Capsid-modified, raav3 vector compositions and uses in gene therapy of human liver cancer

Abstract

Disclosed are next-generation multi-mutated capsid protein-modified rAAV expression vectors, as well as infectious virions, compositions, and pharmaceutical formulations that include them. Also disclosed are methods of preparing and using these high transduction efficiency vector constructs in a variety of therapeutic applications including, inter alia, as delivery agents for the treatment or amelioration of one or more diseases or abnormal conditions in an affected mammal using in vivo and/or ex situ viral vector-based gene therapy protocols. Also disclosed are large-scale production methods for the multi-mutated, capsid-modified rAAV expression vectors, viral particles, and infectious virions, as well as use of the disclosed compositions in the manufacture of medicaments for use in a variety of in vitro and/or in vivo therapeutic methodologies.

1 . An rAAV3 vector comprising a modified capsid protein that comprises:
 (a) a non-tyrosine amino acid residue at one or more positions corresponding to Y252, Y272, Y444, Y701, Y705, and Y731 of the wild-type AAV3 capsid protein as set forth in SEQ ID NO:3;   (b) a non-serine amino acid residue at each of one or more positions corresponding to S459 or S663, of the wild-type AAV3 capsid protein as set forth in SEQ ID NO:3;   (c) a non-threonine amino acid residue at each of one or more positions corresponding to T251 or T492 of the wild-type AAV3 capsid protein as set forth in SEQ ID NO:3;   (d) a non-lysine amino acid residue at each of one or more positions corresponding to K528, K533, or K545 of the wild-type AAV3 capsid protein as set forth in SEQ ID NO:3;   (e) (i) a non-tyrosine amino acid residue at position Y701, or Y705; and (ii) a non-tyrosine amino acid residue at position Y705 or Y731, or a non-serine amino acid residue at position S663 of the wild-type AAV3 capsid protein as set forth in SEQ ID NO:3;   (f) a combination of three or more amino acid substitutions listed in (a), (b), (c), and (d); each with a non-native amino acid;   (g) a combination of four or more amino acid substitutions listed in (a), (b), (c), and (d); each with a non-native amino acid; or   (h) a combination of five or more amino acid substitutions listed in (a), (b), (c), and (d); 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, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, and AAV10, as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively.   
     
     
         2 . The rAAV3 vector in accordance with  claim 1 , wherein 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); or the non-tyrosine, non-lysine, 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). 
     
     
         3 . The rAAV3 vector in accordance with  claim 1 , wherein the combination of three or more amino acid substitutions include a non-native amino acid substitution at one or more of the following combination of acid residues:
 (a) Y701F, Y705F, and Y731F;   (b) Y705F, Y731F, and S663V;   (c) Y705F, Y731F, and T492V;   (d) Y705F, Y731F, K533R;   (e) S663V, T492V, and K533R;   (f) Y705F, Y731F, S663V, and T492V; or   (g) Y705F, Y731F, S663V, T492V, and K533R, of the wild-type AAV3 capsid protein as set forth in SEQ ID NO:3, or at the equivalent surface-exposed amino acid residues in any one of the corresponding wild-type AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 capsid proteins, as set forth in SEQ ID NO:1, SEQ ID NO:2, 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, respectively, or in any combination thereof.   
     
     
         4 . The rAAV3 vector in accordance with  claim 1 , wherein the transduction efficiency of a virion comprising the vector is about 2- to about 50-fold higher in a selected mammalian host cell than that of a virion that comprises a corresponding, unmodified, rAAV3 vector. 
     
     
         5 . The rAAV3 vector in accordance  claim 1 , wherein the transduction efficiency of a virion comprising the vector is about 6- to about 40-fold higher in a selected mammalian host cell than that of a virion that comprises a corresponding, unmodified, rAAV3 vector. 
     
     
         6 . The rAAV3 vector in accordance with  claim 1 , wherein the transduction efficiency of a virion comprising the vector is about 8- to about 30-fold higher in a selected mammalian host cell than that of a virion that comprises a corresponding, unmodified, rAAV3 vector. 
     
     
         7 . The rAAV3 vector in accordance with  claim 1 , wherein the virion comprising the vector is less susceptible to ubiquitination when introduced into a mammalian cell than that of a virion that comprises a corresponding, unmodified, rAAV3 vector. 
     
     
         8 . The rAAV3 vector in accordance with  claim 1 , wherein the vector further comprises a nucleic acid segment that encodes a diagnostic, therapeutic, or chemotherapeutic agent operably linked to a promoter capable of expressing the nucleic acid segment in a suitable host cell comprising the vector. 
     
     
         9 . The rAAV3 vector in accordance with  claim 8 , 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. 
     
     
         10 . The rAAV3 vector in accordance with  claim 8 , further comprising at least a first mammalian intron sequence operably linked to the nucleic segment. 
     
     
         11 . The rAAV3 vector in accordance with  claim 8 , 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. 
     
     
         12 . The rAAV3 vector in accordance with  claim 10 , wherein the promoter is a liver-specific promoter, a tumor cell-specific promoter, or a combination thereof. 
     
     
         13 . The rAAV3 vector in accordance with  claim 8 , 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. 
     
     
         14 . The rAAV3 vector in accordance with  claim 8 , wherein the at least a first nucleic acid segment encodes a chemotherapeutic agent. 
     
     
         15 . The rAAV3 vector in accordance with  claim 8 , wherein the diagnostic, therapeutic or chemotherapeutic 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 kinase 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 cytotoxic agent, a cytostatic agent, an anti-inflammatory agent, or any combination thereof. 
     
     
         16 . The rAAV3 vector in accordance with  claim 1 , comprised within an adeno-associated viral particle or infectious rAAV3 virion. 
     
     
         17 - 29 . (canceled) 
     
     
         30 . A method for providing a mammal in need thereof with a diagnostically- or 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 rAAV3 vector in accordance with  claim 1 ; and for a time effective to provide the mammal with a diagnostically- or a therapeutically-effective amount of the selected biological molecule. 
     
     
         31 . A method for diagnosing, preventing, treating, or ameliorating at least one or more symptoms of liver cancer, including HCC, in a mammal, the method comprising, administering to a mammal in need thereof the rAAV3 vector in accordance with  claim 1 , in an amount and for a time sufficient to diagnose, prevent, treat or ameliorate the one or more symptoms of the liver cancer in the mammal. 
     
     
         32 . The method in accordance with  claim 31 , wherein the mammal is human. 
     
     
         33 . A method of transducing a population of liver cells or liver tumor cells in a human diagnosed with, having, or suspected of having HCC; the method comprising administering to the human, a composition that comprises an effective amount of the rAAV3 vector in accordance with  claim 1 , for a time effective to transduce the population of liver cells or liver tumor cells.