Capsid-modified raav vector compositions having improved transduction efficiencies, and methods of use

Abstract

Disclosed are capsid-modified rAAV expression vectors, as well as infectious virions, compositions, and pharmaceutical formulations that include them. Also disclosed are methods of preparing and using novel capsid-protein-mutated rAAV vector constructs in a variety of diagnostic and therapeutic applications including, inter alia, as delivery agents for diagnosis, treatment, or amelioration of one or more symtpoms of disease or abnormal conditions via in situ and/or ex vivo mammalian gene therapy methods. 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 . A modified AAV capsid protein, comprising:
 a non-tyrosine amino acid residue at one or more positions corresponding to Y252, Y272, Y444, Y500, Y700, Y704, Y730, and Y731 of the wild-type AAV2 capsid protein as set forth in SEQ ID NO:2;   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 AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 capsid proteins, as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively.   
     
     
         2 .- 5 . (canceled) 
     
     
         6 . The modified AAV capsid protein of  claim 1 , comprising (i) a non-tyrosine amino acid residue at position Y252, Y272, Y444, Y500, Y700, Y704, Y730; and (ii) a non-tyrosine amino acid residue at position 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 wild-type AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 capsid proteins, as set forth, respectively, in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, or any combination thereof. 
     
     
         7 .- 9 . (canceled) 
     
     
         10 . The modified AAV capsid protein of  claim 1  wherein the protein comprises a combination of three or more amino acid substitutions, the combination of three of more amino acid substitutions including a non-native amino acid substitution at each of amino acid residues:
 (a) Y272, Y444, Y500, and Y730; 
 (b) Y272, Y444, Y500, Y700, and Y730; 
 (c) Y272, Y444, Y500, Y704, and Y730; 
 (d) Y252, Y272, Y444, Y500, Y704, and Y730; 
 (e) Y272, Y444, Y500, Y700, Y704, and Y730; or 
 (f) Y252, Y272, Y444, Y500, Y700, Y704, and Y730; 
 of the wild-type AAV2 capsid protein as set forth in SEQ ID NO:2, or at equivalent amino acid positions corresponding thereto in any one of the wild-type AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 capsid proteins, as set forth, respectively, in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, or any combination thereof. 
 
     
     
         11 . The modified AAV [[VP3]] capsid protein of  claim 1 , wherein the non-tyrosine 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). 
     
     
         12 . An isolated nucleic acid segment that encodes the modified AAV capsid protein of  claim 1 . 
     
     
         13 . A recombinant adeno-associated viral (rAAV) particle comprising the modified AAV capsid protein of  claim 1 . 
     
     
         14 . The rAAV particle of  claim 13 , wherein the particle further at least comprises a nucleic acid segment that encodes a diagnostic or therapeutic molecule operably linked to a promoter capable of expressing the nucleic acid segment in a suitable host cell comprising the particle. 
     
     
         15 .- 18 . (canceled) 
     
     
         19 . The rAAV particle of  claim 14 , wherein the nucleic acid segment further comprises an enhancer, a post-transcriptional regulatory sequence, a polyadenylation signal, or any combination thereof, operably linked to the nucleic acid segment. 
     
     
         20 . The rAAV particle of  claim 14 , wherein the promoter is a heterologous promoter, a tissue-specific promoter, a cell-specific promoter, a constitutive promoter, an inducible promoter, or any combination thereof. 
     
     
         21 . The rAAV particle of  claim 14 , wherein the nucleic acid segment expresses or encodes a polypeptide, a peptide, a ribozyme, a peptide nucleic acid, an siRNA, an RNAi, an antisense oligonucleotide, an antisense polynucleotide, an antibody, an antigen binding fragment, or any combination thereof. 
     
     
         22 . The rAAV particle of  claim 14 , wherein the therapeutic agent is an agonist, an antagonist, an anti-apoptosis factor, an inhibitor, a receptor, a cytokine, a cytotoxin, an erythropoietic agent, a glycoprotein, a growth factor, a growth factor receptor, a hormone, a hormone receptor, an interferon, an interleukin, an interleukin receptor, a nerve growth factor, a neuroactive peptide, a neuroactive peptide receptor, a protease, a protease inhibitor, a protein decarboxylase, a protein kinase, a protein kinsase inhibitor, an enzyme, a receptor binding protein, a transport protein or an inhibitor thereof, a serotonin receptor, or an uptake inhibitor thereof, a serpin, a serpin receptor, a tumor suppressor, a chemotherapeutic, or any combination thereof. 
     
     
         23 .- 26 . (canceled) 
     
     
         27 . An isolated mammalian host cell comprising the modified AAV capsid protein of  claim 1  or a nucleic acid encoding the modified AAV capsid protein. 
     
     
         28 . The isolated mammalian host cell of  claim 27 , 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 stomach cell, or a CD34+ cell. 
     
     
         29 . A composition comprising:
 (I)
 a viral particle that comprises the modified AAV capsid protein of  claim 1  and a nucleic acid segment that encodes a therapeutic agent; and 
   (II) a pharmaceutically-acceptable buffer, diluent, or excipient.   
     
     
         30 . The composition of  claim 29 , comprised within a kit. 
     
     
         31 . A method for providing a mammal in need thereof with a therapeutically-effective amount of a selected biological molecule, the method comprising providing to a cell, tissue or organ of a mammal in need thereof, an amount of the the composition of  claim 29 ; and for a time effective to provide the mammal with a therapeutically-effective amount of the selected biological molecule. 
     
     
         32 . A method for diagnosing, preventing, treating, or ameliorating at least one or more symptoms of a disease, a disorder, a dysfunction, an injury, an abnormal condition, or trauma in a mammal, the method comprising, administering to a mammal in need thereof the rAAV particle of  claim 14 , in an amount and for a time sufficient to diagnose, prevent, treat or ameliorate the one or more symptoms of the disease, disorder, dysfunction, injury, abnormal condition, or trauma in the mammal. 
     
     
         33 . The method of  claim 32 , wherein the mammal is human. 
     
     
         34 . 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 13 . 
     
     
         35 . The method of  claim 34 , wherein the population of cells comprise a CD34+ cell, a stem cell, a hematopoietic cell, an endothelial cell, an epithelial cell, a vascular cell, a dendritic cell, a blood cell, a fibroblast, a cancer cell, or a cell from the liver, the lung, the heart, the pancreas, the intestines, the kidney, or the brain of a mammal.