Compositions for targeting conducting airway cells comprising adeno-associated virus constructs

Abstract

An artificial AAV capsid comprising a heterologous conducting airway targeting sequence is provided. The artificial AAV is useful as a targeting moiety, for delivery of heterologous molecules which are associated therewith. The artificial AAV is also useful in the generation of AAV vectors having the artificial capsid. Also described are methods of modifying the native tropism and transduction efficiency of vectors by improving and/or ablating their ability to transduce conducting airway cells. Methods of targeting conducting airway cells and delivering therapeutic and other molecules thereto are also provided.

1 . An artificial AAV capsid comprising a heterologous airway conducting targeting peptide derived from the amino acid fragment spanning variable loop region IV and V of an AAV Clade A capsid selected from AAV1, AAV6, hu48R3, and hu44R2 flanked by AAV capsid sequences from another AAV. 
     
     
         2 . The artificial AAV capsid according to  claim 1 , wherein variable loop region IV and V are in the native location of corresponding variable loop regions of the heterologous capsid sequence, which corresponding variable loop regions have been functionally deleted. 
     
     
         3 . The artificial AAV capsid according to  claim 1 , wherein the artificial AAV capsid further comprises an optional spacer flanking the carboxy and/or the amino terminus of the amino acid sequences of the AAV6 variable loop regions. 
     
     
         4 . The artificial AAV capsid according to  claim 3 , wherein the optional spacer comprises one to five amino acids. 
     
     
         5 . The artificial AAV capsid according to  claim 1 , wherein the heterologous airway conducting targeting peptide consists of the full-length variable loop region IV and V of an AAV Clade A capsid, at least one to twelve amino acids from the region flanking the amino terminus of region IV of the AAV Clade A capsid and at least one to twelve amino acids from the region flanking the carboxy terminus of region V of the AAV Clade A capsid. 
     
     
         6 . The artificial AAV capsid according to  claim 1 , wherein the AAV capsid sequences are provided by a single AAV. 
     
     
         7 . The artificial AAV capsid according to  claim 1 , wherein the single AAV is selected from among the group consisting of AAV9, AAV5, AAV2, AAV8 and AAVrh32.33. 
     
     
         8 . The artificial AAV capsid according to  claim 1 , wherein the artificial AAV capsid has no functional AAV sequences packaged therein. 
     
     
         9 . The artificial AAV capsid according to  claim 1 , wherein the artificial AAV capsid has no functional sequences packaged therein. 
     
     
         10 . The artificial AAV capsid according to  claim 1 , wherein the heterologous airway conducting targeting peptide has the amino acid sequence of SEQ ID NO: 4: NRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPG. 
     
     
         11 . The artificial AAV capsid according to  claim 1 , wherein the capsid comprises:
 (a) a fragment of the AAV6 capsid consisting of about amino acid 447 to about 472 through about amino acids 489 to about 510 of AAV6 (SEQ ID NO:1) flanked by AAV9 capsid sequences;   (b) at least one fragment of the AAV6 capsid within the region spanning variable loops consisting of IV-V comprising about amino acid 447 to about amino acid 469 of AAV6 (SEQ ID NO:1); about amino acid 447 to about amino acids 476 of AAV6 (SEQ ID NO:1); about amino acid 487 to about amino acid 510 of AAV6 (SEQ ID NO:1).   
     
     
         12 . An AAV vector comprising an artificial AAV capsid comprising a heterologous airway conducting targeting peptide derived from variable loop region IV and V of an AAV Clade A capsid selected from AAV1, AAV6, hu44R2 and hu48R3, and an expression cassette packaged in the capsid, wherein said expression cassette comprises at least one AAV inverted terminal repeat sequence and a heterologous gene operably linked to regulatory sequences which permit expression of the heterologous gene in conducting airway cells. 
     
     
         13 . The AAV vector according to  claim 12 , wherein the artificial AAV capsid further comprises an optional spacer flanking the carboxy and/or the amino terminus of the amino acid sequences of the AAV6 variable loop regions. 
     
     
         14 . The AAV vector according to  claim 13 , wherein the optional spacer comprises one to five amino acids. 
     
     
         15 . The AAV vector according to  claim 12 , wherein the heterologous airway conducting targeting peptide consists of the full-length variable loop region IV and V of an AAV Clade A capsid, at least one to twelve amino acids from the region flanking the amino terminus of region IV of the AAV Clade A capsid and at least one to twelve amino acids from the region flanking the carboxy terminus of region V of the AAV Clade A capsid. 
     
     
         16 . The AAV vector according to  claim 12 , wherein the heterologous gene encodes a product useful for treating or ameliorating the symptoms of cystic fibrosis. 
     
     
         17 . The AAV vector according to  claim 12 , where the heterologous gene encodes a product is selected from the group consisting of a functional cystic fibrosis transmembrane regulator (CFTR) sequence, alpha-1 antitrypsin (A1AT), surfactant protein C (SPC), and surfactant protein D (SPD). 
     
     
         18 . The AAV vector according to  claim 12 , wherein the airway targeting peptide comprises:
 (a) a fragment of the AAV6 capsid consisting of about amino acid 447 to about 472 through about amino acids 489 to about 510 of AAV6 (SEQ ID NO:1) flanked by AAV9 capsid sequences;   (b) at least one fragment of the AAV6 capsid within the region spanning variable loops consisting of IV-V comprising about amino acid 447 to about amino acid 469 of AAV6 (SEQ ID NO:1); about amino acid 447 to about amino acids 476 of AAV6 (SEQ ID NO:1); about amino acid 487 to about amino acid 510 of AAV6 (SEQ ID NO:1);   (c) a fragment of the AAV6 capsid consisting of about amino acid 447 to about amino acid 521 of AAV6, SEQ ID NO: 1.   
     
     
         19 . An airway targeting molecule comprising the artificial AAV capsid according to  claim 1  linked to a moiety for delivery to the conducting airway epithelium. 
     
     
         20 . A pharmaceutical composition comprising the AAV vector according to  claim 12  and a physiologically compatible carrier.