Constructs and methods for delivering molecules via viral vectors with blunted innate immune responses

Abstract

A CpG-modified recombinant adeno-associated viral (AAV) vector is described. The vector carries a nucleic acid molecule comprising AAV inverted terminal repeat (ITR) sequences and an exogenous gene sequence under the control of regulatory sequences which control expression of the gene product, in which the nucleic acid sequences carried by the vector are modified to significantly reduce CpG di-nucleotides such that an immune response to the vector is reduced as compared to the unmodified AAV vector. Also provided are methods and regimens for delivering transgenes using these AAV viral vectors, in which the innate immune response to the vector and/or transgene is significantly modulated.

1 . A recombinant adeno-associated viral (AAV) vector comprising AAV capsid having packaged therein a nucleic acid molecule comprising at least one copy of AAV inverted terminal repeat (ITR) sequences and an exogenous gene sequence under the control of regulatory sequences which control expression of the gene product, wherein said sequences of said nucleic acid molecule are modified to reduce CpG di-nucleotides such that in immune response to the vector is reduced as compared to the unmodified AAV vector. 
     
     
         2 . The recombinant AAV vector according to  claim 1 , wherein the vector is deleted of functional AAV capsid coding sequences and functional AAV rep coding sequences. 
     
     
         3 . The recombinant AAV vector according to  claim 1 , wherein the exogenous gene sequence contains a reduced number of CpG di-nucleotides as compared to the native coding sequence and the untranslated regions for the gene product. 
     
     
         4 . The recombinant AAV vector according to  claim 1 , wherein the regulatory sequences are mutated to reduce or eliminate CpG di-nucleotides. 
     
     
         5 . The recombinant AAV vector according to  claim 1 , wherein the regulatory sequences are selected from the group consisting of one or more of: a promoter, an enhancer, an intron, microRNAs, and polyA sequence. 
     
     
         6 . The recombinant AAV vector according to  claim 1 , wherein the one or both of the 5′ and the 3′ AAV ITRs are mutated to reduce or eliminate native CpG di-nucleotides. 
     
     
         7 . The recombinant AAV vector according to  claim 1  comprising a 5′ AAV ITRs selected from the group consisting of a CpG-depleted AAV ITR, a wild-type AAV ITR, a CpG-modified self-complementary ITRs, and a self-complementary ITR. 
     
     
         8 . The recombinant AAV vector according to  claim 1  comprising a 3′ AAV ITRs selected from the group consisting of a CpG-depleted AAV ITR, a wild-type AAV ITR, a CpG-modified self-complementary ITRs, and a self-complementary ITR. 
     
     
         9 . The recombinant AAV vector according to  claim 1 , wherein untranslated regions and/or any vector DNA sequences are CpG-depleted. 
     
     
         10 . The recombinant AAV vector according to  claim 1 , wherein the number of CpG di-nucleotides in the nucleic acid molecule is reduced by at least 30% as compared to a nucleic acid molecule having the corresponding native ITRs, native exogenous gene sequence and native regulatory sequence. 
     
     
         11 . The recombinant AAV vector according to  claim 1 , wherein the AAV vector retains at least 95% of the protein expression levels as compared to a nucleic acid molecule having the corresponding native ITRs, native exogenous gene sequence and native regulatory sequence. 
     
     
         12 . The recombinant AAV vector according to  claim 1 , wherein the AAV vector is completely depleted of CpG di-nucleotides. 
     
     
         13 . The recombinant AAV vector according to  claim 1 , wherein said AAV vector has a capsid selected from AAV8 and rh32/33. 
     
     
         14 . A pharmaceutical composition comprising a recombinant AAV vector according  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         15 . The composition according to  claim 14 , wherein said composition is formulated for parenteral delivery. 
     
     
         16 . The composition according to  claim 14 , wherein said composition is formulated for intramuscular delivery. 
     
     
         17 . A method for improving adeno-associated virus (AAV)—mediated gene expression, said method comprising:
 a) generating an AAV viral particle comprising a modified packaging insert, wherein said packaging insert comprises a nucleic acid molecule comprising two AAV inverted terminal repeat (ITR) and an exogenous gene sequence under the control of regulatory sequences which control expression of the gene product, wherein said sequences of said nucleic acid molecule are modified to reduce CpG di-nucleotides such that in immune response to the vector is reduced as compared to the unmodified AAV vector without significant reduction in expression of the gene product; and 
 b) delivering the AAV to a subject. 
 
     
     
         18 . The method according to  claim 17 , wherein the AAV ITRs are mutated to eliminate native CpG di-nucleotides. 
     
     
         19 . The method according to  claim 17 , wherein the regulatory sequences are mutated to eliminate CpG di-nucleotides. 
     
     
         20 . The method according to  claim 17 , wherein the exogenous gene sequence contains a reduced number of CpG di-nucleotides as compared to the native coding sequence for the gene product. 
     
     
         21 . A regimen for repeat administration of a gene product, said regimen comprising:
 a) delivering to a subject an adeno-associated viral (AAV) vector having an AAV capsid having packaged therein a nucleic acid molecule comprising two AAV inverted terminal repeats (ITR) and an exogenous gene sequence under the control of regulatory sequences which control expression of the gene product, wherein said sequences of said nucleic acid molecule are modified to reduce CpG di-nucleotides such that in immune response to the vector is reduced as compared to the unmodified AAV vector without significant reduction in expression of the gene product; and   b) delivering to the subject a second vector comprising the exogenous gene sequence.