Capsid-modified raav vector compositions and methods therefor

Abstract

Disclosed are capsid-modified rAAV expression vectors, as well as infectious virions, compositions, and pharmaceutical formulations containing them. Also provided are methods of preparing and using the disclosed capsid-protein-mutated rAAV constructs in a variety of diagnostic and therapeutic modalities, including, inter alia, as mammalian cell-targeting delivery agents, and as human gene therapy vectors. Also disclosed are large-scale production methods for capsid-modified rAAV expression vectors, viral particles, and infectious virions having improved transduction efficiencies over those of the corresponding, un-modified, rAAV vectors, as well as use of the disclosed compositions in the manufacture of medicaments for a variety of in vitro and/or in vivo applications.

1 - 25 . (canceled) 
     
     
         26 . A modified AAV VP3 capsid protein, comprising:
 (A) a non-tyrosine amino acid residue at (i) one or more positions corresponding to Y445, Y705 and Y731 of the wild-type AAV6 capsid protein as set forth in SEQ ID NO:6; or (ii) one or more positions corresponding to Y444, Y500 and Y730 of the wild-type AAV2 capsid protein as set forth in SEQ ID NO:2;   (B) a non-serine amino acid residue at a position corresponding to S663 of the wild-type AAV6 capsid protein as set forth in SEQ ID NO:6;   (C) a non-threonine amino acid residue at a position corresponding to T492 of the wild-type AAV6 capsid protein as set forth in SEQ ID NO:6;   (D) a combination of two or more amino acid substitutions listed in (A), (B), and (C); each with a non-native amino acid; or   (E) a combination of tree or more amino acid substitutions listed in (A), (B), and (C); each with a non-native amino acid;   or alternatively, wherein each of the amino acid substitutions is at an equivalent amino acid position corresponding thereto in any one of the other wild-type vector serotypes selected from the group consisting of AAV1, AAV3, AAV4, AAV5, AAV7, AAV9, and AAV10, as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO: 10, respectively.   
     
     
         27 . The modified AAV VP3 capsid protein of  claim 26 , comprising: (i) a non-tyrosine amino acid residue at one or more positions corresponding to Y445, Y705 and Y731 of the wild-type AAV6 capsid protein as set forth in SEQ ID NO:6; or (ii) one or more positions corresponding to Y444, Y500 and Y730 of the wild-type AAV2 capsid protein as set forth in SEQ ID NO:2;
 or at an equivalent amino acid position corresponding thereto in any one of the other wild-type vector serotypes selected from the group consisting of AAV1, AAV3, AAV4, AAV5, AAV7, AAV9, and AAV10, as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:10.   
     
     
         28 . The modified AAV VP3 capsid protein of  claim 26 , comprising a non-serine amino acid residue at a position corresponding to S663 of the wild-type AAV6 capsid protein as set forth in SEQ ID NO:6; or at an equivalent amino acid position corresponding thereto in any one of the other wild-type vector serotypes selected from the group consisting of AAV1, AAV3, AAV4, AAV5, AAV7, AAV9, and AAV10, as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:10. 
     
     
         29 . The modified AAV VP3 capsid protein of  claim 26 , comprising a non-threonine amino acid residue at a position corresponding to T492 of the wild-type AAV6 capsid protein as set forth in SEQ ID NO:6; or at an equivalent amino acid position corresponding thereto in any one of the other wild-type vector serotypes selected from the group consisting of AAV1, AAV3, AAV4, AAV5, AAV7, AAV9, and AAV10, as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:10. 
     
     
         30 . The modified AAV VP3 capsid protein of  claim 26 , comprising a combination of two or more amino acid substitutions listed in (A), (B), and (C); each with a non-native amino acid. 
     
     
         31 . The modified AAV VP3 capsid protein of  claim 26 , comprising a combination of three or more amino acid substitutions listed in (A), (B), and (C); each with a non-native amino acid. 
     
     
         32 . The modified AAV VP3 capsid protein of  claim 26 , wherein
 (a) the non-tyrosine or non-threonine amino acid residue is selected from the group consisting of serine (S), phenylalanine (F), valine (V), histidine (H), isoleucine (I), alanine (A), leucine (L) aspartic acid (D), asparagine (N), glutamic acid (E), arginine (R), and isoleucine (I); or   (b) the non-serine amino acid residue is selected from the group consisting of phenylalanine (F), valine (V), histidine (H), isoleucine (I), alanine (A), leucine (L) aspartic acid (D), asparagine (N), glutamic acid (E), arginine (R), and isoleucine (I).   
     
     
         33 . The modified AAV VP3 capsid protein of  claim 26 , wherein
 (a) the non-tyrosine amino acid residue at one or more positions corresponding to Y445, Y705 and Y731 of the wild-type AAV6 capsid protein as set forth in SEQ ID NO:6 or   (b) the non-tyrosine amino acid residue at one or more positions corresponding to Y444, Y500 and Y730 of the wild-type AAV2 capsid protein as set forth in SEQ ID NO:2, is phenyalanine.   
     
     
         34 . The modified AAV VP3 capsid protein of  claim 26 , wherein the non-serine amino acid residue at a position corresponding to S663 of the wild-type AAV6 capsid protein as set forth in SEQ ID NO:6 is valine. 
     
     
         35 . The modified AAV VP3 capsid protein of  claim 26 , wherein the non-threonine amino acid residue at a position corresponding to T492 of the wild-type AAV6 capsid protein as set forth in SEQ ID NO:6 is valine. 
     
     
         36 . An isolated nucleic acid segment that encodes the modified AAV VP3 capsid protein of  claim 26 . 
     
     
         37 . A recombinant adeno-associated viral (rAAV) particle comprising a modified AAV VP3 capsid protein of  claim 26 . 
     
     
         38 . The rAAV particle of  claim 37 , wherein the transduction efficiency of a particle comprising the modified AAV VP3 capsid protein is about 6- to about 30-fold higher in a selected mammalian host cell than that of a particle that comprises a corresponding, unmodified, VP3 capsid protein. 
     
     
         39 . The rAAV particle of  claim 38 , wherein the particle comprises a nucleic acid segment comprising a promoter, an enhancer, a post-transcriptional regulatory sequence, a polyadenylation signal, or any combination thereof, each operably linked to the nucleic acid segment. 
     
     
         40 . An isolated mammalian host cell comprising the modified AAV VP3 capsid protein of  claim 26 . 
     
     
         41 . The isolated mammalian host cell of  claim 40 , wherein the host cell is a stem cell, a hematopoietic cell, a blood cell, a neural cell, a retinal cell, an epithelial cell, an endothelial cell, a pancreatic cell, a cancer cell, a muscle cell, a vascular cell, a diaphragm cell, a smooth muscle cell, a stromal cell, a cardiac cell, a renal cell, a liver cell, a splenic cell, a cell of the digestive tract, a CD34+ cell, or any combination thereof. 
     
     
         42 . A composition comprising:
 (I) (a) the modified AAV VP3 capsid protein of  claim 26 ;
 (b) a nucleic acid segment that encodes the modified AAV VP3 capsid protein of  claim 26 ; or 
 (c) a viral particle comprising a nucleic acid segment that encodes the modified AAV VP3 capsid protein of  claim 26 ; and 
   (II) a pharmaceutically-acceptable buffer, diluent, or excipient.   
     
     
         43 . A method for providing a mammal in need thereof with a therapeutically-effective amount of a mammalian β-globin protein, the method comprising providing to a cell, a tissue or an organ of a mammal in need thereof, the rAAV particle of  claim 37 , wherein the rAAV particle comprises a nucleic acid segment that encodes the mammalian β-globin protein operably linked to a promoter that expresses the nucleic acid segment in one or more cells of the mammal. 
     
     
         44 . A method for preventing, treating, or ameliorating one or more symptoms of a hemoglobinopathy, such as β-thalassemia or sickle cell disease, in a mammal, the method comprising, administering to a mammal in need thereof an rAAV particle comprising a modified AAV VP3 capsid protein that comprises:
 (A) a non-tyrosine amino acid residue at (i) one or more positions corresponding to Y445, Y705 and Y731 of the wild-type AAV6 capsid protein as set forth in SEQ ID NO:6; or (ii) one or more positions corresponding to Y444, Y500 and Y730 of the wild-type AAV2 capsid protein as set forth in SEQ ID NO:2; 
 (B) a non-serine amino acid residue at a position corresponding to S663 of the wild-type AAV6 capsid protein as set forth in SEQ ID NO:6; 
 (C) a non-threonine amino acid residue at a position corresponding to T492 of the wild-type AAV6 capsid protein as set forth in SEQ ID NO:6; 
 (D) a combination of two or more amino acid substitutions listed in (A), (B), and (C); 
 each with a non-native amino acid; or 
 (E) a combination of tree or more amino acid substitutions listed in (A), (B), and (C); each with a non-native amino acid; 
 or alternatively, wherein each of the amino acid substitutions is at an equivalent amino acid position corresponding thereto in any one of the other wild-type vector serotypes selected from the group consisting of AAV1, AAV3, AAV4, AAV5, AAV7, AAV9, and AAV10, as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO: 10, respectively, 
 wherein the vector further comprises a nucleic acid segment that encodes a mammalian 3-globin protein operably linked to a promoter that expresses the nucleic acid segment in one or more cells of the mammal, in an amount and for a time sufficient to prevent, treat or ameliorate the one or more symptoms of the hemoglobinopathy in the mammal. 
 
     
     
         45 . The method of  claim 44 , wherein the mammal is human. 
     
     
         46 . A method of transducing a population of mammalian cells, comprising introducing into one or more cells of the population, a composition that comprises an effective amount of the rAAV particle of  claim 37 .