Adeno-associated variants, formulations and methods for pulmonary delivery
The present disclosure provides a variant AAV capsid protein that confers tropism to lung cells and recombinant adeno-associated viruses comprising the variant AAV and pharmaceutical compositions comprising same and their use in the delivery of heterologous nucleic acids to lung cells for the treatment of pulmonary disorders.
1. A method of delivering a heterologous nucleic acid to a lung cell in a primate comprising administering to the primate a composition comprising a recombinant adeno-associated virus (rAAV) vector, said rAAV vector comprising (i) a capsid comprising a capsid protein comprising the amino acid sequence 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 gene product, wherein the step of administering comprises pulmonary, endobronchial, intranasal, intratracheal, and/or intrabronchial administration.
2. The method according to claim 1 , wherein the lung cell is selected from an airway epithelial cell, a smooth muscle cell, and an endothelial cell.
3. The method according to claim 2 , wherein the airway epithelial cell is a basal cell, a goblet cell or a cilia cell.
4. The method according to claim 3 , wherein the airway epithelial cell is a lung alveolar epithelial type 1 (AECI) cell, a lung alveolar epithelial type 2 (AECII) cell, a bronchial epithelial cell or a tracheal epithelial cell.
5. The method according to claim 1 , wherein the composition is formulated as an aerosol.
6. The method according to claim 1 , wherein the method comprises administering the composition by a nebulizer.
7. The method according claim 1 , wherein the composition comprises 10 11 to 10 14 vector genomes (vg) of the rAAV per ml.
8. The method according to claim 1 , wherein the nucleotide sequence encoding the gene product is operably linked to a promoter.
9. The method according to claim 8 , wherein the promoter is a constitutive promoter.
10. The method according to claim 8 , wherein the promoter is a tissue-specific promoter.
11. The method according to claim 8 , wherein the heterologous nucleic acid comprises a nucleotide sequence encoding a gene product selected from: cystic fibrosis transmembrane conductance regulator (CFTR protein) or a biologically active fragment thereof, SFTPA1 (surfactant A1), Caveolin-1, alpha-1-antitrypsin, alpha-1-antichymotrypsin, alpha-1-macroglobulin, matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 12 (MMP12), microsomal epoxide hydrolyase, CYP1A1, Glutathione S-transferase, heme oxygenase-1, TGF-beta-1, TNF-alpha, IL-1 complex, IL-8, IL-13, human leukocyte antigen, vitamin D binding protein, beta-2-adrenergic receptor.
12. The method according to claim 11 , wherein the gene product is 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.
13. The method according to claim 12 , wherein the gene product is a biologically truncated CFTR protein lacking amino acids 708-759 of the human CFTR protein sequence.
14. The method according to claim 13 , wherein the heterologous nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO:43 or a sequence at least 80% identical thereto.
15. The method according to claim 1 , wherein the primate is a human.
16. A nucleic acid sequence comprising a nucleotide sequence set forth in SEQ ID NO:43 or a sequence at least 80% identical thereto.
17. A pharmaceutical composition comprising the nucleic acid sequence of claim 16 .
18. A pharmaceutical composition comprising an rAAV vector, said rAAV vector comprising (i) a capsid comprising a capsid protein of 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 CFTR or a biologically active truncated CFTR protein lacking amino acids 708-759 of the human CFTR protein sequence, said nucleotide sequence operably linked to an expression control sequence.
19. The pharmaceutical composition according to claim 18 , wherein the nucleotide sequence encoding the CFTR protein is at least 80% identical to the nucleotide sequence set forth as SEQ ID NO:43.
20. The pharmaceutical composition according to claim 19 , wherein the nucleotide sequence encoding the CFTR comprises the nucleotide sequence set forth as SEQ ID NO:43.
21. The pharmaceutical composition according to claim 20 , wherein the capsid comprises a capsid protein comprising the amino acid sequence set forth as SEQ ID NO:12 and wherein the expression control sequence comprises a CMV173 promoter.
22. The pharmaceutical composition according to any claim 18 , wherein the composition is formulated for inhalation.
23. A pharmaceutical composition comprising an rAAV virus, wherein the rAAV virus is formulated in a buffer comprising about 10 mM to about 50 mM citrate, about 70 mM to about 150 mM NaCl and a surfactant and having a pH of about 5.0 to about 7.0.
24. The pharmaceutical composition according to claim 23 , comprising about 20 mM to about 50 mM citrate, about 85 mM to about 125 mM NaCl and about 0.005% Pluronic F68 and having a pH of about 6.0.
25. The pharmaceutical composition according to claim 24 , comprising about 20 mM citrate, about 125 mM NaCl and about 0.005% Pluronic F68 and having a pH of about 6.0.
26. The pharmaceutical composition according to claim 23 , wherein the rAAV vector comprises (i) a capsid protein comprising the amino acid sequence 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 the nucleotide sequence set forth as SEQ ID NO: 43 or a nucleotide sequence at least 80% identical to SEQ ID NO: 43.
27. A method of treating cystic fibrosis in a primate comprising administering to the primate a pharmaceutical composition according to claim 18 , wherein the step of administering comprises pulmonary, endobronchial, intranasal, intratracheal, and/or intrabronchial administration.
28. The method according to claim 27 , wherein the primate is a human.