreased DNA-cleavage activity (even no DNA-cleaving activity) can still be guided to a target DNA and can block RNA polymerase activity. Thus enzymatically inactive Cas9-like proteins can be targeted to a specific location in a target DNA by a DNA-targeting RNA in order to block transcription of the target DNA. Detailed information regarding CRISPR agents can be found, for example in (a) Jinek et. al., Science. 2012 Aug. 17; 337(6096): 816-21: “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity”; (b) Qi et al., Cell. 2013 Feb. 28; 152(5): 1173-83: “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression”, and (c) U.S. patent application Ser. No. 13 / 842,859 and PCT application numberPCT / US13 / 32589; all of which are hereby incorporated by reference in their entirety. Thus,the term “CRISPR agent” as used herein encompasses any agent (or nucleic acid encoding such an agent), comprising naturally occurring and / or synthetic sequences, that can be used in the Cas9-based system (e.g., a Cas9 or Cas9-like protein; any component of a DNA-targeting RNA, e.g., a crRNA-like RNA, a tracrRNA-like RNA, a single guide RNA, etc.; a donor polynucleotide; and the like).

[0121] (iv) Zinc finger nuclease (ZFN) agents. Zinc-finger nucleases (ZFNs) are artificial DNA endonucleases generated by Rising a zinc finger DNA binding domain to a DNA cleavage domain. ZFNs can be engineered to target desired DNA sequences and this enables zinc-finger nucleases to cleave unique target sequences. When introduced into a cell, ZFNs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks. For more information on the use of ZFNs, see, for example: Asuri et al., Mol Ther. 2012 February; 20(2):329-38; Bibikova et al. Science. 2003 May 2; 300(5620):764; Wood et al. Science. 2011 Jul. 15; 333(6040):307; Ochiai etal. Genes Cells. 2010 August;15(8):875-85; Takasu et. al., Insect Biochem Mol Biol. 2010 October; 40(10):759-65; Ekker et al, Zebrafish 2008 Summer; 5(2):121-3; Young et al, Proc Natl Acad Sci USA. 2011 Apr. 26; 108(17):7052-7; Goldberg et al, Cell. 2010 Mar. 5; 140(5):678-91; Geurts etal, Science. 2009 Jul. 24; 325(5939):433; Flisikowska et al, PLoS One. 2011; 6(6):e21045. doi: 10.1371 / joumal.pone.0021045. Epub 2011 Jun. 13; Hauschild et al, Proc Natl Acad Sci USA. 2011 Jul. 19; 108(29):12013-7; and Yu et al, Cell Res. 2011 November; 21(ll):1638-40; all of which are herein incorporated by reference for their teachings related to ZFNs. The term “ZFN agent” encompasses a zinc finger nuclease and / or a polynucleotide comprising a nucleotide sequence encoding a zinc finger nuclease.

[0122] (v) Transcription activator-like effector nuclease (TALEN) agents. Transcription activator-like effector nucleases (TALENs) are artificial DNA endonucleases generated by fusing a TAL (Transcription activator-like) effector DNA binding domain to a DNA cleavage domain. TALENS can be quickly engineered to bind practically any desired DNA sequence and when introduced into a cell, TALENs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks. For more information on the use of TALENs, see, for example: Hockemeyer et al. Nat Biotechnol. 2011 Jul. 7; 29(8):731-4;Wood et al. Science. 2011 Jul. 15; 333(6040):307; Tesson et al. Nat Biotechnol. 2011 Aug. 5; 29(8):695-6; and Huang et. al., Nat Biotechnol. 2011 Aug. 5; 29(8):699-700; all of which are herein incorporated by reference fortheir teachings related to TALENs. The term “TALEN agent” encompasses a TALEN and / or a polynucleotide comprising a nucleotide sequence encoding a TALEN.

[0123] A “control element” or “control sequence” is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide,including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3' direction) from the promoter.

[0124] “Operatively linked” or “operably linked” refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.

[0125] An “expression vector” is a vector comprising a region which encodes a polypeptide of interest, and is used for effecting the expression of the protein in an intended target cell. An expression vector also comprises control elements operatively linked to the encoding region to facilitate expression of the protein in the target. The combination of control elements and a gene or genes to which they are operably linked for expression is sometimes referred to as an “expression cassette,” a large number of which are known and available in the art or can be readily constructed from components that are available in the art.

[0126] “Heterologous” means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter. Thus, for example, an rAAV that includes a heterologous nucleic acid encoding a heterologous gene product is an rAAV that includes a nucleic acid not normally included in a naturally-occurring, wild-type AAV, and the encoded heterologous gene product is a gene product not normally encoded by a naturally-occurring, wild-type AAV.

[0127] A “2A peptide” refers to “self-cleaving” peptides of about 20 amino acids that produce equimolar levels of multiple genes from the same mRNA and may be used in place of IRES elements in multicistronic vectors. Non-limiting examples include T2A, P2A, E2A and F2A peptides sequences.

[0128] The terms “genetic alteration” and “genetic modification” (and grammatical variants thereof), are used interchangeably herein to refer to a process wherein a geneticelement (e.g., a polynucleotide) is introduced into a cell oilier than by mitosis or meiosis. The element may be heterologous to the cell, or it may be an additional copy or improved version of an element already present in the cell. Genetic alteration may be effected, for example, by transfecting a cell with a recombinant plasmid or other polynucleotide through any process known in the art, such as electroporation, calcium phosphate precipitation, or contacting with a polynucleotide-liposome complex. Genetic alteration may also be effected, for example, by transduction or infection with a DNA or RNA virus or viral vector. Generally, the genetic element is introduced into a chromosome or mini-chromosome in the cell; but any alteration that changes the phenotype and / or genotype of the cell and its progeny is included in this term.

[0129] A cell has been “genetically modified” or “transformed” or “transfected” by exogenous DNA (e.g. via a recombinant virus), when such DNA has been introduced inside the cell. The presence of the exogenous DNA results in permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.

[0130] A cell is said to be “stably” altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during extended culture of the cell in vitro and / or for an extended period of time in vivo. Generally, such a cell is “heritably” altered (genetically modified) in that a genetic alteration is introduced which is also inheritable by progeny of the altered cell.

[0131] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. Polypeptides such as anti-angiogenic polypeptides, neuroprotective polypeptides, and the like, when discussed in the context of delivering a gene product to a mammalian subject, and compositions therefor, refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein. Similarly, references to nucleic acids encoding anti-angiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for use in delivery of a gene product to a mammalian subject (which may be referred to as “transgenes” to be delivered to a recipient cell), include polynucleotides encoding the intact polypeptide or any fragment or genetically engineered derivative possessing the desired biochemical function.

[0132] An “isolated” plasmid, nucleic acid, vector, virus, virion, host cell, protein, or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from. Thus, for example, an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this disclosure are increasingly more isolated. An isolated plasmid, nucleic acid, vector, virus, host cell, or other substance is in some embodiments purified, e.g., from about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99%, or more, pure.

[0133] The term “unit dosage form” refers to physically discrete units suitable as a unitary dosage for human subjects and other animals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

[0134] The term "delivered dose" refers to the percentage of the drug in a pharmaceutical dosage form employing an aerosol based delivery system that is delivered from the mouthpiece of the device. For example, a delivered dose of 70% indicates that 70% of the total amount of drug in the dosage form was delivered from the mouthpiece of the device.

[0135] As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease (and / or symptoms caused by the disease) from occurring in a subject which may be predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease (and / or symptoms caused by the disease), i.e., arresting its development; and (c) relieving the disease (and / or symptoms caused by the disease), i.e., causing regression of the disease (and / or symptoms caused by the disease).

[0136] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, humans; non-human primates, including simians; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0137] In some embodiments, the individual is a human who has previously been naturally exposed to AAV and as a result harbors anti-AAV antibodies (i.e., AAV neutralizing antibodies). In some embodiments, the individual is a human who has previously been administered an AAV vector (and as a result may harbor anti-AAV antibodies) and needs re-administration of vector for treatment of a different condition or for further treatment of the same condition. Based on positive results in clinical trials involving AAV gene delivery to, for example, liver, muscle, and retina — all tissues affected by neutralizing antibodies against this vehicle — there are many such therapeutic applications / disease targets.

[0138] The term “effective amount” as used herein is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. For purposes of this disclosure, an effective amount of a compound (e.g., an infectious rAAV virion) is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of (and / or symptoms associated with) a particular disease state (e.g., cancer). Accordingly, an effective amount of an infectious rAAV virion is an amount of the infectious rAAV virion that is able to evade the neutralizing activity of an individual's anti-AAV antibodies, thus effectively delivering the heterologous nucleic acid to a target cell (or target cells) of the individual.

[0139] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0140] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Ranges can be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another case includes from the one particular value and / or to the other particular value.Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another case. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint,and independently of the other endpoint. The term "about" as used herein refers to a range that is 15% plus or minus from a stated numerical value within the context of the particular usage. For example, about 10 would include a range from 8.5 to 11.5. The term "about" also accounts for typical error or imprecision in measurement of values.100141] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0142] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0143] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an infectious recombinant adeno-associated virus (rAAV) virion” includes a plurality of such virions and reference to “the infectious recombinant adeno-associated virus (rAAV) virion” includes reference to one or more such virions and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only" and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0144] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each andevery combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such subcombination was individually and explicitly disclosed herein.

[0145] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0146] The present disclosure, in several embodiments, provides a combination therapy for treating cystic fibrosis (CF) comprising administering a therapeutically effective amount of each of A) a pharmaceutical composition comprising an rAAV vector, said rAAV vector comprising a capsid comprising a capsid protein comprising the amino acid sequence as set forth as SEQ ID NO: 12 or an amino acid sequence at least 90% identical to SEQ ID NO: 12 and comprising a Thr at amino acid 469 and an Ala at amino acid 598 based on the amino acid numbering set forth in SEQ ID NO: 12 and a heterologous nucleic acid comprising a nucleotide sequence encoding a cystic fibrosis transmembrane conductance regulator (CFTR protein) gene product operably linked to a promoter and (B) one or more CFTR modulators.

[0147] CFTR Modulators

[0148] CFTR modulators are active pharmaceutical ingredients (APIs) designed to correct malfunctioning CFTR proteins. CFTR modulators are a specialized group of therapies, as different modulators are designed to address the underlying defect in the CFTR protein caused by a specific CFTR mutation or class of CFTR mutations.

[0149] There are three main types of CFTR modulators: CFTR potentiators, CFTR correctors and CFTR amplifiers.

[0150] Class III CFTR mutations, such as G55 ID, are gating mutations, which prevent normal opening of the CFTR protein to facilitate chloride transport. CFTR potentiators mitigate this defect by opening the CFTR protein gates and keeping them open longer to facilitate the smooth flow of chloride ions. Ivacaftor (Kalydeco®) is an example of a CFTR potentiator developed by Vertex Pharmaceuticals. It is an oral medication approved by the U.S. Food and Drug Administration (FDA), the EU, and Health Canada for CF patients as young as 1 year with at least one mutation (such as G551D) that impairs chloride ion flow. Another example of a CFTR potentiator is the experimental treatment PTI-808 being developed by Proteostasis Therapeutics.

[0151] Class II CFTR mutations are protein processing mutations, which result in misfolding of the CFTR protein, which can affect transport of the misfolded CFTR to the cell surface. CFTR correctors assist the CFTR protein in folding correctly into its 3D conformation, allowing it to be successfully transported to the cell membrane so that it can function. Lumacaftor (VX-809) and tezacaftor (VX-661) are two therapies by Vertex Pharmaceuticals that function as correctors. Another CFTR corrector is elexacaftor. These correctors help the CFTR protein fold correctly and reach the cell surface, but fall short in alleviating CF symptoms by themselves. Therefore, they are not approved as a monotherapy for CF. Another CFTR corrector under development (by Proteostasis Therapeutics) is PTI- 801.

[0152] Class V CFTR mutations result in reduced levels of CFTR protein being present at the cell surface, for example because they result in lower levels of CFTR protein being expressed, or increase the rate of degradation of CFTR protein. Amplifiers are a type of CFTR modulator that enhances the production of CFTR protein by the cells. PTI-428 is an investigational first-generation CFTR amplifier by Proteostasis Therapeutics, which is being tested as a single and combination therapy for CF.

[0153] CFTR potentiators, correctors and amplifiers are described in the art for use independently as monotherapies. In addition, combinations of these CFTR modulators are also known, with three of the four currently approved CFTR modulator therapies being combination therapies. By way of non-limiting example, combining a potentiator with a corrector may improve CFTR activity by correcting the CFTR conformation and opening the gate to allow chloride transport. In particular, a combination of the potentiator ivacaftor and corrector lumacaftor is authorized as a combination therapy and is marketed as Orkambi® by Vertex for use in the treatment for CF patients with two F508del CFTR mutations. Another example of an authorized combination treatment is the potentiator ivacaftor and corrector tezacaftor, which is marked by Vertex as Symdeko® in the US. Another example of an authorized combination treatment is the two "correctors" vanzacaftor and tezacaftor and the "potentiator" deutivacaftor, marketed as Alyftrek®, indicated for patients with CF aged 6 years and older who have at least one F508del mutation or another responsive mutation in the CFTR gene.

[0154] Amplifiers can also be used in combination with other CFTR modulators. Combining CFTR amplifiers with other CFTR modulators may be advantageous as the CFTR amplifier can result in more CFTR protein being expressed, which can then be acted upon by the other CFTR modulator(s).

[0155] In addition to these first-generation CFTR modulators, so-called next-generation modulators may combine multiple CFTR correctors to produce combination therapies with three or more APIs. An example of an approved next-generation CFTR modulator is Trikafta®, which is a combination of the potentiator ivacaftor and two correctors, tezacaftor and elexacaftor. In some preferred embodiments, the pharmaceutical combination comprises (A) a combination of ivacaftor, tezacaftor and elexacaftor and (B) a pharmaceutical composition comprising an rAAV vector, said rAAV vector comprising a capsid comprising a capsid protein comprising the amino acid sequence as set forth as SEQ ID NO: 12 or an amino acid sequence at least 90% identical to SEQ ID NO: 12 and comprising a Thr at amino acid 469 and an Ala at amino acid 598 based on the amino acid numbering set forth in SEQ ID NO: 12 and a heterologous nucleic acid comprising a nucleotide sequence encoding a CFTR protein gene product operably linked to a promoter.

[0156] Another example of an approved next-generation CFTR modulator is Alyftrek®, which is a combination of the potentiator deutivacaftor and two correctors, vanzacaftor and tezacaftor. In some preferred embodiments, the pharmaceutical combination comprises (A) a combination of deutivacaftor, vanzacaftor and tezacaftor, and (B) a pharmaceutical composition comprising an rAAV vector, said rAAV vector comprising a capsid comprising a capsid protein comprising the amino acid sequence as set forth as SEQ ID NO: 12 or an amino acid sequence at least 90% identical to SEQ ID NO: 12 and comprising a Thr at amino acid 469 and an Ala at amino acid 598 based on the amino acid numbering set forth in SEQ ID NO: 12 and a heterologous nucleic acid comprising a nucleotide sequence encoding a CFTR protein gene product operably linked to a promoter.

[0157] Any reference to a CFTR modulator herein encompasses any and all salts, derivatives and analogues of said CFTR modulator, unless expressly stated to the contrary. Thus, the invention relates to the combination of a known CFTR modulators such as those individualized herein (particularly ivacaftor) or a salt, derivative or analogue thereof. Salt forms preferably include pharmaceutically acceptable salts. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or with organic acids such as acetic, oxalic, tartaric, maleic, and the like. An “analogue” of a CFTR modulator, as the term is used herein, refers to a chemical structure that preserves substantial similarity with the CFTR modulator, although it may not be readily derived synthetically from the CFTR modulator. A related chemical structure that is readily derived synthetically from a CFTR modulator structure is referred to as a “derivative.” These are all within the scope of the invention. By way of non-limiting example, a reference to “ivacaftor” includes salt forms, analogues and derivatives of ivacaftor, such as deuterated ivacaftor (D-ivacaftor).

[0158] One or more CFTR modulators for use in combination with a pharmaceutical composition as described herein may include one or more CFTR potentiators, one or more CFTR correctors and / or one or more CFTR amplifiers, or combinations thereof. Non-limiting examples of CFTR modulators that may be used in combination with a pharmaceutical composition as described herein, preferably comprising an rAAV comprising capsid protein of SEQ ID NO: 12 and a heterologous nucleic acid comprising the sequence of SEQ ID NO:43, according to the present invention include ivacaftor (Kalydeco®), PTI-808, VX-809 (Lumacaftor) and VX-661 (tezacaftor), elexacaftor, PTI-801, PTI-428, as well as combinations such as ivacaftor+lumacaftor (Orkambi®), ivacaftor+tezacaftor (Symdeko® or Symkevi®) and ivacaftor+tezacaftori-elexacaftor)(Trikafta®), and deutivacaftor + vanzacaftor+tezacaftor (Alyftrek®).

[0159] By way of non-limiting example, ivacaftor may be administered at a dose of from about 5 mg to about 150 mg, preferably from about 25 mg to about 150 mg, such as about 150 mg every 12 hours. By way of a further non-limiting example, for paediatric dosing ivacaftor may be administered at a dose of about 75 mg every 12 hours,

[0160] By way of a further non-limiting example, Trikafta® (elexacaftor+tezacaftor+ivacaftor) may be administered eveiy 12 hours, with a first (typically morning) dose of Trikafta® typically comprising about 200 mg elexacaftor, about 100 mg tezacaftor and about 150 mg ivacaftor (e.g. in the form of 2 tablets each containing elexacaftor+tezacaftor+ivacaftor), and a second (typically evening) dose of about 150 mg ivacaftor (e.g. in the form of 2 tablets).

[0161] By way of a further non-limiting example, Orkambi® (lumacaftor+ivacaftor) may be administered every 12 hours, with each dose of Orkambi® typically comprising about 400 mg lumacaftor and about 250 mg ivacaftor (e.g. in the form of 2 tablets each containing lumacaftor+ivacaftor). By way of a further non-limiting example, for paediatric dosing Orkambi® may be administered eveiy 12 hours, with each dose of Orkambi® typically comprising about 200 mg lumacaftor and about 250 mg ivacaftor (e.g. in the form of 2 tablets each containing lumacaftor+ivacaftor).

[0162] By way of a further non-limiting example, Symdeko® (tezacaftor+ivacaftor) may be administered every 12 hours, with a first (typically morning) dose of Symdeko® typically comprising about 100 mg tezacaftor and about 150 mg ivacaftor (e.g. in the form of a single tablet containing tezacaftor+ivacaftor), and a second (typically evening) dose of about 150 mg ivacaftor (e.g. in the form of 1 tablet).

[0163] By way of a further non-limiting example, Alyftrek®(deutivacaftor+vanzacaftor+tezacaftor) may be administered every 24 hours (once daily oral),as three tablets containing 4mg vanzacaftor, 20 mg tezacaftor and 50 mg deutivacaftor for weight less than 40 kg or two tablets containing lOmg vanzacaftor, 50 mg tezacaftor and 125 mg deutivacaftor for weight greater than or equal to 40 kg.

[0164] Preferably the invention relates to the combination of a pharmaceutical composition as described herein, with a CFTR modulator selected from ivacaftor, tezacaftor, elexacaftor or lumacaftor, of a combination thereof. The combination of a pharmaceutical composition comprising an rAAV comprising a capsid protein of SEQ ID NO: 12 and a heterologous nucleic acid comprising the sequence of SEQ ID NO:43, with the CFTR modulator (specifically the CFTR potentiator) ivacaftor, and optionally one or more additional CFTR modulators, is preferred. The combination of a pharmaceutical composition comprising an rAAV comprising a capsid protein of SEQ ID NO: 12 and a heterologous nucleic acid comprising the sequence of SEQ ID NO:43, with the CFTR modulators ivacaftor, tezacaftor and elexacaftor is particularly preferred as is the combination of a pharmaceutical composition comprising an rAAV comprising a capsid protein of SEQ ID NO: 12 and a heterologous nucleic acid comprising the sequence of SEQ ID NO:43, with the CFTR modulators deutivacaftor, vanzacaftor and tezacaftor.

[0165] Therapeutic Indications

[0166] A pharmaceutical composition as presently described, comprising an rAAV vector, said rAAV vector comprising a capsid comprising a capsid protein comprising the amino acid sequence as set forth as SEQ ID NO: 12 or an amino acid sequence at least 90% identical to SEQ ID NO: 12 and comprising a Thr at amino acid 469 and an Ala at amino acid 59S based on the amino acid numbering set forth in SEQ ID NO: 12 and a heterologous nucleic acid comprising a nucleotide sequence encoding a cystic fibrosis transmembrane conductance regulator (CFTR protein) gene product operably linked to a promoter, are capable of efficient airway transduction and persistent gene expression. Typically, a pharmaceutical composition as described herein, as a monotherapy or as part of a combination therapy as described herein, may transduce airway epithelial cells at a transduction rate of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more. As defined herein, the term “airway epithelial cell” encompasses any cell found within the airway epithelium, as described herein, including but not limited to basal cells and submucosal gland duct cells in the upper airways, goblet, club cells and neuroendocrine cells in the bronchiolar airways, bronchioalveolar stem cells in the terminal bronchioles and type II pneumocytes in the alveoli, and any combination thereof

[0167] As such, in some embodiments, a pharmaceutical composition comprising an rAAV vector, said rAAV vector comprising a capsid comprising a capsid protein comprisingthe amino acid sequence as set forth as SEQ ID NO: 12 or an amino acid sequence at least 90% identical to SEQ ID NO: 12 and comprising a Thr at amino acid 469 and an Ala at amino acid 598 based on the amino acid numbering set forth in SEQ ID NO: 12 and a heterologous nucleic acid comprising a nucleotide sequence encoding CFTR protein gene product operably linked to a promoter, can be administered as a single unit dose to a subject with CFTR, either alone or as part of a combination therapy with one or more CFTR modulators as herein described, to achieve long-term therapeutic expression of the CFTR gene product. In some preferred embodiments, the single unit dose is about 5x1014vg or about 2.5x1014vg or about 1.25x1014vg of the rAAV vector, more preferably is about 8x1013vg to about 3x1014vg, or about 8x1013vg, about 1.6x1014vg or about 3x1014vg of the rAAV. In related embodiments, the single unit dose is effective to provide a total clinical delivered dose of about 5x1013vg to about 4-7x1014vg, preferably about 6x1013vg, about 1.2x101+vg or about 2.3x1 O’4vg of the rAAV to the lungs of the subject.

[0168] In other embodiments, the pharmaceutical composition can be administered multiple times to a subject with CF either as a monotherapy (regardless of the underlying CFTR mutation) or as part of a combination therapy with one or more CFTR modulators as herein described. In some embodiments, the subject with CF has antibodies against the rAAV capsid prior to administering the pharmaceutical composition. In some embodiments, the subject with CF has antibodies against a capsid protein of SEQ ID NO: 12 prior to administering the pharmaceutical composition.

[0169] The present inventors have discovered that the therapeutic effect of combining one or more CFTR modulators with a pharmaceutical composition as herein described is at least additive relative to either monotherapy alone. When administered to a subject with CF, a combination therapy as herein described can cure, delay, reduce the severity of, or ameliorate one or more symptoms, and / or prolong the survival of a patient beyond that expected in the absence of such treatment and / or beyond that expected using a conventional CF treatment (e.g. a CFTR modulator, particularly a CFTR potentiator, alone). Thus, a combination therapy as herein described may be administered to a patient with CF to ameliorate the disease and / or prolong the survival of a patient with CF beyond that expected in the absence of such treatment and / or beyond that expected using a conventional CF treatment (e.g. a CFTR modulator or CFTR potentiator alone).

[0170] Combination therapy as herein described encompasses both concurrent and sequential administration of the pharmaceutical composition and one or more CFTR modulators. Concurrent administration of the pharmaceutical composition and one or more CFTR modulators is also encompassed.

[0171] For “concurrent” administration, the administration of the pharmaceutical composition may still be occurring when the delivery of the CFTR modulator begins, or the delivery of CFTR modulator may still be occurring when the delivery of the pharmaceutical composition begins, so that there is overlap in terms of administration. Concurrent administration may encompass delivery of the pharmaceutical composition and CFTR modulator within weeks to months or even years of each other, typically so that delivery of the pharmaceutical composition overlaps with the delivery of the CFTR modulator.

[0172] Alternatively, the delivery of the pharmaceutical composition may end before the delivery of the CFTR modulator begins, or the delivery of the CFTR modulator may end before delivery of the pharmaceutical composition begins. Sequential administration may involve the pharmaceutical composition and CFTR modulator being administered within 30 minutes, I hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours or 24 hours, 1 week, 2 weeks, 1 month, 2 months, or longer of each other.

[0173] The CFTR modulator may be administered at an interval of once every hour, once every 2 hours, once every 3 hours, once every 4 hours, once every 6 hours, once every 8 hours, once every 12 hours, daily, once every 2 days or more. Typically the CFTR modulator is administered about every 12 hours or about once per day.

[0174] The pharmaceutical composition may be administered once every month, once every 2 months, once every 3 months, once every 4 months, once every 6 months, once every 8 months, once every 12 months or more. As the frequency of administration of the pharmaceutical composition is lower than the frequency of administration of the CFTR modulator, administration of the combination therapy is typically by sequential administration.

[0175] Treatment with a pharmaceutical composition and / or CFTR modulator at the desired dosing frequency may be continued for as long as required, for example, for at least six months, at least one year, two years, three years, four years, five years, ten years, fifteen years, twenty years, or more, up to for the lifetime of the patient to be treated.

[0176] Typically the treatment is more effective because of combined administration. For example, treatment with the CFTR modulator may be more effective, e.g., an equivalent effect is seen with less of the CFTR modulator, or the CFTR modulator reduces symptoms to a greater extent, than would be seen if the CFTR modulator were administered in the absence of the pharmaceutical composition, or the analogous situation is seen with the pharmaceutical composition. Typically, delivery is such that the reduction in a symptom, or other parameter related to CF is greater than what would be observed with the CFTR modulator delivered inthe absence of the pharmaceutical composition, or the analogous situation is seen with the pharmaceutical composition.

[0177] The duration of action of a combination therapy according to the invention may be for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 12 weeks, at least six months, at least 1 year or more. Typically this is assessed relative to the last administration of the pharmaceutical composition and / or CFTR modulator, particularly the last administration of the pharmaceutical composition.

[0178] A pharmaceutical composition according to the invention is typically administered by inhalation. Accordingly, the pharmaceutical composition may be formulated for inhalation, as described herein. The one or more CFTR modulator may be administered by any appropriate route, and may be formulated accordingly. In particular, the one or more CFTR modulator may be administered orally, and may be formulated for oral administration.|00179] The combination therapies of the invention may restore CFTR expression and / or activity to a level , which provides a therapeutic benefit A combination therapy may restore (increase) CFTR expression and / or activity to a level which matches or exceeds CFTR expression and / or activity in a healthy control. However, restoration of healthy CFTR expression and / or activity is not essential to achieve a therapeutic benefit. Rather, the therapeutic threshold, i.e. the level above which a therapeutic benefit is achieved may be lower than the level of CFTR expression and / or activity in a healthy control. Indeed, patients, particularly those with a class I CFTR mutation resulting in null CFTR expression, may receive a therapeutic benefit from even relatively small increases in CFTR expression and / or activity (e.g. 5% or 10% CFTR expression and / or activity compared to healthy control expression levels).

[0180] A pharmaceutical composition as described herein, particularly in the context of a combination therapy as herein described may increase (restore) CFTR expression (particularly cellular CFTR expression levels and / or global expression in the lungs or respiratory tree) to at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 120% or more of CFTR expression in a healthy control. Typically a pharmaceutical composition as described herein, particularly in the context of a combination therapy as herein described may increase (restore) CFTR expression, particularly CFTR cellular expression levels, to at least 20%, preferably at least 50%, more preferably at least 75% of CFTR expression in a healthy control. A pharmaceutical composition as described herein, particularly in the context of a combination therapy as herein described may increase (restore) CFTR expression, particularly global CFTR expression in the lungs, to at least 5%,at least 10%, preferably at least 20% of CFTR expression in a healthy control. Expression levels of a transgene and / or the encoded therapeutic protein of the invention may be measured in the lung tissue, A high and / or therapeutic expression level may therefore refer to the concentration in the lung. CFTR expression may be quantified using one or more of the following techniques: CFTR RNA expression in lung tissue, CFTR protein expression in lung tissue, PK assays (vector copy number, integration), DNA content in sputum (as surrogate for NETosis).

[0181] Other endpoints for efficacy assessment of treatment according to the embodiments herein described include: improvement in FEV1 (lung function); MRI and / or CT for efficacy assessment; reduced expression of one or more biomarker of inflammation, such as IL-8, IL 1 beta, IL-6, TNFalpha (typically measured in sputum), calprotectin (typically measured in serum); differential cell count in sputum; reduced surfactant protein D (SP-D) in serum as marker for reduced epithelial injury; reduction of pulmonary exacerbations; improvement of Lung Clearance index, Quality of Life as patient reported outcome via CFQ- R respiratory domain (and / or other appropriate questionnaires as well), exploratory imaging endpoints (to show improved lung ventilation, mucus plugging and / or others, e.g. via Eichinger score of mRI).

[0182] Alternatively or in addition, a combination therapy as herein described may increase (restore) CFTR activity (particularly cellular CFTR activity and / or global activity in the lungs or respiratory tree) to at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 120% or more of CFTR activity in a healthy control. Typically a combination therapy as herein described may increase (restore) CFTR activity, particularly CFTR cellular activity, to at least 20%, preferably at least 50%, more preferably at least 75% of CFTR activity in a healthy control. A pharmaceutical composition as described herein, particularly in the context of a combination therapy as herein described may increase (restore) CFTR activity, particularly global CFTR expression in the lungs, to at least 5%, at least 10%, preferably at least 20% of CFTR activity in a healthy control.

[0183] As used herein, typically a healthy control is an equivalent individual or population who do not have CF. Preferably a healthy control is an equivalent individual or population who do not have CF and is otherwise in good health. A healthy control may be matched with the subject using standard clinical methodology (e.g. age / sex matching, or matching based on other criteria). Other controls include an equivalent individual with CF who has been treated with either the pharmaceutical composition or CFTR modulator alone.

[0184] CFTR activity may be defined in terms of the CFTR RNA expression in lung tissue; CFTR protein expression in lung tissue and / or activity of the CFTR channel itself (e.g. by electrophysiological measurements using patient's bronchial brushings).

[0185] A combination therapy as herein described may increase (restore) CFTR current by at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 2 fold or more compared with treatment with the pharmaceutical composition alone (i.e. compared with the increase in CFTR current achieved when treating with the pharmaceutical composition alone). A combination therapy as herein described may increase (restore) CFTR current by between about 1 .3 fold to about 3 fold or between about 1.2 fold to about 2 fold compared with treatment with the pharmaceutical composition alone (i.e. compared with the increase in CFTR current achieved when treating with the pharmaceutical composition alone).Preferably, a combination therapy as herein described may increase (restore) CFTR current by at least about 1.2 fold, at least about 1.3 fold, at least about 1.5 fold, at least about 1.8 fold, such as by about 1.3 fold to about 1.8 fold compared with treatment with the pharmaceutical composition alone.

[0186] A combination therapy as herein described may increase (restore) CFTR current to at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 120% or more of CFTR current in a healthy control. Typically a combination therapy as herein described may increase (restore) CFTR cellular current to at least 20%, preferably at least 50%, more preferably at least 75% of CFTR current in a healthy control. A pharmaceutical composition as described herein, particularly in the context of a combination therapy as herein described may increase (restore) CFTR current, particularly globally in the lungs, to at least 5%, at least 10%, preferably at least 20% of CFTR current in a healthy control.

[0187] The combination therapy herein described for use in treating CF may be administered to a subject with at least one class II CFTR mutation.

[0188] Treatment of CF according to the present disclosure encompasses CF caused by any combination of class I, II, III, IV, V and / or VI mutations. A patient to be treated may have CF caused by one or more class I mutation, one or more class II mutation, one or more class III mutation, one or more class IV mutation, one or more class V mutation and / or one or more class VI mutation. A patient to be treated may have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6 at least 7, at least 8, at least 9, at least 10 or more mutations in the CFTR gene, which may each be independently selected from a class I, class II, class III, class IV and / or class V mutation as described herein.

[0189] A patient to be treated according to the invention may have at least one class I and / or class II CFTR mutation, such as those described herein. A patient to be treated may have (i) at least one class I CFTR mutation that is optionally selected from G542X, W1282X and / or R553C; and / or (ii) at least one class II CFTR mutation that is optionally selected from F508del, N1303K and / or I507del.

[0190] The present disclosure further provides infectious recombinant adeno-associated virus (rAAV) virions that comprise a variant capsid protein and a heterologous nucleic acid. The present disclosure further provides the variant adeno-associated virus (AAV) capsid proteins (and / or a nucleic acid encoding the variant AAV capsid proteins), which confer to an infectious rAAV virion an increased resistance to human AAV neutralizing antibodies. The present disclosure further provides host cells comprising an infectious rAAV virion and / or a nucleic acid encoding a subject variant AAV capsid protein. The present disclosure further provides libraries of the above virions, capsid proteins, nucleic acids, and / or host cells; where the variant AAV capsid protein of at least one member of the library comprises an amino acid sequence having at least one amino acid substitution relative to the amino acid sequence set forth in one of SEQ ID NOs:10-13 and 26-33.

[0191] The present disclosure further provides methods of delivering a heterologous nucleic acid to a target cell where the target cell is contacted with a subject infectious rAAV virion. The present disclosure further provides methods of delivering a gene product to an individual, the methods generally involving administering an effective amount of a subject rAAV virion to an individual in need thereof. Also provided herein are compositions and kits for practicing the subject methods. In many embodiments, a subject infectious rAAV virion, a subject nucleic acid, a subject variant AAV capsid protein, a subject host cell, etc., is isolated.

[0192] Variant AAV Capsid Polypeptides

[0193] A subject variant AAV capsid polypeptide (or the variant AAV capsid protein encoded by a subject nucleic acid) confers to an infectious rAAV virion comprising the variant / AAV capsid polypeptide an increased resistance to human AAV neutralizing antibodies compared to the resistance exhibited by a wild type AAV (e.g., AAV2 (wild type AAV serotype 2)) or an AAV comprising a wild-type capsid protein. In some embodiments, the increased resistance is at least about 1.5-fold (e.g., at least about 1.5-fold, at least about 2- fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold, at least about 17-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50- fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, etc.) greater than theresistance exhibited by a wild type AAV (e.g., AAV2 (wild type AAV serotype 2)) or an AAV comprising a wild-type capsid protein.

[0194] A subject variant AAV capsid protein (or the variant AAV capsid protein encoded by a subject nucleic acid) can be said to confer to an infectious rAAV virion an increased transduction of mammalian cells in the presence of human AAV neutralizing antibodies compared to the transduction exhibited by a wild type AAV (e.g., AAV2 (wild type AAV serotype 2)) or an AAV comprising a wild-type capsid protein. In some embodiments, the increased transduction is at least about 1.5-fold (e.g., at least about 1 .5-fold, at least about 2-fold, at least about 3 -fold, at least about 4-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold, at least about 17-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, etc.) greater than the transduction exhibited by a wild type AAV (e.g., AAV2 (wild type AAV serotype 2)) or an AAV comprising a wild-type capsid protein.

[0195] In some embodiments, a subject variant AAV capsid protein (or the variant AAV capsid protein encoded by a subject nucleic acid) exhibits decreased binding to a neutralizing antibody that binds a wild-type AAV capsid protein. For example, a subject variant AAV capsid protein can exhibit at least about 1.5-fold (e.g., at least about 1.5-fold, at least about 2- fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold, at least about 17-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, etc.) reduced binding (e.g., reduced affinity) to a neutralizing antibody that binds a wild-type capsid AAV protein, compared to the binding affinity of the antibody to wild-type AAV capsid protein.

[0196] In some embodiments, an anti-AAV neutralizing antibody binds to a subject variant AAV capsid protein (or the variant AAV capsid protein encoded by a subject nucleic acid) with an affinity of less than about 107M, less than about 5 * 10~6M, less than about 10~6M, less than about 5x10-5M, less than about 10-5M, less than about 10-J*M, or lower.

[0197] The term “variant capsid protein” does not encompass wild type AAV capsid proteins. A “variant AAV capsid protein” does not comprise an amino acid sequence present in a naturally occurring AAV capsid protein. For example, a subject variant capsid protein does not comprise an amino acid sequence having 100% sequence identity to any of the sequences set forth in SEQ ID NOs: 1-9. In other words, a subject variant capsid protein doesnot comprise an amino acid sequence as set forth in any of SEQ ID NOs:l-9. A variant capsid protein can differ in amino acid sequence from a “starter” or “parental” AAV capsid protein, which parental AAV capsid protein may be a wild-type AAV capsid protein or non-wild-type AAV capsid protein.

[0198] In some embodiments a subject variant AAV capsid protein (or the variant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to amino acids 203-736 of the amino acid sequence set forth in one of SEQ ID NOs:10-13 and 26-33.

[0199] In some embodiments a subject variant AAV capsid protein (or the variant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence having at least about 90% (e.g., at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to the amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26- 33.

[0200] In some embodiments a subject variant AAV capsid protein (or the variant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence having at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to amino acids 203- 736 of the amino acid sequence set forth in SEQ ID NO: 10, and includes the amino acid substitutions N3I2K, N449D, D472N, N551S, I698V, and L735Q relative to the AAV capsid protein of AAV2 (e.g., SEQ ID NO: 2), or the corresponding positions in another AAV parental serotype.

[0201] In some embodiments a subject variant AAV capsid protein (or the variant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence having at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, and includes the amino acid substitutions N312K, N449D, D472N, N551 S, I698V, and L735Q relative to the AAV capsid protein of AAV2 (e.g., SEQ ID NO: 2), or the corresponding positions in another AAV parental serotype.

[0202] In some embodiments a subject variant AAV capsid protein (or the variant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence having at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to amino acids 203-736 of the amino acid sequence set forth in SEQ ID NO:31, and includes the amino acid substitutions N312K, N449D, N551 S, and I698V relative to the AAV capsid protein of AAV2 (e.g., SEQ ID NO;2), or the corresponding positions in another AAV parental serotype.

[0203] In some embodiments a subject variant AAV capsid protein (or the variant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence having at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:31 , and includes the amino acid substitutions N312K, N449D, N551 S, and I698V relative to the AAV capsid protein of AAV2 (e.g., SEQ ID NO:2), or the corresponding positions in another AAV parental serotype.

[0204] In some embodiments a subject variant AAV capsid protein (or the variant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence having at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to amino acids 203- 736 of the amino acid sequence set forth in SEQ ID NO:32, and includes the amino acid substitutions D180N, N312K, Q385R, N449D, N551S, 1698 V, and S721T relative to the AAV capsid protein of AAV2 (e.g., SEQ ID NO:2), or the corresponding positions in another AAV parental serotype.

[0205] In some embodiments a subject variant AAV capsid protein (or the variant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence having at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:32, and includes the amino acid substitutions DI 80N, N312K, Q385R,N449D, N551S, I698V, and S721T relative to the AAV capsid protein of AAV2 (e.g., SEQ ID NO:2), or the corresponding positions in another AAV parental serotype.

[0206] In some embodiments a subject variant AAV capsid protein (or the variant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence having at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to amino acids 203- 736 of the amino acid sequence set forth in SEQ ID NO:33, and includes the amino acid substitutions N312K, N449D, T450A, N551S, and 1698 V relative to the AAV capsid protein of AAV2 (e.g., SEQ ID NO:2), or the corresponding positions in another AAV parental serotype.

[0207] In some embodiments a subject variant AAV capsid protein (or the variant AAV capsid protein encoded by a subject nucleic acid) comprises an amino acid sequence having at least about 95% (e.g., at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or 100%) amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:33, and includes the amino acid substitutions N312K, N449D, T450A, N551S, and I698V relative to the AAV capsid protein of AAV2 (e.g., SEQ ID NO:2), or the corresponding positions in another AAV parental serotype.

[0208] Exemplary variant AAV capsid proteins include, but are not limited to (see FIGS. 8-10 for selected exemplary sequence alignments):

[0209] SM 10-2 (amino acid sequence)(SEQ ID NO: 10); SM 10-2 (nucleotide sequenceXSEQ IDNO:22); Shuffle 100-1 (amino acid sequence) (SEQ ID NO: 11); Shuffle 100-1 (nucleotide sequence) (SEQ ID NO: 23);

[0210] Shuffle 100-3 (amino acid sequence) (SEQ ID NO: 12):MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPF NGLDKGEPVNEADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQQRLQGDTSFG GNLGRAVFQAKKRVLEPLGLVEQAGETAPGKKRPLIESPQQPDSSTGIGKKGKQPAK KRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNA SGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPW GYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNL TSTVQVFSDSDYQLPYVLGSAHEGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFY CLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQN QSGSAQNKDLLFSRGSPTGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGAS KYNLNGRESIINPGTAMASHKDDKDKFFPMSGVMIFGKESAGASNTALDNVMITDEE EIKATNPVATERFGTVAVNLQSSSTDPATGDVHAMGALPGMVWQDRDVYLQGPIW AKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQ VSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRP L;

[0211] Shuffle 100-3 (nucleotide sequence) (SEQ ID NO: 24): atggctgctgatggttatcttccagattggctcgaggacactctctctgaaggaataagacagtggtggaagctcaaacctggcccacc accaccaaagcccgcagagcggcataaggacgacagcaggggtcttgtgcttcctgggtacaagtacctcggacccttcaacggact cgacaagggagagccggtcaacgaggcagacgcagcggccctcgagcacgacaaggcctacgaccagcagctcaaggccggtg acaacccctacctcaagtacaaccacgccgacgcggagttccagcagcggcttcagggcgacacatcgtttgggggcaacctcggc agagcagtcttccaggccaaaaagagggttcttgaacctcttggtctggttgagcaagcgggtgagacggctcctggaaagaagaga ccgttgattgaatccccccagcagcccgactcctccacgggtatcggcaaaaaaggcaagcagccggctaaaaagagactcaattttg gtcagactggcgactcagagtcagtccccgacccacaacctctcggagaacctccagcaacccccgctgctgtgggacctactacaatggcttcaggtggtggcgcaccaatggcagacaataacgaaggcgccgacggagtgggtaatgcctcaggaaattggcattgcgatt ccacatggctgggcgacagagtcatcaccaccagcacccgcacctgggccttgcccacctacaataaccacctctacaagcaaatctc cagtgcttcaacgggggccagcaacgacaaccactacttcggctacagcaccccctgggggtatttgacttcaacagattccactgcc acttttcaccacgtgactggcagcgactcatcaacaacaattggggattccggcccaagagactcaacttcaaactcttcaacatccaag tcaaggaggtcacgacgaatgatggcgtcacaaccatcgctaataaccttaccagcacggttcaagtcttctcggactcagactatcag ctcccgtacgtgctcgggtcggctcacgagggctgcctcccgccgttcccagcagacgtcttcatggtgccacagtatggatacctcac cctgaacaacgggagtcaggcagtaggacgctcttcattttactgcctggagtactttccttctcagatgctgcgtaccggaaacaacttt accttcagctacacttttgaggacgttcctttccacagcagctacgctcacagccagagtetggaccgtctcatgaatcctctcatcgacc agtacctgtattacctgaacagaactcagaatcagtccggaagtgcccaaaacaaggactgctgtttagccgggggtctccaactggc atgtctgttcagcccaaaaactggctacctggaccctgttatcggcagcagcgcgtttctaaaacaaaaacagacaacaacaacagca actttacctggactggtgcttcaaaatataaccttaatgggcgtgaatctataatcaaccctggcactgctatggcctcacacaaagacga caaagacaagttctttcccatgagcggtgtcatgatttttggaaaggagagcgccggagcttcaaacactgcattggacaatgtcatgat cacagacgaagaggaaatcaaagccactaaccccgtggccactgaaagattgggactgtggcagtcaatctccagagcagcagca cagaccctgcgaccggagatgtgcatgccatgggagccttacctggaatggtgtggcaagacagagacgtatacctgcagggtccta tttgggccaaaattcctcacacggatggacactttcacccgtctcctctcatgggcggctttggactcaagaacccgcctcctcagatcct catcaaaaacacgcctgttcctgcgaatcctccggcggagttttcagctacaaagtttgcttcattcatcacccagtattccacaggacaa gtgagcgtggagattgaatgggagctgcagaaagaaaacagcaaacgctggaatcccgaagtgcagtatacatctaactatgcaaaa tctgccaacgttgatttcactgtggacaacaatggactttatactgagcctcgccccattggcacccgttacctcacccgtcccctgtaa;

[0212] Shuffle 100-7 (amino acid sequence) (SEQ ID NO: 13); Shuffle 100-7 (nucleotide sequence) (SEQ ID NO: 25); Shuffle 10-2 (amino acid sequence) (SEQ ID NO: 26); Shuffle 10-2 (nucleotide sequence) (SEQ ID NO: 34); Shuffle 10-6 (amino acid sequence) (SEQ ID NO: 27); Shuffle 10-6 (nucleotide sequence) (SEQ ID NO: 35); Shuffle 10-8 (amino acid sequence) (SEQ ID NO: 28); Shuffle 10-8 (nucleotide sequence) (SEQ ID NO: 36); Shuffle 100-2 (amino acid sequence) (SEQ ID NO: 29); Shuffle 100-2 (nucleotide sequence) (SEQ ID NO: 37); SM 10-1 (amino acid sequence) (SEQ ID NO: 30); SM 10-1 (nucleotide sequence) (SEQ ID NO: 38); SM 10-8 (amino acid sequence) (SEQ ID NO: 31); SM 10-8 (nucleotide sequence) (SEQ ID NO: 39); SM 100-3 (amino acid sequence) (SEQ ID NO: 32); SM 100-3 (nucleotide sequence) (SEQ ID NO: 40); SM 100-10 (amino acid sequence) (SEQ ID NO: 33); and SM 100-10 (nucleotide sequence) (SEQ ID NO: 41).

[0213] Nucleic Acids and Host Cells

[0214] The present disclosure provides nucleic acids comprising nucleotide sequences encoding a variant AAV capsid protein (as described above), as well as host cells comprising a subject nucleic acid. The nucleic acids and host cells are useful for generating rAAV virions (as described below).

[0215] The present disclosure provides host cells, e.g., isolated host cells, comprising a subject nucleic acid. A subject host cell can be referred to as a “genetically modified host cell” and is typically an isolated cell, e.g., a cell in in vitro culture. A subject host cell is useful for producing a subject rAAV virion, as described below. Where a subject host cell is used to produce a subject rAAV virion, it is referred to as a “packaging cell.” In some embodiments, a subject host cell is stably genetically modified (i.e., stably transfected) with a subject nucleic acid. In other embodiments, a subject host cell is transiently genetically modified (i.e., transiently transfected) with a subject nucleic acid.

[0216] A subject nucleic acid is introduced stably or transiently into a host cell, using established techniques, including, but not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, and the like. For stable transformation, a subject nucleic acid will generally further include a selectable marker, e.g., any of several well-known selectable markers such as neomycin resistance, and the like.

[0217] A subject host cell is generated by introducing a subject nucleic acid into any of a variety of cells, e.g., mammalian cells, including, e.g., murine cells, and primate cells (e.g., human cells). Suitable mammalian cells include, but are not limited to, primary cells and cell lines, where suitable cell lines include, but are not limited to, 293 cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1 / 2 fibroblasts, CHO cells, and the like.

[0218] In some embodiments, a subject host cell includes, in addition to a nucleic acid comprising a nucleotide sequence encoding a mutant capsid protein, a nucleic acid that comprises a nucleotide sequence encoding one or more AAV rep proteins. In other embodiments, a subject host cell further comprises an rAAV vector, as described below. As described in more detail below, an rAAV virion is generated using a subject host cell.

[0219] Infectious rAAV Virions

[0220] A subject infectious rAAV virion comprises a variant AAV capsid protein and a heterologous nucleic acid (described in greater detail below), and exhibits an increased resistance to human AAV neutralizing antibodies compared to the resistance exhibited by a wild type AAV (e.g., AAV2 (wild type AAV serotype 2)) or an AAV comprising a wild-type capsid protein. By “increased resistance” it is meant that a subject infectious rAAV virionexhibits an increased infectivity in the presence of human anti-AAV antibodies. As described above, viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Thus in increased infectivity means an increased ratio of infectious viral particles to total viral particles. To determine resistance of an AAV to human anti-AAV antibodies, infectivity of the AAV is measured in the presence of various concentrations of human anti- AAV antibodies in order to obtain the antibody concentration (e.g., serum concentration, IVIG concentration, etc.) (mg / mL) required to reduce gene delivery efficiency (i.e., infectivity) to 50% of that in the absence of human anti-AAV antibodies. A virus that requires a higher antibody concentration to reduce gene delivery efficiency to 50% of that in the absence of human anti-AAV antibodies is said to have increased resistance to antibody neutralization. Thus, a two-fold increase in resistance means a two-fold increase in the antibody concentration required to reduce gene deliveiy efficiency to 50% of that in the absence of human anti-AAV antibodies. In some embodiments, a subject infectious rAAV virion exhibits at least about 1 ,5-fold (e.g., at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold, at least about 17-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100- fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, etc.) greater resistance to human AAV neutralizing antibodies than the resistance exhibited by a wild type AAV (e.g., AAV2 (wild type AAV serotype 2)) or an AAV comprising a wild-type capsid protein.

[0221] A subject infectious rAAV virion can be said to exhibit increased transduction of mammalian cells in the presence of human AAV neutralizing antibodies. In some embodiments, a subject infectious rAAV virion exhibits at least about 1.5-fold (e.g., at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 7.5-fold, at least about 10-foid, at least about 12-fold, at least about 15- fold, at least about 17-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, etc.) greater transduction of mammalian cells in the presence of human AAV neutralizing antibodies than the transduction exhibited by a wild type AAV (e.g., AAV2 (wild type AAV serotype 2)) or an AAV comprising a wild-type capsid protein.

[0222] In some embodiments, a subject infectious rAAV virion exhibits decreased binding to a neutralizing antibody that binds a wild-type AAV capsid protein. For example, a subject infectious rAAV virion can exhibit at least about 1.5-fold (e.g., at least about 1.5-fold, at least about 2-fold, at least about 3 -fold, at least about 4-fold, at least about 5-fold, at leastabout 7.5-fold, at least about 10-fold, at least about 12-fold, at least about 15-fold, at least about 17-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 150- fold, at least about 200-fold, at least about 250-fold, at least about 300- fold, etc.) reduced binding (e.g., reduced affinity) to a neutralizing antibody that binds a wild-type capsid AAV protein, compared to the binding affinity of the antibody to wild-type AAV capsid protein.

[0223] In some embodiments, an anti-AAV neutralizing antibody binds to a subject infectious rAAV virion with an affinity of less than about 107M, less than about 5 * 10“6M, less than about 106M, less than about 5x105M, less than about 105M, less than about 10”4M, or lower.

[0224] In some embodiments, a subject infectious rAAV virion exhibits increased in vivo residence time compared to a wild-type AAV. For example, a subject infectious rAAV virion exhibits a residence time that is at least about 10%, at least about 25%, at least about 50%, at least about 100%, at least about 3-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more, longer than the residence time of a wild-type AAV.

[0225] Whether a given subject infectious rAAV virion exhibits reduced binding to a neutralizing antibody and / or increased resistance to neutralizing antibody can be determined using any convenient assay known to one of ordinary skill in the art.

[0226] In some embodiments, a subject infectious rAAV virion comprises wild-type Rep78, Rep68, Rep52, and Rep40 proteins. In other embodiments, a subject infectious rAAV virion comprises, in addition to one or more variant capsid proteins, one or more mutations in one or more of Rep78, Rep68, Rep52, and Rep40 proteins.

[0227] Heterologous Nucleic Acids

[0228] A suitable heterologous DNA molecule (also referred to herein as a “heterologous nucleic acid”) for use in a subject rAAV vector (e.g., a subject infectious rAAV virion) can be any heterologous nucleic acid. In some embodiments, the heterologous nucleic acid comprises a nucleotide sequence encoding a polypeptide (e.g., a protein that imparts some desired characteristic to the target cell, e.g., a fluorescent protein that allows for cell tracking, an enzyme that provides an activity missing or altered in the target cell, etc.). In some embodiments, the heterologous nucleic acid comprises an RNA interfering agent (as defined above).

[0229] A subject heterologous nucleic acid will generally be less than about 5 kilobases (kb) in size and will include, for example, a gene (a nucleotide sequence) that encodes a protein that is defective or missing from a recipient individual or target cell; a gene that encodes a protein having a desired biological or therapeutic effect (e.g., an antibacterial, antiviral or antitumor / anti-cancer function); a nucleotide sequence that encodes an RNA that inhibits or reduces production of a deleterious or otherwise undesired protein (e.g., a nucleotide sequence that encodes an RNA interfering agent, as defmed above); and / or a nucleotide sequence that encodes an antigenic protein.

[0230] Suitable heterologous nucleic acids include, but are not limited to, those encoding proteins used for the treatment of endocrine, metabolic, hematologic, cardiovascular, neurologic, musculoskeletal, urologic, pulmonary and immune disorders, including such disorders as inflammatory diseases, autoimmune, chronic and infectious diseases, such as acquired immunodeficiency syndrome (AIDS), cancer, hypercholestemia, lysosomal storage diseases such as Activator Deficiency / GM2 Gangliosidosis, Alpha-mannosidosis, Aspartylglucosaminuria, Cholesteryl ester storage disease, Chronic Hexosaminidase A Deficiency, Cystinosis, Danon disease, Fabry disease, Farber disease, Fucosidosis, Galactosialidosis, Gaucher Disease, GM1 gangliosidosis, I-Cell disease / Mucolipidosis II, Infantile Free Sialic Acid Storage Disease / ISSD, Juvenile Hexosaminidase A Deficiency, Krabbe disease, Lysosomal acid lipase deficiency, Metachromatic Leukodystrophy, Mucopolysaccharidoses disorders (including Pseudo-Hurler polydystrophy / Mucolipidosis IIIA, MPSI Hurler Syndrome, MPSI Scheie Syndrome, MPS I Hurler-Scheie Syndrome, MPS II Hunter syndrome, Sanfilippo syndrome Type A / MPS III A, Sanfilippo syndrome Type B / MPS III B, Sanfilippo syndrome Type C / MPS III C, Sanfilippo syndrome Type D / MPS III D, Morquio Type A / MPS IVA, Morquio Type B / MPS IVB, MPS IX Hyaluronidase Deficiency, MPS VI Maroteaux-Lamy, MPS VII Sly Syndrome, Mucolipidosis I / Sialidosis, Mucolipidosis IIIC, and Mucolipidosis type IV), Multiple sulfatase deficiency, Niemann-Pick Disease, Neuronal Ceroid Lipofuscinoses, Pompe disease / Glycogen storage disease type II, Pycnodysostosis, Sandhoff disease / Adult On$et / GM2 Gangliosidosis, Sandhoff disease / GM2 gangliosidosis — Infantile, Sandhoff disease / GM2 gangliosidosis — Juvenile, Schindler disease, Salla disease / Sialic Acid Storage Disease, Tay-Sachs / GM2 gangliosidosis, and Wolman disease, insulin disorders such as diabetes, growth disorders, various blood disorders including various anemias, thalassemias and hemophilia; genetic defects such as cystic fibrosis, Gaucher’s Disease, Hurler's Disease, adenosine deaminase (ADA) deficiency, emphysema, or the like.

[0231] Suitable heterologous nucleic acids include, but are not limited to, those encoding any of a variety of proteins, including, but not limited to: an interferon (e.g., IFN-y, IFN-a,IFN-0, IFN-OJ; IFN-T); an insulin (e.g., Novolin, Humulin, Humalog, Lantus, Ultralente, etc.); an erythropoietin (“EPO”; e.g., Procrit®, Eprex®, or Epogen® (epoetin-a); Aranesp® (darbepoietin-a); NeoRecormon®, Epogin® (epoetin-0); and the like); an antibody (e.g., a monoclonal antibody) (e.g., Rituxan® (rituximab); Remicade® (infliximab); Herceptin® (trastuzumab); Humira™ (adalimumab); Xolair® (omalizumab); Bexxar® (tositumomab); Raptiva™ (efalizumab); Erbitux™ (cetuximab); Avastin® (bevacizumab); and the like), including an antigen-binding fragment of a monoclonal antibody (e.g., Lucentis® (ranibizumab)); a blood factor (e.g., Activase® (alteplase) tissue plasminogen activator; NovoSeven® (recombinant human factor Vila); Factor Vila; Factor VIII (e.g., Kogenate®); Factor IX; 0-globin; hemoglobin; and the like); a colony stimulating factor (e.g., Neupogen® (filgrastim; G-CSF); Neulasta (pegfilgrastim); granulocyte colony stimulating factor (G-CSF), granulocyte-monocyte colony stimulating factor, macrophage colony stimulating factor, megakaryocyte colony stimulating factor; and the like); a growth hormone (e.g., a somatotropin, e.g., Genotropin®, Nutropin®, Norditropin®, Saizen®, Serostim®, Humatrope®, etc.; a human growth hormone; and the like); an interleukin (e.g., IL-1; IL-2, including, e.g., Proleukin®; IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9; etc.); a growth factor (e.g., Regranex® (beclapermin; PDGF); Fiblast® (trafermin; bFGF); Stemgen® (ancestim; stem cell factor); keratinocyte growth factor; an acidic fibroblast growth factor, a stem cell factor, a basic fibroblast growth factor, a hepatocyte growth factor; and the like); a soluble receptor (e.g., a TNF-a-binding soluble receptor such as Enbrel® (etanercept); a soluble VEGF receptor; a soluble interleukin receptor; a soluble y / 8 T cell receptor; and the like); an enzyme (e.g., a-glucosidase; Cerazyme® (imiglucarase; 0-glucocerebrosidase, Ceredase® (alglucerase;); an enzyme activator (e.g., tissue plasminogen activator); a chemokine (e.g., IP- 10; Mig; Groa / IL-8, RANTES; MIP-la; MIP-lp; MCP-t; PF-4; and the like); an angiogenic agent (e.g., vascular endothelial growth factor (VEGF); an anti-angiogenic agent (e.g., a soluble VEGF receptor); a protein vaccine; a neuroactive peptide such as bradykinin, cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dyndrphin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, a sleep peptide, etc.; other proteins such as a thrombolytic agent, an atrial natriuretic peptide, bone morphogenic protein, thrombopoietin, relaxin, glial fibrillary acidic protein, follicle stimulating hormone, a human alpha- 1 antitrypsin, a leukemia inhibitory factor, a transforming growth factor, an insulin-like growth factor, a luteinizing hormone, a macrophage activating factor, tumor necrosis factor, a neutrophil chemotactic factor, a nerve growth factor a tissue inhibitor of metalloproteinases; a vasoactive intestinal peptide, angiogenin, angiotropin, fibrin; hirudin; a leukemia inhibitoryfactor; an IL- 1 receptor antagonist (e.g., Kineret® (anakinra)); an ion channel, e.g., cystic fibrosis transmembrane conductance regulator (CFTR); dystrophin; utrophin, a tumor suppressor; lysosomal enzyme acid a-glucosidase (GAA); and the like. Suitable nucleic acids also include those that encode a functional fragment of any of the aforementioned proteins; and nucleic acids that encode functional variants of any of the aforementioned proteins.

[0232] Suitable heterologous nucleic acids also include those that encode antigenic proteins. A subject rAAV vector that comprises a heterologous nucleic acid that encodes an antigenic protein is suitable for stimulating an immune response to the antigenic protein in a mammalian host. The antigenic protein is derived from an autoantigen, an allergen, a tumor / cancer-associated antigen, a pathogenic virus, a pathogenic bacterium, a pathogenic protozoan, a pathogenic helminth, or any other pathogenic organism that infects a mammalian host. As used herein, the term “a nucleic acid encoding an antigenic protein derived from” includes nucleic acids encoding wild-type antigenic proteins, e.g., a nucleic acid isolated from a pathogenic virus that encodes a viral protein; synthetic nucleic acids generated in the laboratory that encode antigenic proteins that are identical in amino acid sequence to a naturally-occurring antigenic protein; synthetic nucleic acids generated in the laboratory that encode antigenic proteins that differ in amino acid sequence (e.g., by from one amino acid to about 15 amino acids) from a naturally-occurring antigenic protein, but that nonetheless induce an immune response to the corresponding naturally-occurring antigenic protein; synthetic nucleic acids generated in the laboratory that encode fragments of antigenic proteins (e.g., fragments of from about 5 amino acids to about 50 amino acids, which fragments comprises one or more antigenic epitopes), which fragments induce an immune response to the corresponding naturally-occurring antigenic protein; etc.

[0233] Similarly, an antigenic protein “derived from” an autoantigen, an allergen, a tumor / cancer-associated antigen, a pathogenic virus, a pathogenic bacterium, a pathogenic protozoan, a pathogenic helminth, or any other pathogenic organism that infects a mammalian host, includes proteins that are identical in amino acid sequence to a naturally-occurring antigenic protein, and proteins that differ in amino acid sequence (e.g., by from one amino acid to about 15 amino acids) from a naturally-occurring antigenic protein, but that nonetheless induce an immune response to the corresponding naturally-occurring antigenic protein; and fragments of antigenic proteins (e.g., fragments of from about 5 amino acids to about 100 amino acids, e.g., from about 5 to about 50 amino acids, which fragments comprises one or more antigenic epitopes), which fragments induce an immune response to the corresponding naturally-occurring antigenic protein.

[0234] In some embodiments, an immune response to an antigenic protein encoded by a subject rAAV vector will stimulate a protective immune response to a pathogenic organismthat displays the antigenic protein or antigenic epitope (or a protein or an epitope that is cross- reactive with the rAAV-encoded antigenic protein or antigenic epitopes) in the mammalian host. In some embodiments, a cytotoxic T lymphocyte (CTL) response to the rAAV-encoded antigenic protein will be induced in the mammalian host. In other embodiments, a humoral response to the rAAV-encoded antigenic protein will be induced in the mammalian host, such that antibodies specific to the antigenic protein are generated. In many embodiments, a TH1 immune response to the rAAV-encoded antigenic protein will be induced in the mammalian host. Suitable antigenic proteins include tumor / cancer-associated antigens, viral antigens, bacterial antigens, and protozoal antigens; and antigenic fragments thereof. In some embodiments, the antigenic protein is derived from an intracellular pathogen. In other embodiments, the antigenic protein is a self-antigen. In yet other embodiments, the antigenic protein is an allergen.

[0235] Tumor / cancer-specific antigens include, but are not lim ited to, any of the various MAGEs (Melanoma-Associated Antigen E), including MAGE 1 (e.g., GenBank Accession No. M77481), MAGE 2 (e.g., GenBank Accession No. U03735), MAGE 3, MAGE 4, etc.; any of the various tyrosinases; mutant ras; mutant p53 (e.g., GenBank Accession No. X54156 and AA494311); and p97 melanoma antigen (e.g., GenBank Accession No. M12154). Other tumor / cancer-specific antigens include the Ras peptide and p53 peptide associated with advanced cancers, the HPV 16 / 18 and E6 / E7 antigens associated with cervical cancers, MUCI1-KLH antigen associated with breast carcinoma (e.g., GenBank Accession No. J03651), CEA (carcinoembryonic antigen) associated with colorectal cancer (e.g., GenBank Accession No. X98311), gplOO (e.g., GenBank Accession No. S73003) or MARTI antigens associated with melanoma, and the PSA antigen associated with prostate cancer (e.g., GenBank Accession No. X14810). The p53 gene sequence is known (See e.g., Harris et al. (1986) Mol. Cell. Biol., 6:4650-4656) and is deposited with GenBank under Accession No. Ml 4694. Thus, subject proteins, nucleic acids, and / or virions can be used as immunotherapeutics for cancers including, but not limited to, cervical, breast, colorectal, prostate, lung cancers, and for melanomas.

[0236] Viral antigens are derived from known causative agents responsible for diseases including, but not limited to, measles, mumps, rubella, poliomyelitis, hepatitis A, B (e.g., GenBank Accession No. E02707), and C (e.g., GenBank Accession No. E06890), as well as other hepatitis viruses, influenza, adenovirus (e.g., types 4 and 7), rabies (e.g., GenBank Accession No. M34678), yellow fever, Japanese encephalitis (e.g., GenBank Accession No. E07883), dengue (e.g., GenBank Accession No. M24444), hantavirus, and human immunodeficiency virus (e.g., GenBank Accession No. U18552).

[0237] Suitable bacterial and parasitic antigens include those derived from known causative agents responsible for diseases including, but not limited to, diphtheria, pertussis (e.g., GenBank Accession No. M35274), tetanus (e.g., GenBank Accession No. M64353), tuberculosis, bacterial and fungal pneumonias (e.g., Haemophilus influenzae, Pneumocystis carinii, etc.), cholera, typhoid, plague, shigellosis, salmonellosis (e.g., GenBank Accession No. L03833), Legionnaire's Disease, Lyme disease (e.g., GenBank Accession No. U59487), malaria (e.g., GenBank Accession No. X53832), hookworm, onchocerciasis (e.g., GenBank Accession No. M27807), schistosomiasis (e.g., GenBank Accession No. L08198), trypanosomiasis, leshmaniasis, giardiasis (e.g., GenBank Accession No. M33641), amoebiasis, filariasis (e.g., GenBank Accession No. J03266), borreliosis, and trichinosis.

[0238] Suitable heterologous nucleic acids that encode heterologous gene products include non-translated RNAs, such as an RNAi agent (as described in greater detail above) (e.g., an antisense RNA; an siRNA; an shRNA; a double stranded RNA (dsRNA); a CRISPR agent, e.g., a Cas9 or Cas9-like protein, a crRNA-like RNA, a tracrRNA-like RNA, a single guide RNA, and / or a donor polynucleotide; and the like), a ribozyme, etc. RNAi agents can be used to inhibit gene expression. Some RNAi agents provide a tool that can be subsequently used to inhibit gene expression (e.g., a CRISPR agent such as a cas9 or cas9-like protein).

[0239] Target genes include any gene encoding a target gene product (RNA or protein) that is deleterious (e.g., pathological), for example, a target gene product that is malfunctioning (e.g., due to a mutation in the encoded protein sequence, due to a mutation in the non-coding sequences that control the steady state level of the gene product, etc.). Target gene products include, but are not limited to, huntingtin; hepatitis C virus; human immunodeficiency virus; amyloid precursor protein; tau; a protein that includes a polyglutamine repeat; a herpes virus (e.g., varicella zoster); any pathological virus; and the like.

[0240] As such a subject rAAV that includes a heterologous nucleic acid encoding an RNAi agent is useful for treating a variety of disorders and conditions, including, but not limited to, neurodegenerative diseases, e.g., a trinucleotide-repeat disease, such as a disease associated with polyglutamine repeats, e.g,, Huntington's disease, spinocerebellar ataxia, spinal and bulbar muscular atrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA), etc.; an acquired pathology (e.g., a disease or syndrome manifested by an abnormal physiological, biochemical, cellular, structural, or molecular biological state) such as a viral infection, e.g., hepatitis that occurs or may occur as a result of an HCV infection, acquired immunodeficiency syndrome, which occurs as a result of an HIV infection; cancer; and the like.

[0241] In many embodiments, a heterologous nucleic acid encoding an RNAi agent is operably linked to a promoter. Suitable promoters are known those skilled in the art and include the promoter of any protein-encoding gene, e.g., an endogenously regulated gene or a constitutively expressed gene. For example, the promoters of genes regulated by cellular physiological events, e.g., heat shock, oxygen levels and / or carbon monoxide levels, e.g., in hypoxia, may be operably linked to an siRNA-encoding nucleic acid.

[0242] The selected heterologous nucleotide sequence, such as EPO-encoding or nucleic acid of interest, is operably linked to control elements that direct the transcription or expression thereof in the nucleotide sequence in vivo. Such control elements can comprise control sequences normally associated with the selected gene (e.g., endogenous cellular control elements). Alternatively, heterologous control sequences can be employed. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, an endogenous cellular promoter that is heterologous to the gene of interest, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. In addition, sequences derived from nonviral genes, such as the murine metallothionein gene, will also find use herein. Such promoter sequences are commercially available from, e.g., Stratagene (San Diego, Calif.).

[0243] In some embodiments, cell type-specific or tissue-specific promoter will be operably linked to the heterologous nucleic acid encoding the heterologous gene product, such that the gene product is produced selectively or preferentially in a particular cell type(s) or tissue(s). In some embodiments, an inducible promoter will be operably linked to the heterologous nucleic acid.

[0244] For example, muscle-specific and inducible promoters, enhancers and the like, are useful for delivery of a gene product to a muscle cell. Such control elements include, but are not limited to, those derived from the actin and myosin gene families, such as from the myoD gene family; the myocyte-specific enhancer binding factor MEF-2; control elements derived from the human skeletal actin gene and the cardiac actin gene; muscle creatine kinase sequence elements and the murine creatine kinase enhancer (mCK) element; control elements derived from the skeletal fast-twitch troponin C gene, the slow-twitch cardiac troponin C gene and the slow-twitch troponin I gene; hypoxia-inducible nuclear factors; steroid-inducible elements and promoters, such as the glucocorticoid response element (GRE); the fusionconsensus element for RU486 induction; and elements that provide for tetracycline regulated gene expression.

[0245] The AAV expression vector which harbors the DNA molecule of interest (the heterologous DNA) bounded by AAV ITRs, can be constructed by directly inserting the selected sequence(s) into an AAV genome which has had the major AAV open reading frames (“ORFs”) excised therefrom. Other portions of the AAV genome can also be deleted, so long as a sufficient portion of the ITRs remain to allow for replication and packaging functions. Such constructs can be designed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92 / 01070 (published Jan. 23, 1992) and WO 93 / 03769 (published Mar. 4, 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol, and Immunol. 158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shelling and Smith (1994) Gene Therapy 1:165- 169; and Zhou et al. (1994) J. Exp. Med. 179:1867-1875.

[0246] Alternatively, AAV ITRs can be excised from the viral genome or from an AAV vector containing the same and fused 5' and 3' of a selected nucleic acid construct that is present in another vector using any convenient method known to one of ordinary skill in the art. For example, one suitable approach uses standard ligation techniques, such as those described in Sambrook et al., supra. For example, ligations can be accomplished in 20 mM Tris-Cl pH 7.5, 10 mM MgCh, 10 mM DTT, 33 pg / ml BSA, 10 mM-50 mM NaCl, and either 40 pM ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. to 16° C. (for “sticky end” ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C. (for “blunt end” ligation). Intermolecular “sticky end” ligations are usually performed at 30-100 pg / ml total DNA concentrations (5-100 nM total end concentration). AAV vectors which contain ITRs have been described in, e.g., U.S. Pat. No. 5,139,941. In particular, several AAV vectors are described therein which are available from the American Type Culture Collection (“ATCC") under Accession Numbers 53222, 53223, 53224, 53225 and 53226.

[0247] Additionally, chimeric genes can be produced synthetically to include AAV ITR sequences arranged 5rand 3' of one or more selected nucleic acid sequences. Preferred codons for expression of the chimeric gene sequence in mammalian muscle cells can be used. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods. See, e.g., Edge, Nature (1981) 292:756; Nambair et al. Science (1984) 223:1299; Jay et al. J. Biol. Chem. (1984) 259:6311.

[0248] Generation of Subject Infectious rA AV Virions

[0249] By way of introduction, it is typical to employ a host or “producer” cell for rAAV vector replication and packaging. Such a producer cell (usually a mammalian host cell) generally comprises or is modified to comprise several different types of components for rAAV production. The first component is a recombinant adeno-associated viral (rAAV) vector genome (or “rAAV pro-vector”) that can be replicated and packaged into vector particles by the host packaging cell. The rAAV pro-vector will normally comprise a heterologous polynucleotide (or “transgene”), with which it is desired to genetically alter another cell in the context of gene therapy (since the packaging of such a transgene into rAAV vector particles can be effectively used to deliver the transgene to a variety of mammalian cells). The transgene is generally flanked by two AAV inverted terminal repeats (ITRs) which comprise sequences that are recognized during excision, replication and packaging of the AAV vector, as well as during integration of the vector into a host cell genome.

[0250] A second component is a helper virus that can provide helper functions for AAV replication. Although adenovirus is commonly employed, other helper viruses can also be used as is known in the art. Alternatively, the requisite helper virus functions can be isolated genetically from a helper virus and the encoding genes can be used to provide helper virus functions in trans. The AAV vector elements and the helper virus (or helper virus functions) can be introduced into the host cell either simultaneously or sequentially in any order.

[0251] The final components for .AAV production to be provided in the producer cell are “AAV packaging genes” such as AAV rep and cap genes that provide replication and encapsidation proteins, respectively. Several different versions of AAV packaging genes can be provided (including rep-cap cassettes and separate rep and / or cap cassettes in which the rep and / or cap genes can be left under the control of the native promoters or operably linked to heterologous promoters. Such AAV packaging genes can be introduced either transiently or stably into the host packaging cell, as is known in the art and described in more detail below.

[0252] 1. rAAV Vector

[0253] A subject rAAV virion, including the heterologous DNA of interest (where “heterologous DNA of interest” is also referred to herein as “heterologous nucleic acid”), can be produced using standard methodology, known to those of skill in the art. The methods generally involve the steps of (1) introducing a subject rAAV vector into a host cell; (2) introducing an AAV helper construct into the host cell, where the helper construct includes AAV coding regions capable of being expressed in the host cell to complement AAV helper functions missing from the AAV vector; (3) introducing one or more helper viruses and / or accessory function vectors into the host cell, wherein the helper virus and / or accessoiyfunction vectors provide accessory functions capable of supporting efficient recombinant AAV (“rAAV”) virion production in the host cell; and (4) culturing the host cell to produce rAAV virions. The AAV expression vector, AAV helper construct and the helper virus or accessory function vectors) can be introduced into the host cell, either simultaneously or serially, using standard transfection techniques.

[0254] AAV expression vectors are constructed using known techniques to at least provide as operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the DNA of interest and a transcriptional termination region. The control elements are selected to be functional in a mammalian muscle cell. The resulting construct which contains the operatively linked components is bounded (5' and 3') with functional AAV ITR sequences.

[0255] The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Berns, K. I. “Parvoviridae and their Replication” in Fundamental Virology, 2nd Edition, (B. N. Fields and D. M. Knipe, eds.) for the AAV-2 sequence. AAV ITRs used in the vectors of the invention need not have a wild-type nucleotide sequence, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides. Additionally, AAV ITRs may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-7, etc. Furthermore, 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV expression vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the DNA molecule into the recipient cell genome when AAV Rep gene products are present in the cell. ITRs allow replication of the vector sequence in the presence of an appropriate mixture of Rep proteins. ITRs also allow for the incorporation of the vector sequence into the capsid to generate an AAV particle.

[0256] In order to produce rAAV virions, an AAV expression vector is introduced into a suitable host cell using known techniques, such as by transfection. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13: 197. Particularly suitable transfection methods include calcium phosphate co-precipitation (Graham et al. (1973) Virol. 52:456-467), direct micro-injection into cultured cells (Capecchi, M. R. (1980) Cell 22:479-488), electroporation (Shigekawa et al. (1988) BioTechniques 6:742-751), liposome mediated gene transfer (Mannino et al. (1988) BioTechniques 6:682-690), lipid-mediated transduction (Feigner et al. (1987) Proc. Natl.Acad. Sci. USA 84:7413-7417), and nucleic acid delivery using high-velocity microprojectiles (Klein et al. (1987) Nature 327:70-73).

[0257] For the purposes of this disclosure, suitable host cells for producing rAAV virions include microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of a heterologous DNA molecule. The term includes the progeny of the original cell which has been transfected. Thus, a “host cell” for producing rAAV virions generally refers to a cell which has been transfected with an exogenous DNA sequence. Cells from the stable human cell line, 293 (readily available through, e.g., the American Type Culture Collection under Accession Number ATCC CRL1573) are used in many embodiments. Particularly, the human cell line 293 is a human embryonic kidney cell line that has been transformed with adenovirus type-5 DNA fragments (Graham et al. (1977) J. Gen. Virol. 36:59), and expresses the adenoviral Ela and Elb genes (Aiello et al. (1979) Virology 94:460). The 293 cell line is readily transfected, and provides a particularly convenient platform in which to produce rAAV virions.

[0258] 2. AAV Helper Functions

[0259] Host cells containing the above-described AAV expression vectors must be rendered capable of providing AAV helper functions in order to replicate and encapsidate the nucleotide sequences flanked by the AAV ITRs to produce rAAV virions. AAV helper functions are generally AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication. AAV helper functions are used herein to complement necessary AAV functions that are missing from the AAV expression vectors. Thus, AAV helper functions include one, or both of the major AAV ORFs, namely the rep and cap coding regions, or functional homologues thereof. In the context of the instant disclosure, the cap functions include one or more mutant capsid proteins, wherein at least one capsid protein comprises at least one mutation, as described above.

[0260] By “AAV rep coding region” is meant the art-recognized region of the AAV genome which encodes the replication proteins Rep 78, Rep 68, Rep 52 and Rep 40. These Rep expression products have been shown to possess many functions, including recognition, binding and nicking of the AAV origin of DNA replication, DNA helicase activity and modulation of transcription from AAV (or other heterologous) promoters. The Rep expression products are collectively required for replicating the AAV genome. For a description of the AAV rep coding region, see, e.g., Muzyczka, N. (1992) Current Topics in Microbiol, and Immunol. 158:97-129; and Kotin, R. M. (1994) Human Gene Therapy 5:793- 801 . Suitable homologues of the AAV rep coding region include the human herpesvirus 6(HHV-6) rep gene which is also known to mediate AAV-2 DNA replication (Thomson et al. (1994) Virology 204:304-311).

[0261] AAV cap proteins include VP1, VP2, and VP3, wherein at least one of VP1, VP2, and VP3 comprises at least one mutation, as described above.

[0262] AAV helper functions are introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of the AAV expression vector. AAV helper constructs are thus used to provide at least transient expression of AAV rep and / or cap genes to complement missing AAV functions that are necessary for productive AAV infection. AAV helper constructs lack AAV ITRs and can neither replicate nor package themselves. These constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45 which encode both Rep and Cap expression products. See, e.g., Samulski et al. (1989) J. Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945. A number of other vectors have been described which encode Rep and / or Cap expression products. See, e.g., U.S. Pat. No. 5,139,941.

[0263] Both AAV expression vectors and AAV helper constructs can be constructed to contain one or more optional selectable markers. Suitable markers include genes which confer antibiotic resistance or sensitivity to, impart color to, or change the antigenic characteristics of those cells which have been transfected with a nucleic acid construct containing the selectable marker when the cells are grown in an appropriate selective medium. Several selectable marker genes that are useful in practicing methods of the disclosure include the hygromycin B resistance gene (encoding Aminoglycoside phosphotranferase (APH)) that allows selection in mammalian cells by conferring resistance to hygromycin; the neomycin phosphotranferase gene (encoding neomycin phosphotransferase) that allows selection in mammalian cells by conferring resistance to G418; and the like. Other suitable markers are known to those of skill in the art.

[0264] 3. AAV Accessory Functions

[0265] The host cell (or packaging cell) must also be rendered capable of providing non AAV derived functions, or “accessory functions,” in order to produce rAAV virions. Accessory functions are non AAV derived viral and / or cellular functions upon which AAV is dependent for its replication. Thus, accessory functions include at least those non AAV proteins and RNAs that are required in AAV replication, including those involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNAreplication, synthesis of Cap expression products and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses.

[0266] Particularly, accessory functions can be introduced into and then expressed in host cells using methods known to those of skill in the art. Commonly, accessory functions are provided by infection of the host cells with an unrelated helper virus. A number of suitable helper viruses are known, including adenoviruses; herpesviruses such as herpes simplex virus types 1 and 2; and vaccinia viruses. Nonviral accessory functions will also find use herein, such as those provided by cell synchronization using any of various known agents. See, e.g., Buller et al. (1981) J. Virol. 40:241-247; McPherson et al. (1985) Virology 147:217-222; Schlehofer et al. (1986) Virology 152:110-117.

[0267] Alternatively, accessory functions can be provided using an accessory function vector. Accessory function vectors include nucleotide sequences that provide one or more accessory functions. An accessory function vector is capable of being introduced into a suitable host cell in order to support efficient AAV virion production in the host cell. Accessory function vectors can be in the form of a plasmid, phage, transposon, cosmid, or another virus. Accessory vectors can also be in the form of one or more linearized DNA or RNA fragments which, when associated with the appropriate control elements and enzymes, can be transcribed or expressed in a host cell to provide accessory functions.

[0268] Nucleic acid sequences providing the accessory functions can be obtained from natural sources, such as from the genome of an adenovirus particle, or constructed using recombinant or synthetic methods known in the art. In this regard, adenovirus-derived accessory functions have been widely studied, and a number of adenovirus genes involved in accessory functions have been identified and partially characterized. See, e.g., Carter, B. J. (1990) “Adeno-Associated Virus Helper Functions,” in CRC Handbook of Parvoviruses, vol. I (P. Tijssen, ed.), and Muzyczka, N. (1992) Curr. Topics. Microbiol, and Immun. 158:97- 129. Specifically, early adenoviral gene regions El a, E2a, E4, VAI RNA and, possibly, E lb are thought to participate in the accessory process. Janik et al. (1981) Proc. Natl. Acad. Sci. USA 78:1925-1929. Herpesvirus-derived accessory functions have been described. See, e.g., Young et al. (1979) Prog. Med. Virol. 25:113. Vaccinia virus-derived accessory functions have also been described. See, e.g., Carter, B. J. (1990), supra., Schlehofer et al. (1986) Virology 152:110-117.

[0269] As a consequence of the infection of the host cell with a helper virus, or transfection of the host cell with an accessory function vector, accessory functions are expressed which transactivate the AAV helper construct to produce AAV Rep and / or Cap proteins. The Rep expression products excise the recombinant DNA (including the DNA ofinterest, e.g., the heterologous nucleic acid) from the AAV expression vector. The Rep proteins also serve to duplicate the AAV genome. The expressed Cap proteins assemble into capsids, and the recombinant AAV genome is packaged into the capsids. Thus, productive AAV replication ensues, and the DNA is packaged into rAAV virions.

[0270] Following recombinant AAV replication, rAAV virions can be purified from the host cell using a variety of conventional purification methods, such as C$C1 gradients, affinity chromatography, and ion-exchange chromatography. Further, if infection is employed to express the accessory functions, residual helper virus can be inactivated, using known methods. For example, adenovirus can be inactivated by heating to temperatures of approximately 60° C. for, e.g., 20 minutes or more. This treatment effectively inactivates only the helper virus since AAV is extremely heat stable while the helper adenovirus is heat labile.

[0271] The resulting rAAV virions are then ready for use for DNA delivery, such as in gene therapy applications, or for the delivery of a gene product to a mammalian host.

[0272] Delivering a Heterologous Nucleic Acid

[0273] The present disclosure further provides methods of delivering a heterologous nucleic acid to a target cell and / or to an individual in need thereof. In some embodiments, an individual in need thereof is a human who has previously been naturally exposed to AAV and as a result harbors anti-AAV antibodies (i.e., AAV neutralizing antibodies). Based on positive results in clinical trials involving AAV gene delivery to, for example, liver, muscle, and retina — all tissues affected by neutralizing antibodies against this vehicle — there are many such therapeutic applications / disease targets.

[0274] A subject method generally involves: (i) administering an effective amount of a subject rAAV virion to an individual, and / or (ii) contacting a target cell with a subject virion. Generally, rAAV virions are administered to a subject using either in vivo (“direct”) or in vitro (“indirect”) transduction techniques. If transduced in vitro (“indirectly”), a desired recipient cell (i.e., “target cell”) can be removed from the individual, transduced with rAAV virions and reintroduced into the individual. Alternatively, syngeneic or xenogeneic cells can be used where those cells will not generate an inappropriate immune response in the individual.

[0275] Suitable methods for the delivery and introduction of transduced target cells into an individual have been described. For example, cells can be transduced in vitro by combining recombinant AAV virions with cells e.g., in appropriate media, and screening for those cells harboring the DNA of interest using conventional techniques such as Southern blots and / or PCR, or by using selectable markers. Transduced cells can then be formulatedinto pharmaceutical compositions, described more fully below, and the composition introduced into the subject by various techniques, such as by intramuscular, intravenous, subcutaneous and intraperitoneal injection.

[0276] For in vivo (i.e., “direct”) delivery, the rAAV virions will be formulated into pharmaceutical compositions and will generally be administered parenterally (e.g., administered via an intramuscular, subcutaneous, intratumoral, transdermal, intrathecal, intravenous, etc.) route of administration.

[0277] Pharmaceutical compositions will comprise sufficient genetic material to produce a therapeutically effective amount of the gene expression product of interest, i.e., an amount sufficient to reduce or ameliorate symptoms of the disease state in question or an amount sufficient to confer the desired benefit. The pharmaceutical compositions will also contain a pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, & Wilkins Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds., 7thed„ Lippincott, Williams, & Wilkins and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3rded. Amer. Pharmaceutical Assoc.

[0278] Appropriate doses will depend on the mammal being treated (e.g., human or nonhuman primate or other mammal), age and general condition of the subject to be treated, the severity of the condition being treated, the particular therapeutic protein in question, its mode of administration, among other factors. An appropriate effective amount can be readily determined by one of skill in the art.

[0279] Thus, a “therapeutically effective amount” will fall in a relatively broad range that can be determined through clinical trials. For example, for in vivo injection, i.e., injection directly to skeletal or cardiac muscle, a therapeutically effective dose will be on the order offrom about 106to about 10*5of the rAAV virions, e.g., from about 108to 10!2rAAV virions. For in vitro transduction, an effective amount of rAAV virions to be delivered to cells will be on the order of from about 108to about 1013of the rAAV virions. Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves.

[0280] Dosage treatment may be a single dose schedule or a multiple dose schedule. Moreover, the subject may be administered as many doses as appropriate. One of skill in the art can readily determine an appropriate number of doses.

[0281] The cells of interest (i.e., “target cells”) are typically mammalian, where the term refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some embodiments, the target cell is a human cell.

[0282] Target cells of interest include any cell susceptible to infection by a subject rAAV virion. In some cases, e.g., when the method is a method of delivering a heterologous nucleic acid to a target cell, the target cell can be a cell removed from an individual (e.g., a “primary” cell), or the target cell can be a tissue culture cell (e.g,, from an established cell line).

[0283] Exemplary target cells include, but are not limited to, liver cells, pancreatic cells (e.g., islet cells: alpha cells, beta cells, delta cells, gamma cells, and / or epsilon cells), skeletal muscle cells, heart muscle cells, fibroblasts, retinal cells, synovial joint cells, lung cells, T cells, neurons, glial cells, stem cells, hematopoietic progenitor cells, neural progenitor cells, endothelial cells, and cancer cells. Exemplary stem cell target cells include, but are not limited to, hematopoietic stem cells, neural stem cells, neural crest stem cells, embryonic stem cells, induced pluripotent stem cells (iPS cells), mesenchymal stem cells, mesodermal stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, and retinal stem cells.

[0284] The term “stem cell” is used herein to refer to a mammalian cell that has the ability both to self-renew, and to generate differentiated progeny (see, e.g., Morrison et al. (1997) Cell 88:287-298). Generally, stem cells also have one or more of the following properties: an ability to undergo asynchronous, or symmetric replication, that is where the two daughter cells after division can have different phenotypes; extensive self-renewal capacity; capacity for existence in a mitotically quiescent form; and clonal regeneration of all the tissue in which they exist, for example the ability of hematopoietic stem cells to reconstitute all hematopoietic lineages. As is appreciated by one of ordinary skill in the art, “progenitor cells” differ from stem cells in that they typically do not have the extensive self-renewal capacity, and often can generate a more restricted subset of the lineages in the tissue from which theyderive, for example only lymphoid, or erythroid lineages in a hematopoietic setting. As used herein, the term “stem cell” encompasses both “stem cells” and “progenitor cells” as defined above.

[0285] Stem cells may be characterized by both the presence of markers associated with specific epitopes identified by antibodies and the absence of certain markers as identified by the lack of binding of specific antibodies. Stem cells may also be identified by functional assays both in vitro and in vivo, particularly assays relating to the ability of stem cells to give rise to multiple differentiated progeny.

[0286] Suitable stem cells of interest include, but are not limited to: hematopoietic stem cells and progenitor cells derived therefrom (U.S. Pat. No. 5,061,620); neural crest stem cells (see Morrison et al. (1999) Cell 96:737-749); neural stem cells and neural progenitor cells; embryonic stem cells; mesenchymal stem cells; mesodermal stem cells; liver stem cells, muscle stem cells, retinal stem cells, induced pluripotent stem cells (iPS cells), etc. Other hematopoietic “progenitor” cells of interest include cells dedicated to lymphoid lineages, e.g. immature T cell and B cell populations.

[0287] Purified populations of stem or progenitor cells may be used. For example, human hematopoietic stem cells may be positively selected using antibodies specific for CD34, thy- 1 ; or negatively selected using lineage specific markers which may include glycophorin A, CD3, CD24, CD16, CD14, CD38, CD45RA, CD36, CD2, CDI9, CD56, CD66a, and CD66b; T cell specific markers, tumor / cancer specific markers, etc. Markers useful for the separation of mesodermal stem cells include FcyRII, FcyRIII, Thy- 1 , CD44, VLA-4a, LFA-113, HSA, ICAM-1, CD45, Aa4.1, Sca-1, etc. Neural crest stem cells may be positively selected with antibodies specific for low-affinity nerve growth factor receptor (LNGFR), and negatively selected for the markers sulfatide, glial fibrillaiy acidic protein (GFAP), myelin protein Po, peripherin and neurofilament. Human mesenchymal stem cells may be positively separated using the markers SH2, SH3 and SH4.

[0288] Target cells which are employed may be fresh, frozen, or have been subject to prior culture. They may be fetal, neonate, adult. Hematopoietic cells may be obtained from fetal liver, bone marrow, blood, particularly G-CSF or GM-CSF mobilized peripheral blood, or any other conventional source. The manner in which stem cells are separated from other cells of the hematopoietic or other lineage is not critical to this disclosure. As described above, a substantially homogeneous population of stem or progenitor cells may be obtained by selective isolation of cells free of markers associated with differentiated cells, while displaying epitopic characteristics associated with the stem cells.

[0289] Nucleic acids that can be delivered to an individual include any of the above defined heterologous nucleic acids. Proteins that can be delivered using a subject method also include a functional fragment of any of the aforementioned proteins; and functional variants of any of the aforementioned proteins.

[0290] In some embodiments, a therapeutically effective amount of a protein is produced in the mammalian host, Whether a therapeutically effective amount of a particular protein is produced in the mammalian host using a Subject method is readily determined using assays appropriate to the particular protein. For example, where the protein is EPO, hematocrit is measured.

[0291] Where the rAAV encodes an antigenic protein, suitable antigenic proteins that can be delivered to an individual using a subject method include, but are not limited to, tumor / cancer-associated antigens, autoantigens (“self’ antigens), viral antigens, bacterial antigens, protozoal antigens, and allergens; and antigenic fragments thereof. In some embodiments, a cytotoxic T lymphocyte (CTL) response to the rAAV-encoded antigenic protein will be induced in the mammalian host. In other embodiments, a humoral response to the rAAV-encoded antigenic protein will be induced in the mammalian host, such that antibodies specific to the antigenic protein are generated. In many embodiments, a TH1 immune response to the rAAV-encoded antigenic protein, will be induced in the mammalian host. Whether an immune response to the antigenic protein has been generated is readily determined using well-established methods. For example, an enzyme-linked immunosorbent assay can be used to determine whether antibody to an antigenic protein has been generated. Methods of detecting antigen-specific CTL are well known in the art. For example, a detectably labeled target cell expressing the antigenic protein on its surface is used to assay for the presence of antigen-specific CTL in a blood sample.

[0292] Whether a therapeutically effective amount of a heterologous nucleic acid (e.g., a nucleic acid encoding a polypeptide, an RNAi agent, etc.) has been delivered to a mammalian host using a subject method is readily determined using any appropriate assay. For example, where the gene product is an RNAi agent that inhibits HIV, viral load can be measured.

[0293] Methods of Generating and Identifying Modified rAAV Virions

[0294] The present disclosure provides a method of generating and identifying a modified infectious recombinant adeno-associated virus (rAAV) virion that comprises a variant capsid protein comprising an amino acid sequence with at least one amino acid substitution (including deletions, insertions, etc.) compared to a starter AAV capsid protein. A starter AAV capsid protein comprises an amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26-33.

[0295] The method generally involves generating a mutant rAAV virion libraiy; and selecting the library for modified rAAV virions with altered properties relative to a starter rAAV virion. The starter rAAV virion comprises a variant AAV capsid protein that comprises an amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26-33. The present disclosure further provides libraries and compositions comprising the libraries.

[0296] In some embodiments, a given selection step is repeated two, three, four, or more times to enrich a subject AAV library for altered virion properties. In some embodiments, following selection of an AAV libraiy, individual clones are isolated and sequenced.

[0297] Generation of a Mutant AAV Library

[0298] A mutant AAV libraiy is generated that comprises one or more mutations relative to a starter AAV cap gene. A starter cap gene is a cap comprising a nucleotide sequence that encodes a variant AAV capsid protein that comprises an amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26-33. Mutations in the rAAV cap gene are generated using any known method. Suitable methods for mutagenesis of a starter AAV cap gene include, but are not limited to, a polymerase chain reaction (PCR)-based method, oligonucleotide-directed mutagenesis, saturation mutagenesis, loop-swapping mutagenesis, fragment shuffling mutagenesis (i.e., DNA shuffling), and the like. Methods for generating mutations are well described in the art. See, e.g., Zhao et al. Nat Biotechnol. 1998 March; 16(3):234-5; Koerber et. al.; Mol Ther. 2008 October; 16(10): 1703-9; Koerber et. al.; Mol Ther. 2009 December;17(12):2088-95; U.S. Pat. No. 6,579,678; U.S. Pat. No. 6,573,098; and U.S. Pat. No. 6,582,914; all of which are hereby incorporated by reference fortheir teachings related to mutagenesis.

[0299] In some embodiments, a mutant AAV library comprising mutations in the cap gene will be generated using a staggered extension process. The staggered extension process involves amplification of the cap gene using a PCR-based method. The template cap gene is primed using specific PCR primers, followed by repeated cycles of denaturation and very short annealing / polymerase-catalyzed extension. In each cycle, the growing fragments anneal to different templates based on sequence complementarity and extend further. The cycles of denaturation, annealing, and extension are repeated until full-length sequences form. The resulting full-length sequences include at least one mutation in the cap gene compared to a wild-type AAV cap gene.

[0300] The PCR products comprising AAV cap sequences that include one or more mutations are inserted into a plasmid containing a wild-type AAV genome. The result is a libraiy of AAV cap mutants. Thus, the present disclosure provides a mutant AAV cap gene library comprising from about 10 to about IO10members, and comprising mutations in theAAV cap gene. A given member of the library has from about one to about 50 mutations in the AAV cap gene. A subject library comprises from 10 to about 109distinct members, each having a different mutation(s) in the AAV cap gene.

[0301] Once a cap mutant library is generated, viral particles are produced that can then be selected on the basis of altered capsid properties. Library plasmid DNA is transfected into a suitable host cell (e.g., 293 cells), followed by introduction into the cell of helper virus. Viral particles produced by the transfected host cells (rAAV library particles) are collected.

[0302] Library Selection

[0303] Once a library is generated, it is selected for a particular virion property (i.e., an altered property of infection). Viral particles are generated as discussed above (thus producing a library of modified rAAV virions), and subjected to one or more selection steps to identify a modified rAAV virion with an altered property of infection (relative to an infectious rAAV virion comprising a variant capsid protein that comprises an amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26-33). Properties of infection that are selected for can include, but are not limited to: 1) altered binding (e.g., decreased binding) to AAV neutralizing antibodies; 2) increased evasion of AAV neutralizing antibodies; 3) increased infectivity of a cell that is resistant to infection with AAV; and 4) altered heparin binding.

[0304] 1. Selection for Reduced Binding to AAV Neutralizing Antibodies

[0305] In some embodiments, a subject AAV library is selected for altered (e.g., reduced) binding to neutralizing antibodies that bind to and neutralize wild-type AAV virions, compared to the binding of such antibodies to wild-type AAV virions and neutralization of wild-type AAV virions (or relative to an infectious rAAV virion comprising a variant capsid protein that comprises an amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26- 33). AAV library particles (AAV library virion) are contacted with neutralizing antibodies and the ability of the AAV library particles to infect a permissive host cell is tested.Typically, AAV library particles are contacted with various concentrations of neutralizing antibodies. The higher the concentration of neutralizing antibodies that is required to reduce infectivity of the AAV library particles, the more resistant the AAV particles are to neutralization. Any convenient assay known to one of ordinary skill in the art may be used to directly measure the binding (e.g., measure the binding affinity) of an AAV library virion to neutralizing anti-AAV antibodies.

[0306] 2. Selection for Increased Evasion of AAV Neutralizing Antibodies

[0307] In some embodiments, a subject AAV library is selected for increased evasion of neutralizing antibodies (i.e. increased resistance to human neutralizing AAV antibodies)relative to an infectious rAAV virion comprising a variant capsid protein that comprises an amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26-33. AAV library particles are contacted with targets cells in the presence of neutralizing AAV antibodies (usually human neutralizing anti-AAV antibodies). After a suitable amount of time to allow for infection of the cells with AAV library particles, helper virus is added, and AAV library particles that successfully infected the cell(s) are harvested. In some embodiments, infectivity is measured (e.g., as described above) for those virions exhibiting successful infection. In some embodiments, the cycle of infection, addition of helper virus, and harvesting of AAV particles is repeated one, two, three, or more times. The selection can occur with varying amounts (concentrations) of neutralizing AAV antibodies to select for various degrees of evasion (e.g., each repeated round can utilize an increased concentration of antibodies relative to the previous round).

[0308] 3. Selection for Increased Infectivity of Non-Permissive Cells

[0309] In some embodiments, a subject AAV library is selected for increased infectivity of non-permissive cells (relative to an infectious rAAV virion comprising a variant capsid protein that comprises an amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26- 33). AAV library particles are contacted with a non-permissive cell (e.g., a population of non- permissive cells). After a suitable amount of time to allow for infection of the cells with AAV library particles, helper virus is added, and AAV library particles that successfully infected the non-permissive cell(s) are harvested. In some embodiments, the cycle of infection, addition of helper virus, and harvesting of AAV particles is repeated one, two, three, or more times.

[0310] 4. Selection for Altered Heparin Binding

[0311] In some embodiments, a subject library is selected for altered heparin binding, including increased heparin binding and decreased heparin binding relative to wild-type AAV virion heparin binding (or relative to an infectious rAAV virion comprising a variant capsid protein that comprises an amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26- 33). AAV library particles are contacted with a heparin affinity matrix. For example, AAV library particles are loaded onto a heparin affinity column under conditions that permit binding of the AAV library particles to the heparin. Exemplary conditions include equilibration of the column with 0.15 M NaCl and 50 mM Tris at pH 7.5. After allowing the AAV library particle to bind to the heparin affinity matrix, the AAV library particle / heparin affinity matrix complex is washed with volumes of buffer containing progressively increasing concentrations of NaCl, and at each NaCl concentration, eluted AAV library particles are collected. For example, after binding the AAV library particle / heparin affinity matrixcomplex is washed with a volume of 50 mM Tris buffer, pH 7.5, containing 200 mM NaCl, and eluted AAV library particles are collected. The elution step is repeated with a 50 mM Tris buffer, pH 7.5, containing about 250 mM NaCl, about 300 mM NaCl, about 350 mM, about 400 mM NaCl, about 450 mM NaCl, about 500 mM NaCl, about 550 mM NaCl, about 600 mM NaCl, about 650 mM NaCl, about 700 mM NaCl, or about 750 mM NaCl.

[0312] AAV library particles that elute at NaCl concentrations lower than about 450 mM NaCl exhibit decreased heparin binding properties relative to wild-type AAV. AAV library particles that elute at NaCl concentrations higher than about 550 mM NaCl exhibit increased heparin binding properties relative to wild-type AAV.

[0313] In some embodiments, eluted AAV library particles are amplified by co-infection of permissive cells with a helper virus, and are re-fractionated on heparin affinity matrix. This step can be repeated a number of times to enrich for AAV library particles with altered heparin binding properties.

[0314] In the present methods, one or more selection steps may follow generation of AAV library particles. For example, in some embodiments, the method comprises selecting for increased heparin binding, followed by selecting for decreased binding to neutralizing antibodies. In other embodiments, the method comprises selecting for decreased binding to neutralizing antibodies, followed by selecting for increased heparin binding. In other embodiments, the method comprises selecting for decreased heparin binding, followed by selecting for decreased binding to neutralizing antibodies. In other embodiments, the method comprises selecting for decreased binding to neutralizing antibodies, followed by selecting for decreased heparin binding. In other embodiments, the method comprises selecting for decreased binding to neutralizing antibodies, followed by selecting for increased infectivity of a stem cell. In other embodiments, the method comprises selecting for decreased binding to neutralizing antibodies, followed by selecting for increased evasion of neutralizing antibodies. In other embodiments, the method comprises selecting for increased evasion of neutralizing antibodies, followed by selecting for decreased binding to neutralizing antibodies.

[0315] Thus, the present disclosure provides an adeno-associated virus (AAV) library that includes a plurality of nucleic acids, each of which nucleic acid includes a nucleotide sequence that encodes a variant AAV capsid protein. The encoded variant AAV capsid protein includes at least one amino acid substitution relative to a sequence set forth in one of SEQ ID NOs: 10-13 and 26-33. The present disclosure provides a library of mutant adeno- associated virus (AAV) particles, including a plurality of AAV particles each of which includes an AAV capsid protein that includes at least one amino acid substitution relative to a sequence set forth in one of SEQ ID NOs: 10-13 and 26-33. Nucleic acids encoding mutantAAV capsid proteins are described above, as are the properties of the encoded mutant AAV capsid proteins.

[0316] The present disclosure further provides a library comprising at least one of: (i) two or more infectious rAAV virions, each comprising a variant adeno-associated virus (AAV) capsid protein and a heterologous nucleic acid; (ii) two or more isolated nucleic acids, each comprising a nucleotide sequence that encodes a variant AAV capsid protein; (iii) two or more host cells, each comprising a nucleic acid that comprises a nucleotide sequence that encodes a variant AAV capsid protein; and (iv) two or more variant AAV capsid proteins; where the variant AAV capsid protein of at least one member of the library comprises an amino acid sequence having at least one amino acid substitution relative to the amino acid sequence set forth in one of SEQ ID NOs: 10-13 and 26-33.

[0317] Compositions and Kits

[0318] Also provided are compositions and kits for use in the methods of the present disclosure. The subject compositions and kits include at least one of: a subject infectious rAAV virion, a subject rAAV vector, a subject nucleotide acid comprising a nucleotide sequence encoding a subject variant AAV capsid protein, an isolated host cell comprising a subject nucleic acid (i.e., a subject genetically modified host cell comprising a nucleic acid that comprises a nucleotide sequence encoding a subject variant AAV capsid protein); a subject library (e.g., any of the above described libraries); and a subject variant AAV capsid protein. A composition or kit can include any convenient combination of the above. A composition or kit can also include helper virus and / or a nucleic acid comprising a nucleotide sequence that encodes a helper virus. A kit may also include reagents for the generation of nucleic acids (i.e., “mutant” nucleic acids) encoding modified variant AAV capsid proteins.

[0319] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.

[0320] The invention now being fully described, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.EXAMPLES

[0321] Example 1

[0322] Adeno-associated virus (AAV) gene therapy vectors have demonstrated considerable promise in several clinical trials to date. However, circulating anti-AAV antibodies, resulting from childhood exposure or prior administration of an AAV vector, have prevented the implementation of AAV gene therapy for many potential patients. We have isolated novel AAV variants that are capable of enhanced anti-AAV antibody evasion, both in vitro and in vivo. The stringent pressure resulting from selections using low and high potency human sera pools and human IVIG evolved AAV variants capable of evasion of antibody neutralization from individual human sera, human IVIG, and mouse sera, the most broadly evasive variants to date.

[0323] Materials and Methods

[0324] Cell Lines

[0325] Cell lines were cultured at 37° C. and 5% CO2, and unless otherwise mentioned, were obtained from the American Type Culture Collection (Manassas, Va.). HEK293T, HeLa, and HT1080 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Gibco, Carlsbad, Calif.) and 1% penicillin / streptomycin (Invitrogen, Carlsbad, Calif.). CHO KI and CHO pgsA cells were cultured in F-12K medium (ATCC) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin / streptomycin (Invitrogen). Pro5 and Lecl cells were cultured in MEM-alpha medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin / streptomycin (Invitrogen).

[0326] Human Sera Pools for Selection

[0327] Eighteen individual human serum samples were obtained from Innovative Research, Inc. (Southfield, Mich.) and the neutralizing antibody titer for wild type AAV2 was determined for each sample (Table 2). Since individual samples likely possess variations in both the affinities and epitope specificities of the antibodies, three potent sera pools (a-A > F+G, p=B+H+M, and y^I+J+N) were generated by mixing equal volumes of individual serum samples. Selection in the presence of these variations of antibodies should result in a general enhancement of resistance to many pre-existing human antibodies. Later selectionswere performed in the presence of Gamimune N, 10% Human IVIG (Bayer, Elkhart Ind.) to select for resistance to an even broader range of antibodies.

[0328] Table 2: Neutralizing Antibody Titers of Individual Human Serum SamplesNeutralizing antibody (NAb) titers for each sample are reported as the reciprocal of the volume fraction of serum necessary to reduce infectivity to 37% of the value measured in the absence of serum. Three sera pools (a=A+F+G, p=B+H+M, and y=I+J+N) were then generated by mixing equivolume amounts of three individual serum samples.

[0329] TABLE 2Human Serum -NABSample titerA 500B 275C 200D <75E <75F 350G 425H 450I 200J 500K 172L <75M 2200N 5000O <75P <75Q <75R 120

[0330] Library Generation and Viral Production

[0331] To create the saturation mutagenesis library, an AAV2 cap library was generated by error-prone PCR followed by the staggered extension process described by Zhao et al. using S'-GCGGAAGCTTCGATCAACTACGC-S' (SEQ ID NO: 14) and 5'- GGGGCGGCCGCAATTACAGATTACGAGTCAGGTATCTGGTG-3' (SEQ ID NO: 15) as forward and reverse primers, respectively. Selections using pooled individual human sera revealed a variant containing four point mutations (described in the results section) that served as the basis for the saturation mutagenesis library. The cap gene for this variant was subjected to further mutagenesis by changing the amino acids at specific sites. Primer 5'- cattNNKgaccaEtctaggaactge-3 Y SEO ID NO: 16) and the corresponding reverse complement primer were used to mutagenize the R471 amino acid site. Primer 5' gccacaaggacgatgaagaaNNKttttttcctcagagcggggttctcatctttgggaagcaaggctcaNNKaaaacaagt gtggacattg-3'(SEQ ID NO: 17) and the corresponding reverse complement primer were used to mutagenize the K532 and E548 amino acid sites. Primer 5'- ccaacctccagagaggcNNKagacaagcagctacc-3' (SEQ ID NO: 18) and the corresponding reverse complement primer were used to mutagenize the N587 amino acid site. Primer 5'- ccaactacaacaagtctNNKaatgtggactttactgtggacNNKaatggcgtgtatt-SVSEQ ID NO: 19) and the corresponding reverse complement primer were used to mutagenize the V708 and T716 amino acid sites. A library consisting of AAV2 containing randomized cap loop regions and a library containing shuffled DNA from the wild type AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, AAV9 cap genes were packaged and pooled for initial selection steps (Koerber et. al.; Mol Then 2008 October; 16( 10): 1703-9; and Koerber et. al.; Mol Ther. 2009 December; 17(12): 2088-95; both of which are hereby incorporated by reference in their entirety).

[0332] For the second and third rounds of evolution, random mutagenesis libraries were generated by subjecting cap genes from the Loop-Swap / Shuffle library and the Saturation Mutagenesis library to error-prone PCR using 5r-CATGGGAAAGGTGCCAGACG-3' (SEQ ID NO: 20) and 5'-ACCATCGGCAGCCATACCTG-3'(SEQ ID NO: 21) as forward and reverse primers, respectively, as previously described. The replication competent AAV libraries and recombinant AAV vectors expressing GFP under the control of a CMV promoter were packaged using HEK293T cells (ATCC) using the calcium phosphate transfection method, and the viruses were purified by iodixonal gradient centrifugation. Recombinant AAV vectors expressing GFP or luciferase under the control of a CMV promoter for use in vivo were further purified by Amicon filtration. DNase-resistant genomic titers were determined via quantitative PCR. (Excoffon et al, Proc Natl Acad Sci USA. 2009 Mar. 10; 106(10):3865-70; and Maheshri et al., NatBiotechnol. 2006 February; 24(2): 198-204; both of which are hereby incorporated by reference in their entirety).

[0333] Library Selection and Evolution

[0334] One round of selection is defined as HEK293T cell infection using the AAV starting library (incubated for 30 minutes at room temperature for the pooled individual human sera or for 1 hour at 37° C. with heat inactivated IVIG prior to infection), followed by adenovirus rescue and harvest of successful variants. Each round of evolution consists of mutagenesis of the cap gene to create the starting library and three rounds of selection. Three rounds of evolution were performed with each library, with clonal analysis performed between each round of evolution. The starting libraries for each round of evolution were generated as described above. Following the third round of selection, AAV cap genes were isolated from the pool of successful AAV variants and amplified via PCR. Cap genes were inserted into the pXX2 recombinant AAV packaging plasmid using Notl and Hindlll. Cap genes were then sequenced at the University of California, Berkeley DNA sequencing facility, and analyzed using Geneious software (Biomatters, Auckland, New Zealand). Three- dimensional models of the AAV2 capsid (Protein Databank accession number 1LP3) were rendered in Pymol (DeLano Scientific, San Carlos, Calif.).

[0335] In Vitro Transduction Analysis of Antibody-Evading Variants

[0336] HEK293T were plated at a density of 3x104cells / weIl 24 hours prior to infection. Variants were incubated at 37° C. for 1 hour with heat inactivated IVIG, individual human sera, or individual mouse sera prior to infection, and cells were then infected with rAAV-GFP at a genomic MOI of 2000. The percentage of GFP positive cells was assessed 48 hours post infection using an ImageXpress Micro Cellular Imaging and Analysis System (Molecular Devices, Sunnyvale, Calif.) and MetaXpress Image Analysis Software, version 3.1,0, Multi Wavelength Cell Scoring Application Module (Molecular Devices).

[0337] In Vitro Transduction Analysis

[0338] To determine the relative transduction efficiencies the selected mutants compared to parental wild-type AAV serotypes, HEK293T, CHO KI, CHO pgsA (lacking all surface glycosaminoglycans), CHO Pro5 (the parental line for several glycosylation mutants, including Led cells), CHO Led (glycosylation defective), HeLa, and HT1080 cells (a human fibrosarcoma cell line) were plated at a density of 2.5x104cells per well 24 hours prior to infection. Cells were infected with rAAVl-GFP, rAAV2-GFP, rAAV6-GFP, Shuffle 100.1-GFP, Shuffle 100.3-GFP, SM 10.2-GFP, or Shuffle 100.7-GFP at a range of MOI of 100-1000. The percentage of GFP positive cells was assessed 48 hours post infection using a Beckman-Coulter Cytomics FC500 flow cytometer (Beckman-Coulter, Brea, Calif.).

[0339] In Vivo Analysis of Antibody-Evading Variants

[0340] For analysis of gene expression in vivo, eight week old, female, Balb / c mice were primed with 4 mg IVIG per mouse or phosphate buffered saline (for control mice) via tail vein injection 24 hours prior to administration of recombinant Shuffle 100-3 (see SEQ ID NO: 12), SM 10-2 (see SEQ ID NO: 10), or AAV2 vectors. Mice were infected with 1011viral genomes of recombinant AAV vectors encoding luciferase under the control of a CMV promoter via tail vein injection. For bioluminescence imaging, mice were anesthetized with 2% isofluorane and oxygen. D-luciferin substrate (GOLD Biotechnology, St. Louis, Mo.) was injected intraperitoneally, at a dose of 500 pg / g of body weight. Images were generated using a VivoVision I VIS Lumina imager (Xenogen, Alameda, Calif.). For each mouse, ventral images were taken 7-10 minutes after the substrate injection, eveiy week for four weeks. Five weeks post-infection, serum was collected via cardiac puncture and mice were then perfused with 0.9% saline solution. Heart, liver, lungs, kidney, spleen, brain, spinal cord, and hind limb muscle were harvested and frozen. Frozen tissue samples were homogenized and resuspended in reporter lysis buffer (Promega, Mannheim, Germany) for in vitro luciferase analysis. Lysate containing luciferase was clarified by centrifugation for 10 minutes at 10,000 g. To assay the samples, 20 jiL of the lysate was added to 100 pL of the luciferase assay buffer, mixed, incubated for 5 minutes, and placed in the luminometer. The signal was integrated for 30 seconds with a 2 second delay and was reported in Relative Light Units (RLU) detected by a TD 20 / 20 luminometer (Turner Designs, Sunnyvale, Calif). The luciferase signal was normalized to the total protein content determined by a bicinchoninic acid assay (Pierce).

[0341] Results

[0342] Our results demonstrate that AAV can evolve to significantly overcome neutralization by anti-AAV antibodies, both in vitro and in vivo. Novel AAV variants were isolated that required 2- to 35-fold higher neutralizing antibody titers (using human IVIG) than wild-type AAV in vitro. The antibody neutralization properties also translated to enhanced transduction in vivo in the presence of neutralizing antibodies. The isolation of such novel clones resistant to anti-AAV antibodies allows for the broader implementation of treatments based on AAV as a nucleic acid delivery vector (including individuals with high antibody titers that are currently ineligible for AAV gene therapy).

[0343] AAV Library Generation and Selection Through Directed Evolution

[0344] FIG. la shows a schematic of the directed evolution approach used to isolate novel AAV variants capable of evading human antibody neutralization. Libraries of viruses were created using the DNA mutagenesis techniques described in the following paragraphs (FIG. la, steps 1 and 2). During initial selections, pools of viral libraries developed fromerror-prone PCR mutations to AAV2 cap genes were incubated with various dilutions of the low potency a human sera pool for 30 minutes at room temperature prior to infection of HEK293T cells (step 3). Following three rounds of selection against the low potency a human sera pool (FIG. la, steps 4 and 5), several variants with enhanced resistance to this neutralizing sera pool were obtained (FIG. la, step 6, FIG. 7a). Variant 1.45, contained two point mutations (N312K, N449D), which resulted in >10-fold more resistance to neutralization by the a pool compared to wild type AAV2.

[0345] The cap gene from variant 1.45 was subjected to additional random mutagenesis and the resulting library was selected for three additional rounds of selection against the p and y pools, in parallel. As only minor improvements in antibody evasion were observed (data not shown), the recovered cap genes were pooled and subjected to additional diversification via DNA shuffling and EP PCR. Three more rounds of selection against increasing amounts of sera from both the p and y pools resulted in substantial enrichment in the amount of recovered virus from the viral library compared to wild type AAV2 (FIG. 1b, c). Sequencing of the successful cap genes from both pools revealed several low frequency mutants and a single dominant mutant, variant y4.3, which contained four point mutations (N312K, N449D, N551 S, and 1698V), present within both libraries. In the presence of human IVIG, variant 1.45 demonstrated a modest 1.2-fold enhanced resistance to neutralization, whereas y4.3 demonstrated 3.1 -fold enhanced resistance to neutralization (FIG. 7 J). This observation confirms the hypothesis that pools of individual human sera can be used to isolate AAV variants capable of enhanced evasion of antibodies present in the general human population.

[0346] The moderate success of variant y4.3 in resisting neutralization by anti-AAV antibodies prompted the development of a library based on the y4.3 cap gene. Amino acid sites R471, K532, E548, N587, V708, T716, previously determined to be immunogenic sites on the AAV2 capsid, were subjected to saturation mutagenesis in an attempt to find amino acid mutations that may improve upon the antibody resistance of y4.3. This “saturation mutagenesis” library, along with a “shuffled” library composed of random cap chimeras of 7 parent AAV serotypes and a “loop-swap” library composed of AAV2 cap with substituted loop regions were subjected to three additional rounds of selection, in which the pools of viral libraries were incubated with various dilutions of human IVIG for one hour at 37° C. prior to infection of HEK293T cells. Following infection with AAV libraries, and amplification of the infectious AAV variants through adenovirus superinfection, the number of viral genomes, or viral titer, from each library condition was quantified and compared to titers of wild-type AAV2 as a method for determining the success of the selection (FIG. I b\ For each round of selection using the saturation mutagenesis and loop-swap / shuffled libraries, viral pools from the 1:10 and 1:100 IVIG dilution conditions that produced higher viral titers than wild-typeAAV2 were used as the starting point for the subsequent round of selection. After three rounds of selection, the successful viral cap genes were isolated and tested individually to determine the virus with the most efficient gene delivery. In addition, the cap genes isolated from the third round of selection were subjected to additional rounds of error-prone PCR mutagenesis, and the process was repeated to iteratively increase the fitness of the virus.100347] FIG. 1 depicts directed Evolution of AAV for Enhanced Antibody Evasion, (a) Schematic of Directed Evolution. 1) A viral library is created by genetically diversifying the cap gene using several complementary approaches. 2) Viruses are packaged in HEK293T cells using plasmid transfection, then harvested and purified. 3) The viral library is incubated with human IVIG at several concentrations and introduced to HEK293T cells in vitro. 4) Successful viruses are amplified and recovered via adenovirus superinfection. 5) Successful clones are enriched through repeated selections at lower MOIs. 6) Isolated viral DNA reveals successful cap genes, 7) Successful cap genes are mutated again to serve as a new starting point for selection, (b) Selection of Antibody Evading Mutants from Loop-Swap / Shuffled, and Saturation Mutagenesis libraries. HEK293T cells were infected with viral libraries for 24 hours. Viral particles that productively infected cells were amplified by adenovirus infection, and the rescued AAV was quantified by qPCR (quantitative polymerase chain reaction). A 1: 10 dilution of IVIG corresponds to a concentration of 10 mg IVIG / mL. Error bars indicate the standard deviation (n=3).

[0348] FIG. 7 demonstrates the generation of human antibody evaders based on AAV2. (a) Four viral clones selected after three rounds of selection against the low stringency a pool demonstrate enhanced resistance to 1 pL of a serum at MOI of 1. Two additional rounds of diversification (i.e. mutagenesis and DNA shuffling) and selection (3 rounds of increasing serum amounts) resulted in significantly enhanced viral recovery in the presence of large amounts of highly potent (b) p and (c) y pools, (d) Additionally, two viral clones (1.45 and y4.3) demonstrate 1.23- and 3.10-fold enhanced resistances to a highly diverse pool of preexisting antibodies present with pooled human intravenous immunoglobulin (IVIg) from ”100,000 individuals compared to wild-type AAV2.

[0349] Increased Antibody Evasion of the Novel Evolved AAV Variants In Vitro

[0350] Of the twelve clones selected and packaged for individual analysis from the saturation mutagenesis and loop-swap / shuffled libraries after nine rounds screening against human IVIG, all twelve required higher neutralizing antibody titers than both wild-type AAV1 and AAV2 (FIG. 2a and Table 1). Variant Shuffle 100-3 (see SEQ ID NO: 12), which required a 35-fold higher in vitro IVIG concentration for neutralization than wild-type AAV2, was still capable of transducing approximately 10% of cells in the presence of 1 mg / mL IVIG(FIG. 2b), In addition, variant SM 10-2 from the AAV2 saturation mutagenesis library required a 7.5-fold higher in vitro WIG concentration for neutralization than wild-type AAV2. Furthermore, variants Shuffle 100-3 and SM 10-2 (see SEQ ID NO: 10) showed enhanced transduction in the presence of sera samples from individual patients excluded from a hemophilia B clinical trial (FIG. 3) (Nathwani et al., N Engl J Med. 2011 Dec. 22; 365(25):2357-65).

[0351] FIG. 2 depicts the neutralization profiles of antibody evading variants. The cap genes of antibody evading mutants isolated after three rounds of evolution were used to package recombinant AAV encoding GFP and incubated with human IVIG before infection of HEK293T cells. The fraction of remaining infectious particles was determined using high content fluorescence imaging and normalized to the infectious titer in the absence of IVIG. Two clones from each library with resistance to IVIG are shown. Data for the other clones analyzed are displayed in Table 1 . (a) Neutralization curves. Error bars indicate the standard deviation (n=3). (b) Representative fluorescence images from several IVIG dilutions show that mutants are capable of HEK293T transduction in the presence of high concentrations of neutralizing antibodies.

[0352] FIG. 3 depicts the neutralization profiles of antibody evading variants. Human sera were acquired from individuals that were excluded from hemophilia B clinical trials due to the presence of high neutralizing antibody titers against AAV. Recombinant / AAV encoding GFP was incubated with individual human serum samples before infection of HEK293T cells. The fraction of remaining infectious particles was determined using fluorescence microscopy and normalized to the infectious titer in the absence of human sera. Error bars indicate the standard deviation (n=3).

[0353] Sequence analysis of the twelve clones revealed that the two variants with the highest neutralizing antibody resistance, Shuffle 100-3 (see SEQ ID NO: 12) and Shuffle 100- 1 (see SEQ ID NO: 11), are almost identical shuffled capsids containing fragments of AAV1- 4, AAV6, and AAV9 (FIG. 4). Differences in amino acids 469 (AAV6 residue to AAV7 residue) and 598 (AAV6 residue to AAV1 residue) between the two variants translate to almost a 3-fold increase in neutralizing antibody titer for Shuffle 100-3 (see SEQ ID NO: 12) (Table 1). Variant Shuffle 100-7 (see SEQ ID NO: 13), which had the fourth highest neutralizing antibody resistance (Table 1), is also a shuffled capsid containing fragments of AAV1, AAV6, and AAV8 (FIG. 4), which agrees well with reported data showing that wildtype AAV1 and AAV8 are effective at evading anti-AAV2 antibodies. Interestingly, variant SM 10-2 (SEE SEQ ID NO: 10) retained the point mutations acquired by variant y4.3 and also retained wild type residues at the saturation mutagenesis sites. Variant SM 10-2 (SEE SEQ ID NO: 10) did acquire additional point mutations at surface residue D472N and internalresidue L735Q. FIG. 4 depicts the amino acid sequences of loop-swap / shuffle and saturation mutagenesis clones, (a) Schematics of the capsid protein are shown for the two clones from each library with the highest neutralizing IVIG concentrations. Each region is shaded according to the parent serotype from which it is derived. Black arrows denote (from left to right) the start codons of VP1, VP2, and VP3 capsid proteins. Gray arrows denote (from left to right) surface loop regions I, II, III, IV, and V based on the AAV2 capsid, (b) Molecular models of the full AAV2 capsid, based on the solved structure, are shown for the two clones from each library with the highest neutralizing IVIG concentrations. Each region is shaded according to the parent serotype from which it is derived. For variant Shuffle 100-3 (see SEQ ID NO: 12), black arrows indicate differences from variant Shuffle 100-1 (see SEQ ID NO: 11). For variant SM 10-2 (SEE SEQ ID NO; 10), mutations N449D, D472N, N551 S, and 1698V are surface mutations (black).

[0354] Table 1: IVIG Neutralizing Antibody Titers of Library Clones and Parent Serotypes

[0355] Human IVIG was used to neutralize recombinant AAV-GFP vectors with capsids from wild-type AAV1, AAV2, AAV8, and variants recovered from the loop-swap / shuffled and saturation mutagenesis libraries. The IVIG concentration (mg / mL) required to reduce gene delivery efficiency to 50% of that in the absence of IVIG is shown, and compared to the concentration required to reduce delivery of AAV2. All variants analyzed required higher concentrations of IVIG than wild-type AAV1 and AAV2. The neutralizing antibody titer was determined by fitting the curves in FIG. 2 to an exponential curve. SEQ ID NOs are listed as “amino acid, nucleotide.”(00356]TABLE 1Fold ResistanceSEQ ID Neutralizing IVIG Relative toClone NO: concentration mg / mlAAV2AAV1 1 0.026 1.757AAV2 2 0.015 1.000AAV8 8 0.092 6.113Shuffle 10-2 26, 34 0.037 2.443Shuffle 10-6 27, 35 0.028 1.842Shuffle 10-8 28, 36 0.084 5.583Shuffle 100-1 11, 23 0.183 12.178Shuffle 100-229, 37 0.073 4.831Shuffle 100-3 12, 24 0.529 35.227Shuffle 100-7 13, 25 0.090 6.025SM 10-1 30, 38 0.071 4.732SM 10-2 10, 22 0.113 7.519SM 10-8 31, 39 0.051 3.409SM 100-3 32, 40 0.074 4.941SM 100-10 33, 41 0.066 4.393

[0357] Variants Shuffle 100-3 (see SEQ ID NO: 12), Shuffle 100-1 (see SEQ ID NO: 11), and Shuffle 100-7 (see SEQ ID NO: 13) have transduction profiles that mimic the transduction profiles of parent serotypes AAV1 and AAV6 (FIG. 5). In addition, the mutations in SM 10-2 (see SEQ ID NO: 10) do not prevent a heparin dependence (as seen in parent serotype AAV2) leading to a profile similar to AAV2 (FIG. 5).

[0358] FIG. 5 demonstrates the in vitro tropism of novel aav variants. Recombinant AAV vectors expressing green fluorescent protein were used to transduce a panel of cell lines: CHO, pgsA (lacking all surface glycosaminoglycans), Pro5, Led (lacking sialic acid), HEK293T, HeLa, and HT1080 (human fibrosarcoma cell line) to profile the transduction properties of the new AAV variants. Error bars indicate the standard deviation (n— 3).

[0359] Increased Antibody Evasion of the Novel Evolved AAV Variants In Vivo

[0360] To determine the localization pattern of variants Shuffle 100-3 and Shuffle 100-7, luciferase enzyme activity was examined in various tissues of naive mice injected with AAV2, Shuffle 100-3, or Shuffle 100-7 (FIG. 6a). Variant Shuffle 100-7 displayed similar in vivo tropism to AAV2, except for 7-fold higher transduction of the heart, 5-fold higher transduction of the lungs, and 4.5-fold lower transduction of the liver. The Shuffle 100-3 variant exhibited over 4-fold higher transduction of the brain, over 3-fold higher transduction of the lungs, and 27-fold higher transduction of muscle than AAV2. Analysis of the serum from these mice showed that variant Shuffle 100-3 required equal or higher in vitro serum concentrations for neutralization than AAV1 and AAV8 for serum from mice given AAV1, AAV2, AAV8 or Shuffle 100-3 gene delivery vectors (FIG. 11). Shuffle 100-7 required equalor higher in vitro serum concentrations for neutralization than AAV1 for serum from mice given AAV1, AAV2, AAV8, Shuffle 100-3, or SM 10-2 gene delivery vectors (FIG. 11). Furthermore, both variants were less neutralized by serum from mice given AAV2 gene delivery vectors than all wild-type AAV serotypes tested. Interestingly, variant Shuffle 100-3 was also less neutralized by serum of mice immunized against it than any of the other serotypes or variants tested (FIG. 11). This data illustrates the possibility that these variants could be used in combination with wild-type AAV serotypes or the other variant in applications requiring multiple vector administrations.

[0361] FIG. 11 shows the neutralizing antibody titers of library clones and parent serotypes in immunized mouse sera. Sera from mice administered library clones or wild-type AAV was used to neutralize recombinant AAV-GFP vectors with capsids from wild-type AAV1, AAV2, AAV8, and variants recovered from the loop-swap / shuffled and saturation mutagenesis libraries. The serum dilution required to reduce gene delivery efficiency to 50% of that in the absence of serum is shown.

[0362] To determine the ability of variants Shuffle 100-7 and Shuffle 100-3 to evade antibody neutralization in vivo, mice were passively immunized with human IVIG prior to AAV injection. Variant Shuffle 100-7 had significantly higher heart, liver, and muscle transduction than AAV2, as measured by luciferase enzyme activity (FIG. 6b). Variant Shuffle 100-3 had significantly higher heart and muscle transduction compared to AAV2 (FIG. 6b).

[0363] FIG. 6 shows the in vivo localization and neutralization of novel AAV variants, (a) Recombinant AAV vectors encoding luciferase were administered via tail vein injection to female B ALB / c mice. After 5 weeks, levels of luciferase activity were determined and normalized to total protein for each sample analyzed, (b) Recombinant AAV vectors expressing luciferase were administered via tail vein injection to female BALB / c mice 24 hours after tail vein injection of 4 mg of human IVIG. After 5 weeks, levels of luciferase expression were normalized to total protein for each sample analyzed. Error bars indicate the standard deviation (n=3), *=p<0.05. RLU, relative luciferase unit.

[0364] Variant ?4.3, isolated from an AAV2-based error-prone library selected against a pool of individual human sera, contained four point mutations (N312K, N449D, N551S, and I698V). Interestingly, two of these positions (N449 and N551) were previously identified as immunogenic residues using other pools of human serum, demonstrating that antigenic epitopes involving these sites are targeted by many different neutralizing antibodies. Thus, these sites are interesting and valuable targets for mutation. Pairing directed evolution and rational design in the saturation mutagenesis library resulted in the isolation of variant SM 10-2, which was capable of higher antibody resistance than both AAV1 and AAV2 in vitro. Variant SM 10-2 incorporates two additional point mutations (D472N and L735Q) to those found on variant y4.3. The D472N mutation was previously shown to increase the level of capsid synthesis in HEK293 cells. Similarly, the replacement of the positively charged lysine side chain at amino acid position 735 with the uncharged glutamine side chain may function to stabilize the capsid, as it is also present in variant Shuffle 100-7 despite being located within the interior of the assembled capsid (FIG. 4).

[0365] The creation of chimeric AAV capsids allows for the creation of viral variants that can merge desirable properties from multiple AAV serotypes. Although AAV8 and AAV9 have also been shown to be much more resistant to neutralization by IVIG than AAV2, amino acids specific to these capsids were only present in small spans on the surface of the shuffled variants isolated during our selections (FIG. 4). The variant displaying the more efficient evasion of antibody neutralization in vitro, Shuffle 100-3, displayed similar in vitro tropism to its parental serotypes AAV1 and AAV6, but at a higher rate of infectivity than either wild-type serotype. Differences in amino acids 469 and 598 between variants Shuffle 100-1 and Shuffle 100-3 translate to almost a 3-fold increase in neutralizing antibody titer for Shuffle 100-3. A study by Lochrie et al. reported that the immunogenic residues recognized by human sera and IVIG are different, suggesting that different humans can produce various neutralizing antibodies to different sets of epitopes on the AAV capsid and complete escape from neutralization is not easy (Lochrie et al., J Virol. 2006 January;80(2):821-34). Our work demonstrates that the use of multiple rounds of directed evolution using several different serum pools containing various amounts and potencies of anti- AAV antibodies will result in the isolation of novel AAV variants that are capable of enhanced cellular transduction, both in vitro and in vivo, in the presence of multiple anti-AAV antibody pools.

[0366] Adaptive immune responses to AAV vector components in animals and humans often prevent re-administration of AAV vectors of the same serotype, making gene delivery applications requiring multiple vector administrations difficult. In vitro neutralization assays using the serum from the mice used in the biodistribution studies demonstrate that the variants are less neutralized by these sera than wild-type AAV (FIG. 11), which may be useful for gene therapy strategies in which vector readministration is necessary. For example, Shuffle 100-3 was not neutralized by serum from mice injected with AAV2, and AAV2 was not neutralized by serum from mice injected with Shuffle 100-3, suggesting this variant can be used in combination with wild-type AAV serotypes or in applications requiring multiple vector administrations. In conclusion, we have used directed evolution to isolate novel AAVvariants that are capable of reduced neutralization by anti-AAV antibodies derived from individual human patients, pooled human serum, and mouse serum, both in vitro and in vivo.EXAMPLE !Identification of a Capsid Variant Suitable for Use in Gene Therapy to the Primate Lung

[0367] Introduction

[0368] A directed evolution strategy was used to identify AAV Capsid Variants with enhanced gene delivery efficiency to non-human primate (NHP) lung alveolar epithelial type II (AT II) cells following intratracheal aerosol administration in the presence of human neutralizing antibodies (NAbs). Briefly, wild-type adeno-associated virus (AAV) cap genes were diversified by several approaches to create large genetic libraries that were packaged to generate libraries of viral particles, and selective pressure was then applied to isolate novel variants that can overcome gene delivery barriers, including but not limited to anti-capsid immune responses, limited transduction of certain tissues, and inability for targeted delivery to specific cell types.

[0369] Methods

[0370] Cell Lines and Library Production

[0371] HEK293T cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured at 37°C and 5% CCh in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA) and 1% penicillin / streptomycin (Invitrogen, Carlsbad, CA). Viral libraries were produced in HEK293T cells using triple transfection, and viruses were purified by iodixanol gradient centrifugation and Amicon filtration. DNase-resistant genomic titers were determined via quantitative PCR (qPCR).

[0372] Intratracheal Injection and Tissue Harvesting

[0373] For each delivery device used in the selection, a single male cynomolgus macaque (macaca fascicidaris) between 4-6 years of age and weighing between 5.5-6.0 kg was dosed. The animals were anesthetized with 10 mg / kg ketamine and 15 pg / kg dexmedetomidine delivered intramuscularly (IM). Five mL of the library was pre-complexed with 1.75 mg / mL of human intravenous immunoglobulin (IVIG) and administered as described below. Each animal was intubated with a 5 mm endotracheal tube, with the tip of the tube positioned at the level of the clavicle (approximately 5 cm above the carina), and its position confirmed by fluoroscopy.

[0374] The nebulizer device was connected to the distal end of the endotracheal tube, and a bird respirator was used to deliver breaths at a rate of 15± 1 breaths / minute with a pressure of 20 cm HjO. The AeroProbe® catheter (Trudell Medical International) was fitted with a piece of 0.144 inch star tubing to facilitate proper location within the endotracheal tube. The tip of the catheter was positioned just above the tip of the endotracheal tube. The AeroProbe® catheter was connected to the AeroProbe Catheter Control System, and a ventilator (Harvard Appartatus) was used to deliver breaths at a rate of 20 breaths / minute with a pressure of 18-20 cm H2O, Following the completion of dosing, each animal was extubated and received 0.15 mg / kg atipamezole IM to reverse sedation. The animals were visually monitored until fully recovered from anesthesia prior to returning to their home cages.

[0375] Euthanasia was performed by trained veterinary staff using 100 mg / kg pentobarbital sodium delivered intravenously on day 15±1. The lungs, including the trachea, were removed and dissected as detailed below. DNA was isolated from the AT II cells and stored at -20°C until viral genome amplification.

[0376] Alveolar Epithelial Type II (AT II) Cell Isolation

[0377] AT II cells were isolated from non-human primate lungs, as described by Fang et al., Measurement of Protein Permeability and Fluid Transport of Human Alveolar Epithelial Type II Cells Under Pathological Conditions. Humana Press, New York, NY, 2018, pp 121— 128. Briefly, lungs were flushed with 500 mL of PBS containing 5 mM EDTA and 5 mM EGTA using a syringe placed within the trachea. Lungs were then filled with 250 mL of a 1.2 mg / mL elastase solution and incubated for 1 hour at 37°C. Lungs were homogenized to release cells lining the lungs, and the airways were discarded. AT II cells were isolated following a series of inclusion and exclusion steps, including a Percoli gradient and CD14 / CD45 Dynabeads. AT II cells were plated on collagen IV-coated inserts for 24 hours prior to DNA isolation. Characterization was done examining Lysotracker and surfactant protein C by flow cytometry and immunocytochemistry to insure purity of the cell isolate.

[0378] Therapeutic Vector Evolution

[0379] The Vector Evolution process employed is shown in Figure 12. Briefly, a viral capsid library comprising proprietary combinations of DNA mutation techniques and cap genes was created (a). Viruses were then packaged (b) such that each particle is composed of a mutant capsid surrounding the cap gene encoding that capsid and purified. The capsid library was placed under selective pressure in vivo. The tissue or cell type of interest was harvested to isolate AAV variants that have successfully localized to the target. Successful viruses were recovered by PCR amplification. Successful clones were enriched through repeated selection (Stage I - (c)). Selected cap genes then underwent proprietary rediversification and were enriched through further selection steps to iteratively increase viral fitness (Stage 2 - (d)). Variants identified as hits during Vector Selection Stages 1 and 2 were assessed to identify capsid variants with the desired properties (e).

[0380] Motifs were declared “Hits” when the following criteria were met: 1) a motif represents approximately 5% of the sequenced population in two or more consecutive rounds of the selection; or 2) a motif representing at least 10% of the sequenced population in one or more rounds of the selection.

[0381] Results

[0382] Pilot Studies for Delivery Device Parameters and AT II Cell Isolation

[0383] Two delivery devices, an AeroProbe® catheter (Trudell Medical International) and CRO’s in-house nebulizer, were employed to enable downstream compatibility with multiple clinically translatable devices. Both delivery devices were evaluated in pilot studies delivering Evans blue dye to ensure that ventilation parameters resulted in adequate distribution to all lung lobes and the alveolar sacs. Both delivery devices demonstrated good distribution to all lobes, including the alveolar compartment, with more intense dye observed in the dependent lobes.

[0384] The AT II cell isolation protocol was optimized using a total of 6 NHP lungs. The protocol optimization resulted in high yield and purity of AT II cells isolated from both NHP lungs used during Therapeutic Vector Evolution.

[0385] Therapeutic Vector Evolution

[0386] Prior to initiation of Round 1 of the Therapeutic Vector Evolution program, 37 Vector libraries were synthesized, manufactured, and characterized. As shown in Figure 13 A, the diversity of the plasmid libraries is estimated to include approximately 1x10sto >lx108individual unique variants per library. This represents a high quality, highly diverse starting library of AAV variants. Next, production of each individual library was completed in orderto generate enough material for the first round of selection. As shown in Figure 13B, all libraries manufactured at a level sufficient to produce material for an in vivo Therapeutic Vector Evolution selection.

[0387] All 37 libraries were combined and successfully administered to both NHPs via a single dose aerosol administration using either the AeroProbe® or nebulizer. Prior to administration, the libraries were incubated with 1.75 mg / mL human intravenous immunoglobulin. This represents a high, yet physiologically-relevant lung mucus concentration of human NAbs. The library dose of 1.7 x 1012vg perNHP represents a dose that is approximately 10-fold lower than the current maximum feasible dose based on manufacturing considerations. Therefore, this represents stringent selective pressure to enable discovery of a vector capable of transducing AT II cells in the alveolar space in the presence of NAbs. The NHP lungs were harvested two weeks post-administration. AT II cells were isolated from the lungs, and DNA was isolated from the AT II cells.

[0388] Successful Amplification of AAV Capsid Genomes

[0389] Amplification of capsid genes from tissue represents successful localization of library vectors into the cell type of interest. The capsids amplified from each delivery device (Figures 14A-B) were cloned into an AAV library packaging plasmid for sequence analysis and to initiate the subsequent round of selection (if necessary).

[0390] Sequencing Analysis

[0391] Sequencing was performed on individual clones within the library to determine the frequency of variants within the population. Sequencing on a minimum of 90 clones from each delivery device was performed. Variants were evaluated for the presence of motifs within the sequencing data. Variants were grouped into motifs based on the presence of a unifying variation (for example, a specific point mutation or specific peptide insertion sequence in a consistent location within the capsid) that occurred in multiple sequences. A motif was progressed for further evaluation only if it represents at least 5% of the sequenced population in two or more consecutive rounds of the selection or at least 10% of the sequenced population in one or more rounds of the selection. The motif that met the latter criterion is represented in Figure 15A-B. The selection was considered complete following the first round, as strong convergence to the A 101 variant (comprising a capsid protein of SEQ ID NO: 12) was observed using both delivery devices.

[0392] Based on the ranking criteria above, Al 01 capsid variant comprising a capsid protein of SEQ ID NO: 12 was identified as conferring enhanced gene delivery efficiency to the primate lung following intratracheal aerosol administration in the presence of humanneutralizing antibodies (NAbs). Al 01 is a chimera consisting primarily of AAV1 but also including amino acids from AAV2, AAV4, AAV6 and AAV9.EXAMPLE 3

[0393] Although initial attempts at developing AAV-based gene therapy for the lung demonstrated clinical safety, the use of the AAV2 vector ultimately did not efficiently transduce lung cells to show clinical benefit in cystic fibrosis. More recently, additional AAV serotypes, including AAV1 and AAV5, have demonstrated improved, but still not optimal, transduction of primate lungs following aerosolized administration. The experimental data below confirms the surprising suitability of rAAV comprising a capsid comprising a capsid protein of SEQ ID NO: 12 as a vehicle in which to efficiently deliver transgenes such as human CFTR throughout the primate lung in the presence of human neutralizing antibodies.

[0394] Gene delivery of recombinant AAV (rAAV) comprising (i) a capsid comprising a capsid protein of SEQ ID NO: 12 and (ii) a nucleic acid comprising a nucleotide sequence encoding reporter transgene (GFP or EGFP) operably linked to a CAG promoter was characterized (including evaluation of histopathology) following aerosol administration to three non-human primates (NHP). Nebulized delivery of the rAAV resulted in robust delivery of viral genomes to all regions of the lung, including the peripheral (bronchioalveolar regions), with minimal systemic biodistribution, and the rAAV mediates protein expression to all regions of the lung, including the alveoli.

[0395] MATERIALS AND METHODS

[0396] Neutralizing Antibody Assay

[0397] HEK2v6.11 cells (obtained from John Hopkins University) were plated on black opaque 96 well plates at a cell density of 30,000 cells / well in Dulbecco’s modified Eagle medium (DMEM; Coming) with 1% heat inactivated fetal bovine serum (FBS; GE Healthcare Life Sciences) and 1% penicillin / streptomycin (Invitrogen). Cells were allowed to adhere to the plate for 24 hours prior starting the experiment.

[0398] Each NHP serum sample was assayed at dilutions of 1 : 10, 1 :25, 1 :50. Each plate contained positive and negative controls for transduction. NHP serum samples were incubated with rAAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO: 12 and (ii) a nucleic acid comprising a CAG promoter operably linked to a lucerifase gene at an MOI of 1,000 at 37°C for 1 hour. Following a 1 hour incubation, each NHP serum sample dilution plus the rAAV was added to individual wells of black opaque 96 well plates containing 2V6.11 cells. Luciferase was detected by ONE-Glo EX Luciferase assay kit (Promega) 48 hours post-transduction. With the addition of the ONE-Glo EX, cells were lysed, andluciferase substrate was added to the cells in a single step. Luminescence was read using a Cytation 3 microplate reader (BioTek).

[0399] Coefficients of variation (CV) and standard deviations were calculated for allNHP serum sample dilutions and each point of the standard curve. NHP serum samples were normalized to the positive transduction control. Each NHP was assigned a neutralizing antibody titer. The neutralizing antibody titer for each NHP serum sample was defined as the lowest serum dilution at which > 50% transduction was observed. NHPs for which > 50% transduction at 1 : 10 serum dilution was observed were considered for inclusion in the study.

[0400] Test System & Immunosuppression

[0401] Three male cynomolgus macaques were included in the study. Animals ranged in age from 4 years, 10 months to 9 years, 8 months and ranged in weight from 4.73 kg to 7.09 kg. Animals received methylprednisolone (20 mg / kg, intramuscular) immunosuppression once weekly starting on day -7.

[0402] Test Article Preparation and Administration

[0403] Test article lots of rAAV comprising (i) a capsid comprising a capsid protein of SEQ ID NO: 12 and (ii) a nucleic acid comprising a CAG promoter operably linked to a EGFP were thawed on ice, pooled together, and diluted in formulation buffer to deliver a final dose of 2.80 x 1012vg / kg in 5 mL to each NHP. The test article dilutions for each animal are provided in Table 3. Animals were sedated with Ketamine and Dexmedetomidine. Animals were intubated with the tip of the intubation tube located approximately 5 cm above the carina. Animals were placed in a chair in a seated position for administration. For each animal, 5 mL of diluted test article was loaded into the AeroEclipsell nebulizer reservoir (Trudell Medical). During dosing, pressure was adjusted to 15-20 cm H2O. Test article was administered at a rate of 12-24 breaths / minute until no visible mist was generated for 10 pulses (animals were dosed continuously for < 40 minutes). Following the completion of dosing, animals were extubated, and sedation was reversed.

[0404] Table 3: Test: Article Dose Preparation

[0405] In-Life, Necropsy, and Tissue Harvesting

[0406] Cage-side observations were performed twice daily by CRO staff from Day -7 to necropsy. Body weights were assessed weekly, and blood samples were collected at defined timepoints for hematology and clinical chemistry. Euthanasia was performed on Day 57 + 1 by trained veterinary staff using by intravenous injection of pentobarbital sodium (100 mg / kg) followed by bilateral thoracotomy and transcardial perfusion with heparinized phosphate buffered saline. Following perfusion, the lungs (including trachea), brain, spinal cord (cervical, thoracic, and lumbar regions), heart (ventricle and atrial regions), liver, spleen, skeletal muscle (triceps brachii, vastus lateralis), diaphragm, and kidney were collected. Samples of tissue were collected and flash frozen for subsequent DNA and protein isolation. Additional samples were collected and fixed in 4% paraformaldehyde (for lung and neural tissues) or 10% neutral buffered formalin (for peripheral tissues) for subsequent paraffin embedding and sectioning for immunofluorescence.

[0407] The trachea and lungs were sampled extensively to provide multiple samples for each analysis process. The lungs were harvested and clamped as superior on the trachea as possible. The right lung was clamped twice, approximately 1 mm apart, on the mainstem bronchi. The right lung was removed by cutting between the clamps. Sixteen samples each for DNA and protein isolation were collected from regions of the right lung encompassing the primary / secondary bronchi, tertiary bronchi, and alveoli, as described in Figure 16. The trachea and left lung were inflated with 4% paraformaldehyde and fixed in a 1 Ox volume of 4% paraformaldehyde. The trachea and left lung were then sectioned to encompass samples of the trachea, primary / secondary bronchi, tertiary bronchi, and alveoli, as described in Figure 16.

[0408] Viral Genome Biodistribution

[0409] Viral genome biodistribution was performed by the Mattawan site of Charles River Laboratories using a qualified assay for AAV viral genomes containing the EGFP transgene sequence. Total DNA was extracted from tissue samples using a QIAsymphony (Qiagen) and associated DSP DNA mini kit. qPCR reactions were performed on 96-well plates, with each plate containing a standard curve, a set of QC samples, and study samples.Duplicate QC samples were prepared at high, medium, and low copies / reaction in a background of 1,000 ng NHP matrix DNA per reaction. When possible, the tissue DNA samples were tested at 1,000 ng per reaction. If it was not possible to load the amounts specified above for a specific sample (because the DNA concentration was too low or the sample volume was limiting), a smaller amount of sample DNA was analyzed.

[0410] All sample reactions were run in triplicate, and the third reaction was spiked with 200 copies of pAAV-CAG-EGFP-SV40 DNA to evaluate potential qPCR inhibition. If qPCR inhibition was observed as shown by the measured value in the third spiked well at less than 110 copies of the target DNA, the sample DNA were reanalyzed at lower amount.

[0411] Protein Expression Biodistribution

[0412] Total protein was extracted from tissue samples using a gentleMACS tissue dissociator (Miltenyi Biotec) and associated reagents. EGFP was quantified using a GFP ELISA kit (Abeam), and total protein was quantified using a Pierce BCA protein assay kit (ThermoFisher). For both GFP and total protein, reactions were performed in triplicate, and each kit contained a standard curve.

[0413] Immunofluorescence Imaging

[0414] Tissue was processed to paraffin blocks using a Sakura VIP 5, using a standard program for canine, NHP, and porcine tissues by Seventh Wave Laboratory. Slides were cut at 10 pm thickness by Seventh Wave Laboratory stored at 4 C. Immunohistochemistry was performed on 6 sections of lung (including alveolar and bronchial regions) and 2 sections of trachea from each study animal (n - 3) and an additional control animal. Paraffin slides were dehydrated using standard paraffin antibody staining protocol. Briefly, sections were deparaffinized with xylene and rehydrated with decreasing concentrations of ethanol in water (100%, 90%, 70%, 50% and 30%) followed by PBS wash. Antigen retrieval was performed prior to staining with antibody using a combination of heat-induced epitope retrieval (HEIR) and pressure. Slides were incubated for 10 minutes in boiling sodium citrate buffer, then incubated for 3 minutes under pressure. Slides were cooled to room temperature prior to antibody staining. Following antigen retrieval, slides were stained using a primary chicken polyclonal anti-GFP antibody (Abeam #13970) at 1:1000 dilution, a secondary goat antichicken IgY antibody (Abeam #175779) at 1:1000 dilution, and DAPI. Fluorescence imaging was performed using a Zeiss AxioObserver microscope. Anti-GFP signal was acquired in the far-red channel (647 nm) at 1000 ms. DAPI signal was detected in blue channel (355 nm) at 100 ms. All images were processed using the Zeiss ZenPro software using the same parameters and pixel intensity values in the anti-GFP channel.| 00415] Histopathology Assessment

[0416] Tissue trimming, embedding, sectioning, and H&E staining performed by Seventh Wave Laboratory. Tissue was processed to paraffin blocks using a Sakura VIP 5, using a standard program for canine, NHP, and porcine tissues. Slides were cut at 4 pm thickness and stained for hematoxylin and eosin (H&E) using a Leica XL Autostainer, A histopathology assessment was performed on 16 sections of lung, 4 sections of trachea, and 1 section of carina from each study animal (n = 3) and an additional control animal, with the histopathologist blinded to treatment condition. Slides scored for the nature and severity of the findings using standard assessment scale.100417] COMPUTERIZED SYSTEMS

[0418] For serum neutralizing antibody screening, data was generated and analyzed using the Cytation 3 microplate reader (Biotek), Gen5plus software version 3.03.14, and Microsoft Excel version 15.32.

[0419] For quantification of viral genomes within tissue samples, data was generated and analyzed using the QuantStudio 7 Flex Real-Time PCR System, QuantStudio Real Time PCR Software vl.4, Microsoft Excel, and GraphPad Prism version 8,1.2.

[0420] For quantitation of EGFP expression within tissue samples, data was generated and analyzed using the Cytation 3 microplate reader (Biotek), Gen5plus software version 3.03.14, Microsoft Excel version 15.32, and GraphPad Prism version 8.1.2.

[0421] For representative immunofiuorescent imaging, images were acquired using a Zeiss Axio Observer zl microscope and ZenPro software. Images were processed using ZenPro software. Images were transferred into Microsoft PowerPoint version 15.32 for presentation.

[0422] All data analysis and compilation were carried out on a Macbook Pro running OSX (10.12.6).

[0423] RESULTS and DISCUSSION

[0424] Anti- AAV Neutralizing Antibody Screen Identifies NHP for Study Inclusion

[0425] A neutralizing antibody assay was used in order to assess levels of neutralizing antibodies against AAV capsid having a capsid protein of SEQ ID NO: 12 in non human primate (NHP) serum. Each NHP serum sample was assigned a neutralizing antibody titer. An animal was considered seronegative and passed the study inclusion criteria if > 50% transduction was observed at a 1:10 serum dilution.

[0426] In total, 20 NHP serum samples were evaluated across four 96-weIl plates. Assay acceptance criteria was set for 1) the coefficient of variance (CV) of the standard curve, 2) CV of the unknown serum samples, and 3) percent deviation from actual input protein for the standard curve. Acceptable CVs for the standard curve were defined as < 25%, but actual CVs did not exceed 5%. Acceptable CVs for the unknown serum samples within the limit of quantification were defined as < 30%, but actual CVs did not exceed 19%. Acceptable percent deviation from input protein for the standard curve was defined as < 25%, but actual percent deviation did not exceed 19%. All plates met all assay acceptance criteria, and the data from these plates were used for evaluation. Overall, 11 (55%) NHP serum samples evaluated were seronegative for capsid having a capsid protein of SEQ ID NO: 12 (Figure 17). The NHPs with the top three highest percent transductions at the 1: 10 serum dilution were selected for study inclusion. All selected NHPs demonstrated transduction above the transduction observed in the absence of NHP serum. This observation has been noted in previous studies and is likely a result of favorable interactions with an unknown serum protein. The selected NHP IDs and the percent transduction at the 1 :10 serum dilution are reported in Table 4,

[0427] Table 4: NHPs Included in Study

[0428] Nebulized Delivery of Variant Capsid Comprising a Capsid Protein of SEO IDNO: 12 is Well-Tolerated in NHP

[0429] The study design is summarized in Table 5. All animals recovered normally following test article administration and survived to the scheduled necropsy dates. No significant clinical findings were reported in any animal at any point during the in-life portion of the study. Minor changes outside the references ranges for hematology and clinical chemistry findings were noted in some animals at some timepoints, but these variations were interpreted as normal physiological variations. No major findings were reported during gross examination at necropsy.

[0430] Table 5 - Study Design Summary

[0431] Variant Capsid Comprising a Capsid Protein of SEO ID NO: 12 Mediates Robust Gene Delivery to All Regions of Lung

[0432] Viral genomes were quantified by qPCR for all samples obtained during necropsy to determine the genomic biodistribution of rAAV comprising a variant capsid (comprising a capsid protein of SEQ ID NO: 12) and a nucleic acid comprising an EGFP transgene operably linked to a CAG promoter.

[0433] A high quantity of viral genomes, ranging from approximately IO4- 10svg / pg was observed in all 48 lung samples (n - 16 samples per NHP; n = 3 NHP), which represented samples from the alveolar sacs, tertiary bronchi, and primaiy / secondary bronchi (Figure 18). For all three animals, no significant differences in the quantity of viral genomes within different lung lobes or different lung regions were noted. A small number of heart and liver samples, 3 (out of 15) heart samples from NHP V003424 and 10 (out of 10) liver samples from NHP V002969, had detectable viral genomes present. However, the quantity of viral genomes present per pg of DNA was 1,000- to 10, 000- fold less than the quantity of viral genomes present in the lungs. All samples tested from skeletal muscle (triceps brachii, vastus lateralis), diaphragm, kidney, spleen, brain and spinal cord were below the lower limit of quantification (BLQ). Therefore, nebulized delivery of the rAAV results in robust delivery of viral genomes to all regions of the lung, with minimal systemic exposure.

[0434] Variant Capsid (Comprising a Capsid Protein of SEO ID NO: 12) Mediates Protein Expression to All Regions of Lung and Transduces Multiple Cell Types

[0435] EGFP protein expression was quantified for all samples for which qPCR demonstrated presence of viral genomes above the lower limit of quantitation of the assay. This sample set included all lung samples from all three NHPs, 3 (out of 15) heart samples from NHP V003424, and 10 (out of 10) liver samples from NHP V002969. EGFP expression was observed in all 48 lung samples (n = 16 samples per NHP; n = 3 NHP), whichrepresented samples from the alveolar sacs, tertiary bronchi, and primary / secondary bronchi (Figure 19), Expression was highest for animal V002969 across all regions and all lung lobes. For all three animals, no significant differences in the amount of EGFP protein expression within different lung lobes were noted. In general, samples from alveolar regions contained average or above average quantities of EGFP, but this trend was not significant. The qPCR+ liver samples from NHP V002969 did result in detectable EGFP protein expression, but expression levels were multiple orders of magnitude lower than expression levels in the lungs.

[0436] These results are consistent with genome biodistribution data demonstrating that genome localization to the liver was multiple orders of magnitude lower than genome localization to the lungs (Figure 18). The qPCR+ heart samples from NHP V003424 did not contain any detectable EGFP protein expression. These results demonstrate that nebulized delivery of rAAV comprising a capsid with a capsid protein of SEQ ID NO: 12 and a nucleic acid encoding a transgene, mediates protein expression to all regions of the lung.

[0437] Immunofluorescent imaging was performed to further define the extent of transduction within different regions of the lung. Slides representing 6 sections of lung (including alveolar and bronchial regions) and 2 sections of trachea from each study animal (n = 3) and an additional control animal were scanned, and representative images were acquired for each region. In general, EGFP expression was highest for animal V002969 across all regions and all lung lobes, which corresponds to the observed relative expression across animals as determined by ELISA. Within the trachea and bronchi, EGFP expression was observed primarily in cells of the ciliated epithelial layer (Figure 20). Within the alveoli, broad EGFP expression was observed (Figure 20), but ATI and ATII cells cannot be determined without the use of specific cell markers. These results demonstrate that nebulized delivery of rAAV comprising a capsid comprising a capsid protein of SEQ ID NO: 12 and a nucleic acid encoding a transgene mediates protein expression to all regions of the lung.

[0438] Administration of rAAV Comprising a Capsid Comprising a Capsid Protein of SEO ID NO: 12 Is Safe and Does Not Result in Inflammation in Lung Tissue

[0439] Cageside observations were performed twice daily throughout the in-life portion of the study, beginning one week prior to dosing. No significant clinical signs were observed in any of the animals throughout the study in-life portion of the study. Hematology and clinical chemistry analyses of blood samples were performed biweekly throughout the in-life portion of the study and one week prior to dosing. Although some individual hematology and / or clinical chemistry values fell outside of the reference range, these values were largely interpreted as physiological variations.

[0440] Following necropsy, a histopathology assessment was performed on lung tissue and compared to a control (non-administered) animal. Focal hemorrhage, black pigment and minimal mononuclear cell infiltrates in the alveolar spaces were observed in all animals and are common incidental findings in monkeys. In addition, the submucosal lymphoid infiltrates in the trachea and inflammation on the mucosal surface of the carina are also likely incidental findings and unrelated to test article administration. None of the observations were considered adverse. No findings in the treated animals were different or of greater severity than those observed in the control animal.

[0441] CONCLUSIONS rAAV comprising a capsid with a variant capsid protein of SEQ ID NO: 12 and a nucleic acid encoding a reporter transgene (EGFP), was characterized by aerosol delivery of a reporter gene to cynomolgus macaques. Sera was pre-screened to identify animals that were seronegative for pre-existing neutralizing antibodies to the test article capsids. Animals (n = 3) received the maximum feasible dose of rAAV, delivered using the AeroEclipsell device, a clinically relevant actuated nebulizer.

[0442] A high quantity of viral genomes (by qPCR) and resulting EGFP expression (by ELISA and immunostaining) was observed in all lung samples across all 3 NHPs in the study, which represented samples from the alveolar sacs, tertiary bronchi, and primary / secondary bronchi. A small number of heart and liver samples had low but detectable viral genomes present, and all samples from all other tissues showed no detectable viral genomes. These data demonstrate that nebulized delivery of the rAAV results in robust delivery of viral genomes to all regions of the lung, with minimal systemic biodistribution, and the rAAV mediates protein expression to all regions of the lung, including the alveoli. rAAV delivery and EGFP expression was consistent among animals, with even distribution across multiple bronchial levels and alveoli and even distribution across cranial, middle and caudal sections. The protein expression data are consistent with the genome biodistribution data demonstrating that genome localization to the liver was multiple orders of magnitude lower than genome localization to the lungs.

[0443] Additional experiments are performed to determine the efficiency and specificity for AECII cells in the primate lung. If further specificity is desirable beyond what is inherent to the rAAV comprising a capsid comprising a capsid protein of SEQ ID NO: 12, the use of a cell-specific promoter is used to drive expression of the transgene of interest. The experiments described herein indicate that rAAV comprising a capsid protein of SEQ ID NO: 12 can be used to safely and effectively deliver CFTR transgene to the lungs of subjects with cystic fibrosis and to deliver therapeutic genes to treat other pulmonary disorders.EXAMPLE 4

[0444] SUMMARY

[0445] A cell culture model of lung alveolar epithelial type 2 (AECII) cells from freshly isolated non-human primate lungs and human donor lungs rejected for transplant was established. This model was used in the characterization of rAAV comprising capsid comprising a variant capsid protein of SEQ ID NO: 12 identified to target AECII cells. The rAAV, and natural serotype AAV5 were evaluated for transduction efficiency in AECII cell air liquid interface (ALI) culture. rAAV comprising a capsid comprising a variant capsid protein of SEQ ID NO: 12, following apical transduction with a multiplicity of infection (MOI) of 35,000, showed enhanced transduction of AECII cells compared to AAV5. The rAAV also demonstrated strong resistance to human anti-AAV antibodies.

[0446] To confirm that the rAAV capsid targets AECII cells and that infectivity translates to human AECII, AECII cells were isolated from NHP lungs and human donor lungs, rejected from transplant, and cultured in air liquid interface to mimic the lung environment. rAAV transduction efficiency was determined through reporter enhanced green fluorescent protein (eGFP) expression driven by the CAG promoter at multiple time points following infection and compared to AAV5.CAG-eGFP. This data demonstrates that rAAV variants comprising a capsid comprising a variant capsid protein of SEQ ID NO: 12 are more infectious than naturally occurring serotypes (i.e. are superior transducers than AAV5 capsid), potentially providing improved treatments for genetic diseases.

[0447] One general challenge in pre-clinical and clinical gene therapy studies with AAV is that pre-existing neutralizing antibodies can inhibit successful transduction. To understand the ability of rAAV comprising a capsid comprising a variant capsid protein of SEQ ID NO:12 to evade neutralizing antibodies within the human population in comparison to wildtype AAVs, the rAAV and wild-type AAV1, AAV2, AAV5, AAV8, and AAV9 were analyzed against human IVIG in an in vitro luciferase assay. The data provided here reports that rAAV comprising a capsid comprising a variant capsid protein of SEQ ID NO: 12 has neutralizing antibody resistance when exposed to human IVIG (4-fold over AAV5 and 32- fold over AAV2), a critical component for treatment via aerosol delivery and a selective pressure applied during the Therapeutic Vector Evolution process.

[0448] MATERIALS AND METHODS

[0449] Lung Alveolar Epithelial Type 2 Cell Isolation

[0450] Non-human primate (NHP, Cynomolgus macaque, CRO) or human donor lungs rejected for transplant (Donor Network West, Donor ID: AGES430) were used to isolate alveolar epithelial type 2 cells (AECII) according to Fang et al1,2. For NHP cell isolation, the entire lung was utilized; for human donor lungs, the right middle lobe was dissected out and used for cell isolation. The bronchi were flushed with PBS without Calcium or Magnesium (ThermoFisher) containing EDTA (Sigma) and EGTA (Sigma) followed by inflation with elastase (Worthington Chemicals). The tissue was incubated for 1 hour at 37°C. The lung was homogenized, and the trachea and bronchi removed. The cell homogenate was passed through layers of gauze to eliminate large remaining pieces. The cells were sequentially passed through 100 pm and 20 pm strainers. The cells were loaded onto a two-step Percoll (ThermoFisher) density gradient, (70% and 30%), and centrifuged at 1800 rpm for 20 minutes. The intermediate layer was removed, centrifuged, and washed twice with PBS without Calcium or Magnesium. The monocytes and macrophages were removed using CD14 and CD45 Dynabeads (ThermoFisher). The remaining cells were incubated overnight at 37°C on IgG coated plates to remove T cells. The following day, the non-adherent cells were collected from the plates, centrifuged and subjected to hypotonic solution to lyse red blood cells (ACK Lysis Buffer 1:10, ThermoFisher). Resulting cells were plated on human collagen IV coated inserts (Sigma, human placental collagen IV, 18-24 hours at 25°C) at a density of 1.5x106cells / cm2and incubated at 37°C, 5% COj. Cells were cultured in Airway Epithelial Cell Basal Medium (ATCC) with commercial supplements, Fetal Bovine Serum (10%, HyClone, ThermoFisher) and insulin, transferrin, and selenium ( 1 :200, ThermoFisher). One day after seeding, the inserts were washed twice with PBS to remove non-adherent cells. The top of the insert was maintained dry to ensure the formation of an air liquid interface (ALI).(00451 ] LysoTracker Staining

[0452] Cells achieving ALI were examined for LysoTracker staining in culture. LysoTracker (ThermoFisher) is an indicator dye that gets absorbed by highly acidic components of live cells, such as lysosomes and the lamellar bodies of AECII cells. It is routinely used to mark AECII cells.

[0453] LysoTracker on Adherent Cells and Microscopy

[0454] Cells on inserts identified for staining were washed twice with PBS. LysoTracker concentrate was diluted in media (1 : 1000). One hundred microliters of diluted LysoTracker was added to the insert for live cell staining. Cells were incubated for 5 minutes at 37°C. Following incubation, the insert was washed three times with PBS and imaged on a Zeiss Axio Observer D.l fluorescent microscope. A non-stained well was used as a control and to set exposure.

[0455] LysoTracker on Cell Suspension and Flow Cytometry

[0456] Cells are incubated with Trypsin-EDTA 0.05% (ThermoFisher) for 10 minutes at 37°C. Trypsin was deactivated with Defined Trypsin Inhibitor (ThermoFisher), and cells were collected from the inserts and centrifuged at 300xg for 4 minutes. LysoTracker concentrate was diluted in media (1:1000). One hundred microliters of diluted LysoTracker was added to each cell pellet and vortexed at half speed to mix. A non-stained cell sample was used as a control and to set flow cytometry gates. Cells were incubated for 5 minutes at 37°C.Following incubation, cells were centrifugated and washed twice with PBS. Cells were resuspended in PBS and run on a BD Accuri C6 Plus Flow Cytometer. The LysoTracker positive population was identified as a right shifting population from the unstained control.

[0457] EdU Incorporation

[0458] Cell proliferation was determined using a Click-iT EdU Alexa Fluor Kit (ThermoFisher) according to manufacturer’s instructions. Briefly, cells were pulsed for 2 hours with EdU, washed twice with PBS and fixed with 4% paraformaldehyde (15 minutes at 4 °C). The Click-iT reaction cocktail including an Alexa Fluor azide was prepared and incubated with the cells for 30 minutes at 25°C. Following the reaction, the cells were washed twice with PBS and counterstained with DAP! for 10 minutes at 25°C. Cells were imaged on a Zeiss Axio Observer D.1 fluorescent microscope.

[0459] Vector Transduction

[0460] NHP AECII cells were transduced two days after seeding. Human AECII cells were transduced one day after seeding. On the day of transduction, three inserts were incubated with Trypsin-EDTA 0.05% (ThermoFisher) for 10 minutes at 37°C. Trypsin was deactivated with Defined Trypsin Inhibitor (ThermoFisher). Cells were collected from the insert and counted on a hemocytometer. An average cell number was determined per insert and used to calculate total viral genomes required per insert. A multiplicity of infection (MOI) of 35,000 was used for all experiments in a total volume of 100 pl per insert. Cells were exposed apically for 48 hours with rAAV comprising capsid with capsid protein of SEQ ID NO: 12 and a GFP gene operably linked to a CAG promoter or native AAV of serotype 5 comprising a GFP gene operably linked to a CAG promoter. Two days post-infection, virus was removed from the insert to regain air liquid interface. Three days post-infection, NHP cells were harvested for analysis, a total of five days in culture. Six- and ten-days postinfection, human cells were harvested for analysis, a total of seven and eleven days in culture.

[0461] Immunocytochemistry

[0462] Cells were washed twice with PBS and fixed with 4% paraformaldehyde (15 minutes at 4°C). Cells were blocked with 5% goat serum and 2% bovine serum albumin (BSA) in 0.2% Triton X-100 in PBS for 30 minutes. Cells were incubated with Surfactant Protein C antibody or IgG control (MilliporeSigma, 1 : 100) for 2 hours at 25°C. Primary antibody was washed three times with 0.2% Triton in PBS, followed by secondary antibody incubation (Goat anti-Rabbit Alexa Fluor 555, 1:500) for 30 minutes at 25°C. Cells were counterstained with DAPI for 10 minutes at 25°C and washed three times with PBS. Cells were imaged on a Zeiss Axio Observer D.l fluorescent microscope using IgG control to set exposure.

[0463] Flow Cytometry

[0464] Post-transduction cells were lifted with Trypsin-EDTA 0.05% (ThermoFisher) for 10 minutes at 37°C. Trypsin was deactivated with Defined Trypsin Inhibitor (ThermoFisher), and cells were collected from the inserts and centrifuged at 300xg for 4 minutes. Cells were resuspended in PBS and run on a BD Accuri C6 Plus Flow Cytometer. The transduced (eGFP positive) cells were identified as a right shifting population from the non-transduced control.

[0465] Neutralizing Antibody Resistance Assay

[0466] HEK 2V6.11 (obtained from John Hopkins University) cells were plated onto 96- well plates at a density of 3x104cells per well Twenty-four hours after seeding, rAAV comprising (i) capsid comprising capsid protein of SEQ ID NO: 12 (“A 101”) and (ii) a luciferase transgene operably linked to a CAG promoter and wild-type AAVs (serotypes AAV1, AAV2, AAV5, AAV8, and AAV9, all carrying a luciferase reporter transgene) were incubated at37°C for 1 hour with five dilutions 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600 of human intravenous immunoglobins (IVIG) prior to infection. Cells were then infected with the rAAV and the wild-type AAV at a MOI of 1 ,000. Each plate contained positive and negative controls for transduction. The positive control was either the rAAV or wild-type AAV in the absence of IVIG. The negative control for transduction was media without serum or AAV.CAG-Luciferase. Luciferase activity was measured 48 hours post infection using a Cytation 3 (Biotek) plate reader.

[0467] COMPUTERIZED SYSTEMS

[0468] FloJo, LLC Software was used to analyze flow cytometry data.

[0469] Microsoft Excel was used to calculate averages and standard deviations fromFloJo outputs and make histograms.

[0470] For serum neutralizing antibody screening, data was generated and analyzed using the Cytation 3 microplate reader (Biotek), Gen5plus software version 3.03.14, and Microsoft Excel.

[0471] RESULTS and DISCUSSION

[0472] Non-Human Primate AECII ALT Culture Characterization

[0473] Lung alveolar epithelial type 2 cells (AECII) were isolated from a non-human primate (NHP) lung and cultured in air liquid interface (ALI). Cells were cultured on collagen coated inserts and analyzed for AECII specific markers, LysoTracker dye and Surfactant Protein-C (SPC). Cells at day 1 and day 5 contained over 90% LysoTracker positive cells, shown by ICC and quantified by flow cytometry (Figures 21 A and 2 IB), AECII cultured cells were also examined for SPC, a mature AECII marker at day 1 and day 5 in culture. Cells expressed SPC by ICC at the time-points analyzed (Figure 21 C). To further characterize the AECII culture system, cell mitosis was examined over time. Mitosi...

Claims

We Claim:

1. A method of treating cystic fibrosis (CF) in a primate in need thereof, comprising administering to the primate a therapeutically effective amount of each of(i) a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant adeno-associated virus (rAAV) vector, said rAAV vector comprising a capsid comprising a capsid protein comprising the amino acid sequence as set forth as SEQ ID NO: 12 or an amino acid sequence at least 90% identical to SEQ ID NO: 12 and comprising a Thr at amino acid 469 and an Ala at amino acid 598 based on the amino acid numbering set forth in SEQ ID NO: 12 and a heterologous nucleic acid comprising a nucleotide sequence encoding a cystic fibrosis transmembrane conductance regulator (CFTR protein) gene product operably linked to a promoter, and(ii) one or more CFTR modulators).

2. The method according to claim 1, wherein the pharmaceutical composition is formulated as an aerosol.

3. The method according to claim 1 or 2, wherein the pharmaceutical composition is administered to the primate in amount that is effective to provide a total clinical delivered dose of about 5x1013vg to about 4.7x1014vg, preferably about 6x1013vg, about 1.2x1014vg or about 2.3x1014vg of the rAAV to the lungs of the primate.

4. The method according to any one of claims 1-3, wherein the capsid protein comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth as SEQ ID NO: 12.

5. The method according to claim 4, wherein the capsid protein comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth as SEQ ID NO:12.

6. The method according to claim 5, wherein the capsid protein comprises the amino acid sequence set forth as SEQ ID NO: 12.

7. The method according to any one of claims 1 -6, wherein the heterologous nucleic acid comprises a nucleotide sequence encoding a human CFTR protein or a biologically active truncated CFTR protein lacking amino acids 708-759 of the human CFTR protein sequence.

8. The method according to claim 7, wherein the gene product is a biologically truncated CFTR protein lacking amino acids 708-759 of the human CFTR protein sequence.

9. The method according to claim 8, wherein the nucleotide sequence encoding a human CFTR protein or a biologically active truncated CFTR protein lacking amino acids 708-759 of the human CFTR protein sequence comprises the nucleotide sequence set forth in SEQ ID NO:43 or a sequence at least 80% identical thereto.

10. The method according to claim 9, wherein the nucleotide sequence encoding a human CFTR protein or a biologically active truncated CFTR protein lacking amino acids 708-759 of the human CFTR protein sequence comprises the nucleotide sequence set forth.in SEQ ID NO:43.

11. The method according to any one of claims 1-10, wherein the promoter is a constitutive promoter.

12. The method according to claim 11, wherein the promoter is a CMV173 promoter.

13. The method according to any one of claims 1-12, wherein the heterologous nucleic acid comprises a nucleotide sequence at least 80% identical to the nucleotide sequence set forth as SEQ ID NO:45.

14. The method according to claim 13, wherein the rAAV comprises (i) a capsid comprising a capsid protein comprising the amino acid sequence as set forth as SEQ ID NO: 12 and (ii) a heterologous nucleic acid comprising the nucleotide sequence set forth as SEQ ID NO:45.

15. The method according to any one of claims 1-14, wherein the one or more CFTR modulators) is selected from elexacaftor, tezacaftor, ivacaftor, lumacaftor, vanzacaftor, deutivacaftor and a combination thereof.

16. The method according to claim 15, wherein the one or more CFTR modulator(s) comprises ivacaftor.

17. The method according to claim 16, wherein ivacaftor is administered alone or in combination with elexacaftor, tezacaftor, and / or lumacaftor.

18. The method according to claim 16 or 17, comprising administering at least one CFTR modulator at least once per day, preferably about every twelve hours.

19. The method according to claim 15, wherein the one or more CFTR modulator(s) comprises(i) vanzacaftor, tezacaftor, and deutivacaftor(ii) ivacaftor, elexacaftor and tezacaftor(iii) ivacaftor and tezacaftor(iv) ivacaftor and lumacaftor(v) ivacaftor alone20. The method according to claim 19, wherein the one or more CFTR modulator(s) comprises vanzacaftor, tezacaftor, and deutivacaftor.

21. The method according to claim 20, wherein the one or more CFTR modulators are administered once per day.

22. The method according to any one of claims 1-21, wherein the pharmaceutical composition and the one or more CFTR modulators) are administered simultaneously or sequentially.

23. The method according to any one of claims 1-22, wherein the pharmaceutical composition is administered by inhalation, preferably by nebulizer and the one or more CFTR modulator(s) is administered orally.

24. The method according to claim 15, wherein ivacaftor is administered to the primate according to one or more of the following dosage regimens:(i) about 5.8 mg, about 13.4 mg, or about 25 mg administered orally about every 12 hours, wherein the primate is >1 to <2 months old and >3 kg body weight, >2 to <4 months old and >3 kg body weight, or Age >2 to <6 months old and >5 kg body weight, respectively;(ii) about 25 mg, about 50 mg or about 75 mg administered orally about eveiy 12 hours, wherein the primate is >6 months to <6 years old and is 5 kg to <7 kg body weight, 7 kg to <14 kg body weight, or >14 kg body weight, respectively; or(iii) about 150 mg administered orally about eveiy 12 hours, wherein the primate is >6 years.

25. The method according to claim 15, wherein the one or more CFTR modulator(s) are administered to the primate according to one or more of the following dosage regimens:(i) about 50 mg tezacaftor and about 75 mg ivacaftor co-administered orally once per day and about 75 mg ivacaftor administered separately once per day, preferably about 12 hours subsequent to co-administering tezacaftor and ivacaftor, wherein the primate is at least 6 years old and less than 30 kg; or(ii) about 100 mg tezacaftor and about 150 mg ivacaftor co-administered orally once per day and about 150 mg ivacaftor administered separately once per day, preferably about 12 hours subsequent to co-administering tezacaftor and ivacaftor, wherein the primate is at least 6 years old and at least 30 kg.

26. The method according to claim 15, wherein the one or more CFTR modulator(s) are administered to the primate according to one or more of the following dosage regimens:(i) about 75 mg lumacaftor and about 94 mg ivacaftor co-administered orally about every 12 hours, wherein the primate is between 1 and 2 years old and 7 to <9 kg(ii) about 100 mg lumacaftor and about 125 mg ivacaftor co-administered orally about every 12 hours, wherein the primate is between 1 and 2 years old and 9 to <14 kg(iii) about 150 mg lumacaftor and about 188 mg ivacaftor co-administered orally about every 12 hours, wherein the primate is between 1 and 2 years old and >14 kg27. The method according to claim 15, wherein the one or more CFTR modulators) are administered to the primate according to one or more of the following dosage regimens:(i) about 80 mg elexacaftor, about 40 mg tezacaftor and about 60 mg ivacaftor co-administered once per day to the primate and about 59.5 mg ivacaftor separately administered once per day to the primate, wherein the primate is between 2 and 6 years of age and weighs less than 14 kg;(ii) about 100 mg elexacaftor, about 50 mg tezacaftor and about 75 mg ivacaftor co-administered once per day to the primate and about 75 mg ivacaftor separately administered once per day to the primate, wherein the primate is between 2 and 6 years of age and weighs at least 14 kg or wherein the primate is between 6 and 12 years of age and weighs less than 30 kg;(iii) about 200 mg elexacaftor, about 100 mg tezacaftor and about 150 mg ivacaftor co-administered once per day to the primate and about 150 mg ivacaftor separately administered once per day to the primate, wherein the primate is between 6 and 12 years of age and weighs at least 30 kg or wherein the primate is at least 12 years of age.

28. The method according to claim 15, wherein the one or more CFTR modulators) are administered to the primate according to one or more of the following dosage regimens:(iv) about 12 mg vanzacaftor, about 60 mg tezacaftor and about 150 mg deutivacaftor co-administered once per day to the primate, wherein the primate is aged 6 to less than 12 years of age and weighs less than 40 kg;(v) about 20 mg vanzacaftor, about 100 mg tezacaftor and about 250 mg deutivacaftor co-administered once per day to the primate, wherein the primate is at least 12 years of age or is under 12 years of age and weighs at least 40 kg;29. The method according to any one of claims 1-28, wherein the primate is administered a single unit dose of the pharmaceutical composition with no subsequent administration of the pharmaceutical composition for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or at least 12 months.

30. The method according to any one of claims 1-28, wherein the primate is administered multiple unit doses of the pharmaceutical composition.

31. The method according to claim 30, wherein the primate is administered a first unit dose of the pharmaceutical composition followed by administration of at least a second unit dose.

32. The method according to claim 31, comprising repeatedly administering the unit dose with an interval of less than or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or less than or about 12 months between administrations.

33. The method according to claim 31, comprising repeatedly administering the unit dose with an interval of less than or about 1, 2, 3, 4, or 5 years between administrations.

34. The method according to claim 31, wherein the pharmaceutical composition is administered to the primate about once per month, about once evety six months, about once per year, about once per two years, about once per three years, about once per four years, or about once per five years.

35. The method according to any one of claims 1-34, wherein the pharmaceutical composition is formulated as an aerosol.

36. The method according to any one of claims 1-35, wherein the primate has preexisting antibodies to the rAAV prior to administration of the pharmaceutical composition.

37. The method according to any one of claims 1-36, wherein the primate is a human.

38. The method according to claim 37, wherein the human has at least one CFTR F5O8del mutation.

39. The method according to claims 37 or 38, wherein the human demonstrates an improvement in one or more respiratory symptoms at least 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months following administration of the pharmaceutical composition and one or more CFTR modulator(s) relative to a pre-administration baseline, preferably as measured by the Cystic Fibrosis Questionnaire-Revised (CFQ-R).

40. The method according to claim 39, wherein said treatment increases said improvement compared with treatment with the pharmaceutical composition alone.

41. The method according to claim 40, wherein said improvement is at least additive.

42. The method according to any one of claims 37-41, wherein the method further comprises administering one or more corticosteroids to the primate before, simultaneous with or after administration of the pharmaceutical composition, preferably wherein the corticosteroid is orally administered.

43. The method according to claim 42, wherein the one or more corticosteroids comprises prednisone.

44. The method according to claim 42 or 43, wherein the primate is administered multiple doses of the one or more corticosteroids.

45. The method according to claim 42, wherein the one or more corticosteroids are administered to the human about once per day for an administration period comprising at least one, at least two, at least three or at least four weeks, preferably wherein the dose of the one or more corticosteroids is tapered over the administration period.

46. The method according to claim 45, wherein a first dose of the one or more corticosteroids is administered to the primate about one day prior to administering the pharmaceutical composition.

47. The method according to claim 45 or 46, wherein the primate is administered one or more corticosteroids once per day for about four weeks, wherein the first dose of the one or more corticosteroids is administered about one day prior to administering the pharmaceutical composition.

48. The method according to any one of claims 45-47, wherein the primate is administered prednisone or a corticosteroids equivalent to prednisone according tothe following administration schedule, wherein the first dose of the corticosteroid is administered about one day prior to administering the pharmaceutical composition:(i) corticosteroids equivalent to 40 mg prednisone or 40 mg prednisone once per day for about 7 days; followed by(ii) corticosteroids equivalent to 30 mg prednisone or 30 mg prednisone once per day for about 7 days; followed by(iii) corticosteroids equivalent to 20 mg prednisone or 20 mg prednisone once per day for about 7 days; followed by(iv) corticosteroids equivalent to 10 mg prednisone or 10 mg prednisone once per day for about 7 days, wherein the administration period is about 28 days.

49. A method of treating cystic fibrosis (CF) in a primate in need thereof, comprising administering to the primate multiple unit doses of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant adeno-associated virus (rAAV) vector, said rAAV vector comprising a capsid comprising a capsid protein comprising the amino acid sequence as set forth as SEQ ID NO: 12 or an amino acid sequence at least 90% identical to SEQ ID NO: 12 and comprising a Thr at amino acid 469 and an Ala at amino acid 598 based on the amino acid numbering set forth in SEQ ID NO: 12 and a heterologous nucleic acid comprising a nucleotide sequence encoding a cystic fibrosis transmembrane conductance regulator (CFTR protein) gene product operably linked to a promoter, preferably wherein the capsid protein comprises the amino acid sequence of SEQ ID NO: 12 and / or the heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:45 or a nucleotide sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

50. The method according to claim 49, wherein the primate is administered a first unit dose of the pharmaceutical composition followed by administration of at least a second unit dose of the pharmaceutical composition.

51. The method according to claim 50, wherein the 1stunit dose of the pharmaceutical composition is an amount that is effective to provide a total clinical delivered dose of about 5x1013vg to about 4.7x1014vg, preferably about 6x1013vg, about 1.2x1014vg or about 2.3x1014vg of the rAAV to the lungs of the primate, and wherein the 2ndor more unit dose is an amount that is effective to provide a total clinical delivered dose of about 1.2x1014vg of the rAAV to the lungs of the primate.

52. The method according to claim 51 , wherein the primate is administered a second unit dose at least about 12 months after administration of the first unit dose and within about 2 years, 3 years, or 4 years from administration of the first unit dose, preferably wherein the second unit dose is the same as the first unit dose.

53. The method according to claim 51 , wherein the unit dose is repeatedly administered to the primate with an interval of less than or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or less than or about 12 months between administrations.

54. The method according to claim 51, comprising repeatedly administering the unit dose with an interval of less than or about 1, 2, 3, 4, or 5 years between administrations.

55. The method according to claim 51 , wherein the pharmaceutical composition is administered to the human about once per month, about once every six months, about once per year, about once per two years, about once per three years, about once per four years, or about once per five years.

56. The method according to any one of claims 49-55, wherein the pharmaceutical composition is formulated as an aerosol.

57. The method according to any one of claims 49-56, wherein the primate has preexisting antibodies to the rAAV prior to administration of the pharmaceutical composition.

58. The method according to any one of claims 1-57, wherein the rAAV comprises (i) a capsid protein comprising the amino acid sequence set forth as SEQ ID NO: 12 and (ii) a heterologous nucleic acid comprising the nucleotide sequence of SEQ ID NO:45.

59. The method according to any one of claims 49-58, wherein the primate is a human.

60. A kit comprising:(a) a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant adeno-associated virus (rAAV) vector, said rAAV vector comprising a capsid comprising a capsid protein comprising the amino acid sequence as set forth as SEQ ID NO: 12 or an amino acid sequence at least 90% identical to SEQ ID NO: 12 and comprising a Thr at amino acid 469 and an Ala at amino acid 598 based on the amino acid numbering set forth in SEQ ID NO: 12 and a heterologous nucleic acid comprising a nucleotide sequence encoding acystic fibrosis transmembrane conductance regulator (CFTR protein) gene product operably linked to a promoter, and(b) one or more CFTR modulator(s) in combination with written instructions for use of (a) and (b) for treating cystic fibrosis (CF), preferably wherein the one or more CFTR modulators) comprises(i) vanzacaftor, tezacaftor, and deutivacaftor(ii) ivacaftor, elexacaftor and tezacaftor(iii) ivacaftor and tezacaftor(iv) ivacaftor and lumacaftor or(v) ivacaftor alone.

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